tissue, which predominantly expresses α₂A adrenoceptors, and striatal tissue, which expresses both α₂A and α₂C adrenoceptors. Binding events were further studied with computer modeling of ligand docking. The results not only provide conclusive evidence for α₂A and α₂C adrenoceptors being both norepinephrine and dopamine receptors, but also for being common targets for compounds previously characterized as Gi/o-coupled dopamine receptor ligands. Furthermore, the present study demonstrates the Gαi/o protein subtype is fundamental in the determination of the unique pharmacological profile of Gi/o-coupled catecholamine receptor ligands.

P-21

INTERLEUKIN-1 RECEPTOR ANTAGONIST (IL-1RA) AND BRAIN ENDOCANNABINOIDS CROSSTALK TO CONTROL NEUROGENESIS

Molina-Holgado F., de Coteau Volker Behrends K.S., Lacroix L.

Health Science Research Centre, Department of Life Sciences,
University of Roehampton, London, UK

Crosstalk between neuroimmune networks and the brain endocannabi-
noid (eCB) system contribute to the maintenance of neurogenesis.
Moreover, the eCB system directs cell fate specification of neural stem
cells (NSC) in the central nervous system (CNS). We have previously
shown that the activation of CB1 and CB2 cannabinoid receptors sup-
pressed chronic inflammatory responses through the attenuation of pro-
inflammatory mediators such as interleukin-1β (IL-1β) by increasing
the expression of IL-1 receptor antagonist (IL-1RA), an endogenous
antagonist for the actions of IL-1 in the CNS. Endogenous IL-1RA
mediates the neuroprotective and anti-inflammatory actions of CBs in
primary neurons and glia. These effects appear to be mediated by both
CB1 and CB2 receptors. CB-induced IL-RA release may negatively
regulate IL-1 actions in the brain, via IL-1RA blocking the IL-1 recep-
tor (IL-1RI), after inflammatory or excitotoxic insults. Interestingly,
receptors for cannabinoids (CB1 and CB2 receptors) and interleukin-1
are co-expressed in NSC. In order to further explore the effects of IL-
1RA on endocannabinoid signalling in NSC the levels of the endo-
cannabinoids 2-arachidonoylglycerol (2-AG), 1-AG and anandamide
(AEA) were detected using liquid chromatography-mass spectrometry
(LC-MS) on a Waters Acquity H-Class UPLC coupled to TQSmicro
triple quadrupole mass spectrometer following IL-1RA treatment.
Treatment with IL-1RA caused marked increases in the levels of AEA
(approximately three-fold) and 2-AG (approximately three-fold) in
NSC nuclear extracts respectively, compared to the control group.
Whereas, in supernatants the levels of 2-AG and 1-AG and AEA were
similar to that obtained in the control group. In this study we show for
the first time that acute administration with IL-1RA significantly
increases levels of AEA and 2-AG in NSC. Thus it may be hypothe-
sised that IL-1RA increases proliferation by increasing the levels of
endocannabinoids, which acts via CB1 or CB2 receptors. These results
provide crucial new insights into the effects of IL-1RA in regulating
NSC proliferation and the pathways involved, and highlight the thera-
peutic potential of their interplay with eCB signalling in brain repair.

P-22

SEROTONIN 5-HT₃ RECEPTOR ANTAGONISM POTENTIATES THE ANTIDEPRESSANT ACTIVITY OF CITALOPRAM

Perez-Palomar B.¹,², Mollinedo-Gajate I.¹,², Meana J.J.¹,²,³, Ortega J.E.¹,²,³

¹Department of Pharmacology, University of the Basque Country
UPV/EHU, Leioa, Bizkaia, Spain; ²Centro de Investigación Biomédica
en Red de Salud Mental CIBERSAM, Spain; ³BioCruces Health
Research Institute, Bizkaia, Spain

Selective serotonin reuptake inhibitors (SSRIs) are able to regulate the activity of different neurotransmission pathways involved in

depression. The administration of the SSRI citalopram increases sero-
tonin release in the locus coeruleus (LC), contributing to modulate the
function of this noradrenergic nucleus. It has been shown that local 5-
HT₃ receptor activation in LC decreases noradrenaline (NA) release in
prefrontal cortex (PFC). In this context, the blockade of 5-HT₃ recep-
tors in co-administration with SSRIs has been proposed to avoid coun-
tertherapeutic effects of SSRIs in the early stages of treatment.

In the present study, the role of 5-HT₃ receptors in the effect exerted
by citalopram was investigated by dual-probe microdialysis in rats.
Systemic administration of the 5-HT₃ agonist SR57227 (10 mg/kg i.p.)
increased NA in LC (Emax = 200 ± 27%; *p* < 0.0001) and PFC
(Emax = 133 ± 2%; *p* < 0.05). Such cortical NA increase was
enhanced in the presence in LC of the 5-HT₃ antagonist Y25130
(50 µM) (Emax = 296 ± 41%; *p* < 0.0001). Local administration in
PFC of SR57227 (1–100 µM) increased NA in the area
(Emax = 815 ± 148%; *p* < 0.0001). This effect was attenuated by the
cortical presence of Y25130 (Emax = 366 ± 58%; *p* < 0.0001), which
is compatible with the existence of cortical 5-HT₃ receptors involved
in NA release.

Systemic citalopram (10 mg/kg i.p.) increased NA in LC (Emax = 185 ± 11%; *p* < 0.01) and decreased it in PFC (Emax = -35 ± 7%; *p* < 0.001). In the presence in LC of the 5-HT₃ antagonist Y25130, both effects were blocked. When Y25130 (10 mg/kg i.p.) was pre-administered systemically with citalopram, the increment of NA in LC was blocked, whereas in PFC, NA switched from an inhibition (Emax = -40 ± 5%; *p* < 0.001) to an increase (Emax = 117 ± 8%; *p* > 0.05).

These results show that the addition of a 5-HT₃ receptor antagonist is able to reverse the decrease of NA release in PFC exerted by systemic citalopram. Therefore, co-administration of a 5-HT₃ receptor antagonist with SSRIs could represent a strategy to improve antidepressant response.

Key words: locus coeruleus; citalopram; noradrenaline; 5-HT3

P-23

SYNTHESIS OF NEW 1-SUBSTITUTED ISOQUINOLINES WITH POTENTIAL ANTI-PARKINSON ACTIVITY

Silva A.G.¹, Parraga J.², Vila L.³, Sanz M.J.²,³, Loza M.I.¹, Castro M.A.¹, Cortes D.², Cabedo N.³

¹Bioarma Research Group (GI-1685), Universidade de Santiago de Compostela, Galicia, Spain; ²Pharmacology Department, Universitat de València, Valencia, Spain; ³Health Research Institute – INCLIVA, Valencia, Spain

**Introduction/Objectives:** Dopaminergic ligands can bind to D₁-like (D₁ and D₅) and D₂-like (D₂, D₃ and D₄) dopamine receptors (DR) to “restore” the dopaminergic pathway. Agonists can be useful in the treatment of Parkinson’s disease. Tetrahydroisoquinolines (THIQs) display important pharmacological activities including DR binding. Therefore, the aim of this study was to obtain new 1-substituted THIQs with dopaminergic activity.

**Materials and Methods:** (E)-1-Styryl-THIQs and (E)-1-(propenyl)-THIQs were synthesized *via* Bischler–Napieralski cyclization, and tested *in vitro* for their affinity towards DR in rat striatum. Functional assays to agonist activity was performed by measuring inhibition of forskolin-stimulated cyclic AMP production in CHO-K1 cells stably expressing human D₂ receptors. Cytotoxicity studies were carried out in both human neutrophils and HUVEC by MTT assay. Molecular modelling studies (MM) on DRs were performed to determine ligand/receptor complex interactions.

**Results:** 1-Substituted IQs were synthesized bearing 1-styryl or 1-pro-
pene substituent. Catecholic IQs displayed affinity towards D₁-DR and
D₂-DR at µM and nM concentrations, respectively. N-methyl or N-allyl
groups improved considerably the affinity towards D₂-like DR. The
most active compounds, 1e and 2e, also showed high selectivity
(Kᵢ = 41 nM and 18 nM; Kᵢ D₁/D₂ ratio = 147 and 95, respectively).
The cAMP assays indicated that 1e and 2e behaved as full agonist
(EC₅₀ = 500 nM and 555 nM, respectively) with maximal efficacy
values similar to quinpirole at 10 µM. None of these THIQs displayed relevant cytotoxicity in human cells. In agreement with the experimental data, MM studies on DRs revealed stronger molecular interactions with D₂-DR than with D₁-DR.

**Conclusions:** The catechol group and *N*-substitution at the *IQ* nucleus improved the affinity towards D₂-DR. Therefore, 1e and 2e, are potential candidates to be used in the treatment of PD.

<figure><img src="image_2.png" /></figure>

1-(Styryl)-THIQ 1e
*(series 1)*

<figure><img src="image_3.png" /></figure>

1-(Prop-1-en-yl)-THIQ 2e
*(series 2)*

**Key words:** Tetrahydroisoquinolines; synthesis; dopamine receptors; Parkinson disease

P-24
OPTICAL CONTROL OF PAIN IN VIVO WITH A PHOTOACTIVE MGLU<sub>5</sub> RECEPTOR NEGATIVE ALLOSTERIC MODULATOR

López-Cano M.<sup>1,2,*</sup>, Font J.<sup>3,4,*</sup>, Notartomaso S.<sup>5</sup>, Scarselli P.<sup>5</sup>, Di Prieto P.<sup>5</sup>, Bresoló-Obach R.<sup>6</sup>, Battaglia G.<sup>5</sup>, Malhaire F.<sup>7,8</sup>, Rovira X.<sup>7,8</sup>, Catena J.<sup>3</sup>, Giraldo J.<sup>4</sup>, Pin J.P.<sup>7,8</sup>, Fernández-Dueñas V.<sup>1,2</sup>, Goudet C.<sup>7,8</sup>, Nonell S.<sup>6</sup>, Nicoletti F.<sup>5,9</sup>, Llebaria A.<sup>3,†</sup>, Ciruela F.<sup>1,2,†</sup>

¹Unitat de Farmacologia, Departament Patologia i Terapèutica
Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain; ²Institut de Neurociències, Universitat de Barcelona, Spain; ³Institute for Advanced Chemistry of Catalonia (IQAC-CSIC), Barcelona, Spain; ⁴Institut de Neurociències and Unitat de Bioestadística, Universitat Autònoma de Barcelona, Network Biomedical Research Center on Mental Health (CIBERSAM), Bellaterra, Spain; ⁵I.R.C.C.S. Neuromed, Pozzilli, Italy; ⁶Institut Químic de Sarrià, Universitat Ramon Llull, Barcelona, Spain; ⁷Institute of Functional Genomics, Université de Montpellier, Unité Mixte de Recherche 5302 CNRS, Montpellier, France; ⁸Unité de recherche U1191, INSERM, Montpellier, France; ⁹Department of Physiology and Pharmacology, University Sapienza, Rome, Italy

Optopharmacology is a very promising approach to manipulate physi-
logical processes with high spatiotemporal resolution. Indeed, the use of
light-activatable drugs (i.e. caged-compounds) targeting G protein-
coupled receptors (GPCRs) in space and time will provide extraordinary
pharmacological precision. Interestingly, experimental evidences shown
that the metabotropic glutamate type 5 receptor (mGlu5 receptor) is
widely expressed through the pain neuraxis, thus playing a key role in
pain transmission. Importantly, the specific mGlu5 receptor anatomical
location within the pain neuraxis determines its pro- or antinociceptive
action, thus the precise spatial manipulation of these receptors will deter-
mine the ultimate pain sensation. Thereby, selective mGlu5 receptor
negative allosteric modulators (NAMs) have consistently shown anal-
gesic activity in experimental animal models of inflammatory pain.
Accordingly, we synthesized a caged derivative compound, namely the
JF-NP-26, using the chemical structure of raseglurant, an mGlu5 recep-
tor NAM, as a molecular scaffold. JF-NP-26 can be photo-activated by
violet light (405 nm) irradiation to release active raseglurant. We evalu-
ated the light-dependent ability of JF-NP-26 to preclude the mGlu5
receptor function both in cultured cells and striatal neurons. Importantly,
JF-NP-26 showed antinociceptive activity in an animal model of pain
upon irradiation both at the peripheral (i.e. hind paw) and central level
(i.e. thalamus) but did not show this specific effect when irradiated in
unaffected areas (i.e. contralateral hind paw) or brain areas not involved
in pain sensation (i.e. Striatum). Overall, these results demonstrated for
the first time the usefulness of using light in combination with photoac-
tive drugs for pain treatment.

**Acknowledgements:** This work was supported by MINECO/ISCIII (SAF2014-55700-P and PIE14/00034), the Catalan government (2014 SGR 1054), ICREA (ICREA Academia-2010), Fundació la Marató de TV3 (Grant 20152031) and IWT (SBO-140028).

*These authors contributed equally.

†These authors jointly directed this work.

P-25
THE USE OF LICOCHALCONE A AS PRE-EMPTIVE PHARMACOLOGICAL INHIBITION OF JNK1, LEADS TO NEURONAL PROTECTION AGAINST EXCITOTOXIC INSULTS DERIVED OF KAINIC ACID

Busquets O.<sup>1,2,3</sup>, Ettcheto M.<sup>1,2,3</sup>, Verdaguer E.<sup>3,4,5</sup>, Dario R.<sup>4,6</sup>, Auladell C.<sup>3,4,5</sup>, Beas-Zarate C.<sup>6</sup>, Pallàs M.<sup>2,3,5</sup>, Folch J.<sup>1,3</sup>, Camins A.<sup>2,3,5</sup>

¹Departament de Bioquímica i Biotecnologia, Facultat de Medicina i Ciències de la Salut, Universitat Rovira i Virgili, Reus, Tarragona, Spain; ²Departament de Farmacologia i Farmacognòsia, Facultat de Farmàcia i Ciències de l’Alimentació, Universitat de Barcelona, Barcelona, Spain; ³Biomedical Research Networking Centre in Neurodegenerative Diseases (CIBERNED), Madrid, Spain;

⁴Departament de Biologia Cellular, Fisiologia i Immunologia, Facultat de Biologia, Universitat de Barcelona, Barcelona, Spain; ⁵Institut de Neurociències, Universitat de Barcelona, Barcelona, Spain;

⁶Departamento de Biologia Celular y Molecular, C.U.C.B.A., Universidad de Guadalajara y División de Neurociencias, Sierra Mojada 800, Col. Independencia, Guadalajara, Jalisco, Mexico

The mitogen-activated protein kinase family (MAPK) is an important
group of enzymes involved in cellular responses to diverse external
stimuli. One of the members of this family is the c-Jun-N-terminal
kinase (JNK). In particular, JNK1 is involved in neurodegenerative dis-
eases like epilepsy and Alzheimer's disease. Kainic acid (KA) is a
chemoconvulsant that mimics temporal lobule epilepsy (TLE) in
humans. This molecule triggers neuronal death and glial reactivity,
mechanisms modulated by the JNK1.

The root of *Glycyrrhiza inflata* is known to be a source for characteris-
tic phenolic compounds that have various pharmacological applica-
tions. One of them, Licochalcone A (Lic-A), which inhibits the
activity of the JNK1 isoform, is a suitable compound to prevent the
neuronal degeneration induced by epileptic seizures. The aim of this
study was to analyse the effect of Lic-A as a new approach for the
treatment of epilepsy. Our results showed that an intraperitoneal pre-
treatment of Lic-A, previous to the KA administration caused a reduc-
tion in the convulsive pattern. Therefore, it was confirmed that Lic-A
reduced the phosphorylation levels of the JNK. Also, several biomark-
ers were evaluated and it was observed that the neuronal degeneration
associated with the KA administration completely disappeared, along
with a significant reduction of apoptotic signals (BAX, BIM, calpain
and caspase 3 activity), glial reactivity and cellular stress agents
release like NO and TNFα. Additionally, the response of multiple pro-
teins related to survival responses like AKT and CREB, was evaluated,
and the data provided that Lic-A was able to reduce the activation of
these pathways.

In conclusion, the effects of Lic-A give neuroprotection against the
damage derived of KA administration. Therefore, this molecule could
be a new potential drug for the treatment of epilepsy.

**Keywords:** Licochalcone A; epilepsy; kainic acid; JNK; apoptosis; neuroinflammation

P-26
IN VIVO PHOTOMODULATION OF GABA AND GLYCINE RECEPTOR CHANNELS

Gomila A.<sup>1</sup>, Wutz D.<sup>2</sup>, Maleeva G.<sup>3</sup>, Rovira X.<sup>1</sup>, Camarero N.<sup>1</sup>, Mukhametov E.<sup>3,4</sup>, Rustler K.<sup>2</sup>, Koenig B.<sup>2</sup>, Bregestovski P.<sup>3</sup>, Gorostiza P.<sup>1</sup>

¹Institute for Bioengineering of Catalonia, Barcelona, Spain; ²Institute of Organic Chemistry, University of Regensburg, Regensburg,
Germany; ³Institut de Neurosciences des Systèmes, Aix-Marseille University, Marseille, France; ⁴Open Lab of Motor Neurorehabilitation, Kazan Federal University, Kazan, Russia

The photochromic compound, UR-DW290, composed of diazepam and azobenzene groups, was studied *in vivo* and *in vitro*.

Whole-cell patch-clamp recordings were used to study ionic currents of GABA and glycine receptors and Danio rerio larvae (7 and 8 days postfertilization) swimming behaviour was studied and analysed using a ViewPoint ZebraLab system.

Under visible light, currents mediated by GABA receptors formed by α1/β2/γ2 subunits were weakly inhibited by 50 µM UR-DW290 (on ≈15% and ≈5% at GABA 5 and 300 µM, respectively), while at UV illumination the inhibition was entirely absent. GlyRs were more effectively and subunit-specifically inhibited by UR-DW290. On mammalian or zebrafish α2 GlyRs, at visible light, UR-DW290 provoked inhibition of currents induced by 20 µM glycine on 38 ± 7% and 32 ± 3%, respectively. It caused even much stronger suppression upon UV illumination: on 73 ± 6% and 64 ± 2% for mammalian or zebrafish α2 GlyRs, respectively. Action on α1 GlyRs exhibited similar tendency, although with less efficiency. With increase of glycine concentration inhibitory power of UR-DW290 decreased, suggesting a competitive mechanism of the antagonist action. The behavioural analysis of zebrafish larvae (7 and 8 days post fertilization) showed an excitatory effect of UR-DW290. Animals treated with UR-DW290 (100 µM) experienced higher activity behaviour during the relaxation period (20 min in absence of light stimuli) in comparison to controls and vehicle animals. Notwithstanding their permanent excitation state, under 365 nm light animals treated with UR-DW290 evoked an overreaction to light changes which was reduced to control animal levels during blue light (455 nm) application.

We found significant differences for different behavioural parameters between UR-DW290 treated groups and controls including total animal activity and swimming distance, startle responses to light changing conditions and habituation time. We observed how in the presence of UR-DW290 animal responses to light stimuli can be tuned in a dose dependent way and how swimming behaviour, such as exploratory capacities, can be triggered.

**Key words:** photopharmacology; behaviour; GABA; glycine

P-27

THE MITOCHONDRIAL SUBTYPE-2 ANGIOTENSIN II RECEPTOR EXPRESSION DECLINES WITH AGEING, AND MODULATES THE CATECHOLAMINE SECRETION IN CHROMAFFIN CELLS

Fernando Padín J. <fnref n="1" />,²,<fnref n="3" />, Méndez-López I. <fnref n="1" />,², Musial D.C. <fnref n="1" />,<fnref n="4" />, Garcia A.G. <fnref n="1" />,²,<fnref n="3" />

Renin-Angiotensin-Aldosterone System regulates cardiovascular and renal function, where angiotensin II is the main final effector molecule. Renin is released from the juxtaglomerular cells in response to urine osmolarity. Moreover, adrenaline secreted in response to sympathetic stimulation from chromaffin cells (CCs) can also release renin throughout β₁-adrenergic receptor. Angiotensin actions are mediated by angiotensin type-1 (AT₁) and -2 (AT₂) receptors, which are localized in cytoplasmic membrane of different cell types. In addition, the AT₂ receptors could be also located in mitochondrial membrane (mAT₂), releasing nitric oxide (NO), and regulating the mitochondrial respiration. Interestingly, the major expression of mAT₂ occurs during foetal life and it decline with ageing, throughout the maturation of the adrenal gland innervation by the splanchnic nerve. The importance of the absence of the innervation in foetal life is that the main

physiological stimulus is the hypoxia. The sense is that the adrenaline released by hypoxic stimulus protects the newborn from the tissue damage by means of changes in vascular perfusion and pulmonary maturation. In spite of the importance, it remains already unexplored in CCs, and we have wanted to research it. In our study, we have observed that the embryo-CCs shows with respect to adult: i) three-folds increased adrenaline secretion; ii) overall AT₁ expression does not change, but AT₂ receptor is three-folds decreased; iii) AT₂ receptor is localized in mitochondria, but not in adult; iv) mitochondrial depolarization induced by FCCP rose in six-folds the secretion; v) mAT₂ receptor stimulation is associated with NO-releasing in embryo-CCs. We conclude that mAT₂ receptor regulates adrenaline secretion in embryo-CCs by means of NO-pathway. These results are important to understanding the physiological or pathological hypoxic effect in either the foetus, newborn, or in the senescence, and could inspire new mechanism for anti-ageing drugs as well as for arterial hypertension based on angiotensin control.

P-28

UNRAVELLING THE CONTRIBUTION OF ADENOSINE A₂A RECEPTOR TO THE NEUROBIOLOGY OF SCHIZOPHRENIA

Valle-León M. <fnref n="1" />,², López-Cano M. <fnref n="1" />,², Altafaj X. <fnref n="1" />,², Contreras F. <fnref n="3" />,

Fernández-Dueñas V. <fnref n="1" />,², Valverde O.<fnref n="4" />, Menchón J.M.<fnref n="3" />, Ciruela F. <fnref n="1" />,²,<fnref n="4" />

Schizophrenia is a severe neuropsychiatric disorder with an unknown aetiology. Its pharmacotherapy mostly relies on restoring dysregulated striatal dopamine and prefrontal cortex glutamate neurotransmission. However, current treatments are often insufficient to coat all the sympathotology, thus the search for alternative and/or complementary neurotransmitter systems constitutes a big contest in psychiatry. Adenosine has been highlighted because of its relationship with both dopaminergic and glutamatergic neurotransmission. Thus, it has been postulated that restoring adenosine concentrations within schizophrenia-related brain areas might have beneficial antipsychotic properties. Here, we studied the relationship between adenosine A₂A receptor (A₂AR) and dopamine D₂ receptor (D₂R) expression, which may be related to the hyperdopaminergic hypothesis of schizophrenia. First, we used a pharmacological animal model of schizophrenia, using chronic administration of phencyclidine. Interestingly, while PCP treatment induced psychotic-like symptoms in mice, it also promoted a significant increase of D₂R expression in the striatum. On the other hand, as we recently demonstrated, the deletion of A₂AR also promotes psychotic-like symptoms. And similar to PCP-treated mice, we observed an increased D₂R expression in KO-A₂AR when compared to wild type (WT) littermates. Next, in an attempt to manipulate D₂R content in KO-A₂AR mice, which may correlate with the psychotic-like symptoms described, we assessed the effects electroconvulsive therapy (ECT). ECT increased striatal D₂R expression in WT animals, while promoting a significant reduction in KO-A₂AR mice. Importantly, this ECT-mediated D₂R reduction was followed by a concomitant improvement of the KO-A₂AR mice's sensorimotor gating impairment, which was measured by the pre-pulse inhibition test. Finally, we used an adeno-associated virus to express A₂AR in the striatum of KO-A₂AR mice, in which we confirmed the relationship between A₂AR presence/absence and the development of psychotic-like symptoms. Overall, a good correlation between striatal D₂R expression and psychotic like symptoms was established, in which it played a key role A₂AR content.

**Acknowledgements:** This work was supported by MINECO/ISCIII (SAF2014-55700-P, PCIN-2013-019-C03-03 and PIE14/00034), the

<footnote n="1">¹Instituto Teófilo Hernando y, Universidad Autónoma de Madrid, Madrid, España; ²Departamento de Farmacología, Facultad de Medicina, Universidad Autónoma de Madrid, Madrid, España;</footnote>

<footnote n="3">³Facultad de Medicina, Departamento de Ciencias Médicas (Farmacología), Universidad de Castilla-La Mancha, Ciudad Real, España; ⁴Departamento de Farmacología, Universidade Federal de São Paulo, Brazil</footnote>

<footnote n="1">¹Unitat de Farmacologia, Departament Patologia i Terapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Barcelona, Spain; ²Institut de Neurociències, Universitat de Barcelona, Spain; ³Servei de Psiquitria, Hospital Universitari de Bellvitge-IDIBELL, Universitat de Barcelona, CIBERSAM, L'Hospitalet de Llobregat, Barcelona, Spain;</footnote>

<footnote n="4">⁴Neurobiology of Behavior Research Group (GReNeC), Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain</footnote>
Catalan government (2014 SGR 1054), ICREA (ICREA Academia-2010), Fundació la Marató de TV3 (Grant 20152031) and IWT (SBO-140028).

**References:**

(1) Moscoso-Castro, M., Gracia-Rubio, I., Ciruela, F., and Valverde, O. (2016) Genetic blockade of adenosine A2A receptors induces cognitive impairments and anatomical changes related to psychotic symptoms in mice. *Eur. Neuropsychopharmacol.* 26, 1227–40.

## P-29
COCAINE ADMINISTRATION DURING A WINDOW OF ADOLESCENT VULNERABILITY INDUCED LONG-TERM CONSEQUENCES ON NEGATIVE AFFECT IN ADULT RATS

García-Fuster M.J., Keller B., García-Cabrerizo R.

IUNICS, University of the Balearic Islands, and IdISBa, Palma de Mallorca, Spain

Many studies investigated vulnerability factors that predispose to cocaine addiction, but few have focused on adolescence—a critical period for neurobehavioral plasticity and drug use. Prior studies from our laboratory demonstrated a time-specific window during early-mid adolescence (postnatal day PND 33–39) in which cocaine administration induced immediate neurotoxic effects and long-term consequences on addiction-like behaviour. Contrarily, the long-term effects of adolescent cocaine exposure on inducing a pro-depressive phenotype were not clear and thus were the aim of this study. Adolescent male Sprague-Dawley rats were exposed to the forced swim test (FST, pre-test, PND 28 and test, PND 29: for baseline immobility levels). Then, rats were treated with saline (0.9% NaCl, 1 ml/kg, n = 21) or cocaine (5 mg/kg, n = 23) for 7 days (PND 33–39). Following prolonged withdrawal, rats were exposed to the FST (5 min test, PND 71) 45 min after a challenge dose of saline (saline-saline, n = 9; cocaine-saline, n = 11) or cocaine (saline-cocaine, n = 12; cocaine-cocaine, n = 12). Rats were then exposed to a two-bottle choice test (1% sucrose preference, PND 76–77). The main results showed that cocaine administration during adolescence (PND 33–39) induced long-term consequences on negative affect in adulthood as measured by: (1) a pro-depressive phenotype (i.e., increased immobility time in the FST: +63 ± 17 s, *p* < 0.001), and (2) a pro-anhedonic phenotype (i.e., decreased sucrose consumption on PND 77: -17 ± 7 g, *p* < 0.05) when comparing saline-cocaine vs. cocaine-cocaine groups. These results extend our prior data, which showed that cocaine administration at this window of adolescent vulnerability (PND 33–39) induced long-term effects on addiction-like behaviour, to also include long-term consequences on negative affect, and therefore confirming that early drug use leads to enduring behavioural negative consequences. Supported by ‘Delegación del Gobierno para el Plan Nacional sobre Drogas’ (MSSSI, Grants 2012/011, 2016/002) and ‘Ramón y Cajal’ Program to M.J.G.-F. (MINECO), and by RTA-RD16/0017/0010.

**Key words:** adolescence; cocaine; consequences; affect

## P-30
DUAL-SPECIFICITY PHOSPHATASE DUSP6 REGULATES LONG-TERM MEMORY AND ERK/MAP-KINASE SIGNALING IN HIPPOCAMPUS

Alcon C., Pellín C., Sindreu C.

Department of Clinical Foundations, University of Barcelona, Barcelona, Spain

Transient activation of the Erk/MAP-kinase pathway in hippocampal CA1 neurons is essential for contextual memory. However, little is known about the phosphatase(s) that directly regulate Erk1/2 during memory formation. Here we show that Dusp6 (a dual-specificity Y/T-phosphatase) is abundant in hippocampus, localizing in postsynaptic compartments of CA1 and CA3 pyramidal cells, including dendritic spines. Dusp6-KO mice showed marked deficits in contextual fear

conditioning, object recognition, and reversal of spatial reference memory. Notably, expression of short hairpin RNAs against Dusp6 via adeno-associated vectors in area CA1 reproduced the memory deficits observed in global KO mice. Binding between Dusp6 and Erk1/2 transiently increased after contextual training, suggesting that Dusp6 regulates the time course of Erk1/2 activation. In keeping with this, the allosteric inhibitor of Dusp6, (E)-2-Benzylidene-3-(cyclohexylamino)-2,3-dihydro-1H-inden-1-one, prolonged increases in Erk1/2 and CREB phosphorylation in hippocampal neurons following synaptic stimulation. Furthermore, Dusp6-KO mice showed increased basal activation of Erk1/2 and downstream CREB, as well as impaired induction by training. Interestingly, Dusp6-KO mice also showed elevated levels of PSD-95, GluN2A and pY-GluN2B, indicating possible effectors underlying the memory deficits. Collectively, the results indicate that Dusp6 plays a critical role in hippocampus-dependent long-term memory, and underscore the importance of Dusp6 in Erk1/2 regulation in neurons.

**Key words:** MAP kinase; Dusp6; hippocampus; memory;

## P-31
ACTIONS OF LITHIUM TREATMENT ON OXIDATIVE STRESS MARKERS IN MITOCHONDRIAL COMPLEX I AND COMPLEX III INHIBITION AND AFTER CO₂ EXPOSURE IN SH-SY5Y CELLS

Martinez-Pinteño A., Sanchez J., Ferrero Y., Marmol F.

Departament de Fonaments Clínicss, Unitat de Farmacologia, Facultat de medicina, Universitat de Barcelona, Barcelona, Spain

Bipolar disorder is a life-threatening psychiatric illness characterized by mood disturbances with recurrent periods of mania, hypomania and depression. Lithium is the classic mood stabilizer, but the basis of its therapeutic effect remains unclear. There is evidence that lithium exerts neuroprotective effects by increasing resistance to oxidative stress and it has been shown to stimulate cell proliferation. This study aims to evaluate the effects of lithium on resistance to oxidative stress and cell proliferation in the absence or presence of carbon dioxide (CO₂), rotenone (0.15 µM) an inhibitor of mitochondrial complex I or antimycin A (10 nM) an inhibitor of mitochondrial complex III. SH-SY5Y cells were treated for 72 h with lithium (0.5–5 mM). Superoxide anion (O₂⁻) and hydrogen peroxide (H₂O₂) production were measured for fluorescence. All the experiments were carried out under standard conditions, after 4 h of CO₂ exposure or after 24 h of incubation with rotenone and antimycin A. At very low concentrations, lithium decreased O₂⁻ production under normoxic conditions and after exposure to CO₂ and rotenone. Lithium was not observed to produce any changes in H₂O₂ levels under standard conditions or after either the rotenone or antimycin A treatments; but changes were observed after CO₂ exposure. In addition, lithium increased cell proliferation. Our findings demonstrate the specific potential capacity of lithium at very low concentrations to protect SH-SY5Y cells against oxidative stress. The protection afforded by lithium might be due, at least in part, to its inhibition of O₂⁻ from the complex I; although it seems likely that multiple sites affected by lithium contribute to its antioxidant action. Also, lithium could favour neurogenesis and decrease the vulnerability of neuronal cells to cell injury.

**Keywords:** Lithium; SH-SY5Y; COX-2; oxidative stress; CO₂; rotenone; antimycin A

## P-32
α₂A⁻ AND α₂C-ADRENOCEPTOR SUBTYPES EXPRESSION IN POSTMORTEM PREFRONTAL CORTEX OF SUBJECTS WITH SCHIZOPHRENIA

Brocos-Mosquera I., Gabilondo A.M., Meana J.J., Erdozain A.M., Callado L.F.

Department of Pharmacology, University of the Basque Country, UPV/EHU, and Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Spain

α₂A⁻ and α₂C-adrenoceptor subtypes are present in the human prefrontal cortex (PFC) and could play a role in schizophrenia. Moreover,
$\alpha_2$-adrenoceptors are targets for different antipsychotic drugs. In this context, the density of both $\alpha_2$-adrenoceptor subtypes and their pre/postsynaptic expression has not been deeply evaluated in the PFC of schizophrenic subjects.

$\alpha_{2A}^-$ and $\alpha_{2C}$-adrenoceptor protein expression was determined by Western Blot in postmortem PFC of 24 subjects with an antemortem diagnosis of schizophrenia, and 24 controls. Both groups were matched by age, gender, and postmortem delay. Twelve of the schizophrenic subjects were taking antipsychotics at death (based on positive blood toxicological analysis), while the other 12 where antipsychotic-free (negative toxicology). $\alpha_{2A}^-$ and $\alpha_{2C}$-adrenoceptor expression was measured both in a preparation of synaptosomes and in postsynaptic membrane fractions, and was normalized for actin immunoreactivity as loading control.

$\alpha_{2A}$-adrenoceptor protein expression in synaptosomes showed a non-significant trend to increase (+37%, $p$ = 0.114) in schizophrenia subjects compared with controls. When subjects were divided regarding antipsychotic treatment, there was a significant increase in $\alpha_{2A}$-adrenoceptor expression in antipsychotic-treated (+78%, $p$ = 0.025) but not in antipsychotic-free subjects compared with controls. $\alpha_{2A}$-adrenoceptor expression in postsynaptic fraction was significantly increased in schizophrenia subjects vs controls (+71%, $p$ = 0.026). Again, the increase was significant in antipsychotic-treated subjects (+131%, $p$ = 0.014) but not in antipsychotic-free subjects. $\alpha_{2C}$-adrenoceptor protein expression was not significantly different between schizophrenia subjects and controls in synaptosomes and postsynaptic fraction, neither in antipsychotic-treated or antipsychotic-free subjects.

In conclusion, $\alpha_{2A}$-adrenoceptor protein expression was increased in PFC of schizophrenia subjects receiving antipsychotic treatment. This increase was stronger in the postsynaptic fraction compared to synaptosomes (which include both pre- and postsynaptic membranes). These results might be a consequence of the $\alpha_{2A}$-adrenoceptor antagonistic properties of some antipsychotic drugs.

Supported by grants from the Spanish MINECO (SAF2013-48586-R) and the Basque Government (IT616/13).

**Keywords:** schizophrenia, $\alpha_2$ adrenoceptor, antipsychotics, human brain.

## P-33
EFFECT OF FINGOLIMOD PHOSPHATE ON NEURONAL OXIDATIVE STRESS PRODUCED BY MENADIONE

Pavia J.¹, Martín-Montañez E.¹, García-Fernández M.², Lara E.², Boraldi F.³, Oliver B.⁴, Leyva L.⁴, Fernández O.⁴

¹Department of Pharmacology, Biomedical Research Institute of Malaga (IBIMA), University of Malaga, Malaga, Spain; ²Department of Human Physiology, Biomedical Research Institute of Malaga (IBIMA), University of Malaga, Malaga, Spain; ³Department of Life Sciences, University of Modena e Reggio Emilia, Modena, Italy;

⁴Clinical Neurosciences Unit, Biomedical Research Institute of Malaga (IBIMA), Regional University Hospital of Malaga, Malaga, Spain

**Introduction:** Oxidative stress is considered to be an important component of various neurodegenerative diseases such as Parkinson's disease or multiple sclerosis, where the mitochondrial dysfunction is a major source of ROS production. Menadione induces toxic oxidant stress associated with tissue injury, mitochondrial damage and cell death.

**Aim:** To study the antioxidant effects of fingolimod phosphate (FP) on neuronal cell cultures against the oxidative damage produced by menadione.

**Methods:** SN4741 neuronal cells were grown and used as control (non-treated cells), or treated with 15 µM menadione alone or in presence of 50 nM FP during 6 h to study the following: cellular toxicity, intracellular levels of ROS production, levels of oxidative stress markers, and expression of antioxidant enzymes. Statistical differences were determined using one-way ANOVA. Statistical significance was set at $p$ < 0.05.

**Results:** Menadione produces a 50% decrease in neuronal viability being FP capable of recovering half of the damage ($p$ < 0.05). FP also reverted the effect of menadione on both intracellular and mitochondrial $O_2^-$ production measured via flow cytometry ($p$ < 0.05). Studying oxidative stress markers, such as total thiol levels (TTL) or advanced oxidation protein products (AOPPs), menadione produces a decrease of 25% in TTL and an increase of 34% in AOPPS compared to control; these changes are reversed by co-incubation with FP ($p$ < 0.05). The expression of mitochondrial antioxidant enzymes, Thioredoxin-2 (Trx2) and Superoxide dismutase-2 (SOD2) behaves in a similar way. Thus, menadione induces a decrease of 53% (Trx2 levels) and 42% (SOD2 levels) compare to control; these decreases are reduced to 8.5% (Trx2 levels) and 20% (SOD2 levels) in the presence of FP ($p$ < 0.05).

**Conclusion:** FP exerts a modulating effect on the ROS production, oxidative stress markers levels and the expression of antioxidant enzymes that maintains the balance of neuronal oxidative status.

**Key words:** fingolimod phosphate; oxidative stress; antioxidant; neuroprotective.

## P-34
ACUTE TREATMENT WITH 8-OH-DPAT IMPROVES COGNITION IN RATS BY CHANGING THE STRATEGY USED TO RESOLVE A MAZE AT DIFFERENT AGES

Hernández-Hernández E., Miralles A., Esteban S., García-Fuster M.J.

IUNICS, University of the Balearic Islands, and IdISBa, Palma de Mallorca, Spain

Aging is a complex, heterogeneous, and multifactorial process in which a plasticity deficit in neuronal circuits such as in hippocampus is presented. Additionally serotonin (5-HT) is found to be involved in many physiological or pathophysiological processes including cognitive function. In this regard, while some studies reported enhanced memory following 8-OH-DPAT (a 5-HT₁A receptor agonist) administration others reported impaired memory functions. In this context, the present study evaluated whether acute treatment with a low dose of 8-OH-DPAT would improve cognitive function and whether this improvement would be conditioned by aging. To do so, male Sprague-Dawley rats were treated with 8-OH-DPAT (0.3 mg/kg, i.p.) or saline (0.9% NaCl, i.p.) at three different ages (3, 12 and 18 months old: $n$ = 10 rats per age and treatment group) and cognitive function was evaluated in the Barnes maze 1 h later. The behavioural results showed that 8-OH-DPAT improved cognitive function by reducing the time spent (3 months: -61 ± 7%, $p$ < 0.05; 12 months: -75 ± 4%, $p$ < 0.01; 18 months: -31 ± 15%, $p$ > 0.05) and the number of errors committed (3 months: -70 ± 11%, $p$ < 0.05; 12 months: -88 ± 3%, $p$ < 0.05; 18 months: -43 ± 19%, $p$ > 0.05) to complete the maze as compared to vehicle-treated rats. Moreover, the strategy used to resolve the maze varied with age, fluctuating from a more serial/direct strategy towards a mixed one at later ages. Interestingly, 8-OH-DPAT improved cognition by decreasing mixed strategies while increasing either direct and/or serial strategies. Thus, the present results support a role for 8-OH-DPAT in improving cognition in rats at different ages. Supported by SAF2014-55903-R and 'Ramón y Cajal' Program to M.J.G.-F (MINECO).

**Key words:** rat; age; cognition; 5-HT₁A receptor.
P-35
BEHAVIOURAL EFFECTS OF NOVEL MULTITARGET ANTICHOLINESTERASIC DERIVATIVES IN AGING AND ALZHEIMER'S DISEASE: STUDIES IN 3XTG-AD MICE

Giménez-Llort L.¹,², Pérez B.¹,³, Ratia M.¹,³, Camps P.⁴,⁵,
Muñoz-Torrero D.⁴,⁵, Badia A.¹,³, Clos M.V.¹,³

¹Institut de Neurociències, Universitat Autònoma de Barcelona, Spain;

²Departament de Psiquiatria i Medicina Legal, Universitat Autònoma de Barcelona, Spain; ³Departament de Farmacologia, de Terapèutica i de Toxicologia, Universitat Autònoma de Barcelona, Spain;

⁴Laboratori de Química Farmacèutica (Unitat Associada al CSIC),

Facultat de Farmàcia, Universitat de Barcelona, Spain;

⁵Institut de Biomedicina (IBUB), Universitat de Barcelona, Spain

Maintenance of cholinergic activity remains one of the primary objec-
tives in cognitive-enhancing treatments for individuals with cognitive
deficits associated to aging, mild cognitive impairment and Alzhei-
mer's Disease (AD). Current drug development provides new AChEIs
derivatives, hybrid molecules with multitarget actions aimed to result
in a better clinical management outcome than the transitory and symp-
tomatic therapy offered by classical AChEIs. Thus, they are expected
to induce long-lasting amelioration of impaired neurotransmission and
clinical symptoms but also to exert disease-modifying effects. Our
multidisciplinary research consortium has synthesized and evaluated
tacrine-huperzine A hybrids and their derivatives showing that Huprine
X, a reversible AChE inhibitor hybrid of tacrine and huperzine A,
affects the amyloidogenic process *in vitro*, and the AD-related neu-
ropathology *in vivo* in mice models of AD. Besides, AVCRI104P3, a
new donepezil-huprine heterodimer, exerts highly potent and selective
inhibitory action on hAChE, hBuChE and BACE-1 activities as well
as on the AChE-induced and self-induced Aβ aggregation, both
*in vitro* and *ex vivo*. Here, we summarize their behavioural effects,
both at cognitive (learning and memory) and neuropsychiatric-like
(anxiety-like behaviours) levels and as assessed in three distinct
*in vivo* biological scenarios: middle-age, cognitive deficits associated
to aging, and AD-like phenotype. Furthermore, we provide new exper-
imental data of the comparative assessment of chronic (i.p., 21 days)
Huprine X (0.12 µmol/kg) and AVCRI104P3 (0.6 µmol/kg) treatment
in 12 month-old (advanced stages) 3xTg-AD mice in two temporal cir-
cadian frames: after nocturnal activity or after 4 h of diurnal sleep.
Besides the improvement of the hallmark cognitive symptomatology
without inducing side effects, these drugs were able to modulate emo-
tional and anxiety-like behaviours even in poor-performance circadian
activity frames. Overall, the studies show that these novel multitarget
AChEIs provide symptomatic and disease-modifying benefits of poten-
tial interest for the management of AD. Financial support: DGICYT-
FEDER CTQ2011-22433, SAF2014-57094, ISC3-PI10/00283, 2014-
SGR-52.

**Key words:** Huprine X, AVCRI104P3, cognition, learning and memory, anxiety, circadian activity, 3xTg-AD mice, Behaviour.

P-36
STUDY OF THE EFFECT OF EARLY POSTNATAL ADMINISTRATION OF A NMDA RECEPTOR ANTAGONIST IN MOUSE ON DIFFERENT FUNCTIONAL AND NEUROMORPHOLOGICAL SCHIZOPHRENIC PHENOTYPES

García-Cerro S.¹, Martínez-Pinteño A.¹, Cheng-Zhang J.¹, Boloc D.²,
Gortat A.¹, Parellada E.¹,³,⁴,⁵, Rodríguez N.¹, Sindreu C.¹,⁶, Torres
T.¹, Lafuente A.¹,³,⁴

¹Department of Clinical Foundations, Pharmacology Unit, University of Barcelona, Barcelona, Catalonia, Spain; ²Department of Medicine, University of Barcelona, Barcelona, Catalonia, Spain; ³The August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, Catalonia, Spain; ⁴Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Carlos III Health Institute, Madrid, Spain;

⁵Barcelona Clinic Schizophrenia Unit (BCSU), Barcelona, Catalonia, Spain; ⁶Institute of Neuroscience UB, University of Barcelona, Barcelona, Catalonia, Spain

Schizophrenia is a chronic and severe mental disorder. It is characterized by hallucinations and cognitive symptoms such as attention and memory deficits. Recent studies have pointed at a dysfunction of the glutamatergic neurotransmission as a possible source of cognitive deficits in schizophrenia. The decreased action of NMDA receptors (NMDAR) on GABAergic interneurons reduces the inhibitory control of excitatory pyramidal neurons leading to increased glutamate release. This situation results in excitotoxic damage and cognitive impairment. The stimulation of NMDAR affects the maturation of GABAergic interneurons during a critical period of development, for this reason, the blockade of these receptors during the early period implies changes similar to the neuropathophysiology of schizophrenia. The aim of this study was to characterize a mouse model of schizophrenia in the adult stage with early brain damage induced by ketamine. We assayed two experimental groups: a) C57BL/6 mice treated with ketamine (30 mg/kg, s. c.) on postnatal days 7, 9 and 11 and b) C57BL/6 mice treated with vehicle on postnatal days 7, 9 and 11. The experimental groups of mice were assessed in several cognitive and motor tasks in adulthood (PND 90-120). In addition, a neuromorphological analysis of the parvalbumin interneurons cellularity in prefrontal cortex and hippocampus was performed by immunohistochemistry. Mice treated with early ketamine administration showed hypoactivity, an impaired recognition memory, deficits in their working memory and a decreased social memory in adulthood. Moreover, ketamine treatment reduced the parvalbumin cell number in prefrontal cortex and hippocampus. Our results provide new evidence for the alterations induced by early treatment with ketamine in mice.

**Key words:** Schizophrenia; mouse model; cognitive impairment; interneurons.

P-37
KETAMINE-INDUCED HYPNOSIS IN MICE REGULATES FADD AND MEK-ERK SIGNALING IN BRAIN CORTEX

Salort G., Álvaro-Bartolomé M., García-Fuster M.J., García-Sevilla
J.A.

Laboratory of Neuropharmacology, IUNICS, University of the Balearic Islands (UIB), and IdISBa, Palma de Mallorca, Spain

Ketamine (KET) is an antagonist at excitatory NMDA receptors that at low doses induces rapid antidepressant effects and at a higher dosage hypnosis in rodents. Recently, midazolam (MDZ, an agonist at inhibitory GABA<sub>A</sub> receptor)-induced hypnosis in mice was associated with upregulation of cortical p-FADD/FADD ratio and disruption of MEK (increased)-ERK (decreased) phosphorylation. This work assessed whether ketamine-induced hypnosis can regulate, similarly to MDZ, FADD and ERK signaling. Male CD1 Swiss mice were treated (i.p.) with KET (150 mg/kg, n = 5–7) or saline (2 ml/kg, n = 5). The sleeping time induced by KET was monitored according to the loss (LORR) or recovery (RORR) of the righting reflex. During KET-induced hypnosis and
after wake-up, subgroups of mice were sacrificed and the cerebral cortex prepared for protein analysis by Western blot. In KET-treated mice, compared with saline, cortical p-FADD, but not FADD, was increased (*p* < 0.05) in parallel with the hypnotic time course (at LORR: +16%; 20 min: +103%; RORR: +95% and 1 h post-RORR: +65%). Thus, KET increased cortical p-FADD/FADD ratios (LORR: +1.2-fold; 20 min: +2.7-fold; RORR: +2.3-fold and post-RORR: +2.1-fold). On the other hand, activated MEK1/2 (p-MEK/MEK ratio) was also increased (*p* < 0.05) following KET hypnotic time course (at LORR: +41%; 20 min: +75%; RORR: +58% and 1 h post-RORR: +81%). Paradoxically, the expected parallel activation of ERK1/2 (p-ERK/ERK ratio) was not observed but instead it was downregulated (*p* < 0.05) (LORR: no change; 20 min: -47%; RORR: -40% and 1 h after RORR: -8%). During KET-induced hypnosis in mice, p-FADD/FADD ratio (a survival index) was upregulated and the sequential activation of MEK to ERK was disrupted (ERK downregulation could lead to adverse effects including amnesia). The results also revealed the induction of very similar molecular mechanisms for excitatory KET and inhibitory MDZ mediating hypnosis in mice. Supported by SAF2014-55903-R and 'Ramón y Cajal' Program to M.J.G.-F. (MINECO).

## P-38
EARLY EXPOSURE OF MICE TO MDPV INCREASES THE VULNERABILITY TO COCAINE IN ADULTHOOD

Luján M.A.¹, López-Arnau R.², Duart-Castells L.², Pubill D.², Camarasa J.², Valverde O.¹,³, Escubedo E.²

¹Neurobiology of Behaviour Research Group (GReNeC), Department of Experimental and Health Sciences, Universitat Pompeu Fabra, Barcelona, Spain; ²Department of Pharmacology, Toxicology and Therapeutic Chemistry, Pharmacology Section and Institute of Biomedicine (IBUB), Faculty of Pharmacy, University of Barcelona, Barcelona, Spain; ³Neuroscience Research Programme, IMIM-Hospital del Mar Research Institute, Barcelona, Spain

3,4-Methylenedioxypyrovalerone (MDPV) is a synthetic cathinone which has recently emerged as a designer drug of abuse. It acts as a dopamine uptake blocker 10–50-fold more potent than cocaine, which suggests a high potential of abuse. Due to the rapid increase in the abuse of MDPV among the young population, the aim of this work was to investigate the influence of an early exposure to MDPV during adolescence on the psychostimulant, rewarding and reinforcing effects of cocaine in adulthood.

Adolescent (aged 41–44 days) male Swiss CD-1 mice were treated for 7 days with 1.5 mg/kg (s.c.) MDPV twice daily, followed by 21 days of withdrawal. Thereafter, when reached adulthood, they were tested to the psychostimulant, rewarding and reinforcing effects induced by cocaine assessing locomotor activity, conditioned place preference (CPP) and self-administration (SA). Moreover, the dopamine D₂ receptors density and the expression of c-Fos and ΔFosB in the striatum were determined. Differences between groups were compared using three-, two-way ANOVA or Student's *t*-test where appropriate. Subsequent Tukey's *post-hoc* tests were calculated when required. Breaking point was analysed using Mann-Whitney *U*-test.

After withdrawal, mice pretreated with MDPV showed a higher locomotor response and place conditioning to cocaine than those that had received saline. Acquisition of cocaine SA was similar in both groups. However, MDPV-treated animals showed a higher breaking point achieved under a progressive-ratio programme. MDPV pretreatment produced a significant decrease (20%) in D₂ receptor density and an increase in ΔFosB expression (3-fold), 24 h after treatment. The levels of ΔFosB declined during withdrawal but still remained elevated when measured 2 h after cocaine or saline challenge. Cocaine challenge induced an increase in c-Fos which was attenuated in MDPV-pre-treated animals.

These results suggest that MDPV consumption during adolescence induces long-lasting adaptive changes leading to a higher vulnerability to cocaine abuse.

**Key words:** MDPV; cocaine; adolescence; self-administration.

## P-39
IONOTROPIC GLUTAMATE RECEPTORS PHOSPHOPATTERN IS ALTERED IN SYNAPTOPATHY CONDITIONS

Gómez de Salazar M., Grau C., Santos A., Ciruela F., Altafaj X.

IDIBELL, Neuropharmacology and Pain Unit, L'Hospitalet de Llobregat, Spain

The glutamatergic synapse is an exquisite dynamic structure involved in excitatory neurotransmission. The plasticity of the system is mediated, at least in part, by post-translational modifications. In particular, phosphorylation events control the subcellular location of glutamate receptors, a mechanism underlying synaptic strength and neuron survival. In line with this, our group has recently shown that DYRK1A – a protein kinase overexpressed in Down syndrome, DS and Alzheimer's disease, AD – directly phosphorylates GluN2A subunit of NMDA receptors (NMDARs), at serine residue 1048 (S1048). DYRK1A-mediated phosphorylation of GluN2A subunit increases the surface expression of NMDARs and alters NMDA evoked currents. Given the overexpression of DYRK1A kinase in synaptopathy conditions and the critical role of ionotropic glutamate receptors in cognitive performance, we hypothesized that the alterations on NMDARs phosphopattern might be underlying neuronal dysfunction, ultimately contributing to cognitive disorders. To address this hypothesis, we characterized the phosphoproteomic profile of synaptic and extrasynaptic NMDAR-associated protein complexes in the hippocampus of adult Ts65Dn mice (DS murine model) and in the entorhinal cortex and hippocampus of post-mortem AD biopsies. The subsynaptic fractions of the different brain regions were analyzed by Mass Spectrometry and further analysis revealed a specific subsynaptic and disease-associated NMDARs phosphopattern. Importantly, we have detected a significant increase of GluN2A (pS1048) in the postsynaptic density of hippocampus of adult Ts65Dn mice (n = 8 per condition; **p** = 0.0011; Student's *t* test), as well as in the entorhinal cortex of AD individuals (n = 3 controls and n = 7 AD; *p* = 0.0167; Student's *t* test). In agreement with this, our biochemical data supported the presence of Dyrk1A in the postsynaptic density, enabling the direct phosphorylation of GluN2A -containing NMDARs in a synaptic activity-dependent manner. Overall, our results demonstrate an altered phosphopattern in the pathological glutamatergic synapse, which can represent a therapeutic target.

This work has been supported by Grants PI16/00851PI13/00135 (ISCIII), La Marató (Project N. 20140210), PCIN-2014-105 (MINECO), AGAUR (SGR14-297) and Miguel Servet (CP10/00548) to XA.

**Key words:** Glutamate receptors; phosphorylation; DYRK1A; synaptopathy.

## P-40
ASSOCIATION OF REGULATORY *TPH2* POLYMORPHISMS WITH HIGHER REDUCTION IN DEPRESSIVE SYMPTOMS IN CHILDREN AND ADOLESCENTS TREATED WITH FLUOXETINE

Torres T.¹, Gassó P.¹,²,*, Rodríguez N.¹,*, Boloc D.¹, Blázquez A.³, Gortat A.¹, Plana M.T.³, Lafuente A.¹,²,⁴, Mas S.¹,²,⁴, Lázaro L.²,³,⁴,⁵

¹Department of Basic Clinical Practice, Unit of Pharmacology, University of Barcelona, Spain; ²Institut d'Investigacions Biomèdiques August Pi i Sunyer (IDIBAPS), Barcelona, Spain; ³Department of Child and Adolescent Psychiatry and Psychology, Institute of Neurosciences, Hospital Clinic de Barcelona, Spain; ⁴Centro de Investigación Biomédica en Red de Salud Mental (CIBERSAM), Spain; ⁵Department of Psychiatry and Clinical Psychobiology, University of Barcelona, Spain

Genetic variability related to the brain serotonergic system has a significant impact on both the susceptibility to psychiatric disorders, such as major depressive disorder, and the response to antidepressant drugs,
such as fluoxetine. *TPH2* is one of the most important serotonergic candidate genes in selective serotonin reuptake inhibitors (SSRIs) pharmacogenetic studies. The aim of the present study was to evaluate the influence of regulatory polymorphisms that are specifically located in human *TPH2* transcription factor binding sites (TFBSs), and therefore could be functional by altering gene expression, on clinical improvement in paediatric population treated with fluoxetine for the first time. The selection of SNPs was also based on their linkage disequilibrium with *TPH2* rs4570625, a polymorphism with questionable functionality, which was previously associated with clinical response in our population. Eighty-three children and adolescents were diagnosed 12 weeks after initiating fluoxetine treatment. Clinical improvement was assessed by reduction in Children's Depression Inventory (CDI) scale score. To estimate the independent contribution of each SNP to clinical improvement, general linear models were used. Three polymorphisms were, for the first time, significantly associated (applying Bonferroni correction) with clinical improvement. Higher reductions in CDI scale scores were found in minor allele homozygotes for rs34517220 (GG: 20.7 ± 3.1 vs AA+AG: 7.6 ± 0.8; *p* = 0.0000007) and for rs60032326 (AA: 21.7 ± 1.2 vs GG+GA: 8.4 ± 0.9; *p* = 0.006, and also in minor allele carriers for rs11179002 (TT: 9.4 ± 1.1 vs CT: 16.8 ± 3.9 vs CC: 6.4 ± 1.2; *p* = 0.00059). Interestingly, rs34517220 is located in a *TPH2* TFBS for two relevant transcription factors in the serotoninergic neurons, Foxa1 and Foxa2, which together with the high level of significance found for this SNP, could indicate that it is in fact the crucial functional polymorphism related to the fluoxetine response. These results provide new evidence for the role of regulatory genetic variants that could modulate *TPH2* expression in the SSRI antidepressant response.

**Keywords:** Fluoxetine; Pharmacogenetics; Children; *TPH2*; Polymorphism; rs34517220

## P-41

### LONG-TERM CONSEQUENCES OF TREATING RATS WITH METHAMPHETAMINE AT A WINDOW OF VULNERABILITY DURING YOUNG ADULTHOOD ON HIPPOCAMPAL CELL DAMAGE

Bis-Humbert C., García-Cabrerizo R., García-Fuster M.J.

IUNICS, University of the Balearic Islands, and IdISBa, Palma de Mallorca, Spain

A recent study from our laboratory demonstrated certain degree of hippocampal cell damage (i.e., impaired neurogenesis) exerted by methamphetamine when administered specifically at a window of vulnerability during young adulthood (postnatal day, PND 54–57) as measured 24 h after the last injection (PND 58). As a follow up, the present study aimed at evaluating the long-term consequences to drug-induced brain changes of treating rats with methamphetamine at this period of vulnerability. To do so, male Sprague-Dawley rats were pretreated with BrdU (2 × 50 mg/kg, i.p., 3 days) which marks new born cells (PND 48–50). Then, rats were treated following a binge paradigm (3 pulses per day, i.p., every 3 h, for 4 days) with saline (0.9% NaCl, 1 ml/kg, n = 12) or methamphetamine (5 mg/kg, n = 14) (PND 54–57). Following prolonged withdrawal (PND 91), rats were killed 45 min after a challenge dose of saline (saline-saline, n = 6; methamphetamine-saline, n = 7) or 5 mg/kg of methamphetamine (saline-methamphetamine, n = 6; methamphetamine-methamphetamine, n = 7). The left hippocampus was cryostat cut and slide-mounted for evaluation of Ki-67 + cell proliferation by immunohistochemistry. The right hippocampus was dissected to study the cell fate regulator BDNF (i.e., pro- and mature-BDNF forms) by western blot analysis. The main results showed that methamphetamine (PND 54–57) induced enduring hippocampal cell damage on cell proliferation (Ki-67+ cells/sections: ~ 21 ± 5%, *p* < 0.05) and mature-BDNF (~24 ± 6%, *p* < 0.01), but not pro-BDNF (~8 ± 5%, *p* > 0.05), as observed when comparing control-saline vs. methamphetamine-saline groups. The main results showed that a history of methamphetamine during a window of vulnerability in young adulthood (PND 54–57) induced enduring hippocampal cell

damage that persisted in time (PND 91) as observed by decreases on recent cell proliferation and on the cell fate marker mature-BDNF, which is associated with neuronal survival, growth and differentiation. Supported by 'Fundación Alicia Koplowitz' and 'Ramón y Cajal' Program to M.J.G.-F. (MINECO), and by RTA-RD16/0017/0010.

**Key words:** methamphetamine; withdrawal; consequences; hippocampal neurogenesis.

## P-42

### THE FIFTH SUBUNIT MODULATES THE FUNCTION OF CANONICAL AGONIST SITES IN α4β2 NICOTINIC RECEPTORS

García del Villar S., New K., Mazzaferro S., Bermúdez S.

Department of Biological and Medical Sciences, Oxford Brookes University, Oxford, UK

Nicotinic receptors (nAChR) containing the α4 and β2 subunits are the most prevalent type of nAChR in the brain, where they modulate an assortment of physiological functions such as cognition, mood, reward and analgesia. The α4β2 receptors assemble in two functional forms, (α4β2)₂α4 and (α4β2)₂β2. These two receptors have different physiological properties, which is partly accounted for by the presence of an additional agonist site at the signature α4/α4 interface of the (α4β2)₂α4 nAChR. Previous studies found that the canonical agonist sites of these receptors function asymmetrically, even though they are structurally equivalent. These findings suggested that the fifth subunit (α4 in the (α4β2)₂α4 and a β2 in the (α4β2)₂β2 may asymmetrically modulate the agonist sites.

We study the impact of the fifth subunit on receptor function by using two-electrode voltage-clamp electrophysiology, along with subunit-targeted mutagenesis and the substituted cysteine scanning method applied to fully linked (α4β2)₂β2 receptors.

By incorporating a cysteine residue in the fifth subunit and measuring its rate of derivatization by a thiol-reactive reagent (MTS) in the presence or absence of ACh, we found that the fifth subunit increases ACh efficacy. This effect is dependent on the presence of ACh and impairment of the agonist sites perturbs the rate of MTS-modification of the fifth subunit asymmetrically. For the (α4β2)₂α4 receptors, we examined the effects of mutations introduced in the fifth subunit or flanking subunits on Zn²⁺ potentiation of (α4β2)₂α4 receptors. Zn²⁺ potentiation of the agonist responses of the (α4β2)₂α4 receptors is mediated by a site located on the fifth subunit (α4). We found that Zn²⁺ potentiation is inhibited by alanine substitutions of amino acids linking the fifth subunit to neighbouring agonist sites.

Overall, we propose that the fifth subunit allosterically communicates with adjacent subunits to modulate agonist binding site function.

**Key words:** Nicotinic receptor, α4β2 receptors, Cys-loop receptors.

## P-43

### SELENOSUGARS ARE A PROMISING CLASS OF SELENIUM DERIVATIVES WITH VASCULO-PROTECTIVE EFFECTS

Onetti Y.¹, Jiménez-Altayó F.¹, Salvadó M.², Pérez B.¹, Dantas A.P.³, Tarrés F.¹, Vila E.¹, Castillón S.², Boutureira O.²

¹Departament de Farmacologia, de Terapèutica i de Toxicologia, Institut de Neurociències, Universitat Autònoma de Barcelona, Bellaterra, Spain; ²Departament de Química Analítica i Química Orgànica, Universitat Rovira i Virgili, Tarragona, Spain; ³Institut Clínic del Tòrax, Institut d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain

Peroxynitrite (ONOO⁻) is crucially involved in the initiation and progression of human cardiovascular disease. Selenium is an essential micronutrient that plays a key role in redox regulation and antioxidant defence. Synthetic organoselenium compounds such as ebselen can mimic the function of endogenous antioxidant selenoenzymes, but

<footnote>*These authors contributed equally to this work.</footnote>
ebselen can be toxic to cells at high concentrations. In this study, we aimed to analyse the antioxidant properties of two different reactivity profile selenosugars, βGlcSeMe and βGlcSe2, that we hypothesize they have vasculo-protective effects. The parallel artificial lipid membrane permeation assay was used to predict passive membrane permeability. ONOO⁻ scavenging activity was evaluated by measuring the consumption of Pyrogallol red. The effect of preincubation with selenosugars on contractile responses to phenylephrine (1 nM–100 µM) was evaluated by wire myography in aortic rings from Oncins France 1 mice (34.2 ± 0.1 g) in the absence or presence of ONOO⁻ (50 µM; 45 min). Superoxide anion (O₂⁻) formation, and inducible nitric oxide synthase (iNOS) or protein tyrosine nitrosylation levels were determined by ethidium fluorescence and immunofluorescence, respectively, in mice aorta or human cultured microvascular endothelial cells (HMVEC) submitted to hypoxia (overnight)/reoxygenation (24 h). Either ebselen or both selenosugars showed high predictive lipid membrane permeability. Both selenosugars exhibited lower ONOO⁻ scavenging activity than ebselen, and βGlcSe₂ was a more potent (*p* < 0.001) ONOO⁻ scavenger than βGlcSeMe. Ebselen (3 µM) failed to ameliorate ONOO⁻-induced impairment of phenylephrine contractions and rather decreased (*p* < 0.05) maximal contractions. In contrast, βGlcSe₂ (10 µM) but not βGlcSeMe (10 µM) attenuated (*p* < 0.05) aortic contractile dysfunction triggered by ONOO⁻. Furthermore, βGlcSe₂ reduced (*p* < 0.05) the ONOO⁻-mediated increase in aortic O₂⁻ and iNOS expression levels. Hypoxia/reoxygenation in HMVEC augmented O₂⁻ (*p* < 0.05) and nitrosylation (*p* < 0.01) levels, an effect prevented (*p* < 0.001) by βGlcSe₂. In conclusion, we suggest that selenosugar βGlcSe₂ can mitigate the ONOO⁻-induced detrimental effects on the vasculature.

**Key words:** selenosugars; antioxidant; peroxynitrite; aortic reactivity.

P-44

INVOLVEMENT OF ENDOPLASMIC RETICULUM STRESS AND MITOCHONDRIAL DYSFUNCTION IN ABDOMINAL AORTIC ANEURYSM DISEASE

Navas M.¹, Escudero J.R.¹,², Vila L.¹, Martínez-González J.²,³,
Camacho M.¹,², Rodriguez C.²,³, Galán M.¹,²

¹Laboratorio de Angiología, Biología Vascular e Inflamación y Servicio de Cirugía Vascular. Instituto de Investigación Biomédica (IIB-Sant Pau), Barcelona, Spain; ²CIBER de Enfermedades Cardiovasculares, ISCIII, Spain; ³Centro de Investigación Cardiovascular (CSIC-ICCC), Instituto de Investigación Biomédica (IIB-Sant Pau), Barcelona, Spain

**Aim:** Clinical management of abdominal aortic aneurysm (AAA) is limited to elective surgical repair while an effective pharmacotherapy is awaited. We aimed to elucidate the involvement of endoplasmic reticulum (ER) and mitochondrial stress in vascular wall degeneration during the development of AAA.

**Methods and results:** We determined the expression of ER stress, mitochondrial dysfunction and oxidative stress markers in a cohort of AAA samples from 100 patients and 20 healthy donors.

The mRNA and protein levels of ER stress markers such as ATF6, IRE-1, XBP-1 and CHOP were up-regulated in AAA samples compared with donors and this was accompanied by an exacerbated apoptosis measured by TUNEL and by cleaved caspase-3. Higher expression of NOX2, p22phox and NRF2 and an enhanced superoxide anion production, measured by dihydroethidium staining, were also found in AAA. Furthermore, mitochondrial biogenesis measured by the ratio between the expression of cytochromes b/c and Beta-actin was decreased by 50% in AAA. However, the expression of mitochondrial dysfunction markers (NRF1, PGC1-alpha and TFAM) remained unchanged between patients and donors. Additionally, in abdominal aorta of a mouse model of AAA (ApoE⁻/⁻ infused with angiotensin II), the expression of ATF4, GRP78 and IRE-1 was increased while no differences were found in ATF6 or CHOP between AAA and control mice. Similarly to human, mitochondrial biogenesis was decreased by 60% in murine AAA.

**Conclusion:** Our results provide evidence supporting a role for chronic ER stress and mitochondrial stress in the development of AAA and identify them as potential therapeutic targets.

P-45

CHANGES IN THE EXPRESSION OF THE NT-3/TRKC PATHWAY IN VESSELS AND LEFT VENTRICLE DURING CULTURE

Montó F., Zambrano A., Arce C., Bové M., Martí F., Noguera M.A., Ivorra M.D., D'Ocon P.

Departamento de Farmacología, Facultad de Farmacia, ERI BIOTECMED, Universidad de Valencia, Valencia, Spain

**Background:** The expression of neurotrophin-3 (NT-3), a regulator of survival, development and function of sympathetic neurons during embryonic development, decreases in the adult central nervous system (1) but remains constant in the cardiovascular system from development to adulthood (2).

**Aims:** To quantify mRNA levels for NT-3 and its receptor TrkC in aorta (Ao), left ventricle (LV) and different cardiovascular cell types (freshly isolated or cultured) from human origin or Wistar rats.

**Methods:** Human Ao, LV, aortic smooth muscle cells (SMC), cardiac or aortic fibroblasts (FB), cardiomyocytes, endothelial cells (EC) from aorta, coronary artery and coronary microvasculature, were used. SMC and EC from Ao and cardiac FB from Wistar rats were isolated by incubation with collagenase (fresh cells) or cultured until 80% confluence (cultured cells). Some rat Aos were maintained in culture medium for 7 days (quiescent Ao). mRNAs encoding NT-3, TrkC and GAPDH (as a housekeeping gene) were quantified by RT-PCR as previously described (3).

**Results:** Human Ao and LV expressed significant levels of NT-3 and TrkC genes. This expression was observed in lower proportion in all the cultured human cell types. Rat Ao and LV express similar levels of NT-3 and TrkC than human tissues. This expression dramatically decreased in quiescent Ao and cultured vs freshly isolated cells. The decrease was more marked for TrkC gene which almost disappears in some cells.

**Conclusions:** The significant decrease in the expression of NT-3 and TrkC in cultured tissues and cells suggests a role for the NT-3/TrkC pathway in cell homeostasis that would become unnecessary when “ideal” culture conditions were established.

1. Physiol Rev, 89: 279–308, 2009:

2. Life Sci 81, 385–392, 2007:

3. J Cardiovasc Pharmacol 68, 230–40, 2016: Founded by Ministerio de Economía y Competitividad (SAF2013-45362-R). AZ received a fellowship from the Santiago Grisolía Foundation.

**Key words:** neurotrophin-3, trkC, endothelial cells, smooth muscle cells, fibroblasts, aorta, left ventricle.

P-46

CHONDROITIN SULFATE AND GLUCOSAMINE COMBINATION TREATMENT FOR MUSCULOSKELETAL DISEASES AND OSTEOARTHRITIS: A CHANCE TO KILL TWO BIRDS WITH ONE STONE? RESULTS IN A RAT-INJURY MODEL

Montell E.¹, Contreras-Muñoz P.²,³, Torrent A.¹, Vergés J.¹, de la Varga M.², Rodas G.²,⁴, Marotta M.²,³

¹Pre-Clinical R&D Area, Bioibérica S.A.U., Barcelona, Spain; ²Leitat Foundation, Leitat Technological Center, Barcelona, Spain;

³Bioengineering Cell Therapy and Surgery in Congenital Malformations Laboratory, Vall D'Hebron Institute, Barcelona, Spain;

⁴FC Barcelona Medical Services, Barcelona, Spain

**Background:** Skeletal muscle injuries and osteoarthritis (OA) are prevalent in middle-aged active individuals. The similarities between
cartilage and muscle tissue may have clinical implications. There is an acute need to develop effective therapeutic strategies on sports-related muscle lesions.

**Objective:** Our aim was to evaluate the beneficial effects of chondroitin sulfate (CS) and glucosamine hydrochloride (GLU) administration (both compounds used for the symptomatic OA treatment) on muscle healing in a recently established rat model of skeletal muscle injury which reproduces the lesions seen in human athletes.

**Methods:** Male 8-weeks-old Wistar rats with a medial gastrocnemius injury received daily treatment for 3 weeks with CS + GLU by oral gavage administration (140 and 175 mg/kg of CS and GLU, respectively) or intraperitoneal injection (400 and 500 mg/kg, respectively). Healthy and untreated animals were used as controls.

**Results:** CS + GLU administration stimulated the growth of newly formed regenerating myofibers (1.35- and 1.77-fold increase after oral ($p < 0.01$) and i.p. ($p < 0.001$) administration, respectively), that was accompanied by 1.22- and 1.28-fold ($p < 0.01$) of improvement in gastrocnemius muscle force. Both treatments showed a clear anti-fibrotic effect by reducing 21% (oral, $p < 0.05$) and 28% (i.p., $p < 0.01$) the intramuscular collagen-I deposition. Oral ($p < 0.01$) and i.p. ($p < 0.05$) administration induced an increase of more than 30% in CS intramuscular deposition. Treated animals also revealed a tendency in accelerating the muscle regeneration process by showing a decrease of approximately 15% of developmental Myosin Heavy Chain (dMHC)-positive regenerating fibers.

**Conclusions:** CS and GLU administration improves muscle healing and force recovery of the injured skeletal muscle in rats, thus suggesting an important role of these products as potential new therapies for the treatment of muscle injuries in sports medicine. These results could help clinicians to account effects of comorbidities on musculoskeletal diseases and OA, as well as to consider new treatment options that show potential in both conditions.

**Key words:** osteoarthritis; skeletal muscle; chondroitin sulfate; glucosamine.

## P-47
ENDOTHELIUM-DERIVED HYPERPOLARIZATION (EDH)-INDUCED RELAXATION OF RENAL ARTERIES: ROLE OF $H_2O_2$

Pinilla E., Muñoz M., Sánchez A., García Sacristán A., Prieto D., Rivera L.

Department of Physiology, Facultad de Farmacia, Universidad Complutense de Madrid, Madrid, Spain

**Background and purpose:** The endothelium mediates vasodilation through endothelium-derived hyperpolarization (EDH) and hydrogen peroxide ($H_2O_2$) appears to play a pivotal role. Thus, we sought to investigate whether $H_2O_2$ is involved in the EDH-mediated relaxations of rat renal arteries.

**Material and methods:** Renal interlobar arteries of male Wistar rats were used. Changes in isometric wall tension of the vessels were also studied using wire myography. We studied the relaxant effect of $H_2O_2$ under conditions of NOS- and COX-blockade. Moreover, electrophysiological recording of $K^+$ currents were made in freshly isolated endothelial cells (EC) under the whole cell configuration of the patch clamp technique (E. Pinilla, et al., J. Physiol. Biochem. 72 (2016) S48).

**Key results:** Under conditions of NOS and COX blockade, relaxations to ACh were largely inhibited by catalase (200 UI/ml) in renal arteries suggesting the involvement of endothelium-derived $H_2O_2$. Furthermore, ACh-stimulated $H_2O_2$ generation measured by Amplex Red fluorescence assay as well, and exogenous $H_2O_2$ elicited relaxations that were accompanied by simultaneous decreases in VSM [Ca$^{2+}$]i thus mimicking catalase-sensitive renal vasodilation. Under voltage-clamp conditions we observed a marked change in the shape of the currents in response to $H_2O_2$, reflecting selective activation of $K^+$ channels. The SERCA inhibitor cyclopiazonic acid (CPA) evoked a change in the shape of the outward currents similar to that evoked by $H_2O_2$.

However, in the presence of CPA $H_2O_2$ did not further modify the amplitude of these currents.

**Conclusions and implications:** The results show that $H_2O_2$ is involved in the EDH relaxant responses of intrarenal arteries. $H_2O_2$ induced a slow, pronounced and long-lasting activation of outward KCa currents in EC, which indicates that $H_2O_2$ released by endothelial stimulation hyperpolarizes EC and may thus initiate an EDH vasodilator response in renal arteries.

**Keywords:** Renal arteries, EDH, $H_2O_2$, $K_{Ca}$ channels.

**Supported by:** MINECO SAF2016-77526;

## P-48
GUT MICROBIOTA CONTROLS ARTERIAL BLOOD PRESSURE IN RATS BY ALTERING GUT-BRAIN-BONE MARROW INTERACTION

Toral M.¹, Romero M.¹,², Robles-Vera L.¹, Sánchez M.¹,², Gómez-Guzmán M.¹, Rodriguez-Nogales A.¹,², Algieri F.¹,², de la Visitación N.¹, Gálvez J.¹,²,³, Duarte J.¹,²,⁴,⁵

¹Department of Pharmacology, School of Pharmacy, University of Granada, Granada, Spain; ²Instituto de Investigación Biosanitaria de Granada, ibs.GRANADA, Granada, Spain; ³CIBER-EHD, Center for Biomedical Research (CIBM), University of Granada, Granada, Spain; ⁴CIBERCV, Granada, Spain; ⁵Department of Pathology, School of Medicine, University of Granada, Granada, Spain

The aim of this study was to investigate whether changes in gut microbiota alter brain-gut-bone marrow (BM) interaction inducing changes in vascular tone and SBP. Fecal contents were collected and pooled from 20 week old spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. Recipient WKY and SHR (25 weeks old) were orally gavaged with donor fecal contents for 3 consecutive days, and weekly during 4 weeks. Four groups were involved in this study: WKY with WKY microbiota (W-W), WKY with SHR (W-S), SHR with SHR (S-S) and SHR with WKY (S-W). Fecal microbiota transplantation (FMT) from WKY rats to SHR rats reduced basal SBP. Similarly, FMT from SHR to WKY increased basal SBP. The higher reduction on SBP after ganglionic blockade and plasma noradrenaline levels found in S-S group as compared to W-W group were reduced in S-W group, whereas in W-S these variables were also higher than that in W-W group. In S-S group mRNA levels of gap junction proteins and pro-inflammatory cytokines in the proximal colon were reduced and increased, respectively, as compared to W-W group and these changes were inhibited in S-W group. Relaxation induced by acetylcholine was impaired in aortic rings from S-S as compared to W-W and improved in S-W. S-W restored imbalance between Th17/Treg found S-S in mesenteric lymph nodes (MLN) and BM. Relaxation induced by acetylcholine was higher in aortic rings from Wistar rats after incubation with the condition media of lymphocytes from MLN of S-W group, as compared with the condition media from S-S. In conclusion, gut dysbiosis disrupts gut barrier function and increases sympathetic outflow. The central and endothelial effects, that increase vascular tone and SBP, seem to be associated with T cell infiltration, possibly as a result of a change in T cells polarization in MLN and BM.

**Key words:** bone marrow, endothelial dysfunction, fecal microbiota transplantation, gut dysbiosis, hypertension, T cells.
P-49

HIV TRANSGENE EXPRESSION INDUCES ENDOTHELIAL
DYSFUNCTION AND KV7 CHANNEL IMPAIRMENT IN THE
PULMONARY VASCULATURE

Mondejar-Parreño G.¹, Morales-Cano D.¹, Barreira B.¹, Callejo M.¹,
Moreno L.¹, Mathie A.², Butrous G.², Perez-Vizcaino F.¹, Cogolludo A.¹

¹Department of Pharmacology, School of Medicine, CIBERES, Insto
Investigación Sanitaria Gregorio Marañón (IISGM), University
Complutense of Madrid, Spain; ²Medway School of Pharmacy,
University of Kent, Chatham, UK

**Introduction:** Pulmonary arterial hypertension (PAH) is a severe disease that results in increased pulmonary vascular resistance, right heart failure and death (1). The vascular dysfunction that leads to PAH is likely to be multifactorial, but endothelial dysfunction and downregulation of K⁺ channels are considered common abnormalities in most forms of PAH. Human immunodeficiency virus (HIV) infection is an established risk factor for PAH, however the pathogenesis of HIV-related PAH remains unclear. Our aim was to analyse if the expression of HIV proteins is associated with impairment of endothelial or K⁺ channel function in the pulmonary circulation.

**Methods:** HIV transgenic mice (Tg26) expressing seven of the nine HIV viral proteins and wild type (Wt) mice were used in this study. Right ventricular systolic pressure and systolic, diastolic and mean pulmonary arterial pressures were measured in open chest mice. Vascular reactivity was studied in endothelium-intact pulmonary arteries mounted in a wire myograph. K⁺ currents were recorded in freshly isolated pulmonary artery smooth muscle cells using the patch-clamp technique. K⁺ channel gene expression was assessed using rt-PCR. Data are expressed as mean ± sem.

**Results:** Right ventricular systolic pressure and systolic, diastolic and mean pulmonary arterial pressures were similar in Tg26 and Wt mice. Interestingly, pulmonary arteries from Tg26 mice showed impaired endothelium relaxation (11 ± 5% vs 33 ± 5% relaxation to Acetylcholine 1 µM; n = 6 *p* < 0.05). Likewise, pulmonary arteries and pulmonary artery smooth muscle cells derived from Tg26 mice had decreased Kv7 channel activity assessed by vascular reactivity and patch-clamp experimental approaches. This was associated with reduced expression of Kv7.1 and Kv7.4 channels, which have recently emerged as protective in the pulmonary circulation (2).

**Conclusion:** Our data indicate that HIV proteins induce endothelial dysfunction and Kv7 channel impairment, which could contribute to the vascular dysfunction in HIV-associated PAH.

**References:**

1. Humbert M et al. (2014). Circulation; 130: 2189-2208. 2. Sedivy V et al. (2015). Am J Physiol Lung Cell Mol Physiol; 308:L48-57. Supported by SAF 2014-55399 and SAF2016-77222-R.

**Key Words:**

Pulmonary arterial hypertension, Kv7 channels, endothelial dysfunction.

P-50

PGE₂ DERIVED FROM MPGES-1 FACILITATES EXCESSIVE
ALDOSTERONE PRODUCTION FROM ADIPOSE TISSUE IN
OBESITY, ROLE IN VASCULAR FUNCTION

González-Amor M.¹, Beltrán L.M.², Rodrígues-Díez R.¹, García-Redondo A.B.¹,³, Mata A.⁴, López-Andrés N.⁵, Cachofeiro V.³,⁶, Aras-López R.¹, Salaices M.¹,³, Briones A.M.¹,³

¹Department of Pharmacology, Faculty of Medicine, Universidad Autónoma de Madrid, IdiPaz, Madrid, Spain; ²Servicio de Medicina Interna, Hospital Universitario La Paz, UAM, IdiPaz, Madrid, Spain; ³Ciber CV; ⁴Sección de Hepatobiliopancreática, Hospital Universitario La Paz, Madrid, Spain; ⁵Navarrabiomed (Miguel Servet Foundation), IdiSNA, Pamplona, Spain; ⁶Department of Physiology, Faculty of Medicine, Universidad Complutense de Madrid, IiSGM, Madrid, Spain

Adipocytes are more than fat storage cells since they release a number of factors contributing to energy homeostasis and vascular tone and

structure. We demonstrated that adipocytes are a source of aldosterone in response to Ang II and that this is facilitated by mPGES-1-derived Prostaglandin E₂ (PGE₂) (unpublished). However, whether this pathway is activated in obesity is unknown. Mineralocorticoid-Receptor (MR), is involved in the stiffness and the endothelial dysfunction observed in obesity. We determined if mPGES-1 participates in aldosterone production from adipocytes in obesity and whether this is involved in endothelial dysfunction and vascular stiffness observed in this pathology.

Epididymal fat from DBA mPGES-1⁺/+ and mPGES-1⁻/⁻ mice fed with normal or high fat diet (HFD) was analyzed. CYP11B2 and MR expression was studied by qRT-PCR. Changes in vascular function and stiffness were studied using wire and pressure myographs. 3T3-L1 adipocytes were stimulated with 16,16-Dimethyl Prostaglandin E₂ (DPGE₂). Furthermore, visceral fat was obtained from patients and gene data were correlated with parameters of vascular stiffness. We found that CYP11B2 mRNA expression is augmented in the adipose tissue of the mPGES-1⁺/+ HFD mice, but not in mPGES-1⁻/⁻. However, we did not find differences in the MR. Moreover, DPGE₂ increases CYP11B2 mRNA expression in 3T3-L1 adipocytes. HFD provokes endothelial dysfunction in both genotypes, which is prevented by eplerenone. HFD induces vascular stiffness in mPGES-1⁺/+ but not in mPGES-1⁻/⁻ mice. Preliminary data in patients show positive correlations between mPGES-1, CYP11B2 and MR gene expression. Moreover, there is a positive correlation between pulse wave velocity and CYP11B2 gene expression.

Our study suggests that mPGES-1-derived PGE₂ is involved in the excessive aldosterone synthase expression observed in adipose tissue in obesity and this might have a role in the vascular damage observed in this pathology.

**Key words:** mPGES-1; aldosterone; obesity; vascular function.

Support: ISCIII-Fondo Europeo de Desarrollo Regional (FEDER): PI13/01488; RD12/0042/0024 and MINECO:SAF 2016-80305-P; Roche-IDIPAZ; CIBERCV, COST-ADMIRE:BM1301.

P-51

NADPH OXIDASE-DERIVED H₂O₂ IS INVOLVED IN THE
ENDOTHELIAL-DEPENDENT RELAXATIONS OF
INTRARENAL ARTERIES

Muñoz, M.¹,²*, Martínez M.P.¹,²*, López-Oliva M.E.¹*, Rodriguez C.¹, Rivera L.¹, Prieto D.¹

¹Departamento de Fisiología, Facultad de Farmacia, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain;
²Departamento de Anatomía y Anatomía Patológica Comparadas, Facultad de Veterinaria, Universidad Complutense de Madrid, Madrid, Spain

The role of reactive oxygen species (ROS) in kidney vascular function has extensively been investigated in the harmful context of oxidative stress in hypertension and diabetes and obesity-associated kidney disease (Sharma, *Diabetes* 64:663, 2015). We have recently demonstrated that H₂O₂ derived from endothelial CYP epoxygenases is involved in the non-nitric oxide (NO) non-prostanoid EDH relaxations of rat intrarenal arteries (Muñoz et al., *Free Radic Biol Med* 106:168, 2017). The present study was sought to investigate whether NADPH oxidases (Nox) may be functional sources of vasodilator H₂O₂ and to assess their role in the endothelium-dependent relaxations of intrarenal arteries. Renal interlobar arteries isolated from the kidney of Wistar rats were mounted in microvascular myographs to assess function. H₂O₂ production was measured by Amplex Red fluorescence and Nox2 and Nox4 enzymes were detected by Western blotting and by immunohistochemistry. Under conditions of cyclooxygenase and NO synthase blockade, acetylcholine (ACh) induced catalase sensitive endothelium-dependent relaxations that were blunted by the NADPH oxidase inhibitor apocynin, by the Nox2 inhibitor gp91ds-tat and by the dual Nox1-Nox4 inhibitor GKT136901. Acetylcholine (10⁻⁵ M) stimulated H₂O₂ production that was reduced by p91ds-tat and GKT 136901. Nox2 and Nox4 proteins were expressed in renal interlobar arteries and cortex,
levels of expression of both enzymes being higher in the cortex. Nox4 was co-localized with eNOS in the endothelium of renal arteries and glomeruli. These results suggest that both Nox2 and Nox4 are physiologically relevant renal endothelial sources of ROS generation and that Nox-derived H₂O₂ is involved in the endothelium-dependent relaxations of renal arteries.

**This work was supported by grant SAF2016-77526 from MINECO (Spain):**

**Key words:** H₂O₂, renal endothelium, Nox2, Nox4.

P-52

EFFECT OF SWIMMING TRAINING ON NEUROGENIC CONTRACTION AND STOCK OF INTRACELLULAR CALCIUM CONCENTRATION IN SPONTANEOUSLY HYPERTENSIVE RATS

Peña-García M., Miranda-Ferreira R., Castro Musial D., Caricatti-Neto A., Luiz da Silva R., Ribeiro Cezaretti M., Jurkiewicz N.H., Jurkiewicz A.

Department of Pharmacology and Department of Neurologic, Federal University of São Paulo (UNIFESP), São Paulo, Brazil

**Introduction:** Calcium (Ca²⁺) is an important second messenger cell; Changes in Ca²⁺ concentrations in the intracellular stocks were linked to the pathogenesis of hypertension. Studies have shown that exercise training has beneficial effects in both humans and animals, such as lowering blood pressure (BP), decreased sympathetic activity, decreased peripheral vascular resistance, and it is known that Ca²⁺ participates in these functions. However it is not known whether the exercise is able to modify the Ca²⁺ concentration of intracellular stores as sarcoplasmic reticulum (SR) and mitochondria (MIT) before the onset of the disease, thus leading to an improvement in blood pressure levels in SHR young and adults.

**Aims:** To study the effect of swimming training (ST) on BP, neurogenic contraction in vas deferens (VD) and the concentration basal of calcium in adrenal gland SHR youth and adults.

**Materials and Methods:** All experimental procedures were approved by Ethical Committee of Federal University of São Paulo, Brazil (7866020315). The ST consisted of swimming sessions of 60 min, 5 days per week, for 8 weeks. Using animals NWR and SHR Young (12 weeks) and adults (20 weeks), n = 8; We study: 1) BP Measurement. 2) Neurogenic Contraction through electrical stimulation (0.2 Hz, 60 V) of the sympathetic nerves of the vas deferens (VD); 2a) Using CRT mixture of caffeine 10⁻² M, Ryanodine 3 × 10⁻⁶ M and thapsigargin 10⁻⁶ M to depletion of Ca²⁺ RE. 2b) Neurogenic Contraction using CCCP 10⁻⁶ M to depletion of Ca²⁺ MIT. 3) Fluorescence microscopy with Fura-2AM, fluorescent probe to measure intracellular Ca²⁺ basal unstimulated, in adrenal gland slices in trained and sedentary animals.

**Results and Conclusion:** Compared to the sedentary animals trained animals both NWR and SHR youth and adults showed: 1) Decreased BP (mmHg): Young = NWR (s) 145.5 ± 1.0; NWR (t) 128.6 ± 1.2 and SHR (s) 199.5 ± 1.3; SHR (t) 141.2 ± 1.5; Adults = NWR (s) 151.4 ± 0.56; NWR (t) 144.4 ± 1.1 and SHR (s) 228.5 ± 2.0; SHR (t) 173.5 ± 1.9 2)) Decrease in neurogenic contraction in training groups; 2a) Decrease in neurogenic contraction (expressed in g of tension / g of tissue) using CRT: Young = NWR (s) 33.9 ± 2.1; NWR (t) 25.17 ± 2.1 * and SHR (s) 43.57 ± 0.8; SHR (t) 26.9 ± 1.3 ***; Adults = NWR (s) 28.5 ± 3.7; NWR (t) 18.41 ± 0.7 * and SHR (s) 48.06 ± 4.2; SHR (t) 26.96 ± 1.3. 2b) Decrease in neurogenic contraction using CCCP those from group.3) Decreased basal intracellular Ca²⁺ expressed in [Ca²⁺]i basal (UAF): Young = NWR (s) 0.95 ± 0.08; NWR (t) 0.89 ± 0.11; SHR (s) 1.3 ± 0.1; SHR (t): 1.0 ± 0.2. Adults = NWR (s). 1.3 ± 0.12; NWR (t) 0.99 ± 0.23; SHR (s) 2.4 ± 0.19; SHR (t): 1.99 ± 0.35. With these results we can infer that the ST was able to improve the condition of these animals by reducing all parameters studied before the development of hypertension in young animals as well as in adults, showing the importance of

physical activity, because it leads to improved quality and the life expectancy of animals, results could be extrapolated to humans.

**Financial Support:** FAPESP, CAPES, CNPq.

P-53

SCLERODERMA FIBROBLASTS SUPPRESS ANGIOGENESIS VIA TGF-β/CAVEOLIN-DEPENDENT SECRETION OF PIGMENTED EPITHELIUM DERIVED FACTOR: TGF-β RECEPTOR INHIBITORS AS A NOVEL THERAPEUTIC TARGET FOR THE TREATMENT OF SCLERODERMA

Elies J.<fnref n="1" />,², Liakouli V.<fnref n="1" />,², Abignano G.<fnref n="1" />,², Esteves F.<fnref n="1" />,², Emery P.<fnref n="1" />,², Denton C.<fnref n="1" />,², Giacomelli R.<fnref n="1" />,², Mavria G.<fnref n="1" />,², Del Galdo F.<fnref n="1" />,²

Systemic Sclerosis (SSc) or Scleroderma is an autoimmune condition characterised by vasculopathy and tissue fibrosis of the skin and multiple internal organs, with heterogeneous severity and clinical outcome. Transforming Growth Factor-beta (TGF-β) has been shown to play a major role in the pathogenesis of tissue fibrosis in Scleroderma, whereas its role in the pathogenesis of vasculopathy and defective angiogenesis is less clearly elucidated. SSc fibroblasts secrete Pigmented Epithelium Derived Factor (PEDF), a major antiangiogenic protein implicated in the vascular homoeostasis of the retina and pigmented epithelium. By means of immunohistochemistry we show that PEDF expression is increased in SSc skin biopsies whilst caveolin-1 (Cav-1) expression is downregulated. Importantly we show that 10 ng/ml TGF-β increases PEDF in both healthy and SSc fibroblasts and this effect is associated with downregulation of Cav-1. Additionally, we show that PEDF has a strong anti-angiogenic effect in an organotypic fibroblast/endothelial co-culture angiogenesis model, and demonstrate that fibroblasts lacking Cav-1 suppress angiogenesis through PEDF, without affecting endothelial cell proliferation. Concordantly *in vivo*, mice with transgenic hyperactivation of TGF-β receptor display reduced Cav-1 expression and increased expression of PEDF in the skin, which is accompanied by a reduced capillary density. Altogether, our data show that TGF-β plays an important role in the pathogenesis of vasculopathy in SSc through downregulation of Cav-1 in tissue fibroblasts, and corresponding increased expression of PEDF leading to suppression of angiogenesis. Consequently, mice with fibroblast specific activation of the TGF-β pathway may be a useful pre-clinical model to study the contribution of TGF-β in vasculopathy of Scleroderma. Our studies establish the molecular basis for the therapeutic use of TGF-β receptor inhibitors (currently in phase 2/3) in the treatment of vasculopathy associated with fibrotic diseases.

**Key words:** Systemic Sclerosis; vasculopathy; Transforming Growth Factor β; Pigmented Epithelium Derived Factor.

P-54

HIGH-FAT FEEDING PROMOTES DYSFUNCTION OF PERIVASCULAR ADIPOSE TISSUE

Almabrouk T.A.M.<fnref n="1" />,², Ugusman A.B.<fnref n="1" />,³, White A.D.<fnref n="1" />, Skiba D.<fnref n="1" />, Salt I.P.<fnref n="1" />, *Kennedy S.*<fnref n="1" />

Perivascular adipose tissue (PVAT) releases multiple vasoactive substances and regulates vascular tone. AMPK-activated protein kinase is present in PVAT and involved in the anticontractile effect of PVAT (1). In mice, high fat feeding increases expression of pro-inflammatory genes within the PVAT which may induce dysfunction (2). In this

<footnote>*Equal contribution.</footnote>

<footnote n="1">¹School of Pharmacy, University of Bradford, Bradford, UK; ²School of Medicine, University of L'Aquila, Italy; ³School of Medicine, University of Leeds, Leeds, UK</footnote>

<footnote n="1">¹Institute of Cardiovascular & Medical Sciences, University of Glasgow, Glasgow, United Kingdom; ²Department of Pharmacology, School of Medicine, University of Zawia, Zawia, Libya; ³Department of Physiology, Faculty of Medicine, National University of Malaysia Medical Centre, Kuala Lumpur, Malaysia</footnote>
study we investigated the effect of up to 12 weeks fat feeding on
PVAT function and whether the effects were altered in mice lacking
AMPK.

Wild-type (WT) and knock-out (KO) mice were fed chow or high fat diet (HFD) and blood pressure was monitored every 4 week by tail-cuff plethysmography. At 12 weeks, vascular function in endothelium-denuded rings of thoracic aorta with or without attached PVAT was studied by small artery wire myography. Macrophage markers in PVAT were studied using RT-PCR and release of adiponectin quantified by ELISA. AMPK activity in PVAT was assessed by Western blotting.

HFD caused a significantly greater weight gain in WT and KO mice compared to chow diet but had no effect on blood pressure in either strain. Relaxation to cromakalim was significantly attenuated in PVAT-containing aortic rings from WT mice fed HFD but not KO mice. HFD did not affect relaxation in aortic rings without PVAT in either strain. Fat feeding increased IL-1β and iNOS expression in WT PVAT and significantly reduced adiponectin secretion but this was not observed in KO PVAT. In WT PVAT, the level of phospho-AMPK was significantly greater compared to KO mice. HFD significantly reduced total and phospho-AMPK in WT but not KO mice.

In conclusion, 12 weeks of HFD causes PVAT to lose its anticontrac-
tile effect. This is likely due to reduced adiponectin secretion, possibly
through alterations in AMPK activity in the PVAT.

(1) Almabrouk et al (2016) Br J Pharmacol. https://doi.org/10.1111/
bph.13633.

(2) Chatterjee et al (2009); Circ Res. 104:541-9.

**Keywords:** AMPK; perivascular adipose tissue; fat-feeding; adiponectin

P-55

NEUROTROPHIN-3 AND TRKC EXPRESSION IN BLOOD
VESSELS AND LEFT VENTRICLE OF ADULT RODENTS

Ivorra M.D.¹, Zambrano A.¹, Bové M.¹, Arce C.¹, Moltó F.¹, Noguera M.A.¹, Piquer S.², Fariñas I.², D'Ocon P.¹

¹Departament de Farmacologia and ERI BiotecMed. Universitat de València. Spain; ²Departament de Biologia Cellular and ERI BiotecMed, Universidad de Valencia, Spain

Neurotrophin-3 (NT-3), an established neurotrophic factor that partici-
pates in embryonic heart development (1), has also been detected in
heart and blood vessels of adult animals (2, 3).

In the present study the expression pattern of NT-3 and its receptor
TrkC has been determined in left ventricle (LV) and blood vessels
(aorta and tail artery) as well as in cerebral cortex of adult Wistar rats
by real time quantitative RT-PCR and Western blot analysis. In addi-
tion, mouse expressing the lacZ reporter gene, encoding the enzyme
beta-galactosidase, from the NT-3 locus (Ntf3+/lacZneo) were used for
studying by immunohistochemistry, the distribution of the NT-3 gene
in these tissues.

The results obtained shown that mRNA and protein expression levels of NT-3 were detected in blood vessels in a major proportion than in LV and cerebral cortex. TrkC was expressed at higher levels in blood vessels and cerebral cortex compared to LV. 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining in Ntf3+/lacZneo mice reveals that NT-3 appears in blood vessels of LV and cerebral cortex and is largely observed in the aorta and tail artery wall.

(1) Lin *MI* et al., *Developmental Biology* **226**, 180–191 (2000).

(2) Kawaguchi-Manabe H et al., Life Sciences 81, 385-392 (2007).

(3) *Scarisbrick IA et al.*, *J Neurosci* 13(3), 875-893 (1993).

Keyword: neurotrophin-3, TrkC, blood vessels, heart.

Funded by Ministerio de Economía y Competitividad (SAF2013-45362-R). AZ received a fellowship from the Santiago Grisolía Foundation.

P-56

LACTOBACILLUS FERMENTUM CECT5716 CONSUMPTION PROTECTS KIDNEY AND IMPROVES ENDOTHELIAL DYSFUNCTION IN MURINE LUPUS

Robles-Vera I.¹,*, Toral M.¹,*, Romero M.¹,², Sánchez M.¹,², Jiménez R.¹,²,³, O'Valle F.²,⁴, Rodriguez-Nogales A.¹,², de la Visitación N.¹, Gálvez J.¹,²,⁵, Duarte J.¹,²,³

¹Department of Pharmacology, School of Pharmacy, University of Granada, 18071- Granada, Spain; ²Instituto de Investigación Biosanitaria de Granada, ibs.GRANADA, Granada, Spain; ³CIBERCV, Granada, Spain; ⁴Department of Pathology, School of Medicine, University of Granada, Granada, Spain; ⁵CIBER-EHD, Center for Biomedical Research (CIBM), University of Granada, Granada, Spain

Systemic lupus erythematosus (SLE) is a multisystemic chronic autoim-
mune inflammatory disorder that is associated with a high risk for the
development of renal and cardiovascular diseases associated with intes-
tine dysbiosis. *Lactobacillus fermentum* CECT5716 (LC40) is a probi-
otic bacteria that modulates the human immune system. The aim of this
study was to examine whether LC40 ameliorates disease activity and
cardiovascular complications in a female mouse model of lupus. Eight-
ten-week-old NZBWF1 (lupus) and NZW/LacJ (control) mice were
treated with LC40 (5x10⁸ CFU day⁻¹) during 15 weeks. Blood pres-
sure, plasmatic double-stranded DNA autoantibodies, lipopolysaccha-
rides (LPS) and cytokines, nephritis, spleen and mesenteric lymph nodes
lymphocytic populations, endothelial function, and vascular oxidative
stress were compared in treated and untreated mice. LC40 treatment
reduced lupus disease activity, blood pressure, cardiac and renal hyper-
trophy, splenomegaly, albuminuria, and renal injury in lupus mice.
LC40 reduced the elevated T, B, Treg and Th1 cells in spleen and in
mesenteric lymph nodes from lupus mice. LC40 lowered the higher
plasma concentration of proinflammatory cytokines observed in lupus
mice. Aortae from lupus mice showed reduced endothelium-dependent
vasodilator responses to acetylcholine. Endothelial dysfunction induced
by SLE is related to both increased NADPH oxidase driven-superoxide
production and Rho-kinase mediated eNOS inhibition which were nor-
malized by LC40. In addition, LC40 treatment reduced hypertension,
endothelial dysfunction, and organ damage in severe lupus mice, which
was associated with reduced plasma antidouble-stranded DNA autoanti-
bodies and anti-inflammatory and antioxidant effects in target tissues.
Probiotic administration to lupus mice reduced the metabolic endotox-
emia since it decreased the LPS plasmatic levels, which were related to
a significant improvement of the gut barrier disruption. In conclusion,
our findings identify this gut microbiota manipulation as a promising
target for an alternative approach in the treatment of SLE and its associ-
ated vascular damage.

**Keywords:** probiotics, lupus, endothelial dysfunction, kidney, T cell, nitric oxide, reactive oxygen species

*These authors contributed equally as first author.

P-57

INCREASED CONTRACTILE RESPONSES TO
PHENYLEPHRINE IN THE ASCENDING AORTA OF A
MURINE MODEL OF MARFAN SYNDROME REVEAL
REGIONAL AND SEX DIFFERENCES

Jiménez-Altayó F.¹, Meirelles T.², Bonorino F.², Siegert A.-M.², Vila E.¹, Egea G.²

¹Departament de Farmacologia, de Terapèutica i de Toxicologia, Institut de Neurociències, Facultat de Medicina, Universitat Autònoma de Barcelona, Bellaterra, Spain; ²Departament de Biomedicina, Facultat de Medicina i Ciències de la Salut, Universitat de Barcelona, Barcelona, Spain; IDIBAPS, Barcelona, Spain

Marfan syndrome (MFS) is a hereditary disorder of the connective tissue that results from mutations in the gene for fibrillin 1 (Fbn1) that cause life-threatening aortic aneurysm. MFS is equally prevalent in
men and women, but epidemiologic evidence suggests that men are at higher risk for aortic complications. Aneurysmal expansion initiates at the aortic root and can progress into the ascending portion. Nevertheless, neither the mechanisms involved in sexual dimorphism nor the ascending aortic reactivity have been fully investigated. We aimed to study the influence of sex on aortic function in 6-month-old MFS ($Fbn1^{C1039G+}$) mice. Ascending and descending thoracic aorta reactivity was evaluated by wire myography and elastic fiber integrity was assessed by Verhoeff-Van Gieson histological staining. Contractions to phenylephrine (Phe) were only altered in ascending aorta of males with MFS, where higher (P < 0.05) contractions than wild-type (WT) mice were observed. In contrast, MFS descending thoracic aorta only showed a trend (P = 0.0599) toward decreased contraction in males. In ascending aorta, incubation with indomethacin (10 µM), a non-selective cyclooxygenase inhibitor, removed the MFS-induced enhancement of Phe contractions in males. Although endothelium-dependent acetylcholine (ACh)-induced relaxations were similar, the presence of Nω-nitro-l-arginine methyl ester (300 µM) plus indomethacin revealed augmented (P < 0.05) endothelium dependent hyperpolarization (EDH)-type relaxations in MFS regardless of sex. Consistently, subsequent inhibition of EDH-type relaxations with small (apamin; 100 nM) and intermediate (charybdotoxin; 100 nM) calcium-activated potassium channel inhibitors abolished MFS-related differences on EDH-type responses. As expected, elastic fiber ruptures increased in MFS compared to WT ascending aortas, but no sex differences were observed. These results suggest that $Fbn1^{C1039G+}$ mice show regional and sex differences in aortic contractility. Increased $\alpha_1$-adrenoceptor-dependent vasoconstriction and enhanced EDH-type relaxations could be functional adaptations to counteract excessive aortic enlargement in males with MFS. The potential long-term impact of these alterations during aortic disease development deserves attention.

**Keywords:** Marfan syndrome; sex differences; ascending thoracic aorta reactivity; aneurysm

## P-58
PROTECTIVE EFFECTS OF PPARβ/A ACTIVATION ON ENDOTHELIAL DYSFUNCTION INDUCED BY PLASMA FROM LUPUS PATIENTS: ROLE OF ENDOPLASMIC RETICULUM STRESS

Toral M.<fnref n="1" />, Jiménez R.<fnref n="1" />,²,³, Romero M.<fnref n="1" />,²,³, Robles-Vera I.<fnref n="1" />, Sánchez M.<fnref n="1" />,², Sabio J.M.²,<fnref n="4" />, Duarte J.<fnref n="1" />,²,³

M.T. and R.J. contributed equally as first author. J.M.S. and J.D. contributed equally as last author.

**Abstract:** We tested whether GW0742, a peroxisome proliferator-activated receptor beta/delta (PPARβ/δ) agonist, improves endothelial dysfunction induced by plasma from systemic lupus erythematosus (SLE) patients involving the inhibition of endoplasmic reticulum (ER) stress. 12 lupus and 5 healthy (control) nonpregnant women participated in the study. Cytokines and double-stranded DNA autoantibodies (anti-dsDNA) levels were tested in plasma samples. Endothelial cells, isolated from human umbilical cord veins, (HUVECs) were used to measure nitric oxide (NO), and intracellular reactive oxygen species (ROS) production, NADPH oxidase activity, and ER stress markers. Interferon-γ, interleukin-6, and interleukin-12 levels were significantly increased in plasma from SLE patients with active nephritis (AN), as compared to both SLE patients with inactive nephritis (IN), and control group. The NO production stimulated by both the calcium ionophore A23187 and insulin, was significantly reduced in HUVECs incubated with plasma from AN-SLE patients as compared with control group. Plasma from IN-SLE patients did not modify A23187-stimulated NO production. Increased ROS production and NADPH

oxidase activity were found in HUVECs incubated with plasma from AN-SLE patients, which were suppressed by the ER stress inhibitor 4-PBA and the NADPH oxidase inhibitor apocynin. GW0742 incubation restored the impaired NO production, the increased ROS levels, and the increased ER stress markers induced by plasma from AN-SLE patients. These protective effects were abolished by the PPARβ/δ antagonist GSK0660 and by silencing PPARβ/δ. In conclusion, PPARβ/δ activation may be an important target to control endothelial dysfunction in SLE patients.

**Keywords:** Endothelial dysfunction, PPARβ/δ, Systemic lupus erythematosus, Nitric oxide, ROS, HUVECs, Endoplasmic reticulum

## P-59
ELECTROPHYSIOLOGICAL CHARACTERISATION OF TWO NOVEL KCNK3 (TASK-1) MUTATIONS INDICATED IN PULMONARY ARTERIAL HYPERTENSION

Cunningham K.P.<fnref n="1" />, Veale E.L.<fnref n="1" />, Escribano-Subias P.³, Cogolludo A.L.², Mathie A.<fnref n="1" />

**Introduction:** Pulmonary arterial hypertension (PAH) affects approximately 15-50 people per million population (1). KCNK3, the gene that encodes the two pore domain potassium channel TASK-1 (K2P3.1), has been identified as a possible disease-causing gene within familial PAH (2). Recently two new mutations have been identified in KCNK3, G106R and L214R in PAH patients (3). The aim of this study is to assess the electrophysiological profile of these mutated KCNK3 channels and understand how this might contribute to PAH.

**Method:** Currents through wild-type (WT) and mutated human KCNK3 channels transiently expressed in tsA201 cells were measured using whole-cell patch-clamp electrophysiology. Data are given as mean ± SEM (n=cells) of the current measured between -40 and -80 mV and statistical analysis used one-way ANOVA with post-hoc Dunnett's multiple comparison test.

**Results:** At pH 8.4, where KCNK3 channels are not inhibited by H ions, G106R and L214R mutant KCNK3 channels had a significantly reduced current amplitude compared to WT channels (WT: 374 ± 59 pA, n = 6; G106R 145 ± 35 pA n = 6; L214R 197 ± 44 pA, n = 6; P < 0.05). Similarly, in the presence of the phospholipase A2 inhibitor ONO-RS-082 (10 µM), a compound known to increase KCNK3 current (2), mutant channel current was significantly smaller compared to WT channels (WT: 336 ± 110 pA, n = 5; G106R: 40 ± 7 pA, n = 7; L214R, 106 ± 20 pA n = 7; P < 0.05).

**Conclusion:** The KCNK3 mutants G106R and L214R have been indicated in the pathogenesis of PAH (3). Evidence from this study suggests these mutant channels carry less current than WT channels and, in contrast to other KCNK3 mutations (2) this activity is not recovered by application of ONO-RS-082.

**Keywords:** Pulmonary arterial hypertension, KCNK3 (TASK-1), Electrophysiology, Potassium channel

**References:**

1. Humbert et al. (2014). Circulation 130: 2189-2208

2. Ma et al. (2013). NEJM 369: 351-361

3. Navas-Tejador et al. (2016). Clin Genet. https://doi.org/10.1111/cge. 12869

<footnote n="1">¹Department of Pharmacology, School of Pharmacy, University of Granada, 18071- Granada, Spain; ²Instituto de Investigación Biosanitaria de Granada, ibs.GRANADA, Granada, Spain; ³CIBER de enfermedades cardiovasculares (CIBERCV), Madrid, Spain;</footnote>

<footnote n="4">⁴Department of Internal Medicine, Virgen de las Nieves University Hospital, Granada, Spain</footnote>

<footnote n="1">¹Medway School of Pharmacy, University of Kent, Chatham, United Kingdom; ²Departmento de Farmacologia, Universidad Complutense de Madrid, Madrid, Spain; ³Red de Investigación Cardiovascular, Instituto de Salud Carlos III, Madrid, Spain</footnote>
P-60
OPTICAL CONTROL OF MUSCARINIC ACETYLCHOLINE
RECEPTORS USING PHOTOSWITCHABLE BITOPIC
LIGANDS

Riefolo F.¹, Garrido-Charles A.¹, Matera C.¹, Claro E.², Masgrau R.²,
Camarero N.¹, Gomila-Juanela A.¹, Gorostiza P.¹,³,⁴

¹Institut de Bioenginyeria de Catalunya (IBEC), Barcelona 08028,

Spain; ²Universitat Autònoma de Barcelona (UAB); ³CIBER-bbn;

⁴ICREA

Muscarinic acetylcholine receptors (mAChRs) are class A GPCRs char-
acterized by a widespread tissue distribution and involved in the control
of numerous central and peripheral physiological responses. The high
sequence homology of the different subtypes (M1–M5) in the transmem-
brane region hampers the development of subtype selective orthosteric
agonists. On the other hand, the allosteric site, located in the extracellular
loop, is less conserved, thus muscarinic allosteric agents are commonly
endowed with a more pronounced subtype-selectivity. Recently, a new
strategy was developed towards the selective modulation of mAChRs,
i.e. the development of dualsteric ligands, which are molecules that can
bind simultaneously to both the orthosteric and the allosteric sites of such
receptors. The most interesting bitopic ligands emerging from this inves-
tigation were hybrid derivatives incorporating a) iperoxo, an oxotremor-
ine-related unselective orthosteric superagonist, b) a polymethylene
spacer, and c) a moiety targeting the allosteric site.¹

Inspired by this strategy, in the course of our ongoing development of
photoswitchable ligands for the optical control of (neuro)biological
functions,² we designed and synthesized a new set of light-regulated
muscarinic bitopic ligands by replacing the polymethylene spacer chain
with an azobenzene linker to serve as molecular photoswitch. This
modification enabled the remote control of the mutual position
between the two pharmacophoric moieties with light, thus potentially
modulating affinity and efficacy of our compounds as a function of
their photo isomerization state. One of our ligands (P-Azo-Iper) turned
out to be a potent activator of M2 receptors under UV illumination
(cis isomer), but inactive after relaxation in the dark or under illumina-
tion with blue light or white light (trans isomer). All compounds were
investigated in binding and enzymatic experiments. Their cellular
responses were evaluated in vitro and in vivo in the Xenopus tropicalis
heart. The design, synthesis and pharmacological profile of these new
photopharmacological tools will be discussed.

**Keywords:** photopharmacology; muscarinic receptors; dualsteric ligands; GPCRs

**References:**

1. Disingrini T et al. J Med Chem 2006; 49(1): 366-372; Antony J et al. FASEB J 2009; 23(2): 442-50; Matera C et al. Eur J Med Chem 2014;75:222-232.

2. Izquierdo-Serra M et al. Nat Commun 2016; 7: 12221; Pittolo S et al. Nat Chem Biol 2014; 10(10): 813–815.

P-61
SENESCENCE AND LATE-ONSET ESTROGEN THERAPY
PROMOTE DELETERIOUS EFFECTS IN COMMON
CAROTID ARTERY OF FEMALE MICE: INFLUENCE OF
EPIGENETIC REGULATION

Costa T.J.¹,²,³, Dantas A.P.², Jimenez-Altayó F.³, Akamine E.H.¹,
Vila E.³, Carvalho M.H.C.¹

¹Department of pharmacology. Institute of Biomedical Science.
University of São Paulo, Brazil; ²Institut Clínic del Tórax, Institut
d'Investigacions Biomèdiques August Pi i Sunyer, Barcelona, Spain;

³Facultat de Medicina, Departament de Farmacologia, Terapèutica i Toxicologia, Institut de Neurociències, Universitat Autònoma de Barcelona, Bellaterra, Spain

The incidence of vascular disease increases with aging at alarming
rates in women. Yet, the risk-benefits of estrogen (E₂) therapy in

postmenopausal women are still under debate. Controversies may be
explained by age of postmenopausal women and timing to start E2
therapy. We aimed to determine the effects of early (E2E) and late-
(E2L) onset E2 therapy in carotid of senescent (SAMP8) and non-
senescent (SAMR1) female mice. Six-month-old SAMP8 and SAMR1
were ovariectomized (OVX) and treated with E2 (5 µg/kg) starting at
the first day of OVX (E2E) or 6 weeks after OVX (E2L). Concentra-
tion-response curves to phenylephrine (Phe1 nM-100 µM) were per-
formed in isolated carotid rings in the absence or presence of the non-
selective cyclooxygenase inhibitor, indomethacin (1 µM). Prostaglan-
din I2 (PGI2) and thromboxane A2 (TXA2) were measured by ELISA,
estrogen receptor (ER) α and β expression by immunofluorescence and
the degree of DNA methylation of genes encoding ER by methylation-
sensitive qPCR. In OVX-SAMR1, both E2E and E2L decreased vaso-
constriction. Indomethacin reduced vasoconstriction in non-treated
OVX-SAMR1, but was without effect in E2-treated mice. PGI2 levels
were greater after both E2 treatments, while TXA2 was reduced in E2E
vs. OVX-SAMR1. In SAMP8, E2L, but not E2E, enlarged vasocon-
striction compared to OVX. Indomethacin incubation did not affect
vasoconstriction in either SAMP8 group. E2-treatment in OVX-SAMP8
did not modify PGI2 release, but increased TXA2 levels in E2L mice.
Ratio of ERα/ERβ expression was higher in SAMR1 than SAMP8
after E2E and E2L treatments. These results are in association with a
higher degree of DNA methylation at exon 8 of ERα in SAMP8. In
conclusion, senescence abolishes the beneficial effects of E2 in carotid
arteries. While E2E has no effect on vascular function, E2L worsen
vasoconstriction. These effects are associated with senescence-mediated
modifications on DNA-methylation status of ERα, which may influ-
ence its pattern of expression.

**Keywords:** estrogen; carotid artery; senescence; DNA methylation
**Financial support:** FAPESP (2015/26690-9), CAPES (269/12), Pro-
grama Hispano-Brasileño de Cooperación Interuniversitaria (HBP-
2011-0054_PC); Red de Investigación Cardiovascular – HERACLES
(RD12/0042/0006).

P-62
INFLUENCE OF LYCOPENE ON METABOLIC SYNDROME
INDUCED BY FRUCTOSE IN WISTAR RATS

Ferreira-Santos P.¹,², Áparicio R.¹,², Sevilla M.A.¹,², Carrón R.¹,²,
Monroy-Ruiz J.³, Montero M.J.¹,²

¹Dpto. de Fisiología y Farmacología; ²Instituto de Investigaciones
Biomédicas de Salamanca (IBSAL). Campus Miguel de Unamuno.
37007 Salamanca; ³Dpto de Salud. Universidad Iberoamericana, 01219
Ciudad de México

Overconsumption of fructose results in dyslipidemia, hypertension, and impaired glucose tolerance, which characterizes the metabolic syndrome (MS). Thus, the aim of this study was to investigate the effects of a diet supplemented with lycopene on MS induced by fructose in rats.

**Methods:** Wistar rats were divided into 3 groups: control (C) fed with standard diet, group (F) receiving a supplementation of 20% fructose and group (F+L) fed with a diet supplemented with 20% fructose plus 0.01% of lycopene, for 8 weeks. Blood pressure was monitored by the tail cuff method. Glucose tolerance test and plasmatic triglycerides levels were determined. The liver hypertrophy index was determined using liver weight/body weight ratio.

The endothelial function was evaluated on aortic rings. Concentration-response curves to acetylcholine and phenylephrine were performed in rings with or without perivascular adipocyte tissue.

**Results:** High fructose diet caused a significant increase on blood pressure (from 108 ± 1 to 134 ± 2 mmHg), plasma triglycerides levels, oral glucose tolerance, and liver weight. The endothelium function in presence of perivascular fat was also impaired.

Lycopene supplementation prevented the increase of blood pressure and improved the oral glucose tolerance compared to group (F). Lycopene treatment was able to reduce the triglycerides levels in plasma increased by fructose ingestion. The liver hypertrophy index was
higher in group (F) than group (F+L) and control group (29.3 ± 1.8 vs. 26.3 ± 1.6 and 27.1 ± 1.6 mg/g respectively, *P* < 0.05). In addition, lycopene treatment ameliorated the endothelial function in presence of perivascular adipocyte tissue, recovering the impairment of vasorelaxation and vasoconstriction.

**Conclusion:** Our results indicate that a diet supplemented with lycopene may be a beneficial therapeutic approach for MS. Lycopene was kindly supply by DSM NUTRITIONAL PRODUCTS (México).

**Keywords:** Lycopene; metabolic syndrome; fructose

## P-63
### EFFECT OF THE PROBIOTIC LACTOBACILLUS FERMENTUM CECT5716 IN HYPERTENSIVE RAT INDUCED BY CHRONIC NITRIC OXIDE BLOCKADE

de la Visitación N.¹, Robles-Vera I.¹, Romero M.¹,², Sánchez M.¹,², Toral M.¹, Jiménez R.¹,², Duarte J.¹,²

¹Department of Pharmacology, School of Pharmacy, University of Granada, 18071- Granada, Spain; ²Instituto de Investigación Biosanitaria de Granada, ibs.GRANADA, Granada, Spain;

³Department of Pathology, School of Medicine, University of Granada, Granada, Spain

Lactobacillus fermentum CECT5716 (LC40) reduced blood pressure in spontaneously hypertensive rats. We tested whether the probiotic Lactobacillus fermentum CECT5716 ameliorates hypertension in rats with chronic nitric oxide synthase inhibition. Rats were randomly divided into four different treatment groups for 4 weeks: a) vehicle (control, 1 ml of tap water once daily), b) vehicle plus L-NAME (50 mg 100 ml⁻¹ in drinking water), c) LC40 (10⁹ colony-forming units/day by gavage), and d) LC40 plus L-NAME. The evolution in systolic blood pressure, and morphological variables, proteinuria, lymphocytes populations in mesenteric nodes and vascular NADPH oxidase activity and vascular reactivity at the end of experiment were analysed. LC40 did not inhibit the development of L-NAME-induced hypertension, the increase in the left ventricular hypertrophy, and proteinuria. However, this probiotic partially prevents the impaired endothelium- and NO-mediated relaxation to acetylcholine in aorta, being without effects in small mesenteric arteries. This improvement was suppressed in presence of apocynin in the bath and was accompanied by reduced NADPH oxidase activity in both arterial beds from LC40 plus L-NAME group as compared with L-NAME group. In mesenteric nodes L-NAME increases Th17 cells and reduces Treg cells. These changes were prevented by LC40 treatment. In most cases these effects were not observed in normotensive animals. In conclusion, this study confirms the critical role of NO in the antihypertensive and end-organ protective effects of the probiotic LC40 in animal models of hypertension.

**Keywords:** L-NAME-induced hypertension, Probiotics, LC40, NADPH oxidase, Nitric oxide, immune response

## P-64
### POMACE OLIVE OIL ENRICHED IN OLEANOLIC ACID RESTORES VASCULAR DYSFUNCTION AND METABOLIC PARAMETERS IN MICE FED A HIGH FAT DIET

Claro C.¹, Justo M.L.¹, Quintela J.C.², Alvarez de Sotomayor M.¹, Herrera M.D.¹, Rodríguez-Rodríguez R.³

¹Department of Pharmacology, Faculty of Pharmacy, University of Sevilla, Sevilla, Spain; ²Natac Biotech S.L., Parque Científico de Madrid, Campus de Cantoblanco, Madrid, Spain; ³Basic Sciences Department, Faculty of Medicine and Health Sciences, Universitat Internacional de Catalunya, Sant Cugat del Vallés, Barcelona, Spain

**Background and objective:** Despite the amount of research on the effects of virgin olive oil and its components in cardiovascular disease, little attention has been paid to the effects of Pomace Olive Oil, an

subproduct traditionally used in Spain, which is an important source of triterpenic acids such as oleanolic acid. Our aim was to evaluate the potential effects of a Pomace Olive Oil enriched in oleanolic acid (POMO) on blood biochemical parameters and vascular function related to obesity.

**Methods and results:** Male C57BL/6J mice were fed a high fat diet (HFD) for 10 weeks. Mice were randomized into 2 groups: 1) HFD; 2) HFD supplemented with POMO (17% w/w) for 10 weeks. In parallel, another group was fed a standard diet (SD). Body weight, food and water intake were weekly evaluated. After treatment, functional studies were performed in aortic rings in isometric myograph and serum glucose and total cholesterol (TC) were determined. Introduction of POMO in the diet reduced body weight gain (*P* < 0.0001 vs. HFD). Oral glucose tolerance test revealed significant differences between SD and HFD animals (*P* < 0.01 vs. SD) that were restored by POMO administration (*P* < 0.05 vs. HDF). Serum glucose and TC levels were also attenuated in POMO-fed mice (*P* < 0.001 vs. HFD). POMO also restored endothelial dysfunction in aorta compared to HFD group. In aortas from POMO group, the ACh relaxation was not completely abolished by L-NAME, suggesting the possible involvement of a NO-independent mechanism.

**Conclusion:** These findings suggest that pomace olive oil enriched in oleanolic acid reduces body weight, improves glucose tolerance and hypercholesterolemia in obese mice and ameliorates vascular dysfunction associated to HFD-induced obesity.

**Keywords:** Pomace Olive Oil, Vascular function, Glucose tolerance, High-fat diet

## P-65
### MARCKS REGULATES VASCULAR CONTRACTILITY

Jahan K.S., Shi J., Greenwood I.A., Albert A.P.

Vascular Biology Research Centre, St George's University of London, London, United Kingdom

Myristoylated alanine-rich C-Kinase (MARCKS) is expressed in vascular smooth muscle cells (VSMCs), but its function is unclear. The present work investigates the role of MARCKS in regulating vascular tone.

Experiments were carried out using freshly isolated VSMCs and tissue lysates from rat and mice mesenteric arteries. Expression and interactions between MARCKS, voltage-dependent Ca²⁺ channels (VDCCs), and PIP₂, were studied using western blot, immunocytochemical, co-immunoprecipitation, and dot-blot methods. The cell-permeable MARCKS inhibitor, MANS peptide, and MARCKS knockdown were examined on contractility using isometric tension recordings. The result of MANS peptide on VDCC activity was measured using whole-cell patch clamp techniques.

MARCKS was expressed in mouse and rat mesenteric arteries, predominantly at the plasma membrane. MANS peptide evoked concentration-dependent increases in vascular contractility, which were abolished by VDCC blockers. Knockdown of MARCKS significantly reduced contractions evoked by MANS peptide. In un-stimulated tissue, MARCKS interacted with the L-type VDCC protein, CaV₁.₂, and PIP₂. MANS peptide released PIP₂ from MARCKS and increased interactions between CaV₁.₂ and PIP₂. MANS peptide induced an increase in the peak amplitude, and a negative shift in activation and inactivation of whole-cell VDCC currents. Depletion of PIP₂, with wortmannin, abolished excitatory effects of MANS peptide on whole-cell VDCC activity.

This is the first study to demonstrate that MARCKS is involved in regulating vascular contractility. We propose that in un-stimulated vessels, MARCKS prevents opening of VDCCs by acting as a PIP₂ buffer, but following MARCKS inhibition PIP₂ is released leading to channel gating and contractility.

**Keywords:** MARCKS; PIP₂, VDCCs, Vascular Contractility
P-66

LUPALBIGENIN, A PRENYLATED ISOFLAVONE IN DERRIS SCANDEN EXTRACTS, LACKS VASODILATOR ACTIVITY IN THE PORCINE ISOLATED SPLENIC ARTERY

Sriklung K.¹,², Samosorn S.³, Apiratikul N.³, Wilson V.²,
Watanapokasin R.¹

¹Department of Biochemistry, Faculty of Medicine, Srinakharinwirot University, Bangkok, Thailand; ²School of Life Sciences, Faculty of Medicine & Health Sciences, University of Nottingham, United Kingdom; ³Department of Chemistry, Faculty of Science Srinakharinwirot University, Bangkok, Thailand

Extracts of the isoflavone-rich plant *Derris scandens* are used in S.E. Asia for musculoskeletal pain instead of NSAIDs¹ and the isoflavone genistein has been advocated for the treatment of rheumatoid arthritis because it possesses anti-inflammatory activity². Any benefit of genistein in this condition may be offset by the well-known, oestrogen-like vasodilator effect of isoflavones³. A prenylated metabolite of genistein, lupalbigenin, is a key component of *Derris scandens*, and we compared the vascular activity of this isoflavone with that of genistein, daidzein and 17-β-oestradiol in the porcine isolated splenic artery.

Porcine isolated splenic artery segments (4 mm) were prepared for isometric tension as previously described⁴. After establishment of stable submaximal contractions to the thromboxane-mimetic U46619, cumulatively increasing concentrations of the isoflavones were added. Effects of drugs have been expressed as negative logarithm of the concentration required to reduce contractions by 50% (pIC₅₀).

Genistein (pIC₅₀ 5.30 ± 0.08, *n* = 7), daidzein (pIC₅₀ 5.03 ± 0.15, *n* = 7) and 17-β-oestradiol (pIC₅₀ 4.63 ± 0.14, *n* = 10) caused slow, concentration-dependent inhibition of U46619-induced contractions and abolished tone. In contrast, lupalbigenin (3–30 µM) induced small rhythmic contractions (approx' 30% of U46619 tone) without affecting basal tone. Neither the vasodilator activity of genistein (pIC₅₀ 5.19 ± 0.19), nor the maximum response, were affected by the presence of 30 µM lupalbigenin (genistein pIC₅₀ 5.07 ± 0.20, *n* = 7).

Genistein, and daidzein inhibited vascular tone of the porcine isolated splenic artery in a similar manner to 17-β-oestradiol, while the prenylated isoflavone, lupalbigenin, facilitated contractile activity. Future experiments will establish whether lupalbigenin possesses anti-inflammatory activity, like other isoflavones², and can account for the beneficial effects attributed to *Derris scandens* extracts in musculoskeletal conditions.

1. Puttrak P et al., (2016). *J. Ethnopharmacol.* **194**, 316-323.

2. Li J et al., (2013). *Clin Rheumatol* **32**, 535-540.

3. Seok et al., (2008). *J. Pharmacol. Exp. Ther.* **326**, 991-998.

4. Roberts R et al., (1999). *Br. J. Pharmacol.* **128**, 1705-1712.

**Keywords:** Genistein, Lupalbigenin, porcine splenic artery, oestrogen

P-67

ROLE OF MITOGEN-ACTIVATED PROTEIN KINASE (MAPK) IN THE REGULATION OF L-TYPE CHANNEL CALCIUM ENTRY IN RESISTANCE ARTERIES

Gutiérrez-Cruz A., Sánchez A., García-Sacristán A., Rivera L.,
Prieto D.

Departamento de Fisiología, Facultad de Farmacia, Universidad
Complutense de Madrid, 28040-Madrid, Spain

Ca²⁺ entry through L-type channels plays a key role in arterial vasoconstriction but also in Ca²⁺-dependent gene transcription of vascular smooth muscle (VSM) (Kudryavtseva et al, *FEBS J* 280: 5488, 2013). Extracellular signal regulated (ERK) mitogen activated protein kinase (MAPK) phosphorylates and activates the cardiac L-type Ca²⁺ channel and modulates agonist-induced L-type Ca²⁺ entry in cardiac myocytes (Takashi et al., *Circ Res* 94:1242, 2004). In the present study, we sought to assess whether MAPK plays a role in the regulation of Ca²⁺ entry in resistance arteries. Simultaneous measurements of [Ca²⁺]ᵢ and tension were performed by Fura-2M ratio fluorometry in 3rd order branches of

mesenteric arteries from Wistar rats mounted in a microvascular myograph. Activation of α-1 adrenoceptors with phenylephrine (Phe, 10 μM) induced a pronounced vasoconstriction largely inhibited by blockade of L-type Ca²⁺ channels with nifedipine. Phe stimulated Ca²⁺ entry along with contraction and removal of extracellular Ca²⁺ markedly reduced Phe responses to small transients in [Ca²⁺]ᵢ and tension (8 ± 3 and 5 ± 1% of control, *n* = 5, respectively). Readdition of Ca²⁺ to the medium concentration-dependently restored Phe-induced increases in Ca²⁺ and contraction indicating that α₁-mediated vasoconstriction is largely dependent on Ca²⁺ influx to VSM. Inhibition of the ERK-MAPK with PD98059 (3 μM) reduced by 37 ± 8% (*P* < 0.01, *n* = 5, vs. control) the increase in [Ca²⁺]ᵢ elicited by Phe without altering vasoconstriction. Moreover, PD98059 inhibited by 41 ± 9% (*P* < 0.01, *n* = 4, vs. control) Ca²⁺ entry induced by depolarization of the arteries with a high K⁺ solution. The present results provide evidence for an involvement of the ERK-MAPK pathway in the L-type channel Ca²⁺ entry not coupled to vasoconstriction in resistance arteries.

**Keywords:** L-type calcium channel, ERK-MAP kinase, resistance arteries

This work was supported by grant SAF 2012-31631 from MINECO, Spain.

P-68

PATAGONIA CALAFATE (BERBERIS MICROPHYLLA)

BERRY EXTRACTS HAS A POTENT CELLULAR

ANTIOXIDANT PROFILE AND ELICITS ENDOTHELIUM-
DEPENDENT VASCULAR RELAXATION

Calfío C., Mateluna C., Huidobro-Toro J.P.

Laboratorio de Farmacología, Facultad de Química y Biología,
Universidad de Santiago de Chile. Santiago, Chile

Patagonian calafate (Berberis microphylla) is a native dark purple berry reported with the highest antioxidant profile from a collection of endemic southern chilean fruits; the pharmacology of this fruit has been scarcely investigated. Chemically, the berry has a high ascorbic acid and polyphenolic content which includes anthocyanins, flavonols and phenolic acid derivatives. Since red berries cultured in southern Chile have a beneficial effect in chronic vascular diseases, we proposed that extracts of calafate berries might have be vasoactive and present a favorable cellular antioxidant profile.

To this aim, ethanol or acetone calafate extracts (1–300 µg/ml), were evaluated as mesenteric bed vasodilators previously contracted with 50 µM noradrenaline and assessed whether the effect involves endothelial nitric oxide (NO) production and release. Cellular antioxidant activity was evaluated in isolated endothelial cells.

Calafate extracts elicited a concentration-dependent antioxidant activity comparable to that of quercetin standards. Extracts elicited a concentration-dependent vasodilator response with an EC₅₀ of 3.5 µg/ml; the maximal response was reached with 10–30 µg/mL (which caused a 75–80% vasodilatation in noradrenaline precontracted preparations). The vasodilatation was significantly reduced by either endothelium removal (after a 90 sec 0.1% saponin perfusion) or eNOS inhibition with 150 µM N-nitro-L-arginine. Moreover, relaxation significantly correlated with the concentration-dependent extract NO production (R² =0.8, *P* < 0.05). The chemical polyphenol extract profile (derived from HPLC-ESI-MS/MS analysis), identified delphinidin, petunidin and malvidin as 3-glucosides as the main extract polyphenols. We are in the process of assessing whether these molecules could partially explain the extracts induced-vascular responses.

In conclusion, the calafate berry extracts elicited a potent endothelium-
dependent vascular relaxation coupled NO production, a vascular
response likely linked to its cellular antioxidant properties.

**Keywords:** Vascular relaxation, cellular antioxidant activity, nitric oxide and anthocyanins of calafate

Funding sources FONDECYT 114-1132 and Conicyt doctoral grant 21141226.
P-69

**PIOGLITAZONE ALTERS THE VASCULAR**
**CONTRACTABILITY IN HYPERTENSION BY INTERFERENCE**
**WITH ET-1 SYSTEM**

Palacios R.¹, Hernanz R.¹, Martín A.¹, Barrús M.T.¹, Salaices M.²,
Alonso M.J.¹

¹Depto. de Ciencias Básicas de la Salud, Fac. de Ciencias de la Salud, Universidad Rey Juan Carlos (URJC); ²Depto. de Farmacología, Fac. de Medicina, Universidad Autónoma de Madrid (UAM). Spain

**Introduction:** Hypertension is an inflammatory disease associated with high cytokine levels and proinflammatory mediators such as cyclooxygenase-2 (COX-2) and reactive oxygen species (ROS), which contribute to develop the hypertension-associated vascular alterations. The endothelin (ET) system is an important modulator of vascular tone; alterations in this system contribute to hypertension. Peroxisome proliferator-activated receptors-γ (PPARγ) have significant antiinflammatory actions by transrepression mechanisms. Its activation has cardioprotective effects by decreasing cytokines and ROS production.

**Aim:** To analyse the modulation of the vascular ET-1 system by PPARγ in hypertension. For this, we have used: (1) vascular segments from Wistar-Kyoto (WKY) and spontaneously hypertensive rats (SHR) treated or not with the PPARγ agonist pioglitazone (2.5 mg·kg⁻¹·day⁻¹, 28 days), and (2) vascular smooth muscle cells (VSMC) from both strains stimulated with ET-1 incubated or not with pioglitazone.

**Results:** Pioglitazone reduces the increased vascular COX-2, pre-pro-ET-1 and ETAR expressions observed in hypertension, while increases the ETBR levels. In SHR, but not in WKY, ET-1, through ETAR, potentiates phenylephrine-induced contraction. TP receptors activation and NO bioavailability reduction, associated to the increased ROS, participate in this effect. In arteries from pioglitazone-treated SHR, ET-1 reduces phenylephrine contraction through ETBR by increasing NO bioavailability associated to oxidative stress reduction. In VSMC from SHR, ET-1 induces COX-2 expression through ETAR; activation of AP-1 and NF-κB and the subsequent ROS production plays a role in this effect. Pioglitazone, by interfering with AP-1 and NF-κB and the subsequent ROS production, reduces this COX-2 expression

**Conclusion:** Pioglitazone, by reducing the ET-1 effect, diminishes the increased vascular COX-2 expression in hypertension; in addition, pioglitazone shifts the ETA/ETB ratio expression. In this situation, ET-1 induces an opposing effect on phenylephrine-induced contraction by increasing NO bioavailability. The antioxidant activity contributes to these vascular effects.

Supported by MINECO (SAF2015-69294-R) and ISCIII (CB16/11/00286).

**Keywords:** ET-1; hypertension; vascular responses; PPARγ

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**DECREASED EXPRESSION OF ETS-2 IN EPCS AS AN**
**EARLY MARKER OF CARDIOVASCULAR INSTABILITY IN**
**CABG PATIENTS, BENEFICIAL EFFECTS OF SITAGLIPTIN**

Tejerina T.¹, Medina Ú.¹, Carnero M.², Dorado J.N.¹, Bouza M.G.²,
Reguillo F.²

¹Dpto. Farmacología, Facultad de Medicina, Universidad Complutense de Madrid; ²Servicio de Cirugía Cardiaca, Hospital Clínico San Carlos. Madrid

In patients with cardiovascular disease (CVD), the endothelial progenitor cells (EPCs) play a key role in endothelial repair processes. It has been hypothesized that Ets2 transcription factor could be involved in the instability of CVD/hyperlipidaemia. Our objective was to determine the degree of expression of transcription factor Ets2 and the endothelial repair factors Endoglin and CXCR4 in EPCs from patients undergoing coronary artery-bypass grafting (CABG), and to prove a possible beneficial effect of Sitagliptin.

**Methods:** seventy patients undergoing CABG from the cardiac surgery service were selected for the study and categorized into five CVD stages. The Expression of Ets-2, CXCR4, and Endoglin were measured by Western blot. Sitagliptin effect on EPCs and on the expression of different factors was studied by Wb, ELISA and immunofluorescence.

**Results:** An increase in the expression of the transcription factor Ets-2 in patients, without CV predictor risk factor associated with early stages of CV instability was observed (GI: 2.16 ± 1.4). Ets-2 expression decreased in patients with longer evolution of CVD (GII: 1.16 ± 0.8; GIII:0.6 ± 0.5; GIV: 0.91 ± 0.3; GV: 0.56 ± 0.3). A direct association of expression of Ets-2 with age (P = 0.04) and Endoglin expression (P = 0.008), and indirectly with the evolution of CVD (P = 0.008) and hyperlipidaemia (P = 0.03) was found. A direct effect of Sitagliptin 1 µM on the colony formation of EPCs at 6 and 12 hours (P = 0.0077) and on expression of CXCR at 24 hours (P = 0.0201) and SDF1 at 6 and 24 hours (P = 0.0085) was observed.

**Conclusions:** The transcription factor Ets-2 could be an early marker of cardiovascular instability. Our data suggest that a poor functionality of circulating EPCs could be associated with decreased expression of the transcription factor Ets-2 in advanced stages of cardiovascular disease. Sitagliptin treatment of cultured EPCs, could help to revert EPCs deficient functionality. Therefore, the activatory response might depend on the SDF1-CXCR4 axis.

**Keywords:** cardiovascular disease (CVD), the endothelial progenitor cells (EPCs), Ets2 transcription factor, Endoglin, CXCR4, Sitagliptin

P-71

**HYPOXIC PRECONDITIONING INCREASES THE**
**POTENTIAL OF EXOSOMES DERIVED FROM**
**MESENCHYMAL STEM CELLS TO LIMIT PULMONARY**
**VASCULAR DYSFUNCTION INDUCED BY**
**LIPOPOLYSACCHARIDE (LPS)**

Esquivel-Ruiz S.¹, Álvarez-Fuente M.², Royo F.³, Barreira B.¹,
González-Murillo Á.⁴, Falcón-Pérez J.M.³, Ramírez-Orellana M.⁴,
Cerro M.J.², Pérez-Vizcaíno F.¹, Moreno L.¹

¹Department of Pharmacology, School of Medicine, Universidad Complutense of Madrid, Instituto de Investigación Sanitaria Gregorio Marañón (IISGM), Ciber de Enfermedades Respiratorias (CIBERES), Madrid, Spain; ²Servicio de Cardiología Pediátrica, Hospital Universitario Ramón y Cajal, Madrid, Spain; ³Exosomes Lab. CIC bioGUNE, CIBERehd, Derio, Bizkaia, Spain; ⁴Unidad de Terapia Celular, Hospital Niño Jesús, Instituto Investigación Sanitaria La Princesa, Madrid, Spain

Preclinical studies have demonstrated promising results using mesenchymal stem cells (MSCs) for the treatment of inflammatory lung diseases, including acute respiratory distress syndrome (ARDS), pulmonary arterial hypertension (PAH) or bronchopulmonary dysplasia (BPD). However, recent evidences suggest that their therapeutic activity is mediated by paracrine mediators, most notably exosomes, rather than cell engraftment. Hypoxic preconditioning is thought to enhance the therapeutic potential of MSCs. In this work, we aimed to analyse whether MSCs-derived exosomes are able to prevent the pulmonary vascular dysfunction induced by lipopolysaccharide (LPS) and to enhance their effectiveness by the use of hypoxia-preconditioning.

Exosomes released by umbilical cord blood (UCB)-derived MSCs cultured under normoxic (21% O₂) or hypoxic (3% O₂) conditions were obtained by differential ultracentrifugation. Rat pulmonary artery (PA) smooth muscle cells (PASMCs) were treated with 1 µg/ml of LPS for 48 hours in the absence or presence of MSC-derived exosomes (0.5–5 µg/ml). IL-6 levels were determined by ELISA. Contractile responses were analysed in rat PA mounted in a wire myograph.

Exposure to LPS significantly increased IL-6 release by rat PASMCs (Ctrl = 2.56 ± 0.24; LPS = 75.46 ± 4.16 ng/ml), inhibited hypoxic pulmonary vasoconstriction (HPV), induced endothelial dysfunction and potentiated the contractile effects induced by serotonin in isolated PA. Treatment with normoxic MSC-derived exosomes had no effect in these responses. In contrast, hypoxia-preconditioned MSCs-derived
exosomes significantly prevented the impairment of HPV, the hyperre-
sponsiveness to serotonin and attenuated the reduction of the relaxant
responses to acetylcholine. Neither exosome preparation was able to
decrease the IL-6 release induced by LPS.

Our data show that hypoxia-preconditioned MSCs-derived exosomes
prevent the development of pulmonary vascular dysfunction induced
by LPS in isolated PA. These findings suggest that hypoxic precondi-
tioning increases the therapeutic potential of MSCs-derived exosomes
and may represent a new therapeutic approach in pulmonary vascular
diseases associated with inflammation.

Supported by ISCIII (CP12/03304 and PI15/01100).

Key words: Exosomes, inflammation, pulmonary vascular dysfunction.

P-72
PHARMACOLOGICAL AND GENETIC DISSECTION OF THE ROLE OF TRPA1 AND TRPM8 CHANNELS IN INTRINSIC VASCULAR RESPONSES TO COLD

Alonso-Carabajo L.¹,², Perez-Garcia M.T.², Lopez-Lopez J.R.²,
Talavera K.¹

¹Laboratory of Ion Channel Research, Department of Cellular and Molecular Medicine, KU Leuven, Leuven, Belgium; ²Dpto de Bioquímica, Biología Molecular y Fisiología e Instituto de Biología y Genética Molecular IBGM, Universidad de Valladolid y CSIC, Valladolid, Spain

It is well-known that cold induces vasoconstriction in skin blood vessels as a protective response against heat loss. This phenomenon is thought to be mediated by an efferent reflex to the activation of cold-sensitive afferent nerves in the skin. In contrast to this view, we found in *ex vivo* myography experiments that cooling to 10°C induced a 28 ± 3.01% contraction in endothelium-denuded plantar arteries dissected from wild type (WT) mice. Cold produced a significantly smaller vasoconstriction in the presence of the TRPA1 inhibitor HC030031 (9 ± 2.23%), and in arteries dissected from *Trpa1* (11.04 ± 2.78%) or *Trpm8* (10 ± 1.54%) knockout (KO) animals. Application of HC030031 virtually abolished the responses to cold in arteries from *Trpm8* KO (2 ± 1.13%). Neither TRPA1 nor TRPM8 channels could be detected in vascular smooth muscle cells, suggesting that the effects of cold are mediated by activation of these channels in perivascular sensory and/or sympathetic nerves. Cold-induced vasoconstriction was potentiated in the presence of the CGRP receptor inhibitor BIBN 4096 (40 ± 4.26%) and reduced after depletion of catecholamines from the sympathetic nerve terminals with guanethidine (9 ± 2.6%). Cold had no effect in arteries were incubated with both BIBN 4096 and guanethidine. We detected mRNA of both TRPA1 and TRPM8 channels in sympathetic ganglia isolated from WT mice and we could confirm the presence of perivascular sympathetic nerves in confocal images of plantar arteries labelled with an anti-TH antibody. Taken together, our data demonstrates that cold has a dual action, mediated by both vasodilatory and a vasoconstrictor responses via the activation of TRPA1 and TRPM8 channels. To our knowledge, our results represent the first evidence for an intrinsic response to cold in cutaneous arteries and for the presence and the functional role of sensory TRP channels in efferent nerve fibers.

Keywords: TRPA1, cold, TRPM8, vascular response

P-73
DYNAMIC MASS REDISTRIBUTION PHENOTYPIC ASSAY FOR IDENTIFYING LIGANDS ACTIVE AT GPR35 RECEPTORS

Alejandro G.-G., Laura G.-G., Isabel C.M., Isabel L.M., Manuel B.J.

Drug Screening Platform/BioFarma Research Group. CIMUS Research Center, University of Santiago de Compostela, Health Research Institute of Santiago de Compostela (IDIS)

**Introduction:** GPR35 is an orphan receptor reported to be involved in inflammatory disorders (HU H. 2012), CNS disfunction (Shore DM

2015), pain (Alkondon M 2015), diabetes (Tanoguchi Y 2006) and immunological diseases such as asthma (Yenkins L 2010). Kynurenic acid (HU H. 2012), 2-oleoyl lysophosphatidic acid and recently chemokine CXCL17 (Berlinguer-Palmini R 2013) have been described as potential endogenous ligands but without confirmation; showing differences in efficacy between human and rat orthologues (Yenkins L 2010). Phenotypic assays based on Dynamic Mass Redistribution (DMR) revealed as powerful tool screening libraries in orphan receptors which signalling pathways and biology remain unknown. We aimed to develop a miniaturized phenotypic assay based on a DMR label-free technology that enables the detection of new ligands for human and rat GPR35 receptors.

**Material and Methods:** HT-29 cell line and IEC-6 cell line were seeded in LFC-384 well microplates (PerkinElmer 6057408). 24 hours after seeding, the culture medium was replaced with medium or HBSS buffer to optimize the assay conditions. Plates were incubated before reading a base line and later standard compounds were added. Measurements were done using an EnSpire (PerkinElmer) reader with Corning ® Epic® Label-free technology.

**Results:** A miniaturized phenotypic assay based on a DMR was developed to find new ligands for hGPR35 and rGPR35. For both cell lines, 15000 cells per well were selected as a suitable concentration. McCoy's 5A medium supplemented with 25 mM HEPES pH=7.4 was selected as Buffer Assay for HT-29 whereas HBSS buffer was selected for IEC-6. To validate the feasibility of the method, we obtained concentration-response curves of a synthetic agonist of GPR35 (Taniguchi Y 2008), Zaprinast, with values of EC₅₀=0.50 ± 0.25 µM for human GPR35 and values of EC₅₀ = 4.2 ± 0.30 µM for rat GPR35.

**Conclusions:** We have developed a miniaturized phenotypic assay based on a DMR technology to measure the activity of compounds in human and rat cell lines expressing the orphan receptor GPR35.

P-74
FUNCTIONAL μ-ΟΡΙΟΙΟ-GALANIN RECEPTOR HETEROMERS IN THE VENTRAL TEGMENTAL AREA

Moreno E.¹,²,³, Quiroz C.⁴, Mallol J.¹,²,³, Cortés A.¹,²,³, Lluís C.¹,²,³,
Ferré S.⁴, Canela E.I.*¹,²,³, Casadó V.*¹,²,³

*Equal contribution; ¹Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas, Spain; ²Department of Biochemistry and Molecular Biomedicine, University of Barcelona, Barcelona, Spain 08028; ³Institute of Biomedicine of the University of Barcelona (IBUB), Spain; ⁴Integrative Neurobiology Section, National Institute on Drug Abuse, Intramural Research Program, National Institutes of Health, Baltimore, MD 21224.

The neuropeptide galanin has been shown to interact with the opioid system. More specifically, galanin counteracts the behavioral effects of the systemic administration of µ-opioid receptor (MOR) agonists. Yet the mechanism responsible for this galanin–opioid interaction has remained elusive. Using biophysical techniques in mammalian transfected cells, we found evidence for selective heteromerization of MOR and the galanin receptor subtype Gal1 (Gal1R). Also in transfected cells, a synthetic peptide selectively disrupted MOR–Gal1R heteromerization as well as specific interactions between MOR and Gal1R ligands: a negative cross talk, by which galanin counteracted MAPK activation induced by the endogenous MOR agonist endomorphin-1, and a cross-antagonism, by which a MOR antagonist counteracted MAPK activation induced by galanin. These specific interactions, which represented biochemical properties of the MOR-Gal1R heteromer, could then be identified *in situ* in slices of rat ventral tegmental area (VTA) with MAPK activation and two additional cell signaling pathways, AKT and CREB phosphorylation. Furthermore, *in vivo* microdialysis experiments showed that the disruptive peptide selectively counteracted the ability of galanin to block the dendritic dopamine release in the rat VTA induced by local infusion of endomorphin-1, demonstrating a key role of MOR-Gal1R heteromers localized in the VTA in the direct control of dopamine cell function and their ability to mediate antagonistic interactions between MOR and
Gal1R ligands. The results also indicate that MOR-Gal1R heteromers should be viewed as targets for the treatment of opioid use disorders.

**Keywords:** dopamine; galanin receptor; MAPK; opioid receptor; receptor heteromer; ventral tegmental area

P-75

**CHARACTERIZATION OF THE ACTIONS OF THE NOVEL P2Y₂ RECEPTOR ANTAGONIST, AR-C118925XX**

Muoboghare M.O., Drummond R., Kennedy C.

Strathclyde Institute of Pharmacy and Biomedical Sciences, University of Strathclyde, Glasgow, United Kingdom.

**Introduction:** The endogenous nucleotides, adenosine 5'-triphosphate (ATP), uridine 5'-triphosphate (UTP), and uridine 5'-diphosphate (UDP) act via P2X and P2Y receptors. Due to the limited selectivity of most antagonists, the functions of many individual P2 subtypes are poorly characterised. A putative P2Y₂ antagonist, AR-C118925XX, has recently become available, so the aims were to quantify the action of AR-C118925XX at recombinant P2Y₂ receptors and then to determine the contribution of P2Y₂ receptors to nucleotide-evoked vasoconstriction.

**Methods:** Recombinant human P2Y₁, P2Y₂, P2Y₄ and P2Y₁₁ receptors, stably expressed in 1321N1 cells, were grown on glass coverslips, which were placed in a fluorimeter, and intracellular Ca²⁺ measured using the Ca²⁺-sensitive dye, Cal-520AM. Concentration-response curves (CRC) were constructed by superfusing cells with agonists (P2Y₁-ADP, P2Y₂-UTP, P2Y₄ and P2Y₁₁-ATP), in the absence and presence of AR-C118925XX. The Hill equation was fitted to the data and a Schild plot generated using the EC₅₀ values. 5 mm rings of rat intrapulmonary artery (rIPA) were mounted under isometric conditions *in vitro* at 37°C. Contractions were elicited by addition of P2Y agonists to the bath.

**Results:** In 1321N1 cells expressing hP2Y₂ receptors, UTP (10 nM-3 µM) evoked a concentration-dependent rise in intracellular Ca²⁺ (EC₅₀=54 nM, 95% cl. 43–67 nM, n = 5). Increasing concentrations of AR-C118925XX (10 nM-1 µM), produced a progressive rightward shift in the UTP CRC, with no decrease in maximum response (n = 6 each). These inhibitory effects were fully reversible. Schild analysis produced a pA₂=8.30 and slope=0.985. In contrast, AR-C118925XX (1 µM) had no effect at human P2Y₁, P2Y₄ and P2Y₁₁ receptors (n = 5 each). In rIPA UTP, ATP and UDP (300 µM) evoked reproducible contractions that were unaffected by AR-C118925XX (1 µM) (n = 4 each).

**Conclusion:** AR-C118925XX is a potent, competitive and reversible P2Y₂ antagonist, making the identification of the physiological functions of P2Y₂ receptors possible. P2Y₂ receptors do not appear to play a role in nucleotide-evoked contractions of rIPA.

**Keywords:** P2Y receptors AR-C117825XX pulmonary artery

P-76

**SIGNALING OF THE FRACTALKINE RECEPTOR CX₃CR1 AND ITS NATURAL GENETIC VARIANTS: IMPACT OF RECEPTOR NON-SYNONYMOUS SINGLE NUCLEOTIDE POLYMORPHISMS**

García-Silva A.<fnref n="1" />, de la Fuente R.A.<fnref n="1" />, Izquierdo A.G.<fnref n="1" />, Loza M.I.<fnref n="1" />,
Costas J.², Castro M.<fnref n="1" />

The chemokine receptor CX₃CR1 is a Gᵢ/o G protein-coupled receptor (GPCR) expressed in monocytes, NK cells, T lymphocytes, astrocytes

and microglia, among other cells, and it plays an important role in inflammation and immunity as well as in neuron-microglia communication in the Central Nervous System [1]. Its known ligand is fractalkine (CX₃CL1), the sole member of the CX₃C chemokine subfamily. Genomic studies have identified non-synonymous single nucleotide polymorphisms (nsSNPs) in the CX₃CR1 gene. Specifically, the common receptor genetic variant CX₃CR1-V249I/T280M has been associated with faster progression to disease in HIV-infected patients, cardiovascular atheroprotection, increased risk of age-related macular degeneration, and obesity [2]. We aimed to investigate the possible functional impact of the currently identified nsSNPs of CX₃CR1 on the pharmacology of this receptor.

Receptor interaction with G protein-coupled receptor kinase 2 (GRK2) and beta-arrestins were investigated in transfected HEK293 cells by bioluminescence resonance energy transfer (BRET)-based assays. Our results indicate that the CX₃CR1-V249I/T280M receptor variant interacts with more efficacy than the wild type receptor with beta-arrestins 1 and 2 (Emax 146% and 204% of wild type, respectively) and GRK2 (Emax 310% of wild type) in response to fractalkine. The functional impact of this observation on the dynamics and compartmentalization of the receptor signaling is being further investigated. Being GRKs and beta-arrestins crucial regulators of G protein-dependent and -independent signaling of GPCRs [3], our findings expand our current knowledge on the signaling pathways modulated by CX₃CR1 and their possible implications in physiological and pathological condition.

**Acknowledgements:** This work is funded by the Spanish Ministry of Economy and Competitiveness (MINECO) (SAF2014-57138-C2-1-R).

**References:**

[1] Smit M.J. Lira S.A. and Leurs R. (eds) (2011) Chemokine Receptors as Drug Targets, WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim; Paolicelli R.C. and Gross C.T. (2011) Neuron Glia Biol 7: 77-83; Bachelerie F. et al. (2013) Pharmacol Rev 66: 1-79.

[2] Faure S. et al. (2000) Science 287: 2274-2277; McDermott D.H. et al. (2003) J Clin Invest 111: 1241-1250; Combatière C. et al. (2007) J Clin Invest 117: 2920-2928; Sirois-Gagnon D. et al. (2011) Obesity (Silver Spring) 19: 222-7; Wu J. et al. (2014) Dis Markers 913678.

[3] Schmid C.L. and Bohn L.M. (2009) Pharmacol Ther 121: 285-93; Shukla A.K. et al. (2011) Trends Biochem Sci 36: 457-469.

P-77

**GLIBENCLAMIDE INHIBITS ATP-SENSITIVE K CURRENTS IN THE CANTU SYNDROME**

Nieto-Marín P., Utrilla R.G., Alfayate S., Tinaquero D., Pérez-Hernández M., Matamoros M., Tamargo J., van der Heyden M., Delpón E., Caballero R.

Dpto. de Farmacología. Fac. de Medicina. Universidad Complutense de Madrid. CIBERCV. Madrid. España; Dpt. of Molecular Cell Biology. Utrecht University. Utrecht. Holanda

(KCNJ11) and β-subunits SUR2A (ABCC9). Cantu syndrome (CS) is a rare genetic disorder, which leads to multiple alterations including hypotension and cardiac abnormalities. CS is caused by mutations in KCNJ8 and ABCC9 that reduce channel sensitivity to ATP-blockade, increasing K<sub>ATP</sub> channel activity. Glibenclamide, a K<sub>ATP</sub> blocker, has been proposed as a potential treatment for CS. However, it is unknown whether CS-associated mutations are sensitive to K<sub>ATP</sub> modulating drugs. We analyzed the effects of glibenclamide on pinacidil-activated K<sub>ATP</sub> currents (I<sub>KATP</sub>) generated by channels with Kir6.1 (V65M and G343D) and SUR2A (H60Y, S1020P, and S1054Y) CS-associated mutations. I<sub>KATP</sub> were recorded in Chinese Hamster Ovary (CHO) cells transiently transfected with the cDNA encoding WT or mutant Kir6.x and SUR2A subunits by whole-cell patch-clamping. Pinacidil increased I<sub>KATP</sub> generated by Kir6.1WT+SUR2AWT and Kir6.2WT+-SUR2AWT channels in a concentration-dependent manner, the effective concentration 50 (EC<sub>50</sub>) being 1.9 ± 0.03 and 4.9 ± 1.1 µM, respectively. The EC<sub>50</sub> values for pinacidil were significantly increased

<footnote n="1">¹Universidade de Santiago de Compostela, Center for Research in Molecular Medicine and Chronic Diseases (CIMUS), 15782 Santiago de Compostela, Spain; ²Instituto de Investigación Sanitaria (IDIS) de Santiago de Compostela, Complexo Hospitalario Universitario de Santiago de Compostela (CHUS), Servizo Galego de Saúde (SERGAS), Santiago de Compostela, Spain</footnote>
in the channels composed by V65M (5.5 ± 0.03 µM) and G343D (8.4 ± 0.04 µM) Kir6.1 + SUR2AWT subunits. Pinacidil-induced increase of Kir6.2WT+SUR2A H60Y and Kir6.2WT+SUR2A S1020P was similar to that of Kir6.2WT+SUR2AWT channels. Unexpectedly, pinacidil failed to increase $I_{KATP}$ generated by Kir6.2WT+SUR2A S1054Y channels. Glibenclamide inhibited currents generated by Kir6.1WT+SUR2AWT and Kir6.2WT+SUR2AWT in a similar extent, yielding inhibitory concentration 50 (IC₅₀) values (0.3 ± 0.1 and 0.22 ± 0.06 µM, respectively) within the range of therapeutic concentrations of glibenclamide. The presence of the V65M mutation in Kir6.1 significantly reduced the glibenclamide potency (IC₅₀=1.6 ± 0.04 µM), while the rest of the mutations in Kir6.1 (G343D) or SUR2A (H60Y and S1020P) did not affect the glibenclamide-induced block. These results demonstrate that most of the CS-associated mutations reduced channel response to pinacidil, whereas only the V65M mutation in Kir6.1 affected channel sensitivity to glibenclamide.

**Keywords:** Kir6.x, SUR2A, glibenclamide, Cantu Syndrome, cardiac

P-78

THE PARKINSON'S DISEASE-ASSOCIATED GPR37 RECEPTOR IS AN ADENOSINE A₂A RECEPTOR REPRESSOR IN MICE

Morató X.<sup>1,2</sup>, Luján R.<sup>3</sup>, López-Cano M.<sup>1,2</sup>, Gandia J.<sup>1,2</sup>, Stagljar I.<sup>4</sup>, Watanabe M.<sup>5</sup>, Cunha R.<sup>6</sup>, Fernández-Dueñas V.<sup>1,2</sup>, Ciruela F.<sup>1,2</sup>

<sup>1</sup>Unitat de Farmacologia, Departament Patologia i Terapèutica
Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, L'Hospitalet de Llobregat, Spain; <sup>2</sup>Institut de Neurociències, Universitat de Barcelona, Spain; <sup>3</sup>IDINE, Departamento de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Albacete, Spain; <sup>4</sup>Donnelly Centre, University of Toronto, M5S 3E1, Canada; <sup>5</sup>Department of Anatomy, Hokkaido University School of Medicine, Sapporo 060-0818, Japan; <sup>6</sup>CNC-Center for Neuroscience and Cell Biology, University of Coimbra, Coimbra, Portugal

GPR37, also known as parkin-associated endothelin-like receptor (Pael-R), is an orphan G protein-coupled receptor that has been related with Parkinson's disease (PD) neuropathology. Interestingly, the genetic blockade of GPR37 enhances cell surface expression of striatal dopamine transporter (DAT), resulting in reduced dopamine content in the striatum. In addition, it has been shown that deletion of GPR37 triggers anxiolytic-like effects and sensitizes mice to adenosine A₂A receptors (A₂AR)-mediated signaling. Here we report that GPR37 and A₂AR physically and functionally interact both in living cells and in native tissue. Thus, by using biochemical techniques (i.e. co-immunoprecipitation and proximity ligation assay) we demonstrated a physical interaction between these two receptors in the striatum. Also, by means of post-embedding immunogold-electron microscopy (EM) techniques detection it was demonstrated a close proximity at the postsynaptic level of striatal synapses. On the other hand, GPR37 deletion promoted striatal A₂AR cell surface expression, which correlated well with an increased A₂AR agonist-mediated cAMP accumulation both in primary striatal neurons and synaptosomes from striatum. Furthermore, GPR37-KO mice showed enhanced catalepsy induced by an A₂AR agonist and an increased response to A₂AR antagonist-mediated locomotor activity. Overall, these results demonstrate for the first time an important role for GPR37 controlling A₂AR expression and function in the striatum, which may be of interest for the treatment of PD.

**Acknowledgements:** This work was supported by MINECO/ISCIII (SAF2014-55700-P and PIE14/00034), the Catalan government (2014 SGR 1054), ICREA (ICREA Academia-2010), Fundació la Marató de TV3 (Grant 20152031) and IWT (SBO-140028). Also supported by the PhD fellowship FPI (BES-2012-052722).

P-79

CELLULAR BIOLOGY OF HUMAN CARDIAC NAV1.5-KIR2.1 CHANNEL COMPLEXES

Matamoros M., Pérez-Hernández M., Utrilla R.G., Tinaquero D., Nieto-Marín P., Alfayate S., Tamargo J., Delpón E., Caballero R.

Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid, CIBERCV, Ramón y Cajal square, 28040-MADRID

Cardiac Kir2.1 and Nav1.5 channels generate the inward rectifier K⁺ (I<sub>K1</sub>) and the Na⁺ (I<sub>Na</sub>) currents, respectively. Nav1.5 and Kir2.1 channels exhibit positive reciprocal modulation and the augmented presence of Nav1.5 and Kir2.1 channels at the membrane could be the result of an increase in the protein synthesis and forward trafficking and/or to a decrease in channel internalization. Here we compared the differential characteristics of these processes when Nav1.5 and Kir2.1 channels are expressed together or separately. The proximity ligation assays demonstrated that Nav1.5 and Kir2.1 proteins are in close proximity to each other (<40 nm apart) in the membrane of ventricular myocytes, suggesting that they form complexes. Patch-clamp experiments in heterologous transfection systems demonstrated that the inhibition of endoplasmic reticulum (ER) to Golgi transport with Brefeldin A did not abolish the positive reciprocal modulation between Kir2.1 and Nav1.5 channels and that the internalization time constants of Kir2.1 (5.1 ± 0.5 h) and Nav1.5 (4.9 ± 0.4 h) channels were not modify when they were coexpressed with Nav1.5 and Kir2.1 channels, respectively. Either when they were coexpressed or not, the inhibition of dynamin-dependent endocytosis similarly reduced the internalization of Nav1.5 and Kir2.1 channels. Inhibition of the dynein/dynactin motor suggested that it is involved in the backward and forward traffic of Kir2.1 and Nav1.5, respectively. Conversely, Nav1.5-Kir2.1 complexes were forwarded by the dynein/dynactin motor. Nav1.5 but not Kir2.1 were ubiquitinated by Nedd4-2 ubiquitin-protein ligase and degraded by the proteasome. Nav1.5-Kir2.1 complexes were also degraded following this route as demonstrated by the overexpression of Nedd4-2 and the inhibition of the proteasome with MG132. We concluded that there is a pool of Kir2.1 and Nav1.5 channels that form complexes whose biology is similar to that of the Nav1.5 channels.

**Keywords:** Nav1.5 channel, Kir2.1 channel, cardiac, intracellular traffic

P-80

A METHOD FOR QUANTIFYING BIASED SIGNALLING OF INVERSE AGONISTS

David Hall.<sup>1</sup>, Jesús Giraldo.<sup>2,3</sup>

<sup>1</sup>Fibrosis and Lung Injury DPU, GlaxoSmithKline, Gunnels Wood Road, Stevenage, Herts, SG1 2NY, UK; <sup>2</sup>Institut de Neurociències and Unitat de Bioestadística, Universitat Autònoma de Barcelona, 08193 Bellaterra, Spain; <sup>3</sup>Network Biomedical Research Center on Mental Health (CIBERSAM)

Biased agonism, the ability of an agonist to selectively trigger specific signalling pathways when they act at a given receptor, is an increasingly frequently described pharmacological phenomenon. Several methods to quantify the bias of agonists have been proposed, for example the ΔΔlog(τ/Ka) parameter of Kenakin and co-workers (Kenakin et al, 2011, ACS Chem. Neurosci. 3, 193–203). However, all of the methods used for the analysis of biased signalling focus on the analysis of agonists as they are based on the use of the Operational model. However, the classical Operational model does not account for constitutive receptor signalling and cannot be used to analyse the properties of inverse agonists. Here we report a method for the analysis of ligand bias based on the Operational model of Slack & Hall (Slack & Hall, 2012, Br. J. Pharmacol. 166, 1774–1792) which accounts for receptor constitutive activity and hence can be used to analyse biased signalling in response to inverse agonists as well as agonists. This method also has the advantage of providing a measure of absolute rather relative bias since it is derived from estimates of ligand intrinsic
efficacy and is not, therefore, influenced by the efficiency of the signal transduction cascades involved.

**Keywords:** biased agonism, constitutive activity, inverse agonism, Operational model

## P-81
### A MUTATION IN THE TBX5 TRANSCRIPTION FACTOR DECREASES THE HUMAN CARDIAC SODIUM CURRENT AND IS ASSOCIATED WITH THE BRUGADA SYNDROME

Alfayate S., Utrilla R.G., Nieto-Marín P., Tinaquero D., Pérez-Hernández M., Matamoros M., Jalife J., Tamargo J., Caballero R., Delpón E.

Department of Pharmacology, School of Medicine, Universidad Complutense de Madrid, CIBERCV, Ramón y Cajal square, 28040-MADRID; Center for Arrhythmia Research, University of Michigan, USA

In a proband diagnosed with Brugada syndrome (BrS), in whom screening for mutations in all described BrS genes was negative, we found a missense mutation in the Tbx5 transcription factor (p.F206L) that was predicted as pathogenic. It has been described that Tbx5, besides its effects on cardiac development, drives the expression of Nav1.5 channels in the adult mouse heart. Since BrS is associated to loss-of-function Nav1.5 mutations, here we analyzed the effects of p.F206L Tbx5 on the cardiac sodium current (I<sub>Na</sub>) generated by Nav1.5 channels to unravel whether the mutation can underlie the BrS. Human native (WT) and mutated Tbx5 tagged with GFP were transfected in HL-1 cells or included in lentiviral particles for infecting human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CM). Tbx5 WT doubled the peak I<sub>Na</sub> density recorded in HL-1 cells (from -37.5 ± 5.1 to -62.6 ± 8.2 pA/pF, n ≥ 6, P < 0.05), whereas p.F206L Tbx5 strongly reduced the peak I<sub>Na</sub> density (-6.7 ± 0.2 pA/pF; n = 6; P < 0.01). Importantly, in hiPSC-CM Tbx5 WT and p.F206L significantly increased (-27.6 ± 1.9 pA/pF; n = 7) and decreased (-9.5 ± 1.9 pA/pF), respectively, the I<sub>Na</sub> peak density compared to non-infected cells (-19.4 ± 2.8 pA/pF; n = 10; P < 0.05). Both in HL-1 cells and hiPSC-CM neither WT nor mutated Tbx5 modified the voltage dependence of Nav1.5 channels activation and inactivation. Luciferase reporter assays using the human minimal promoter of the gene encoding Nav1.5 channels (SCN5A) demonstrated that p.F206L Tbx5 completely abolished the Tbx5 pro-transcriptional activity produced by WT Tbx5. We concluded that the p.F206L mutation markedly decreases the I<sub>Na</sub> density by suppressing the remarkable Tbx5 pro-transcriptional activity over the human SCN5A gene and thus, it could be associated with the BrS.

**Keywords:** sodium current, Tbx5, Brugada Syndrome, cardiac

## P-82
### THE LARGE CONDUCTANCE, CA<sup>2+</sup>-ACTIVATED K<sup>+</sup> CHANNEL OPENER VSN16R MODULATES HIPPOCAMPAL CA1 PYRAMIDAL NEURON FIRING

Tabatabaee S.<sup>1</sup>, Baker D.<sup>2</sup>, Stephens G.J.<sup>1</sup>, Whalley B.J.<sup>1</sup>

Ion channels are critical regulators of neuronal excitability and are also implicated in a variety of pathologies such as epilepsy. Large conductance, Ca<sup>2+</sup>-activated K<sup>+</sup> (BK<sub>Ca</sub>) channels play an important functionally modulate neuronal firing and have a known role in seizure aetiology. BK<sub>Ca</sub> are widely expressed in the central nervous system, regulate action potential duration, firing frequency and consequential neurotransmitter release, and are activated by membrane depolarization and increased intracellular Ca<sup>2+</sup>. The unique coupling of Ca<sup>2+</sup> signalling to membrane depolarization associated with BK<sub>Ca</sub> plays a crucial role in controlling neuronal hyperexcitability, since K<sup>+</sup> efflux via

BK<sub>Ca</sub> causes neuronal hyperpolarisation. Moreover, loss-of-function mutations to, or reduced expression of, BK<sub>Ca</sub> contribute to neuronal hyperexcitability that has been associated with temporal lobe epilepsy, tonic-clonic seizures and alcohol withdrawal-induced seizures. Conversely, there is evidence to show that that BK<sub>Ca</sub> can facilitate high-frequency neuronal firing in some neuronal populations (e.g. hippocampal CA1 pyramidal neurons) and some gain of function mutations to BK<sub>Ca</sub> subunits have been associated with the development of idiopathic epilepsy (primarily absence epilepsy). Thus, both loss of function and gain of function of BK<sub>Ca</sub> channels can differentially subserve modulation of seizure phenotypes such as temporal lobe seizures and absence seizures, respectively.

Here, we examined the effects of the BK<sub>Ca</sub> channel opener, VSN16R (50 µM), on the firing properties of hippocampal CA1 pyramidal neurons in acute hippocampal slices obtained from healthy male C57BL/6 mice (P30-P40) using whole-cell current clamp electrophysiological recording. Neuronal outputs in response to a variety of hyperpolarizing and depolarizing stimulation protocols were assessed. Modulate of hippocampal CA1 pyramidal neuron firing via VSN16R effects on BK<sub>Ca</sub> channels results will be presented and discussed.

**Keywords:** BK<sub>Ca</sub> channels; Hippocampal pyramidal neurons

## P-83
### MODELING HUMAN BIPOLAR DISORDER: THE HINT1-DEFICIENT MICE

Rodríguez-Muñoz M., Cortés-Montero E., Sánchez-Blázquez P., Garzón-Niño J.

Neuropharmacology. Cajal Institute, CSIC. Avenida Doctor Arce 37, 28002 Madrid. Spain

Human bipolar disorder (BPD) is a chronic and debilitating illness with alternating periods of mania and depression. Several currently available animal models reproduce select behavioral facets of human mania and depression and can be used to reliably detect novel drugs. However, in the case of BPD, there is no single valid animal model for reproducing the fluctuating moods of affected patients. Mice with histidine triad nucleotide-binding protein 1 (HINT1) deletion exhibit manic-like symptoms that evolve into depressive-like behavior in response to stressful paradigms. Recent studies have indicated that HINT1-/- mice exhibit molecular changes similar to those found in BPD patients, such as increased PKC, PKA, and GSK3β activities, as well as glutamate N-methyl-D-aspartate receptor (NMDAR)/α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic receptor (AMPAR) and NR2B/NR2A subunit ratios<sup>1,2</sup>. Pharmacological interventions stabilized their behavior. GSK3β inhibitors and valproate directly attenuated the expression of the manic-like symptoms, whereas PKC inhibition, lamotrigine, or risperidone promoted NMDAR-mediated depressive-like behaviors that counterbalanced the preexisting manic-like symptoms. Naïve HINT1-/- mice exposed to stressful paradigms rapidly manifested depressive-like behaviors in subsequent stressful situations that persisted for a couple of weeks thereafter. During the depressive-like phase, citalopram, amitriptyline and MK801 precipitated manic-like behaviors in stressed HINT1-/- mice. Notably, as observed in BPD patients, the antagonism of NMDARs prevented HINT1-/- mice from alternating behaviors in response to stress<sup>3</sup>. In HINT1-/- mice, PKC supports manic like symptoms and reduces the expression of depressive-like behaviors via activation of GSK3β and regulation of NR2B-enriched NMDARs. Our data show that HINT1-/- mice represent a suitable model for studying human BPD and may facilitate the identification of novel targets and drugs to treat this mental disorder. (Supported by Plan Nacional de Drogas 2014-012 and MINECO, SAF-2015-65420R)

<footnote n="1"><sup>1</sup>Reading School of Pharmacy, University of Reading, Reading, UK;</footnote>

<footnote n="2"><sup>2</sup>Centre for Neuroscience and Trauma, Blizard Institute, Queen Mary University of London, London, UK</footnote>

<footnote>1-Hahn et al., 1999. *Bipolar disorder. 1: 81-86.</footnote>

<footnote>2-Naughton et al., 2014 *J. Affetc. Disord.* **156**: 24-35.</footnote>

<footnote>3-Ghasemi et al., 2014 *Neurosci. Biobehav. Rev.* **47**: 336-58.</footnote>
P-84
THE P.P888L SAP97 POLYMORPHISM SHORTENS THE CARDIAC ACTION POTENTIAL DURATION AND THE QT INTERVAL

Tinaquero D.<fnref n="1" />,<fnref n="2" />, Alfayate S.<fnref n="1" />,<fnref n="2" />, Nieto-Marín P.<fnref n="1" />,<fnref n="2" />, Utrilla R.G.<fnref n="1" />,<fnref n="2" />,
Matamoros M.<fnref n="1" />,<fnref n="2" />, Pérez-Hernández M.<fnref n="1" />,<fnref n="2" />, Tamargo M.<fnref n="1" />,<fnref n="2" />, Tamargo
J.<fnref n="1" />,<fnref n="2" />, Caballero R.<fnref n="1" />,<fnref n="2" />, Delpón E.<fnref n="1" />,<fnref n="2" />

The *DLG1* gene encodes for SAP97, an scaffolding protein that inter-
acts with several cardiac ion channels including those underlying the
fast Na (Ina) and the transient outward (Ito) currents. By next genera-
tion sequencing we identified a common *DLG1* polymorphism
(p.P888L) in a man and two sisters diagnosed with Brugada Syndrome
(BrS). This work aimed to determine the electrocardiographic and the
cellular electrophysiological consequences of the SAP97 polymor-
phism.

Native (WT) and p.P888L SAP97 tagged with ds-red were cotrans-
fected or not together with the cDNA encoding the alpha and beta sub-
units underlying human Ina and Ito in Chinese hamster ovary (CHO)
cells. Two cardiac-specific transgenic-like mouse models on the basis
of adeno-associated virus gene transfer were created expressing WT
and p.P888L SAP97, respectively.

Co-expression of WT SAP97 significantly increased the I<sub>Na</sub> and I<sub>to</sub> recorded using patch-clamp in CHO cells and in ventricular myocytes from SAP97 overexpressing mice. The SAP97 polymorphism increased the I<sub>Na</sub> similarly as WT SAP97 did, both in CHO cells and in transgenic-like mouse myocytes. Conversely, p.P888L SAP97 further increased the time constant of current inactivation and, thus, the I<sub>to</sub> charge density in both CHO cells (from 18.6 ± 2.3 to 42.2 ± 6.1 pC/pF at +50 mV, n ≥ 8, P < 0.05) and mouse myocytes (from 0.4 ± 0.04 to 0.7 ± 0.06 pC/pF at +50 mV, n ≥ 15, P < 0.05). As a consequence, in p.P888L SAP97 mouse myocytes the AP duration (APD) measured at 20% and 50% of repolarization was significantly shortened. Furthermore, in transgenic-like mice p.P888L overexpression significantly shortened the QT interval compared with WT SAP97 (from 61.5 ± 2.7 to 52.5 ± 2.1 ms, n = 6, P < 0.05).

The SAP97 p.P888L polymorphism shortens the QT interval and the APD as a consequence of a marked increase of the Ito charge. Therefore, this polymorphism could contribute to the phenotypic manifestations of the BrS.

**Keywords:** Sodium current, transient outward potassium current, SAP97, cardiac

P-85
PHARMACOLOGICAL AND BIOPHYSICAL PROPERTIES OF
PLASMA MEMBRANE PORES INDUCED BY SILICA
NANOPARTICLES

Alicia S.L., Yeranddy A.A., Peter H., Karel T.P.

KU Leuven

Amorphous silica nanoparticles (SiNPs) are extensively used for their beneficial properties in cosmetics and food industry as an anti-caking, densifying and hydrophobic agent. However, the high levels of exposure have raised concerns about health hazards, since SiNPs can penetrate tissues and cells resulting in health problems. In this study we first evaluated the effects of commercial 9 nm SiNPs (Ludox®) on intracellular Ca<fnref n="2" />⁺ concentration ([Ca<fnref n="2" />⁺]ᵢ) in HEK293, CHO and mouse airway epithelial cells. We found that extracellular application of SiNPs at 25 °C increased [Ca<fnref n="2" />⁺], when cells were co-stimulated with arachidonic acid or LPS. These effects were not observed when Ca<fnref n="2" />⁺ was omitted in the extracellular solution, indicating that SiNPs induce a Ca<fnref n="2" />⁺ entry pathway through the plasma membrane. Similar results were obtained when cells were exposed to SiNPs and heating from 25 to 35 °C, suggesting that SiNPs are sufficient to induce the Ca<fnref n="2" />⁺ entry pathway at physiological

temperatures. Whole-cell patch-clamp experiments revealed that SiNPs
combined with arachidonic acid trigger large currents that could be
blocked by the cation channel blocker ruthenium red in a voltage-depen-
dent manner. Analysis of the selectivity properties of this current showed
that they are mainly carried by cations. Taken together, our results
demonstrate that SiNPs induce Ca<fnref n="2" />⁺-permeable pores in the plasma
membrane, and that this phenomenon is enhanced by factors that
increase membrane fluidity. We propose that this Ca<fnref n="2" />⁺ entry pathway
may be relevant for the toxicological properties of SiNPs.

P-86
RETT-LIKE SEVERE ENCEPHALOPATHY CAUSED BY A DE
NOVO GRIN2B MUTATION IS ATTENUATED BY D-SERINE
DIETARY SUPPLEMENT

Soto D.<fnref n="1" />,<fnref n="2" />,³*, Olivella M.⁴*, Grau C.⁵, Ciruela F.⁵, Bayés À.⁶,⁷,
Síndreu C.<fnref n="2" />,⁸, López-Sala A.⁹, Armstrong J.¹⁰, García-Cazorla À.⁹,¹⁰,
Altafaj X.⁵

*Equal contribution; ¹Department of Biomedicine, University of Barcelona, 08036 Spain; ²Institute of Neurosciences, University of Barcelona, 08036 Spain; ³August Pi i Sunyer Biomedical Research Institute (IDIBAPS), Barcelona, 08036 Spain; ⁴Bioinformatics and Medical Statistics Group, University of Vic - Central University of Catalonia, 08050 Spain; ⁵Bellvitge Biomedical Research Institute (IDIBELL) - Unit of Neuropharmacology and Pain, Barcelona, 08908 Spain; ⁶Molecular Physiology of the Synapse Laboratory, Biomedical Research Institute Sant Pau (IIB Sant Pau), 08026 Spain;

⁷Autonomous University of Barcelona, Bellaterra, 08193 Spain;

⁸Department of Clinical Foundations, University of Barcelona, 08036 Spain; ⁹Department of Neurology, Neurometabolic Unit, Hospital Sant Joan de Déu and CIBERER, Barcelona, 08950 Spain; ¹⁰Genetics and Molecular Medicine Service, Hospital Sant Joan de Déu and CIBERER, Barcelona, 08950 Spain

N-Methyl-D-aspartic acid subfamily of glutamate ionotropic receptors (NMDARs) are activated during fast excitatory transmission and they have been proved to be key elements in synaptic plasticity, synaptogenesis and neuron survival. Several genetic studies have identified *de novo* NMDAR mutations in patients with neurodevelopmental diseases (including severe encephalopathies, autism, intellectual disability) as well as psychiatric disorders. In this work we report a case study of a 4 years-old Rett-like patient with a severe encephalopathy. The genetic studies of this patient (WES and Sanger sequencing) showed the presence of a missense *de novo* mutation of GRIN2B(p.P553T) coding for the GluN2B subunit of NMDARs.

Given the key role of GluN2B subunit in the very early stages of
synaptogenesis, we hypothesized that this mutation could be leading to
neuronal dysfunction and, subsequently, its normalization would poten-
tially ameliorate the patient's symptomatology. In heterologous expres-
sion systems, GluN2B(P553T) mutant construct do neither affected
NMDAR oligomerization nor their surface expression in primary neu-
ronal cultures. However, electrophysiological studies showed that
although functional, the mutant receptor displayed a significantly
reduced channel conductance concomitant with a strong reduction of
NMDA-evoked current density. These data are in agreement with our
structural molecular model, and strongly suggest the hypo-functionality
of mutant NMDARs that, potentially, could be rescued throughout the
enhancement of their activity. In accordance with this hypothesis,
in vitro administration of D-serine, a physiological NMDAR co-ago-
nist, displayed a significant increase of NMDA-evoked currents of
mutant receptors. Next, a clinical trial with dietary supplement of D-
serine was performed. Importantly, after fourteen-months dietary sup-
plement of D-serine, the patient showed an increase of serine plasma
levels, together with a noteworthy clinical improvement. Our results
show the possibility to enhance the hypofunctionality of glutamatergic
transmission as a therapeutic approach to attenuate cognitive and motor
impairment in early childhood.

This work has been supported by Grants PI16/00851PI13/00135 (ISCIII), La Marató (Project N. 20140210), PCIN-2014-105 (MINECO) and Miguel Servet (CP10/00548) to XA; BFU2014-57562-

<footnote n="1">¹Department of Pharmacology, School of Medicine, Universidad
Complutense de Madrid, CIBERCV, Ramón y Cajal square, Madrid;</footnote>

<footnote n="2">²Department of Cardiology, Hospital General Universitario Gregorio Marañón, Madrid</footnote>
P (MICIN) to DS; PI15/01082 (ISCIII) to AGC; BFU2012-34398 and BFU2015-69717-P (MINECO), Career Integration Grant (ref. 304111), Marie Curie Intra-European Fellowship (ref. 221540), Ramón y Cajal Fellowship (RYC-2011-08391p) to AB; SAF2012-40102 (MINECO/FEDER), FP7 Marie Curie CIG grant (#631035) and Ramón y Cajal (RYC-2011-08026) to CS. CA received a FPI contract (MINECO). We thank the 'Medicinal Computational Laboratory' from Universitat Autònoma de Barcelona for providing computing facilities. XA, CS and AB also benefit from the financial support of AGAUR (SGR14-297).

P-87
HISTOCHEMICAL AND FUNCTIONAL EVIDENCE OF
METABOTROPIC GLUTAMATE 5 RECEPTOR EXISTENCE
IN BRAIN DOPAMINERGIC NEURONS, WITH MODELING
OF RECEPTOR INTERACTIONS

Abass D., Dalton J.¹, Gil C., Ciruela F.², Giraldo J.¹,³, Sabria M.J., Ortiz J.³

¹Neuroscience Institute, Medical Biochemistry and Biostatistic Units,
Universitat Autònoma de Barcelona; ²Pharmacology Unit, Universitat
de Barcelona; ³CIBERSAM, Spain

Glutamatergic neurotransmission is involved in schizophrenia, a chronic disorder currently treated with dopamine D2 receptor antagonists. Metabotropic glutamate 5 receptors (mGlu5R) are class C heptahelical receptors naturally arranged as homodimers, although heteromerization with dopamine D2 receptors has been reported.¹ The majority of brain striatal mGlu5R are expressed postsynaptically in medium spiny projection neurons. Although presynaptic dopaminergic terminals have been reported to be devoid of mGlu5R immunoreactivity,⁵ here we present histochemical evidence of mGlu5R immunoreactivity colocalization with tyrosine hydroxylase-labelled terminals in rat and human brains. Dopamine synthesis, a specific functional property of catecholaminergic terminals, was explored in search for the relevance of mGlu5R expression in this type of nerve endings. Two different mGlu5R negative allosteric modulators (MTEP and fenobam) were able to modify D2 autoreceptor inhibition of dopamine synthesis in tissue minces obtained from rat brain striatum, which lends support to the hypothesis of functional mGlu5R expression in dopaminergic nerve endings. However, the precise role of mGlu5R in these neurons needs further clarification: MTEP and fenobam blocked the decrease of dopamine synthesis elicited by D2 partial agonists aripiprazole and (-)PPP, but not that of full agonist quinpirole. A classical allosteric model can be used to interpret this result, where D2 and mGlu5R interact in the membrane as a result of heteromerization, leading to cooperativity between both receptor sites. *In silico* long-timescale molecular dynamics simulations showed stable direct receptor-receptor interactions. Thus, the simplest explanation for our functional results would be a direct D2/mGlu5R interaction in the membranes of dopaminergic nerve endings. These results could be relevant for the treatment of neuropsychiatric disorders, where D2 receptors are important targets of pharmacotherapy.

⁴Cabello et al. J Neurochem. 2009 Jun; 109(5):1497-507.

⁵Paquet M, Smith Y. J Neurosci. 2003 Aug 20;23(20):7659–69.

Funded by MINECO grant SAF2014-58396.

**Keywords:** GPCR; operational model; psychosis; intrinsic efficacy

P-88
OPTICAL CONTROL OF LOCOMOTOR ACTIVITY IN A
MOUSE MODEL OF PARKINSONISM USING A
PHOTOACTIVE ADENOSINE A₂A RECEPTOR ANTAGONIST

Taura J.¹,², Nolen E.G.³, Cabré G.⁴, Hernando J.⁴, López-Cano M.¹,²,
Fernández-Dueñas V.¹,², Jacobson K.A.⁵, Ciruela F.¹,²

¹Unitat de Farmacologia, Departament Patologia i Terapèutica
Experimental, Facultat de Medicina, IDIBELL, Universitat de
Barcelona, L'Hospitalet de Llobregat, Spain; ²Institut de
Neurociències, Universitat de Barcelona, Spain; ³Colgate University,
Hamilton, USA; ⁴Departament de Química, Universitat Autònoma de
Barcelona, Cerdanyola del Vallès, Spain; ⁵Molecular Recognition
Section, Laboratory of Bioorganic Chemistry, National Institute of
Diabetes and Digestive and Kidney Diseases, National Institutes of
Health, Bethesda, USA

G protein-coupled adenosine receptors are promising therapeutic tar-
gets for a wide range of pathological conditions, such as Parkin-
son's disease. However, the ubiquity of adenosine receptors and
eventual lack of selectivity of most of adenosine-based drugs have
frequently diminished their therapeutic potential. Optopharmacology
is a novel approach that may help sorting out this issue, since it
allows the spatiotemporal control of receptor functioning. We have
developed the first generation of light-sensitive caged adenosine A2A
receptor (A2AR) ligands: MRS7145 is a SCH442416 (an A2AR
antagonist) derivative that is coumarin-blocked at the 5-amino posi-
tion. First, MRS7145 was photochemically characterized by *in vitro*
spectroscopy, monitoring SCH442416 release upon violet light illu-
mination (405 nm). Next, the light-dependent pharmacological profile
of MRS7145 was assessed in living cells (HEK-293T cells perma-
nently expressing the receptor). Thus, upon photoactivation,
MRS7145 precluded A2AR ligand binding and agonist-induced
cAMP accumulation. Thereafter, the ability of MRS7145 to block
A2AR in a light dependent manner was assessed *in vivo*. To this
end, A2AR antagonist-mediated locomotor activity potentiation was
evaluated in brain (striatum) fiber-optic implanted mice. Upon light
irradiation (405 nm) of the dorsal striatum, MRS7145 induced sig-
nificant hyperlocomotion. Finally, it was evaluated the efficacy of
MRS7145 reversing motor impairments in an animal model of
movement disorders, namely the hemiparkinsonian 6-OHDA lesioned
mouse. Thus, photo-activated MRS7145 was able to potentiate the
number of contralateral rotations induced by L-3,4-dihydroxypheny-
lalanine (L-DOPA). Overall, MRS7145 is a new light-operated
A2AR antagonist with potential utility for the treatment of Parkin-
son's disease.

**Acknowledgements:** This work was supported by MINECO/ISCIII (SAF2014-55700-P and PIE14/00034), the Catalan government (2014 SGR 1054), ICREA (ICREA Academia-2010), Fundació la Marató de TV3 (Grant 20152031) and IWT (SBO-140028).

P-89
CAv2.2 (N-TYPE) VOLTAGE-GATED CALCIUM CHANNELS
ARE ACTIVATED BY SUMO-1 PROTEIN

Silveirinha V.C.¹, Lin H.¹, Tanifuji S.², Mochida S.², Cottrell G.S.¹,
Cimarosti H.¹,³, Stephens G.J.¹

¹School of Pharmacy, University of Reading, Reading, RG6 6AJ, UK;

²Dept of Physiology, Tokyo Medical University, Tokyo, Japan;

³Present address: Universidade Federal de Santa Catarina,
Florianópolis, Brazil

SUMOylation is an important post-translational modification process involving covalent attachment of SUMO (Small Ubiquitin-like MOdifier) protein to target proteins, including plasma membrane ion channels and receptors (Silveirinha et al., 2013). Here, we tested the hypothesis that SUMO-1 can modulate function of the Ca<sub>v</sub>2.2 (N-type) voltage-gated calcium channel (VGCC), a protein vital for presynaptic
neurotransmitter release and implicated as a therapeutic target in diseases such as chronic pain. $Ca_{v}2.2$ function was investigated using *in vitro* electrophysiology in recombinant HEK cells (patch clamp recording) and native superior cervical ganglion (SCG) neurons (dual microelectrode recording). Co-expression of SUMO-1 with its conjugating enzyme Ubc9 caused an increase in maximal conductance ($G_{max}$) in current-voltage relationships, accompanied by a hyperpolarizing shift in the midpoint of activation ($V_{1/2}$), an effect not seen in control, conjugation deficient mutant SUMO-1ΔGG (Table 1). In synaptically-coupled SCG neurons, SUMO-1 protein was distributed throughout the cell body, axons and dendrites and presumptive presynaptic terminals, whilst SUMO-1ΔGG protein was largely confined to the cell body. SUMO-1, in comparison to SUMO-1ΔGG, increased paired excitatory postsynaptic potential ratio at short (20 -120 ms) inter-stimuli intervals. Together, these data demonstrate activation of $Ca_{v}2.2$ (N-type) VGCCs by SUMO-1 protein; in native neurons, data are consistent with $Ca_{v}2.2$ SUMOylation causing an increase in residual presynaptic $Ca^{2+}$ current and an increase in release probability of synaptic vesicles.

**References:**

Silveirinha *et al.*, 2013, *J Neurochem.* 127:580–591.

<table><caption>Table 1. Effects of SUMO proteins on $Ca_{v}2.2$ biophysical properties.</caption><thead><tr><th></th><th>Ca<sub>v</sub>2.2<br>(n = 16)</th><th>Ca<sub>v</sub>2.2/<br>SUMO-1<br>(n = 16)</th><th>Ca<sub>v</sub>2.2/<br>SUMO-1/<br>Ubc9<br>(n = 13)</th><th>Ca<sub>v</sub>2.2<br>(n = 16)</th><th>Ca<sub>v</sub>2.2/<br>SUMO-1<br>ΔGG<br>(n = 14)</th><th>Ca<sub>v</sub>2.2/<br>SUMO-1<br>ΔGG/<br>Ubc9<br>(n = 10)</th></tr></thead><tbody><tr><td>Gmax<br>(nS)</td><td>7.7 ± 0.9</td><td>13.4 ± 2.0</td><td>25.8 ± 4.0**</td><td>7.7 ± 0.9</td><td>6.5 ± 0.8</td><td>9.4 ± 1.3</td></tr><tr><td>V<sub>1/2</sub><br>(mV)</td><td>1.2 ± 1.0</td><td>-0.5 ± 1.0</td><td>-3.5 ± 0.9*</td><td>0.3 ± 0.9</td><td>3.5 ± 1.9</td><td>0.0 ± 2.0</td></tr><tr><td>k<br>(mV)</td><td>4.4 ± 0.4</td><td>3.9 ± 0.3</td><td>3.7 ± 0.4</td><td>4.2 ± 0.4</td><td>5.6 ± 0.4</td><td>4.0 ± 0.8</td></tr></tbody></table>

## P-90

### CONTACTIN-ASSOCIATED PROTEIN 1 INTERACTS WITH METABOTROPIC GLUTAMATE RECEPTOR TYPE 5 AND MODULATES ITS FUNCTION IN THE HIPPOCAMPUS

Morató X.¹,², Luján R.³, Heyninck K.⁴, Carvalho A.L.⁵, Craenenbroeck K.V.⁴, Cunha R.⁵, Fernandez-Dueñas V.¹,², Ciruela F.¹,²

¹Unitat de Farmacologia, Departament PatologiaiTerapèutica Experimental, Facultat de Medicina, IDIBELL, Universitat de Barcelona, Spain; ²Institut de Neurociències, Universitat de Barcelona, Spain; ³IDINE, Departamento de Ciencias Médicas, Facultad de Medicina, Universidad Castilla-La Mancha, Albacete, Spain;

⁴Department of Biochemistry and Microbiology, Faculty of Sciences, University of Ghent, Gent, Belgium; ⁵CNC-Center for Neurosciences and Cell Biology and Faculty of Medicine, University of Coimbra, Coimbra, Portugal

Metabotropic glutamate receptor type 5 ($mGlu_{5}$) receptors are G protein-coupled receptors that regulate the excitability of hippocampal CA1 pyramidal neurons and thus being implicated in multiple forms of neuronal plasticity. Accordingly, the pharmacological manipulation of these receptors became valuable for the management of neurological disorders affecting the hippocampus. Here, by means of a modified Membrane Yeast Two-Hybrid (MYTH) approach, we identified contactin-associated protein 1 (CASPR1), a type I transmembrane protein member of the neurexin family, as new $mGlu_{5}$ receptor partner. CASPR1 is expressed on both axons and dendrites of neurons and it is believed to play a key role in the targeting of contactin, $Na^{+}/K^{+}$ channels and AMPA receptors to appropriate sites within the neuronal membrane. Thus, CASPR1 participates in key processes such as myelination and excitatory neurotransmission.

We show that $mGlu_{5}$ receptor and CASPR1 co-distribute and co-assemble both in heterologous expression systems and in rat brain.

Interestingly, down regulation of CASPR1 in rat hippocampal primary cultures decreased intracellular $Ca^{2+}$ accumulation in response to $mGlu_{5}$ receptor agonists. In addition, silencing $mGlu_{5}$ receptor expression in the hippocampus by means of an AAV10 shCASPR1 impaired receptor-dependent spatial memory. Overall, these results demonstrate that CASPR1 plays a key role controlling the functionality of hippocampal $mGlu_{5}$ receptors.

**Acknowledgements:** This work was supported by MINECO/ISCIII (SAF2014-55700-P and PIE14/00034), the Catalan government (2014 SGR 1054), ICREA (ICREA Academia-2010), Fundació la Marató de TV3 (Grant 20152031) and IWT (SBO-140028). Also supported by the PhD fellowship FPI (BES-2012-052722).

## P-91

### EFFECT OF SIDERITIS HYSSOPIFOLIA ON APOPTOSIS IN HUMAN PROSTATE CANCER LNCAP CELLS

García Vieitez J.J., Huerga Mañanes V., Fernández Martínez M.N., Diez Liébana M.J., Sahagún Prieto A.M., Díez Láiz R., Sierra Vega M.

Department of Biomedical Sciences, IBIOMED, University of Leon, León, Spain

**Introduction:** Prostate cancer is the second major cause of cancer-related deaths and the most common cancer diagnosed in men in Europe. The main treatment for most cancers is chemotherapy but it has important adverse effects. In recent years, medicinal plant compounds are being studied and used to treat cancer. Considering the antioxidant properties of *Sideritis hyssopifolia*, the aim of the study was to determine the effects of the ether, methanol and chloroform extracts obtained from the aerial parts of this plant on apoptosis in LNCaP cells of human prostate cancer.

**Material and Methods:** The analysis of apoptosis was conducted by flow cytometry. Apoptosis was measured by using a specific kit (FITC-Annexin V Apoptosis Kit) following the manufacturer's instructions. Briefly, LNCaP cells were seeded (2x10⁵ cells/well) in 2 ml of culture medium. After 24 h, the cells were treated with the extracts and incubated at 37°C for 72 h. Cells harvested by trypsinization were collected and washed twice with ice-cold PBS. After centrifugation, cells were resuspended in 100 µL of binding buffer, and 5 µL of FITC-Annexin V and 1 µL of propidium iodide (1 mg/ml) were added. After 15 minutes of incubation, 400 µL of binding buffer were added and cells were analyzed by flow cytometry using Summit 4.3 software. Data were processed using Flowing Software 2.5.1.

**Results and Conclusions:** The results obtained indicated that all extracts induced apoptosis of LNCaP cells. In the untreated cells (control group), the majority of cells were viable (87%). In contrast, when samples were treated with extracts, the percentage of viable cells were: 17.79, 13.69 and 13.36% (ether, methanol and chloroform extracts, respectively). The highest number of cells in early apoptosis was found for methanol extract (56.17%) and in late apoptosis or necrosis for ether extract (42.52%).

**Keywords:** LNCaP; *Sideritis hyssopifolia*; flow cytometry; apoptosis

## P-92

### EFFECT OF SIDERITIS HYSSOPIFOLIA ON CELL CYCLE DISTRIBUTION IN HUMAN PROSTATE CANCER LNCAP CELLS

Huerga Mañanes V., García Vieitez J.J., Sierra Vega M., Diez Liébana M.J., Sahagún Prieto A.M., Fernández Martínez M.N.

Department of Biomedical Sciences, IBIOMED, University of Leon, León, Spain

**Introduction:** *Sideritis hyssopifolia* is a little woody plant belonging to *Lamiaceae* family with antioxidant properties that contains polyphenols. These compounds possess potentially health-promoting properties

<footnote>* = *P* < 0.05, ** = *P* < 0.01 vs. $Ca_{v}2.2$, ANOVA with Bonferroni post-hoc test.</footnote>
like anticarcinogenic and chemopreventive activities. The aim of the study was to evaluate the effects of the ether, methanol and chloroform extracts, obtained from the aerial parts of this plant on cell cycle in human prostate cancer LNCaP cells.

**Material and Methods:** LNCaP cell line were seeded (2x10⁵ cells/well) in 2 ml of culture medium. After 24 h, the cells were treated with the extracts and incubated at 37°C for 72 h. Cells harvested by trypsinization were collected, centrifuged and resuspended in cold PBS. To determine cell cycle distribution, cells were incubated for 15 min in the dark with Igepal CA-630 (1%), RNase (10 mg/ml) dissolved in sodium acetate (0.01 M at pH 5.2) and propidium iodide (1 mg/ml). Cells were quantified by flow cytometry using Summit 4.3 software, followed by data analysis using Flowing Software 2.5.1.

**Results and Conclusions:** The results were expressed as the percentage of cells in subG0/G1 (apoptotic cells), G0/G1, S and G2/M phases of the cell cycle. The treatment with the extracts induced cell accumulation in the subG0/G1 phase: 2.04% in untreated cells (control group), 12.20% after ether extract treatment, 14.74% with methanol extract and 27.25% with chloroform extract. These results showed the presence of apoptotic cells. It was also observed a reduction in the number of cells in the other phases, being the percentages obtained (G0/G1, S and G2/M phases, respectively): 74.40, 9.43 and 12.67% in the control group; 74.63, 6.65 and 5.01% with ether extract; 67.49, 9.81 and 5.76% with methanol extract and 56.05, 7.54 and 5.62% with chloroform extract.

## P-93

### OPTICAL CONTROL OF ENDOGENOUS RECEPTORS AND CELLULAR EXCITABILITY USING TARGETED COVALENT PHOTOSWITCHES

Garrido-Charles A.¹, Izquierdo-Serra M.¹, Bautista-Barrufet A.¹,²,
Trapero A.¹,³, Díaz-Tahoces A.⁴, Camarero N.¹, Pittolo S.¹, Valbuena
S.⁵, Pérez-Jiménez A.¹, Gay M.⁶, García-Moll A.⁴, Rodríguez-Escrich
C.⁷, Lerma J.⁵, de la Villa P.⁷, Fernández E.⁴,⁸, Pericás M.À.²,⁹,
Llebaria A.³, Gorostiza P.¹,⁸,¹⁰

¹Institut de Bioenginyeria de Catalunya (IBEC), Barcelona, Spain;
²Institute of Chemical Research of Catalonia (ICIQ), The Barcelona
Institute of Science and Technology, Tarragona, Spain; ³Institut de
Química Avançada de Catalunya (IQAC-CSIC), Barcelona, Spain;
⁴Instituto de Bioingeniería, Universidad Miguel Hernández (UMH),
Elche, Spain; ⁵Instituto de Neurociencias (CSIC-UMH), San Juan de
Alicante, Spain; ⁶Institut de Recerca en Biomedicina (IRBB),
Barcelona, Spain; ⁷Universidad de Alcalá de Henares (UAH), Alcalá
de Henares, Spain; ⁸Centro de Investigación Biomédica en Red en
Bioingeniería, Biomateriales y Nanomedicina (CIBER-BBN),
Zaragoza, Spain; ⁹Departament de Química Inorgànica i Orgànica,
Universitat de Barcelona (UB), Barcelona, Spain; ¹⁰Institució Catalana
de Recerca i Estudis Avançats (ICREA), Barcelona, Spain

Light-regulated drugs allow remotely photoswitching biological activity and enable plausible therapies based on small molecules. However, only freely diffusible photochromic ligands have been shown to work directly in endogenous receptors and methods for covalent attachment depend on genetic manipulation. Here we introduce a chemical strategy to covalently conjugate and photoswitch the activity of endogenous proteins and demonstrate its application to the kainate receptor channel GluK1. The approach is based on photoswitchable ligands containing a short-lived, highly reactive anchoring group that is targeted at the protein of interest by ligand affinity. These targeted covalent photoswitches (TCPs) constitute a new class of light-regulated drugs and act as prosthetic molecules that photocontrol the activity of GluK1-expressing neurons, and restore photoresponses in degenerated retina. The modularity of TCPs enables the application to different ligands and opens the way to new therapeutic opportunities.

## P-94

### SYNTHESIS AND BIOLOGICAL EVALUATION OF NOVEL INHIBITORS OF SIGNAL TRANSDUCTOR AND ACTIVATOR OF TRANSCRIPTION (STAT) PROTEINS IN HEMATOLOGICAL MALIGNANCES

Guerra B.¹, Martín-Rodríguez P.¹, Díaz-Chico J.C.¹, Hueso-Falcón
I.², Díaz-Chico B.N.¹, Aranda-Tavío H.¹,², Rodrigo-González L.¹,²,
Estévez-Braun A.², Fernández-Pérez L.¹

¹Instituto de Investigaciones Biomédicas y Sanitarias (IUIBS) – Universidad de Las Palmas de Gran Canaria, España; ²Departamento de Química Orgánica, Instituto de Bio-Orgánica Antonio González (CIBICAN), Universidad de la Laguna, España

**Background:** Aberrant activation of JAK/STAT signaling pathway has been related to several hematological malignances, including Chronic Myelogenous Leukemia (CML). Naphthoquinones (NFQ)-based derivatives have been shown to suppress STAT signaling pathway in non-cancer, inflammatory, and cancer cells. Now, the Ihf-c6, a novel NFQ-based derivative, has been identified as anti-leukemic drug by using a phenotyping screening of chemical library.

**Methods:** Novel NFQ-based derivatives structures were obtained by using multicomponent reactions. The mitochondrial metabolization of the tetrazolium salt 3-(4,5-methylthiazol-2yl-)-2,5diphenyl-tetrazolium bromide] (MTT) was used as indicator of cell viability. Cytostatic and cytotoxic effects were evaluated by using a Live-Cell Imaging System (Incucyte Zoom HD). Phosphorylated and total levels of STAT, MAPK and leukemic-relevant proteins were determined by immunoblotting. Cytokine-stimulated pY-STAT3/5 and STAT binding to DNA were also analyzed in adenocarcinoma breast (T47D) cells.

**Results:** Ihf-c6 showed antitumoral effects in vitro. Ihf-c6 was highly effective in inhibiting human promyelocytic leukemia HL60 cells (IC50 = 0.3 ± 0.1 µM), human erythroleukemia HEL cells (IC50 = 1.3 ± 0.6 µM), and human CML K562 cells (IC50 = 1.4 ± 0.7 µM) whereas non-blood tumor cells (e.g., MCF7 and SKBR3) and non-tumor cells (MRC.5) were higher resistant to Ihf-c6. Ihf-c6 reduced the proliferation of K562 cells in a time and dose-dependent manner. Ihf-c6 (3 µM for 6 h) caused 50% and 30% inhibition of constitutive pY-STAT5 and pY-Bcr-Abl in K562 cells, respectively. Moreover, Ihf-c6 (5 µM for 6-12 h) reduced constitutive pY-JAK2, pY-STAT3, and pY-STAT5 in HEL cells. GH-induced pY-STAT5 or IL-6-induced pY-STAT3, as well as cytokine-induced STAT-DNA interactions, were inhibited by Ihf-c6 in T47D cells in a time (maximal at 30 min)- and dose-dependent (maximal at 3 µM) manner.

**Conclusions:** Ihf-c6 is an effective inhibitor of constitutive as well as cytokine-induced pY-STAT5/3. NFQ-based derivatives might be potential therapeutic agents for STAT5/3-dependent blood cancer. Sociedad Española Farmacología 2017, Barcelona, Spain, June 2017.

## P-95

### IDENTIFICATION OF DIFFERENTIAL KINASE OFF-TARGETS AMONG CLINICAL PARP INHIBITORS: NEW OPPORTUNITIES FOR PRECISION ONCOLOGY?

Antolin A.A.¹,², Mestres J.², Workman P.¹, Al-Lazikani B.¹

¹Cancer Research UK Cancer Therapeutics Unit, Division of Cancer Therapeutics, The Institute of Cancer Research, London, UK;

²Systems Pharmacology, Research Program on Biomedical Informatics, IMIM Hospital del Mar Medical Research Institute and Universitat Pompeu Fabra, Barcelona, Catalonia, Spain

The on-going development of a more comprehensive and systems-based approach to pharmacology is uncovering a far more promiscuous interaction between drugs and the human proteome than was previously anticipated, a behaviour termed polypharmacology with clinical implications that are still not well understood.¹,² While many off-targets of available drugs still remain to be identified, computational