bluuebunny/crossref_metadata_2025 huggingface dataset:
Homologous and heterologous uncoupling of muscarinic M3 and α1B adrenoceptors to Gαq/11 in SH‐SY5Y human neuroblastoma cells
Authors: Bundey R A , Nahorski S R
Published: January 2005

The present study employed a [35S]‐GTPγS binding protocol in conjunction with immunoprecipitation (IP) of the Gα subunits to investigate the desensitization of Gq/11‐coupled receptors at the level of the G‐protein activation. Membranes from SH‐SY5Y cells expressing the recombinant human α1B‐adrenoceptor (α1B‐AR) (and endogenously expressing the M3 muscarinic acetylcholine receptor (M3‐AChR)) exhibited Gq/11 activation in a concentration‐dependent manner in response to noradrenaline or methacholine.

Pre‐treatment of intact cells with agonist prior to membrane preparation and use in the [35S]‐GTPγS IP assay demonstrated that both receptors were homologously desensitized by pre‐treatment with agonist since the Gq/11 activation in response to a secondary challenge with agonist was markedly reduced. Stimulation of α1B‐AR was effective at heterologously desensitizing the M3‐AChR. The PKC inhibitor, Ro‐31‐8220 (10 μM) was ineffective at preventing the agonist‐mediated receptor desensitization.

[32P]Pi‐labelled cells allowed the detection of increases in receptor phosphorylation. Phorbol 12,13 dibutyrate (PDBu) (1 μM) was effective at producing a Ro‐31‐8220 (10 μM)‐sensitive, detectable increase in α1B‐AR but not M3‐AChR phosphorylation. Noradrenaline (30 μM) stimulated α1B‐AR phosphorylation, which could be partially inhibited by Ro‐31‐8220 (10 μM). The phosphorylation of M3‐AChR was increased by methacholine (100 μM) incubation and this effect appeared to be insensitive to Ro‐31‐8220 (10 μM).

These findings demonstrate that [35S]‐GTPγS‐Gα‐subunit IP can be used to estimate receptor desensitization as a decline in receptor‐G‐protein coupling. Both the α1B‐AR and M3‐AChR undergo rapid homologous desensitization that is associated with an increase in receptor phosphorylation. The heterologous desensitization of M3‐AChR produced by α1B‐AR stimulation is not associated with a detectable increase in M3‐AChR phosphorylation, suggesting that receptor phosphorylation is not necessarily a prerequisite for desensitization.

British Journal of Pharmacology (2001) 134, 257–264; doi:10.1038/sj.bjp.0704229
A study on the role of nitric oxide and iron in 3‐morpholino‐sydnonimine‐induced increases in dopamine release in the striatum of freely moving rats
Authors: Serra Pier Andrea , Rocchitta Gaia , Esposito Giovanni , Delogu M Rosaria , Migheli Rossana , Miele Egidio , Desole Maria S , Miele Maddalena
Published: January 2005

We showed previously that interaction between NO and iron (II), both released following the decomposition of sodium nitroprusside (SNP), accounted for the late SNP‐induced dopamine (DA) increase in dialysates from the striatum of freely moving rats; in addition, we showed that co‐infusion of iron (II) with the NO‐donor S‐nitroso‐N‐acetylpenicillamine mimicked SNP effects on striatal DA release.

In the present study, intrastriatal co‐infusion of iron (II) (given as FeSO4, 1 mM for 40 min) with the NO‐donor and potential peroxynitrite generator 3‐morpholinosydnonimine (SIN‐1) (0.2, 0.5, 1.0 or 5.0 mM for 180 min), potentiated the SIN‐1‐induced increase in DA concentration in dialysates from the striatum of freely moving rats. Neither alone nor associated with iron (II) did SIN‐1 induce changes in dialysate ascorbic acid or uric acid concentrations.

Neither co‐infusion of a superoxide dismutase mimetic nor uric acid affected SIN‐1‐induced increases in dialysate DA concentration.

Infusion of the iron chelator deferoxamine (0.2 mM for 180 min) decreased dialysate DA and attenuated SIN‐1‐induced increases in dialysate DA concentrations.

These results suggest that iron plays a key role in SIN‐1‐induced release of striatal DA and do not support any role for either peroxynitrite or superoxide anion in SIN‐1‐induced release of striatal DA.

British Journal of Pharmacology (2001) 134, 275–282; doi:10.1038/sj.bjp.0704232
Apocynin and 1400 W prevents airway hyperresponsiveness during allergic reactions in mice
Authors: Muijsers Richard B R , Van Ark Ingrid , Folkerts Gert , Koster Andries S , Van Oosterhout Antoon J M , Postma Dirkje S , Nijkamp Frans P
Published: January 2005

The contribution of reactive nitrogen species to the development of airway hyperresponsiveness in a mouse model of allergic inflammation was investigated by the use of selective inhibitors of nitric oxide and superoxide formation.

Sensitized mice, repeatedly challenged with ovalbumin showed a significant (P<0.001, n=9) increase in airway responsiveness measured using whole body plethysmography. This hyperresponsiveness was accompanied by an influx of eosinophils into the airway lumen and increased levels of ovalbumin‐specific serum IgE.

Treatment of mice with the iNOS inhibitor 1400 W or the NADPH‐oxidase inhibitor apocynin did not significantly alter cellular influx into the airway lumen nor serum ovalbumin specific IgE. In contrast, apocynin as well as 1400 W inhibited ovalbumin‐induced airway hyperresponsiveness (P<0.001 and P<0.05 respectively, n=9). Furthermore, the airways of allergen challenged animals showed clear 3‐nitrotyrosine staining, which was mainly located in eosinophils. Remarkably, treatment with apocynin or 1400 W did not alter 3‐nitrotyrosine staining.

These data suggest that the development of airway hyperresponsiveness during the airway inflammation upon ovalbumin challenge is dependent on the release of both superoxide and nitric oxide and is therefore likely to be dependent on reactive nitrogen species. This mechanism, however, is not reflected by 3‐nitrotyrosine formation in the airways.

British Journal of Pharmacology (2001) 134, 434–440; doi:10.1038/sj.bjp.0704235
Suppression of granulocyte/macrophage colony‐stimulating factor release from human monocytes by cyclic AMP‐elevating drugs: role of interleukin‐10
Authors: Seldon Paul M , Giembycz Mark A
Published: January 2005

Granulocyte/macrophage colony‐stimulating factor (GM‐CSF) is a pro‐inflammatory cytokine secreted by cells of the monocyte/macrophage lineage and has been implicated in the pathogenesis of bronchitis and asthma.

In the present study we have evaluated the effect of several cyclic AMP‐elevating agents on lipopolysaccharide (LPS)‐induced GM‐CSF release from human monocytes and the extent to which the anti‐inflammatory cytokine, interleukin (IL)‐10, is involved.

LPS evoked a concentration‐dependent generation of GM‐CSF from human monocytes that was inhibited, at the mRNA and protein level, by 8‐Br‐cyclic AMP, cholera toxin, prostaglandin E2 (PGE2) and a number of structurally dissimilar phosphodiesterase (PDE) 4 inhibitors.

Pre‐treatment of monocytes with a concentration of an anti‐IL‐10 monoclonal antibody that abolished the inhibitory action of a maximally effective concentration of exogenous human recombinant IL‐10, significantly augmented LPS‐induced GM‐CSF generation. This effect was associated with a parallel upwards displacement of the concentration‐response curves that described the inhibition of GM‐CSF by PGE2, 8‐Br‐cyclic AMP and the PDE4 inhibitor, rolipram, without significantly changing the potency of any drug. Consequently, the maximum percentage inhibition of GM‐CSF release was reduced. Further experiments established that the reduction in the maximum inhibition of GM‐CSF release seen in anti‐IL‐10‐treated cells was not due to functional antagonism as rolipram, PGE2 and 8‐Br‐cyclic AMP were equi‐effective at all concentrations of LPS studied.

These data indicate that cyclic AMP‐elevating drugs attenuate the elaboration of GM‐CSF from LPS‐stimulated human monocytes by a mechanism that is not mediated via IL‐10. Suppression of GM‐CSF from monocytes may explain, at least in part, the efficacy of PDE4 inhibitors in clinical trials of chronic obstructive pulmonary disease.

British Journal of Pharmacology (2001) 134, 58–67; doi:10.1038/sj.bjp.0704238
Analysis of GABAA‐ and GABAB‐receptor mediated effects on intracellular Ca2+ in DRG hybrid neurones
Authors: Yokogawa Tomonori , Kim Seung U , Krieger Charles , Puil Ernest
Published: January 2005

Using pharmacological analysis and fura‐2 spectrofluorimetry, we examined the effects of γ‐aminobutyric acid (GABA) and related substances on intracellular Ca2+ concentration ([Ca2+]i) of hybrid neurones, called MD3 cells. The cell line was produced by fusion between a mouse neuroblastoma cell and a mouse dorsal root ganglion (DRG) neurone.

MD3 cells exhibited DRG neurone‐like properties, such as immunoreactivity to microtubule‐associated protein‐2 and neurofilament proteins. Bath applications of capsaicin and α, β‐methylene adenosine triphosphate reversibly increased [Ca2+]i. However, repeated applications of capsaicin were much less effective.

Pressure applications of GABA (100 μM), (Z)‐3‐[(aminoiminomethyl) thio] prop‐2‐enoic acid sulphate (ZAPA; 100 μM), an agonist at low affinity GABAA‐receptors, or KCl (25 mM), transiently increased [Ca2+]i.

Bath application of bicuculline (100 nM – 100 μM), but not picrotoxinin (10 – 25 μM), antagonized GABA‐induced increases in [Ca2+]i in a concentration‐dependent manner (IC50=9.3 μM).

Ca2+‐free perfusion reversibly abolished GABA‐evoked increases in [Ca2+]i. Nifedipine and nimodipine eliminated GABA‐evoked increases in [Ca2+]i. These results imply GABA response dependence on extracellular Ca2+.

Baclofen (500 nM – 100 μM) activation of GABAB‐receptors reversibly attenuated KCl‐induced increases in [Ca2+]i in a concentration‐dependent manner (EC50=1.8 μM). 2‐hydroxy‐saclofen (1 – 20 μM) antagonized the baclofen‐depression of the KCl‐induced increase in [Ca2+]i.

In conclusion, GABAA‐receptor activation had effects similar to depolarization by high external K+, initiating Ca2+ influx through high voltage‐activated channels, thereby transiently elevating [Ca2+]i. GABAB‐receptor activation reduced Ca2+ influx evoked by depolarization, possibly at Ca2+‐channel sites in MD3 cells.

British Journal of Pharmacology (2001) 134, 98–107; doi:10.1038/sj.bjp.0704244
Inhibitory effects of brefeldin A, a membrane transport blocker, on the bradykinin‐induced hyperpolarization‐mediated relaxation in the porcine coronary artery
Authors: Ohnishi Yoshinori , Hirano Katsuya , Nishimura Junji , Furue Masutaka , Kanaide Hideo
Published: January 2005

To elucidate the mechanism of the relaxation mediated by endothelium‐derived hyperpolarizing factors (EDHFs), the effect of brefeldin A, a membrane transport blocker, on cytosolic Ca2+ concentration ([Ca2+]i) and tension was determined in the porcine coronary arterial strips. We also examined the effect of brefeldin A on [Ca2+]i in the endothelial cells of the porcine aortic valve.

In the presence of 10 μM indomethacin and 30 μM NG‐nitro‐L‐arginine (L‐NOARG), both bradykinin and substance P induced a transient decrease in [Ca2+]i and tension in arterial strips contracted with 100 nM U46619 (thromboxane A2 analogue). A 6 h pre‐treatment with 20 μg ml−1 brefeldin A abolished the bradykinin‐induced relaxation, while it had no effect on the substance P‐induced relaxation.

In the absence of indomethacin and L‐NOARG, brefeldin A had no effect on the bradykinin‐induced relaxation during the contraction induced by U46619 or 118 mM K+.

The indomethacin/L‐NOARG‐resistant relaxation induced by bradykinin was completely inhibited by 3 mM tetrabutylammonium (non‐specific Ca2+‐activated K+ channel blocker), while that induced by substance P was not inhibited by 3 mM tetrabutylammonium or 1 mM 4‐aminopyridine (voltage‐dependent K+ channels blocker) alone, but completely inhibited by their combination.

Brefeldin A had no effect on the [Ca2+]i elevation in endothelial cells induced by bradykinin or substance P.

In conclusion, bradykinin produce EDHF in a brefeldin A‐sensitive mechanism in the porcine coronary artery. However, this mechanism is not active in a substance P‐induced production of EDHF, which thus suggests EDHF to be more than a single entity.

British Journal of Pharmacology (2001) 134, 168–178; doi:10.1038/sj.bjp.0704246
Pharmacological and biochemical characterization of A3 adenosine receptors in Jurkat T cells
Authors: Gessi Stefania , Varani Katia , Merighi Stefania , Morelli Anna , Ferrari Davide , Leung Edward , Baraldi Pier Giovanni , Spalluto Giampiero , Borea Pier Andrea
Published: January 2005

The present work was devoted to the study of A3 adenosine receptors in Jurkat cells, a human leukemia line.

The A3 subtype was found by means of RT‐PCR experiments and characterized by using the new A3 adenosine receptor antagonist [3H]‐MRE 3008F20, the only A3 selective radioligand currently available. Saturation experiments revealed a single high affinity binding site with KD of 1.9±0.2 nM and Bmax of 1.3±0.1 pmol mg−1 of protein.

The pharmacological profile of [3H]‐MRE 3008F20 binding on Jurkat cells was established using typical adenosine ligands which displayed a rank order of potency typical of the A3 subtype.

Thermodynamic data indicated that [3H]‐MRE 3008F20 binding to A3 subtype in Jurkat cells was entropy‐ and enthalpy‐driven, according with that found in cells expressing the recombinant human A3 subtype.

In functional assays the high affinity A3 agonists Cl‐IB‐MECA and IB‐MECA were able to inhibit cyclic AMP accumulation and stimulate Ca2+ release from intracellular Ca2+ pools followed by Ca2+ influx.

The presence of the other adenosine subtypes was investigated in Jurkat cells. A1 receptors were characterized using [3H]‐DPCPX binding with a KD of 0.9±0.1 nM and Bmax of 42±3 fmol mg−1 of protein. A2A receptors were studied with [3H]‐SCH 58261 binding and revealed a KD of 2.5±0.3 nM and a Bmax of 1.4±0.2 pmol mg−1 of protein.

In conclusion, by means of the first antagonist radioligand [3H]‐MRE 3008F20 we could demonstrate the existence of functional A3 receptors on Jurkat cells.

British Journal of Pharmacology (2001) 134, 116–126; doi:10.1038/sj.bjp.0704254
RNA‐editing of the 5‐HT2C receptor alters agonist‐receptor‐effector coupling specificity
Authors: Berg Kelly A , Cropper Jodie D , Niswender Colleen M , Sanders‐Bush Elaine , Emeson Ronald B , Clarke William P
Published: January 2005

The serotonin2C (5‐HT2C) receptor couples to both phospholipase C (PLC)‐inositol phosphate (IP) and phospholipase A2 (PLA2)‐arachidonic acid (AA) signalling cascades. Agonists can differentially activate these effectors (i.e. agonist‐directed trafficking of receptor stimulus) perhaps due to agonist‐specific receptor conformations which differentially couple to/activate transducer molecules (e.g. G proteins). Since editing of RNA transcripts of the human 5‐HT2C receptor leads to substitution of amino acids at positions 156, 158 and 160 of the putative second intracellular loop, a region important for G protein coupling, we examined the capacity of agonists to activate both the PLC‐IP and PLA2‐AA pathways in CHO cells stably expressing two major, fully RNA‐edited isoforms (5‐HT2C‐VSV, 5‐HT2C‐VGV) of the h5‐HT2C receptor.

5‐HT increased AA release and IP accumulation in both 5‐HT2C‐VSV and 5‐HT2C‐VGV expressing cells. As expected, the potency of 5‐HT for both RNA‐edited isoforms for both responses was 10 fold lower relative to that of the non‐edited receptor (5‐HT2C‐INI) when receptors were expressed at similar levels.

Consistent with our previous report, the efficacy order of two 5‐HT receptor agonists (TFMPP and bufotenin) was reversed for AA release and IP accumulation at the non‐edited receptor thus demonstrating agonist trafficking of receptor stimulus. However, with the RNA‐edited receptor isoforms there was no difference in the relative efficacies of TFMPP or bufotenin for AA release and IP accumulation suggesting that the capacity for 5‐HT2C agonists to traffic receptor stimulus is lost as a result of RNA editing.

These results suggest an important role for the second intracellular loop in transmitting agonist‐specific information to signalling molecules.

British Journal of Pharmacology (2001) 134, 386–392; doi:10.1038/sj.bjp.0704255
Suppression of K+‐induced hyperpolarization by phenylephrine in rat mesenteric artery: relevance to studies of endothelium‐derived hyperpolarizing factor
Authors: Richards G R , Weston A H , Burnham M P , Félétou M , Vanhoutte P M , Edwards G
Published: January 2005

In intact mesenteric arteries, increasing [K+]o by 5 mM hyperpolarized both endothelial and smooth muscle cells. Subsequent exposure to 10 μM phenylephrine depolarized both cell types which were then repolarized by a 5 mM increase in [K+]o. In endothelium‐denuded vessels, increasing [K+]o by 5 mM hyperpolarized the smooth muscle but K+ had no effect after depolarization by 10 μM phenylephrine. On subsequent exposure to iberiotoxin plus 4‐aminopyridine, the repolarizing action of 5 mM K+ was restored. In endothelium‐intact vessels exposed to phenylephrine, pretreatment with a gap junction inhibitor (gap 27) reduced K+‐mediated smooth muscle repolarization without affecting the endothelial cell response. It is concluded that phenylephrine‐induced efflux of K+ via smooth muscle K+ channels produces a local increase in [K+]o which impairs repolarization to added K+. Thus, studies involving vessels precontracted with agonists which increase [K+]o maximize the role of gap junctions and minimize any contribution to the EDHF pathway from endothelium‐derived K+.

British Journal of Pharmacology (2001) 134, 1–5; doi:10.1038/sj.bjp.0704256
A cerebral nitrergic pathway modulates endotoxin‐induced changes in gastric motility
Authors: Quintana Elsa , García‐Zaragozá Eugenia , Angeles Martínez‐Cuesta M , Calatayud Sara , Esplugues Juan V , Barrachina María Dolores
Published: January 2005

This study analyses the neural pathway involved in the modulation of gastric motor function by stress.

Systemic administration of low doses of endotoxin (40 μg kg−1, i.v.) prevents the increase in gastric tone induced by 2‐deoxy‐D‐glucose (200 mg kg−1, i.v., 2‐DG) in urethane‐anaesthetized rats.

Functional inhibition of afferent neurones by systemic administration of capsaicin (20+30+50 mg kg−1, i.m.) in adult rats prevented the inhibitory effects of endotoxin.

Pre‐treatment with the nitric oxide synthase (NOS) inhibitor, NG‐nitro‐L‐arginine methyl ester (L‐NAME), both i.v. (10 mg kg−1) and i.c. (200 μg rat−1), prevented the inhibitory effects of endotoxin on gastric tone induced by 2‐DG.

Immunohistochemical studies show Fos expression in the dorsal vagal complex (DVC) of the brainstem of 2‐DG‐treated animals. Peripheral administration of endotoxin (40 μg kg−1, i.p.) increased the number of Fos‐immunoreactive cells induced by 2‐DG, both in the nucleus tractus solitarii (NTS) and in the dorsal motor nucleus (DMN) of the DVC. Pre‐treatment with L‐NAME prevented the increase in Fos expression induced by endotoxin in both nuclei.

Endotoxin (40 μg kg−1, i.p.) increased Ca2+‐dependent nitric oxide synthase (cNOS) activity in the brainstem. Addition of 7‐nitroindazole (600 μM, 7‐NI) to the assay significantly inhibited the increase in cNOS activity caused by endotoxin. No change in NOS activity of any isoform was observed in the stomach of animals treated with endotoxin.

The present study suggests that inhibition of gastric motor function by low doses of endotoxin involves activation of capsaicin‐sensitive afferent neurones and neuronal NOS in the brainstem.

British Journal of Pharmacology (2001) 134, 325–332; doi:10.1038/sj.bjp.0704258
Effects of S‐2474, a novel nonsteroidal anti‐inflammatory drug, on amyloid β protein‐induced neuronal cell death
Authors: Yagami Tatsurou , Ueda Keiichi , Asakura Kenji , Sakaeda Toshiyuki , Kuroda Takayuki , Hata Satoshi , Kambayashi Yoshikazu , Fujimoto Masafumi
Published: January 2005

The accumulation of amyloid β protein (Aβ) in the brain is a characteristic feature of Alzheimer's disease (AD). Clinical trials of AD patients with nonsteroidal anti‐inflammatory drugs (NSAIDs) indicate a clinical benefit. NSAIDs are presumed to act by suppressing inhibiting chronic inflammation in the brain of AD patients.

In the present study, we investigated effects of S‐2474 on Aβ‐induced cell death in primary cultures of rat cortical neurons. S‐2474 is a novel NSAID, which inhibits cyclo‐oxygenase‐2 (COX‐2) and contains the di‐tert‐butylphenol antioxidant moiety.

S‐2474 significantly prevented neurons from Aβ(25 – 35)‐ and Aβ(1 – 40)‐induced cell death. S‐2474 ameliorated Aβ‐induced apoptotic features such as the condensation of chromatin and the fragmentation of DNA completely.

Prior to cell death, Aβ(25 – 35) generated prostaglandin D2 (PGD2) and free radicals from neurons. PGD2 is a product of cyclo‐oxygenase (COX), and caused neuronal cell death.

S‐2474 significantly inhibited the Aβ(25 – 35)‐induced generation of PGD2 and free radicals.

The present cortical cultures contained little non‐neuronal cells, indicating that S‐2474 affected neuronal survival directly, but not indirectly via non‐neuronal cells. Both an inhibitory effect of COX‐2 and an antioxidant effect might contribute to the neuroprotective effects of S‐2474.

In conclusion, S‐2474 exhibits protective effects against neurotoxicity of Aβ. Furthermore, the present study suggests that S‐2474 may possess therapeutic potential for AD via ameliorating degeneration in neurons as well as suppressing chronic inflammation in non‐neuronal cells.

British Journal of Pharmacology (2001) 134, 673–681; doi:10.1038/sj.bjp.0704261
Inhibitory action of nociceptin/orphanin FQ on functionally different thalamic neurons in urethane‐anaesthetized rats
Authors: Albrecht Doris , Blühdorn Roman , Siegmund Herbert , Berger Hartmut , Calo' Girolamo
Published: January 2005

In this study we administered nociceptin/orphanin FQ (NC) ionotophoretically onto neurons located in functionally distinct thalamic structures of urethane‐anesthetized rats. Extracellular single unit recordings were made in the medial and lateral ventroposterior nucleus, posterior thalamic nucleus, zona incerta, lateral posterior nucleus, laterodorsal nucleus, ventrolateral nucleus and reticular nucleus.

NC decreased the firing rate in 60% of thalamic neurons. This decrease in firing rate was accompanied by a significant reduction in the number of high threshold bursts.

In about 20% of the neurons NC increased the firing rate. In most cells NC‐induced increases in discharge rate could be blocked by the GABAA receptor antagonists bicuculline and SR 95531.

The NC receptor ligands [Phe1Ψ(CH2‐NH)Gly2] nociceptin(1‐13)NH2, Ac‐RYYRIK‐NH2 and [Nphe1]NC(1‐13)NH2 were also evaluated. All these peptides inhibited NC‐induced changes in firing rate. In addition, in some neurons where NC inhibited firing, [Nphe1]NC(1‐13)NH2 and Ac‐RYYRIK‐NH2 elicited per se an increase in firing rate, suggesting the existence of tonic innervation of thalamic neurons by NC‐containing fibres.

In NC‐inhibited neurons nocistatin induced a significant increase in firing rate.

The present study demonstrated that NC regulates various thalamic nuclei related not only to somatosensory, but also to the visual and motor functions.

British Journal of Pharmacology (2001) 134, 333–342; doi:10.1038/sj.bjp.0704264
Vascular smooth muscle relaxation mediated by nitric oxide donors: a comparison with acetylcholine, nitric oxide andnitroxyl ion
Authors: Wanstall Janet C , Jeffery Trina K , Gambino Agatha , Lovren Fina , Triggle Christopher R
Published: January 2005

Vasorelaxant properties of three nitric oxide (NO) donor drugs (glyceryl trinitrate, sodium nitroprusside and spermine NONOate) in mouse aorta (phenylephrine pre‐contracted) were compared with those of endothelium‐derived NO (generated with acetylcholine), NO free radical (NO·; NO gas solution) and nitroxyl ion (NO−; from Angeli's salt).

The soluble guanylate cyclase inhibitor, ODQ (1H‐(1,2,4‐)oxadiazolo(4,3‐a)‐quinoxalin‐1‐one; 0.3, 1 and 10 μM), concentration‐dependently inhibited responses to all agents. 10 μM ODQ abolished responses to acetylcholine and glyceryl trinitrate, almost abolished responses to sodium nitroprusside but produced parallel shifts (to a higher concentration range; no depression in maxima) in the concentration‐response curves for NO gas solution, Angeli's salt and spermine NONOate.

The NO· scavengers, carboxy‐PTIO, (2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethyl‐imidazoline‐1‐oxyl‐3‐oxide; 100 μM) and hydroxocobalamin (100 μM), both inhibited responses to NO gas solution and to the three NO donor drugs, but not Angeli's salt. Hydroxocobalamin, but not carboxy‐PTIO, also inhibited responses to acetylcholine.

The NO− inhibitor, L‐cysteine (3 mM), inhibited responses to Angeli's salt, acetylcholine and the three NO donor drugs, but not NO gas solution.

The data suggest that, in mouse aorta, responses to all three NO donors involve (i) activation of soluble guanylate cyclase, but to differing degrees and (ii) generation of both NO· and NO−. Glyceryl trinitrate and sodium nitroprusside, which generate NO following tissue bioactivation, have profiles resembling the profile of endothelium‐derived NO more than that of exogenous NO. Spermine NONOate, which generates NO spontaneously outside the tissue, was the drug that most closely resembled (but was not identical to) exogenous NO.

British Journal of Pharmacology (2001) 134, 463–472; doi:10.1038/sj.bjp.0704269
Guanosine 3′: 5′‐cyclic monophosphate‐dependent pathway alterations in ventricular cardiomyocytes of spontaneously hypertensive rats
Authors: Mazzetti Luca , Ruocco Carlo , Giovannelli Lisa , Ciuffi Mario , Franchi‐Micheli Sergio , Marra Fabio , Zilletti Lucilla , Failli Paola
Published: January 2005

We investigated the effect of the NO‐donor S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP) on cardiomyocytes isolated from control normotensive Wistar Kyoto (WKY) and spontaneously hypertensive (SHR) rats.

Ventricular cardiomyocytes were isolated from SHR and WKY hearts and imaging analysis of fura‐2‐loaded cells was performed in order to evaluate calcium transient in electrical field paced (0.5 Hz) cells.

In WKY cardiomyocytes, 1 – 200 μM SNAP dose‐dependently increased cyclic GMP content. In basal conditions, cyclic GMP content of SHR cardiomyocytes was significantly higher than in WKY, but SNAP failed to further increase cyclic GMP over the basal level.

In control conditions, the ΔF/F and decay time of the calcium transient were similar in both strains. In WKY cardiomyocytes, SNAP (1 – 100 μM) reduced the decay time. In SHR cardiomyocytes, SNAP was ineffective. Dibutyryl cyclic GMP (10−6 – 10−8 M), a membrane permeable cyclic GMP analogue, behaved similarly to SNAP.

In WKY and SHR cardiomyocytes, 10−8 M isoprenaline similarly increased ΔF/F and decreased the decay time. SNAP and dibutyryl cyclic GMP prevented the effect of isoprenaline in WKY, whereas both molecules were ineffective in SHR cardiomyocytes. In WKY, SNAP effects were blocked by pretreating cells with the cGK inhibitor KT‐5823.

Western blotting analysis of cGK type I showed that the enzyme was expressed in WKY isolated cardiomyocytes, but absent in four out of five SHR preparations.

We concluded that the low expression of cGKI may determine the lack of NO/cyclic GMP‐dependent regulation on calcium transient in SHR cardiomyocytes. This alteration may contribute to the development of heart hypertrophy in hypertensive status.

British Journal of Pharmacology (2001) 134, 596–602; doi:10.1038/sj.bjp.0704275
Protective effects of 5‐HT1A receptor agonists against emotional changes produced by stress stimuli are related to their neuroendocrine effects
Authors: Tsuji Minoru , Takeda Hiroshi , Matsumiya Teruhiko
Published: January 2005

The effects of the 11β‐hydroxylase inhibitor metyrapone on the protective effects of serotonin (5‐hydroxytryptamine; 5‐HT)1A receptor agonists against emotional changes produced by acute restraint stress were examined in mice.

Changes in the emotional state of mice were evaluated in terms of changes in exploratory activity, i.e. total locomotor activity, number and duration of rearing and head‐dipping behaviours, and latency to the first head‐dipping, using an automatic hole‐board apparatus.

Treatment with the 5‐HT1A receptor agonists flesinoxan (1 mg kg−1, i.p.) and R(+)‐2‐di‐n‐propylamino‐8‐hydroxy‐1,2,3,4‐tetrahydronaphthalene hydrobromide (8‐OH‐DPAT; 1 mg kg−1, i.p.) 24 h prior to exposure to stress significantly suppressed the decrease in various exploratory behaviours that was observed immediately after the exposure to acute restraint stress (60 min). The effects of flesinoxan (1 mg kg−1, i.p.) and 8‐OH‐DPAT (1 mg kg−1, i.p.) were antagonized by co‐injection with N‐{2‐[4‐(2‐methoxyphenyl)‐1‐piperazinyl]ethyl}‐N‐(2‐pyridinyl) cyclohexanecarboxamide trihydrochloride (WAY100635; 1 mg kg−1, i.p.), a selective 5‐HT1A receptor antagonist.

Flesinoxan (1 mg kg−1, i.p.) and 8‐OH‐DPAT (1 mg kg−1 i.p.) significantly increased the plasma corticosterone level, and these effects of 5‐HT1A receptor agonists were dose‐dependently blocked by pretreatment with metyrapone (12.5 and 25 mg kg−1, s.c.).

Metyrapone (25 mg kg−1, s.c.) alone did not modify the stress‐induced changes in exploratory behaviours. Pretreatment with metyrapone (12.5 and 25 mg kg−1, s.c.) partly antagonized the protective effects of flesinoxan (1 mg kg−1, i.p.) and 8‐OH‐DPAT (1 mg kg−1, i.p.) with regard to only the number and duration of head‐dipping behaviours.

These results suggest that activation of the adrenocortical system via 5‐HT1A receptors may facilitate some adaptive mechanism(s) involved in the recognition of and/or ability to cope with stressful situations.

British Journal of Pharmacology (2001) 134, 585–595; doi:10.1038/sj.bjp.0704276
Orphanin FQ/nociceptin attenuates the development of morphine tolerance in rats
Authors: Lutfy Kabirullah , Hossain Syed M , Khaliq Imran , Maidment Nigel T
Published: January 2005

Recent evidence from studies in mice lacking the opioid receptor‐like (ORL‐1) receptor and from experiments using antibodies raised against orphanin FQ/nociceptin (OFQ/N) suggest that this peptide may be involved in morphine tolerance. In the present study we sought to investigate if administration of exogenous OFQ/N would modulate the development of tolerance to the antinociceptive effect of morphine.

Rats were treated for 3 days with either saline or morphine (10 mg kg−1, s.c.) followed, 15 and 75 min later, by two intracerebroventricular injections of either artificial cerebrospinal fluid (aCSF) or OFQ/N. The dose of OFQ/N was doubled each day (7.5, 15, 30 nmol). On day 4, rats were tested on a hot plate apparatus before and 30, 60 and 90 min after morphine administration.

Repeated OFQ/N treatment did not affect basal nociceptive responses or morphine‐induced antinociception. However, the same treatment significantly attenuated the development of morphine tolerance.

Since learning and memory could contribute to the development of morphine tolerance, in subsequent studies, we examined the effect of OFQ/N administered in the CA3 region of the hippocampus, where OFQ/N has been shown to block LTP and impair spatial memory. A greater attenuation of morphine tolerance with no alteration of baseline hot plate latency or morphine‐induced antinociception was observed when OFQ/N was administered in this area of the rat brain.

Taken together, our results demonstrate that OFQ/N may act in the hippocampus to attenuate morphine tolerance.

British Journal of Pharmacology (2001) 134, 529–534; doi:10.1038/sj.bjp.0704279
Reply to Dodds & Rivory
Authors: Blandizzi C , De Paolis B , Colucci R , Lazzeri G , Baschiera F , Del Tacca M
Published: January 2005

British Journal of Pharmacology (2001) 134, 449–450; doi:10.1038/sj.bjp.0704283
Effects of agents that inactivate free radical NO (NO•) on nitroxyl anion‐mediated relaxations, and on the detection of NO• released from the nitroxyl anion donor Angeli's salt
Authors: Ellis Anthie , Lu Hong , Li Chun Guang , Rand Michael J
Published: January 2005

The effects of agents that inactivate free radical nitric oxide (carboxy‐PTIO, hydroxocobalamin and pyrogallol) were tested on relaxations produced by the nitroxyl anion (NO−) donor Angeli's salt in rat aortic rings and anococcygeus muscles. The amount of NO• generated from Angeli's salt in the presence of these agents was measured using a NO•‐selective electrode sensor.

Carboxy‐PTIO (100, 300 μM), hydroxocobalamin (30, 100 μM) and pyrogallol (10, 30 μM) significantly reduced relaxations produced by Angeli's salt (0.3 μM) in aortic rings but not in anococcygeus muscles.

NO• generated from Angeli's salt (0.1 – 10 μM), as detected by the sensor electrode, was less than 0.5% of the amount of Angeli's salt added. Carboxy‐PTIO (100 μM) and hydroxocobalamin (30 μM), but not pyrogallol significantly increased the amount of NO• detected.

In the presence of an oxidizing agent copper [II] (as CuSO4 100 μM), the amount of NO• detected from 0.3 μM of Angeli's salt increased from an undetectable level of 142.7±15.7 nM (equivalent to 47.6% of Angeli's salt added). Under these conditions, carboxy‐PTIO, hydroxocobalamin and pyrogallol significantly reduced the amount of NO• detected from Angeli's salt as well as the signal generated by an equivalent amount of authentic NO (0.33 μM).

The difference in effects of these agents on relaxations to Angeli's salt in the aorta and the anococcygeus muscle may be explained by the ready conversion of NO− to NO• in the aorta through an unidentified mechanism, which makes NO− susceptible to inactivation by these agents. Furthermore, in addition to inactivating NO•, carboxy‐PTIO and hydroxocobalamin may themselves oxidize NO− to NO•, albeit slightly.

British Journal of Pharmacology (2001) 134, 521–528; doi:10.1038/sj.bjp.0704287
Changes in extracellular pH and myocardial ischaemia alter the cardiac effects of diadenosine tetraphosphate and pentaphosphate
Authors: Stavrou Brigitte M , Beck Caroline , Flores Nicholas A
Published: January 2005

The structural conformation of diadenosine tetraphosphate (Ap4A) and pentaphosphate (Ap5A) has been reported to alter as pH is reduced. As such, it is possible that the cardiac effects of Ap4A and Ap5A vary during acidosis and myocardial ischaemia due to changes in ligand structure, receptor proteins or intracellular signalling.

We investigated whether the cardiac electrophysiological and coronary vasomotor effects of Ap4A and Ap5A are preserved under conditions of extracellular acidosis (pH 6.5) and alkalosis (pH 8.5) and whether Ap4A has any electrophysiological or antiarrhythmic effects during ischaemia.

Transmembrane right ventricular action potentials, refractory periods and coronary perfusion pressure were recorded from isolated, Langendorff‐perfused guinea‐pig hearts under constant flow conditions. The effects of 1 nM and 1 μM Ap4A and Ap5A were studied at pH 7.4, 6.5 and 8.5. The effects of 1 μM Ap4A were studied during global low‐flow ischaemia and reperfusion.

At pH 7.4, Ap4A and Ap5A increased action potential duration (APD95) and refractory period (RP) and reduced coronary perfusion pressure. The electrophysiological effects were absent at pH 6.5 while the reductions in perfusion pressure were attenuated. At pH 8.5, Ap4A increased RP but the effects of Ap4A and Ap5A on perfusion pressure were attenuated. During ischaemia, Ap4A had no antiarrhythmic or electrophysiological effects.

These data demonstrate the importance of extracellular pH in influencing the effects of Ap4A and Ap5A on the heart and indicate that any potentially cardioprotective effects of these compounds during normal perfusion at physiological pH are absent during ischaemia.

British Journal of Pharmacology (2001) 134, 639–647; doi:10.1038/sj.bjp.0704288
Induction of cyclooxygenase‐2 protein by lipoteichoic acid from Staphylococcus aureus in human pulmonary epithelial cells: involvement of a nuclear factor‐κB‐dependent pathway
Authors: Lin Chien‐Huang , Kuan I‐Hui , Lee Horng‐Mo , Lee Wen‐Sen , Sheu Joen‐Rong , Ho Yuan‐Soon , Wang Chun‐Hua , Kuo Han‐Pin
Published: January 2005

This study investigated the role of protein kinase C (PKC) and transcription factor nuclear factor‐κB (NF‐κB) in cyclooxygenase‐2 (COX‐2) expression caused by lipoteichoic acid (LTA), a cell wall component of the gram‐positive bacterium Staphylococcus aureus, in human pulmonary epithelial cell line (A549).

LTA caused dose‐ and time‐dependent increases in COX‐2 expression and COX activity, and a dose‐dependent increase in PGE2 release in A549 cells. The LTA‐induced increases in COX‐2 expression and COX activity were markedly inhibited by dexamethasone, actinomycin D or cyclohexamide, but not by polymyxin B, which binds and inactivates endotoxin.

The phosphatidylcholine‐phospholipase C (PC‐PLC) inhibitor (D‐609) and the phosphatidate phosphohydrolase inhibitor (propranolol) reduced the LTA‐induced increases in COX‐2 expression and COX activity, while phosphatidylinositol‐phospholipase C inhibitor (U‐73122) had no effect. The PKC inhibitors (Go 6976, Ro 31‐8220 and GF 109203X) and NF‐κB inhibitor, pyrrolidine dithiocarbamate (PDTC), also attenuated the LTA‐induced increases in COX‐2 expression and COX activity.

Treatment of A549 cells with LTA caused an increase in PKC activity in the plasma membrane; this stimulatory effect was inhibited by D‐609, propranolol, or Go 6976, but not by U‐73122.

Exposure of A549 cells to LTA caused a translocation of p65 NF‐κB from the cytosol to the nucleus and a degradation of IκB‐α in the cytosol. Treatment of A549 cells with LTA caused NF‐κB activation by detecting the formation of NF‐κB‐specific DNA‐protein complex in the nucleus; this effect was inhibited by dexamethasone, D‐609, propranolol, Go 6976, Ro 31‐8220, or PDTC.

These results suggest that LTA might activate PC‐PLC and phosphatidylcholine‐phospholipase D to induce PKC activation, which in turn initiates NF‐κB activation, and finally induces COX‐2 expression and PGE2 release in human pulmonary epithelial cell line.

British Journal of Pharmacology (2001) 134, 543–552; doi:10.1038/sj.bjp.0704290
Beneficial effects of the Ca2+ sensitizer EMD 57033 in exercising pigs with infarction‐induced chronic left ventricular dysfunction
Authors: Duncker D J , Haitsma D B , Liem D A , Heins N , Stubenitsky R , Verdouw P D
Published: January 2005

It is unknown how cardiac stimulation by Ca2+ sensitization modulates the cardiovascular response to exercise when left ventricular (LV) function is chronically depressed following a myocardial infarction. We therefore investigated the effects of EMD 57033 at rest and during exercise and compared these to those of the mixed Ca2+‐sensitizer/phosphodiesterase‐III inhibitor pimobendan.

Pigs were chronically instrumented for measurement of cardiovascular performance. At the time of instrumentation, infarction was produced by coronary artery ligation (MI, n=12). Studies in MI were performed in the awake state, 2 – 3 weeks after infarction.

MI were characterized by a lower resting cardiac output (18%), stroke volume (30%) and LVdP/dtmax (18%), and a doubling of LV end‐diastolic pressure, compared to normal pigs (N, n=13).

In 11 resting MI, intravenous EMD 57033 (0.2 – 0.8 mg kg−1 min−1) increased LVdP/dtmax (57±5%) and stroke volume (26±6%) with no effect on heart rate, LV filling pressure, and myocardial O2‐consumption, similar to N.

In MI, the effects of EMD 57033 (0.4 mg kg−1 min−1, IV) on stroke volume and LVdP/dtmax were maintained during treadmill exercise up to 85% of maximal heart rate, while heart rate was lower compared to control exercise (all P<0.05). In contrast, the effects of EMD57033 gradually waned in N at increasing intensity of exercise.

Compared to N, the cardiostimulatory effects of pimobendan (20 μg kg−1 min−1, IV) were blunted in MI both at rest and during exercise compared to N.

In conclusion, the positive inotropic actions of the Ca2+ sensitizer EMD 57033 are unmitigated in resting and exercising MI compared to N, while those of the mixed Ca2+‐sensitizer/phosphodiesterase‐III inhibitor pimobendan are blunted.

British Journal of Pharmacology (2001) 134, 553–562; doi:10.1038/sj.bjp.0704292
Anandamide induces cardiovascular and respiratory reflexesviavasosensory nerves in the anaesthetized rat
Authors: Smith Paula J W , McQueen Daniel S
Published: January 2005

We tested the hypothesis that sensory nerves innervating blood vessels play a role in the local and systemic regulation of the cardiovascular and respiratory (CVR) systems. We measured CVR reflexes evoked by administration of anandamide (86 – 863 nmoles) and capsaicin (0.3 – 10 nmoles) into the hindlimb vasculature of anaesthetized rats.

Anandamide and capsaicin each caused a rapid dose‐dependent reflex fall in blood pressure and an increase in ventilation when injected intra‐arterially into the hindlimb.

Action of both agonists at the vanilloid receptor (VR1) on perivascular sensory nerves was investigated using capsazepine (1 mg kg−1i.a.) a competitive VR1 antagonist, ruthenium red (1 mg kg−1i.a.), a non‐competitive antagonist at VR1, or a desensitizing dose of capsaicin (200 nmoles i.a.). The cannabinoid receptor antagonist SR141716 (1 mg kg−1i.a.) was used to determine agonist activity at the CB1receptor.

Capsazepine, ruthenium red, or acute VR1 desensitization by capsaicin‐pretreatment, markedly attenuated the reflex CVR responses evoked by anandamide and capsaicin (P<0.05; paired Student'st‐test). Blockade of CB1had no significant effect on the responses to anandamide.

Local sectioning of the femoral and sciatic nerves attenuated CVR responses to anandamide and capsaicin (P<0.05). Vagotomy or carotid sinus sectioning had no significant effect on anandamide‐ or capsaicin‐induced responses.

These data demonstrate that both the endogenous cannabinoid, anandamide, and the vanilloid, capsaicin, evoke CVR reflexes when injected intra‐arterially into the rat hindlimb. These responses appear to be mediated reflexlyviaVR1 located on sensory nerve endings within the hindlimb vasculature.

British Journal of Pharmacology(2001)134, 655–663; doi:10.1038/sj.bjp.0704296
Inhibition of interleukin‐12 production by auranofin, an anti‐rheumatic gold compound, deviates CD4+ T cells from the Th1 to the Th2 pathway
Authors: Kim T S , Kang B Y , Lee M H , Choe Y K , Hwang S Y
Published: January 2005

Interleukin‐12 (IL‐12) may play a central role in the development and progression of rheumatoid arthritis by driving the immune response towards T helper 1 (Th1) type responses characterized by high IFN‐γ and low IL‐4 production. In this study we investigated the effect of auranofin (AF), an anti‐rheumatic gold compound, on IL‐12 production in mouse macrophages and dendritic cells, and studied whether AF‐mediated inhibition of IL‐12 production could regulate a cytokine profile of antigen (Ag)‐primed CD4+ Th cells.

Treatment with AF significantly inhibited IL‐12 production in lipopolysaccharide (LPS)‐stimulated macrophages and also in CD40L‐stimulated dendritic cells. AF‐pretreated macrophages reduced their ability to induce IFN‐γ and increased the ability to induce IL‐4 in Ag‐primed CD4+ T cells. AF did not influence the cell surface expression of the class II MHC molecule and the costimulatory molecules CD80 and CD86.

Addition of recombinant IL‐12 to cultures of AF‐pretreated macrophages and CD4+ T cells restored IFN‐γ production in Ag‐primed CD4+ T cells.

The in vivo administration of AF resulted in the inhibition of IL‐12 production by macrophages stimulated in vitro with LPS or heat‐killed Listeria monocytogenes (HKL), leading to the inhibition of Th1 cytokine profile (decreased IFN‐γ and increased IL‐4 production) in Ag‐primed CD4+ T cells.

These findings may explain some known effects of AF including anti‐rheumatic effects and the inhibition of encephalitogenicity, and point to a possible therapeutic use of AF in the Th1‐mediated immune diseases such as autoimmune diseases.

British Journal of Pharmacology (2001) 134, 571–578; doi:10.1038/sj.bjp.0704298
Downregulation of vascular soluble guanylate cyclase induced by high salt intake in spontaneously hypertensive rats
Authors: Kagota Satomi , Tamashiro Akiko , Yamaguchi Yu , Sugiura Reiko , Kuno Takayoshi , Nakamura Kazuki , Kunitomo Masaru
Published: January 2005

Cyclic guanosine monophosphate (cyclic GMP)‐mediated mechanism plays an important role in vasodilatation and blood pressure regulation. We investigated the effects of high salt intake on the nitric oxide (NO) – cyclic GMP signal transduction pathway regulating relaxation in aortas of spontaneously hypertensive rats (SHR).

Four‐week‐old SHR and normotensive Wistar‐Kyoto rats (WKY) received a normal salt diet (0.3% NaCl) or a high salt diet (8% NaCl) for 4 weeks.

In aortic rings from SHR, endothelium‐dependent relaxations in response to acetylcholine (ACh), adenosine diphosphate (ADP) and calcium ionophore A23187 were significantly impaired by the high salt intake. The endothelium‐independent relaxations in response to sodium nitroprusside (SNP) and nitroglycerin were also impaired, but that to 8‐bromo‐cyclic GMP remained unchanged. On the other hand, high salt diet had no significant effects on the relaxations of aortic rings from WKY.

In aortas from SHR, the release of NO stimulated by ACh was significantly enhanced, whereas the production of cyclic GMP induced by either ACh or SNP was decreased by the high salt intake.

Western blot analysis showed that the protein level of endothelial NO synthase (eNOS) was slightly increased, whereas that of soluble guanylate cyclase (sGC) was dramatically reduced by the high salt intake.

These results indicate that in SHR, excessive dietary salt can result in downregulation of sGC followed by decreased cyclic GMP production, which leads to impairment of vascular relaxation in responses to NO. It is notable that chronic high salt intake impairs the sGC/cyclic GMP pathway but not the eNOS/NO pathway.

British Journal of Pharmacology (2001) 134, 737–744; doi:10.1038/sj.bjp.0704300
Adenosine‐mediated hypotension in in vivo guinea‐pig: receptors involved and role of NO
Authors: Nieri P , Martinotti E , Calderone V , Breschi M C
Published: January 2005

Adenosine produced a biphasic lowering of the mean BP with a drastic bradycardic effect at the highest doses. The first phase hypotensive response was significantly reduced by the nitric oxide (NO) synthase inhibitor L‐NAME.

The A2a/A2b agonist NECA produced hypotensive and bradycardic responses similar to those elicited by adenosine, which were not significantly modified by the A2b antagonist enprofylline.

The A2a agonist CGS 21680 did not significantly influence basal HR while induced a hypotensive response antagonized by the A2a selective antagonist ZM 241385, and reduced by both L‐NAME and the guanylate cyclase inhibitor methylene blue.

The A1 agonist R‐PIA showed a dose‐dependent decrease in BP with a drastic decrease in HR at the highest doses. The A1 selective antagonist DPCPX significantly reduced the bradycardic activity and also the hypotensive responses obtained with the lowest doses while it increased those obtained with the highest ones.

The A1/A3 agonist APNEA, in the presence of the xanthinic non‐selective antagonist 8‐pSPT, maintained a significant hypotensive, but not bradycardic, activity, not abolished by the histamine antagonist diphenhydramine.

The selective A3 agonist IB‐MECA revealed a weak hypotensive and bradycardic effect, but only at the highest doses.

In conclusion, in the systemic cardiovascular response to adenosine two major components may be relevant: an A2a‐ and NO‐mediated hypotension, and a bradycardic effect with a consequent hypotension, via atypical A1 receptors. Finally, an 8‐pSPT‐resistant hypotensive response not attributable to A3 receptor‐stimulation or to release of histamine by mastocytes or other immune cells was observed.

British Journal of Pharmacology (2001) 134, 745–752; doi:10.1038/sj.bjp.0704301