bluuebunny/crossref_metadata_2025 huggingface dataset:
The effects of cross‐drug preexposure on cocaine‐ and fluoxetine‐induced conditioned taste aversions
Authors: Serafine Katherine Marie , Riley Anthony Lee
Published: June 2021

Although cocaine‐induced CTAs are well documented, little is known about the basis for cocaine's aversive effects. To address a role of serotonin (5‐HT) in cocaine‐induced aversions, the present experiments used the cross‐drug preexposure design in which the effects of preexposure to fluoxetine (FLU), a selective 5‐HT transporter (SERT) inhibitor, were examined on aversions induced by cocaine (nonselective monoamine transport inhibitor) and the effects of cocaine preexposure were examined on FLU‐induced aversions. A FLU dose‐response function was established in male Sprague‐Dawley rats to determine a dose of FLU that would induce intermediate aversions comparable to those induced by 18 mg/kg cocaine (Experiment 1). Naïve rats were then exposed to FLU (10 mg/kg) prior to aversion conditioning with cocaine (18 mg/kg ; Experiment 2) and with cocaine (18 mg/kg) prior to aversion conditioning with FLU (10 mg/kg; Experiment 3). Although there was no effect of FLU preexposure on either cocaine‐ or FLU‐induced aversions, preexposure to cocaine attenuated aversions induced by itself and by FLU. One interpretation of this attenuation is that exposure to one drug produces cross‐tolerance to the aversion‐inducing effects of the second. These results suggest that these two compounds may induce aversions via a common mechanism, i.e., SERT inhibition. Supported by a Mellon Foundation grant to ALR.
Impulsivity and vulnerability to cocaine self‐administration in adult rhesus monkeys exposed to cocaine in utero
Authors: Hamilton Lindsey R , Wilson Whitney D , Nader Michael A
Published: June 2021

Impulsivity has been associated with increased likelihood to self‐administer (SA) drugs of abuse. In this study, male and female adult rhesus monkeys prenatally exposed to cocaine (n = 10) and controls (n = 10) were assessed across four measures of impulsivity (locomotor activity, novel object reactivity, delay discounting, and perseverative behavior). Each animal was ranked from most (20) to least (1) impulsive on each of the four measures and the average impulsivity ranking was determined. Male monkeys exposed to cocaine in utero were more impulsive than male controls; no differences were observed between the two groups of female monkeys. Next, monkeys were surgically implanted with an intravenous catheter and trained to respond on a fixed‐ratio 30 schedule of food presentations. When responding was stable, saline and increasing doses of cocaine were substituted for food. Male prenatally cocaine exposed monkeys were more likely to acquire cocaine SA at low doses compared to controls. Additionally, increased impulsivity predicted a greater vulnerability to self‐administer cocaine only in the male monkeys (r2 = 0.63, p = 0.006). The link between impulsivity and drug abuse suggests that males, but not females, exposed to cocaine in utero may be more vulnerable to stimulant abuse compared to controls. These findings support differential treatment strategies for male and female substance abusers. DA13370, DA25120.
Adolescent Maturation of D1 and D2 Dopamine Receptor Interactions
Authors: Dwyer Jennifer Buenzle , Martinez Lesly , Ganddini Gabriela , Perez Mayra , Leslie Frances M
Published: June 2021

Adolescence is a developmental period during which neuropsychiatric disorders often emerge. This onset is likely influenced by the continued maturation of mesocorticolimbic dopamine (DA) systems. Consistent with this development, adolescents respond uniquely to drugs that target DA systems, with blunted behavioral responses to indirect agonists like amphetamine. We have previously shown using direct agonists, that the functional sensitivity of D1 and D2‐like receptors changes with age. To evaluate age differences in D1/D2 behavioral interactions, adolescent and adult rats were given combinations of second messenger‐selective direct D1 and D2 agonists. Drug‐induced locomotor and stereotypic behaviors were measured, and brains were collected for analysis of immediate early gene (IEG) expression via in situ hybridization for c‐fos and arc mRNA. Behavioral data suggests that D1/D2 interactions are immature during adolescence, as adults show synergistic behavioral responses to D1/D2 combinations while adolescents do not. IEG analysis suggests age differences in the neural circuitry underlying these behaviors. Interregional IEG correlation further suggests that dopaminergic drugs alter functional connectivity in the limbic system, with distinct effects in adolescence. These data have important implications for pharmacotherapy design and use in this population.

Support: DA19138, PhRMA Foundation
Serotonergic mechanisms underlying nicotine induced alterations in the dopamine system
Authors: Dao Jasmin M , Salgado Patricia , Lee Jenny H , Loughlin Sandra E , Belluzzi James D , Leslie France M
Published: June 2021

Clinical studies have shown that adolescent smoking is associated with a variety of health risk behaviors including the development of anxiety disorders, high‐risk sexual behavior and other types of illicit drug use. Neurotransmitter systems implicated in the modulation of these behaviors are not fully mature during this period, and can be critically altered by external stimuli, such as smoking. Given the importance of dopamine (DA) in central reward functions, the purpose of this study was to determine whether nicotine induces age‐specific alterations in mesocorticolimbic DA function. Using rat as an animal model, we found that four‐day pretreatment with a low dose of nicotine (60μg/kg; i.v.), equivalent to that found in 1‐2 cigarettes, significantly altered locomotion induced by direct DA agonists. Behavioral studies show that locomotor activity and erectile response induced by quinpirole (0.4mg/kg; i.p.) was increased after adolescent nicotine exposure, an effect not seen in adults. Neurochemical studies and double‐labeling in situ hybridization studies showed unique changes in limbic, sex and stress‐related systems. WAY 100,635, a selective 5‐HT1a receptor antagonist, blocked the enhancement of quinpirole‐induced locomotion when co‐administered with daily nicotine treatment. Pretreatment with 8‐OH‐DPAT, a 5‐HT1A agonist (10 μg/kg, i.v.), mimicked nicotine's pretreatment effects indicating that adolescent nicotine alters the 5‐HT system, critically impacting central reward pathways These findings provide evidence that nicotine administration causes unique neural adaptations within adolescence, and elucidates novel pathways altered by nicotine that may increase vulnerability to high‐risk behaviors.
Discriminative stimulus effects of the positive GABAA receptor modulator midazolam in rhesus monkeys are not altered by non‐GABAergic drugs
Authors: Bai Xiang , France Charles P. , Gerak Lisa R.
Published: June 2021

The incidence of exclusive benzodiazepine abuse is low; however, benzodiazepine abuse is very high in polydrug abusers, particularly those who also abuse opioids, stimulants or club drugs. This study investigated whether drugs from pharmacological classes representative of those other abused drugs alter the discriminative stimulus effects of midazolam, a positive γ‐aminobutyric acidA (GABAA) receptor modulator acting at benzodiazepine sites. Three rhesus monkeys discriminated 0.56 mg/kg of midazolam while responding under a fixed ratio 10 schedule of food presentation. Dose‐effect curves were determined for midazolam alone and in the presence of morphine (opioid receptor agonist), amphetamine (stimulant), dizocilpine (N‐methyl‐D‐aspartic acid receptor antagonist), or γ‐butyrolactone (club drug). Doses of midazolam larger than 0.32 mg/kg produced >80% midazolam‐lever responding. When administered alone, the other drugs did not produce midazolam‐lever responding, and when administered in combination with midazolam, they did not alter the midazolam dose‐effect curve. The results suggest that increased abuse of benzodiazepines in polydrug abusers is not due to changes in behavioral effects of benzodiazepines caused by other drugs of abuse. Supported by USPHS DA009157 and DA17918 (CPF).
Development of behavioral and neuronal responses to social stress: early adolescence as a sensitive period
Authors: Bingham Brian , McFadden Kile , Zhang Xiaoyan , Bhatnagar Seema , Beck Sheryl , Valentino Rita
Published: June 2021

The research goal was to determine whether social stress differentially impacts on behavior and brain physiology during critical windows in adolescent development. Early adolescent (EA), mid‐adolescent (MA) and adult rats were exposed to 7 days of a social stressor, the resident‐intruder paradigm. Social stress had divergent effects on behavior in EA and adult rats, promoting active coping behaviors in EA rats and decreasing active coping behaviors in adult rats as determined by the defensive burying test and response to swim stress. Mid‐adolescent rats exposed to social stress showed no changes in the behavioral endpoints. Effects of social stress in EA rats was not mimicked by chronic restraint stress. Because the locus coeruleus (LC)‐norepinephrine system has been implicated in certain active coping behaviors, LC neuronal activity was recorded in EA rats exposed to social stress and matched controls. LC spontaneous firing rates were higher in EA rats exposed to social stress. Moreover, intra‐LC infusion of the CRF antagonist, DPheCRF12‐41, inhibited LC neurons of stressed rats but not controls. These data suggest that exposure to social stress in early adolescence promotes tonic secretion of CRF into the LC to activate this system. This CRF‐induced activation of the LC‐norepinephrine system may underlie the promotion of active‐coping behaviors noted in socially stressed EA rats.

MH058250
Cytokine profiling of chronic social defeat in mice
Authors: Wong Keith , Tan Julia , Cachet Jonathan , Goodspeed Jason , Suciu Chris , Roy Sudipta , Grimes Chelsea , Rosenberg Michael , Jackson Andrew , Tien David , Elegante Marko , Bartels Brett , Elkhayat Salem , Chung Amanda , Gaikwad Siddharth , Duncan Ashley , Kadri Ferdous , Chung Kyung Min , Stewart Adam , Kalueff Allan V.
Published: June 2021

Although the biological basis of affective disorders remains unclear, recent clinical and experimental data implicate various cytokines in anxiety and depression pathogenesis. Here we analyze cytokine expression and correlate it with affective behavioral phenotypes using chronic social defeat stress in C57/BL6/J male mice. Open field and tail suspension tests assessed anxiety and depression in the mice experiencing 20 days of daily 15‐min social confrontations (Kudryavtseva et al., 2002, 2009). Serum samples were collected to assess circulating cytokine levels. Our data demonstrate that pro‐inflammatory cytokines (IL‐6, IL‐7, IL‐15, and VEGF) are elevated in serum of chronic loser mice. We also found a trend towards elevation of anti‐inflammatory cytokines (IL‐4 and IL‐10) in the winner mice. Overall, our results suggest that animal anxiety/depression‐like behaviors correlate with specific cytokine profiles. These findings may foster the search for new therapeutic and preventative strategies (against affective disorders) that target central and peripheral cytokine pathways.
Behavioral inhibition in rats: a model to examine mechanisms underlying the risk to develop anxiety and depression
Authors: Qi Chao , Roseboom Patrick H. , Nanda Steven A. , Lane Jason C. , Speers James M. , Kalin Ned H.
Published: June 2021

Behavioral inhibition (BI) is an adaptive defensive response to threat that has been evolutionarily conserved. Extreme BI in childhood is a temperamental disposition for the development of psychopathology. Our goal was to develop a rodent model of BI that is elicited by predator exposure. The ferret exposure test consists of placing a rat in a housing cage in the ferret colony room for 15 min and rating behavior for BI. We show that predator‐induced BI is stable across development when measured in rats (n = 37) in adolescence and again in adulthood (r = 0.475, p < 0.01). BI is reduced by the anxiolytic diazepam (1 mg/kg, 30 min pretreatment; n = 8/group; p < 0.05), and BI is associated with heightened paraventricular nucleus of the hypothalamus (PVN) and CA3 activity as measured by expression of the immediate early gene homer1a (r = 0.379, p < 0.05; r = 0.309, p < 0.05 respectively). Finally, we show that rats with high levels of BI display disruptions in appetitive behavior as a consequence of prior threat exposure compared to rats with low levels of BI (p < 0.001, n = 7 and 8). Taken together, these findings show that rodent BI is a stable, trait‐like characteristic that is associated with maladaptive responding. This model can be useful in identifying the molecular underpinnings for the childhood risk to develop psychiatric illness. This work was supported by NIH grant MH43454, Meriter Hospital and UW HealthEmotions Research Institute.
Identification of new G protein‐coupled receptor (GPCR) target(s) for the regulation of cardiac fibrosis
Authors: Snead Aaron Nathan , Insel Paul A
Published: June 2021

Cardiac fibrosis occurs in advanced age, after acute injury (e.g. myocardial infarction) and in disease states (e.g. diabetes mellitus). Fibrosis is characterized by the transformation of fibroblasts to myofibroblasts, which contribute to the excessive accumulation of collagen‐rich extracellular matrix resulting in reduced cardiac function. In order to identify novel therapeutic targets for cardiac fibrosis we used an unbiased approach (GPCR RT‐PCR array) to define the most abundantly expressed GPCRs that may function to regulate the fibroblast/myofibroblast transformation in rat cardiac fibroblasts (rCFs). Pharmacological analyses were then used to validate the most abundant of these receptors as a functional target. We identified protease‐activated receptor 1 (PAR1) as the most abundant GPCR in rCFs. Subsequent studies revealed that thrombin activation of PAR1 elevates (with distinct kinetics) the expression of a variety of known profibrotic markers in rCFs. Thrombin functions via at least two distinct post‐receptor mechanisms to regulate fibroblast/myofibroblast transformation, one of which is dependent on the production of the atypical second messenger sphingosine‐1‐phosphate. Ours are the first data to define the level of expression of GPCRs in rCFs; the results provide a proof‐of‐principle that such an approach can identify novel disease‐relevant GPCR targets. Supported by NIH.
Stimulation of [35S]GTPγS binding by GABAB receptor agonists and positive modulators in different mouse brain regions
Authors: Advani Tushar , Hensler Julie G , Cheng Kejun , Rice Kenner C , Koek Wouter
Published: June 2021

GHB is used to treat narcolepsy, but is also abused. Its mechanism of action likely involves GABAB receptors because it shares many behavioral effects with the GABAB receptor agonist baclofen. To further examine the role of these receptors, effects of GHB in different mouse brain regions were compared with those of baclofen and the GABAB receptor positive modulators CGP7930 and BHFF. Agonist‐stimulated [35S]GTPγS binding was measured using quantitative autoradiography to examine drug effects in prefrontal and anterior cingulate cortex, and in hippocampus (CA1, CA2/3, dentate gyrus). Baclofen concentration‐dependently stimulated [35S]GTPγS binding in hippocampal and cortical regions. GHB did not stimulate [35S]GTPγS binding in any region examined. CGP7930 and BHFF stimulated [35S]GTPγS binding in all regions examined. The maximal effects of CGP7930 were smaller than those of baclofen, but those of BHFF were similar to those of baclofen. A concentration of the GABAB receptor antagonist CGP35348 that blocked the effects of baclofen did not appear to attenuate the effects of CGP7930 and BHFF. These results suggest that GHB has lower efficacy than baclofen at GABAB receptors. The results further suggest that the positive GABAB receptor modulators CGP7930 and BHFF exert agonist activity that may not result from potentiation of endogenous GABA at GABAB receptors. Supported by MH052369(JGH), DA15692(WK).
Modulation of the cardiac β‐adrenergic contractile response by adenosine A1 and A2a receptors: evidence for receptor interactions
Authors: Chandrasekera P. Charukeshi , McIntosh Victoria J. , Cao Frank , Lasley Robert D.
Published: June 2021

Mammalian myocardium expresses four adenosine receptor (AR) subtypes (A1, A2a, A2b, & A3). A1AR inhibits the β‐adrenergic receptor‐mediated positive inotropy, but the role of A2aAR in altering cardiac contractility is controversial. The objective of this study was to determine the effects of A2aAR on A1AR‐anti‐adrenergic response. Experiments were conducted in wild type (WT) and A2a‐knock‐out (KO) isolated perfused mouse hearts by measuring left ventricular systolic pressure (LVSP), +dP/dt, & −dP/dt. A2aAR agonist CGS‐21680 alone did not alter contractility or the isoproterenol (ISO)‐induced contractile response. Effects of 200nM CCPA pre‐treatment on 2 nM ISO are shown below (n≥4, data expressed as % increase from baseline, *p<0.05 CCPA+ISO vs ISO, #p<0.05 KO‐CCPA+ISO vs WT‐CCPA+ISO):

LVSP
+dP/dt
−dP/dt

WT‐ISO
76±4
109±7
133±7

WT‐CCPA+ISO
35±4*
48±4*
56±5*

KO‐ISO
86±8
129±10
154±9

KO‐CCPA+ISO
16±3*#
19±4*#
31±5*#

Reversing the sequence of agonist infusion (i.e. ISO, then CCPA) significantly reduced the A1AR anti‐adrenergic response. These results indicate that anti‐adrenergic effects of A1AR are potentiated in the absence of A2aAR, and that A1AR pre‐activation is necessary to elicit the full anti‐adrenergic response. Since A2aAR only alters contractility indirectly, these data suggest that receptor cross‐talk/direct receptor interactions may account for the observed contractile response.
Vascular KCNQ Potassium Channels as Therapeutic Targets in Cerebral Vasospasm
Authors: Mani Bharath Kumar , Brueggemann Lioubov I , Byron Kenneth L
Published: June 2021

Following subarachnoid hemorrhage, chronic narrowing of cerebral arteries, including basilar artery, leads to reduction of blood flow to the brain and results in high morbidity and mortality. During vasospasm, cerebrospinal fluid levels of putative spasmogens, endothelin‐1, serotonin, and vasopressin (AVP) are elevated. However the mechanisms by which they produce vasospasm are not clear. We recently found that AVP suppresses KCNQ (Kv7) potassium currents in mesenteric artery myocytes. This depolarizes the membrane leading to activation of voltage‐gated Ca2+ channels and arterial constriction. Here we propose that KCNQ channels are also central mediators of cerebral vasospasm. We used patch clamp electrophysiology in myocytes and pressure myography in isolated artery segments to characterize the role of KCNQ channels in rat basilar artery vasospasm. The spasmogens all suppress KCNQ currents in basilar artery myocytes. Flupirtine, a KCNQ channel opener, reversed the suppression of KCNQ currents and basilar artery constriction induced by the spasmogens. Celecoxib, a dual KCNQ channel opener and Ca2+ channel blocker was more effective than nimodipine in dilating basilar arteries. These findings provide the first evidence that KCNQ channels are expressed and functional in basilar artery and suggest that these channels are potential novel targets for therapeutic intervention in cerebral vasospasm. This work was supported by the NIH (R01‐HL089564 to KLB) and the AHA (09PRE2260209 to BKM)
Effect of beta‐adrenergic stimulation on the rapid delayed rectifier K current in canine ventricular cardiomyocytes
Authors: Nanasi Peter
Published: June 2021

Activation the PKA pathway has been reported to suppress the cardiac rapid delayed rectifier K current (IKr) in expression systems, however, the phosphorylation‐dependent regulation of IKr is still controversial. In the present study, therefore, the effects of activators and inhibitors of the PKA and PKC pathways were studied on IKr in canine ventricular myocytes using the whole cell patch clamp technique. Superfusion with isoproterenol (ISO), forskolin, or intracellular perfusion with PKA activator cAMP analogues (cAMP, 8‐Br‐cAMP, 6‐Bnz‐cAMP) increased IKr significantly by 30–50 %. Inhibition of PKA (by H‐89) and PKC (by GF‐109203X or chelerythine) decreased IKr significantly by 70–90 %. The IKr stimulator effect of ISO was fully eliminated by H‐89, chelerythine, and PKA activator cAMP analogues – but not by the EPAC activator 8‐pCPT‐2′‐O‐Me‐cAMP. The results indicate that beta‐adrenergic stimulation increases IKr in canine ventricular cells, and this effect involves the activation of both PKA and PKC.

Financial support was provided by the Hungarian Research Fund (OTKA‐K68457).
Molecular Expression and Distribution of Calcium‐Activated Chloride Channel TMEM16A in Mouse Heart
Authors: Zhang Mi , Yoon Carol , Kuhn Andrew R. , Dai Yanping , Ye Linda , Singer Cherie A. , Burkin Dean J. , Duan Dayue Darrel
Published: June 2021

Background

Calcium‐activated Cl− channels (CaCCs) are important in many cellular and physiological functions and in certain cardiac diseases. But the molecular identity of CaCCs in the heart is still elusive. Recent evidence suggests TMEM16A as the gene encoding the pore subunit of CaCCs. In this study, the molecular expression and tissue distribution of TMEM16A in mouse heart were quantitatively analyzed.

Methods

Total RNA was isolated from atria and ventricles of mouse heart (C57/BL6) and subjected to a quantitative real‐time PCR and Western blot analysis. TMEM16A mRNA and protein expression was calculated relative to 18S rRNA and GAPDH expression, respectively, within the same tissues.

Results

The transcriptional level of TMEM16A as estimated by mRNA ratio of TMEM16A/18s rRNA was highest in the left atrium (2.13±1.5, n=3), followed by right atrium (1.31±0.38), right ventricle (0.11±0.01), and left ventricle (0.051±0.004). Immunoblots of atrial and ventricular wall protein extracts demonstrated TMEM16A immunoreactivity at 113 kDa. The protein expression levels of TMEM16A were consistent with the same tissue‐distribution pattern as the transcriptional levels.

Conclusion

These results suggest that TMEM16A is heterogeneously expressed in mouse cardiac tissues, which may be responsible for the different functional activity of CaCCs in different tissues of the heart. (Supported by NCRR P‐20 RR‐15581)
Two intracellular helices of G‐protein coupling receptors as transducer‐attaching entities
Authors: Parker Steven L , Parker Michael S
Published: June 2021

The C‐terminal tract in the third intracellular (ic3) domain, and the juxtamembrane tract in the fourth intracellular (ic4) domain of G‐protein coupling receptors are known to interact with G‐proteins. Both segments show considerable conservation of sequence and size. These tracts have 12–17 residues, large content of basic sidechains, and high potential helicity. This is found regardless of domain size (which can exceed 400 residues), or the preferred G‐protein alpha subunit, and should represent a designated ability to interact with transducers. Association of either tract with transducers could be enhanced by restricted mobility (due to interaction with the bilayer) and broadside orientation, and could involve especially the long C‐terminal anion sidechain‐rich stretches in G‐protein subunits. Both tracts also show little phosphorylation compared to the rest of their domains, indicating a low processing by kinases and phosphatases, and low involvement in disassembly of transductional complexes. Helix 8 could perform as primary G‐protein partner, in the stable precoupling of receptors and G‐proteins as well as in GTPase activation upon agonist attachment. This is evidenced by effect of pertussis toxin on receptor/G‐protein heteropentamers. The ic3 C‐terminal helix could assist G‐protein coupling, and is widely known to perform in transducer activation.
Characterization of a novel Rab11 binding domain (RBD) within the human prostacyclin receptor that is regulated by palmitoylation
Authors: Kinsella B Therese , Mulvaney Eamon P , Reid Helen M
Published: June 2021

The human prostacyclin receptor (hIP) undergoes agonist‐induced internalization and subsequent recycling involving a direct physical interaction with Rab11a. That interaction localizes to a putative Rab11 binding domain (RBD) within the carboxyl‐terminal (C)‐tail of the hIP. Herein, we sought to define the structural determinants of the RBD and examine the possible role of palmitoylation therein in regulating trafficking of the hIP. Through complimentary approaches in yeast and mammalian cells, the RBD was defined as a 14 residue domain, proximal to transmembrane 7 domain, that is structurally organised into an α‐helix 8 (α‐H8) domain adjacent to the palmitoylated residues at Cys308‐ Cys311. Mutational and metabolic labelling studies determined that agonist‐mediated de‐acylation at Cys309 > Cys308 and palmitoylation at Cys311 may dynamically position α‐H8 of the hIP in proximity to Rab11a, to regulate its intracellular trafficking in response to agonist‐stimulation. The identification of a functional role for α‐H8, a common feature of G‐protein coupled receptors, as an RBD as exemplified herein for the hIP is likely to have broader significance for the receptor superfamily. Funded by SFI.
Attenuation of Methylmercury‐induced Neurotoxicity by N‐methyl‐D‐aspartate Antagonist, MK801 in SH‐SY5Y Cells
Authors: Tsai Jeffrey , Petroni Daniel H , George William J
Published: June 2021

Methylmercury (MeHg), a well known neurotoxin, has been reported to alter glutamate homeostasis in the neuronal environment resulting in excitotoxicity. The present study was conducted to investigate whether, and if so, under what conditions, MeHg induced excitotoxicity may be attenuated by N‐methyl‐D‐aspartate (NMDA) receptor blockade. A cell viability tissue culture assay was utilized to study the effects of MeHg, glutamate (glu) and a non‐competitive NMDA antagonist, MK‐801 on a human neuroblastoma cell line, SH‐SY5Y. Treatment of cells with MeHg (50nM) and glu (1mM) alone demonstrated a decrease of cell viability from control of 64%±6.5 and 79%±8.5, respectively. When glu and MeHg were added to cells in combination there was a 45% decrease in cell viability (p<0.005 n=3), as compared to treatment of glu alone. MK‐801 (4μM) significantly protected the cells from the effects of glu alone (p<0.005 n=3) as well as from combination treatments with glu and MeHg (p<0.0001 n=3). This study indicates that MeHg may induce an increase in number of NMDA receptors on the cell surface of neuroblastoma cells, and thus enhance the toxicity of glutamate. This apparent blockade of NMDA receptor with MK801 demonstrated a significant attenuation of the toxicity of both MeHg and glutamate. These results support the hypothesis that NMDA receptor antagonists may provide a potential treatment for MeHg‐induced neurological diseases.
Identification of novel inhibitors of human equilibrative nucleoside transporter 4
Authors: Wang Chunmei , Buolamwini John
Published: June 2021

To investigate the interaction of human nucleoside transporter 4 (hENT4) with dipyridamole analogues for the identification of novel needed potent and selective inhibitors. hENT4 was cloned and stably expressed in PK15NTD cells. A series of dipyridamole analogues synthesized in our laboratory were tested in this newly established hENT4 expressing system as inhibitors of [3H]uridine uptake.. Comparative molecular field analyses three‐dimensional quantitative structure‐activity relationship (3D‐QSAR) modeling was used to rationalize activity and selectivity. Of 73 dipyridamole analogues evaluated, 50 compounds were found to inhibit hENT4 with moderate to high potency (≥30%). About one fourth of compounds inhibited hENT4 with higher potencies than dipyridamole. The most potent compound (WL‐30) displayed an IC50 of 74.4 nM, making it about 38 times more potent than dipyridamole (IC50 = 2.8 μM). The structure‐activity relationships and insights gained into structural determinants of transporter inhibitory potency and selectivity relative to hENT1 and hENT2, by means of 3D‐QSAR modeling of will be presented. The hENT4 inhibitors identified in this study are novel and the most potent inhibitors reported to date, and should serve as useful pharmacological/biochemical tools and/or potential leads for ENT4‐based therapeutics. Supported by NIH grants CA125850, AI065372 and HL095002.
Increased Vascular Generation of Thromboxane A2: an Initiating Condition for Microvascular Rarefaction in Obese Zucker Rats?
Authors: Goodwill Adam G. , Stapleton Phoebe A. , Frisbee Stephanie J. , James Milinda E. , Frisbee Jefferson C.
Published: June 2021

Our studies have demonstrated that rarefaction of the skeletal muscle microcirculation in OZR with evolution of the metabolic syndrome is well predicted by a chronic reduction to vascular nitric oxide (NO) bioavailability. However, prior to any loss in NO, skeletal muscle vessels from OZR exhibit a shift in arachidonic acid metabolism, with an increased production of TxA2 over PGI2. Temporal analyses indicate that this early increase in vascular TxA2 production/action is associated with an initial early rarefaction of 5–7%. If NO bioavailability is maintained (TEMPOL) after the initial increase in TxA2 and microvessel loss, the total rarefaction magnitude is blunted. Alternately, if actions of TxA2 are chronically inhibited, the initial rarefaction never develops and the subsequent secondary pulse (from NO reductions) is strongly muted. Chronic treatment with pentoxyfillene (to prevent TNF‐α production) or HMG CoA reductase inhibitors (atorvastatin/simvastatin) prevented much of the secondary pulse of rarefaction (associated with maintenance of NO bioavailability), yet did not alter either the shift in TxA2 production or the initial rarefaction. These results suggest that microvessel rarefaction in OZR represents the integration of an early mild vessel loss that is dependent on TxA2, which creates an environment for the extensive rarefaction that is dependent on low NO bioavailability. (NIH DK64668)
Increased gracilis artery medial volume and inner diameter following chronic reduction of blood flow
Authors: Gruionu Gabriel , Hoying James B. , Secomb Timothy W.
Published: June 2021

Vascular networks respond to chronic alterations in blood supply by structural remodeling. A previous study showed that the inner diameter of the mouse gracilis artery increased transiently with a peak at 14 days following a permanent reduction in blood flow. Here, the objective was to investigate whether the diameter increase is accompanied by growth of medial tissue. Blood flow was reduced by removing a portion of the saphenous artery, one of the two blood supplies of the gracilis artery. After 7, 14, 21, 28 and 56 days, the vasculature was perfused with India ink for diameter measurements, and processed for immune cytochemistry to mark smooth muscle cells (SMC) and nuclei of SMC and endothelial cells (EC). In the two thirds of the vessel length closest to the injury, the inner diameter was significantly larger at days 7–28. The cross‐sectional medial area was significantly larger at days 21 and 28. The ratio of internal diameter to medial thickness (an index of circumferential wall stress) and the ratio of medial area to number of SMC nuclei were significantly higher only at day 14. The results are consistent with the concept that outward remodeling causes increased circumferential wall stress, which stimulates vessel wall adaptation by a combination of SMC hypertrophy and proliferation to restore circumferential stress to near its initial level. Supported by HL34555 and HL63732.
Renal Microvascular Disease and the Responses to Renal Revascularization
Authors: Chade Alejandro R , Kelsen Silvia , Fails Fredrick , Bailey James
Published: June 2021

Renal angioplasty and stenting (PTRA) is the most frequent therapeutic approach for renal artery stenosis (RAS). However, renal function recovers in only 30% of the cases. The causes of these poor outcomes are still unknown. We hypothesize that preserving the renal microcirculation in the stenotic kidney will improve the responses to PTRA.

RAS was induced in 12 pigs. In 6 pigs, VEGF‐165 was single‐infused intra‐renally (RAS+VEGF, 0.05 ug/kg). Additional animals were used as normal controls (n=6), and all were followed for 10 weeks. Single‐kidney function was assessed in vivo using ultra‐fast CT after 6 weeks of RAS. Then, RAS and RAS+VEGF underwent PTRA, VEGF repeated, and CT studies performed 4 weeks post‐PTRA. Pigs were then euthanized, the stenotic kidney removed, renal microvascular (MV) architecture reconstructed ex‐vivo using 3D micro‐CT, and renal fibrosis quantified.

Degree of RAS and hypertension were similar in RAS and RAS+VEGF. Renal function and MV density were decreased in RAS but improved in RAS+VEGF.

PTRA largely resolved RAS. Improvements in blood pressure and renal function were greater in RAS+VEGF+PTRA than in RAS+PTRA, accompanied by a 29% increase in MV density and decreased fibrosis.

Preservation of the MV architecture and function in the stenotic kidney improved the responses to PTRA, indicating that renal MV rarefaction plays a role in determining the responses to PTRA. This study indicates that the damage and early loss of renal MV is an important determinant of the progression of renal injury in RAS and instigates often irreversible damage.

Support: AHA‐SDG‐0830100N
Oxidative Stress and Endothelial Dysfunction in Pulmonary Arteries of Aged Rats
Authors: Ungvari Zoltan , Ballabh Praveen , Podlutsky Andrej , Csiszar Anna
Published: June 2021

Aging in the systemic circulation is associated with generalized endothelial dysfunction and increased oxidative stress, which are thought to contribute to the increased morbidity and mortality of cardiovascular diseases in the elderly. Previous studies have shown that pulmonary artery pressure and vascular resistance increase with normal aging in humans, yet age‐related functional and phenotypic changes in the pulmonary arteries have not been characterized. To determine whether in the pulmonary circulation aging elicits endothelial dysfunction and oxidative stress, isolated pulmonary arteries of young (3 m.o.) and aged (28 m.o.) F344 rats were compared. We found that aging in rat pulmonary arteries is associated with impaired acetylcholine‐induced relaxation and vascular oxidative stress. Endothelial dysfunction in the aged pulmonary vessels is reversed by inhibition of NAD(P)H oxidase. Expression of gp91phox, Nox‐1 and Nox‐4 tends to increase in aged vessels. In pulmonary arteries of aged rats expression of eNOS, Cu,Zn‐SOD, Mn‐SOD and glutathione peroxidase is unaltered, whereas expression of catalase is significantly decreased. Our results suggest that aging is associated with oxidative stress and endothelial dysfunction in the pulmonary arteries, which may contribute to the age‐related functional alterations in the pulmonary circulation.
Vascular Gap Junction Cx37 Uncoupling By Tumor Necrosis Factor Is Dependent On ZO‐1
Authors: Ouellette Yves , Ermilov Leonid G. , Sieck Gary C.
Published: June 2021

Regulation of gap junctional intercellular communication plays a very important role in many physiological and pathophysiological processes. Our hypothesis is that tumor necrosis factor (TNF) will decrease gap junction dependent cell‐to‐cell communication of vascular connexin by disturbing connexin‐cytoskeleton interactions. Transformed HeLa cells expressing vascular connexin 37 were used in these experiments. After 1 hour, TNF (20 ng/ml) treatment resulted in near total loss of dye‐coupling (Alexa Fluor‐480) in HeLaCx37 (p<0.02, n=16) and remained constant up to 2 hours. siRNA‐mediated knockdown of ZO‐1 restored dye coupling. TNF caused a significant increase in detergent solubility of Cx37. ZO‐1 was co‐IP with Cx37 only after TNF treatment, suggesting that TNF induces a ZO‐1 and Cx37 interaction. Actin was co‐IP with Cx37 but TNF did not affect this association. N‐cadherin was not co‐IP with Cx37. Immunofluorescence double labeling for Cx37/ZO‐1 and Cx37/actin confirm the co‐IP experiments. TNF reduces gap junction coupling of Cx37 when expressed alone in epithelial cells. TNF mediates Cx37 interaction with ZO‐1 but not actin. N‐cadherin does not interact with Cx37. TNF may affect Cx37/ZO‐1 interaction resulting in reduced dye coupling.
PPARs protect against HIV‐induced MMPs overexpression by caveolae‐associated ERK and Akt signaling
Authors: Huang Wen , András Ibolya E , Hennig Bernhard , Toborek Michal
Published: June 2021

Tight junctions (TJs) are the main structural and functional elements that regulate the blood‐brain barrier (BBB) integrity. Our previous data indicated that exposure of human brain microvascular endothelial cells (HBMEC) to human immunodeficiency virus‐1 (HIV) resulted in a decreased expression of TJs. Overexpression of peroxisome proliferator‐activated receptor (PPAR)‐α and γ or exposure to PPAR agonists attenuated HIV‐1‐mediated dysregulation of TJs partly by inhibition of matrix metalloproteinase (MMP) activity. Evidence indicates that MMP expression is regulated by the Ras‐ERK and PI3K‐Akt signaling. In addition, MMPs are closely associated with caveolae. In the present study, we hypothesize that PPARs protect against HIV‐induced MMP overexpression by attenuating cellular oxidative stress and downregulation of redox‐regulated transcription. Exposure to HIV induced protein expression of caveolin‐1, Ras, pERK and pAkt in HBMEC; however, these effects were attenuated in PPAR overexpressing cells. Silencing of caveolin‐1 further protected against HIV‐mediated activation of Ras, pERK and pAkt. These effects were confirmed in cells pretreated with PPAR agonists fenofibrate and rosiglitazone. HIV‐induced MMP promoter activity was inhibited by the Ras‐ERK or PI3K‐Akt inhibitors or by silencing of caveolin‐1. The present data indicate that the Ras‐ERK, PI3K‐Akt signaling and caveolin‐1 are involved in the mechanisms of PPAR‐mediated protection against HIV‐induced MMP expression.
β‐adrenergic stimulation contributes to maintenance of endothelial barrier functions under resting conditions
Authors: Spindler Volker , Waschke Jens
Published: June 2021

Protective effects of cAMP on endothelial barrier properties are well established. We tested the hypothesis whether β‐adrenergic receptor‐induced cAMP increase is involved in maintenance of endothelial barrier function. Inhibition of β‐receptors by propranolol increased hydraulic conductivity in Ringer‐perfused rat postcapillary venules. Similarly, in human cultured microvascular endothelial cells (HDMEC), propranolol reduced transendothelial electrical resistance (TER) and cAMP levels, and induced intercellular gap formation. In contrast, activation of adrenergic receptors by epinephrine both increased cAMP and TER and resulted in linearized VE‐cadherin immunostaining. Forced formation of cAMP by adenylate cyclase activator forskolin but neither epinephrine nor phosphodiesterase inhibition by rolipram prevented propranolol‐induced TER reduction and VE‐cadherin reorganization. Taken together, our data indicate that β‐adrenergic receptor‐mediated generation of cAMP contributes to maintenance of endothelial barrier properties. Furthermore, in addition to epinephrine from systemic circulation locally generated β‐receptor agonists likely participate in this process. Thus, impaired endothelial barrier function may contribute to formation of lung and ankle edema observed in patients treated with β‐blockers. DFG SFB688, A4
Differences in expression and function of ryanodine receptors between arteries and arterioles in the mouse
Authors: Lange Erica , Boerman Erika M. , Segal Steven S. , Jackson William F.
Published: June 2021

Ryanodine receptors (RyR) in smooth muscle cells (SMC) underlie Ca2+ sparks and Ca2+‐induced‐Ca2+ release, contributing to the regulation of myogenic tone in arteries. In contrast, RyR are silent and are not involved in Ca2+ signals in SMC or myogenic tone in hamster arterioles. The purpose of the present study was to investigate the role of RyR in vessels of male C57BL/6 mice. We tested the hypothesis that differences in RyR isoform expression contribute to differences in RyR function in iliac feed arteries (FA) vs. cremaster arterioles (CA). In cannulated pressurized (80 cm H2O) vessels (34 – 37 °C), ryanodine (10 μM) constricted FA (from 53 ± 4 to 40 ± 3 μm; n = 5, p < 0.05) but not CA (24 ± 2 μm vs. 22 ± 2 μm; n = 5, p > 0.05). Transcript levels for RyR2 in freshly isolated SMC assessed by q‐RT‐PCR were 2.1 ± 0.3 ‐fold higher in FA vs. CA (n = 7, p < 0.05), while those for RyR3 were 0.6 ± 0.1 in FA vs. CA (n = 6, p < 0.05). RyR1 transcripts were not detected in SMC from FA or CA. Immunofluorescence with an anti‐RyR1/2 antibody showed clustered labeling in FA SMC (n = 6 SMC) suggestive of RyR2 expression in endoplasmic reticulum. In contrast, more uniform cytoplasmic staining in CA SMC (n = 13 SMC) suggested non‐organelle‐specific staining. These data support our hypothesis and indicate fundamental differences in SMC RyR expression and function between arteries and arterioles across rodent species. Supported by NIH HL086483 & AHA Fellowship 0815778G.
Endothelial TNFR1 shedding by mitochondria
Authors: Rowlands David John , Lindert Jens , Das Shonit , Bhattacharya Jahar
Published: June 2021

Lung inflammation critically results from the binding of the proinflammatory cytokine, tumor necrosis factor alpha (TNFα) to TNFα receptor 1 (TNFR1). Although the binding sheds TNFR1 in leukocytes, responses in lung endothelial cells (EC) are not known. To determine TNFR1 dynamics in lung EC, we considered the role of the mitochondrial Rieske Iron‐Sulphur protein (RISP). We gave tail‐vein injections of liposome‐conjugated, fluorescent siRNA that were specific (siRISP) or non‐specific (siNS) for RISP. To quantify responses in real‐time, after 48 hours we imaged single microvessels of inflated isolated, blood‐perfused mouse lungs by live confocal microscopy. By micropuncture, we infused microvessels with a fluorophore‐conjugated TNFR1 mAb followed by buffer wash. The residual fluorescence denoted EC TNFR1 expression. TNFR1 expression was stable for >1 hr. Line scan analyses revealed no correlation between expressions of TNFR1 and siRISP. In siNS‐treated lungs, TNFα infusion (1 ng/ml) globally decreased TNFR1 fluorescence by >50% within 30 minutes (P<0.05, n=3). By contrast, siRISP expression blocked the decrease (P<0.05). We conclude that in lung EC, TNFα causes TNFR1 shedding, thereby possibly limiting inflammation. Our findings implicating RISP are the first evidence that a mitochondrial protein determines lung expression of a critical proinflammatory receptor (HL57556).
Adenosine is involved in the arteriolar vasodilatory responses to a single skeletal muscle contraction
Authors: Ross Graeme A , Armstrong Marika L , Murrant Coral L
Published: June 2021

To investigate the role of Adenosine (Ado) in response to a single contraction we stimulated 4–5 skeletal muscle fibres in a hamster cremaster preparation and measured the diameter of arterioles at the site of overlap with the stimulated muscle fibres before and after a single contraction (250ms train duration) at 4, 20 and 60Hz. Muscle fibres were stimulated prior to and following 30min incubation with Ado receptor antagonists 1,3‐dipropyl‐8‐sulphophenylxanthine (DPSPX, 10−5M) or xanthine amine congener (XAC,10−6M), or ecto‐5′‐nucleotidase inhibitor adenosine 5′‐[α,β‐methylene]diphosphate (AMPCP,10−5M). A dilation within 10s was observed at 4, 20 and 60Hz (1.0±0.2; 1.5±0.2; 2.0±0.2μm respectively) which was significantly attenuated by DPSPX (4Hz −0.1±0.2; 20Hz −0.2±0.3; 60Hz 0.04±0.2μm), XAC (4Hz 0.5±0.2; 20Hz 1.0±0.2; 60Hz 0.3±0.2μm), and AMPCP (4Hz 0.03±0.1; 20Hz −0.3±0.3; 60Hz −0.3±0.2μm). A secondary dilation at 20s at 20Hz (1.4±0.3μm) and 60Hz (1.1±0.1μm) was significantly attenuated by DPSPX (20Hz −0.1±0.1; 60Hz −0.1±0.3μm), XAC (20Hz 0.3±0.2; 60Hz 0.5±0.2) and AMPCP (20Hz 0.2±0.2; 60Hz 0.02±0.1μm). These findings implicate Ado in the vasodilations in response to a single muscle contraction.

NSERC
Peroxisome proliferator‐activated receptor‐α Reduces Mean Arterial Pressure by Renal‐Dependent Mechanisms during a Slow Pressor Dose of Angiotensin II
Authors: Wilson Justin L. , Lee Dexter L.
Published: June 2021

Peroxisome proliferator‐activated receptor‐α (PPAR‐α) activation reduces blood pressure in some hypertensive models by unclear mechanisms. We tested the hypothesis that the deletion of PPAR‐α would potentiate the slow pressor response to prolonged AngII infusion by a renal‐ dependent mechanism. We tested this hypothesis in PPAR‐α knockout (KO) and wild‐type (WT) male mice infused with AngII (400 ng/kg/min) and fed a normal salt diet for 14 days. Male KO and WT mice were implanted with biotelemetry devices and placed in metabolic cages to measure mean arterial pressure (MAP), heart rate (HR) and collect metabolic data. Male KO mice had normal basal MAP, HR and locomotor activity, sodium excretion, water intake and urine volume compared to WT, suggesting that PPAR‐α has no effect on basal MAP, HR, activity, sodium excretion, water intake and urine volume. On day 12 of AngII infusion, MAP and HR were lower in WT compared to KO mice: (MAP: 105 ± 4 vs 120 ± 3 mmHg, p < 0.001; HR: 395 ± 20 vs 500 ± 50 bpm, p < 0.001). Sodium excretion averaged 1.0 ± 0.5 and 0.8 ± 0.4 meq sodium/2 days in WT and KO, respectively, despite similar sodium intakes. Renal blood flow on day 12 of AngII was increased in WT (9.6 ± 2.3 ml/min/g) when compared to KO (7.0 ± 2.0 ml/min/g). The results suggest that during a slow pressor response to prolonged AngII infusion, the presence of PPAR‐α attenuates MAP via decreased renal vasoconstriction and increased sodium excretion.
Subsensory galvanic vestibular stimulation improves arterial pressure control upon head‐up tilt
Authors: Tanaka Kunihiko , Abe Chikara , Iwata Chihiro , Sakaida Yuzuru , Aoki Mitsuhiro , Morita Hironobu
Published: June 2021

Previously, we demonstrated that strong galvanic vestibular stimulation (GVS) obscured arterial pressure (AP) control upon posture transition from supine to 60 degree of head‐up tilt (HUT). In the present study, we hypothesized contrary that weak GVS might improve AP control during HUT according to a theory of stochastic resonance that noise enhances signal input and improves response. To verify an effect of the the weak GVS as noise for vestibular input, AP was measured continuously during HUT in 21 human subjects with and without GVS. GVS was applied with biphasic, random interval of square wave. The amplitude was changed from 0.1 mA to 0.6 mA randomly. Vestibular function was also evaluated with caloric test and subjective visual vertical test. Change in AP within 15 seconds upon HUT was correlated with degree of deviation in the vestibular function. 13 subjects decreased mean AP more than 5 mmHg upon HUT, but the decrease was not observed with simultaneous application of the GVS with the amplitude of 0.1 mA below the somatosensory threshold. Thus, orthostatic hypotension might be derived by dysfunction or imbalance of the vestibular system, and it might be able to improve using subsensory amplitude of GVS.