bluuebunny/crossref_metadata_2025 huggingface dataset:
Effect of L‐carnitine intake on tolerance to physical exercise, oxidative stress and vascular reactivity in obese trained rats
Authors: Priviero Fernanda B. M. , Rojas‐Moscoso Julio , Gómez‐Campos Rossana , da Silva Carmem Valgas , Carneiro Everardo M , Antunes Edson , Zanesco Angelina
Published: June 2021

We assessed the effect of L‐Car ingestion on tolerance to exercise (EX) and vascular reactivity of rats fed with a hyperlipidic diet (HD) and submitted to EX. Male Wistar rats were divided in sedentary (SD), exercised (EX), SD + L‐Car (SDLC) and EX + L‐Car (EXLC). After 4 weeks of HD intake, rats underwent to supplementation and an EX program of running for further 4 weeks. Tolerance to exercise was tested until exhaustion. Concentration‐response curves for acetylcholine (ACh), sodium nitroprusside (SNP) and phenylephrine (PE) were obtained in mesenteric artery (RMA). Plasma MDA and SOD levels were measured. Body weight gain was reduced in SDLC (Δ: 137 ± 4 g) compared to SD (Δ: 188 ± 10 g). No further reduction was found in EXLC. Tolerance to exercise was higher in EX (15 ± 1 min) compared to SD (7 ± 1 min). L‐Car raised tolerance to exercise in EXLC (22 ± 2 min) and SDLC (12 ± 1 min). In RMA, L‐Car increased the potency (pEC50) of ACh in EXLC (7.89 ± 0.03), but not in SDLC (7.67 ± 0.06), compared to SD (7.65 ± 0.04). Responses to SNP and PE were not altered. L‐Car decreased MDA levels in SDLC (2.4 ± 0.2 μM) and EXLC (2.9 ± 0.4 μM) compared to SD (4.6 ± 0.2 μM). SOD activity was not changed. In conclusion, L‐Car improved body weight loss and tolerance to exercise in SD. Together with EX, L‐Car improved endothelium‐dependent relaxation in RMA probably through decreasing lipid peroxidation.

Financial Support: FAPESP
The role of endothelium‐derived relaxing factors in mesenteric endothelial function in streptozocin‐induced diabetic rats
Authors: Zhang Rui , Anderson Leigh , Rahimian Roshanak
Published: June 2021

We investigated the relative contribution of endothelium‐derived relaxing factors (EDRFs) in mesenteric endothelial function in streptozocin (60 mg/kg, iv)‐induced diabetic female rats. Endothelium dependent vasodilation (EDV) to acetylcholine (10−8 to 10−5 M) in rat mesenteric rings precontracted with phenylephrine (2 μM) were obtained before and after pretreatment with indomethacin (indo, 10 μM), a cyclooxygenase (COX) inhibitor, L‐NAME (200 μM), a nitric oxide synthase inhibitor, or barium chloride (100 μM), a Kir channel blocker, with ouabain (10 μM), a Na+‐K+‐ATPase inhibitor. Rings from diabetic rats relaxed less than those from controls. In controls, indo slightly decreased EDV. However, in diabetic group, indo tended to enhance it. Addition of L‐NAME significantly reduced indo‐resistant vasodilation in diabetes, but the added effect of L‐NAME was not shown in controls. The remaining indo and L‐NAME‐resistant vasodilation was fully abolished by the combination of barium and ouabain in both groups. In conclusion, mesenteric EDV is impaired in diabetic female rats compare to that in controls. The effect of COX metabolites seems to be opposite in diabetic and control groups. Nitric oxide (NO) plays more role in mesenteric EDV in diabetic rats. However, when other pathways are blocked, endothelium‐derived hyperpolarizing factor (EDHF) makes a greater contribution in controls (Supported by NIDCR).
Inhibition of lactate‐producing glycolysis increases antidiabetic drug‐induced vasorelaxations and blocks sildenafil's attenuation of them
Authors: Peuler Jacob D. , Phelps Laura E.
Published: June 2021

Sildenafil is used to treat erectile dysfunction in men with type 2 diabetes (T2D). Hypertension is common in T2D but sildenafil attenuates vasorelaxant actions of metformin, pioglitazone and rosiglitazone: 3 antidiabetic (AD) drugs often used to improve glucose metabolism in T2D. Previously, to assess the glucose dependence of this effect, vascular rings from rat tail arteries were treated with each AD drug ± sildenafil. After 4 hours, all rings were contracted with norepinephrine (NE). With extracellular glucose present, all 3 AD drugs relaxed NE contractions and sildenafil attenuated such relaxations. With glucose omitted, relaxant actions of the AD drugs were notably increased and sildenafil failed to attenuate them. More recently, specific inhibition of only lactate‐producing glycolysis pathways with iodoacetate (30 micromolar) exerted similar results. We conclude that, in addition to vascular smooth muscle relaxant mechanisms, these 3 AD drugs also activate contraction‐enhancing mechanisms, which are dependent on specific glycolysis pathways and mask the full potential of the relaxant mechanisms. Thus, sildenafil's attenuation of AD drug‐induced vasorelaxations may not only involve interference with their relaxant mechanisms but also facilitation of specific glycolysis‐dependent contraction‐enhancing mechanisms.

Support: Midwestern University
Aldehyde dehydrogenase activator attenuates diabetic cardiomyopathy; a role in improving the quality of resident cardiac stem cells?
Authors: Palaniyandi Suresh Selvaraj , Disatnik Marie‐Helene , Sun Lihan , Vishnumangalam Jyothi Jayaram , Xia Xiaoyu , Pavlovic Aleksandra , Bhalla Vivek , Ashley Euan , Mochly‐Rosen Daria
Published: June 2021

Background

Hyperglycemia due to diabetes leads to the production of reactive aldehydes such as 4‐hydroxy‐2‐nonenal (HNE). These toxic aldehydes form adducts on proteins, leading to cell damage and organ dysfunction such as cardiomyopathy. Activation of aldehyde dehydrogenases (ALDHs) which metabolize toxic aldehydes into non‐toxic acids, can ameliorate diabetic cardiomyopathy.

Methods

Neonatal cardiomyocytes were subjected to high glucose stress (25mM) and treated with Alda‐1 (20 μM), a small molecule ALDH activator that we discovered. Diabetic cardiomyopathy was induced in DBA mice at 8 weeks of age by injecting streptozotocin. The mice with a significant hyperglycemia were treated from 12 week onwards for three months without (n=18) or with (n=24) Alda‐1 (16mg/Kg/day) using Alzet osmotic pumps.

Results and Discussion

We found that Alda‐1 treatment increased ALDH activity and decreased HNE‐protein adducts formation in vitro and vivo. Alda‐1 reduced adverse cardiac remodeling and myocardial dysfunction. Alda‐1 increased the number of resident cardiac stem cells in the tissue. Human heart samples from patients with cardiomyopathy and diabetes also showed decreased ALDH activity and increased HNE‐protein adducts formation

Conclusion

Activator of ALDH attenuated diabetic cardiomyopathy in mice. Finally, ALDH activators have potential to be a therapeutic agent to prevent diabetic cardiomyopathy.
Inducible cardiac‐specific RhoA‐expression protects against ischemia/reperfusion injury in mouse hearts
Authors: Xiang Sunny Yang , Ling Haiyun , Del Re Dominic P. , Purcell Nicole H , Miyamoto Shigeki , Matkovich Scott J. , Dorn Gerald W. , Brown Joan Heller
Published: June 2021

The small GTPase RhoA has established roles in regulating cell growth and cytoskeletal dynamics but the role of RhoA in modulating cardiac hypertrophy and cell survival remains elusive. Inducible cardiac‐specific RhoA mice (RhoA DTG) expressing 2–5 fold increase in active RhoA showed no evident basal phenotype. Remarkably, isolated RhoA DTG hearts subjected to 45min global ischemia followed by 2h reperfusion displayed a 60% reduction in infarct size, decreased lactate dehydrogenase release and significantly improved post‐ischemic cardiac function compared to the wild‐type hearts. RhoA DTG hearts subjected to 30min coronary occlusion followed by 24h reperfusion in vivo showed even greater protection with infarct size reduced by more than 70%. The Rho Kinase (ROCK) inhibitor Y‐27632 enhanced rather than blocking the protection in RhoA DTGs, suggesting that ROCK is not downstream of RhoA‐mediated ischemia/reperfusion (I/R) protection. Activated protein kinase C epsilon and protein kinase D were significantly increased in the RhoA DTG heart, and their roles in the protective effect of RhoA are currently under investigation. In conclusion, inducible and cardiac‐specific RhoA‐expression provides strong protection against I/R injury in the mouse heart both ex vivo and in vivo, contrasting with the conventional notion that RhoA/ROCK signaling in the heart is maladaptive. (HL28143)
AT1 Receptor‐specific down‐regulation of Smad7 Plays a Key Role in Atrial Fibrillation‐Induced Myocardial Fibrosis
Authors: Duan Dayue Darrel , He Xuyu , Peng Longyun , Wang Shenming , Ma Hong , Lin Jun , Gao Xiuren
Published: June 2021

Tachycardia‐induced atrial fibrosis is a hallmark for structural remodeling of atrial fibrillation (AF). But the molecular mechanisms for the genesis of atrial fibrosis remain unclear. In this study, we tested whether AT1 receptor‐specific inhibition of Smad7 plays a key role in the AF‐induced atrial fibrosis. Atrial fibrillation and fibrosis was induced by rapid atrial pacing (RAP, 1000 ppm) to the left atrium of rabbit heart. While RAP caused a significant increase in the expression of AngII, hydroxyproline, TGF‐β1, phosphorylated Smad2/3 and Arkadia, it decreased Smad7 expression. These changes were reversed by AT1 receptor antagonist losartan in a dose‐dependent manner. Similar results were obtained in the adult rabbit cardiac fibroblast. In the presence of AngII, AT1 antagonist, but not the AT2 antagonist PD123319, restored Smad7 expression and caused a decrease in P‐Smad2/3, Smad4 collagen I and Arkadia, indicating a key role of AT1 receptor‐specific inhibition of Smad7 in the AngII‐induced fibrosis and suggesting that AT1 receptor‐specific inhibition of Smad7 may be mediated by Arkadia‐induced Smad7 poly‐ubiquitination and degradation. In addition, interruption of both ERK mitogen‐activated protein kinase (MAPK) and TGF‐β1 reversed the AngII‐induced inhibition of Smad7 expression. Thus, AngII may induce atrial fibrosis through an AT1‐receptor specific activation of TGF‐β1, which in turn decreases the inhibitory Smad7 and increases P‐Smad2/3. These results suggest a key role of AT1 receptor specific inhibition of Smad7 in AF‐induced atrial fibrosis. (Supported by Guangdong SFC #06021342 and NCRR P‐20 RR‐15581)
Role of O‐linked β‐N‐acetylglucosamine in isoflurane induced cardiac protection
Authors: Tsutsumi Yasuo M. , Hirose Kayo , Katayama Erika , Tsutsumi Rie , Kinoshita Michiko , Tanaka Katsuya , Oshita Shuzo
Published: June 2021

The administration of isoflurane, volatile anesthetics, before myocardial ischemia‐reperfusion exerts protective effects against myocardial infarction similar to that of ischemic preconditioning (IPC). Although the endogenous signaling mechanisms remains unknown, previous work suggests that the modification of proteins with O‐linked β‐N‐acetylglucosamine (O‐GlcNAc) plays a critical role in IPC. We therefore examined the role of O‐GlcNAc in the cardiac protection induced by anesthetic preconditioning (APC).

Mice were exposed to 30 min isoflurane or oxygen and allowed to recover for 15 min. After washout periods, mice then underwent 30 min ischemia, followed by 2 h of reperfusion. Some isoflurane‐treated mice were also given alloxan, an inhibitor of O‐GlcNAc‐transferase, 20 min prior to isoflurane exposure. The area at risk as a percent of left ventricular were similar among groups. Infarct size after reperfusion was reduced by isoflurane (29 ± 4% vs. 40 ± 4%, p<0.05) but not alloxane pretreated mice (42 ± 5%, n=4). Additionally, hearts excised from APC‐treated mice had a 1.5‐fold increase in O‐GlcNAc level (p<0.05, n=4) and pretreatment with alloxan reversed this effect of APC.

These data demonstrate that cardiac protection produced by isoflurane are sensitive to O‐GlcNAc levels, suggesting O‐GlcNAc signaling may play a vital role in the isoflurane‐induced cardiac protection.
Role of sex steroids in the regulation of the UA myogenic tone during pregnancy at high‐altitude
Authors: Chang Katherine , Xiao Daliao , Xue Qin , Zhang Lubo
Published: June 2021

Previous studies demonstrated that sex steroids mediate a reduction in myogenic tone of the uterine artery (UA) during pregnancy. The present study tested whether chronic hypoxia (CH) alters hormonal effects on the UA. UAs were isolated from nonpregnant and near‐term pregnant (PUA) ewes maintained at ~300 m or 3801 m altitude for 110 days. UAs were treated with 17b‐estradiol (E2b), progesterone (P4), hormone inhibitors for 48 h. In contrast to previous findings in the normoxic animals, E2b and P4 had no effect on the myogenic tone and PKC‐induced contractions in the UA of high‐altitude animals. Additionally, high altitude hypoxia decreased ERá expression in PUA whereas ERb and progesterone receptor expression was unaltered. To determine the direct effect of hypoxia, UAs of normoxic animals were incubated in 10.5% or 21% oxygen for 48h, and our results showed that ERá expression in PUA was lowered in the 10.5% group than in the 21% group. The results suggest that CH differentially regulates the expression of steroid receptors in the UAs, leading to increased myogenic tone and impaired uterine blood flow during pregnancy.(supported in part by NIH grant HL89012)
L‐Tryptophan methyl ester Evokes Endothelium‐, Serotonin‐Independent Biphasic Vasoconstrictor and Vasodilator Responses in Rat Mesenteric Arterioles
Authors: Jadhav Ashok B , Nadithe Laxman Reddy , Balsevich John , Akhtar Jawed , Gopalakrishnan Venkat
Published: June 2021

The vascular effects of L‐tryptophan (L‐W) are unknown. The in vitro responses to L‐W and its cell permeant analog, L‐W methyl ester (L‐Wme), were compared in third order branches of mesenteric arterioles of 12 week old male Sprague Dawley rats. While L‐W had no effect, L‐Wme evoked concentration‐dependent vasoconstriction (10 – 160 μM) followed by vasodilatation (300 μM – 2.5 mM) in both endothelium‐intact and endothelium‐denuded vessels. While 5‐HT antagonist, ketanserin (50 nM), shifted the responses to 5‐HT (EC50 Control 107 ± 10 nM vs. Treatment 32 ± 6 μM; p<0.001), it failed to affect the responses to L‐Wme. The inclusion of transient receptor potential channel (TRPC) blocker, 2‐aminoethoxy phenyl borate (10 μM) or the voltage‐gated Ca2+ channel blocker, verapamil (10 μM), abolished the bipahsic responses to L‐Wme. L‐Wme evoked (1 μM ‐ 2.5 mM) vasodilatation was more sensitive in potassium chloride depolarized (IC50 330 ± 19 μM; Imax 95%) than in phenylephrine constricted vessels (IC50 767 ± 23 μM; Imax 96%). These data suggest that the cell permeant amino acid, L‐Wme, exerts endothelium‐/serotonin‐independent responses that could be due to activation and blockade of Ca2+ influx occurring through either the TRPC and/or the voltage‐gated Ca2+ channels present on vascular smooth muscle cells of resistance type vessel. This confirms a direct role for L‐W analogs in the regulation of vascular smooth muscle tone.
Modeling antidepressant binding at the human serotonin transporter
Authors: Kuntz Charles P , Sealover Natalie R , Mukhopadhyay Soma , Barker Eric L
Published: June 2021

The structure of a bacterial homologue of the biogenic amine transporters, the leucine transporter from Aquifex aeolicus (LeuTAa) has allowed the generation of homology models of the human serotonin transporter (hSERT) by superimposing the known amino acid sequence of hSERT onto the three‐dimensional scaffold of LeuTAa. A number of docking studies have been performed in our lab, using Autodock Vina, that have revealed possible binding modes of the antidepressants sertraline, imipramine, and desipramine which are consistent with and help to explain experimental data previously published by our laboratory. There are two ligand binding sites on the transporter–a high affinity site at the center of the transporter, and a lower‐affinity site located at the extracellular‐facing vestibule above the high‐affinity site. We have identified a distinct position at the low‐affinity site which differs somewhat from the site identified in diffraction studies on LeuTAa bound to tricyclic antidepressants. This finding is consistent with previous experimental data on residues contributing to ligand binding. Molecular dynamics simulations on these models have refined the binding modes identified in the docking studies. Furthermore, we have computationally mutated the residues around the two binding sites in LeuTAa to those found in hSERT, resulting in a gain of antidepressant affinity for the transporter. In future studies, we will use information from ligand docking analyses on our hSERT homology model to probe the structural determinants of the antidepressant binding site.
Validating the Acetylcholine Binding Protein's Conversion Potential to a Human Nicotinic Acetylcholine Receptor.
Authors: Nemecz Ákos , Taylor Palmer
Published: June 2021

The cys‐loop family of ligand‐gated ion channels plays an important role in the chemical‐to‐electrical transduction of neuronal signaling. The well‐studied homomeric α7‐nAChR may be linked to nicotine addiction, and shows possible genetic linkages to such diseases as Alzheimer's and schizophrenia. The discovery of a homologous soluble snail acetylcholine binding protein (AChBP), also homomeric, provided a structural surrogate of the extracellular ligand‐binding domain; however, the binding protein, despite the requisite ligand properties, shows only ~30% residue identity with members of the nicotinic receptor family. We generated systematic mutations of the soluble Aplysia californica to achieve sequences resembling α7‐nAChR at the subunit interfaces and analyzed ligand binding affinities. Key constructs, representing stages of subunit interface conversion, were subsequently characterized using classical α7‐nAChR selective and nAChR ligands, such as peptidic antagonists α‐neurotoxin and α‐conotoxins, (+)‐epibatidine, lobeline, and methyllycaconitine and compared to values obtained on a full‐length human α7‐nAChR. Although no mutational construct fully resembles α7 selectivity, we have found a final construct with comparable α7‐nAChR affinities. Hence such constructs provide better suited templates for α7‐nAChR drug design. (Supported by: U01‐DA 019372, GM07752‐29)
Accuracy and Reliability of Consumer Breath Alcohol Analyzers
Authors: Davis William , Andrews J C , Herb E , Lee J R , Latman N S , Bouma C L
Published: June 2021

Between 1999 and 2007, there were approximately 13,000 fatalities/year in the United States as a result of alcohol‐impaired driving accidents. These accidents are preventable, and there are many personal breath alcohol concentration (BrAC) analyzers being utilized by consumers. The purpose of this study was to determine the accuracy and reliability of five consumer models of BrAC analyzers by comparing their results to blood alcohol concentration (BAC). Fifty‐seven subjects with ages ranging from 21–75 were tested with a %BAC range of 0.03 to 0.14.

Results were:

Standard Line‐of‐ Equality
BACTrac S 50
BACTrac B 70
AlcoHAWK Pro
AlcoHAWK Slim
Alcomate Premium

y‐Intercept
0.00
0.006
0.061
0.026
0.026
0.001

Slope
1.00
1.23
0.52
1.20
1.05
0.77

Correlation Coefficient
1.00
0.52
0.29
0.76
0.75
0.81

Standard Error of Estimate
0.000
0.051
0.043
0.026
0.022
0.014

Mean Absolute Error
0.000
0.036
0.032
0.021
0.017
0.011

Reliability
0.000
0.012
0.019
0.008
0.005
0.007

None of the models of BrAC analyzers were sufficiently accurate to be useful in reliably warning drivers of their state of intoxication.

This study was supported in part by Lifeloc Technologies, Inc.
Cytisine Attenuates Nicotine‐induced Ethanol Consumption in C57BL/6J mice
Authors: Sajja Ravi , Spinar Matthew , Rahman Shafiqur
Published: June 2021

Alcohol and nicotine are frequently co‐abused drugs affecting millions of people in the US and other parts of world. Previous research suggests that high incidence rates of smoking are associated with increased alcohol consumption contributing a major risk factor for alcohol relapse. Accumulated evidence indicates that brain nicotinic receptors (nAChRs) are common points of action for nicotine‐alcohol (ethanol) interactions. Thus targeting nAChRs could be a potential strategy for the management of nicotine‐ethanol co‐dependence. The present study examined the effects of cytisine, a nAChR partial agonist, on chronic nicotine‐induced ethanol intake in C57BL/6J mice. After establishment of ethanol (10%, v/v) preference using two‐bottle choice continuous drinking procedure, mice received subcutaneous injections of saline, nicotine (0.4 mg/kg) or cytisine (0.5, 1.5 or 3 mg/kg) given before nicotine treatment for 10 days. Chronic nicotine treatment significantly increased ethanol intake (g/kg) post 1 h (166%; p < 0.05) and 2 h (152%; p < 0.05) compared to saline. Pretreatment with cytisine (0.5 or 1.5 mg/kg) significantly reduced nicotine‐induced ethanol intake post 1 h (32% or 43% respectively; p < 0.005) and 2 h (50% or 53% respectively; p < 0.005). These results further suggest that brain nAChRs localized in mid‐brain dopamine system are ideal therapeutic targets in regulating ethanol and nicotine consumption. Thus, development of cytisine or new analogs would be a potential pharmacological strategy for the treatment of nicotine‐alcohol co‐dependence. (Supported in part by SDSU Research grant).
Morphine‐induced motor stimulation and ataxia: differences among adolescent, late adolescent, and adult mice
Authors: Koek Wouter , France Charles P.
Published: June 2021

Given evidence for age‐related differences in the effects of drugs of abuse, surprisingly few preclinical studies have explored effects of opioids in adolescents (versus adults). The present study compared the motor stimulating and ataxic effects of morphine in adolescent and adult mice.

Morphine (3.2–56 mg/kg, i.p.) increased locomotion along an inverted U‐shaped dose‐response curve in adolescent, late adolescent, and adult male C57BL/6J mice. In all age groups, the minimum effective dose to enhance locomotion was 10 mg/kg, and maximal stimulation was observed at 17.8 mg/kg. However, maximal stimulation was higher in adolescents than in adults. In contrast, adolescents showed less ataxia than adults when given morphine (5.6–100 mg/kg, i.p.).

The finding that adolescent mice show larger locomotor‐stimulating effects of morphine than adults is consistent with previous findings in rats. This could be taken to indicate that adolescent animals are more sensitive to motor stimulating effects of morphine. Alternatively, larger locomotor effects of morphine could be due to less interference by its ataxic effects. Thus, it is possible that morphine‐induced motor stimulation appears larger in adolescents than in adults not because adolescents are more sensitive to the motor stimulating effects of morphine, but because they are less sensitive to its ataxic effects.

Supported by DA23261 (WK), DA17918 (CF)
Dopamine D2‐like receptors mediate the discriminative stimulus effects of delta‐9‐tetrahydrocannabinol (delta‐9‐THC) in rhesus monkeys
Authors: Stewart Jennifer L , McMahon Lance R.
Published: June 2021

Monoamines are implicated in the behavioral effects of CB1 receptor agonists; dopamine, in particular, could mediate the abuse liability of marijuana. The objective of this study was to investigate the capacity of monoaminergic ligands to modify the discriminative stimulus effects of Δ9‐THC (0.1 mg/kg i.v.) in rhesus monkeys. The Δ9‐THC discriminative stimulus was attenuated by cocaine (0.32–1.78 mg/kg) and amphetamine (0.1–0.56 mg/kg), while acute administration of the norepinephrine and serotonin transporter inhibitors desipramine (10–17.8 mg/kg) and imipramine (10–17.8 mg/kg) did not modify Δ9‐THC. The involvement of dopamine receptor subtypes was then investigated; the dopamine D1‐like receptor agonists (±)‐SKF 38393 (3.2–10 mg/kg) and (±)‐6‐Chloro‐PB (0.56–5.6 mg/kg) did not modify the Δ9‐THC discriminative stimulus. The dopamine D2‐like receptor antagonist haloperidol (0.0032–0.01 mg/kg) and the dopamine D2‐like receptor agonist quinpirole significantly enhanced the discriminative stimulus effects of Δ9‐THC. Drugs that directly or indirectly interact with dopamine D2‐like receptors play an important role in mediating the behavioral effects of cannabinoids; the mechanism responsible for attenuation of Δ9‐THC by cocaine and amphetamine remains unclear. Supported by USPHS grant DA 19222 and DA 26781.
Elevation of Endocannabinoids via Inhibition of their Catabolic Enzymes Attenuates the Precipitated Opioid Withdrawal Syndrome
Authors: Ramesh Divya , Long Jonathan Z , Ahn Kay , Cravatt Benjamin F , Lichtman Aron H
Published: June 2021

It has long been known that cannabis and its primary active constituent, Δ9‐tetrahydrocannbinol (THC), reduce opioid withdrawal symptoms. The endocannabinoids, anandamide (AEA) and 2‐arachidonyl glycerol (2‐AG), also activate CB1 receptors, but they are rapidly metabolized by their respective enzymes, fatty acid amide hydrolase (FAAH) and monoacylglycerol lipase (MAGL). The objective of this study was to determine whether elevating AEA or 2‐AG, via selective pharmacological inhibition of their metabolizing enzymes, would reduce precipitated morphine withdrawal symptoms. ICR mice were implanted subcutaneously with morphine pellets and challenged 72 h later with the opioid receptor antagonist, naloxone, to precipitate withdrawal. The withdrawal signs included number of jumps, paw tremors, head twitches, body weight loss and the occurrence of diarrhea. Pretreatment with the MAGL inhibitor, JZL184 dose‐dependently reduced the intensity of all naloxone‐precipitated withdrawal signs and these effects were reversed by the CB1R antagonist rimonabant, but not by the CB2R antagonist SR144528, suggesting a CB1 receptor specific mechanism of action. The FAAH inhibitor, PF‐3845 reduced the intensity of naloxone precipitated jumps and paw flutters through a CB1 receptor mechanism, but did not affect other withdrawal signs. The results from the present study are the first to show that the elevation of endocannabinoids ameliorates the expression of opioid withdrawal. Thus, the enzymes responsible for endocannabinoid degradation offer promising targets for possible new treatments for opioid dependence.
Acute morphine increased both perseverative and non‐perseverative errors of rats in a modified attentional set‐shifting test
Authors: Chen Yukun , Young Alice M , Schrimsher Gregory W , Evola Marianne
Published: June 2021

Increased perseverative errors (PE, repeated responses to previously correct cues) are a key feature of clinical disorders with frontal lobe damage (e.g., schizophrenia and Alzheimer's disease, substance abuse). To mimic the Wisconsin Card Sorting Test that detects PE in humans, we modified Birrell & Brown's (2000) attentional set‐shifting test to a three‐choice set‐shifting and perseveration test (SSPT). The SSPT utilizes successive sets of three cups that differ in both digging medium and odor. On each trial, only one cue (medium or odor) is paired with food. The rats learn to select the baited cup by either medium or odor, and to switch between cues in a series of discriminations: compound discrimination (CD), intradimensional (ID) shift, extradimensional (ED) shift. To optimize test conditions, we tested rats’ preference for 21 odors, and identified three equally‐preferred odors and an odor intensity that is recognizable but not aversive. In the primary study, we used these three odors and the intensity to evaluate acute effects of morphine on rats’ (N=8) SSPT performance. Morphine (3.2 mg/kg, −15 min) increased both PE and non‐PE in CD and ID1, and none of the rats finished ID1. Given ‐4 hr, the same dose increased PE in CD and non‐PE in CD and ED1. Acute morphine may impair ability to inhibit inappropriate behavior and ignore irrelevant cues when acquiring a new strategy. Supported by TTU Research Enhancement Fund
The interaction of chronic delta‐9‐tetrahydrocannabinol (THC) and estradiol during early adulthood has long‐term effects on learning in female rats
Authors: Winsauer Peter John , Sutton Jessie L
Published: June 2021

Persistent THC abuse by adolescents has been shown to produce long‐term, hormonally‐dependent deficits in complex behavioral processes such as learning. This study examined whether the same deficits would be produced by chronic THC during early adulthood to determine the age sensitivity of these effects. To do this, either sham‐operated (Intact) or ovariectomized (OVX) female rats received 40, daily THC injections of 5.6 mg/kg i.p. during early adulthood. Following THC administration and a drug‐free period for training to respond under a multiple schedule with acquisition and performance components, the baseline response rates and response accuracy for the groups were not significantly different (Intact/Saline, Intact/THC, OVX/Saline, and OVX/THC). However, when 0.56–10 mg/kg of THC was administered acutely, group differences were apparent. With respect to response rate, the rank order for disruption by THC in the acquisition and performance components was Intact/Saline > Intact/THC = OVX/Saline > OVX/THC. With respect to accuracy in both components, the rank order for disruption by THC was Intact/THC > OVX/Saline > Intact/Saline = OVX/THC. These results indicate that chronic THC in female rats during early adulthood, as well as adolescence, can have long‐term, hormonally‐dependent effects on learning, and that these effects show only moderate age sensitivity.
Effect of diacerein alone and combination with aceclofenac in adjuvant induced arthritis and in chronic inflammatory model in rats
Authors: Medhi Bikash , Prasad Byrav D.S. , Patyar Sazal , Prakash Ajay , Saikia Uma Nahar
Published: June 2021

Present study was carried out evaluate the efficacy of diacerein alone and in combination with aceclofenac in different chronic inflammatory models in rats.

48 male rats were divided into two groups of arthritis, and cotton wool granuloma. Each group had 4 subgroups Vehicle, Freund's adjuvant, Diacerein + Freund's adjuvant, Diacerein + Aceclofenac + Freund's adjuvant. And in cotton wool granuloma group Sham operated, Cotton wool pellet, Diacerein + Cotton wool pellet, Diacerein +Aceclofenac+ Cotton wool pellet. Animals were treated with diacerin alone and in combination irrespective to its groups. At the end of seventeen days in arthritis and seven days in cotton wool granuloma group, animals were sacrifice after assessment of inflammation. Blood sample were collected for TNF α and MPO estimation.

The models of arthritis and CWG were effectively produced as evidenced by morphology and histology scores (p<0.001). Diacerein reduced the morphological, histological including cytokine and MPO activity (p<0.002), combination showed better effect on this parameters (p<0.001). In the arthritis group and cotton wool granuloma model TNF α and MPO levels in plasma were reduced following diacerein treatment (p<0.002) and combination showed better effect (p<0.001) in both the model.

Diacerein is effective in above mentioned model however combination showed better efficacy in chronic inflammatory models.
The selective mu opioid antagonist β‐funaltrexamine (β‐FNA) reduces toll‐like receptor‐4 signaling
Authors: Stevens Craig W , Aravind Shruthi , Davis Randall L
Published: June 2021

Recently published data from our labs showed that the selective mu opioid antagonist, β‐FNA (β‐funaltrexamine), inhibits the release of pro‐inflammatory cytokines and the expression of nitric oxide synthase (iNOS) in tumor necrosis factor alpha (TNFα)‐stimulated human astrocytes. The finding that the opioid agonists, morphine and fentanyl, also inhibited these pro‐inflammatory processes suggested that these opioid effects were not mediated at the classical mu opioid receptor. Other recent data primarily from Watkins’ research group, suggest that this non‐opioid receptor mediated effect of opioid agents on glial cell activation may be due to an interaction of opioids on the toll‐like receptor 4 (TLR4) signaling pathway. Thus, the present studies were undertaken to further investigate the effects of β‐FNA on the TLR4 signaling pathway using human astrocytes and a commercially available reporter cell line for TLR4 signaling called HEK‐Blue4 cells. We found that opioid agonists themselves produce a minor but significant stimulation of TLR4 signaling which is not blocked by the general opioid antagonist, naltrexone. When cells were treated with a specific TLR4 agonist, LPS‐EK, opioid agonists and antagonists both decreased TLR4 signaling. β‐FNA was the most potent agent in this regard. Given these preliminary data, β‐FNA may show promise as a new anti‐inflammatory agent. Supported by NIH grants DA12448 (CWS) and NS062664 (RLD).
Lipid rafts and membrane cholesterol are involved in regulating D2 dopamine receptor signaling
Authors: Hazelwood Lisa A , Romanelli Robert J , Free R Benjamin , Roof Rebecca A , Javitch Jonathan A , Neve Kim A , Sibley David R
Published: June 2021

Lipid rafts are specialized membrane microdomains enriched in cholesterol, sphingolipids and caveolin. They are important in the organization of GPCR‐protein complexes and the regulation of signaling. Given the emerging significance of lipids with respect to GPCR structure and activation, we have investigated the role of lipid rafts and membrane cholesterol on D2 dopamine receptor (DAR) localization and function. Sucrose density fractionation revealed that D2 DARs are enriched in lipid rafts from both striatum and transfected HEK293 cells. Depletion of membrane cholesterol with methyl‐β‐cyclodextrin (MCD) diminished cell‐surface D2 DAR expression in HEK293T cells by approximately 75% without altering total receptor number. MCD treatment also completely abolished DA signaling at the D2 DAR. Importantly, when D2 DAR transfections were titrated to achieve surface expression values comparable to MCD‐treatment, a DA‐dependent decrease in cAMP was still observed. Diminished signaling of Gi/o‐coupled muscarinic receptors was also observed following MCD‐treatments. Notably, MCD‐treatment did not inhibit Gs‐linked D1 DAR function or forskolin‐stimulated cAMP accumulation. These results suggest that lipid rafts and/or membrane cholesterol are critical for D2 DAR signaling.
Serine 5.43 of the D4 dopamine receptor plays a key role in its interactions with norepinephrine and Ro10‐4548
Authors: Schetz John , Goetz Angela , Cummings David
Published: June 2021

Conserved serines of transmembrane segment five (TM5) have been shown to be critical for the interactions of endogenous neurotransmitters with catecholamine GPCRs. Interest in the D4 dopamine receptor for its role in attention and erectile dysfunction, and the high affinity interaction of only this dopamine receptor subtype with norepinephrine, lead us to investigate the interactions of ligands with D4 receptor TM5 serines. Serine to alanine mutations at positions 5.42 and 5.46 decreased the affinities of dopamine and norepinephrine for the D4 receptor by greater then 150‐fold. The D4‐S5.43A receptor mutant had drastically reduced affinity for norepinephrine (> 300‐fold), but only a modest loss of affinity for dopamine (< 8‐fold). In functional assays of cAMP accumulation, norephinephrine was unable to activate any of the mutant receptors, even though the agonist quinpirole displayed wild type functional properties for all of them. Dopamine was unable to activate the S5.46A mutant, had reduce potency for the S5.43A mutant and reduced potency and efficacy for the S5.42A mutant. While six D4‐selective ligands had affinities and functions for mutant receptors similar to the wild type receptor, Ro10‐4548, an antagonist of the wild type receptor unexpectedly functions as an agonist of the S5.43A mutant.
Regulation of kappa Opioid Receptor (KOR) Responsiveness on Peripheral Sensory Neurons
Authors: Sanchez Teresa A , Rowan Matthew P , Silva Michelle , Clarke William P , Berg Kelly A
Published: June 2021

We have examined the function of KOR in primary cultures of trigeminal ganglion (TG) sensory neurons and in an animal model of thermal allodynia. The KOR agonist, U50488, did not inhibit PGE2‐stimulated adenylyl cyclase (AC) activity in vitro and did not reduce PGE2‐stimulated thermal allodynia in vivo. However, after a 15 min treatment (priming) with the inflammatory mediator bradykinin (BK) quiescent KOR became functionally competent, capable of inhibiting AC activity and thermal allodynia. The effect of U50488 in BK‐primed tissue was blocked by the KOR antagonist nor‐BNI both in vitro and in vivo. The priming effect of BK was blocked by either indomethacin or bis‐indolylmaleimide, suggesting that an arachidonic acid (AA) metabolite and PKC activation mediate BK‐induced regulation of KOR. Further, KOR function in BK‐primed tissue was blocked by a soluble integrin‐blocking peptide (GRGDSP), but not the inactive reverse sequence peptide (GDGRSP), suggesting that in addition to AA and PKC, RGD‐binding integrins also mediate regulation of KOR signaling. Interestingly, not all signaling elicited by KOR requires inflammatory mediator pretreatment. For example, U50488‐mediated increases in ERK activation occurred in the absence of BK and was not altered by BK pretreatment. We propose that peripheral nociceptor function can be regulated by KOR, especially under conditions of tissue inflammation.
Synergistic α1‐adrenergic receptor mediated increases in IL‐1β from lipopolysaccharide‐challenged human monocytes is β‐arrestin 1‐dependent and NF‐κB‐independent
Authors: Grisanti Laurel A , Porter James E
Published: June 2021

Adrenergic receptors (AR) are expressed on immune cell populations, but their precise function is not well characterized. We previously demonstrated that activation of α1b‐ARs synergistically increases IL‐1β release from lipopolysaccharide (LPS) challenged human monocytes through a PKC/p38 MAPK dependent mechanism. Studies show βarrestin signaling complexes regulate LPS induced NFκB activity as well as AR mediated p38 MAPK activation. Thus, we hypothesized that α1b‐ARs increases IL‐1β production from LPS‐primed monocytes through βarrestin enhancement of NFκB signaling. RNA interference demonstrated βarrestin 1 expression is needed for potentiated IL‐1β release from monocytes treated with phenylephrine (PE) and LPS. Using an NFκB inhibitor, we found a requirement for NFκB activation to increase IL‐1β production in cells treated with LPS alone. However, synergistic increases in IL‐1β from monocytes treated with PE and LPS are NFκB independent. Electrophoretic mobility shift and luciferase reporter assays showed NFκB protein/promoter interactions are disrupted, resulting in decreased transcription. Examination of alternative transcription factors demonstrated increased AP‐1 activation. These results provide evidence for a novel βarrestin 1 dependent, NFκB independent mechanism through which α1b‐AR stimulation potentiates the innate immune response from LPS challenged human monocytes.
AGS3 blocks D2L dopamine receptor‐mediated heterologous sensitization of adenylyl cyclase 2
Authors: Conley Jason Michael , Watts Val J
Published: June 2021

The present study was designed to investigate the function of the activator of G protein‐signaling 3 (AGS3/Gpsm1) in cellular signaling. AGS3 acts in vitro as a guanine nucleotide dissociation inhibitor by binding Gαi in the GDP‐bound state and has been implicated in drug‐seeking or relapse behavior in rats dependent on cocaine, ethanol, or heroin. HEK293 cells expressing adenylyl cyclase type 2 (AC2) and the D2L dopamine receptor were utilized to study the effects of AGS3 expression on G protein signaling following acute or persistent activation of the D2L receptor. AGS3 produced no discernable changes in Gβγ‐dependent stimulation of AC2 upon acute D2L receptor activation. Similar studies revealed analogous results when acute D2L‐stimulated ERK1/2 activation was examined in the presence or absence of AGS3. Subsequent experiments revealed that AGS3 lacked effects on long‐term D2L receptor desensitization. In contrast, the expression of AGS3 completely blocked D2L receptor‐mediated heterologous sensitization. These findings suggest that desensitization and heterologous sensitization depend on distinct mechanisms and that AGS3 may play a role in regulation of D2L receptor‐mediated heterologous sensitization. Our results are consistent with the work of Bohn and colleagues, who showed that tolerance and dependence involve distinct signaling pathways. This work was supported by grant MH060397 to VJW.
Movement of Activator of G‐Protein Signaling 3 within the Aggresome Pathway
Authors: Vural Ali , Oner Sukru Sadik , An Ningfei , Blumer Joe B. , Lanier Stephen M.
Published: June 2021

AGS3, a receptor independent activator of G‐protein signaling, is involved in unexpected functional diversity for G‐protein signaling systems. AGS3 has seven tetratricopeptide (TPR) motifs upstream of four G‐protein regulatory (GPR) motifs, each of which bind and stabilizes the GDP bound conformation of Giα. The positioning of AGS3 within the cell and the intra‐molecular dynamics between different domains of the proteins are likely key determinants of their ability to influence G‐protein signaling. We report that AGS3 enters into the aggresome pathway and that this positioning of the protein is differentially regulated by the AGS3 binding partners Giα and mInsc. Giα rescues AGS3 from the aggresome, whereas mInsc augments the aggresomal distribution of AGS3. The distribution of AGS3 to the aggresome is dependent upon the TPR domain and it is accelerated by disruption of the TPR organizational structure or introduction of a non‐synonymous single nucleotide polymorphism. These data present AGS3, G‐proteins and mInsc as candidate proteins involved in regulating cellular stress associated with protein processing pathologies.
COUPLING OF A G‐PROTEIN COUPLED RECEPTOR TO THE AGS3‐Galphai SIGNALING COMPLEX
Authors: Oner Sukru Sadik , An Ningfei , Vural Ali , Breton Billy , Bouvier Michel , Blumer Joe B. , Lanier Stephen M.
Published: June 2021

AGS3 and related entities serve as alternative binding partners for Gαi independent of the classical heterotrimer Gαβγ and this interaction is mediated by a 20–25 amino acid motif termed the G‐protein regulatory (GPR). Such a signaling module may function in the context of signal processing through G‐protein coupled receptors or actually regulate intracellular events independent of the classical receptor ‐ heterotrimeric G‐protein – effector model. We report the regulation of the GPR‐Gαi signaling module by a cell surface receptor using bioluminescence resonance energy transfer (BRET). AGS3‐Renilla luciferase (Rluc), and Gαi1‐yellow fluorescent protein (YFP) exhibited robust, specific BRET. AGS3‐Rluc ‐ Gαil‐YFP BRET was inhibited by activation of the α2‐adrenergic receptor (α2 ‐AR), a typical G‐protein coupled receptor that couples to the Gαi/Gαo class of heterotrimeric G‐proteins. The AGS3‐Gαi complex is in close proximity to the receptor as Gαi‐dependent and agonist‐sensitive BRET was observed between AGS3‐Rluc and the α2‐AR‐Venus. AGS3‐Gαi appears to be anchored within a receptor signaling complex at the membrane and the biophysical organization of this complex is stabilized or destabilized by specific conformations of the receptor.
Characterization of estradiol‐mediated, rapid enhancement of bradykinin signaling in sensory neurons
Authors: Rowan Matthew P , Berg Kelly A , Hargreaves Kenneth M , Roberts James L , Clarke William P
Published: June 2021

Premenopausal women are at increased risk for numerous pain disorders. Although this sex difference is far from understood, many studies have shown the importance of estrogen in the enhancement of nociceptive signaling. Estrogen receptors (ERs) are present on sensory neurons, and we have previously shown that 17β‐estradiol (E2) rapidly enhances signaling of the inflammatory mediator, bradykinin (BK), in vitro and in vivo. The aim of this study was to further characterize and delineate mechanisms involved in the enhancement of BK signaling by E2. Pretreatment (15 min) with the ERα‐selective agonist, PPT, significantly enhanced BK‐induced PLC activity in cultures of primary sensory neurons and thermal allodynia. The ERβ‐selective agonist, DPN, and the GPR30‐selective agonist, G‐1, had no effect. Enhancement of BK signaling by E2 was seen with pretreatment times ranging from 0 (co‐injection) to 60 min, and persisted for up to 60 min following washout of E2. The effect of E2 was not blocked by the transcription inhibitor anisomycin and was mimicked by E2 conjugated to BSA (membrane impermeable). These data suggest that ERα mediates the rapid onset effect of E2 to enhance BK signaling and that the effect of E2 is non‐genomic.
Pumpkin Seed Oil Supplementation improves HDL and LDL cholesterol in Post‐Menopausal Women: A Random Double‐Blind Pilot Study
Authors: Hyde Coretha Dionne , Gossell‐Williams Maxine , Hunter Tiffany , Simms‐Stewart Donnette , McGrowder Donovan , Fletcher Horace , Choo‐Kang Eric
Published: June 2021

This study aimed to determine the effects of pumpkin seed oil supplementation on lipids in post‐menopausal women. Post‐menopausal females with normal lipid concentrations and blood pressure were randomly assigned to 2 groups to receive capsules of 2 g pumpkin seed oil (PSO, n =16) or wheat germ oil (WGO, n=10) daily for 12 weeks. Fasting blood samples were collected and assayed for total cholesterol (TC), triglycerides, HDL‐cholesterol and LDL‐cholesterol before and at the end of supplementation period. Changes in the mean (± standard deviation) lipids concentrations were analysed using paired student's t‐test. There was no change in TC for both WGO (5.58 ± 1.43 mmol/l to 5.30 ± 1.32 mmol/l) and PSO (5.93 ±1.38 mmol/l to 5.72 ± 1.71 mmol/l). Triglyceride concentration remained the same for the PSO (1.39 ±0.69 mmol/l to 1.37± 0.64 mmol/l), but an increasing trend was seen in the WGO group (1.27± 0.43 mmol/lto 1.45 ±0.42 mmol/l, P =0.09). HDL –cholesterol significantly increased by 16.38% in the WGO group (P < 0.05) and 18.85% in PSO group (P < 0.01). LDL ‐cholesterol concentrations significantly decreased in the PSO group by 13.42% (P < 0.05) and there was a decreasing trend in the WGO group, though not significant (P = 0.10). These preliminary findings suggest PSO supplementation can improve HDL‐cholesterol and LDL‐cholesterol profile in post‐menopausal women, but further investigations are required.
Selective Serotonin Reuptake Inhibitors Influence Agonist‐Induced Platelet Aggregation. Preliminary Results from Co‐morbidity of Depression and Cardiovascular Disease Study
Authors: Litinas Evangelos , Fareed Jawed , Iqbal Omer , Tobin Erin , Piletz John , Meresh Edwin , Halaris Angelos
Published: June 2021

Selective serotonin reuptake inhibitors (SSRI) are a first‐line treatment option for mood disorders such as depression (D). They inhibit neuronal reuptake of serotonin and result in depleted serotonin stores in the dense bodies of platelets. It is hypothesized that depressive illnesses cause platelet activation and endothelial dysfunction, which could be modulated by the use of SSRIs. To validate this hypothesis an institutional‐based co‐morbidity of D and cardiovascular (CV) study was undertaken. 25 healthy control subjects and 25 patients meeting the DSM‐IV criteria for major depressive disorder were recruited in this study. Blood samples were collected at baseline and then 4 and 8 weeks following treatment with Escitalopram. Agonist‐induced platelet aggregation showed varying degrees of inhibition of collagen, ADP, arachidonic acid and epinephrine‐induced platelet aggregation.

Agonist Induced Percent Platelet Aggregation

BL
4 weeks
8 weeks

Collagen
71.5±22.4
63.4±35.1
74.7±21.3

AA
64.1±35.1
67.1±25.1
55.7±36.1

ADP
48.2±30.9
33.0±26.7
52.4±31.0

EPI
55.6±29.2
37.4±25.8
50.4±32.3

Saline
6.9±4.5
9.4±7.8
5.0±3.9

These results indicate that treatment with SSRIs may inhibit platelet aggregation by some agonists, which may have an impact in the prevention of atherothrombotic and CV disease. SSRIs might alter hemostasis in some patients by inhibiting platelet function.