bluuebunny/crossref_metadata_2025 huggingface dataset:
Intrathecal nociceptin displaces a PET ligand for its receptor in the brain at analgesic doses in rhesus monkeys (658.1)
Authors: Saccone Phillip , Shao Xia , Sherman Phillip , Quesada Carole , Woods James , Winger Gail , Kilbourn Michael , Scott Peter
Published: June 2021

The nociceptin receptor (NOP) has been investigated as a potential therapeutic target for pain control. Nociceptin, the endogenous ligand for NOP, produces analgesia in rodents and primates when given intrathecally (i.t.) (Ko et al. 2006, Xu et al. 1996). Recently NOP radiotracers have been developed for use with Positron Emission Tomography (PET) (Kimura et al. 2011, Hostetler et al. 2013). Although behavioral studies have shown that NOP agonists may be therapeutically useful, it is unknown whether nociceptin given i.t. binds to receptors supraspinally, and if so, which areas in the brain display the greatest ligand‐target interaction. The purpose of this study was to measure the amount of radiotracer displacement in the brain at NOP following an analgesic dose of nociceptin. Two female rhesus monkeys were anesthetized, and underwent PET scans with the NOP radiotracer 11C NOP‐1A. Eight minutes following tracer delivery, the monkey was injected i.t. with either saline or 100 nM nociceptin. Preliminary data show that 100 nM nociceptin delivered into the spinal canal displaces the radiotracer 11C NOP‐1A in a variety of brain nuclei. Prominent regions of displacement include the cortex, striatum, and thalamus. These data suggest that analgesia with NOP agonists can be produced with relatively low receptor occupancy and further supports NOP as a novel drug target for analgesia.

Grant Funding Source: Supported by: The Michigan Memorial Pheonix Project and NIDA Traning Grant T32‐DA007281
Differential effects of lipopolysaccharide and cigarette smoke exposure on genes associated with contractile agonist responsiveness in mouse lungs (660.2)
Authors: Donovan Chantal , Seow Huei Jiunn , Royce Simon , Vlahos Ross , Bourke Jane
Published: June 2021

Introduction: Inflammation is a key driver of many lung diseases, including asthma and chronic obstructive pulmonary disease. Exposure to lipopolysaccharide (LPS) and cigarette smoke (CS) are two mouse models that induce inflammation, however studies assessing changes in expression of contractile receptors in these models are limited.

Methods: Female Balb/C mice were exposed to inhaled LPS once a day for 4 days or PBS. Male Balb/C mice were exposed to 9 cigarettes 3 times a day for 4 days and sham mice were exposed to air. On day 5, mice were euthanized, bronchoalveolar lavage fluid (BALF) was taken and lungs were harvested for PCR.

Results: Inflammation was confirmed in both LPS and CS models by an increase in total cell number in the BALF. In both groups, there were no changes in gene expression levels of serotonin 2A, bradykinin, or muscarinic M3 receptors. There were no changes in G‐protein expression or downstream inositol trisphosphate receptor expression. In the LPS‐treated mice, there was an upregulation of muscarinic M2 receptors and ryanodine isoform 2 receptors (RyR2). In contrast, these receptors were down regulated in CS mice.

Discussion: Despite both LPS and CS inducing inflammation, these stimuli have opposite effects on expression of M2 and RyR2 receptors. The influence of these changes on responsiveness to muscarinic and other contractile agonists remain to be determined.

Grant Funding Source: Supported by NHMRC
The effect of a novel TRPA1 antagonist JNJ‐41477670 on models of airway hyperactivity and inflammation in rats (660.6)
Authors: Bhattacharya Anindya , Eckert William , Ao Hong , Lebsack Alec , Rech Jason , Wickenden Alan
Published: June 2021

TRPA1 activation by respiratory irritants and pungent foods causes neurogenic inflammation, possibly contributing to wheezing, cough and dyspnea in humans. In this study we characterized JNJ‐41477670 as a TRPA1 antagonist and tested the efficacy in an allergen challenged model of airway inflammation and hyperreactivity in rats.

The potency of JNJ‐41477670 in blocking AITC induced calcium flux at the recombinant rat TRPA1 was 7.2 ± 0.1. JNJ‐41477670 produced a pharmacodynamic effect of mustard oil‐induced in‐vivo CGRP release and nocifensive behavior at an oral dose of 60 mg/kg. In the airways hyperreactivity study, exposure of rats to allergen caused a statistically significant increase airway resistance (RL) following administration of adenosine indicative of the development of airways hyperreactivity. JNJ舉41477670 (60 mg/kg) had no effect on the airways hyperreactivity to adenosine. In the pulmonary leucocyte accumulation study, exposure of sensitised rats to allergen caused a significant increase in pulmonary leucocyte accumulation. JNJ‐41477670, tended to reduce eosinophil accumulation in recovered BAL fluid; however, this reduction was not statistically significant when data were compared to vehicle treated animals. Our data indicates a pharmacodynamic effect of JNJ‐41477670 in mustard‐oil nocifensive model without any efficacy in the airway model of inflammation and hyperreactivity.
Differential generalization among nicotinic acetylcholine receptor agonists in nicotine, varenicline, and epibatidine drug discriminations in mice (661.11)
Authors: Moura Fernando , McMahon Lance
Published: June 2021

In addition to a nicotine discrimination assay, two novel nicotinic acetylcholine receptor (nAChR) agonist discriminations were established to examine the relative contribution of nAChR agonist efficacy to behavioral effects. Male C57Bl/6J mice were trained to discriminate nicotine (1 mg/kg), the low efficacy nAChR agonist varenicline (3.2 mg/kg), or the high efficacy nAChR agonist epibatidine (0.0056 mg/kg) from saline. Nicotine produced a maximum effect of 86% and 82% drug‐appropriate responding in the nicotine and varenicline discriminations, respectively. In mice trained to discriminate epibatidine, nicotine produced 84% drug‐appropriate responding, but at a dose that reduced the rate of responding to 19% of control. Varenicline produced 95% drug‐appropriate responding in the varenicline discrimination, but only a maximum of 46% and 70% drug‐appropriate responding in the nicotine and epibatidine discriminations, respectively. A t‐test showed that the maximum effect for varenicline was significantly lower (p<0.05) in the nicotine discrimination than in the varenicline discrimination. Epibatidine produced no less than 90% drug‐appropriate responding in all three discriminations. These data suggest that the discriminative stimulus effects of nAChR agonists vary as a function of the efficacy of the training drug. That nicotine only substituted for epibatidine at a dose of nicotine that markedly decreased response rate might suggest that epibatidine and nicotine have overlapping, but not identical, in vivo effects.

Grant Funding Source: USPHS DA25267
Running‐wheel access decreases ±3,4‐methylenedioxymethampetamine self‐administration in rats (661.6)
Authors: Aarde Shawn , Vandewater Sophia , Creehan Kevin , Taffe Michael
Published: June 2021

Running wheel access has been demonstrated to decrease the intravenous self‐administration (IVSA) of methamphetamine (MA). However, the acute effects of MA on running wheel activity and IVSA differ qualitatively from those of ±3,4‐methylenedioxymethampetamine (MDMA). The dose‐response curve for acute, non‐contingent administration of MA on wheel activity is bi‐phasic while MDMA causes monotonic decreases in running as dose increases. To determine the generalizability of wheel access to decrease the IVSA of amphetamines, we trained Wistar rats to self‐administer MDMA (1.0 mg/kg/inf; 2 hr/session M‐F) while vivarium wheel access (7 days/wk) was manipulated between groups. The IVSA of MDMA in the group with wheel access was less than that of the no‐wheel‐access group by the end of the 20‐session acquisition period. Additionally, a smaller percentage of rats within the wheel‐access group showed a clear dose‐response curve as determined over 10 dose‐manipulation sessions that immediately followed the acquisition period. These data indicate that the suppressive effect of running‐wheel access on amphetamine IVSA generalizes across drugs within the amphetamine class despite differences in their acute effects on wheel activity and IVSA.

Grant Funding Source: Supported by NIH Grant DA024105 and DA024705
Development of Gs‐biased modulators of the β2‐adrenergic receptor (662.1)
Authors: Carr Richard , Panettieri Reynold , Benovic Jeffrey
Published: June 2021

Asthma is a complex disease that is manifested by enhanced airway inflammation that leads to airway smooth muscle (ASM) contraction and airway constriction. While β‐agonists serve as a major asthma therapeutic, these drugs may also increase the risk of having a severe asthmatic attack that can result in death. Since several lines of evidence suggest that β2AR interaction with β‐arrestins may play a role in these adverse effects, biased ligands that selectively promote β2AR interaction with Gs may serve as an effective way of treating asthma. While an orthosteric Gs‐biased agonist for the β2AR has yet to be identified, another strategy to stabilize the conformation of a G protein‐coupled receptor (GPCR) is to target the intracellular surface of the receptor using pepducins. Pepducins are lipidated peptides derived from the intracellular domains of a GPCR. A library of β2AR pepducins was synthesized and screened for cAMP production and β‐arrestin recruitment and identified two classes of Gs‐biased pepducins: receptor‐dependent and receptor‐independent. The Gs‐biased pepducins exhibit minimal functional desensitization in cells and do not induce GPCR kinase (GRK) phosphorylation or internalization of the β2AR, although PKA‐mediated phosphorylation was observed. The receptor‐independent pepducins appear to directly activate Gs while the receptor‐dependent pepducins promote cAMP production that is sensitive to the inverse agonist ICI 118,551, suggesting that the pepducin is stabilizing a Gs‐biased conformation of the β2AR. These pepducins represent the first reported Gs‐biased modulators and offer a potential new class of asthma therapeutics.

Grant Funding Source: Supported by P01HL114471 (NHLBI) and T32GM100836 (NIGMS)
High‐throughput screening for novel allosteric modulators of the D1 dopamine receptor (662.4)
Authors: Conroy Jennie , Free R. , Doyle Trevor , Southall Noel , Hazelwood Lisa , Miller Nicole , Ferrer Marc , Sibley David
Published: June 2021

Dopamine is a critically important neurotransmitter in the CNS and peripheral nervous system, with five receptor subtypes mediating its effects. Importantly, D1 receptor (D1R)‐selective agents may prove beneficial in the treatment of many neuropsychiatric disorders. In an effort to identify novel selective allosteric modulators of the D1R we used a high throughput screening approach. 380,000 small molecules in the NIH Molecular Libraries Screening Center Network library were screened using a cell line expressing the D1R coupled to G15 resulting in a robust Ca2+ signal upon receptor stimulation. Hit compounds were triaged through secondary functional and radioligand displacement binding assays to determine subtype selectivity and their allosteric versus orthosteric nature. We initially identified 96 putative positive allosteric modulator (PAM) hits that enhanced the EC20 activity of dopamine in the Ca2+ response ‐ 6 of these were subsequently confirmed during triage and were chosen for further characterization. In addition we found approximately 115 agonist and 125 antagonist hits that failed to completely inhibit radioligand binding and have been classified as potential allosteric agonists and antagonists or negative allosteric modulators (NAMs). These compounds are currently being characterized using additional assays to confirm their selectivity, activity, and classification.

Grant Funding Source: NINDS Intramural Program
FeTTPs treatment reduces secondary injury associated in a mouse model of traumatic brain injury (663.3)
Authors: Impellizzeri Daniela , Bruschetta Giuseppe , Ahmad Akbar , Cordaro Marika , Siracusa Rosalba , Esposito Emanuela , Cuzzocrea Salvatore
Published: June 2021

Controlled cortical impact injury (CCI) is a widely used, clinically relevant model of traumatic brain injury (TBI). The aim of the present study was to assess the contribution of peroxynitrite formation in the pathophysiology of TBI in mice. To this purpose, we administered a peroxynitrite decomposition catalyst, 5,10,15,20‐tetrakis (4‐sulfonatophenyl) porphyrinato iron III chloride (FeTTPs) (100 mg/kg, i.p.) at 24 h after TBI. FeTTPs treatment significantly reduced (1) the degree of brain inflammation and tissue injury (histological score), (2) neutrophil infiltration (myeloperoxidase activity), (3) NO production, (4) proinflammmaory cytokines expression, (5) nuclear factor (NF‐κB) translocation and IκB‐α degradation, (6) apoptosis. Moreover, treatment with FeTTPs resulted in a significant improvement in motor and cognitive recovery after CCI, as well as in marked reduction of lesion volumes. Taken together, our results clearly demonstrated that FeTTPs treatment reduces the development of inflammation and tissue injury associated with TBI.
Application of Einstein matrix medical treatment for HIVv/HCV therapy (668.4)
Authors: Einstein George , Echavarria Louis , Tulp Orien
Published: June 2021

Biophotonic Therapy [Photo‐therapy] uses ultraviolet light, is a safe, non‐intrusive, drug free, cost effective, and nonspecific treatment for a broad spectrum of bacterial and viral infections Biophotonics generates and harnesses light (photons) to image, detect and manipulate biological materials, and can also be applied as a biological probe for molecular mechanisms, function and structure. Einstein Clinical Laboratories [ECL] has perfected a proprietary therapy of purification blood called Einstein's Matrix Treatment (EMT) also referred to as the EMC‐ 2001E [named for its developer & inventor, Dr. George P. Einstein]. The EMT approach is to target and destroy viruses or bacteria by the application of a specific amount of photon energy that is not harmful for healthy cells. The parameters that targeted the pathogens are specific wavelength, intensity and time of exposure to the light targets the physical & chemical structure of bacteria resulting in their death via a UV‐mediated intracellular coagulative process. Bacteria are much smaller than somatic cells, their shells are thinner, and are more susceptible to UV Light. Also, the amino acids phe and tyr are present in the micro‐organisms in 5x greater amounts than in somatic cells which are largely unaffected by the UV process. In pilot studies with HIV or both HIV and HCV viruses, patients remained asymptomatic and virus free throughout a 10 year followup. UVC irradiation of blood had an inhibitory influence on mRNA expression of inflammatory cytokines, IL‐1, IL‐6 whereas it stimulated production of IL‐b. Pro‐inflammatory cytokines are engaged in migration of cells to the inflammation area, as well as in stimulation, proliferation and differentiation of cells and a decrease in inflammatory markers. In conclusion, HVB was found to be safe and beneficial for patients, while UVC lends promise to the treatment of a broad range of infectious illnesses.

Grant Funding Source: Institutional Resources
The effect of cancer cells on angiogenesis: engineering the cancer niche (676.13)
Authors: Sweat Richard , Phamduy Theresa , Chrisey Douglas , Murfee Walter
Published: June 2021

Tumor growth requires uncontrolled angiogenesis, defined as the growth of new capillaries from existing blood vessels. Developing anti‐tumor therapies necessitates understanding how cancer cells and their microenvironment synergistically influence capillary sprouting from existing vascular networks. Recently, we demonstrated the use of the rat mesentery culture model for investigating angiogenesis in an intact microvascular network scenario. The objective of this study was to demonstrate the use of this model to determine the effects of cancer cells on angiogenesis. Mesenteric tissue windows were harvested from adult male Wistar rats and cultured for 5 days according to the following experimental groups: 1) Control (MEM + 10% DMEM + 1% FBS; n = 7 tissues) and 2) Cancer Cell (MEM + 10% DMEM + 1% FBS + cells; n = 6 tissues). For the cancer cell group, 2x10^5 RFP‐transfected metastatic MDA‐MB‐231 breast cancer cells were seeded onto tissues on day 0. On day 5, cancer cells remained viable, as confirmed by live/dead assay. More importantly, cells were observed to be in the same focal plane and region as vessels along intact microvascular networks. The presence of cancer cells decreased vascular length density and the number of capillary sprouts per vascular area compared to the Control (no cell) group. Our results suggest that cancer cells alone are not sufficient to induce angiogenesis. Application of the rat mesentery culture model as a multicellular breadboard system to engineer the cancer cell/vascular niche will offer a new tool for tumor angiogenesis research.

Grant Funding Source: Supported by Tulane Center for Aging and NIH 5‐P20GM103629‐02
Inhibition of the vascular renin‐angiotensin system preserves nitric oxide‐mediated vasodilation in human adipose arterioles after transient high pressure stress (676.9)
Authors: Durand Matthew , Beyer Andreas , Hockenberry Joseph , Gutterman David
Published: June 2021

Increased intraluminal pressure converts the mediator of flow mediated dilation (FMD) from nitric oxide (NO) to hydrogen peroxide (H2O2) in human adipose arterioles. We hypothesized that inhibition of the local renin‐angiotensin system (RAS) would preserve NO‐mediated dilation after acute high pressure (HP) stress. Microvessels were dissected from surgically discarded adipose tissue, cannulated onto glass pipettes, pressurized to 60 mmHg, and constricted with endothelin‐1. Vasodilation to incremental increases in flow was measured followed by exposure to an intraluminal pressure of 150 mmHg for 30 minutes ± the ACE inhibitor captopril (10 µM), the AT1R antagonist losartan (10 µM), or the ACE2 activator DIZE (10 µM). In untreated vessels, PEG‐catalase reduced maximum FMD (24±6.1% vs. 62±5.6%, p<0.05) following exposure to HP, whereas L‐NAME had no effect (72±5.9% vs. 62±5.6%). When HP was generated in the presence of losartan, captopril, or DIZE, L‐NAME inhibited FMD (19±6.8%; 43±20.3%; 14±15.6% respectively; p<0.05 vs. control). These results indicate that the vascular RAS is activated by increased intraluminal pressure, and plays a role in switching the mechanism of FMD from an NO‐mediated response to one mediated by H2O2.
Dichotomous estradiol and genistein effects on cutaneous endothelium‐dependent vasodilation in women with and without insulin resistance (678.6)
Authors: Wenner Megan , Taylor Hugh , Stachenfeld Nina
Published: June 2021

17β‐estradiol (E2) cardioprotection in women includes endothelial dependent dilation (EDD), but this E2 effect is not consistent in insulin resistant women (IR). Genistein (GEN, a phytoestrogen structurally similar to E2) effects on EDD are unclear. We hypothesized E2 and GEN improve cutaneous EDD in healthy women [C, n=8, 23 (6) yr, BMI 25.0 (8.0), AUC insulin 6111 (1368) µUI/ml] but not IR [n=6, 20 (2) yr, BMI 27.3 (8.4), AUC insulin 140006 (4020) µUI/ml]. We measured cutaneous vascular conductance (CVC) with laser Doppler flowmetry during local 42°C heating with E2 or GEN skin microdialysis infusions, each with and without the eNOS inhibitor L‐NMMA; nitroprusside was then infused with 44°C heating to induce CVCmax. In C, E2 enhanced EDD [93.4 (5.8) %max, 85.0 (12.2) %max for E2, saline P<0.05], with no GEN effect [84.5 (22.2) %max], and L‐NMMA reduced EDD [81.9 (21.6) %max, GEN 73.2 % (29.1) %max P<0.05). In IR, E2 and GEN did not affect EDD [93.5 (5.5), 93.6 (5.2), 97.4 (13.0), saline, E2, GEN]; L‐NMMA reduced EDD with GEN [67.1 (18.7) %max P<0.05] but not E2 [90.5 (5.5)]. E2 and GEN did not enhance EDD in women with IR in contrast to healthy women. However, NO contributes to EDD in both groups.

Grant Funding Source: Supported by National Institutes of Health HL109882
Vascular reactivity is altered in mice with a conditional knockout of relaxin receptor in endothelial cells (681.11)
Authors: Woodward Lawrence , Rusnak Margaret , Ramirez Rolando , Huang Zaohua , Lopez Carolina , Agoulnik Alexander , Conrad Kirk , Novak Jacqueline
Published: June 2021

The ovarian hormone relaxin (RLX) has been traditionally thought of as a reproductive hormone; however, the effects of RLX outside the reproductive organs have gained a better appreciation. Relaxin is a vasodilator and increases arterial compliance. These beneficial effects have opened up the therapeutic potential for RLX and have focused our studies on the physiological role for arterial‐derived RLX. The RLX receptor RXFP1 has been identified in both endothelium and vascular smooth muscle. For this study, conditional knockout mice with RXFP1 ablation in endothelial cells were used. Floxed allele of RXFP1 was deleted using endothelial‐specific Cdh5‐cre transgene. The littermate control group maintained functional RXFP1 receptor gene. Mice lacking the RXFP1 receptor in their endothelium displayed an altered vascular reactivity compared to control animals. Isolated resistance sized renal interlobar arties were studied in a isobaric arteriograph. Myogenic reactivity was reduced in control mouse arteries treated with L‐arginine but not in those arteries treated with D‐arginine. Percent change in diameter was 5.8±0.8% with L‐arg compared 0.90±.8% to D‐arg treated arteries. However, the arteries isolated from conditional knockout mice failed to respond to L‐arg and remained very myogenic (‐0.81±.2%). These data suggest that the endothelial RLX receptor is important for the reduced myogenic response to RLX.
Selective activation of agouti‐related protein neurons rapidly decreases arterial pressure and sympathetic nerve activity in mice (682.4)
Authors: Shi Zhigang , Brooks Virginia
Published: June 2021

In addition to stimulating food intake, Neuropeptide Y (NPY) acts in the hypothalamic paraventricular nucleus (PVN) to decrease sympathetic nerve activity (SNA) and arterial pressure (AP). PVN NPY inputs arise from the brainstem and the arcuate nucleus (ArcN), with the latter population co‐expressing agouti‐related protein (AgRP). However, whether ArcN AgRP/NPY neurons are sympathoinhibitory is unknown. To test this hypothesis, we employed designer receptors exclusively activated by designer drugs (DREADD) technology to specifically activate ArcN NPY/AgRP neurons in mice. In this approach, mutated stimulatory muscarinic receptors (hM3Dq) are insensitive to endogenous ligands, but following binding of the otherwise inert ligand, Clozapine‐N‐oxide (CNO), depolarize neurons. Cre‐recombinase‐dependent adeno‐associated virus (AAV)‐hM3Dq‐mCherry was nanoinjected bilaterally into the ArcN of AgRP‐Ires‐cre mice (AgRP‐Cre mice). After ~2 weeks, to functionally confirm AgRP neuronal hM3Dq expression, AgRP‐Cre mice (n=5) received ip injections of CNO (0.3 mg/kg), to activate AgRP neurons, or on another day, ip saline. After 4 hr, compared to ip saline, ip CNO increased food intake (0.95±0.07 g, CNO; 0.59±0.07 g, saline; P<0.05) and body weight (change in body weight: 0.23±0.1 g, CNO; ‐0.38±0.07 g, saline; P<0.05). Next, in AgRP‐Cre α‐chloralose‐anesthetized mice (n=3), ip CNO rapidly decreased mean AP (MAP; ‐14.1±0.3 mmHg; P<0.05), heart rate (HR; ‐42±4 bpm; P<0.05) and splanchnic SNA (SSNA; ‐41±2% control; P<0.05), and these effects were sustained for at least 1 hr; ip saline had no effects. In WT mice, neither saline nor CNO significantly altered MAP, HR or SSNA. In conclusion, direct activation of ArcN AgRP/NPY neurons is sufficient to decrease SNA, HR and MAP.

Grant Funding Source: Supported by HL088552 and AHA.
Oxytocin’s role in salt appetite revealed by PEP7 blockade of angiotensin II action (683.1)
Authors: Yosten Gina , Liu Jun , Ji Hong , Sandberg Kathryn , Samson Willis
Published: June 2021

The paradoxical mechanism by which angiotensin II (Ang II) stimulates saline intake and simultaneously releases oxytocin (OT), which profoundly inhibits Ang II‐induced saline intake, is not known. We discovered that a 7 amino acid peptide (PEP7) encoded by a short open reading frame in the 5’ leader sequence of the angiotensin type 1a receptor (AT1aR) mRNA blocks saline, but not water, intake induced by Ang II by abrogating the non‐G protein‐coupled Erk1/2 signaling pathway. We hypothesized that blockade of the MAP‐kinase‐dependent pathway uncovers the inhibitory action of OT released in response to Ang II. Pretreatment icv with PEP7 blocks the saline intake response and this can be reversed by subsequent icv administration of the OT antagonist, OVT. Thus Ang II controls electrolyte intake via two unique pathways, one stimulatory (MAP‐kinase dependent, blockable by PEP7), and the other inhibitory (G protein‐dependent, via OT release). These results together with in vitro data demonstrating PEP7 selective inhibition of the MAP‐kinase‐dependent signaling pathway suggests that PEP7 is a novel therapeutic target for pathologies involving MAP‐kinase dependent, non‐G protein‐coupled signaling of Ang II.

Grant Funding Source: NIH AG/HL 19291, HL57502, AG039779, AG16902, HL066023
Influence of TRPV1 and/or TRPV4 receptors on osmoregulatory thirst in mice (683.2)
Authors: Kinsman Brian , Cowles James , Browning Kirsteen , Stocker Sean
Published: June 2021

Recent in vitro studies suggest that hypothalamic osmolality‐sensing is mediated by either the transient receptor potential vanilloid‐1 (TRPV1) or vanilloid‐4 (TRPV4) channel expressed by neurons in the organum vasculosum of the lamina terminalis (OVLT). This study assessed whether deletion of TRPV1 and/or TRPV4 channels altered thirst responses to hyperosmolar stimuli. Wild‐type (WT), TRPV1‐/‐, TRPV4‐/‐, and TRPV1‐/‐V4‐/‐ mice (n = 9‐12 per genotype) ingested similar volumes of water 1 hr after sc injection (0.3mL) of 0.5M NaCl (0.37±0.03, 0.39±0.04, 0.42±0.04, 0.38±0.05 mL; P > 0.4) or 1.0M NaCl (0.89±0.06, 0.95±0.04, 1.07±0.11, 1.02±0.15 mL; P > 0.4). Similar results were observed after injection of 0.7M mannitol (0.38±0.06, 0.36±0.02, 0.31±0.04, 0.33±0.05 mL; P > 0.4). In addition, sole access to 2% NaCl for 48 hrs yielded similar increases in water intake across strains (0.22±0.03, 0.31±0.07, 0.21±0.02, 0.18±0.05 mL; P > 0.4). In a final experiment, injection of 0.5M NaCl generated similar increases in the number of OVLT Fos‐positive nuclei (21±4, 23±4, 16±4, 15±2; P > 0.2) across all genotypes. This investigation suggests that TRPV1 and TRPV4 receptors are not the central osmolality‐sensors that mediate osmotically‐stimulated thirst.

Grant Funding Source: NIHHL113270, AHA EIA
Psychobiological assessment in offspring of Wistar rats supplemented with sodium selenite during the period of pregnancy and lactation (684.13)
Authors: Laureano‐Melo Roberto , Imperio Guínever , Almeida Claudio , Kluck George , Seara Fernando , Reis Luis , Ortiga Tania Maria , Cortes Wellington
Published: June 2021

The objective of this study was to evaluate the possible occurrence of behavioral changes in offspring of female rats supplemented during pregnancy and lactation with sodium selenite. Two groups were treated during pregnancy and lactation with: saline or sodium selenite at a dose of 1 mg/kg through oral route. Thyroid status was determined by serum analysis of thyroid hormones and for gene expression analyzes, we used RT‐qPCR. At 60 days‐old, the offspring underwent a behavioral assessment in an open field test, elevated plus maze test, object recognition task and swimming forced test for five minutes. Statistical analysis was performed by t student test, considering the significant differences between means at p <0.05. According to the results, it was demonstrated that supplementation with Se causes increased levels of triiodothyronine (25%, p = 0.0001) and the expression of type 2 deiodinase in brown adipose tissue (351%, p = 0.002). Moreover, Se treatment promotes reduction of TPH2 expression (12%, p = 0.007), but increases the levels of mRNA for TH (200%, p = 0.002) in the hippocampus. It was also found that the offspring of females supplemented with Se have higher locomotor activity (50%, p = 0.004) and more rearing (77%, p <0.0001) in the open field test. In the elevated plus maze, the treated group had higher time spent in the open arms (143%, p = 0.005). Finally, through the object recognition task, the programmed offspring had a higher absolute index of discrimination (230%, p = 0.02). The results demonstrate that maternal supplementation with sodium selenite affects the physiological systems involved with learning and expression of affective behavior in adulthood. Therefore, we believe that the behavioral profile observed can be related to neurochemical changes during the critical period of the central nervous system ontogeny.

Grant Funding Source: FAPERJ/CNPq
Maternal omega‐3 fatty acid consumption decreases offspring reactivity to the stress of a novel environment (684.3)
Authors: Jackson Corey , Barrett Douglas , Shumake Jason , Gonzalez‐Lima Francisco , Lane Michelle
Published: June 2021

Prenatal supplementation with omega‐3 fatty acids (O3FAs) is common because O3FAs are integral to fetal brain development. Our objective was to assess effects of prenatal O3FA consumption on offspring behavior using a rat model. Pups were obtained from the second litter of mothers that consumed diets with or without O3FAs for two cycles of gestation and lactation. The without‐O3FA diet contained 7% sunflower oil, which depletes maternal brain O3FA levels. The with‐O3FA diet contained 7% menhaden oil (26% O3FAs). Pups were weaned at age 21 days to the facility chow diet, which contained 0.2 g O3FA/100 g. Behavioral testing began at 8 weeks of age. The offspring were tested in the open field (OF) to examine locomotor reactivity to the stress of being placed in a novel environment, the holeboard task (HBT) to assess memory, and the forced swim test (FST) to assess depression‐related behavior. Offspring from O3FA‐consuming mothers were less reactive to novel OF stress than pups from O3FA‐deficient mothers, F(1,16) = 9.613, p = 0.007. In the HBT, offspring whose mothers ate O3FAs tended to have improved working memory, F(1,15) = 3.03, p = 0.10, but more subjects are needed for statistical power. Maternal O3FA intake did not influence offspring immobility in the FST. Taken together, these data indicate that maternal consumption of O3FAs during gestation and lactation may dampen stress reactivity and improve the memory of offspring.

Grant Funding Source: Research Enhancement Program of Texas State University
Stimulation of endoplasmic reticulum stress and inflammation by neuronal (pro)renin receptor is mediated by Toll‐like receptor 4 activation (686.32)
Authors: Shan Zhiying , Yuan Wei , Qi Xaioli , Chen Qinghui
Published: June 2021

Our previous studies demonstrated that brain neuronal prorenin receptor (PRR) contributes to hypertension via NF‐KappB (KB)‐cytokine signaling. This finding coupled with the important role of toll like receptor 4 (TLR4) in inflammation and endoplasmic reticulum (ER) stress suggest that brain neuronal PRR activation, in part, stimulates TLR4 and subsequently induces ER stress and NF‐KB‐cytokine signaling. Therefore blocking TLR4 activation should attenuate the cytokines and ER stress response induced by PRR activation. To test this hypothesis, primary neuronal cultures from rat brain were incubated with 20 nM of renin for 6 hours. Real time PCR was performed to analyze the expression of inflammatory cytokines, NF‐KB and ER stress markers. Renin treatment resulted in significant increases in IL1β (1200‐fold), IL6 (33‐fold), TNFα (17‐fold), NF‐KB (6‐fold) and I‐kappa B kinase (IKK) (7‐fold). In addition, the mRNA levels of ER stress marker Ddit3 and Hspa3 increased by 47%and 41% in renin treated neurons. Quinazoline (10µM), a NF‐KB activation inhibitor, effectively blocked renin‐induced increases in IL1β, IL6, TNFα and NF‐KB, by more than 85%, but failed to inhibit IKK, Ddit3 and Hspa3. In contrast, renin stimulation on all these genes was almost entirely blocked (>90%) by a TLR4 inhibitor TAK‐242 (2 µM), indicating an important role of TRL4 in this newly identified PRR mediated activation of ER stress and inflammation signaling. Further study will probe whether NF‐KB‐inflammation is mediated by ER stress in the context of hypertension.

Grant Funding Source: AHA 11SDG7420029
Exogenous thyroxine (T4) attenuates glucose intolerance and insulin resistance in obese Otsuka Long‐Evans Tokushima Fatty rats (688.12)
Authors: Vazquez‐Anaya Guillermo , Martinez Bridget , Nakano Daisuke , Nishiyama Akira , Ortiz Rudy
Published: June 2021

Thyroid hormones (TH) regulate metabolism and their levels are altered with the development of metabolic syndrome (MetS), which is associated with insulin resistance (IR) and hypertension. TH analogues and derivates may be effective treatments for glucose intolerance and IR, but their effects on the hypertension associated with MetS are not well described. To assess the effects of thyroxine (T4) on glucose regulation and systolic blood pressure (SBP), an oral glucose tolerance test (oGTT) was performed and arterial pressure monitored on four groups of rats (n=7‐8): 1) lean control strain, LETO, 2) obese OLETF, 3) LETO + T4 (8.0µg/100g BW/d x 5wks), and 4) OLETF + T4. While a strain effect on SBP was detected, T4 did not alter the pressure. T4 attenuated glucose intolerance in OLETF rats by 15 %, and improved insulin clearance by 33%. IR index (IRI) decreased 34% in treated OLETF compared to untreated OLETF. TH profiles were measured to assess the status of the hypothalamus‐pituitary‐thyroid axis. Total T4 (60 ± 11 vs. 30 ± 7 nmol/L) and total triiodothryonine (T3)(1.5± 0.2 vs. 0.67 ±0.13 nmol/L) were higher in OLETF compared to LETO. The results suggest that despite a hyperthyroid state in OLETF rats, improvements in glucose intolerance and IR with exogenous T4 were not sufficient to ameliorate the hypertension associated with the development of MetS. Furthermore, the data suggest that the associated hypertension in this model is not a consequence of metabolic derangement, but likely other factors independent of glucoregulation.

Grant Funding Source: National Institute of Health (NIH)
High mobility group box protein 1 as a novel vascular permeability factor derived from endothelial inflammasome activation during obesity (688.5)
Authors: Chen Yang , Pitzer Ashley , Li Xiang , Xu Ming , Zhang Yang , Li Pin‐Lan
Published: June 2021

The early onset of type II diabetes in obese individuals is characteristic of increased vascular permeability leading to diabetic vasculopathy. However, it remains unknown how obesity instigates endothelial dysfunction resulting in vascular hyperpermeability. Here, we tested a hypothesis that visfatin, an injurious adipokine activates endothelial Nlrp3 inflammsome to produce high mobility group box protein 1 (HMGB1) and that HMGB1 serves as a novel vascular permeability factor to mediate the onset of obesity‐associated vascular hyperpermeability. In coronary arteries of Nlrp3+/+ mice fed high fat diet (HFD) for 6 weeks, confocal microscopic analysis showed significantly lower expression of gap junction proteins (GJPs) including VE‐Cadherin, ZO‐2 and occludin in the coronary arterial endothelium compared to mice on normal diet (ND). In vivo assessment of vascular integrity demonstrated that more Evans blue was extravasated into the myocardium in mice on HFD compared to mice on ND. However, such HFD‐induced decrease in endothelial GJP level and increase in vascular permeability were significantly attenuated in Nlrp3‐/‐ mice. Accompanied with these vascular permeability alterations, interestingly, HMGB1 expression was elevated in coronary arterial endothelium of HFD‐treated Nlrp3+/+ mice, but not in Nlrp3‐/‐ mice. In cultured mouse microvascular endothelial cells (MVECs), visfatin markedly decreased expression of GJPs and increased endothelial permeability. These visfatin‐induced changes in GJPs and endothelial permeability were blocked when MVECs were treated with HMGB1 inhibitor glycyrrhizin, caspase‐1 inhibitor WEHD or siRNA targeting HMGB1 receptor RAGE, respectively. Together, these data suggest that during obesity Nlrp3 inflammasome is activated due to the action of increased adipokines such as visfatin, which produces HMGB1 to serve as a vacular permeability factor leading to the onset of diabetic vasculopathy and ultimately resulting in atherosclerosis.

Grant Funding Source: Supported by NIH grants HL057244, HL091464 and HL075316
Abstract withdrawn. (689.7)
Authors: Withdrawn Abstract
Published: June 2021

Urinary Sodium Excretion Decreases In the Early Stages of Insulin Resistance In Model of Metabolic Syndrome Melissa E. Williams1, Jacqueline N. Minas1, Ruben Rodriguez1, Daisuke Nakano2, Akira Nishiyama2, and Rudy M. Ortiz11Department of Molecular & Cellular Biology, School of Natural Science, University of California Merced 2Department of Pharmacology, Kagawa Medical University, Kagawa, Japan Type II diabetes is associated with abnormal activation of the renin‐angiotensin aldosterone system and impaired sodium (Na+) regulation contributing to volume dependent hypertension. However, the mechanisms contributing to elevated systolic blood pressure (SBP) during the development of insulin resistance (IR) are not well defined. To examine the effects of the onset of IR to impaired UNaV and SBP, three groups of rats were studied for 6 weeks (n=6/group); 1) non‐IR, Long Evans Tokushima Otsuka (LETO), 2) untreated, IR Otsuka Long Evans Tokushima Fatty (OLETF), 3) OLETF + angiotensin receptor blocker (ARB; 10 mg olmesartan/kg/d for 3 weeks). Systolic blood pressure (SBP), urine Na+ excretion (UNaV), and insulin resistance index (IRI) were measured at 9 and 15 weeks of age. At 9 weeks of age, the OLETF group exhibited a 43% increase in IRI compared to the LETO group. However, no differences in SBP and UNaV between LETO and OLETF were observed. At 15 weeks of age, the OLETFs experienced a 54% increase in IRI, accompanied by a 15% (145 mmHg) increase in mean SBP at 15 weeks, which was attributed to the 46% decrease in mean UNaV. This suggests that IR causes impaired renal Na+ regulation, contributing to the onset of hypertension. Conversely, ARB treatment in OLETFs reduced SBP by 25% and increased UNaV by 50%, as well as a 24% decrease in IRI. This indicates that inappropriate activation of AT1 contributes to IR‐associated hypertension observed in OLETF.
Role of soluble epoxide hydrolase in experimental obstructive nephropathy (690.11)
Authors: Chiang Chin‐Wei , Lee Tzong‐Shyuan
Published: June 2021

Inhibition of soluble epoxide hydrolase (sEH), is abundantly expressed in kidney, retard the inflammatory disease including acute kidney disease. However, the role of sEH in progression of chronic kidney disease, such as obstructive nephropathy, is still elusive. In current study, 8‐week‐old wild‐type (WT) and sEH knockout (KO) mice were subjected to the unilateral ureteral obstruction (UUO) surgery and the pathology was analyzed by western blotting, ELISA, and histological examination. The protein level of sEH in kidney was increased in UUO group compared to the sham group. Additionally, UUO induced an increase in hydronephrosis, renal tubular injury, glomerulosclerosis, inflammation and fibrosis in WT mice; however, this increase but not glomerulosclerosis was ameliorated in sEH KO mice. Moreover, sEH KO mice with UUO showed lower levels of fibrosis‐related or inflammation‐related protein such as collagen1A1, α‐actin, transforming growth factor‐β, interlukin‐1β (IL‐1β), IL‐6, monocyte chemoattractant protein‐1, macrophage inflammatory protein‐2 and inducible nitric oxide synthase than in WT mice. Collectively, our findings suggest that sEH may play an important role in the pathogenesis of experimental obstructive nephropathy and inhibition of sEH may be a therapeutic target for the treatment of obstructive nephropathy‐related diseases.
Effect of bee’s propolis on fasting blood sugar and its antioxidant properties in diabetic rats (693.6)
Authors: Usman Umar
Published: June 2021

In Africa, hundreds of plants and plant products are used traditionally for the management of diabetes mellitus. This study was undertaken to evaluate the anti hyperglycemic effects and anti oxidative properties of Bee’s Propolis in diabetic Wister rats. Thirty five adult male Wister rats weighing (120‐ 180) grams of about 18 to 20 weeks of age were used. The animals were assigned into seven groups of five rats each. Group VII(untreated DM), served as the positive control group receiving 0.9% normal saline( 5mls/kg) alone via oral rout. Group 1 (normoglycemic) while groups II, III, IV, V and VI were treated with alloxan and after the induction of diabetes received orally, 250mg/kg(Metformin), 100mg/kg, 200mg/kg, 400mg/kg(Bee’s Propolis) and 100mg/kg( Vitamin C) daily for four weeks. FBS are recorded weekly and serum is taken on the last day for oxidative stress markers. The results showed 100mg/kg and 400mg/kg of Bee’s Propolis Significantly decrease fbs Levels to (4.62±0.32mmol/l) and (4.22±0.14mmol/l) compared with group II as (5.94±0.21mmol/l) and group vii (22.67±1.14mmol/l). While 200mg/kg and 400mg/kg of Bee’s Propolis significantly decreased Malandoaldehyde to (1.91±0.14nmol/ml) and (1.89±0.14nmol/ml), compared to group II and group V as (2.39 ±0.15) and (1.83±0.16). Serum activities of catalase significantly increased in group III, IV and V (57.14±2.48ug/mgprot), (55.29±4.71u/mgprot) and (58.71±2.24u/mgprot) compared with group VI and group II as (39.86±3.03u/mgprot) and (54.86±1.08u/mgprot). Serum glutathione peroxidate was significantly lower in the group II and group VI (36.86± 3.91u/mgprot) and (46.43±4.25u/mgprot) compared with the groups treated with different doses of Propolis as (44.86±1.82u/mgprot), (42.14±2.96u/mgprot) and (43.00±2.50u/mgprot). The results of the study showed that Bee’s Propolis Possesses anti‐ hyperglycemic and ant oxidative potentials which doe’s not depend on the dose administer.

Grant Funding Source: USMANU DANFODIYO UNIVERSITY SOKOTO, NIGERIA
Endurance exercise protects cardiac muscle against doxorubicin‐induced damage via mitochondrial adaptations (706.1)
Authors: Kavazis Andreas , Smuder Ashley , Powers Scott
Published: June 2021

Doxorubicin (DOX) is an antitumor agent used in cancer treatment. Unfortunately, DOX can induce cardiotoxicity that limits its clinical use. While endurance exercise training protects against DOX‐induced cardiotoxicity, the mechanisms for this exercise‐induced cardioprotection remain elusive. Therefore, we tested the hypothesis that exercise‐induced increases in the activity of cardiac AMPK can mediate alterations to downstream pathways protecting the heart from DOX‐induced damage. Rats were assigned to sedentary or exercise groups and paired with either placebo or DOX treatment. Exercise prior to DOX administration induced significant increases in the ratio of cardiac pAMPK/AMPK resulting in increased mRNA and protein levels of PGC‐1α in the heart. Cardiac PGC‐1α levels remained elevated in exercised animals treated with DOX. Also, exercise increased expression of the PGC‐1α‐sensitive mitochondrial fusion protein Mfn2 in the heart, which remained elevated in hearts of exercised animals treated with DOX. These findings reveal that exercise‐induced protection against DOX‐induced cardiomyopathy is associated with increases in myocardial levels of PGC‐1α that regulate the expression of proteins essential for mitochondrial function.
Claudin‐5 increases alveolar permeability in alcoholic lung syndrome by destabilizing claudin‐18/zonula occludens‐1 interactions (716.4)
Authors: Schlingmann Barbara , Ward Christina , Molina Samuel , Overgaard Christian , Guidot David , Koval Micheal
Published: June 2021

We found that alcohol abuse increases the severity of acute respiratory distress syndrome (ARDS) by impairing alveolar epithelial cell (AEC) tight junctions. In a rat model of chronic alcohol ingestion, decreased AEC barrier function was accompanied by increased claudin‐5 (cldn‐5) and claudin‐18 (cldn‐18) expression. Immunofluorescence of model type I AECs cultured on Transwell permeable supports showed that paracellular leak in cells from alcohol fed rats was accompanied by disrupted tight junction morphology. We then used Stochastic Optical Reconstruction Microscopy (STORM) which increases the resolution of immunofluorescence to 20 nm. STORM revealed that alcohol caused a significant decrease in co‐localization between AEC cldn‐18 and ZO‐1 along with increased co‐localization between cldn‐18 and cldn‐5. Thus, cldn‐5 binding to cldn‐18 inhibits binding to ZO‐1 which destabilizes tight junctions. We found that shRNA targeting to decrease cldn‐5 expression improved AEC tight junction morphology and function. Conversely, transfecting cells to increase expression of YFP‐tagged cldn‐5 altered tight junction morphology and impaired barrier function. Thus, increased cldn‐5 was necessary and sufficient to inhibit cldn‐18 assembly and function. These data suggest that targeting cldn‐5 may provide a therapeutic strategy to improve barrier function in ARDS.

Grant Funding Source: Emory URC, NIH‐NIAAA, NIH‐NHLBI, DAAD
Histologic and biochemical alterations predict mechanical dysfunction in aging and chronically inflamed mice (717.4)
Authors: Massa Christopher , Groves Angela , Laskin Debra , Gow Andrew
Published: June 2021

Aging produces structural and biochemical alterations to the lung, which impact work of breathing. Mice deficient in surfactant protein D (SP‐D) develop progressive inflammation characterized by increased tissue destruction/remodeling, surfactant composition alterations and retention of foamy macrophages. We have previously reported such alterations in 8, 27 and 80 week old C57BL/6 and congenic SP‐D‐/‐ mice (Groves et al., 2012). In prior studies, alterations to pulmonary function have been inferred from changes in estimated parameters from simple models, however interpretation has been confounded by the simultaneous presence of conflicting pathophysiologic processes. In contrast to the inverse modeling approach, this study uses first‐principles simulation from experimental observations to recapitulate how aging and inflammation alter respiratory impedance (Zrs) at airway opening. An anatomic computational model was developed based on airway morphometry in the literature and Zrs simulated between 0.5 and 20 Hz. Upper airway dimensions were derived from microCT (Thiesse et al., 2010), while lower airways were based on casts (Gomes and Bates, 2002). Airway caliber and patency varied with end expiratory pressure and parenchymal tethering based on elastance changes. Tissue elements were simulated using the constant phase model of viscoelasticity. Baseline elastance was estimated in 8 wk WT mice, and varied for each experimental condition based on alteration in parenchymal composition, alveolar geometry and surfactant composition. Reduction in model error is weighed against increasing model complexity to determine essential features underlying mechanical pathology and their contribution to Zrs.

Grant Funding Source: NIH HL086621, ES004738, ES007148