bluuebunny/crossref_metadata_2025 huggingface dataset:
Buprenorphine analgesia requires exon 11‐associated mu opioid receptor splice variants (659.11)
Authors: Grinnell Steven , Majumdar Susruta , Ansonoff Michael , Pintar John , Pan Ying‐Xian , Pasternak Gavril
Published: June 2021

Buprenorphine is a potent analgesic with an attractive side effect profile. Although it is considered a partial mu opioid receptor (MOR) agonist and a weak partial agonist or antagonist at kappa opioid receptors, it also competes MOR exon 11‐associated IBNtxA binding with high affinity. We therefore sought to examine the roles of exon 11‐ and exon 1‐associated MOR splice variants in buprenorphine analgesia. Exon 11 knockout animals showed a profound loss of analgesia in the tail flick assay at doses over 300 times the analgesic ED50 in wildtype C57 controls, despite the fact that these animals have intact full length MOR‐1 receptors and other opioids such as morphine and methadone retain full analgesic efficacy. Furthermore, administration of a high dose of buprenorphine actually reversed morphine analgesia in these animals. In exon 1 or triple opioid knockout (MOR exon 1, DOR, and KOR) animals, analgesia was also significantly reduced relative to wildtype controls, suggesting that full buprenorphine analgesic efficacy requires both exon 11‐ and exon 1‐associated MOR splice variants, and that the pharmacology of buprenorphine is more complicated than previously believed.

Grant Funding Source: Supported by DA02615, DA06241, DA07242 (GWP); DA013997 (YXP); and the PhRMA Foundation (SGG)
A glycosylation‐specific interaction between the Sigma‐1 and Neuorkinin‐1 receptors (659.13)
Authors: Weaver Sara , Ruoho Arnold , Yang Jay
Published: June 2021

The putative chaperone protein Sigma‐1 Receptor (S1R) has been implicated in chronic pain via pharmacological and gene knockout studies, however, how S1R affects pain is not known. Activation of the GPCR Neurokinin‐1 Receptor (NK1R) is implicated in pain signaling. Herein we demonstrate a novel robust interaction between S1R (FLAG‐tagged) and NK1R (HA‐tagged) using co‐immunoprecipitation (co‐IP) in transiently transfected HEK293 cells. NK1R are glycoproteins with several mobility‐shifted immunoreactive bands present on a Western blot. Co‐IP demonstrates specific interaction between S1R and only the non‐glycosylated NK1R. This specific interaction was confirmed by tunicamycin‐treatment to eliminate the glycosylation and by expression of a glycosylation‐deficient NQ‐NK1R mutant. Additionally, we assay a panel of GPCRs for S1R interaction, (including the NK2R, NK3R, MOR, DA1, and MRGR), and demonstrate that all of the interactions are restricted to the non‐glycosylated GPCR. We conclude that S1R acts as a protein chaperone by a selective interaction with the non‐glycosylated GPCR proteins possibly providing insight on how S1R may influence nociceptive transmission.

Grant Funding Source: Supported by the National Institute of General Medical Sciences
Effects of isoflurane on dopaminergic neurotransmission (659.8)
Authors: Bonvicino Christina , Baumgart Joel , Zhou Zhenyu , Hemmings Hugh
Published: June 2021

Volatile anesthetics have poorly understood mechanisms of action, despite their wide usage in medicine. Current knowledge of the molecular and cellular mechanisms of anesthetic action is insufficient to explain amnesia, unconsciousness or immobilization, the principal features of general anesthesia. The volatile anesthetic isoflurane alters synaptic transmission by blocking ionotropic glutamate receptors postsynaptically and/or by enhancing fast inhibitory transmission and tonic currents mediated by GABAA receptors. However, dopaminergic neurons, which are primarily found in the ventral tegmental area and the substantia nigra pars compacta, play roles in cognition, attention, voluntary movement, among other functions, pose another potential site of anesthetic action. Here we test the hypothesis that isoflurane perturbs the stimulus‐secretion coupling between [Ca2+]i and exocytosis in dopaminergic neurons by probing synaptic vesicle exocytosis and nerve terminal [Ca2+]i. Laser‐ scanning fluorescence image measurements of [Ca2+]i in neonatal rat dopaminergic neurons were made using the cell permeant calcium indicator Fluo‐5N and action potential‐evoked exocytosis was measured with vesicular monoamine transporter‐ pHluorin (vMAT‐pH). The effects of isoflurane on [Ca2+]i and exocytosis at varying external [Ca2+] reveal a change in the Ca2+ sensitivity of exocytosis. These data suggest that isoflurane inhibits dopamine release from synaptic vesicles by shifting the coupling between [Ca2+]i and exocytosis.
Autotaxin mRNA and protein expression and regulation by salmeterol xinafoate and fluticasone propionate in lung cells (660.13)
Authors: Toews Myron , Schulte Nancy
Published: June 2021

Autotaxin (ATX) is the lyso‐phospholipase D enzyme that catalyzes the final step in the synthesis of the lipid mediator lysophosphatidic acid (LPA). ATX, LPA, and LPA receptors in the lung are all strongly associated with the pathology of pulmonary fibrosis, and increasing evidence implicates these same mediators as contributors to asthma. Accordingly, we hypothesized that the asthma drugs salmeterol xinafoate (SX), a long‐acting beta‐2 agonist bronchodilator, and fluticasone propionate (FP), an anti‐inflammatory steroid, would reduce the expression of ATX in lung cells. Cells were treated for 72 hr in the absence or presence of the drugs, alone or in combination. ATX protein assessed by Western blotting of culture medium and ATX mRNA assessed by qPCR were not detected in primary human bronchial epithelial cells or the Beas‐2B bronchial epithelial cell line. ATX protein but not mRNA was detected for human A549 alveolar adenocarcinoma cells and mouse LA4 cells, both of which represent alveolar rather than airway epithelium. Both SX and FP decreased ATX protein expression in these alveolar cells. Stronger ATX protein and mRNA signals were observed in mesenchymal cells, both primary human airway smooth muscle (HASM) cells and the HFL‐1 human fetal lung fibroblast cell line. In these cells, the level of mRNA was decreased moderately by SX, to a greater extent by FP, and decreased yet further by SX + FP. However, ATX protein was minimally decreased or not at all, despite the clear decrease in mRNA. These data reveal interesting cell type‐specific differences in ATX mRNA and protein expression, and they show that asthma therapeutic agents can reduce the expression of ATX in at least some of these cells, which may contribute to their overall therapeutic benefit in patients with asthma.

Grant Funding Source: Supported by investigator‐initiated grant 115165 from GlaxoSmithKline
Identification of allosteric modulators of the D2 dopamine receptor (662.6)
Authors: Free R , Southall Noel , Doyle Trevor , Meade Julie , Conroy Jennie , Javitch Jonathan , Ferrer Marc , Sibley David
Published: June 2021

The D2 dopamine receptor (DAR) is central in the etiology and/or therapy of numerous neuropsychiatric disorders. Unfortunately, truly specific drugs for this receptor have been difficult to obtain, primarily due to high conservation of the orthosteric binding site. We have employed a high throughput‐screening approach using the 380,000 small molecule NIH MLPCN library to identify novel allosteric modulators of the D2 DAR. The primary screen was conducted using a cell line expressing an inducible D2 DAR coupled to a chimeric Gqi5 protein, thus linking receptor activation to Ca2+ mobilization. Hits were subjected to an extensive triage strategy to characterize DAR activity and selectivity. On the basis of these analyses, 745 agonist and 499 antagonist compounds were selected and evaluated using radioligand binding competition assays to identify the nature of their receptor interactions (orthosteric or allosteric). Compounds that are ineffective in competing for binding likely exert their functional activity via allosteric mechanisms. These experiments resulted in the identification of 47 agonists and 48 antagonists that had insignificant effects on radioligand binding when tested at concentrations up to 40 μM, despite exhibiting maximal functional effects at significantly lower concentrations. These compounds would thus appear to be allosteric agonists and negative allosteric modulators of the D2 DAR.

Grant Funding Source: NIH Intramural Program
Mitoquinone exerts antioxidant effects independent of mitochondrial targeted effects in phorbol‐12‐myristate‐13‐acetate stimulated polymorphonuclear leukocyte superoxide release (667.3)
Authors: Lepera Matthew , Pesikan Dragana , Voeun Jenifer , Perkins Kerry‐Anne , Chen Qian , Barsotti Robert , Young Lindon
Published: June 2021

MitoQ is a mitochondrial‐targeted coenzyme Q antioxidant analog that dose‐dependently restored cardiac function and reduced infarct size in isolated perfused rat hearts subjected to ischemia reperfusion (I/R). Moreover, mitoQ also dose‐dependently attenuated PMA stimulated PMN superoxide (SO) release at the same concentration (10uM) as the cardioprotective dose. NADPH oxidase is the principle source of PMN SO release. We speculate that mitoQ may exert antioxidant effects independent of the mitochondria. Therefore, we hypothesized that inhibition of mitoQ on PMN‐SO release will be similar as other coenzyme Q analogs: coenzyme Q1 and decylubiquinone without affecting cell viability. SO release was measured spectrophotometrically from isolated rat PMNs measured by the reduction of ferricytochrome c and were stimulated with 100nM PMA. The absorbance was measured at 550 nm up to 360sec. Positive control samples were given SO dismutase (SOD; 10ug/ml) which inhibited PMA induced SO release by >90%. MitoQ significantly inhibited SO release by 56 + 3% (10uM, n=10 , P<0.05); coenzyme Q1 by 49 + 9% (40uM, n=6) 68 + 7% (80uM, n=5),( both P<0.05); and decylubiquinone by 61 + 9% (80uM, n=8, P<0.05). Additionally, there was no significant change in cell viability. These preliminary results suggest that mitoQ may exert its antioxidant effects independent of the mitochondria. .
Increased systemic low grade inflammation in high altitude native rats mediated by adrenergic receptors (669.7)
Authors: AL‐Hashem Fahaid , Assiri Abdullah , Shatoor Abdullah , Elrafaey Hisham , Alessa Riyad , Alkhateeb Mahmoud
Published: June 2021

Background: There is paucity information on the effect ofhypoxiagenerated by high altitude (HA) on the systemic inflammation and its forcing mechanism in native dwellers Objectives:To comparethe serum levels of themain inflammatorycytokines and mediators in HA native rats and to search for the possible underling mechanism(s).

Methods:The study was carried out between January and April 2013. Fifty male rats were bred andreared at either HA in Abha city or low altitude (LA)in Riyadh city, Saudi Arabia. The study was carried out in two stages. In the first stage, serum levels of Inflammatory markers, cell adhesive molecules, lipids profile,catecholamine,magnesium (Mg+2)and lipid peroxidation were analysed and compared between HA and LA native rats. In the second stages, inflammatory response and lipid peroxidation levels were analysed in HA native rats after treat them with either α (Prazocin)or β (propranolol) adrenergic blockage.

Results: Rats of HA native area showed significant increases in the levels of inflammatory cytokines,lipid profile as well as significant increase in norepinepherine and dopamine with a concomitant decrease in Mg+2 levels and increased lipid peroxidation. Blockage of the adrenergic receptors of HA native rats is either partially (propranolol)or completely (Prazosin) ameliorated the increase in inflammatory cytokine and oxidative stress.

Conclusion: our study shows that living under HA conditions results in increased systemic inflammatory reaction an effect that is mediated through sympathetic nervous system mainly viaα adrenergic receptors and could be attributed to low Mg+2 levels.
Cholinergic signaling through the alpha 7 nicotinic receptor inhibits atherosclerosis in hypercholesterolemic mice (671.7)
Authors: Ulleryd Marcus , Johansson Maria , Lundberg Anna , Folkersen Lasse , Fogelstrand Linda , Yan Zhong‐Qun , Hansson Göran
Published: June 2021

Objective: The aim was to examine the expression of the alpha 7 nicotinic acetylcholine receptor (α7R) in human atherosclerotic plaques and to investigate α7R effects on progression of atherosclerosis in hypercholesterolemic Ldlr‐/‐ mice.

Methods: Human carotid plaques and aortic lesions of hypercholesterolemic mice were stained for α7R, using immunohistochemistry. To study the role of cholinergic signaling in atherosclerosis, male Ldlr‐/‐ mice were lethally irradiated and reconstituted with bone marrow from wildtype (WT) or α7R deficient animals.

Results: α7R was detected on T cells and macrophages in human carotid plaques. Ablation of hematopoietic cell α7R in Ldlr‐/‐ mice increased aortic atherosclerosis by 38%. This was accompanied by increased aortic interferon‐γ mRNA, implying increased Th1 activity in the absence of α7R signaling.

Conclusion: α7R is expressed by T cells and macrophages in human plaques. Lack of α7R in bone marrow dramatically accelerates atherosclerosis in LDLr‐/‐ mice. The present study suggests that cholinergic signaling through hematopoietic α7R inhibits atherosclerosis by modulating immune inflammation.
Activation of histone deacetylase 2: a novel strategy for reversing vascular dysfunction in atherogenesis (671.9)
Authors: Pandey Deepesh , Sikka Gautam , Bergman Yehudis , Romer Lewis , Berkowitz Dan
Published: June 2021

Although they are targeted by therapies for cancer, the implications of epigenetic modifications of histone tails by Histone Deacetylases (HDACs) for vascular homeostasis and cardiovascular diseases including atherosclerosis, diabetes and hypertension are unclear. The use of HDAC inhibitors in the treatment of cardiovascular disease is controversial, since the global HDAC inhibitor Trichostatin (TSA) was shown to accelerate atheroma formation in LDL‐receptor knockout mice.

We have demonstrated that TSA causes impairment (~40%) of endothelium‐dependent vascular relaxation in isolated vascular rings. Using class‐specific biochemical HDAC inhibitors and siRNA‐mediated gene knockdown, we have identified HDAC2 as a specific, critical regulator of homeostasis in the vascular endothelium. We have also identified Arginase 2 (Arg2) as a major transcriptional target for HDAC2 that contributes to intimal dysfunction, by showing that vascular dysfunction mediated by HDAC inhibition was abolished by either pharmacologic inhibiton or genetic knockout of Arg2. Finally, inhibition of neddylation, a post‐translational modification that enhances ubiquitin‐mediated proteosomal degradation, increased the abundance of HDAC2 and caused decreased Arg2 activity. Hence, we hypothesize that atherogenic stimuli such as OxLDL and inflammation downregulate HDAC2 expression in vascular EC through NEDD8‐mediated enhancement of the proteosomal degradation of HDAC2. This reduction in HDAC2 results in the following cascade: increased Arg2 expression in EC, decreased L‐arginine concentration, impaired NO bioavailability via eNOS substrate depletion, and increased ROS. These events culminate in EC dysfunction and atherogenesis. In conclusion, activation of HDAC2 by pharmacologic agents or small molecule activators could serve as a potent strategy in an atheroprotective therapeutic regimen.

Grant Funding Source: NIH
Vasoactive effects of endothelin‐1 in the mesentery and cremaster muscle of leptin deficient mice (674.11)
Authors: Vigilance Jacqueline , Frame Mary
Published: June 2021

Leptin has been shown to up‐regulate endothelin‐1 production. We investigated the responses to endothelin‐1 (ET) in leptin deficient diabetic mice (db/db) in comparison to C57BL/6J (C57) genetic background mice in the absence or presence of endogenous NO (LNNA), and with blockade of the ETB receptor (PD142893, BQ788). The cremaster muscle of anesthetized (50 mg/kg Nembutal or 4% isoflurane) mice (n=33) was prepared for intravital microscopy. Arteriole(A)/venule(V) pairs were chosen for study. Baseline diameters in the cremaster (A/V, 30/35 um) were ~20‐30% larger than in db/dbs; in the mesentery (60/120 um) they were similar for the 2 strains. Vasodilatory tone (adenosine, 10‐4M) was not significantly different between strains, and was lower in mesentery vs. cremaster. Vasoconstrictor tone (Phenylephrine, 10‐4M) was decreased in db/db compared to C57 in the cremaster, but not mesentery. ET1 dose dependent constriction in the C57 or db/db cremaster (logM EC50: C57 ‐11.9±29.6 A, ‐11.7±2.8 V; db/db ‐11.7±4.4 A, ‐10.0 V) was suppressed by LNNA, and enhanced with ETB antagonism to a greater extent in the A vs. V. In the mesentery, baseline constriction to ET1 was diminished to a greater extent in the db/db vs C57 and was suppressed by LNNA and enhanced with ETB antagonism. Thus, the leptin deficient mouse exhibits a tissue specific loss of ET1 mediated constriction. Yet, the potential role for ETB mediated vasodilation appears similar for C57 and db/db in both tissues

Grant Funding Source: NIH DK68401, The UWI
Transmural variation and anisotropy of microvascular flow conductivity in the rat left ventricular myocardium (675.4)
Authors: Smith Amy , Shipley Rebecca , Lee Jack , Sands Gregory , LeGrice Ian , Smith Nicolas
Published: June 2021

Three‐dimensional (3D) ex vivo data on microvascular anatomy in the myocardium is used to determine the transmural gradient in fluid transport properties via mathematical averaging methods. The 3D capillary network is modeled as a porous medium, whose conductivity is quantified in terms of a permeability tensor. This metric is directly computed by volume‐averaging blood flow solutions in model networks with geometrical and topological properties derived from an anatomically‐detailed data set recently extracted from the rat left ventricular myocardium using confocal microscopy, high‐precision sectioning and image processing techniques.

Results showed that the permeability was strongly anisotropic: the network was approximately ten times more permeable in the principal direction of capillary alignment (the ‘longitudinal’ direction) than perpendicular to this direction. Structural properties of the network varied smoothly from subepicardium to subendocardium, with a 30% transmural increase in capillary diameter translating to a 130% rise in permeability in the longitudinal direction. This supports the hypothesis that perfusion is preferentially facilitated during ventricular diastole in the subendocardial microvasculature to compensate for severely‐reduced systolic perfusion in the subendocardium.

Grant Funding Source: Supported by NIH GM094503‐1, EPSRC EP/F043929/1 and EP/G007527/2, and Award KUK‐C1‐013‐0 from KAUST.
Adipose‐derived cell treatment is associated with decreased ROS production and improved coronary microvascular function in advanced age (676.11)
Authors: LeBlanc Amanda , Aird Allison , Nguyen Quang , Nevitt Christopher
Published: June 2021

In aged rats and humans, coronary blood flow reserve (CFR) is significantly decreased and may contribute to the increased risk for CVD as people age. Adipose‐derived stromal vascular fraction (SVF) cells have shown an innate capacity for angiogenesis and regeneration in the periphery. We wanted to assess the therapeutic potential of SVF to improve poor CFR in aged rats and evaluate the effect on underlying ROS signaling mechanisms.

SVF was isolated from fat pads of young (4 mos) rats through mincing, enzyme digestion, and removal of buoyant adipocytes. SVF (1x10(6) cells/cm2) was plated on Vicryl® and cultured for 14 days before implantation on epicardial surface of old rats (22 mos). After 6 weeks, microspheres were injected during baseline and dobutamine infusion to evaluate CFR (hyperemic BF/ baseline BF). Coronary arterioles were isolated, pressurized and infused with ROS‐specific fluorescent dyes to detect basal levels of O2‐ (DHE, 5 uM) and NO (DAF, 5 uM). Another set of arterioles were evaluated for NO‐dependent vasodilatory function.

CFR in the apical region of the LV was increased by 78+/‐10% following treatment with a SVF epicardial patch, while the papillary region was increased by 28+/‐12% compared to untreated hearts (n=8, SVF; n=3, Control). DHE and DAF mean fluorescence (measures of H2O2 and NO production, respectively) and NO‐dependent vasodilation were significantly lower in arterioles isolated from SVF‐treated hearts compared to untreated old females.

These results suggest that this adipose‐derived cell‐based therapy could improve age‐related declines in the reactivity of the coronary microvasculature through ROS‐related mechanisms.

Grant Funding Source: Supported by American Heart Association BGIA 120066
Myosin phosphatase isoforms in the microvasculature during endotoxemia (678.2)
Authors: Reho John , Fisher Steven
Published: June 2021

Sepsis is characterized by widespread inflammation and severe hypotension caused by the induction of iNOS as well as other vasodilators. We hypothesized that altered contractility of resistance artery smooth muscle could play a role in the later stages of sepsis. Sepsis was modeled via a single IP injection of lipopolysaccharides (LPS; 20mg/kg), and mouse vascular tissues were harvested 24hrs post injection. We examined the expression of the myosin phosphatase (MP) regulatory (Mypt1) and inhibitory (CPI‐17) subunits. Expression of Mypt1 Exon24/leucine zipper variants may determine sensitivity to vasodilator NO, while the level of expression of CPI‐17 may determine sensitivity to vasoconstrictor NE. Mesenteric and femoral arteries and portal veins switched from the E24+/LZ‐ isoform to the E24‐/LZ+ isoform of Mypt1 (p<0.05) with decreases in total Mypt1 and CPI‐17 transcript expression (p<0.05). Relaxation to cGMP via wire myography was enhanced in α‐toxin permeabilized mesenteric arteries from LPS injected mice compared to saline injected control. Constriction to phenylephrine activation was significantly depressed in LPS injected mice compared to control. We propose that endotoxemia triggers specific changes in MP subunit expression thereby enhancing vasodilatory mechanisms and suppressing vasoconstrictor mechanisms thereby contributing to the severe hypotension of the later stages of sepsis.

Grant Funding Source: HL066171
Evidence that cutaneous microvascular dysfunction in peripheral arterial disease is due to reduced nitric oxide (678.9)
Authors: Hodges Gary , Tew Garry , Nawaz Shah
Published: June 2021

We investigated the role of nitric oxide (NO) in cutaneous microvascular dysfunction in patients with symptomatic unilateral peripheral arterial disease (PAD) and in limbs of disease‐free, age‐matched controls in response to local skin warming. In 5 patients with unilateral PAD and 10 age‐matched controls, skin blood flow (SkBF) was measured using laser‐Doppler flowmetry. Local skin temperature was controlled with heater probe holders. Microdialysis was used to administer L‐NAME to inhibition NO synthase activity. Ringer’s was perfused at control sites. Data are presented as cutaneous vascular conductance (SkBF / blood pressure). Vasodilation (plateau phase) at the untreated skin sites was significantly lower in the diseased limb of the PAD patients (70.20 ± 12.41 %max) compared to the healthy contralateral limb (85.14 ± 9.26 %max, P = 0.021) and the response achieved in the control participants (86.58 ± 6.99 %max, P = 0.003). There were no differences between the NO‐inhibited skin sites of the PAD patients and healthy participants (38.32 %max vs. 38.72 %max, P = 0.395) or between the diseased and healthy leg of the PAD patients (37.19 %max, P = 0.393). The primary findings of this study are that cutaneous vasodilation is markedly reduced in response to local skin warming and this decreased response appears to be due to NO.
Loss of gender‐based protection against chronic stress‐induced impairments to cerebrovascular reactivity by pre‐existence of metabolic syndrome (680.10)
Authors: Brooks Steven , Stanley Shyla , Chantler Paul , Butcher Joshua , Skaff Paulina , D'Audiffret Alexandre , Frisbee Jefferson
Published: June 2021

Previous studies demonstrate that imposition of depressive symptoms via unpredictable chronic mild stress (UCMS) or metabolic syndrome (MetSyn) impairs vascular reactivity across organ systems, including the cerebrovasculature. Consistently, the degree of altered vascular reactivity is blunted in females compared to males, regardless of the severity of depressive symptoms (with UCMS) or of the elevated risk factors (with MetSyn). We imposed 8 weeks of UCMS on 9 week old lean (LZR) and obese (OZR) Zucker rats (both genders) and determined behavioral and vascular outcomes. UCMS caused depressive symptoms in both genders; development of MS resulted in similar, but blunted, effects. UCMS (in LZR) and MetSyn (in OZR) impaired endothelial dilator reactivity and mildly increased constrictor responses in middle cerebral arteries (MCA) of both genders, but severity of the alterations was reduced in females. When UCMS was combined with MetSyn, vascular reactivity was impaired to a comparable extent regardless of gender, and ROS/inflammatory biomarker levels were comparable as well. These results suggest that gender‐based protection against UCMS or MetSyn‐induced vasculopathy in females is lost when these risks are combined.

Grant Funding Source: Supported by National Institutes of Health and American Heart Association
Leptin induces CD16 expression on human monocytes in a sex‐specific manner (680.13)
Authors: Cannon Joseph , Sloan Gloria , Cortez‐Cooper Miriam
Published: June 2021

In addition to a centrally‐mediated influence on blood pressure, leptin stimulates monocyte migration, cytokine secretion, and other functions that contribute to hypertension and atherosclerotic plaque development. These functions are characteristics of CD16‐positive monocytes implicated in the clinical progression of atherosclerosis. This investigation sought to determine if leptin promoted the development of CD16‐positive monocytes in healthy men and women ranging in age from 20‐59 years. Circulating numbers of CD14+16++ monocytes, which preferentially migrate into nascent plaques, correlated with plasma leptin in men (R = 0.68, P = 0.01), but not women. In vitro, exogenous leptin increased percentages of CD14+16++ monocytes in cultures of male cells and reduced them in cultures of female cells (P=0.03 for leptin*sex interaction). For all subjects, circulating CD14+16++ cell counts correlated with systolic pressure (R=0.45, P=0.02) and carotid intima‐media thickness (R=0.36, P=0.04). There were no sex‐related differences in total expression of leptin receptors, measured by flow cytometry, or in relative expression of different isoforms of the receptor, measured by western blots. These results support the concept that leptin promotes the development of an atherogenic monocyte phenotype and has a stronger influence in men than in women.

Grant Funding Source: Supported by NIH grant HL093663
Coronary ischemia increases corticotropin releasing hormone receptor expression in locus coeruleus of female rats (680.5)
Authors: Scrogin Karie , Lawrence Flora , Samarel Allen , Koshman Yevgeniya
Published: June 2021

Women with coronary artery disease have two times the rate of anxiety as men, and anxiety is a better predictor of future cardiovascular events in women than men. We found that coronary artery ligation (CAL) impairs memory of fear extinction in female, but not male rats. Αdrenergic receptors in the forebrain regulate fear expression and extinction. The locus coeruleus (LC) is the sole source of norepinephrine in cortical and subcortical regions that govern fear expression. Our evidence suggests that adrenergic neurotransmission in the female rat cortex is increased after CAL. The corticotropin releasing hormone receptor, CRF1, stimulates LC activity during stress. Here, we examined the density of CRF1 in tyrosine hydroxylase‐positive LC neurons in male and female rats 8 weeks after CAL. Females showed a higher percentage of LC cells with high density CRF1 label (p<0.05). CAL surgery further increased high density CRF1 label in females (p<0.05). The percentage of LC neurons with high density CRF1 was inversely correlated with fractional shortening in females (p<0.05), but not males. Increased CRF1 expression may contribute to elevated cortical adrenergic neurotransmission after CAL in females.
Endothelium‐dependent vasodilation in cerebral resistance arteries: effects of age and estrogen status (680.8)
Authors: Kilar Cody , Reyes Rafael , Kang Lori , LeBlanc Amanda , McCroskey Casey , Bryan Robert , Muller‐Delp Judy
Published: June 2021

Postmenopausal women have increased risk of cerebrovascular disease, possibly related to loss of cardioprotective estrogen. We investigated the effects of age and estrogen status on flow‐induced dilation (FID) and the role of nitric oxide (NO) in FID of cerebral resistance arteries. Young (Y), middle‐aged (MA) and old (O) female rats were placed in the following groups: intact, ovariectomized (ovex) and ovariectomized with estrogen replacement (ovex +E). FID was assessed in posterior communicating arteries (PCoA) with and without L‐NAME to block NO synthase. FID declined with age in PCoA of intact rats. Ovex reduced FID in PCoA of Y and MA rats, but not in PCoA of O rats. Ovex +E restored FID in Y rats but decreased FID in O rats. L‐NAME reduced FID in PCoA of intact Y and MA rats, but not in PCoA of intact O rats. In contrast, L‐NAME reduced FID in MA and O ovex rats, but did not affect PCoA of Y ovex rats. In ovex + E groups, L‐NAME inhibited FID in PCoA of Y rats, but did not alter FID in PCoA of MA or O rats. Thus, NO contributes to FID in PCoA of both Y and MA, but not O intact rats. Estrogen removal eliminates NO‐mediated FID in PCoA of Y rats, but upregulates NO signaling in PCoA of O rats. Estrogen replacement restores FID in PCoA of Y rats, but exacerbates declining FID in PCoA of O rats. Age‐related loss of ovarian estrogen contributes to impairment of NO‐mediated FID in PCoA; however, exogenous estrogen cannot restore NO‐mediated FID in old PCoA.
Hydrogen sulphide acts in the subfornical organ and paraventricular nucleus to increase blood pressure (681.6)
Authors: Smith Pauline , Ferguson Alastair
Published: June 2021

Hydrogen sulfide (H2S) is as an endogenous gaseous transmitter that has been shown to act in the central nervous system to influence variety of functions including cardiovascular function. Administration of H2S in the lateral ventricle has been shown to increase blood pressure although the specific nuclei responsible for mediating these effects have not been identified. The subfornical organ (SFO) in the forebrain and the hypothalamic paraventricular nucleus (PVN) are central nervous system structures with well‐documented roles in cardiovascular regulation. The present study was undertaken to determine the effects of direct administration of the hydrogen sulfide donor, sodium hydrogen sulfide (NaH2S),into the SFO and PVN in urethane anesthetized male Sprague Dawley rats. Microinjection of NaH2S (10mM, 0.5µl) into either the SFO or PVN resulted in increases in BP (SFO mean AUC = 663.3 ± 51.6 mmHg*s, n=5; PVN mean AUC 447.4 ± 66 mmHg*s, n=4) and HR (SFO mean AUC = 29.6 ± 5.9 beats, n=5; PVN mean AUC 10.7 ± 2.1 beats, n=4). These cardiovascular responses were of rapid onset (responses began within 10s of microinjection) and short duration, returning to baseline within 90s. In both cases, these responses were shown to be dose related and site specific. These observations identify the SFO and PVN as possible central nervous system locations at which H2S may act to influence cardiovascular regulation.
Postoperative cognitive dysfunction: effects of previous pathology (684.7)
Authors: Hovens Iris , Schoemaker Regien , van der Zee Eddy , Nyakas Csaba , van Leeuwen Barbara
Published: June 2021

Surgery patients may experience postoperative cognitive dysfunction (POCD), potentially depending on age and pre‐operative health status. The study objective was to research the effect of prior infection on POCD in aged rats.

Cognitive function after abdominal surgery was compared between healthy aged (AH, 18 months) and aged rats that had recovered from an active mycoplasma infection (AI). Healthy young rats (YH, 3 months) and un‐operated rats served as control. Cognition was assessed 10 ‐ 14 days post‐surgery in the novel object ( NO) and location (NL) test and the Morris water maze (MWM).

Cognitive performance of AH was comparable to YH. In these groups surgery only led to a decline in spatial memory. AI showed impaired spatial learning and memory and impaired cognitive flexibility compared to YH, which was further impaired following surgery. Additionally, surgery led to impaired object recognition in AI. Velocity in the MWM did not differ significantly between groups.

Age in itself did not lead to cognitive impairment. In healthy young and aged rats abdominal surgery only impaired spatial, hippocampal dependent, cognition. Prior infection however caused cognitive impairment and more generalized and severe POCD in aged rats, indicating that patients with a history of infection may be more prone to develop POCD. Analysis of (neuro)inflammatory markers may provide insight in the influence of prior infection on POCD.
Altered susceptibility of the blood‐brain barrier to disruption in type 1 diabetes (685.2)
Authors: Scott Jasmine , Yang Shu , Arrick Denise , Mayhan William
Published: June 2021

Our previous studies have shown that nitric oxide synthase‐dependent responses of cerebral arterioles are impaired in Type 1 diabetic (T1D) rats by a mechanism that appears to involve an increase in oxidative stress. Since the integrity of the blood‐brain barrier (BBB) may be influenced by nitric oxide and oxidative stress, we reasoned that the susceptibility of the BBB to disruption may be altered by T1D as well. Thus, our goal was to examine basal and agonist‐induced changes in permeability of the BBB in nondiabetic and diabetic (streptozotocin; 50 mg/kg IP) rats. On the day of the experiment (2‐3 months after streptozotocin), a craniotomy was made over the parietal cortex. We measured the permeability of the BBB to FITC‐10K dextran in nondiabetic and diabetic rats under basal conditions and during topical application of histamine (eNOS‐dependent) or glutamate (nNOS‐dependent). In addition, we examined the levels of superoxide anion in brain tissue using lucigenin chemiluminescence. Finally, we measured tight junctional protein levels in brain tissue of nondiabetic and diabetic rats. We found that basal permeability of the BBB was increased in diabetic compared to nondiabetic rats. In addition, eNOS‐ and nNOS‐dependent changes in permeability of the BBB were less in diabetic compared to nondiabetic rats. Finally, tight junctional protein and superoxide anion levels were altered by T1D. We suggest that T1D can influence the susceptibility of the BBB to disruption by altering the balance between nitric oxide and superoxide anion. We speculate that altered permeability characteristics of the BBB may contribute to cognitive impairment/impaired neurovascular coupling observed in diabetic patients.

Grant Funding Source: NIH HL090657
Mitochondria influence NMDA‐induced calcium dynamics in hypothalamic magnocellular neurosecretary cells (686.28)
Authors: Potapenko Evgeniy , Javier Stern
Published: June 2021

The spatiotemporal characteristics of NMDA receptor‐mediated changes in intracellular calcium ([Ca2+]i) (NMDAR‐[Ca2+]i) is critical in determining the strength of NMDA effects on neuronal excitability. Here we investigated the role mitochondria in shaping NMDAR‐[Ca2+]i dynamics. We activated NMDARs before and after the mitochondrial uncoupler CCCP (10µM for 10 sec), which prevents [Ca2+]i uptake by disruption of the proton gradient across the mitochondrial inner membrane. In both, identified and non‐identified eGFP‐VP SON neurons, focal application of NMDA (10µM for 1 sec) following CCCP resulted in a significantly larger and slower Δ[Ca2+]i response both in soma and dendrites, and a more robust membrane depolarization and firing discharge compared to the control application. Conversely, a similar underlying NMDAR‐mediated current (INMDA) was observed. Direct depolarization of SON neurons by current injection (0.15 nA for 2 sec) was used to evoke a similar number of action potentials and associated increase in [Ca2+]I as those evoked NMDAR activation. In this condition however, CCCP failed to potentiate the action potential‐evoked Δ[Ca2+]i response. Moreover In slices preincubated with Ru360 (10 µM, 30 min), a selective and potent inhibitor of the mitochondrial Ca2+ uptake uniporter, subsequent application of CCCP failed to potentiate the NMDA‐[Ca2+]i response. Taken together, our study support the hypothesis that mitochondria play a critical role in regulating NMDA‐[Ca2+]i dynamics, and therefore, the degree of membrane excitability and firing activity of MNC neurons following NMDAR activation.

Grant Funding Source: NIH HL090948‐2
GRK5 overexpression in the RVLM of CHF rats improves autonomic imbalance and restores AT1R and Kv4.3 expressions (686.33)
Authors: Haack Karla , Becker Bryan , Zucker Irving
Published: June 2021

Chronic heart failure (CHF) is characterized by an overactive Angiotensin II type 1 receptor (AT1R) signaling axis and autonomic imbalance. AT1R is regulated by its phosphorylation by G protein coupled receptor kinase 5 (GRK5). We have shown previously that AT1R and GRK5 are paradoxically increased and that Kv4.3, a potassium channel involved in neuronal excitability, is decreased in the rostral ventrolateral medulla (RVLM) of rats with CHF. We tested the hypothesis is that GRK5 overexpression in the RVLM improves autonomic imbalance and normalizes AT1R and Kv4.3 expression in CHF. CHF was induced by coronary artery ligation and confirmed by echocardiography. Following implantation of radiotelemetry transducers, two weeks of heart rate (HR), mean arterial pressure (MAP) and metabolic cage data were collected before and after either vehicle (Veh) or lentiviral GRK5 (GRK5) injection into the RVLM. HR was decreased in GRK5 rats compared to Veh (293±6 vs 316±2 bpm) while MAP was unchanged. Urine norepinephrine was decreased in GRK5 vs Veh (113±51 vs 252±89 ng/24hrs). Western blot analyses of RVLM micropunches indicated that GRK5 decreased AT1R expression vs Veh (1.2±0.1 vs 1.8±0.4 a.u.) and increased Kv4.3 expression by almost 50% compared to Veh. Taken together, these data suggest that GRK5 overxpression in the RVLM may be protective in CHF by decreasing sympathetic tone and normalizing AT1R and Kv4.3 imbalance.

Grant Funding Source: Supported by NHLBI F32HL116172‐01
Excitatory amino acid receptors in the dorsomedial hypothalamus are not involved in the regulation of the tachypneic response induced by chemoreflex (686.34)
Authors: Haibara Andrea , Silva Nathália , Nalivaiko Eugene
Published: June 2021

EAA receptors into the DMH are involved in the control of sympathoexcitatory component of the chemoreflex, because kynurenic acid into DMH reduces the chemoreflex pressor response. Stimulation of the DMH elicited an increase in respiratory frequency. Considering that activation of inspiratory neurons on the ventral surface of the brainstem can increase the activity of sympathetic preganglionic neurons, we cannot rule out that the blockade of the DMH by kynurenic acid affects the chemoreflex ventilatory responses and consequently the pressor response. This study evaluated the effect of EAA receptors blockade in the DMH on the ventilatory and cardiovascular responses induced by intravenous injection of potassium cyanide (KCN) in conscious rats. Bilateral microinjection of kynurenic acid (2.7 nmol, n=6) into the DMH produced no significant changes on the tachypneic (+198±27 vs +163±19) and bradycardic responses (‐214±7 vs ‐244±17 bpm), but reduced the pressor response (+49±4 vs +23±11 mmHg) of the chemoreflex. Furthermore, the bilateral microinjection of kynurenic acid also produced no changes on baseline cardiovascular parameters (104±3 vs 105±3 mmHg; 330±5 vs 358±12 bpm) and baseline respiratory rate (129±12 vs 120±8 cpm). These results indicate that EAA receptors in the DMH are not involved in the tachypneic response of the chemoreflex, suggesting that the reduction on chemoreflex pressor response induced by kynurenic acid was not a consequence of reduction in respiratory activity.

Grant Funding Source: Supported by INCT‐CNPq/ CAPES/ FAPEMIG
Activated pathway from the parabrachial nucleus to rostal ventrolateral medulla during stimulation of cardiac sympathetic afferents (686.8)
Authors: Guo Zhi‐Ling , Longhurst John
Published: June 2021

Our previous studies have shown that stimulation of cardiac sympathetic afferents activates pontine nuclei like the parabrachial nucleus (PBN) and locus coeruleus (LC) as well as rostral ventrolateral medulla (rVLM), a critical area that controls cardiac sympathoexcitatory responses. However, there is no information on direct influence of pontine nuclei on the rVLM during stimulation of cardiac sympathetic afferents. Thus, the present study evaluated neuronal activation of the pontine nuclei in response to stimulation of cardiac sympathetic afferents, with regard to their projections to the rVLM. Seven to ten days after unilateral microinjection of rodamine‐conjugated microspheres retrograde tracer (100 nl) into the rVLM, rats with bilateral cervical vagotomy were subjected to epicardial application of α, β‐meATP (1.1 to 6.5 mM, in 40 µl) or 0.9% normal saline for six times over 90 min. Repetitive increases in blood pressure were observed in the rats following repeated stimulation of the heart with α, β‐meATP, but not with normal saline. Perikarya containing the microsphere tracer were found in the PBN and, but not in the LC of both groups. Compared to three controls, c‐Fos immunoreactivity and neurons double‐labeled with c‐Fos, an immediate early gene and the tracer were more frequently found in the PBN, but not LC of rats with cardiac stimulation (n=5), particularly in the lateral PBN and Kölliker‐Fuse nucleus. These results suggest that PBN neurons responsive to cardiac sympathetic afferent stimulation frequently project directly to the rVLM, which is known to participate in processing of excitatory cardiovascular responses during stimulation of cardiac sympathetic afferents.

Grant Funding Source: NIH grant HL‐66217
Selective regulation of voltage‐gated potassium channel subunits in presympathetic PVN neurons of rats with myocardial infarction (686.9)
Authors: Lee Seul Ki , Ryu Pan Dong , Lee So Yeong
Published: June 2021

Hypothalamic paraventricular nucleus (PVN) neurons contain presympathetic neurons that project to the rostral ventrolateral medulla (RVLM; PVN‐RVLM neurons) and control cardiovascular sympathetic tones. In this neuronal population, cardiovascular disorders such as hypertension modulate voltage‐gated potassium (Kv) channels, but under myocardial infarction (MI), modulation of Kv channels has not been extensively studied. In the present study, we investigated differences in mRNA expressions of the Kv channel subunits in PVN‐RVLM neurons between sham and MI rats, using single‐cell real‐time RT‐PCR. The results showed that the mRNA level of Kv4.2 was significantly up‐regulated after MI, and this regulation was exclusively found in the dorsal cap subdivision. There was no change in Kv4.3 mRNA level between sham and MI rats in any of the subdivisions. These results suggest that heterogeneous cell populations exist in each subdivision of PVN‐RVLM neurons and that MI selectively regulates the mRNA levels of Kv4.2 in PVN‐RVLM neurons distributed in the dorsal cap subdivision.

Grant Funding Source: Supported by the National Research Foundation of Korea (2010‐0015531)
Curcumin ameliorates nephrotoxicity and histopathological alterations induced by chlorpyrifos in albino rats (690.5)
Authors: Al‐Amoudi Wael
Published: June 2021

In the present investigation an attempt was made to assess the possible ameliorative role of curcumin on nephrotoxicity and histopathological alterations caused by Chlorpyrifos in the kidney tissue of albino rats. Rats were given daily doses of intragastric and suble‐ thal concentrations of CPF. Histological examination showed alterations in the treated kidney such as degeneration of the renal tubules, atrophy and hypertrophy of glomeruli and intertubular leucocytic infiltrations. The histopathological changes were more pro‐ nounced with higher concentrations of CPF. The degree of degeneration of renal tubules found in higher concentration treatment of CPF was more pronounced. These alterations of the renal tubules and glomeruli affected the functioning of motor coordination of the rat body. Moreover, histochemical examination of the renal tubules showed depletion of glycogen and total proteins content and marked elevation in serum creatinine and blood urea nitrogen. However, treating animals with CPF and curcumin (150 mg/kg b. w. 5 days / week for 6 weeks) caused an improvement in histological and histochemical appearance of the kidney together with decrease of serum creatinine and blood urea nitrogen. It is concluded from the obtained results that CPF is highly toxic to albino rats by causing nephrotoxicity and may leads to oxidative stress. This effects appeared to be prevented and ameliorated by curcumin.
Enhanced dedifferentiation and injury in mouse podocytes lacking CD38 gene (691.4)
Authors: Boini Krishna , Xia Min , Abais Justine , Li Guangbi , Li Pin‐Lan
Published: June 2021

The absence of CD38 in lymphocytes has been used as a dedifferentiation or low differentiation marker for diagnosis of leukemia. Our recent studies have reported that CD38 deficiency leads to phenotypic plasticity of arterial smooth muscle cells. In the present study, we hypothesized that CD38 and its signaling pathway may participate in the regulation of podocyte differentiation and proliferation and its gene defect or dysfunction may lead to podocyte injury and glomerular sclerosis. CD38 gene targeted mice and their wild type littermates were used to test this hypothesis. Western blot, confocal and RT‐PCR analysis demonstrated that synaptopodin (a marker of differentiation) expression was significantly lower in CD38‐/‐ mice compared to CD38+/+ littermates. In contrast, the proliferation marker cyclin E was significantly higher in CD38‐/‐ mice compared to CD38+/+ littermates. Functional studies showed that the urinary albumin and protein excretion were significantly higher in CD38‐/‐ mice than in CD38+/+ mice. Fluorescent Immunohistochemical analyses also illustrated podocyte damages in the glomeruli from CD38‐/‐ mice, as shown by decreased expression of podocin and enhanced expression of desmin. Visfatin treatment further decreased the expression of synaptopodin and increased the abundance of cyclin E, which were accompanied by more increased glomerular damage in CD38‐/‐ than in CD38+/+ mice. In vitro studies showed that inhibition of CD38 enhances the synaptopodin expression, but decreases the level of cyclin E in podocytes. In conclusion, our observations reveal that the normal expression of CD38 importantly contributes to the regulation of podocyte proliferation and dedifferentiation and that defect of this gene may be a critical mechanism resulting in podocyte injury and glomerular sclerosis.

Grant Funding Source: supported by NIH grants DK54927 and HL‐75316