bluuebunny/crossref_metadata_2025 huggingface dataset:
Amino acid residues in transmembrane domain 1 and in the membrane proximal C‐tail of muscarinic M1receptors are necessary for transport‐competent folding
Authors: Sawyer Gregory William , Shults Crystal Alane
Published: June 2021

The principle goal of this investigation was to identify the functional role of a conserved C‐terminal tail motif, 423‐F(X)6LL‐431, in muscarinic M1 receptors. Point mutations were introduced into this motif, and at the base of transmembrane spanning domain 1 (TM1), using site‐directed mutagenesis. Wild‐type and mutant M1 receptors were transiently expressed in CHO cells and the amount of receptor expressed on the plasma membrane was determined using intact, whole cell [3H]‐N‐methylscopolamine ([3H]NMS) binding assays. Mutation of Leu pair 430‐LL‐431 in the F(X)6LL motif to an Ala pair (hM1LL430‐431) caused a 92% reduction in the plasma membrane expression of M1 receptors. Similarly, mutation of 46‐VL‐47 at the base of TM1 to 46‐AA‐47 (hM1VL46‐47) caused a 98% reduction in plasma membrane expression. The plasma membrane expression of hM1LL430‐431 and hM1VL46‐47 receptors increased 5.3‐ and 18.2‐fold, respectively, when incubated with atropine (0.1 μM) for 18 h. Unlike GFP‐tagged M1 receptors, GFP‐tagged M1LL430‐431 receptors colocalized extensively with the ER marker DsRed‐ER. When incubated with atropine (0.1 mM) for 18 h, GFP‐tagged M1LL430‐431 receptors acquired a cellular localization consistent with that of wild‐type receptors (i.e., expressed on the plasma membrane). Collectively, these data suggest that 430‐LL‐431 and 46‐VL‐47 are necessary for transport‐competent folding of M1 receptors.
Protective Efficacy and Potency of Neuroactive Steroids and Benzodiazepines in the Amygdala Kindling Model of Epilepsy
Authors: Reddy Doodipala S. , Rogawski Michael A.
Published: June 2021

Although neuroactive steroids protects against experimental seizures, the role of GABA‐A receptor‐modulating neuroactive steroids in the development and expression of amygdala kindling remains unclear. In this study, the neuroactive steroid ganaxolone was examined on behavioral and electrographic seizure expression in the amygdala kindling model of epilepsy in mice, and the protective activity was compared with the antiseizure benzodiazepine clonazepam. Mice were kindled by daily stimulation of amygdala until they exhibit consistent behavioral and electrographic seizures. In fully kindled mice showing stage 5 seizures, ganaxolone (1.25‐20 mg/kg, s.c.) significantly reduced both clonic and generalized seizures elicited by amygdala stimulation. The protective effect of ganaxolone was dose‐dependent with an ED50 of 6.6 mg/kg. Ganaxolone treatment was associated with significant reduction in the afterdischarge duration. As expected, there was a substantial suppression of behavioral and electrographic seizures in mice treated with clonazepam. Clonazepam was more potent than ganaxolone, but the overall maximal efficacy of both clonazepam and ganaxolone was similar. These studies provide strong evidence that ganaxolone is highly effective antiseizure agent in the amygdala kindling model, which is a clinically‐relevant model of complex partial epilepsy.
Response of NO levels in the rat spinotrapezius muscle following systemic hemorrhage
Authors: Carvalho Helena , Meliagros Pete D , Pittman Roland N
Published: June 2021

The loss of blood produces profound effects on homeostasis of an organism, including reduction in mean arterial pressure (MAP). The control of MAP involves many factors including nitric oxide (NO) production associated with shear stress at the vessel wall. The aim of this study was to investigate the effect of decreased perfusion pressure on NO production in the microcirculation. NO concentration was measured using carbon fiber microelectrodes in the spinotrapezius muscle of anesthetized male Sprague Dawley rats. An NO microelectrode was positioned in the perivascular space of arterioles, venules and capillaries. Baseline values of NO were recorded before and after blood withdrawal. The hemorrhage was done in a stepwise manner at the rate of 1 ml every 2 min, until 40% of the blood volume was withdrawn. MAP and microvessel diameter were monitored throughout. A 40% reduction MAP from baseline lead to a decrease in NO concentration from 800 nM to 200 nM in arterioles and capillaries. However, in the venules there was no significant variation in NO concentration after hemorrhage. These results suggest that NO production in arterioles and capillaries is more affected by reduced perfusion than it is in venules, corroborating the observation that shear stress is more pronounced in resistance vessels. Supported by NIH grant HL18292 and HL079087.
Descending outer medullary vasa recta are constricted by perfusion with iodinated contrast media
Authors: Sendeski Mauricio Michalak , Patzak Andreas , Pallone Thomas L. , Cao Chunhua , Persson Erik , Persson Pontus B.
Published: June 2021

Objective

Contrast induced nephropathy (CIN) is a frequent complication of coronary angiography. Outer medullary descending vasa recta (DVR) are contractile microvessels supplying blood flow to the renal medulla ‐ the area of kidney at risk for CIN. Our objective was to test whether perfusion of DVR with a contrast medium (CM) modifies DVR vasoactivity.

Methods

DVR from rats were microscopically isolated and perfused with iodixanol (23 mg iodine/ml, calculated after usual doses of CM). DVR luminal diameter was determined by microscopy and nitric oxide (NO) was measured by fluorescent techniques.

Results

CM led to 50% reduction of luminal diameter, and increased vasoconstriction of DVR by angiotensin II (ANG II). Dismutation of superoxide by Tempol prevented these effects. CM also decreased NO bioavailability by >60 percent.

Discussion

Our data suggest that CM may lead to renal circulatory derangements described in CIN through a direct effect on DVR. This effect seems to happen via increased oxidative stress. Scavenging of reactive oxygen species might exert a therapeutic effect on CIN by mildering such derangements.

Supported by the German Research Foundation, Werner Jackstaedt Foundation, and NIH
Microfluidic device functionalized with P‐selectin reveals discontinuous rolling of leukocytes in mouse whole blood
Authors: Sundd Prithu , Gutierrez Edgar , Groisman Alex , Ley Klaus
Published: June 2021

Intravital studies of mouse cremaster have shown evidence that leukocytes roll discontinuously on endothelial P‐selectin. We use a microfluidic device to study the rolling of leukocytes in heparinized mouse blood over a coverglass with adhered P‐selectin at known site density. The microfluidic device has an array of 300×30 μm channels, and is reversibly sealed against the coverglass using a magnetic clamp. Leukocytes are observed to undergo periods of stable rolling separated by jumps indicating brief periods of detachment from the substrate. During the jumps, cells are observed to move at velocities comparable with the theoretical estimates for a cell freely flowing in a shear flow. In the entire range of wall shear stress (2‐8 dyes/cm2), the average rolling velocities of leukocytes linearly increase with the shear stress. This increase is due to faster translocation of leukocytes during the jumps rather than increased velocity of rolling. The velocity of leukocytes during the periods of stable rolling remains nearly unchanged in the shear stress range studied. The rolling is completely inhibited by a mAb against mouse P‐selectin, demonstrating molecular specificity. We conclude that the apparent variation of rolling velocity with wall shear stress is mainly caused by increased distances traveled by leukocytes during periods of detachment. This study was supported by NIH EB 02185 (K.L) and NSF NIRT 0608863 (A.G).
Temporal and spatial investigations of the cellular mechanisms of H2O2‐induced increases in microvessel permeability in individually perfused rat mesenteric venules
Authors: Zhou Xueping , He Pingnian
Published: June 2021

We previously showed that H2O2 induced delayed but progressive increases in microvessel permeability, a pattern different from agonist‐induced immediate and transient permeability increase. This study aims to investigate the cellular mechanism of H2O2‐induced permeability increase. We hypothesized that oxidation‐induced apoptosis contributes to H2O2‐induced permeability increase. The temporal and spatial occurrence of apoptosis in H2O2 perfused rat mesenteric venules was examined with fluorescence labeled annexin‐V, a phosphatidylserine‐binding protein, and caspase‐3,7 binding peptide using confocal imaging. No annexin‐V and caspase‐3,7 staining was found in Ringer‐albumin perfused vessels. Apoptosis did not occur in vessels perfused with 100 μM H2O2 until after 60 min of perfusion, in which apoptosis occurred only in pericytes, not endothelial cells (ECs). Apoptosis occurred in both ECs and pericytes after perfusion of 500 μM H2O2 for 45 min. The application of Ca2+ channel blocker LaCl3 (50 μM) that blocked H2O2‐induced Ca2+ influx significantly reduced H2O2‐induced apoptosis in both ECs and pericytes. These results indicated that Ca2+ influx‐dependent apoptosis of ECs and/or pericytes might be the mechanism for H2O2‐induced increases in microvessel permeability. Pericytes showed higher sensitivity to lower concentration of H2O2 than ECs in apoptosis. Supported by HL56237 and HL084338.
In vivo glycocalyx degradation induces proteinuria and insulin resistance without affecting atherogenesis in apoE knockout mice on a Western‐type diet
Authors: van den Berg Bernard M , Meuwese Marijn C , Stroes Erik S G , Vink Hans
Published: June 2021

Four weeks of hyaluronidase (HYAL) infusion was used to study the role of the endothelial glycocalyx in vascular circulatory‐ and barrier properties, and atherogenic lesion progression in apoE knockout (apoE−/−) mice on a Western‐type diet (HFC). Male apoE−/− on HFC for 10 weeks and Hyal (10 U/hr, i.v. in the last 4 weeks) revealed a significantly reduced systemic endothelial glycocalyx volume of 11.3 ± 7.7 mL/kg BW, compared to pre diet‐ and Hyalinactive groups (21.6 ± 6.3 and 17.9 ± 9.8 mL/kg BW, respectively). Red blood cell volume (27.6 ± 4.0 vs. 27.9 ± 1.6 and 30.5 ± 6.0 mL/kg BW, Hyal vs. pre diet and Hyalinactive, respectively) and hematocrit (0.43 ± 0.03 vs. 0.45 ± 0.02 and 0.41 ± 0.03) were not changed. Hyal infusion induced a significant renal protein leakage (urine protein/creatinin ratio 0.27 ± 0.02 vs. 0.06 ± 0.03 and 0.15 ± 0.01), without changes in renal morphology, and increased insulin resistance (HOMA‐IR 6.6 ± 5.7 vs. 1.7 ± 0.1 and 3.3 ± 2.5). There was no effect on atherogenic lesion progression (innominate artery lesion area 14.9 ± 6.2 vs. 0.74 ± 1.0 and 16.4 ± 9.1 x 10,000 µm2 ). Our findings indicate that accelerated glycocalyx degradation in an atherogenic mouse model contributes to loss of glycocalyx volume and in turn modest changes in renal function and insulin homeostasis without affecting atherogenesis.

Supported by NHF 2005T037 to H. Vink
Endothelial ephrin‐B2 is essential for arterial dilation
Authors: Wang Rong
Published: June 2021

The Efnb2 gene encodes the cell surface protein ephrin‐B2, which is expressed in the arteries but not veins. While ephrin‐B2 plays essential roles in vascular development, its function in adult vasculature remains unknown. We have generated mice lacking Efnb2 specifically in endothelial cells (Efnb2ΔEC), using Tetracycline‐regulated Cre excision. About 60% of the mutants survived into adulthood and exhibited no obvious abnormalities. However, immediately following femoral artery occlusion, the Efnb2ΔEC mice exhibited lower residual foot blood flow. Foot gangrene seldom seen in controls developed in all mutants starting one day after occlusion. No obvious vascular structural abnormalities were detected to explain the blood flow defect. Acute arterial dilation induced by increased blood flow, however, in both cremaster arterioles and common carotid arteries, was impaired in Efnb2ΔEC mice. Acetylcholine‐induced arterial dilation was also reduced in Efnb2ΔEC cremasters examined. Decreased eNOS protein level and Nitric Oxide (NO) production were evident in Efnb2−/− arterial endothelial cells. S‐Nitrosoglutathione NO, a more stable NO derivative, partially rescued gangrene progression in Efnb2ΔEC mice. Our data suggest that ephrin‐B2 plays an essential function in arterial dilation, through an NO‐mediated mechanism, and thus ephrin‐B2 is a novel molecular regulator for arterial specific dilation, crucial to vascular physiology.
A theoretical study of myoendothelial communication: K+‐mediated EDHF signaling and the role of myoendothelial projections
Authors: Kapela Adam , Nagaraja Sridevi , Tsoukias Nikolaos M.
Published: June 2021

Experiments indicate that a small increase in the extracellular potassium concentration ([K+]o) may act as endothelium‐derived hyperpolarizing factor (EDHF). This hypothesis has been challenged by some studies and has received little theoretical analysis. Here we examine K+‐induced hyperpolarizations in mathematical models of isolated and integrated endothelial and smooth muscle cells. The effect of various components on the response is examined, including the role of channels, pumps, and the presence of distinct microdomains in myoendothelial projections. Activation of the sodium‐potassium pump by K+ generates only transient hyperpolarization and a rebound depolarization when [K+]o is restored. Activation of inward rectifying potassium channels by K+ could produce strong and sustained hyperpolarization, but the cell had to be initially depolarized. A large number of inward rectifying potassium channels introduces a bistable behavior and prevents return to resting state. K+‐mediated EDHF was small under most of the examined scenarios, including localization of important components in the myoendothelial projections. These results suggest a rather minor role for extracellular K+ as EDHF, but may also indicate limitations of the current electrophysiological models. [Supported by AHA grant NSDG043506N and NIH grant HL095101]
Impact of P450 eicosanoids on bioavailability of NO in the vasculature of mice
Authors: Gollasch Maik , Hercule Hantz C. , Schunck Wolf‐Hagen , Gross Volkmar , Seringer Jasmin , Leung Fung Ping , da Costa Goncalves Andrey Ch. , Huang Yu , Luft Friedrich C.
Published: June 2021

Epoxyeicosatrienoic acids (EETs) serve as endothelial‐derived hyperpolarizing factors (EDHF), but may also affect vascular function by other mechanisms. We identified a novel interaction between EETs and endothelial NO release using soluble epoxide hydrolase (sEH) ‐/‐ and +/+ mice. EDHF responses to acetylcholine in pressurized isolated mesenteric arteries were neither affected by the sEH inhibitor, N‐adamantyl‐N′‐dodecylurea (ADU), nor by sEH gene deletion. However, the EDHF responses were abolished by catalase and by apamin plus charybdotoxin (ChTx). All four EETs (5,6‐EET, 8,9‐EET, 11,12‐EET, 14,15‐EET) (total 243 +/‐ 56 ng/g, ratio 52:16:11:21) were present in the vessel wall of mesenteric arteries. All four EETs (order of potency: 8,9‐EET>14,15‐EET~5,6‐EET>11,12‐EET) and all four dihydroxy derivatives (14,15‐DHET~8,9‐DHET~11,12‐DHET>5,6‐DHET) produced dose‐dependent vasodilation. Endothelial removal or L‐NAME blocked 8,9‐EET and 14,15‐DHET‐dependent dilations. The effects of apamin/ChTx were minimal. 8,9‐EET and 14,15‐DHET induced NO production in endothelial cells. ADU (100 μg/ml in drinking water) lowered blood pressure in angiotensin II‐infused hypertension, but not in L‐NAME‐induced hypertension. Blood pressure and EDHF responses were similar in L‐NAME‐treated sEH +/+ and ‐/‐ mice. Our data indicate that the EDHF response in mice is caused by hydrogen peroxide, but not by P450 eicosanoids. Moreover, P450 eicosanoids are vasodilatory, largely through their ability to activate endothelial NO release. This pathway may increase in importance during cardiovascular disease, when NO production is reduced.
Physical activity prevents endothelial dysfunction induced by sedentary life style and high fat diet in murine coronary microcirculation
Authors: Park Yoonjung , Lee Sewon , Booth Frank W , Laye Matthew J , Zhang Cuihua
Published: June 2021

Sedentary life style and high fat diet increase a risk of diabetes and cardiovascular diseases, but physical activity counteracts high fat diet‐induced vascular dysfunction. Our goal was to investigate the mechanisms by which physical activity prevents endothelial dysfunction caused by high fat diet. Female mice (C57BL/6) were fed either control low‐fat diet (LF; 15% fat) or high‐fat diet (HF; 45% fat) for 10 weeks. HF mice were randomly assigned to either a sedentary group or a group given access to a voluntary running wheel (HF‐Run). HF sedentary group was divided to HF‐Lean (HF‐LN; body weight<28g) and HF‐Obese (HF‐OB; body weight>28g) group. Endothelium‐dependent dilation (isolated coronary arterioles) to acetylcholine and to flow were impaired in HF‐OB mice, but were greater in HF‐Run and HF‐LN mice compared to HF‐OB mice. Leptin‐mediated vasodilation was higher in HF‐LN compared to HF‐OB. Compared to LF, HF‐LN, and HF‐Run mice, leptin levels and insulin resistance were higher in HF‐OB mice. These results suggest that the leptin and insulin resistance caused by high fat diet were reversed by physical activity. Also physical activity prevented high fat‐induced endothelial dysfunction, which may be mediated and improved leptin and insulin sensitivity.
MRP4 as a regulator of β1‐adrenergic receptor signaling in cardiomyocytes
Authors: Sellers Zachary M , Naren Anjaparavanda P , Xiang Yang , Best Philip M
Published: June 2021

Modulation of β‐adrenergic receptor (β‐AR) stimulated contraction rate in cardiomyocytes involves distinct pools of cAMP. Multi‐drug resistance protein 4 (MRP4) regulates local cAMP concentrations in epithelial cells resulting in control of the cystic fibrosis transmembrane conductance regulator (CFTR). Since the role of MRP4 in cardiomyocytes is unknown, we examined MRP4 involvement in cAMP‐dependent processes through investigation of β‐AR stimulated cardiomyocyte contraction rate and CFTR function. Spontaneous beating cardiomyocytes were generated by isolation and culture of mouse neonatal ventricular myocytes. MRP4 was detected by Western blot. Inhibition of MRP4 significantly enhanced submaximal β‐AR stimulated contraction rate. Use of β‐AR knockout mice indicated this response was mediated by β1‐AR, not β2 or β3. Additionally, this potentiation was abolished by inhibition or knockout of CFTR. In contrast, phosphodiesterase 4 (PDE4)‐dependent potentiation did not require CFTR activity or expression. We propose that while MRP4 and PDE4 are both involved in β1‐AR signaling, the influence of MRP4 is constrained to local regions near CFTR, whereas PDE4 can influence broad changes. Investigation into this is ongoing, however, these results present MRP4 as an additional player in the complex and dynamic control of β1‐AR signaling in cardiomyocytes. Funded by the AHA, NIH, University of Illinois.
ATM plays a protective role in β‐adrenergic receptor (β‐AR)‐stimulated cardiac myocyte apoptosis and myocardial remodeling
Authors: Foster Cerrone R. , Madireddy Sreedhar R. , Singh Mahipal , Joyner William L. , Singh Krishna
Published: June 2021

β‐AR stimulation induces cardiac myocyte apoptosis and plays an important role in myocardial remodeling. We tested the hypothesis that ATM (ataxia telangiectasia mutated kinase; a protein involved in cell cycle checkpoints, DNA repair and apoptosis) plays a protective role in myocyte apoptosis and myocardial remodeling following β‐AR stimulation. Left ventricular structure and function, myocyte apoptosis, and fibrosis were examined in wild‐type (WT) and ATM heterozygous knockout mice (hKO) 28 days after L‐isoproterenol (ISO; 400µg/kg/h) infusion. Heart weight to body weight ratio was higher in hKO group with or without ISO‐infusion. Echocardiographic parameters were not different between the two sham groups. ISO‐infusion increased percent fractional shortening (%FS) and ejection fraction (EF) in both groups. Interestingly, the increase in %FS and EF was lower in hKO‐ISO group (p<0.01). Myocyte apoptosis and fibrosis were higher in hKO‐sham vs WT‐sham. ISO‐infusion increased apoptosis and fibrosis in both groups. However, the increase in fibrosis and apoptosis was higher in hKO‐ISO (p<0.01).Western blot analysis revealed increased p53 and reduced MMP‐9 protein levels in hKO‐ISO. Thus, deficiency of ATM induces remodeling at basal levels. Chronic β‐AR stimulation adversely affects heart function in ATM deficient mice with increased myocyte apoptosis and myocardial fibrosis.
Decreased knee extensor torque and power production in human heart failure
Authors: Toth Michael J. , Shaw Anthony O. , Ades Philip A. , Miller Mark S.
Published: June 2021

Exercise intolerance is a hallmark symptom of chronic heart failure. The goal of this study was to determine if heart failure impairs skeletal muscle torque, power production or contractile velocity. Heart failure patients (n=11; 70.4 ± 4.3 yrs) and controls (n=11; 70.3 ± 3.4 yrs) matched for age, sex and medications that may affect muscle function were evaluated for knee extensor contractile performance, body composition and physical activity level. Groups did not differ in total or regional body composition or physical activity level. Despite similarities in muscle size and use, heart failure patients were characterized by 21‐29% lower (P<0.05 to P<0.01) isometric knee extensor torque throughout a range of knee angles, 15‐33% lower (P=0.05 to P<0.01) peak torque and power output at various isokinetic speeds. These differences in muscle function were evident after adjustment of data for muscle mass. Expression of peak isokinetic torque data relative to isometric torque eliminated group differences, suggesting that impaired function under dynamic conditions is explained by deficits in the force generating capacity of muscle. No group differences were found in the time required to reach target velocity during isokinetic contractions, a proxy of contractile velocity. These results suggest that heart failure reduces skeletal muscle peak torque and power production, but does not alter contractile velocity.
A proteomics‐based approach to changes in skeletal muscle gene expression with age‐related sarcopenia
Authors: Chin Eva R , Ibebunjo Chikwendu , McDowell Michael T
Published: June 2021

The purpose of this study was to identify proteins that are differentially expressed in sarcopenic rat skeletal muscle. Gastrocnemius muscle was obtained from 18 and 27 mos. old Harlan Sprague Dawley rats (n=3). These ages were chosen based on the 50% reduction in muscle mass and maximum force that were observed over this period. Muscle homogenates were prepared, run on 2‐D gels (n=3‐4 gels per muscle) and optimum gels chosen for overlay and identification of altered proteins. Twenty‐six proteins, some with multiple isoforms, were differentially regulated with 16 being down‐regulated and 10 being up‐regulated in 27 vs. 18 mos. gastrocnemius muscle. The altered proteome included changes in: i) contractile proteins reflecting a decrease in fast muscle‐specific and an increase in slow muscle‐specific proteins; ii) enzymes of metabolic pathway reflecting an increase in PCr metabolism but decreases in glycolytic and oxidative metabolism; and iii) increased anti‐oxidant enzymes reflecting a compensatory response to oxidative stress. These data support previous reports of a loss of fast twitch fibres and demonstrate concomitant decreases in oxidative and glycolytic capacities. These data suggest that altered muscle fibre type and metabolic rate along with increased cellular oxidative stress contribute to the age‐related decrease in muscle function.
Sensitivity of neuromuscular junctions to unloading and pre‐habilitation
Authors: Sherman Elizabeth Grace , Glass Emily , Deschenes Michael
Published: June 2021

It has been well documented that muscle unloading elicits significant decrements in muscle strength and size. In contrast, the effects of muscle unloading on the neuromuscular junction (NMJ) are less elucidated and the potential of pre‐habilitative conditioning remains unknown. The aim of this study was to examine the impact of unloading on the NMJs of aged rats, as well as the capacity of pre‐habilitation to affect those synapses. Thirty aged (25 mo) male Fischer 344 rats were assigned to 3 groups; 1) control, 2) unloaded (UL), 3) pre‐habilitation before unloading (PH‐UL). UL rats were subjected to hindlimb suspension for 2 weeks, and PH‐UL rats completed a 2 week (5days/wk) treadmill running program before 2 weeks of hindlimb suspension. Following all interventions, all rats were euthanized and soleus muscles were removed and frozen at resting length. Cytofluorescent techniques were used to visualize pre‐synaptic nerve terminal branching, and post‐synaptic acetylcholine receptors. Confocal microscopy was used to collect images of pre‐ and post‐synaptic morphology. Our results indicated that neither pre‐ nor post‐synaptic morphological parameters were altered in UL or PH‐UL rats. Thus, a 2 week unloading period is insufficient to modify synaptic structure among aged rats. Moreover, the combination of 2 weeks of run training and 2 weeks of unloading also failed to provoke changes in NMJ morphology.

Supported by the National Institute on Aging, and The Foundation for Aging Studies and Exercise Science Research.
Exercise‐induced increases in trabecular bone volume are associated with increased nitric oxide‐mediated vasodilation in osseous vasculature of young and old rats
Authors: Dominguez James Michael , Prisby Rhonda D. , Allen Matthew R. , Delp Michael D.
Published: June 2021

Old age‐associated osteoporosis is related to diminished bone blood flow and impaired nitric oxide (NO)‐mediated vasodilation. The purpose of this study was to determine whether exercise training enhances endothelium‐dependent vasodilation and correspondingly improves bone volume in aged animals. Young adult (4‐6mo) and old (24‐26mo) male Fischer‐344 rats remained sedentary or were exercise trained (15m/min, 15o incline, 5days/wk, 10‐12wk). Endothelium‐dependent vasodilation of the femoral principle nutrient artery was assessed in vitro using acetylcholine (ACh) and inhibitors of NO synthase (NOS) and cyclooxygenase (COX) were used to determine the signaling pathways involved. Endothelium‐dependent vasodilation was greater after exercise training (young: 87±3%; old: 73±5% relaxation) compared to sedentary (young: 75±6%; old: 59±5% relaxation). In old rats, the NOS‐mediated contribution to endothelium‐dependent vasodilation was enhanced by exercise training. Distal femur trabecular bone volume (BV/TV) was significantly reduced with old age in sedentary animals (young: 27±2%; old: 23±1%). Exercise training increased BV/TV in both groups (young: 34±2%; old: 26±1%) compared to sedentary. These data are the first to demonstrate that exercise training impacts the osseous vasculature. Moreover, a training‐induced elevation in the NOS signaling pathway is associated with greater trabecular bone volume.
Baroreflex sensitivity is impaired in Sprague‐Dawley rats with spontaneous hydronephrosis independent of angiotensin II actions in the solitary tract nucleus
Authors: Arnold Amy C , Diz Debra I
Published: June 2021

Hydronephrosis, associated with ureteral obstruction, activates the circulating renin‐angiotensin system (RAS). Elevations in the RAS impair baroreflex sensitivity (BRS) for control of heart rate (HR) involving central sites of action. We hypothesized that central cardiovascular regulation is altered in this condition. Resting arterial pressure (AP), HR and BRS for control of HR were assessed in urethane/chloralose anesthetized Sprague‐Dawley (SD) rats with normal or hydronephrotic (HN) kidneys defined by established criteria. BRS was significantly lower in rats with severe HN (0.56 ± 0.08 vs 1.06 ± 0.06 msec/mm Hg in normal rats; p < 0.01, n = 7, 8) with a trend for lower BRS in rats with mild/moderate HN (0.80 ± 0.13 msec/mm Hg, n = 8). While resting AP was similar among conditions, resting HR was significantly elevated in mild/moderate and severe HN rats (290 ± 12 normal vs 344 ± 11 mild/moderate vs 355 ± 13 bpm severe; p < 0.01). A possible site for Ang II‐mediated BRS impairment is the solitary tract nucleus (NTS). NTS administration of the Ang II AT1 receptor blocker candesartan had no significant effects on AP, HR or BRS in severe HN rats, but enhanced BRS by ~ 40% in normal rats. Thus, hydronephrosis is associated with impairments in BRS and autonomic regulation. Since Ang II provides inhibitory tone to the BRS in normal rats, the lack of Ang II actions in hydronephrosis may suggest dysregulation of the local NTS RAS. HL51952
Effect of water deprivation on ACE binding and AT1 angiotensin receptor expression in regions of the brain that regulate hemodynamics
Authors: Bourassa Erick A , Speth Robert C
Published: June 2021

The rostral ventrolateral medulla (RVLM) is a brainstem region critical to the maintenance of blood pressure. A period of water deprivation leads to increased sensitivity of the RVLM and paraventricular nucleus of the hypothalamus (PVH) to angiotensin II (Ang II). This Ang II helps sustain blood pressure. It has been proposed that peripheral Ang II can influence the RVLM via an angiotensinergic neural connection involving circumventricular organs and the PVH. We studied the differences in angiotensin type 1 (AT1) receptor and angiotensin converting enzyme (ACE) expression in these and other brain regions involved in blood pressure regulation and water intake following a 48 hr period of water‐deprivation. Brains were sectioned and used for quantitative AT1 receptor and ACE autoradiography. AT1 receptor expression was increased in the subfornical organ and periventricular nucleus of the hypothalamus, but not in other brain regions measured. ACE expression was increased in the RVLM, PVH, choroid plexus, median preoptic nucleus, and organosum vasculosum of the lamina terminalis. These findings suggest that increased brain‐derived Ang II production, but not increased receptor expression or sensitivity is the mechanism by which Ang II in the brain helps to sustain systemic blood pressure during water‐deprivation. Supported by the Peptide Radioiodination Service Center of the University of Mississippi.
Expression and angiotensin II‐induced modulation of KCNQ K+ channels in rat visceral sensory neurons
Authors: Tolstykh Gleb , Shapiro Mark
Published: June 2021

Neurons of the nodose ganglion (NG) play an essential role in transmitting barosensory information to the nucleus tractus solitarius (NTS) in the brainstem, a nucleus critical to autonomic cardiovascular regulation. Recently, NG neurons were found to express M‐type (KCNQ) K+ channels (Wladyka et al., 2006; 2007). We now show that KCNQ channels are expressed in the NTS as well. M‐current densities in NTS neurons (at ‐60 mV) were 0.98 ± 0.1 pA/pF (n=3) and immunostaining analysis revealed expression of at least KCNQ2 subunits. In the NG, M‐current densities of A‐type and C‐type neurons were 1.2 ± 0.2 (n=7) and 0.95 ± 0.11 pA/pF (n=12), with time constants of deactivation of 65 ± 12 and 51 ± 8 ms, respectively. Of 16 cells, angiotensin II (AngII, 500 nM) suppressed M‐current amplitude in 7 neurons by 42 ± 9%. AngII caused translocation of the PLC‐PH‐EGFP probe from membrane to cytoplasm, with an increased cytosolic fluorescence of 46 ± 30% (n=4), demonstrating AngII‐induced hydrolysis of PIP2. Ca2+ imaging showed 9 of 40 NG neurons challenged with AngII to respond with substantial [Ca2+]i increases (340 nm/380 nm ratio 0.15 ± 0.01). The Ca2+ imaging results in NG neurons are similar to those obtained from NTS neurons reported earlier (Tolstykh et al., FASEB 2006). Our data suggest an important role of KCNQ K+ channels in autonomic control of the heart and vascular tone. Supported by AHA grant 0865151F (G.T.) and NIH NS043394 (M.S).
Insights into novel host factors required for HIV‐1 replication in human cells
Authors: Jeang Kuan‐Teh
Published: June 2021

Comprehensive genomewide screening of genes needed by various viruses for replication in human cells has become achievable with the advent of large libraries of siRNAs or shRNAs. To study genes needed for HIV‐1 to replicate in human T‐cells, a lentivirus‐vector based shRNA library that targets 47,400 human mRNAs using 200,000+ discrete shRNAs was employed. This approach allowed for the generation of independent cell clones, each chronically and individually expressing a single shRNA. When a pool of these cell clones was infected with high titer HIV‐1, cells that survived virus‐infection were ones that have knocked‐down a human mRNA whose loss interrupted productive HIV‐1 infection. The analyses of survivor cells revealed a set of candidate mRNAs that are needed for HIV‐1 replication. This approach and some of the gene candidates and their functions revealed from this study will be discussed.
Glial contribution to the modulation of preBötzinger Complex rhythm generating networks by ATP
Authors: Huxtable Adrianne G , Zwicker Jennifer D , Alvares Tuca S , Ruangkittisakul Araya , Fang X , Ballanyi Klaus , Funk Gregory D
Published: June 2021

Activation of P2Y1 receptors in the preBötzinger Complex (preBötC) potently increases frequency (f) (J Neurosci, 27, 993‐05, '07). This could reflect the ubiquitous but weak sensitivity of preBötC neurons to ATP (J Physiol, 586, 129‐46, '08). The present study used rhythmic medullary brainstem slices from neonatal rats to test if glial cells contribute to the ATP‐evoked f increase. Control ATP responses were compared to those after glial function was disrupted by bath‐applied glial toxins (>60 min, fluoroacetate, FA, or methionine sulfoximine, MSO), and rhythm restored with glutamine (1.5 mM). In control, f doubled in response to ATP (0.1 mM, 10 s). However in toxin, ATP was either without effect (MSO, n=7), or 1.5‐fold less potent (FA, n=10). Substance‐P responses were unaffected by the toxins. We next tested whether the glial contribution could involve their direct sensitivity to ATP. Two‐photon imaging of fluo‐4 Ca2+ fluorescence revealed small cells in the preBötC with somatic areas = 100 μm2 (n=126, 5 slices) are sensitive to bath‐applied ATP (0.1 mM). Whole‐cell recordings confirmed the presence of small, non‐excitable, low input resistance (23±2 MO) cells in this region are ATP‐sensitive (n=4). These data indicate that glial cells contribute to the f increase evoked by ATP in the preBötC, and that glial P2 receptors are likely involved. Supported by NSERC, CIHR‐MFN, CIHR, AHFMR, CFI, ASRA.
Local microejection of mu‐opioids into the pre‐Bötzinger complex (pBC) region produces opposite effects on breathing rate to systemic mu‐opioid infusion in decerebrate dogs
Authors: Mustapic Sanda , Radocaj Tomislav , Stucke Astrid G. , Stuth Eckehard A. , Dogas Zoran , Zuperku Edward J.
Published: June 2021

Systemic administration of mu‐opioids at clinical doses typically produces bradypnea. This study evaluated whether mu‐opioid receptors within the pBC region mediate this response. The effects of local microinjections of the selective mu‐opioid agonist [D‐Ala2,N‐Me‐Phe4,Gly‐ol5]‐enkephalin (DAMGO;100 μM) into the pBC region on the phrenic neurogram were studied in a decerebrate, vagotomized, mechanically‐ventilated, paralyzed canine preparation during hyperoxia. The location of the pBC was determined by ventral respiratory column neuronal recordings and the maximum tachypneic phrenic response to glutamate agonist (DLH) microejections. Following unilateral DAMGO microejections (190±13 nl), the maximum increase in respiratory rate was 44±5% (SE) in 16 dogs. Bilateral DAMGO microejections (4 dogs) did not cause additional increases in respiratory rate compared to unilateral ejection. Subsequent i.v. infusion of the mu‐agonist remifentanil typically produced a marked reduction in rate to 44.5±8.0% of control that more than offset the DAMGO induced tachypnea. These findings support our previous study indicating that clinical doses of mu‐opioids produce their bradypneic effect via mu‐opioid receptors that are located outside the pBC. In contrast activation of mu‐opioid receptors within the pBC region produces tachypnea in vivo. Supported by VA Medical Research and NIH GM059234 funds.
Episodic respiratory motor output is decreased by 5‐HT3 receptor agonists and increased by 5‐HT3 receptor antagonists in isolated adult turtle brainstems
Authors: Bartman Michelle Ellen , Johnson Stephen Mark
Published: June 2021

Episodic breathing is a respiratory pattern marked by episodes of several consecutive breaths that are separated by non‐ventilatory pauses. Episodic breathing occurs naturally in some ectothermic vertebrates, but the underlying mechanisms are not known. Approximately 56% of isolated turtle brainstems produce single bursts of respiratory motor output on hypoglossal nerves (bursts/episode < 1.25), while 25% of isolated turtle brainstems produce episodic respiratory bursts (bursts/episode > 1.75; n=171; Johnson & Creighton, Am J Physiol Regul Integr Comp Physiol, 2005). Bath‐application of 5‐HT3 agonists, such as phenylbiguanide (PBG; 20 µM; n=8) or 1‐(m‐Chlorophenyl)biguanide hydrochloride (mCPBG; 50 µM; n=6), onto isolated turtle brainstems switched episodic respiratory bursts (2.04 ± 0.06 bursts/episode) into singlet bursts (1.31 ± 0.09 bursts/episode) after 1.0 h (P<0.001). Bath‐application of 5‐HT3 antagonists, such as tropisetron (50 µM; n=5) and MDL‐72222 (50 µM; n=3) tended to switch singlet respiratory bursts (1.05 ± 0.03 bursts/episode) into episodic bursts (1.52 ± 0.06 bursts/episode) after 2.0 h (P>0.05). These data suggest that 5‐HT3 receptors in the brainstem are involved in regulating episodic breathing in turtles.
Developmental neurotoxicity impairs ventilatory control: insight from an amphibian model
Authors: Taylor Barbara E , Brundage Cord , Audie Spencer , Lamb Jessica
Published: June 2021

Neurotoxins (nicotine, ethanol) elicit deficits in neuroventilation by impairing responses to CO2. This may explain why these toxins are risk factors for SIDS. We recorded neuroventilation from tadpole hindbrains before and during CO2 stimulation. Recordings were made during acute toxin exposure of hindbrains from naïve animals or following chronic toxin exposure of intact animals. Acute nicotine exposure decreased baseline neuroventilation in some tadpole stages but did not alter CO2 responses. Acute ethanol exposure depressed baseline neuroventilation but did not alter CO2 responses. Chronic exposure (nicotine or ethanol) abolished CO2 responses. These data indicate that chronic and acute toxin exposures differentially impair the neuroventilation. To determine the potential for recovery, tadpoles were returned to control conditions following chronic toxin exposure. Impairment by chronic nicotine disappeared over a 3 wk recovery period, but impairment by chronic ethanol persisted despite 6 wk recovery. Chronic ethanol causes a persistent developmental deficit in the neuroventilatory CO2 response, whereas chronic nicotine produces transient impairment. To determine whether neurogenesis underlies recovery, we exposed recovering tadpoles to BrDU. BrDU labeling unique to nicotine‐recovery animals could indicate regeneration of neurons critical to the CO2 response.
Gestational stress delays maturation of the hypoxic ventilatory response: an in vivo and in vitro study
Authors: Kinkead Richard , Fournier Stéphanie , Fournier Sébastien , Voituron Nicolas , Hilaire Gérard
Published: June 2021

Exposure to stress during gestation disrupts CNS development; however, its effects on the respiratory control system are unknown. We tested the hypothesis that exposing dams to a predator odor during gestation disrupts the maturation of the hypoxic ventilatory response (HVR) of newborn rats. From the 9th to the 19th day of pregnancy, females subjected to gestational stress (GS) were placed in a cage containing 35 μl of TMT, a component of fox odor for 20 min. For sham treatment, gestating females were exposed to a strong odor (butyric acid, 105 μl). Controls were undisturbed. Ventilatory activity was measured at post natal days 0, 2 and 4 using plethysmography (in vivo) and "en bloc" brainstem spinal cord preparations (in vitro). Measurements were performed under baseline conditions and hypoxia. In vivo: unlike controls, P4 male pups born to GS dams could not sustain a significant increase in breathing frequency through out the hypoxic period (12% O2, 20 min). This effect of GS was not observed in females. In vitro: at the end of hypoxia (7% O2, 20 min), preparations from GS pups showed a smaller phrenic burst frequency decrease than controls (‐58% vs ‐76%). No sexual dimorphism was observed in vitro. GS delays maturation of the hypoxic ventilatory response. Comparison of in vivo vs in vitro data suggests that GS affects peripheral chemoreceptor development and/or integration of afferent signal in the CNS. Supported by the CIHR.
TRPC1‐Mediated Ca2+ Entry Increases Lung Microvascular Permeability
Authors: Tauseef Mohammad , Knezevic Nebojsa , Malik Asrar B , Abramowitz Joel , Birnbaumer Lutz , Mehta Dolly
Published: June 2021

Despite the evidence that transient receptor potential canonical (TRPC1) channel can mediate Ca2+ entry in endothelial cells the role of TRPC1 activity in the mechanism of increased lung vascular permeability remains unclear. We isolated lungs from TRPC1−/− and wild type (WT) mice and activated Ca2+ entry by two independent mechanisms to determine the role of TRPC1 in regulating lung microvascular permeability. We perfused lungs with either thapsigargin, that activates Ca2+ entry by depleting intracellular stores or diacylglycerol (DAG), that activate Ca2+ entry independent of store depletion. We determined lung microvascular permeability by determining filtration co‐efficient (Kfc), a measure of hydraulic conductivity across endothelial exchange barrier. We show that in WT lungs thapsigargin increased lung Kfc whereas thapsigargin failed to increase Kfc in lungs isolated from TRPC1−/− mice. Interestingly, OAG, a cell permeable analog of DAG, increased Kfc in TRPC1−/− mice but the Kfc response was significantly lower than that observed in WT mice lungs. We next determined the contribution of TRPC1 in regulating lung microvascular permeability in response to thrombin ligation of protease activating receptor 1 (PAR‐1) which activate Ca2+ entry both by store‐dependent and DAG‐dependent manner. We show that PAR‐1 activation by specific PAR‐1 peptide increased Kfc in WT lungs whereas it failed to induce a response in lungs isolated from TRPC1−/− mice. We also address the role of TRPC1 in the lung inflammatory response to sepsis induced by i.p. injection of LPS. Prevention of lung injury in TRPC1−/− mice was associated with decreased mortality in response to LPS challenge. Thus, TRPC1 through its ability to regulate Ca2+ entry is a crucial determinant of increased lung microvascular permeability and inflammatory response.
Exocytosis of Endothelin‐1 from Endothelial Weibel‐Palade Bodies Contributes to Vasoconstriction in Aged Arteries
Authors: Goel Aditya A , Lowenstein Charles , Berkowitz Dan , Flavahan Nicholas
Published: June 2021

Endothelin‐1 (ET) has been localized to Weibel‐Palade Bodies (WPB) in endothelial cells. Experiments were performed to analyze the role of WPB‐derived ET in arterial regulation. Aortas from young (3 months) and aged F344 rats (19 months) were isolated and processed for isometric tension recording, ET release and immunofluorescent analysis. After inhibition of NO synthase (L‐NAME, 100 µM), thrombin (1U/ml) caused rapid contraction of aged aortas that was abolished by endothelial‐denudation, the ETA receptor antagonist BQ123 (1 µM) or by TAT‐NSF (1 µM), a cell‐permeable inhibitor of N‐ethylmaleimide sensitive factor (NSF) and inhibitor of WPB exocytosis. In young aortas, thrombin (1 U/ml) evoked a small contraction that was not inhibited by BQ123 or endothelial denudation. The response to thrombin in aged aortas was associated with a 5‐fold increase in ET release that was prevented by TAT‐NSF or endothelial‐denudation. WPBs were visualized by immunofluorescent imaging of von Willebrand factor using laser scanning microscopy of aortic segments. In aged aortas, thrombin caused a marked translocation of WPB to the cell surface. In conclusion, the acute release and exocytosis of ET from endothelial cells can contribute to vascular regulation. The dramatic increase in this activity in aged arteries might contribute to thrombogenic and inflammatory events associated with aging. (NIH grants # HL 80119 and OH 8531)
Membrane and Soluble Adenylyl Cyclases Generate Discrete cAMP Pools which Discriminate between Cytoskeletal Binding Proteins to Regulate Endothelial Barrier
Authors: Sayner Sarah L. , Prasain Nutan , Balczon Ron , Stevens Troy
Published: June 2021

Transmembrane adenylyl cyclases generate a cAMP pool restricted to the sub‐plasma membrane compartment, which strengthens endothelial barrier integrity. In contrast, a eukaryotic dependent bacterial adenylyl cyclase, ExoY of P. aeruginosa, generates cytosolic cAMP that disrupts the endothelial barrier. Similarly, a soluble chimeric mammalian adenlyl cyclase generates forskolin sensitive cAMP in both the subplasma membrane and bulk cytosolic compartments and disrupts the barrier. The molecular targets responsible for the diverse actions of these two distinct cAMP microdomains ‐ plasma membrane versus cytosolic ‐ are unknown. Filamin, an actin binding protein, is PKA phosphorylated at Ser2152, thus stabilizing its association with actin, which strengthens the barrier. In contrast, tau stabilizes microtubules and is PKA phosphorylated at Ser214 destabilizing microtubules sufficient to disrupt the endothelial barrier. We found that activation of transmembrane adenylyl cyclase leads to phosphorylation of filamin2152, while activation of cytosolic adenylyl cyclase induces phosphorylation of tau214. These data suggest that the barrier enhancing properties of subplasma membrane cAMP are transduced via filamin mediated stabilization of actin while barrier disruptive properties of cytosolic cAMP are transduced via tau mediated disruption of microtubules. Supported by HL66299 and F32HL091651promotes orthogonal branching of F‐actin and links microfilaments to membrane glycoprotein.
Retinoic acid decreases endothelial permeability and fenestrae‐associated protein PV‐1 expression in human umbilical vein endothelial cells (HUVEC)
Authors: Bodor Csaba , Németh Adrienn , Sebe Attila , Rosivall László
Published: June 2021

Vascular endothelial growth factor (VEGF), recognized as a growth factor secreted by tumor cells, increases PV‐1 expression and endothelial permeability. Retinoic acid (RA), a vitamin A derivative, regulates cell differentiation and is implicated in obesity, diabetes and cardiovascular disease. We postulated that RA may alter the effects of VEGF on HUVEC. To test this hypothesis we measured if administration of RA modulates PV‐1 expression and endothelial permeability in cell culture. HUVEC cultures were treated with 1 and 10 μM of RA. mRNA and protein levels of PV‐1 were detected with real‐time PCR and western blot, respectively. Permeability of endothelial monolayer was determined using 40 kDa FITC‐labeled dextran. 10 μM of RA significantly decreased the mRNA and protein level of PV‐1 and this effect was most pronounced after 48 hours. 10 μM of RA also decreased significantly the permeability of endothelial monolayer. Preincubation of HUVEC for 30 minutes with RA increased cell proliferation and blocked the endothelial permeability induced by 100 ng/ml VEGF. We demonstrated that RA decreased permeability and PV‐1 expression and blocked the effect of VEGF on endothelial permeability. Our results also suggest that RA may modulate the endothelial permeability effect of VEGF which at least partly induced by PV‐1 protein. Further studies needed to clarify the functional consequences of this striking phenomenon.