bluuebunny/crossref_metadata_2025 huggingface dataset:
Pharmacological characterization of the P2 receptors profile in the podocytes of the Sprague Dawley rat glomeruli
Authors: Ilatovskaya Daria V. , Palygin Oleg , Levchenko Vladislav , Staruschenko Alexander
Published: June 2021

Renal P2 purinoreceptors are widely distributed throughout glomeruli, vasculature and tubular segments of the kidney. Extracellular ATP can mobilize calcium in the podocytes by activating these receptors and therefore contribute to glomeruli functions. The goal of this study was to identify specific P2 receptor subtypes in the podocytes of the rat glomeruli. The glomeruli of the Sprague Dawley rats were isolated by differential sieving, loaded with Fluo‐4/Fura Red calcium dyes and analysed with real‐time ratiometric fluorescent measurements under the confocal scope. Various specific chemical agonists and antagonists of the P2Y and P2X receptor subfamilies were utilized to determine responsible receptors under zero or 2 mM Ca2+ extracellular conditions. In contrast to previously published data, we have identified P2Y1 receptor as the main functional P2 receptor in the podocytes. Furthermore, we have assessed the contraction rates of the glomeruli in response to ATP and other P2Y1 receptors agonists therefore having shown the role of the P2 receptors, particularly, P2Y1, in the regulation of the glomerular volume dynamics. As a result of this study we identified P2Y1 as the main purinergic receptor localized in the podocytes, which might be involved in the pathogenesis of glomerular injury and in contraction and relaxation of glomeruli. Supported by the NIH (HL108880).
Sodium Transport in a Mathematical Model of a Macula Densa Cell
Authors: Nadal‐Quiros Monica , Nieves‐Gonzalez Aniel , Moore Leon C. , Marcano Mariano
Published: June 2021

Macula Densa (MD) cells, located at the boundary of the cortical thick ascending limb and the distal convoluted tubule of the nephron, act as a signal sensor‐transmitter complex for glomerular filtration rate regulation. We developed a mathematical model of an MD cell based on solute and water conservation and electroneutrality constraints. The model includes representations of major transport pathways across the cell membranes: Na+‐K+‐2Clcotransporter, Na+/H+‐exchangers, H+ (Na+)/K+‐ ATPase, aquaporins, and renal outer medulla K+, maxi‐anion, and non‐selective cation channels. We simulated two cases: (1) an increase in luminal [NaCl] from 25 to 150 mM, and (2) a decrease from 150 to 25 mM, and obtained steady‐state cytosolic [Na+] of ~70 mM and ~ 30 mM for case (1) and (2), respectively. These results are in agreement with in vitro experiments from MD cells in rabbits.

Research supported by MBRS‐RISE grant 2R25GM061151–09 and by NIH‐NIGMS grant SC1GM084744.
Characterization of the trafficking of a Ca2+‐activated K+ channel (KCa3.1) in polarized epithelial cells
Authors: Bertuccio Claudia Alejandra , Devor Daniel
Published: June 2021

While the intermediate conductance, Ca2+‐activated K+channel (KCa3.1) is targeted to the basolateral membrane in several polarized epithelial cells, an alternatively spliced form has been shown to traffic to the apical membrane in colonic epithelia. Thus, the aim of this work was to study how KCa3.1 traffics to the plasma membrane (PM) in polarized epithelial cells. We expressed Biotin Ligase Acceptor Peptide (BLAP)‐tagged KCa3.1 in polarized MDCK and CACO‐2 cells grown on Transwell filters and labeled PM channels with streptavidin. KCa3.1 was localized exclusively to the basolateral PM in both cell types and was shown to be functional in Ussing chamber studies by activation with DCEBIO and inhibition with TRAM‐34. Degradation of PM KCa3.1 had a half‐life of ~5 hrs in MDCK cells and ~10 hrs in CACO‐2 cells; being inhibited by leupeptin/pepstatin, indicative of lysosomal degradation. Further, lactacystin inhibited degradation of endocytosed KCa3.1 suggesting a role for proteaosmes in this degradation process. We further demonstrated that KCa3.1 is ubiquitylated following endocytosis in polarized epithelial cells. Finally, in HEK cells we demonstrate a role for Rab1 and Rab8 in the PM expression of KCa3.1 and that both Rab1 and Rab8 coimmunoprecipitate with KCa3.1. (Supported by NIH HL092157)
Acute effects of angiotensin II on TRPC6 channels in the podocytes of the freshly isolated glomeruli
Authors: Ilatovskaya Daria V. , Chubinskiy‐Nadezhdin Vladislav , Negulyaev Yuri A. , Staruschenko Alexander
Published: June 2021

There has been proposed a key role of the podocytes in the pathogenesis of nephrotic diseases since the injury of these cells leads to heavy proteinuria. It was also shown that angiotensin II (Ang II) caused release of intracellular Ca2+ in podocytes; members of the TRPC‐family channels emerged as prime candidates for this effect. Mutations in the gene encoding the TRPC6 channel result in development of focal segmental glomerulosclerosis. Here, we examined the effects of Ang II (0.1 to 10 μM) on Ca2+ levels in intact podocytes of freshly isolated Sprague Dawley rat glomeruli. Changes in intracellular Ca2+ stimulated by Ang II in the presence or absence of thapsigargin or TRPC inhibitor 2‐APB were measured in podocytes of the glomeruli loaded with Fura‐2AM. Depletion of internal Ca2+ stores did not affect the calcium flux activated by Ang II, whereas 2‐APB completely blocked it. Patch‐clamp analysis demonstrated that 100 nM Ang II acutely activates native TRPC‐like channels in the podocytes of freshly isolated glomeruli as well as TRPC6 channels transiently overexpressed in CHO cells, and this effect is mediated by changes in the channel open probability. Thus, we provide direct evidence of Ang II‐dependent activation of TRPC6 channels in podocytes, which might play a significant role in the development of kidney diseases. This research was supported by the NIH (HL108880) and Zeiss‐OPTEC LLC grant’2012.
Divergent Phenotype of Rat Thoracic and Abdominal Perivascular Adipose Tissues
Authors: Padilla Jaume , Jenkins Nathan T , Vieira‐Potter Victoria J
Published: June 2021

Perivascular adipose tissue (PVAT) is implicated as a source of pro‐atherogenic adipokines. It is possible that phenotypic differences in local PVAT depots may contribute to differences in disease susceptibility among arteries and even regions within an artery. In this regard, it has been proposed that PVAT around the abdominal and thoracic aorta share characteristics of white and brown AT, respectively; however, a detailed comparison between these PVAT depots has not been performed. Using young (3 months) and older (13 months) adult rats, we compared the phenotype of PVATs surrounding the abdominal and thoracic aorta to each other and also to epididymal white and subscapular brown ATs. We found that expression of inflammatory genes and markers of immune cell infiltration were greater in abdominal PVAT than in thoracic PVAT, and that overall, abdominal and thoracic PVATs resembled the phenotype of white and brown ATs, respectively. Immunohistochemistry and electron microscopy indicated structural similarity between white and abdominal PVAT and between brown and thoracic PVAT. Our data provide evidence that abdominal PVAT is more inflamed than thoracic PVAT independent of aging. Interestingly, these phenotypic differences in PVAT depots associate with known differences in disease susceptibility of the underlying aortic regions. Support: AHA 11POST5080002, T32‐AR048523, R01‐HL036088.
Antihypertensive and renoprotective effects of ABT‐627 and chlorthalidone treatment in Dahl S rats on a high salt high fat diet.
Authors: Jin Chunhua , Pollock David M.
Published: June 2021

Over the last two decades, it has become increasingly evident that endothelin‐1 (ET‐1) is important in blood pressure regulation and may be a good target for the treatment and prevention of hypertension; however, the use of ETA receptor antagonists can produce fluid retention and may be better suited for use in combination with other antihypertensive medications, especially diuretics. Therefore, we determined the antihypertensive and renoprotective effects of an ETA receptor antagonist (ABT‐627, 5 mg/kg/day), a thiazide diuretic (chlorthalidone, 5 mg/kg/day), and combination of both in high fat (59% fat)/high salt (4% NaCl) fed Dahl S rats, a rodent model of salt sensitive hypertension. First, we found that after 4 weeks of treatment, mean arterial pressure (MAP; telemetry) was significantly reduced by both ABT‐627 and chlorthalidone (CH), an effect that was significantly enhanced by combination treatment (vehicle: 176±8, ABT‐627: 146±3, CH: 135±2, ABT‐627+CH: 115±6 mmHg; p<0.05). Associated with the reduction in blood pressure, ABT‐627 and CH alone both reduced proteinuria while the combination treatment significantly reduced proteinuria further (vehicle: 578±145, ABT‐627: 297+29, CH: 138+10, ABT‐627+CH: 95+13 mg/day; p<0.05). Finally, we found that urinary nephrin excretion, a marker of glomerular damage, was significantly decreased in CH and ABT‐627+CH (vehicle: 1072+218, CH: 423+45, ABT‐627+CH: 315+54 μg/day; p<0.05). These results indicate that simultaneous blockade of ETA receptors and diuretic treatment provides greater antihypertensive and renoprotective effects than either drug alone in Dahl salt sensitive rats maintained on a Western diet.
eNOS and nNOS Mediate Isoflurane Preconditioning
Authors: Ge Zhi‐Dong , Liu Yanan , Xia Zhengyuan , Warltier David C. , Kersten Judy R.
Published: June 2021

Anesthetic preconditioning (APC) has been shown to protect the heart against ischemia and reperfusion injury via a nitric oxide‐dependent mechanism. However, the role of nitric oxide synthase (NOS) isoforms in the cardioprotective effect of APC remains elusive. We examined the role of endothelial (eNOS) and neuronal NOS (nNOS) isoforms in cardioprotection by isoflurane. Pentobarbital‐anesthetized C57BL/6 and eNOS−/− mice underwent APC or equivalent amounts of air/oxygen (control) prior to 30 min of coronary occlusion followed by 2 h of reperfusion. APC was produced with 1.0 minimum alveolar concentration of the volatile anesthetic isoflurane for 30 min followed by a 15 min washout period. Infarct area and area at risk were delineated by 2,3,5‐triphenyltetrazolium chloride and phthalo blue dye, respectively. There were no significant differences in area at risk between groups. APC decreased infarct size in C57BL/6 mice (41±3% vs. 54±3% in control, n=8 mice/group, P<0.05), which was partially blocked by 7‐nitroindazole (25 mg/kg), a selective inhibitor for nNOS, administered intraperitoneally 15 min prior to APC. Disruption of eNOS gene did not significantly increase infarct size compared with C57BL/6 mice and partially blocked the cardioprotective effect of APC. Furthermore, APC‐elicited decrease in infarct size in eNOS−/− mice was abolished by 7‐nitroindazole. Cardiomyocytes were co‐cultured with endothelial cells and subsequently exposed to isoflurane (0.5 mM) or air (control) for 60 min prior to 2 h of hypoxia and 2 h of reoxygenation. Isoflurane significantly increased phosphorylated eNOS compared with control. The results demonstrate that eNOS and nNOS synergistically mediate cardioprotection against ischemia and reperfusion injury by isoflurane.
Smooth muscle cell transient receptor potential polycystin (TRPP)2 channels contribute to the myogenic response in cerebral arteries
Authors: Narayanan Damodaran , Bulley Simon , Leo M Dennis , Gabrick Kyle S , Boop Frederick A , Jaggar Jonathan H
Published: June 2021

Intravascular pressure‐induced vasoconstriction is a smooth muscle cell (myocyte)‐specific mechanism that controls systemic blood pressure and regional organ blood flow. Myocyte polycystin (TRPP)‐1 and ‐2 proteins modulate the myogenic response in mesenteric arteries, but involvement in other vascular beds is unclear. Here, we examined myocyte TRPP2 expression, cellular distribution, cation currents (ICat), and contribution to the myogenic response in cerebral arteries. Real‐time RT‐PCR amplified transcripts for TRPP1 and TRPP2, with TRPP2 being the predominant message in isolated rat cerebral artery myocytes. Western blot analysis indicated that TRPP2 protein was present in rat and human cerebral arteries. Arterial surface biotinylation and immunofluorescence indicated that TRPP2 is located primarily within the myocyte plasma membrane. Cell swelling induced by hyposmotic bath solution stimulated ICat in myocytes that were inhibited by RNAi‐mediated selective TRPP2 channel knockdown. TRPP2 knockdown did not alter myogenic tone at 20 mmHg but reduced myogenic tone at pressures between 40 and 100 mmHg. In contrast, TRPP2 knockdown did not alter membrane depolarization‐induced (60 mmol/L K+) vasoconstriction. These data indicate that TRPP2 contributes to stretch‐activated ICat in myocytes of resistance‐size cerebral arteries, leading to vasoconstriction. NIH/NHLBI.
Involvement of TRPM4 in Pressure and Agonist‐Induced Vasoconstriction in the Cerebral Microcirculation
Authors: Li Yao , Baylie Rachael L. , Brayden Joseph E.
Published: June 2021

Cerebral parenchymal arterioles (PAs) play a critical role in regulating blood flow and perfusion pressure in the brain. These arterioles are centrally involved in neurovascular coupling and are substantially more sensitive to mechanical and chemical stimulants compared to cerebral arteries on the surface of the brain, i.e. the pial arteries. Intravascular pressure, an essential vasomotor stimulant, causes membrane depolarization and vasoconstriction (myogenic tone). Previous research and recent published work from our laboratory indicates the involvement of a transient receptor potential channel (TRPM4) and purinergic receptors (P2Y4 and P2Y6) in myogenic tone development of pial arteries and PAs, respectively. In the current study, the relationship between TRPM4 and P2Y receptors, and their contribution to myogenic tone development in PAs were investigated. Here we report that TRPM4 mRNA signal is present in PAs. In functional (pressure myography) studies, the selective TRPM4 channel blocker 9‐phenanthrol substantially inhibited myogenic tone of endothelium‐denuded PAs with an IC50 of 28 μM. Suppression of TRPM4 expression using siRNA constructs significantly reduced myogenic constriction; PAs treated with control siRNA and TRPM4 siRNA developed 45% ± 2% and 33% ± 4% myogenic tone, respectively. Moreover, in denuded PAs, 9‐phenanthrol (30 μM) attenuated P2Y4 (UTPγS, 0.5 μM) and P2Y6 (MRS2693, 1 μM) agonist‐induced vasoconstriction by 67% and 68%, respectively. Consistent with this finding, TRPM4 ion channel activity in myocytes isolated from PAs was significantly elevated by UTPγS (0.5 μM). These results indicate that TRPM4 contributes to pressure‐induced, purinergic receptor‐mediated vasoconstriction of PAs.
Azilsartan, New Angiotensin II Type 1 Receptor Blocker, Would Suppress Morning Surge in Hypertension
Authors: Isegawa Kengo , Hirooka Yoshitaka , Kishi Takuya , Sunagawa Kenji
Published: June 2021

Background

Recent investigations on circadian variations of blood pressure have shown that the suppression of morning surge is crucial in optimal management of hypertension. The pressure surge is often observed in the neurohormonal transitional phase from the rest to active phase. Azilsartan (AZL), a new angiotensin II type 1 receptor blocker (ARB), is a powerful and long‐acting antihypertensive agent. We examined if AZL suppress the elevation of blood pressure from the light‐rest to dark‐active phase in spontaneously hypertensive rats.

Methods and Results

SHR were divided into three groups, treated at 5:00 for 28 days with orally administered azilsartan (AZL; 1mg/kg/day), candesartan (CAN; 1mg/kg/day), or vehicle (VEH). Telemetric mean arterial pressures (MAP) for 24 hours were similarly lower in AZL and CAN, than vehicle (AZL 108±4, CAN 116±7, VEH 134±5 mmHg, p<0.01). Both in the light‐rest (from 6:00 AM to 6:00 PM) and dark‐active phase (from 6:00 PM to 6:00 AM), MAP were similarly lower in AZL and CAN than in VEH. However, the changes in MAP between the last 4 hours in light‐rest phase and the first 2 hours in dark‐active phase were significantly lower in AZL than in CAN (−2±2 vs. 3±1 mmHg, p<0.05).

Conclusion

AZL is capable of lowering the elevation of blood pressure from the rest to active phase, indicating that AZL may benefit hypertensive patients with pressure surge in the early morning.
Increased arrhythmogenesis and expression of left ventricular pS368‐Connexin43 following acute myocardial ischemia (AMI) in female ovariectomized rats
Authors: Jarrett Erica , Firkins Rachel M. , Henry Matthew K. , Moffitt Julia A.
Published: June 2021

Estrogen has been previously shown to be protective against ventricular arrhythmias following myocardial infarction possibly through changes in connexin 43 (Cx43) expression and phosphorylation. Phosphorylation of Cx43 at S368 is induced with AMI, suggesting that phosphorylation at this site plays a role in arrhythmogenesis. The goal of the current study was to determine if there is a direct correspondence between phosphorylation of left ventricular (LV)‐Cx43 at S368 and the arrhythmogenic response to AMI, and to further examine if this effect is augmented following loss of estrogen. Anesthetized, ventilated female ovariectomized (n=3) and intact (n=3) Sprague‐Dawley rats were subjected to 20‐minutes of AMI produced through ligation of the left coronary artery. The arrhythmic burden in response to AMI was measured using a standard lead II ECG, and quantified through an established scoring method. Total LV‐Cx43 expression and pS368‐Cx43 expression were measured through standard Western blotting techniques. Ovariectomized animals had a greater arrhythmic burden versus intact animals following AMI. Correspondingly, ovariectomized animals also had an increase in the pS368‐Cx43/Total Cx43 ratio. When data were further examined using a linear regression analysis, results show a direct positive correlation between the level of pS368‐Cx43 expression and arrhythmic burden (R2 = 0.94). IOER‐3726.
Activation of the unfolded protein response in vascular endothelial cells of non‐diabetic obese middle‐aged and older adults
Authors: Kaplon Rachelle Elizabeth , Seals Douglas R , Gentile Christopher L
Published: June 2021

Obesity is associated with vascular endothelial dysfunction, but the underlying mechanisms are incompletely understood. Activation of the unfolded protein response (UPR) is emerging as an important molecular signature of cardio‐metabolic disease. We tested the hypothesis that UPR activation occurs in the vascular endothelium of obese adults. Brachial artery flow‐mediated dilation, a measure of endothelial function, was 31% lower in nondiabetic obese (Ob, BMI≥30, n=9) vs. non‐obese (NOb, BMI<30, n=13) adults (31–74 years) (3.97±0.52 vs. 5.79±0.62%, P<0.05). In endothelial cells (EC) obtained from antecubital veins, expression (quantitative immunofluorescence) of the UPR stress sensor IRE1 was greater in Ob vs. NOb (0.78±0.12 vs. 0.46±0.02 IRE1 intensity/human umbilical vein EC [HUVEC] intensity, n=7/7, P<0.05). Ob also had greater EC activation of UPR stress sensors PERK and ATF6, as indicated by increased expression of phosphorylated PERK (pPERK; 0.42±0.04 vs. 0.33±0.02 pPERK [threonine 981] intensity/HUVEC intensity, n=7/12, P<0.05) and nuclear localization of ATF6 (0.38±0.05 vs. 0.23±0.02 nuclear ATF6 intensity/HUVEC intensity, n=5/9, P<0.01), respectively, compared with NOb. These results provide novel evidence for UPR activation in endothelial cells of non‐diabetic obese middle‐aged and older adults with vascular endothelial dysfunction. NIH TR000154, AG000279, K01DK087777, AG013038
Relief of endoplasmic reticulum (ER) stress in the brain subfornical organ (SFO) selectively prevents high fat diet (HFD)‐induced hepatic dysfunction but not the development of adiposity or hypertension
Authors: Young Colin N. , Horwath Julie A. , Butler Scott D. , Mark Allyn L. , Davisson Robin L.
Published: June 2021

Fatty liver, in concert with obesity and hypertension, are hallmarks of metabolic syndrome. ER stress in the brain has been suggested to contribute, although the precise neural regions involved remain unclear. We tested the hypothesis that ER stress in the SFO mediates HFD‐induced obesity, hypertension, and hepatic dysfunction. C57Bl/6 mice were instrumented with radiotelemeters for measurement of mean arterial pressure (MAP) and underwent SFO‐targeted microinjections of an adenovirus encoding the ER chaperone GRP78 (AdGRP78), an approach we have shown to reduce ER stress. Mice were fed normal chow (NC) or HFD for 10 weeks. Relative to NC, HFD control (AdLacZ, n=7) and SFO‐targeted AdGRP78 (n=7) mice demonstrated similar significant increases in body weight (Δ19±1 vs 18±1 g, AdLacZ vs AdGRP78, p>;0.05), adiposity and MAP (Δ7±2 vs 8±2 mmHg, AdLacZ vs AdGRP78, p>;0.05). However, HFD induced profound hepatomegaly (0.98±0.02 vs 1.88±0.11 g, NC vs AdLacZ, p<0.05), that was prevented with SFO‐targeted AdGRP78 (1.31±0.08 g). SFO‐targeted AdGRP78 also prevented obesity‐induced lipid accumulation in the liver and upregulation of hepatic gluconeogenic markers (e.g. Pepck mRNA 1.6±0.1 vs 0.7±0.2 fold NC, AdLacZ vs AdGRP78, p<0.05). These findings suggest that ER stress in the SFO selectively mediates obesity‐induced hepatic dysfunction, but not the development of adiposity or hypertension.

HL63887, HL96571, HL84207
Prediction of inverse relationship between compression phase duration and expulsive airflow during voluntary cough in humans by a joint neural network biomechanical computational model
Authors: Bolser Donald C , Pitts Teresa E , O'Connor Russell , Segers Lauren S , Sapienza Christine M , Davenport Paul W , Morris Kendall F , Lindsey Bruce G
Published: June 2021

Previous computational modeling efforts of the cough motor control system have focused on neural networks and predicting motor output patterns. We speculated that a recent joint neuromechanical model of the cough central motor control system that incorporated airway and chest wall mechanics would be useful in predicting mechanical aspects of cough in humans. Simulations of cough with this model produced large airflows and pressures with mechanical and temporal features very similar to those recorded during cough in humans. In particular, compression phase durations and airflows during simulated coughing varied by approximately 60%. Compression phase durations were longer during simulated coughs with low airflows. In human studies, healthy subjects performed a magnitude production protocol by producing weak, moderate, or strong voluntary coughs. Peak expiratory airflows increased by approximately 40% and abdominal electromyogram amplitudes approximately doubled from weak to strong voluntary coughs. Compression phase durations shortened significantly from weak to strong coughs (weak‐351±33 ms, moderate‐253±86 ms, strong‐217±39 ms, p<0.05). The simulation results represent an emergent property and support the predictive value of a joint neuromechanical computational model for human airway protective behaviors. Supported by NIH HL103415, HL89104, HL89071, NS19814.
Characterization of breathing following four weeks of prolonged dietary Bisphenol A exposure in mice
Authors: Baker Kristin , Liang Chen , Benson Marisa , DeRuisseau Keith , DeRuisseau Lara
Published: June 2021

Bisphenol A (BPA) is a chemical used in the production of various plastics. It is classified as an endocrine disruptor since it can exert estrogen‐like effects in the body. Although estrogen can play a role in the control of breathing, it is unknown if BPA influences conscious ventilation. We tested the hypothesis that 4 weeks of dietary BPA administration would result in an altered pattern of breathing compared to the response of dietary controls. At 8 weeks of age, male CD‐1 mice were housed in polypropylene cages and administered a soybean oil‐free diet (AIN‐93G) for 2 weeks. At 10 weeks of age the mice were placed onto control (C: AIN‐93G; n=4), C+ethinyl estradiol (EE: 0.1 ppb; n=5) or C+BPA (50 mg BPA/kg diet; n=5) diets. Unrestrained barometric plethysmography was used to quantify frequency (F; breaths/min), tidal volume (TV; mL/breath), and minute ventilation (MV; mL/min) in 14 week old mice during exposure to room air (MEAN±SEM). Control mice displayed similar F (C: 150±28 vs. C+BPA: 129±11 vs. C+EE: 150±25), TV (C: 0.35±0.01 vs. C+BPA: 0.49±0.05 vs. C+EE: 0.48±0.07) and MV (C: 54.4±9.5 vs. C+BPA: 62.5±10.3 vs. C+EE: 69.8±9.6). These preliminary findings indicate no difference in quiet breathing following 4 weeks of dietary BPA or EE administration in mice. Supported by LMC start‐up funds (LRD) and the Hill Collaboration on Environmental Medicine (KCD).
Respiratory Sensory Gating in Children Measured by the Respiratory‐Related Evoked Potentials
Authors: Chan Pei‐Ying Sarah , Li Chieh‐Wen , Chen Ying‐Jhen
Published: June 2021

Respiratory neural gating function in the cortices was measured in healthy children using the paired respiratory‐related evoked potential (RREP) measured elicited by inspiratory occlusions. We hypothesized that respiratory sensory gating of healthy children would be normal as represented by a reduced amplitude of the second stimulus (S2) compared by the first stimulus (S1) for the short latency Nf, P1, and N1 peaks. We further hypothesized that the gating peak would be normal as represented by a N1 peak S2/S1 ratio of less than 0.5.

Paired 150‐msec inspiratory occlusions with an inter‐stimulus‐interval of 500 milliseconds were given to the subjects. The RREP was recorded with a 40‐channel electroencephalography system. A minimum of 100 paired occlusions were collected for data analysis. The RREP Nf, P1, and N1 peaks were identified in latencies and amplitudes. The S2/S1 ratio was calculated for the amplitudes for each peak.

Our preliminary data showed that the averaged latencies for the Nf, P1, and N1 peaks were 40 and 42, 55 and 48, 94 and 81 msec, respectively. The averaged S2/S1 ratio for the RREP Nf, P1, and N1 peak amplitudes were 0.97, 1.13, and 0.64, respectively.

The result suggested that young children may have a decreased respiratory gating function represented by increased P1 and N1 peak S2/S1 ratios for the first order neural information processing.

Supported by: NSC100–2320‐B‐182–018, CMRPD1A0021, CMRPD1A0022
Intrapleural siRNAs targeting TrkB attenuate acute intermittent hypoxia (AIH)‐induced phrenic long‐term facilitation (pLTF)
Authors: Dale Erica Arden , Fields Daryl P , Mitchell Gordon S
Published: June 2021

Activation of cervical spinal TrkB (the high affinity BDNF receptor) is necessary for pLTF (Baker‐Herman et al., Nature Neurosci, 2004), a long‐lasting increase in phrenic motor output following AIH. However, the specific cell type expressing relevant TrkB receptors is not yet known. Here, we tested the hypothesis that relevant TrkB receptors are expressed in phrenic motor neurons using a novel RNAi technique to knock down TrkB specifically within phrenic motor neurons (Mantilla et al., 2012). Adult, male, Sprague‐Dawley rats received successive intrapleural injections (3 days) of: 1) vehicle (30uL/side; n=2); 2) siRNAs targeting TrkB mRNA (30uL, 100pmol/side; n=6), 3) siPKCζ, a molecule involved in other forms of phrenic plasticity, but not pLTF (30uL, 100pmol/side; n=1), or 4) no treatment (n=6). One day later, anesthetized, paralyzed and ventilated rats were given AIH or normoxia. Vehicle (49%) and uninjected rats (51% ±14%) exhibited pLTF 60 min post‐AIH (p<0.05). Intrapleural siTrkB attenuated pLTF (20%±16%; p<0.05 vs. other treatments), whereas siPKCζ did not (68%). Although experimental verification of selective TrkB knock‐down is necessary, these preliminary data strongly suggest that the TrkB receptors relevant for pLTF are expressed in phrenic motor neurons.
Medullary post‐inspiratory neuron activities are selectively driven by NMDA receptor‐dependent neurons in pontine Kölliker‐Fuse nucleus
Authors: Song Gang , Ning Yinghui , Poon Chi‐Sang
Published: June 2021

We have previously shown that post‐I phase duration (TE1) and vagal post‐I motor activity exhibit post‐hypoxic STP that is mediated by NMDA receptor‐expressing early‐E neurons in pontine Kölliker‐Fuse nucleus (KFN), presumably through these neurons’ excitatory influences on medullary post‐I neuronal pools that modulate TE1 and the activity of laryngeal adductive muscles. In this study we examined the changes in firing pattern of various medullary respiratory neurons upon the induction of apneusis by microinjection of NMDA receptor antagonist AP5 at KFN. We found that after apneusis was induced: 1) all medullary post‐I neurons were severely depressed or completely silenced and their post‐hypoxic STP was greatly reduced or completely abolished; 2) discharge period of E‐aug/late‐E neurons was lengthened to span the entire expiratory period; 3) firing patterns of inspiratory‐related neurons (early‐I, aug‐I, pre‐I, and late‐I) were not significantly altered but their firing frequencies were slightly or moderately reduced. These findings indicate that the activities of medullary post‐I neurons (but not other medullary respiratory‐related neurons) are dependent on excitatory drive from NMDA receptor‐expressing KFN early‐E neurons. In the absence of such KFN post‐I excitatory drive, the expiratory phase is solely under the control of medullary E‐aug neurons. (Supported by HL093225)
Sympathoexcitation Induced by SK Channel Blockade in PVN Requires Activation of NMDA Receptors
Authors: Larson Robert A , Chapp Andrew D , Gu Mingjun , Chen Qing‐Hui
Published: June 2021

Small conductance calcium activated potassium channels (SK) are abundantly expressed in the paraventricular nucleus (PVN). SK channel blockade in the PVN augments sympathetic nerve activity (SNA), yet the mechanism remains unclear. We hypothesized that the increased SNA evoked by SK channel blockade in PVN requires local activation of glutamate receptors. In anesthetized rats, bilateral PVN microinjection of SK channel blocker Apamin (25 pmol, 50 nl) significantly (n=5; p<0.01) increased splanchnic SNA (SSNA) (304±60%) and mean arterial pressure (MAP) (33±6mmHg). Pre‐treatment with PVN injection of KYN (7.2 nmol), a non‐selective glutamate receptor blocker, significantly attenuated (n=4) the elevated SSNA (137±31%; p<0.05 vs vehicle control) response to Apamin. Similarly, pre‐treatment with AP5 (6.0 nmol), a NMDA receptor blocker, also attenuated (n=4) the SSNA (155±17%; p<0.05 vs vehicle control) response to Apamin. Pre‐treatment with KYN (n=4) and AP5 (n=4) obviously attenuated MAP responses to Apamin (17±6mmHg, p=0.08 for KYN; 14±11mmHg, p=0.06 for AP5) but didn't reach statistical significance. Immunohistochemistry studies demonstrated SK channel expression in PVN neurons with projections to the RVLM. This data indicates that the sympathoexcitatory response elicited by SK channel blockade in PVN, at least partly, requires local activation of NMDA receptors. Support: AHA2640130 (QHC).
Astrocyte damage but not microglial infiltration in the nucleus tractus solitarii attenuates cardiovascular reflexes
Authors: Talman William , Jones Susan , Lin Li‐Hsien
Published: June 2021

Nucleus tractus solitarii (NTS) injections of saporin (SAP) conjugates that kill neurokinin‐1 receptor (NK1R) or tyrosine hydroxylase (TH)/dopamine‐β‐hydroxylase (DBH) neurons attenuate baroreflexes, lead to loss of NTS astrocytes with glial fibrillary acidic protein (GFAP), and lead to increased microglial staining with OX42. We sought to determine if SAP alone altered cardiovascular reflexes due to loss of astrocytes, loss of neurons or proliferation of microglia. NTS injection of SAP led to loss of GFAP as well as connexin 43 (CX43) immunofluorescent labeling but did not affect neuronal markers NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), neuronal nitric oxide synthase (nNOS), TH, DBH, vesicular glutamate transporter 2 (VGluT2), NK1R, and protein gene product 9.5 (PGP 9.5). In contrast, SAP led to increased OX42. Bilateral NTS injections of SAP decreased baroreflex, chemoreflex, and von Bezold Jarisch reflex responses. Decreases in baroreflex responses seen in rats treated with SAP were comparable to responses when SAP conjugates targeted TH/DBH neurons or NK1R neurons. Baroreflexes were unaffected by 6‐hydroxydopamine (6‐OHDA), which did not reduce GFAP staining, but did increase OX42. Thus, full cardiovascular reflex expression relies on intact NTS astrocytes. Reflex changes after SAP are independent of microglial proliferation. Support: NIH R01 HL088090 and VA Merit Review.
mTOR signaling in the dorsal motor nucleus of vagus
Authors: Zhang Weizhen , Zhang Chao , Li Ziru , Mulholland Michael
Published: June 2021

Previous studies demonstrate that mTOR signaling in the hypothalamus is involved in the control of energy homeostasis. The aim of this study is to characterize the effect of mTOR signaling in the dorsal motor nucleus of vagus (DMNV) on the energy intake. Phosphor‐mTOR was detected in the DMNV neurons and its levels increased by energy deprivation. Rapamycin significantly inhibited mTOR activity and reduced food intake when administrated into 4th ventricle. Exposure of DMNV neurons to ghrelin increased the phosphorylation of mTOR. Fourth ventricle injection of ghrelin significantly increased food intake relative to the control vehicle. Pre‐treatment with rapamycin attenuated the orexigenic effect of ghrelin. Reduction in the phosphorylation of mTOR was observed following 4th intracerebroventricular injection of nesfatin‐1. When administrated by 4th ICV injection, nesfatin‐1 suppressed the food intake as compared with the control. The anorexigenic effect of nesfatin‐1 was significantly attenuated by pre‐treatment with leucine. All these studies suggest that mTOR signaling in the DMNV neurons regulates both the nutrient and hormonal signals for the modulation of food intake.
NOS uncoupling and redox‐dependent baroreflex inhibition in neural mechanism of metabolic syndrome‐associated hypertension
Authors: Chan Julie Y.H. , Chan Samuel H.H.
Published: June 2021

Up to 75% of patients with metabolic syndromes also exhibit hypertension, although the underlying mechanism is not fully understood. Alterations in neural control of cardiovascular functions are evident in both disease conditions. We tested the hypothesis that oxidative stress because of nitric oxide synthase (NOS) uncoupling in the nucleus tractus solitarii (NTS), terminal sites of the baroreceptor afferents in the brain stem, is involved in the reduced baroreflex function and hypertension under metabolic syndromes. Normotensive Wistar‐Kyoto rats subjected to 8‐weeks high fructose (HF) diet developed hyperglycemia, hyperlipidemia, insulin resistance and hypertension, with concurrent increases in protein expression of neuronal and endothelial NOS (nNOS and eNOS), and a NOS inhibitor, asymmetric dimethylarginine (ADMA), as well as tissue levels of the reactive oxygen species (ROS) but not NO in NTS. Baroreflex sensitivity was significantly reduced in the animals; and was reverted by intracisternal infusion of a ROS scavenger, tempol, or an ADMA inhibitor, dimethylarginine dimethylaminohydrolase (DDAH). DDAH treatment also attenuated ROS production in NTS and promoted antihypertension in the experimental rats. These results suggest that increase in ROS production due to NOS uncoupling in NTS inhibits baroreflex sensitivity and promotes hypertension under metabolic syndromes.
Identification of cationic and anionic nicotinic acetylcholine receptor subunits in Helisoma trivolvis
Authors: Zhong Lei , Wu Yang , Jiang Chun , Rehder Vincent
Published: June 2021

Many actions of acetylcholine (ACh) are mediated by cation‐selective nicotinic ACh receptors (nAChRs). Our lab found that ACh activates cation‐selective nAChRs to elongate growth cone filopodia on B5 neurons from the pond snail Helisoma trivolvis in vitro. Interestingly, pharmacological evidence revealed the existence of anion‐selective nAChRs in B19 neurons, mediating inhibitory cholinergic responses. Unfortunately, little is known about the molecular nature of these two distinct nAChRs in Helisoma. Using a Helisoma trivolvis transcriptome database, we found putative nAChR partial coding sequences. We cloned two nAChR subunits from Helisoma nervous tissue, HsAChR A and K. HsAChR A was most similar to LnAChR A, a cation‐selective subunit from Lymnaea stagnalis. HsAChR K shared the highest sequence identity with LnAChR K, a predicted anionic receptor subunit. Analysis of the full length sequences showed the putative ACh binding sites, transmembrane domains, and residue motifs for ion selectivity. Single‐cell PCR results revealed the differential expression of HsAChR A and K in B5 and B19 neuron, which might account for their distinct electrophysiological responses to ACh. Identification of the cationic and anionic nAChR subunits is a critical step towards genetic manipulation of this receptor and eventually understanding its roles in brain function and controlling behavior.
Alpha‐1 adrenoreceptors modulate GABA release onto ventral tegmental area dopamine neurons
Authors: Velasquez Maria Carolina , Vazquez Rafael , Sanabria Priscila , Jimenez‐Rivera Carlos A.
Published: June 2021

The ventral tegmental area (VTA) plays an important role in reward and motivational processes involved in drug addiction. Previous studies have shown that alpha1‐adrenoreceptors (α1‐AR) are primarily found pre‐synaptically at this area. We hypothesized that GABA released onto VTA‐dopamine (DA) cells is modulated by pre‐synaptic α1‐AR. Recordings were obtained from putative VTA‐DA cells of male Sprague‐Dawley rats (28–50 days postnatal) using whole‐cell voltage clamp technique. Phenylephrine (10uM; α1‐AR agonist) decreased the amplitude of GABAA receptor‐mediated inhibitory postsynaptic currents (IPSCs) evoked by electric stimulation of afferent fibers (n=7; p<0.05). Prazosin (1uM, α1‐AR antagonist), blocked this effect. Paired‐pulse ratios were increased by phenylephrine application (n=13; p<0.05). Spontaneous IPSCs frequency but not amplitude, were decreased in presence of phenylephrine (n=7; p<0.05). However, frequency or amplitude of miniature IPSCs were not different (n=9; p>;0.05). Phenylephrine in low Ca2+ (1mM) concentration decreased IPSC amplitude (n=7; p<0.05). These results demonstrate that α1‐ARs at presynaptic terminals can modulate GABA release onto VTA‐DA cells. Drug‐induced changes in α1‐AR could contribute to the modifications occurring in the VTA during the addiction process. (Supported by GM‐08224 to CAJR; UIS to MCVM).
Relationship of Internet Game Addiction and Autonomic Function in Adolescents
Authors: Kim Na‐Hyun , Kong In Deok
Published: June 2021

The purpose of this study was to identify the relationship of internet game addiction and autonomic function in adolescents. The subjects of this study were 140 high school students. Data were collected from July to September, 2012, using a questionnaire for measuring internet game addiction, stress and anxiety, and blood serum cortisol level for autonomic function. Data were analyzed using descriptive analysis, t‐test, ANOVA and Pearson correlation coefficient with SPSS/WIN 15.0. The stress level of internet game addiction group was significant higher than the stress level of the non‐addiction group (t=−5.06, p<.001). The anxiety score was also significant high in the group of internet game addiction (t=−4.90, p<.001). Serum cortisol level was higher in internet game addiction group than non‐addiction adolescents. There were significant positive correlations between daily internet game time and the score of stress and anxiety.

These results showed that internet game addiction could increase sympathetic activities in high school adolescents. Thus, nursing approach to relieve the internet game addiction could be helpful to prevent cardiovascular distress due to autonomic imbalance in their future.

[This research was supported by Basic Science Research Program through the National Research Foundation of Korea funded by the Ministry of Education, Science and Technology (2012R1A1A4A01012884)]
Aldosterone‐mediated cardiac regulation of microRNA‐21
Authors: Romero Damian G. , Price Jana E. , Hall Michael E , Bandyopadhyay Sibali , Yanes Licy L.
Published: June 2021

Excess aldosterone (ALDO), along with high salt intake, causes cardiac damage. The molecular mechanisms of ALDO‐mediated cardiac injury are poorly understood. MicroRNAs (miRNAs) are endogenous, non‐coding RNAs that have important roles in cell proliferation and differentiation.

We previously reported that treatment of rats with ALDO/SALT increases cardiac left ventricle miR‐21 expression and downregulation of miR‐21 exacerbated cardiac injury.

We aimed to identify the cardiac cells in which miR‐21 is regulated by ALDO/SALT. Uninephrectomized male Sprague Dawley rats were treated with ALDO/SALT or vehicle for 2 weeks. Cardiomyocytes and fibroblasts were isolated and sorted using cell‐specific markers and miR‐21 expression quantified by qPCR. Cardiac fibroblasts were isolated from control rats, cultured, treated with ALDO for 24 h and miR‐21 expression quantified by qPCR.

ALDO/SALT treatment increased miR‐21 expression only in the fibroblasts. In situ hybridization confirmed these results. In vitro treatment of cardiac fibroblasts with ALDO dose‐dependently increased miR‐21 expression.

In summary, these results suggest that cardiac fibroblasts are a direct target of ALDO both in vitro and in vivo and modulation of cardiac fibroblast miR‐21 levels may be a novel therapeutic approach to mitigate ALDO/SALT‐mediated cardiac injury.

Supported by American Heart Association Grant 12SDG8980032.
Effects of menopausal hormone treatments on prostanoid levels and platelet sensitivity to cyclic AMP activation in healthy, recently menopausal women
Authors: Hunter Larry , Raz Limor , Jayachandran Muthuvel , Miller Virginia M
Published: June 2021

Estrogen modulates the prostanoids prostacyclin (PGI2) and thromboxane A2 (TXA2) which contribute to vascular hemostasis in endothelium and platelets. Estrogen decreases in women at menopause, and menopausal hormone therapy (MHT) may increase risk of thrombosis. This study aimed to determine effects of MHT on serum prostanoid levels and on prostanoid‐related platelet functions in healthy, recently‐menopausal women. Women (age 52.3±2.3 yrs; n=115) enrolled in the Kronos Early Estrogen Prevention Study (KEEPS) were randomized to either: 1) oral conjugated equine estrogen, 2) transdermal 17β estradiol each with intermittent oral progesterone or 3) placebo, for 48 months. Serum PGI2 and TXA2 levels were assessed by quantification (ng/ml) of their stable metabolites 6‐k‐PGF1α and TXB2, respectively. Initial TXB2 levels (46.9±7.5) were unchanged (P>; 0.05) after 48 months in each treatment group. However, 6‐k‐PGF1α decreased from 2.1±0.2 to 1.4±0.1 at 48 months in combined groups, but was statistically significant (1.5±0.3) only in the transdermal group. Inhibition of platelet ATP secretion by PGE1 did not correlate with serum 6‐k‐PGF1α levels. These results suggest that serum PGI2 levels decrease with age in menopausal women and are unaffected by MHT. Factors other than changes in serum prostanoids may impose thrombotic risk associated with MHT. (Supported by the Aurora Fnd, NIH HL88988 and Mayo Clinic).
Stress‐triggered regulation of the adrenomedullary angiotensin II type 2 receptor
Authors: Nostramo Regina , Tillinger Andrej , Saavedra Juan M. , Serova Lidia , Kumar Ashok , Pandey Varunkumar , Kvetnansky Richard , Sabban Esther L.
Published: June 2021

The renin‐angiotensin system is involved in the adrenomedullary stress response, however the role of specific angiotensin II receptor subtypes is unclear. We examined gene expression changes of angiotensin II type 1A (AT1A) and type 2 (AT2) receptors in rat adrenal medulla following immobilization stress (IMO). AT1A receptor mRNA levels were unaltered, whereas AT2 receptor mRNA levels decreased after single and repeated (6x) IMO by as much as 90%. To elucidate the role of the hypothalamic‐pituitary‐adrenocortical axis, cultured cells were treated with dexamethasone, which elicited a transcriptionally‐mediated decrease in AT2 receptor mRNA levels. However, glucocorticoids are not required for the stress‐triggered decrease in AT2 receptor mRNA levels, as shown in corticotropin‐releasing hormone knockout mice and hypophysectomized rats, although they can regulate basal gene expression. cAMP and pituitary adenylate cyclase‐activating polypeptide also reduced AT2 receptor mRNA levels and may mediate this response via splanchnic innervation. Overall, the effects of stress on adrenomedullary AT1A and AT2 receptor expression may contribute to allostatic changes, such as regulation of catecholamine biosynthesis. Supported by American Heart Association grant 10GRNT4420001 and grants APVV‐0088–10 and VEGA 2/0036/11.
Exploring acid secretion machinery in embryos of marine mollusks cephalopod under hypercapnic environment
Authors: Lee Jay‐Ron , Tseng Yung‐Che , Hu Marian Y. , Liu Sian‐Tai , Lin Li‐Yih , Lee Mong‐Fang , Hwang Pung‐Pung
Published: June 2021

The present studies investigated effects of elevated seawater pCO2 on acid secretion mechanism in early life stages of squid Sepioteuthis lessoniana. Although cephalopods have already proved as powerful acid‐base regulators, which tightly regulate their pH homeostasis; however the acid‐base regulatory machinery is virtually unexplored. Thus, a major focus of this work has been dedicated to the mechanistic understanding of proton secretion pathways in epithelia of squid embryos during environmental hypercapnia. We cloned a Na+/H+ exchanger 3 (slNHE3), V‐type H+‐ATPase (slVHA) and Rhesus protein (slRhP) from squid, S. lessoniana, which are hypothesized to represent key players in proton secretion pathways of marine organisms. In situ hybridization and immunohistochemical analyses demonstrate the expression of slNHE3 and slVHA in epidermal ionocytes, which are rich in mitochondria, and thus, can be referred to as mitochondria‐rich cells (MRCs). Gene expression analysis demonstrates that putative acid‐base transporters, including slNHE3, slVHA and slRhP are up‐regulated in response to environmental hypercapnia (pH 7.4; 0.46 kPa pCO2) in body and yolk tissues. This observation is further supported by selective ion electrode measurements, which demonstrate increased proton gradients on the yolk epithelium of CO2 treated embryos. The present work suggests that in convergence to teleosts, cephalopod embryos have evolved sophisticated physiological mechanisms to cope with acid‐base disturbances during their oviparous development.
Acid and base secreting cells in leopard shark (Triakis semifasciata) gills: mitochondrial richness, ATPase specificity, and vacuolar H+ ATPase (VHA) translocation
Authors: Munevar Christian Ludwig , Roa Jinae N. , Tresguerres Martin
Published: June 2021

Sharks maintain stable blood pH by way of acid and base secreting cells in the gill. Using immunofluorescence, we confirmed the existence of two distinct gill cell types characterized by large abundance of either: vacuolar H+ ATPase (VHA) or Na+/K+ ATPase. Further analysis using antibodies against complex IV, confirmed that both cell types are also mitochondria rich. In VHA‐rich gill cells from dogfish sharks (Squalus acanthias) VHA moves from cytoplasmic vesicles to the basolateral membrane in order to rectify blood alkalosis experienced post feeding by pumping protons into alkaline blood. We observed similar VHA translocation occurs in fed leopard sharks. Western blot analyses showed a comparatively greater concentration of VHA in the membrane fraction of fed sharks over starved sharks. Western blots of gill crude homogenate fractions showed steady amounts of VHA, suggesting that the increased VHA content in the membrane of fed sharks result from translocation as opposed to generation of new gill cells with high membrane VHA content. VHA immunofluorescence microscopy of gill sections confirmed that fed sharks have more basolaterally located VHA. Because this phenomenon occurs in both leopard and dogfish sharks, the results suggest that VHA translocation may be a universal mechanism for combating blood alkalosis in marine elasmobranchs. Research was funded by the American Physiological Society UGSR Fellowship to Christian Munévar, the San Diego Fellowship to Jinae N. Roa, and Scripps Startup Funds to Martin Tresguerres.