bluuebunny/crossref_metadata_2025 huggingface dataset:
Disruption of Intermediate‐conductance Calcium‐activated Potassium Channels, KCa3.1, Unchanges NO‐dependent Relaxations in Mouse Aortas
Authors: Ohta Masanori , Miura Hiroto
Published: June 2021

Endothelial NO release and vasorelaxation are preceded by KCa channel activation causing hyperpolarization, facilitated influx of extracellular calcium and NO synthesis in endothelial cells. The aim of this study was to investigate the precise role of KCa3.1 in vascular responses in mouse aortas.

Methods

Wire myography was performed using KCa3.1+/+ (WT) and −/− (KO) mouse aortic rings precontracted with phenylephrine.

Results

Relaxation to acetylcholine (10−10 to 10−4 M) was identical between strains (%max relaxation: KO 78±5 p=ns vs WT 87±5% and EC50; KO 4.4±1.4 p=ns vs WT 5.0±2.0 x 10−8 M, n=5‐6) and was abolished by endothelial denudation. Nω‐nitro‐L‐arginine methyl ester significantly reduced the relaxation in both to a similar extent (WT 34±9 and KO 36±9%, p<0.05 vs control, respectively, n=7‐8), while indomethacin had no effect. No further reduction in relaxations was seen with the combination of inhibitors. Relaxation to sodium nitroprusside (10−12 to 10−4 M) was also unaltered in KO compared to WT (%max relaxation: KO 90±4 p=ns vs WT 83±8% and EC50; KO 2.9±1.5 p=ns vs WT 21.3±13.6 x 10−8 M, n=5‐6).

Conclusion

KCa3.1 alone plays no discernible role in mediating NO‐dependent vasorelaxation in mice, supporting a role for small‐conductance KCa channel either alone or in combination with KCa3.1. Supported by an NIH grant.
Altered EDHF‐mediated dilation in spontaneously hypertensive rats following enalapril treatment
Authors: Ellis Anthie , Goto Kenichi , Brackenbury Therese D , Meaney Kate R , Falck John R , Wojcikiewicz Richard J.H. , Hill Caryl E
Published: June 2021

Endothelium derived hyperpolarising factor (EDHF) plays a prominent role in resistance arteries via a variety of different mechanisms. We examined these mechanisms in first order mesenteric arterial branches from Wistar‐Kyoto (WKY), spontaneously hypertensive rats (SHR) and age‐matched SHR with blood pressure normalized by enalapril (30 mg/kg/day, 8 wks), using serial section electron microscopy and wire myography in the presence of L‐NAME (100 microM) and indomethacin (10 microM). EDHF‐mediated dilations to acetylcholine (ACh) were sensitive in all three groups to the combination of BKCa, IKCa and SKCa channel inhibitors, iberiotoxin (100 nM), apamin (500 nM) and TRAM‐34 (500 nM). However, only responses in WKY were sensitive to the combined gap junction antagonists, 37,43gap 27, 40gap 40 and 43gap 26 (100 microM each), while responses in both WKY and enalapril treated SHR were sensitive to the selective inhibitor of epoxyeicosatrienoic acid derivatives (EETs), 14,15‐EEZE (10 microM). Electron microscopy showed significantly fewer myoendothelial gap junctions (MEGJs) in SHR than in WKY and no change following enalapril treatment. In conclusion, responses in WKY are dependent on EETs, MEGJs and BKCa, while responses in SHR are dependent on BKCa but independent of EETs or gap junctions. Reduction in angiotensinII with enalapril restores the involvement of EETs but not gap junctions.
Mechanistic Basis of Differential Conduction in Skeletal Muscle Arteries
Authors: Tran Cam Ha , Vigmond Edward , Plane Frances , Welsh Donald
Published: June 2021

This investigation probed intercellular conduction in skeletal muscle feed arteries and addressed why smooth muscle‐initiated responses fail to robustly spread like their endothelial counterpart. Using computational and experimental approaches, two interrelated rationales were developed to explain this apparent discrepancy in cell‐to‐cell communication. The first rationale stressed that smooth muscle electrical responses, if initiated, will be actively dissipated as they spread from cell‐to‐cell along the arterial wall. Charge dissipation is promoted within arteries by the structural and connectivity properties of vascular cells. The second rationale centered on the idea that when agents other than KCl stimulate a limited number of smooth muscle cells, they fail to generate the currents required for a localized membrane potential (VM) response. This insufficiency is the result in part from charge loss, via gap junctions, to neighboring unstimulated cells. Experiments confirmed the latter rationale by showing that focal phenylephrine application: 1) elicited a localized constriction insensitive to L‐type Ca2+ channel blockade; and 2) failed to substantively depolarize vascular smooth muscle cells. The preceding findings illustrate that by using computational modeling and experimentation in a complementary manner, one can isolate key cellular properties and rationally examine their role in limiting the conduction of smooth muscle‐initiated responses. Functionally, these observations enable investigators to assign the concept of "local and global" blood flow control to the electrical and/or non‐electrical behavior of specific cell types.
The positive force‐frequency relationship is maintained in absence of sarcoplasmic reticulum function in rabbit, but not in rat myocardium
Authors: Monasky Michelle M. , Janssen Paul M.L.
Published: June 2021

Myocardial calcium handling differs between species. To investigate the role of myofilaments and [Ca2+]i decline in governing cardiac muscle relaxation, and to elucidate determinants other than the sarcoplasmic reticulum (SR) at various frequencies within the in vivo range, the present study was performed by altering the Ca2+ handling in rat and rabbit. Trabeculae at optimal preload and 37 °C were iontophoretically loaded with bis‐fura‐2 to monitor cytoplasmic Ca2+ levels before subjection to ryanodine and cyclopiazonic acid to inhibit SR function, after which simultaneous force and [Ca2+]i measurements were obtained at 1‐4 Hz in rabbit and at 4‐8 Hz in rat. Inhibition of the SR resulted in increased diastolic and peak Ca2+ levels and slower kinetics. Also, Ca2+ amplitude increased in rabbit but decreased in rat. Developed force increased with frequency in rabbit but decreased in rat after inhibition of SR function, despite both species normally exhibiting a positive force‐frequency relationship. The response of the myofilaments to alterations in Ca2+ handling plays a critical role in the final determination of force, and may differ between species. These results imply the balance between force relaxation and Ca2+ decline is significantly different in larger mammals, necessitating a critical re‐evaluation of how myocardial relaxation is governed, specifically regarding frequency‐dependent activation.
The role of cellular calcium homeostasis in the improved contractile function associated with high‐fat feeding in heart failure
Authors: Cheng Yi‐Hsin , McElfresh Tracy A , Chen Xiaoqin , Rutledge Cody A , Chandler Margaret P
Published: June 2021

Heart failure (HF) is characterized by deteriorating left ventricular (LV) function. High saturated fat diets (SAT) are associated with increased risks for lipotoxicity and contractile dysfunction. However, HF animals fed SAT for 8 weeks post coronary artery ligation exhibited improved LV contractility in vivo compared to HF animals fed normal chow (NC). Ca2+ handling proteins (e.g. sarcoplasmic reticulum (SR) Ca2+ ATPase) regulate myocardial contractility, and Ca2+ handling properties (e.g. Ca2+ transients) are known to be altered in HF. Since SAT improved LV contractility in HF, it is possible that alterations in Ca2+ handling properties have been restored. Male Wistar rats underwent coronary ligation or sham surgery and fed NC (10% kcal fat) or SAT (60% kcal saturated fat) for 8 weeks. In vivo contractile function was assessed by echocardiography and LV cannulation 7‐8 weeks post surgery. Hearts were harvested and cardiomyocytes were isolated enzymatically, following by measurements of cell shortening (digitally), Ca2+ transients (with fura‐2AM), and SR Ca2+ store (by caffeine). Preliminary results suggest that cell shortening, Ca2+ transients, and SR Ca2+ store are improved in HF+SAT relative to HF+NC. However, SAT had no effect in sham animals. Thus, high saturated fat diet improves myocardial contractility by reversing altered Ca2+ handling properties in HF. (Grant: NHLBI HL‐081857 & AHA 0535361N)
Qualitative and quantitative changes in skeletal muscle from heart failure patients
Authors: Miller Mark Stuart , Braddock Joan M , Moulton David , Ward Kimberly A , VanBuren Peter , LeWinter Martin M , Ades Philip A , Maughan David W , Toth Michael J
Published: June 2021

The objective of this study was to determine if functional deficits in skeletal muscle of heart failure (HF) patients are related to changes at the single fiber level. We examined protein content, ultrastructure and mechanical properties of single fibers from HF patients and sedentary controls. Overall, myosin heavy chain (MHC) protein content was reduced 19% (p<0.01) with HF. In single fibers, MHC content was reduced 15% in Type I (p=0.06) and 19% in Type IIA (p<0.04) with HF. These qualitative decreases in MHC content were associated with a 12% reduction in force production per cross‐sectional area in Type I fibers (p<0.05). No differences in thick to thin filament ratio or A‐band length were found suggesting that MHC loss occurs along the thick filament length. At low [Ca2+] (pCa 8), Type I fibers from HF examined with sinusoidal analysis produced less oscillatory work and had a higher frequency of maximum work (p<0.05), indicating an increase in myosin kinetics. At high [Ca2+] (pCa 4.5), Type I and IIA HF fibers showed similar myosin kinetics, larger elastic moduli at low oscillation frequencies and greater oscillatory work than controls (p<0.05). These results indicate that HF qualitatively modifies skeletal muscle performance at the single fiber level. Together, these qualitative and quantitative changes may conspire to impair whole muscle function in heart failure patients. Support: NIH HL‐077418 and AG‐031303.
Effects of age on training‐induced adaptations of the neuromuscular junction
Authors: Roby Mackenzie Anne , Glass Emily , Deschenes Michael
Published: June 2021

Unlike skeletal muscle, the effect of aging on training induced adaptations of the neuromuscular junction (NMJ) has received scant attention. This study aimed to determine whether this important synapse exhibits age‐specific adaptations to endurance training. Twenty young adult (8 mo) and 20 aged (24 mo) male Fischer 344 rats were assigned to either exercise trained or sedentary control groups (4 groups, N=10/group). All trained rats participated in the same 10 week program of treadmill running (5days/wk). Following the 10 week intervention, all rats were euthanized and soleus muscles were removed and quickly frozen at resting length. Muscles were sectioned and stained with cytofluorescent procedures to visualize pre‐synaptic nerve terminal branches, and post‐synaptic acetylcholine receptors. Pre‐ and post‐synaptic images were collected with confocal microscopy and used to quantify various parameters of NMJ morphology. Results showed that young, but not aged rats demonstrated significant (P<0.05) training‐induced increases in pre‐synaptic nerve terminal branch number, total branch length, and branching complexity. Similarly, only the NMJs of young rats displayed significant training‐induced increments in perimeter length around endplate regions, and expanded areas occupied by acetylcholine receptors. Our data show that aging blunts the ability of the NMJ to adapt to endurance exercise training.

Supported by the National Institute on Aging, and The Foundation for Aging Studies and Exercise Science Research.
Muscle contractile dysfunction and oxidative injury are observed in the soleus muscle of metallothionein deficient mice following acute spinal cord injury
Authors: DeRuisseau Lara Roberts , Zoccolillo Michelle , Recca Daniel M , Mogle Jacqueline A , Keslacy Stefan , DeRuisseau Keith C
Published: June 2021

Metallothioneins (Mts) are small molecular weight proteins possessing metal binding and free radical scavenging properties that are upregulated in skeletal muscle following acute spinal cord injury (SCI). We tested the postulate that deficiency of Mt (Mt1 and Mt2; Mt−/−) would exacerbate the development of atrophy, contractile dysfunction, and the oxidative stress response of the soleus following SCI (7 days, T9) compared to control strain mice. Four groups of mice were studied: control transected (CT, n=12); Mt−/− transected (MT; n=12); control laminectomy (CL, n=7); and Mt−/− laminectomy (ML, n=5). CT and MT mice experienced similar % body weight losses following SCI vs. pre‐surgery values. Soleus muscle mass: body mass ratio was lower (p<.05) for CT vs. CL and MT vs. ML groups, with no difference between MT and CT groups. MT mice showed reduced maximal specific tension values at high stimulation frequencies compared to ML animals (p<.05). Furthermore, oxidative stress (4‐hydroxynoneal) and protein levels for the antioxidant catalase were greater (p<.05) in MT vs. ML, but not in CT vs. CL groups. No differences in manganese superoxide dismutase (SOD) or copper‐zinc SOD protein levels were detected between groups. In summary, Mt−/− does not impact muscle mass loss but leads to lower maximal force generating ability and increased oxidative stress of the soleus following acute SCI. Support: Syracuse University SOE
Effect of pre‐habilitative conditioning on aged muscles
Authors: Glass Emily Katharine , Sherman Elizabeth Grace
Published: June 2021

It is well established that muscle unloading results in decreased strength and myofiber atrophy. The objective of this study was to assess the capacity of exercise training performed prior to unloading (pre‐habilitation) to modulate the atrophy caused by unloading. Thirty aged (25 mo) male Fischer 344 rats were assigned to 3 treatment groups (10/group). These groups were: 1) control, 2) unloaded (UL), 3) pre‐habilitation before unloading (PH‐UL). Muscle unloading was imposed for 2 weeks via hindlimb suspension. Pre‐habilitation involved 2 weeks (5 days/wk) of treadmill running. Following intervention all rats were euthanized and soleus muscles were obtained and frozen at resting length. Muscle cross sections were collected and stained for mATPase activity. Results showed that compared to controls, myofibers from UL rats evinced significant (P<0.05) atrophy of 45%. Although the myofibers of PH‐UL rats also exhibited significant atrophy relative to controls, it was significantly less than that of UL rats (32% vs. 45%). Our data confirm that in aged rats ‐ who have been previously found to be acutely susceptible to unloading‐induced atrophy ‐ pre‐habilitative conditioning effectively attenuates unloading‐induced myofiber atrophy.
Chronic resistance training attenuates age‐associated endothelial dysfunction in coronary arteries of aging rats
Authors: Araj Christina A. , Richards Margaret A. , Harris M. Brennan
Published: June 2021

Age is associated with reduction endothelial‐dependent vasorelaxation in human and animal coronary arteries. Endurance exercise training has been shown to reverse this and to protect against myocardial ischemia/reperfusion in isolated rat hearts in part through improved coronary flow. Although resistance exercise training may be prescribed in aged individuals to enhance quality of life, little is known regarding the effects of this type of exercise on coronary endothelial function. Therefore, aged (24 mo) male, Fisher 344 rats were divided into two groups: Aged Sedentary (AS, n=8), and Aged Trained (AT, n=10). Resistance exercise training consisted of climbing a 1 m wire ladder, at an 85o angle, 3d/wk for 7 weeks with increasing weight added to the tail. Coronary flow was measured in isolated, perfused hearts using the Langendorff model and constant pressure. After a 30 minute stabilization period, changes in coronary flow were measured following bolus injections of bradykinin (BK, 10−12‐10−4) using an in‐line flow meter. Coronary flows were significantly (P<0.05) improved at high doses of BK in AT versus AC. In conclusion, these results suggest that resistance exercise training in aged rats attenuates age‐associated endothelial dysfunction in coronary arteries. Supported by NIH R15 HL082649‐01 (MBH) and the Borgenicht Program for Aging Studies and Exercise Science (MBH).
Scleraxis is expressed in adult tendons and is upregulated in response to mechanical loading
Authors: Mendias Christopher L , Bakhurin Konstantin I , Arruda Ellen M , Brooks Susan V , Faulkner John A , Larkin Lisa M
Published: June 2021

Scleraxis is a bHLH transcription factor that is critical for the embryonic formation of limb tendons. Scleraxis promotes the proliferation of tendon fibroblasts and enhances the synthesis of type I collagen. In the absence of scleraxis during embryonic development limb tendons fail to form properly. While scleraxis plays a critical role in regulating the formation of embryonic tendons, the role of scleraxis in the regulation of adult tendon tissue is not known. We hypothesized that scleraxis continues to be expressed in tendons following development and that scleraxis is an important factor in the adaptation of tendons during adulthood. To measure scleraxis expression, we analyzed tissues from a line of transgenic mice that produce GFP under the control of the scleraxis promoter. Scleraxis is detected in whole adult tendons and ligaments for at least twelve months following birth. Scleraxis is also expressed in monolayers of cultured fibroblasts and in three‐dimensional tendon constructs engineered from fibroblasts obtained from adult tendon tissues. In response to mechanical loading an upregulation of scleraxis expression is observed. Further studies will determine whether the absence of scleraxis affects the ability of tendons to adapt to mechanical loading.
Exercise training improves heat balance during exercise depending on tail vasodilatation mediated by modification in vascular reactivity
Authors: Guimarães Juliana Bohnen , Rezende Bruno A. , Lima Daniel C. , Rodovalho Gisele V. , Patel Kaushik P. , Lemos Virgínia S. , Coimbra Cândido C.
Published: June 2021

Exercise induced‐hyperthermia results from imbalance between heat production and heat dissipation. In rats, artery tail dissipates 45% of exercise heat. Therefore, the aim of this study was to assess the influence of exercise training in heat balance during exercise and in tail artery reactivity. Male Wistar rats (weaning) were submitted to 8 weeks of training (Tr, n=6) or kept untrained (Un, n=6). The exercise training was done in a treadmill during 8 weeks in a gradual increase intensity until achieve 25 m/min, 60 min/day with 5% of inclination. After training, body (Tb) and skin tail (Ts) temperatures were evaluated during a session of submaximal exercise (60% VO2 max). The vascular reactivity in isolated tail artery was evaluated in a bath organ system. The data showed that exercise training increases running performance (30%) and decreases ratio of heat storage/workload in 48%, indicating that training improves heat loss mechanisms. This evidence was reinforced by reduced latency (6.41± 0.71 min, Tr vs 9.75 ± 0.61 min, Un, p<0.01) and by 0.43 oC decrease in Tb threshold to tail vasodilation. Training improved ACh‐induced relaxation in isolated tail artery (P<0,01) that was partially impaired by L‐NAME pretreatment (p<0,05). Our results indicate that exercise training improves heat loss by increasing relaxation of tail artery and decreasing sympathetic tonus to this vessel during exercise.
LKB1/AMPK Signaling in the Diaphragm
Authors: Brown Jacob Daniel , Mongillo Anthony D. , DiGiovanni Ryan A. , Lane Matt D. , Winder William W. , Thomson David M.
Published: June 2021

The LKB1/AMP‐Activated Protein Kinase (AMPK) signaling pathway is a major regulator of skeletal muscle metabolic processes. During exercise, LKB1‐mediated phosphorylation of AMPK leads to its activation, promoting mitochondrial biogenesis and glucose transport, among other effects. The roles of LKB1 and AMPK have not been characterized in the diaphragm. Two methods of AMPK activation were used to characterize LKB1/AMPK signaling in diaphragms from muscle‐specific LKB1 knockout (KO) and littermate control (C) mice: (1) acute injection of 5‐aminoimidazole‐4‐carboxamide ribonucleoside (AICAR) and (2) 5‐min direct electrical stimulation (ES) of the diaphragm. Diaphragms were excised 60 minutes post‐AICAR injection and immediately after ES. AMPK phosphorylation increased with AICAR and ES in C but not KO mice. Acetyl CoA Carboxylase (ACC) phosphorylation increased with AICAR in C but not KO mice, but increased in both genotypes with ES. No differences in Hexokinase 2 (HK2) levels were evident. CREB phosphorylation was lower in KO diaphragms. While Cytochrome C (Cyto C) levels were lower in KO diaphragms, Uncoupling Protein 3 (UCP‐3) levels were not different between genotypes. In conclusion, AMPK is activated in an LKB1‐dependent manner in the diaphragm by AICAR injection and direct ES.

This study was supported by National Institute of Arthritis and Musculoskeletal and Skin Diseases Grant AR‐051928.
nNOS mediates relaxation in corpus cavernosum mice strips improved by Tx2‐6 toxin from Phoneutria nigriventer spider via cGMP increase
Authors: nunes kenia p , Carneiro Fernando S , Cordeiro M N , Privierio Fernanda , Tostes Rita C , De Lima Maria Helena , Leite Romulo , Webb R. Clinton
Published: June 2021

A toxin from the venom of Phoneutria nigriventer spider, Tx2‐6, causes priapism in mice and relaxation of rat cavernosal tissue. The aim of this study is to investigate the involvement of NOS isoforms and cGMP in the action of the toxin Tx2‐6 on erectile tissue. We isolated cavernosum strips from eNOS and nNOS knock out (KO) mice. The strips were incubated with bretylium tonsilate (3x10−5M) to block adrenergic neurotransmission. Following contraction induced by phenylephrine (10−5 M), the strips were made to relax in response to electrical field stimulation (EFS). Cumulative concentration‐response curves to acetylcholine (10−9 to 10−5M) were performed in absence and presence of Tx2‐6. cGMP levels were analyzed using kit (Cayman) in presence of Tx2‐6 and this toxin plus L‐NAME (10−4M). The action of the N‐type calcium channels antagonist, ω‐conotoxin GVIA (10−6M) on EFS was evaluated. Results showed relaxation increased in the presence Tx2‐6 toxin (10nM) in wild type and eNOS KO mice but not in nNOS KO mice. Acetylcholine seems not be involve in the relaxation improved by Tx2‐6. cGMP levels significantly increased in the presence of Tx2‐6. This increase was abolished by L‐NAME and ω‐conotoxin GVIA. This study indicates that Tx2‐6 improves the erectile response, probably via activation of calcium channels by slowing the inactivation of Na+ channels in nitrergic nerves facilitating NO production from nNOS and consequently increased cyclic GMP.
Augmented sympatho‐excitatory reflexes in rats exposed to chronic intermittent hypoxia
Authors: Silva Ana Q , Schreihofer Ann M.
Published: June 2021

Chronic intermittent hypoxia (CIH) yields persistent elevations in sympathetic nerve activity (SNA) and arterial pressure (AP). In addition, acute hypoxia evokes a larger rise in SNA in rats after CIH due to enhanced sensory and central mechanisms. We examined the hypothesis that other sympatho‐excitatory reflexes are also augmented after CIH. Male Sprague‐Dawley rats (4 wks old) were exposed to CIH (6% O2 for 40s, every 9 min, 8h/day, 3 wks). Isoflurane‐anesthetized, ventilated, paralyzed rats were prepared for recording AP, splanchnic SNA, phrenic nerve discharge and then switched urethane anesthesia (1.5 g/kg, iv). To activate the somatic pressor reflex a sciatic nerve was stimulated (5 sec, 100 μA, 33 Hz). To activate the diving reflex, water (2 ml) was passed through the nose via a tracheal catheter. Compared to control rats (n=17), rats with CIH (n=13) had elevated mean AP (134±5 vs.115±3 mmHg, P<0.05), SNA (63±7 vs. 38±6 mV, P<0.05) and hematocrit (45.1±0.5 vs. 42.4±0.6%, P<0.05). Stimulation of the sciatic nerve evoked a greater increase in SNA in CIH rats (67%±16 vs. 15±8%, P<0.05). Likewise, stimulation of the diving reflex induced an exaggerated rise in SNA in rats with CIH compared to control rats (n=6/group; 674±236% vs. 123±41%, P<0.05). These data suggest that exposure to CIH leads to adaptations that facilitate activation of the sympathetic nervous system. Support: AHA0755292B, NIH‐HL075174.
Chronic intermittent hypoxia alters the respiratory and autonomic responses to activation of glutamate receptors in the caudal NTS of juvenile rats
Authors: Zoccal Daniel B , Machado Benedito H
Published: June 2021

Alterations in glutamatergic mechanisms in the caudal NTS (cNTS) were evaluated after chronic intermittent hypoxia (CIH) exposure. Using the in situ working heart‐brainstem preparation we recorded the changes in phrenic (PNA), cervical vagus (cVN) and thoracic sympathetic (tSNA) nerve activities evoked by microinjections of different concentrations of L‐glutamate (L‐glu: 25, 50 and 250 mM) into cNTS of juvenile Wistar rats submitted to CIH (6% O2 for 40 s every 9 min, 8 h/day) for 10 days and of control rats. L‐glu into cNTS of CIH (n=10) and control (n=7) rats produced an increase in post‐inspiratory vagal activity and sympathoexcitation. The cVN responses was smaller in CIH rats than in controls at the all concentrations tested (P<0.05), while the tSNA response was higher in CIH only at the higher concentration of L‐glu microinjected (53±9 vs 27±2 %, P<0.05). Besides, L‐glu into cNTS evoked significant reductions in the frequency of PNA in the control but not in the CIH group (P<0.05). The higher sympathoexcitation and the blunted apnea in response to L‐glu into cNTS of CIH juvenile rats suggest that glutamatergic mechanisms in or downstream to cNTS are altered after CIH exposure. Further studies are required to verify the involvement of these alterations in the autonomic and respiratory dysfunctions observed after CIH. Supported by FAPESP and CNPq.
Influence of endurance training on the neural and hemodynamic responses to a mixed meal
Authors: Deo Shekhar Hari , Young Colin N. , Kim Areum , Mikus Catherine R. , Uptergrove Grace M. , Thyfault John P. , Fadel Paul J.
Published: June 2021

Previous studies have indicated that nutrient intake is accompanied by increases in muscle sympathetic nerve activity (MSNA) and limb blood flow; responses that have been mainly attributed to insulin. However, whether an insulin sensitive state, such as endurance training, alters neural and hemodynamic responses to a meal remains unknown. Thus, MSNA, femoral blood flow (FBF) and plasma glucose and insulin concentrations were obtained in 3 high fit (HF; VO2 65±3 ml/kg/min) and 5 average fit (AF; VO2 46±1 ml/kg/min) subjects after ingestion of a mixed meal drink (57% CHO, 28% Fat, 15% Protein). In both groups, MSNA was significantly increased from baseline at 30 (Δ+10±1 HF, Δ+7±3 AF, bursts/min; P=0.02) and FBF at 45 (Δ+7±6 HF, Δ+17±11 AF, ml/min/kg; P<0.01) minutes and both remained elevated up to 120 minutes after the meal. Plasma glucose was similarly elevated between groups. However, HF subjects tended to exhibit greater insulin sensitivity by a lower AUC for insulin after the meal (1650±501 HF vs. 3263±630 AF; P=0.09). Thus, despite an 85% greater increase in plasma insulin from baseline in AF subjects (+836±254% HF vs. +1551±179% AF, uIU/ml, 60 min), no fitness‐group differences were found in MSNA (P=0.81) or FBF (P=0.93) responses. These preliminary data suggest that endurance training may enhance the central sympathetic (MSNA) and peripheral vascular (FBF) effects of insulin following consumption of a mixed meal.
Sympathetic innervation of the splanchnic region is necessary for the beneficial effects of 5HT‐1A receptor agonists in rats during hypovolemic shock
Authors: Tiniakov Ruslan L , Scrogin Karie E
Published: June 2021

The 5‐HT1A agonist, 8‐OH‐DPAT, improves blood gases following hemorrhagic shock via sympathetic‐dependent increases in venous return. To determine if this response requires sympathetic innervation of the splanchnic region, effects of 8‐OH‐DPAT (30 nmol/kg, iv) or saline on arterial pressure (AP), mean circulatory filling pressure (MCFP, an index of venous tone) and blood gases were determined in splanchnic‐denervated (SD) and sham‐operated rats during hemorrhagic shock (50 mmHg, 25 min). 8‐OH‐DPAT produced a rapid pressor effect in intact‐ (n= 7) and SD (n= 7) rats (+26 ± 3 and +21 ± 2 mmHg; P<0.01). However, the pressor effect was only transient in SD rats (~ 5 min). A persistent pressor effect was paralleled by increased MCFP in intact‐ (+2.2 ± 0.4 mmHg, 35 min after injection; P<0.01), but not SD rats. Saline had no effect on AP or MCFP. 8‐OH‐DPAT‐treated, intact rats had higher total bicarbonate (23 ± 2 vs. 14 ± 2, 12 ± 2, 16 ± 3 mmol/L; P<0.01) and lower lactate (7.7 ± 1.6 vs. 14.9 ± 1.5, 16.5 ± 1.8, 12.9 ± 2.1 mmol/L; P<0.01), compared to all other groups. The response to 8‐OH‐DPAT in hemorrhagic shock is dependent upon sympathetic innervation of the splanchnic region. Supported by HL 076162.
Discharge of RVLM Vasomotor Neurons is Increased in Angiotensin II – Salt Hypertensive Rats: Selective Modulation of a Functionally Identified Group of Neurons
Authors: Pedrino Gustavo R , Calderon Alfredo S , Andrade Mary Ann , Cravo Sergio L , Toney Glenn M
Published: June 2021

In rats consuming a 2% NaCl diet and treated with ANG II (150 ng/kg/min, s.c.; n = 28), MAP increased from 103 ± 1.6 to 133 ± 3.5 mmHg. Extracellular recording was used in anesthetized rats to assess effects of ANG II + Salt on the discharge of RVLM vasomotor neurons. Among cells with baro‐inhibitable and cardiac rhythmic discharge, spontaneous firing rate (SFR) was similar in hypertensive (HT: 8.0 ± 2.1 Hz; n=20) and normotensive (NT: 9.5 ± 1.8 Hz; n = 35) rats. However, a greater (P<0.01) increase of MAP was needed to silence discharge in HT (44 ± 4.9 mmHg) than NT (29 ± 3 mmHg) rats. The maximum firing rate (MFR) achieved when arterial baroreceptors were unloaded was similar across groups (HT: 15.3 ± 4.1 Hz, NT: 17.4 ± 4.1 Hz). In another group of cells discharge was baro‐inhibitable, but was not cardiac rhythmic. Among these cells, SFR was significantly (P<0.0001) higher in HT (19.6 ± 2.6 Hz; n=34) than NT (8.1 ± 1.1 Hz; n=35) rats. The increase in MAP needed to silence discharge, and the MFR during baro‐unloading were also higher (P<0.01) in HT (58 ± 5.6 mmHg; 38.9 ± 6.2 Hz) than NT (39 ± 4.1 mmHg; 12.5 ± 2.2 Hz) rats. This group consisted mostly of non‐C1cells with bursting discharge. Burst discharge was phase‐locked to the frequency of respiratory drive. We conclude that discharge of non‐cardiac rhythmic RVLM vasomotor neurons with respiratory‐driven bursting activity is increased in rats with ANG II + Salt hypertension.
Altered balance of GABAergic and glutamatergic afferent inputs in PVN‐RVLM neurons of renovascular hypertensive rats
Authors: Biancardi Vinicia Campana , Campos Ruy Ribeiro , Stern Javier Eduardo
Published: June 2021

Altered hypothalamic synaptic function contributes to exacerbated neurohumoral drive in hypertension. Using immunohistochemistry and tract‐tracing techniques, we determined if remodeling of afferent GABAergic and glutamatergic inputs in PVN‐RVLM neurons contributes to the imbalanced excitatory/inhibitory function observed in renovascular hypertensive rats (RVH). The density of GAD67ir and VGLUT2ir boutons increased in PVN‐RVLM neurons from RVH rats (P< 0.001). Despite this concomitant increase, time‐dependent and compartment‐specific differences in their reorganization resulted in an altered balance of excitatory/inhibitory inputs. In SHAM and early stage RVH rats (3 weeks), cell body VGLUT2ir predominated over GAD67ir (P< 0.001); no differences were observed in proximal dendrites. Conversely, in late‐stage RVH rats (6 weeks), the somatic VGLUT2/GAD67 predominance diminished (P>0.5), while this relationship in proximal dendrites increased (P< 0.05). Our results support that anatomical remodeling of GABA and glutamate afferent inputs in the PVN, resulting in a net predominance of excitatory over inhibitory inputs, may contribute as an underlying mechanism to enhanced neurohumoral drive in renovascular hypertension. This work was supported by NIH HL090948‐01 to Stern JE and CAPES BEX0325/03‐1 (Brazil).
Neurochemical changes in rat nucleus tractus solitarii after ablation of neurons with neurokinin‐1 receptors
Authors: Lin Li‐Hsien , Taktakishvili Otar M. , Dragon Deidre N. , Talman William T.
Published: June 2021

Ablation of nucleus tractus solitarii (NTS) neurons that express neurokinin‐1 (NK1) receptors attenuates baroreflexes, increases blood pressure lability and causes cardiac damage. Mechanisms for these effects are not clear. NK1 receptors colocalize with N‐methyl‐D‐aspartate (NMDA) receptors and alpha‐amino‐3‐hydroxy‐5‐methyl‐4‐isoxazolepropionat (AMPA) receptors in NTS. Decreased glutamate transmission in NTS through loss of NK1 cells could be responsible for the effects. We tested this hypothesis by fluorescent immunohistochemistry after injecting stabilized substance P‐saporin (SSP‐SAP) unilaterally into the NTS. We assessed changes in NMDAR1 (NMDA receptor subunit 1), GluR2 (AMPA receptor subunit 2), gamma‐aminobutyric acid (GABA) receptor type a and b, neuronal nitirc oxide synthase (nNOS), tyrosine hydroxylase (TH), vesicular glutamate transporter (VGluT), choline acetyl transferase (ChAT), glial fibrillary acidic protein (GFAP), and protein gene product 9.5 (PGP 9.5). Immunoreactivity (IR) for NMDAR1, GluR2, GABAb, VGluT3, ChAT, GFAP, and PGP9.5 decreased in the injected NTS. In contrast, IR for VGluT2, nNOS and TH increased. We conclude that physiologic and pathologic effects following ablation of neurons with NK1 receptors in NTS may result from interruption of neurotransmission produced by neurochemicals other than SP. Support: NIH HL59593, NIH HL088090 and VA Merit Review.
Dehydration switches emphasis from hypothalamus to medulla oblongata for maintenance of sympathetic nerve activity (SNA)
Authors: Colombari Debora Simoes Almeida , Colombari Eduardo , Murphy David , Paton Julian F. R.
Published: June 2021

The mechanisms underlying the increase in SNA after dehydration are not fully established. In the present study, we investigated the sequential effects of systemic administration of Losartan (20 μM, AT1 receptor antagonist), brain transections and inhibition of commissural nucleus tractus solitarii (cNTS) on ongoing thoracic SNA after water deprivation for 3 days. Male Wistar rats (65‐85 g) were decorticated to make insentient and perfused intra‐arterially. In euhydrated (EH) rats (290 mOsmol perfusate, n=5), systemic application of Losartan and subsequent pre‐collicular transection (to remove the hypothalamus) reduced SNA by ‐20 ± 3% and ‐44 ± 2% relative to baseline respectively. In contrast, in 3 day dehydrated (DH) rats (340 mOsmol perfusate, n=6) Losartan, subsequent pre‐collicular and pontine transections failed to reduce SNA (‐1 ± 4%, ‐3 ± 8% and ‐12 ± 4% respectively). However, transection at the medulla‐spinal cord junction reduced SNA by ‐70±8%. In intact DH, but not EH rats, reversible inactivation of cNTS using isoguvacine, (a GABAA receptor agonist; 100 mM, 100 nl), reduced significantly baseline SNA (‐33 ± 7% vs 3 ± 8% in EH). These data indicate that in the EH rat baseline SNA is dependent on both the hypothalamus and AT1 receptors. Following chronic dehydration, the regulation of SNA transfers to the medulla oblongata, particularly cNTS. Funded by CNPq and CAPES (Brazil), The Royal Society.
Presynaptic inhibition of pre‐Bötzinger complex neurons in high calcium and disturbed seizure‐like inspiratory bursting in low calcium in newborn rat brainstem slices
Authors: Ruangkittisakul Araya , Panaitescu Bogdan , Bornes Troy D , Ballanyi Klaus
Published: June 2021

Inspiratory pre‐Bötzinger complex (preBötC) rhythm in brainstem slices in physiological (3mM) K+ is depressed by raising superfusate Ca2+ from 1mM to 1.5mM. Here, we studied cellular mechanisms of such Ca2+ block of preBötC rhythm and quantified extracellular preBötC interneuron and preBötC‐driven hypoglossal (XII) motoneuron responses to varied Ca2+ and Mg2+. In 400µm thick newborn rat brainstem slices, preBötC/XII rhythm stopped after <2h in 3K+‐1.2Ca2+, but was restored in 7mM K+. Stable preBötC/XII rhythm in 7K+‐1.2Ca2+ was blocked by 2‐3mM Ca2+ without effect on resting potential or input resistance of preBötC and non‐preBötC inspiratory neurons or tonic neurons. preBötC/XII burst rates increased while amplitudes decreased in <1.2Ca2+ until rhythms stopped in <0.25Ca2+. =0.8mM Ca2+evoked seizure‐like activity, partially masking inspiratory XII bursts. Lowering Mg2+ from 1mM to 0.25mM in 7K+‐1.2Ca2+ did not affect preBötC/XII rhythms, whereas 3mM Mg2+ depressed their rate and amplitude to 43% and 82% of control, respectively. Findings show that inspiratory interneuron and XII motoneuron networks in brainstem slices are relatively insensitive to extracellular Mg2+, but generate stable rhythm only in 0.8‐1.2mM Ca2+. Lack of postsynaptic membrane effects suggests that Ca2+ block of isolated preBötC networks involves primarily presynaptic mechanisms. Supported by CIHR‐MFN, CIHR, AHFMR.
Autoresuscitation responses to hypoxia‐induced apnea are prolonged in newborn 5HT‐deficient Pet‐1 homozygous mice
Authors: Erickson Jeffery T , Sposato Brian C , Mecker Samantha V
Published: June 2021

Autoresuscitation is a critical survival‐promoting mechanism in mammals that allows recovery from primary apnea via hypoxia‐induced gasping. Serotonin (5HT) neurotransmission has been implicated in gasp generation and autoresuscitation although the role of endogenous 5HT in these processes in vivo is not yet clear. In this study we used body plethysmography to compare autoresuscitation responses to hypoxia‐induced apnea in unanesthetized newborn wild type (WT) mice and Pet‐1 knockout (KO) mice that have only 30% of the normal complement of brainstem 5HT neurons. When exposed to 97% N2/3% CO2 on postnatal day 4.5, the time to first gasp following primary apnea was significantly longer in the KO compared to WT mice. During unrelenting hypoxia the gasp frequency, the total number of gasps generated, the duration of the gasping period, and the time to last gasp was similar between genotypes. However, if O2 was made available before the first gasp, allowing for autoresuscitation to occur, gasping frequency was decreased and the duration of the gasping period was prolonged in KO mice, relative to the WT, resulting in a significant increase in the time needed for a successful autoresuscitation response. We conclude that 5HT‐deficient Pet‐1 KO mice can gasp and autoresuscitate from a single episode of hypoxia‐induced apnea, but autoresuscitation is inefficient, relative to WT controls. Supported by a TCNJ SOSA award (JTE).
Interaction between Nicotine and Estradiol in Regulation of Gene Expression in Substantia Nigra (SN) in Animal Model of Parkinson's Disease (PD)
Authors: Serova Lidia I , Nostramo Regina , Maharjan Shreekrishna , Sabban Esther L
Published: June 2021

The incidence of PD is two fold higher in men than in women. Nicotine (N), which reduces the risk of PD, exhibits sex specific effects. We found that injections of estradiol benzoate (EB) to ovariectomized (OVX) rats change the sensitivity of GTP cyclohydrolase I (GTPCH), the rate‐limiting enzyme for biosynthesis of essential co‐factor for tyrosine hydroxylase (TH), to N. We hypothesize that N and EB have interactive effects on physiology of SN in rats with PD at the level of regulation of GTPCH gene.

PD rats were generated by infusion of 6‐OHDA in striatum of OVX rats. The control group received ascorbic acid. 6‐OHDA rats were injected with EB or vehicle (V) once daily for 13 days. EB or V treated rats were divided into two subgroups and from 14th day, in addition to EB or V, were administered saline or N every 12 hrs. Rats with 6‐OHDA lesions developed forelimb and sensory‐motor deficits while those with EB itself, and in combination with N, improved apomorphine induced rotation behavior. TH and GTPCH mRNA levels were lower in the SN of 6‐OHDA rats compared to controls. N restored both mRNAs in SN only in EB treated rats, with significant interactive effects for GTPCH mRNA. The improvement might be mediated by regulation of nAChR subtype expression. Significant interactive effects of N and EB were found on mRNA levels of α3, α6, α7 subunits of nAChR in SN of the PD rats.

Supported by American Parkinson Disease Association.
Orai1 interacts with the endogenous endothelial ISOC channel both constitutively and dynamically
Authors: Cioffi Donna L , Stevens Troy
Published: June 2021

The endothelial ISOC channel is a calcium selective store‐operated calcium entry channel whose activation is an important step leading to intercellular gap formation and increased permeability. The transient receptor potential canonical (TRPC) proteins 1 and 4 contribute to ISOC channel structure. More recently orai1 has been implicated in store‐operated calcium entry channel structure and function. However, the contribution of orai1 to endothelial ISOC channel structure and function is currently unknown. We sought to determine whether orai1 interacts with the ISOC channel subunits TRPC1 and TRPC4 using Förster Resonance Energy Transfer (FRET). Protein 4.1 immunoprecipitation was performed to isolate a population of endothelial ISOC channels. TRPC1, TRPC4 and orai1 were tagged with fluorophore (Cy3 and Cy5) labeled antibodies. In an unactivated state, positive FRET was observed between orai1 and TRPC4 revealing direct and constitutive interaction. On the other hand, FRET was not observed between orai1 and TRPC1 until ISOC was activated by thapsigargin. Collectively, these data reveal that orai1 interacts with the endothelial ISOC channel through constitutive interaction with TRPC4 and dynamic interaction with TRPC1. Supported by HL60024 and F32HL086124.
The peroxisome proliferator‐activated gamma (PPARγ) ligand, rosiglitazone, attenuates lung barrier dysfunction following chronic alcohol ingestion
Authors: Wagner Matthew Christian , Hart C. Michael , Brown LouAnn S. , Kleinhenz Dean Jason
Published: June 2021

Chronic alcohol consumption increases the incidence of the Acute Respiratory Distress Syndrome in part through alcohol‐induced, NADPH oxidase‐mediated increases in oxidative stress in the lung. We hypothesized that rosiglitazone, a PPAR‐γ ligand that attenuates NADPH oxidase expression, would attenuate alcohol‐induced oxidative stress and alveolar capillary barrier dysfunction in the lung. To explore this hypothesis, C57Bl/6 mice consumed standard chow ± ethanol (EtOH, 20% w/v) in drinking water for 12 weeks. After 11 weeks, selected mice were gavaged daily with rosiglitazone (10 mg/kg) for 1 week. Mice were then treated ± lipopolysaccahride (LPS, 2mg/kg IP) 6 h prior to sacrifice. LPS treatment stimulated lung barrier dysfunction evidenced by increases in bronchoalveolar lavage (BAL) protein levels. Chronic EtOH ingestion enhanced LPS‐mediated increases in BAL protein, whereas rosiglitazone attenuated BAL protein levels in EtOH and EtOH + LPS‐treated mice. In vitro studies employing HUVEC monolayers on transwell filters confirmed that EtOH (0.1% for 72 h)‐induced endothelial barrier dysfunction was attenuated by rosiglitazone. These findings indicate that PPARγ may represent a novel therapeutic target that can reduce the barrier disruptive effects of alcohol on the lung despite continued EtOH ingestion. This work is supported by grants from NIAAA and the VA Research Service.
Protein interactions with cdc37 in agonist stimulated bovine aortic endothelial cells
Authors: Horst Kyle E , Wilson Christopher G , Harris M Brennan
Published: June 2021

Previously we have demonstrated that the cell division cycle 37 homolog, cdc37, interacts directly with the endothelial nitric oxide synthase (eNOS) and inhibits eNOS activity in vitro. In addition, cdc37 regulates interactions between other key regulators of eNOS activity including Hsp90 and Akt. Therefore, we investigated changes cdc37 interactions with eNOS, Hsp90, and Akt in bovine aortic endothelial cells (BAECs) stimulated with either vascular endothelial growth factor (VEGF) or bradykinin (BK). Treatment of BAECs with VEGF or BK for 0, 1, 5, 15, 30, or 60 min resulted in a biphasic change in cdc37/eNOS interaction. Cdc37 interaction with eNOS decreased upon stimulation but returned to basal levels by 15 min and then decreased by 30 and 60 min. Agonist stimulation also resulted in a transient increase in Hsp90 association with cdc37, however, no significant changes in cdc37 and Akt interaction were observed. These data coupled with our previous findings suggest that cdc37 initially inhibits basal eNOS activity through direct interaction and may play a role in agonist stimulated eNOS activity through its interaction with Hsp90. Supported by NIH R15 HL082649 (MBH) and an American Physiological Society Summer Undergraduate Research Fellowship (KEH).
N‐acetyl Glucosamine probe upon binding to endothelial lectins selectively modulate flow‐induced; cardiac contractility, A‐V delay and vascular smooth muscle tone
Authors: Ramiro‐Diaz Juan M , Barajas‐Espinosa Alma , Castillo‐Hernandez Jesus R , Torres‐Tirado David , Rubio Rafael
Published: June 2021

Coronary flow modifies cardiac and vascular functions. Stress exerted by flow on the luminal capillary wall may be transduced by glycosidic and lectinic structures in the endothelial glycocalyx. To test this, we synthesized a monosaccharide polymer formed by N‐acetyl Glucosamine (Pol‐GlcNac) covalently linked to a 70 KDa dextran. This polymer was intracoronarily administered in perfused guinea pig hearts at increasing concentrations. Left intraventricular pressure (CC), and the Auricular‐Ventricular delay was (A‐VD) measured. Pol‐GlcNac increases the A‐VD and decreases the CC. The effect of this polymer was not easily washed. To visualize polymer binding sites, PolNGlc covalently linked to a dextran‐FITC was synthesized. Histological slices were made on polymer‐FITC perfused and subsequently washed hearts. Confocal microscopy revealed the polymer retained on vascular endothelium and coincided with factor VIII: an endothelial marker. These results demonstrate the presence of lectinic receptors with affinity to GlcNac.

To establish that Pol‐GlcNac binding to endothelium can regulate flow‐induced release of paracrine agonists, we used isolated guinea pigs aortas perfused at different flows. Flow induced relaxation was measured and blocked by a combination of L‐NAME and indometacin, indicating the mediation of paracrine effectors. Pol‐GlcNac also blocked flow induced relaxations. This suggests that

Pol‐GlcNac binding to the endothelial lumen blocks the flow‐induced paracrine mediators release.

Funded by CONACyT SEP‐42567, CONACyT‐SALUD 2004‐C01‐156