bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of cation‐chloride cotransporter activity in glial tumor proliferation and migration
Authors: Gagnon Kenneth B. , Delpire Eric
Published: June 2021

Primary central nervous system tumors account for 2.2% of all cancer‐related deaths. Glioblastoma multiformes, the most aggressive form of brain tumor have relatively short median survival times (9–12 months in most cases). Like most aggressive cancers, malignant gliomas exhibit a strong tendency to migrate through narrow extracellular spaces in brain tissue by the functional shape‐volume changes mediated by net salt fluxes across cell membranes. The electroneutral‐coupled movement of ions by the cation‐chloride cotransporter family may, therefore, have an important role in this process. In this study, we used the ‘scratch wound’ assay and time‐lapse microscopy to examine the role of cotransporter activity on the proliferation and migration of rat C6 glioma cells, an immortalized astrocytoma tumor cell line. Untreated C6 cells completely filled the ‘wound area’ within 6 hours, significantly faster than their doubling time. Application of 2 mM furosemide (loop diuretic) completely inhibited the ‘closure’ of the wound area. Four hours after removal of the loop diuretic, the C6 cells were able to migrate into the ‘wound area’, similar to untreated cells. These studies suggest a significant role for cation‐chloride cotransporter activity in the physiology of primary CNS tumors and may help identify novel targets for the development of drugs and therapies to combat these aggressive and fatal neoplasms.
Functional Characterization of Pufferfish Slc26a6A and Slc26a6B
Authors: Kato Akira , Chang Min‐Hwang , Kurita Yukihiro , Nakada Tsutomu , Hirose Shigehisa , Romero Michael F.
Published: June 2021

Euryhaline pufferfish (Takifugu obscurus, mefugu) can live in freshwater (FW) with ion concentration less than 1/100 of its body fluid or acclimate to seawater (SW) which has more than 3 times the ion concentration of its body fluid. However, the body‐fluid ion concentrations are maintained stable by the function of epithelial cells in the gill, intestine, and kidney which transport ions directionally and selectively. To understand the mechanisms of the fish body‐fluid homeostasis at the molecular level, there are several essentials: identification of transporters expressed in the epithelia, determination of transporter localization, and characterization of transporter activities. Analyses of various transporter mRNA levels in mefugu tissues identified increases for Slc26a6A and Slc26a6B in the intestine and the kidney of SW‐acclimated mefugu. To study their functions for SW acclimation, we subcloned the cDNAs into a Xenopus expression vector and assayed their functions using microelectrodes in oocytes.

The electrophysiological analyses of the oocytes revealed that (a) Slc26a6A and Slc26a6B are electrogenic Cl−/nHCO3− exchangers; (b) Slc26a6A and Slc26a6B are also exchangers of Cl−/formate, Cl−/oxalate, and Cl−/SO42−; and (c) the slope of I–V relationship of Slc26a6A in the presence of SO42− is 70 times higher than that of Slc26a6B. These results suggest significant roles for Slc26a6A and Slc26a6B in intestinal HCO3− secretion and a role for Slc26a6A in renal SO42− excretion during SW acclimation.

This work was supported by MEXT Japan, NIH (EY017732) and CFF grant (Romero‐06G0).
ENaC/Degenerin family members reduces glioblastoma whole well current and inhibits cell migration
Authors: Kapoor Niren , Fuller Catherine M , Benos Dale J
Published: June 2021

High‐grade gliomas display a basally active, amiloride sensitive current not seen in normal astrocytes or low‐grade gliomas. We hypothesize that this glioma current is mediated by a hybrid channel composed of a mixture of Epithelial Sodium Channel (ENaC)/ Degenerins (Deg) subunits. Knocking down ASIC1 using siRNA in U251‐ MG glioma cells reduced the amiloride sensitive whole cell patch clamp current by 60–70% and ASIC1 protein expression by 50%. Knocking down γENaC using dominant negative mutation (S155X) in D54‐MG glioma cells reduced the whole cell patch clamp current by over 50%. γENaC protein expression was nearly completely abolished as shown by Western blot. αENaC dominant negative mutation (E34X) had a similar effect upon glioma cell whole cell current as γENaC knockdown. Using co‐immunoprecipitation, we also demonstrated an interaction between ASIC1 and γENaC suggesting that these subunits interact with each other to form an ion channel in D54‐MG glioma cell. Our study also suggests that knocking down either γ‐ or αENaC inhibits glioma cell migration by 30–40% consistent with the hypothesis that these channels play a role in glioma cell migration. Therefore ENaC/Deg members play an important role in glioblastoma pathophysiology, and knocking down these different subunits changes important characteristics of glioma cells. This study was supported by NIH Grant CA101952.
TRPM3 channel expression and block in vascular smooth muscle cells
Authors: Naylor Jacqueline , Li Jing , Milligan Carol J , Zeng Fanning , Porter Karen E , Beech David J
Published: June 2021

TRPM3 is a calcium‐permeable channel activated by sphingolipids, including D‐erythro‐sphingosine (Grimm et al 2005, Mol Pharmacol 67, 798–805). Its cellular functions are largely unknown. Using methods to develop isoform‐specific channel blockers (Xu et al 2005, Nat Biotechnol 23, 1289–93) we produced a TRPM3‐specific blocking agent (TM3E3). Preincubation of cells with TM3E3 produced about 40% block of TRPM3‐mediated calcium‐entry or ionic current. TM3E3 had no effect on the closely related channel TRPM2, or other TRP family members TRPC5 and TRPV4, confirming its specificity for TRPM3. Recently, novel activators for TRPM3 have been suggested. In conjunction with TM3E3, these activators have been used to study a potential role of TRPM3 in human vascular smooth muscle cells. The cells express mRNA encoding TRPM3 and the presence of TRPM3 is indicated by labelling with anti‐TRPM3 antibodies. Sphingosine and its analogues evoked calcium responses that could be inhibited by gadolinium, 2‐APB, TM3E3 or siRNA targeting TRPM3. The data suggest TRPM3 is a novel calcium‐entry channel of vascular smooth muscle cells.

The research was supported by a BBSRC Industrial Co‐operative Award in Science and Engineering, the Wellcome Trust and the British Heart Foundation.
Female mRen2.Lewis Exhibit Higher Intrarenal Renin But Lower Renal and Circulating Angiotensin II Than Male Littermates
Authors: Pendergrass Karl , Chappell Mark C
Published: June 2021

The mRen2.Lewis rat (mRen), a congenic hypertensive model, exhibits marked gender differences in blood pressure and tissue injury. Male mRen have 2‐fold higher plasma and renal levels of Angiotensin II (Ang II) compared to female mRen or Lewis controls. Plasma Ang I was also higher in male mRen, but renal Ang I content was significantly reduced. Cortical ACE activity was similar for male and females, however, renal neprilysin protein was 10 fold higher in female mRen possibly contributing to the metabolism of intrarenal Ang II. The present studies further characterized gender differences in the mRen focusing on the expression of renin. Rat renal renin levels (pH 6.5) were 55% [M: 3.6±1 vs. F: 8.0±2 μg AngI/mg/hr; p≤0.01] and 80% [M: 0.5±0.1 vs. F: 2.9±0.3 μg AngI/mg/hr; p≤0.01] lower in males versus females for Lewis and mRen rats, respectively. Female rats also displayed higher mouse renin levels (pH 8.5). Correlation analysis of all groups revealed a negative association between renal renin and plasma Ang II [r2=0.4, p≤0.05], but not renal Ang II [p≥0.05]. Negative feedback of renal renin is evident in the mRen strain, although the mechanism for gender differences in renin regulation is not known. Moreover, the greater Ang II content in the male mRen kidneys may arise from a nonrenal source. Supported by NIH grants HL56973 & HL51952.
Rats with congestive heart failure have increased anxiety
Authors: Henze Marcus P , Eckert Laurie , Tiniakov Ruslan L , Henderson Kyle , Barakat John , Samarel Allen , Scrogin Karie E
Published: June 2021

Sham rats (n=7) and rats that developed congestive heart failure (CHF) 7 weeks after left coronary‐artery ligation (CAL, n=5) were exposed to an elevated plus maze (EPM) to determine anxiety. Rats subjected to CAL went on to develop congestive heart failure (CHF), as determined by elevated left ventricular pressure (29.7 ± 2.7 vs. 6.5 ± 2.1 mmHg, P<0.01), decreased fractional shortening (16.9 ± 2.7 vs. 48.1 ± 3.1%, P<0.01), and elevated heart‐ (5.4 ± 0.2 vs. 3.3 ± 0.1 g/kg, P<0.01) and lung/body weight ratios (13.5 ± 0.4 vs. 5.1 ± 0.2 g/kg, P<0.01). All CHF rats chose to enter an open arm of the EPM first, compared with one sham rat (100% vs. 14%, P<0.025). In addition, CHF rats spent more time in the open arm (36 ± 15 vs. 5 ± 2%, P<0.01). Entry into open arms was increased in CHF rats (4.2 ± 1.2 vs 1.0 ± 0.4 arm entries, P<0.01). Two CHF rats repeatedly jumped from the open arm, while no sham animals jumped from the apparatus (40 vs. 0%, P=0.067). Though these results are suggestive of reduced anxiety based on validation studies of the EPM test, previous studies show that adult rats exposed to sporadic stress during adolescents show increased activity in the open arm of the EPM and jump from the apparatus suggestive of escape behaviors (Pohl, J, 2007). Research suggests that increased activity in the open arm can represent a prelude to escape behavior in relatively severe anxiety. As such, the paradoxical increased open arm activity coupled with purposeful jumps among CHF rats may represent a more severe form of anxiety than that normally characterized by reduced open arm activity in the EPM. Supported by AHA 507025.
Collecting duct‐specific gene inactivation of αENaC in the mouse kidney does not attenuate rosiglitazone‐induced weight gain
Authors: Vallon Volker , Hummler Edith , Rieg Timo , Schroth Jana , Cheng Vickie Pei‐Chun , Rossier Bernard , Cunard Robyn
Published: June 2021

PPARγ agonists (thiazolidinediones) induce fluid retention and weight gain, but the involved mechanisms remain unclear; a role for activation of PPARγ and the epithelial sodium channel ENaC in the collecting duct has been proposed (PNAS 102:9406, 2005; Nat Med. 11:861, 2005). To further test this hypothesis, studies were performed in mice with and without conditional inactivation of αENaC (Scnn1a) in the collecting duct. Scnn1aloxlox mice (control) were crossed with a transgenic line expressing Cre recombinase driven by the Hoxb7 promoter, Scnn1aloxloxcre (JCI 112:554, 2003). As expected, rosiglitazone (320mg/kg diet) rapidly increased body wt (BW) in Scnn1aloxlox mice compared with vehicle‐treatment (ΔBW day 11: 4.5±0.8 vs. 1.1±0.6%, P<0.005) and lowered hematocrit (44±1 vs. 47±1, P<0.0005). In the αENaC collecting duct knockout mice, rosiglitazone still increased body weight (ΔBW: 7.3±0.9 vs. 0.9±0.7%, P<0.0005) and decreased hematocrit (42±2 vs. 47±1, P<0.05). Real‐time PCR in wild‐type mice revealed that rosiglitazone modestly lowered αENaC expression in kidney cortex (to 76±7%, P<0.05)(but not medulla) without affecting γENaC expression. The data argue against a significant role of ENaC activity in the collecting duct in rosiglitazone‐induced weight gain.
NFkB blockade attenuate oxidative stress and improves metabolic abnormalities in Obese Zucker rats
Authors: Ebenezer Philip Jesudason , Mariappan Nithya , Haque Masudul , Elks Carrie , Francis Joseph
Published: June 2021

Obesity plays an essential role in the pathogenesis of metabolic syndrome (MS) and is directly linked with hypertension, type 2 diabetes and dyslipidemia. We investigated the effect of pyrrolidine dithiocarbamate (PDTC), an inhibitor of nuclear factor kappa‐B (NF‐kB), in obese Zucker rat (OZR) fed with high fat diet. OZR (n = 8) were fed a high fat diet and allowed free access to drinking water (control) or water containing PDTC (150mg/kg for 10 weeks). Biochemical, biophysical and gene expression profile were examined. Increased levels (P<0.05) of total ROS generation in kidney cortex, plasma lipid profile, insulin, C‐Reactive protein (CRP), blood urea nitrogen (BUN), creatinine, urine albumin excretion (UAE), accompanied by a decreased (P<0.05) plasma high density lipoprotein (HDL), were observed in the OZR fed with a high fat diet (OZR‐HF) when compared with the OZR fed with regular diet (OZR‐RD).The mRNA expression of desmin, TNF‐α, NFkB and NOX‐1 was significantly increased (P<0.05) in the kidney cortex of OZR‐HF group animals. However PDTC therapy blocked oxidative stress. Our results show that PDTC therapy ameliorates metabolic abnormalities, hypertension and early renal injury by attenuating oxidative stress. The inhibition of NFkB pathway may serve as a potential tool for treatment of metabolic syndrome associated with the early stage of diabetic nephropathy.
Electrolyzed water reduces urinary protein excretion in the streptozotocin induced diabetic Dahl salt sensitive rats
Authors: Ohsaki Yusuke , Mori Takefumi , Yoneki Yoshimi , Endo Satoshi , Hosoya Takuma , Zhu Wanjun , Nakayama Masaaki , Ito Sadayoshi
Published: June 2021

Background: Electrolyzed water generated at the cathode side (EW) has been shown to have anti‐oxidative properties. Present study was designed to investigate the anti‐oxidative and renal protective effect of EW in the progression of renal injury in streptozotocin induced diabetic Dahl salt sensitive rats.

Methods: Four weeks old male Dahl salt sensitive rats were given either tap water (Tap) or EW for 15 weeks. Streptozotocin was administered (65mg/kg intraperitoneally) at 6th week, and rats were assigned to two groups by the blood glucose (BG) level below (STZ‐) and above (STZ+) 300 mg/dL. Plasma biological antioxidant potential (BAP) level was assayed. Urine samples were collected in a metabolic cage.

Results: Urinary protein excretion largely increased in STZ+ group and reached to 516±56mg/day at end of study compared to those of 256±19mg/day in STZ‐ group. EW significantly attenuated the increase in urinary protein excretion at level of 375±65mg/day at the end of the study. Plasma BAP level was significantly reduced in STZ+ group while no reduction of was observed in STZ‐ group. EW abolished the reduction of BAP in STZ+ group.

Conclusion: The results in the present study indicate that EW could reduce renal injury by increasing plasma anti‐oxidative activity in streptozotocin induced diabetic Dahl salt sensitive rats.
Obesity Increases Renal Cortical Neovascularization in Zucker Rats
Authors: Iliescu Radu , Reckelhoff Jane F , Chade Alejandro R.
Published: June 2021

Growing evidence supports the role of obesity as an independent risk factor for chronic kidney disease. Obesity promotes inflammation. Inflammatory cytokines can induce neovascularization in different organs, including the kidneys. However, whether obesity triggers renal neovascularization and, if so, the physiological significance has never been investigated.

Renal microvascular architecture was studied by 3D micro‐computer tomography in lean and obese Zucker rats (LZR and OZR, n=6/group). Mean arterial pressure (MAP), renal blood flow (RBF), and proteinuria were measured in vivo. Systemic inflammation was assessed by measuring levels of interleukin (IL)‐6 and tumornecrosis‐factor (TNF)‐α. In addition, renal histology was studied in cross sections stained with trichrome.

Renal microvascular density was significantly elevated in OZR (Figure). Despite mild proteinuria and increased tubulo‐interstitial and glomerular damage in OZR, RBF and MAP were similar in both groups (Figure). Furthermore, OZR showed a substantial elevation in systemic IL‐6 and TNF‐α (3–4 fold), suggesting increased inflammation.

The current study shows for the first time increased renal cortical vascularization in an animal model of obesity, which possibly reflects a compensatory mechanism that sustains RBF. However, increased inflammation in obese rats may promote further renal injury, as has been observed in obese humans, as the disease progresses.

(Supported by HL‐69194 and Intramural Research Grant Support from the University of Mississippi)
Mitochondrial Acyl‐CoA synthase activity is related to intramyocellular triglyceride and oxidative capacity in lean and obese rhesus monkeys
Authors: Koepper Lindsay M , George Jonelle G , Ryan Alice S , Hansen Barbara C , Ortmeyer Heidi K
Published: June 2021

Acyl‐CoA synthase (ACS) plays a pivotal role in fatty acid metabolism by activating long‐chain fatty acids for either lipid synthesis or lipid oxidation. Older obese, type 2 diabetic (DM) and chronically calorie‐restricted (CR) monkeys have higher intramuscular triglyceride (IMTG) compared to younger lean monkeys. We sought to determine whether (1) obese, DM and/or CR monkeys have reduced ACS activity and (2) ACS activity is associated with IMTG and/or oxidative capacity (citrate synthase [CS]). Fasting ACS and CS activities were measured in mitochondria isolated from skeletal muscle (vastus lateralis) in 4 groups of monkeys: young lean (9 yrs, 13% body fat, n=9), obese non‐DM (17 yrs, 32% body fat, n=7), obese DM (22 yrs, 30% body fat, n=8) and CR (20 yrs, 21% body fat, n=6). ACS activity was 37% lower in the obese non‐DM (54 ± 21 pmol/min/mg protein, p<0.05) and 48% lower in the CR (45 ± 10, p<0.05) compared to the normal monkeys (86 ± 16), but only 10% lower in the DM monkeys (77 ± 16, NS). ACS activity was strongly associated with CS activity (r=0.68, p<0.0001); IMTG was inversely related to ACS activity (r=−0.58, p<0.005) and CS activity (r=−0.61, p=0.002). We conclude that low ACS activity leads to low mitochondrial oxidative capacity, resulting in high IMTG. The mechanisms for the reduced ACS activity in obese and CR monkeys and near‐normal ACS activity in the DM monkeys warrant further study.
Salsalate lowers fasting blood glucose concentrations without improving insulin resistance in non‐diabetic older adults with the metabolic syndrome
Authors: Lesniewski Lisa , Folian Brian , Durrant Jessica , Beske Stacy , Stob Nicole , Seals Douglas
Published: June 2021

Insulin resistance (IR) is a key feature of the metabolic syndrome (MetS), the prevalence of which increases with age. Chronic low‐grade inflammation may contribute to IR, but direct evidence for a role in non‐diabetic, but at risk older adults is lacking. We determined if short‐term (5‐day) treatment with the anti‐inflammatory agent Salsalate (S) (4500 mg/day; placebo‐controlled [PC] randomized crossover design) improves glucose‐insulin metabolism in obese older (BMI=31.7±1.8; 61± 2 yrs; n=7) men with MetS. Subjects demonstrated features of MetS and moderately elevated C‐reactive protein levels (1.82±0.54 mg/L) at baseline. Compared with PC, S produced reductions in fasting blood glucose concentrations (84±3 vs. 95±2 mg/dl, P=0.005) that were strongly related to corresponding increases in fasting plasma insulin levels (absolute Δ: r=−0.90, P=0.01; % Δ: r=−0.96, P=0.003). Consistent with this, the homeostasis model assessment (HOMA) of β‐cell function was greater during S vs. PC (212±41 vs. 99±19, P=0.024). In contrast, S had no effect on the HOMA of IR or on intravenous glucose tolerance test‐derived measures of insulin sensitivity (Si) or glucose effectiveness (Sg) (all P>0.6). These results support the concept that the anti‐inflammatory compound Salsalate lowers fasting blood glucose concentrations in non‐diabetic adults with the MetS by enhancing insulin secretion. Supported by AG006537 and AG015897
Structural Remodeling of Baroreceptor Afferent Innervation of Aortic Arch in Transgenic Type 1 Diabetic Mice (OVE 26)
Authors: Huang Chenghui , Li Lihua , Yan Binbin , Epstein Paul N , Cheng Zixi (Jack)
Published: June 2021

Baroreflex control of heart rate is impaired in OVE26 mice (Lin et al. 2008, EB abstract). The location and the nature of such a neurally‐mediated change are not clear. TMR‐D was injected into the left nodose ganglion to anterogradely label baroreceptor afferent in the aortic arch of OVE 26 diabetic and FVB control mice (6–9 months, n = 9/group). A confocal microscope and a Neurolucida 3‐D digitization system were used to examine baroreceptor axons and terminals. In the aortic arch of FVB and OVE26 mice, we found large and small baroreceptor afferent axons which formed 4 different types of baroreceptor afferent terminals: large and small axons formed large and small flower‐sprays and end‐nets, respectively. In addition, the size of baroreceptor afferent terminals (flower‐sprays and end‐nets) in OVE26 mice was decreased (p<0.01) compared to FVB mice. The overall baroreceptor afferent innervation of the aortic arch appeared sparser in OVE26 than in FVB mice. Our observation demonstrates that diabetes induces a significant structural remodeling of baroreceptor afferent innervation of the aortic arch. Since baroreceptor afferent activity in response to the arterial pressure increase is not changed (Gu et al., 2008, EB abstract), we postulate that morphological remodeling precedes the functional deficit of baroreceptor afferent axons. *The first two authors equally contributed to the present work. NIH HL‐79636.
Functional Changes in Baroreceptor Afferent, Central, and Efferent Components of the Baroreflex Circuitry in Type 1 Diabetic Mice (OVE26)
Authors: Gu He , Epstein Paul N , Li Lihua , Wurster Robert D , Cheng Zixi Jack
Published: June 2021

Baroreflex control of heart rate (HR) is impaired in diabetes mellitus. We hypothesized that diabetes induced functional changes of the neural components at multiple sites within the baroreflex arc. OVE26 diabetic and FVB control mice were anesthetized. Baroreflex‐mediated HR responses to sodium nitroprusside (SNP)‐ and phenylephrine (PE)‐induced mean arterial blood pressure (MAP) changes were measured. Baroreceptor afferent function was characterized by measuring the percent change of the baseline of integrated aortic depressor nerve activity (Int ADNA) in response to MAP changes. The HR responses to electrical stimulation of the left aortic depressor nerve and the right vagus nerve were assessed. Compared to FVB control mice, we found in OVE26 mice that (1) Reflex‐mediated bradycardia and tachycardia were reduced (p<0.05). (2) The baroreceptor afferent function in response to MAP increase did not differ (p>0.05), as assessed by the parameters of the logistic function curve. But, the inhibition of Int ADNA in response to MAP decrease was attenuated (p<0.05). (3) The maximum amplitude of HR responses to right vagal efferent stimulation was augmented (P<0.05). (4) The maximum amplitude of HR responses to left ADN stimulation was decreased (p<0.05). We conclude that a selective deficit of central mediation of reflex bradycardia contributes to the overall attenuation of baroreflex control of HR in OVE26 mice.
Dynamic Cardiac Output Regulation during Exercise, Muscle Metaboreflex Activation and Heart Failure
Authors: Ichinose Masashi , Sala‐Mercado Javier A , Coutsos Matthew , Ichinose Tomoko , O’Leary Donal S
Published: June 2021

We tested whether moderate dynamic exercise (EX) and muscle metaboreflex activation (MMA) affects dynamic baroreflex control of heart rate (HR), cardiac output (CO), and the influence of stroke volume (SV) fluctuations on CO regulation in normal (N) and pacing‐induced heart failure (HF) dogs by employing transfer function analyses of the relationships between spontaneous changes in left ventricular systolic pressure (LVSP) and HR, LVSP and CO, HR and CO, and SV and CO at low and high frequencies (Lo‐F, 0–0.25 Hz; Hi‐F 0.25‐0.6 Hz). In N dogs, EX significantly decreased transfer function gains for LVSP‐HR and LVSP‐CO in Hi‐F and reduced the LVSP‐CO gain in Lo‐F. MMA further reduced LVSP‐HR gain in Lo‐F. Coherence for HR‐CO in Hi‐F was decreased by EX and MMA, whereas that in Lo‐F was sustained at high levels (> 0.8) in all the settings. HF significantly reduced LVSP‐HR gain in Hi‐F. LVSP‐CO gain was decreased in both Lo‐F and Hi‐F. EX and MMA caused similar effects as observed in N conditions. In HF, the coherence for HR‐CO in Lo‐F decreased significantly in all settings, the coherence for SV‐CO in Lo‐F was significantly higher. We conclude that EX and MMA reduces dynamic baroreflex control of HR and CO and those are substantially diminished in HF. In N conditions HR modulation plays a major role in CO regulation. In HF, influence of HR modulation wanes and fluctuations of SV dominates in CO variations. NIH HL 55473
Exercise training normalizes ACE and ACE2 in the brain of rabbits with pacing induced chronic heart failure
Authors: Kar Sumit , Gao Lie , Zucker Irving H
Published: June 2021

Activation of the renin‐angiotensin system (RAS) in the brain plays a critical role in sympatho‐excitation of chronic heart failure (CHF). Exercise training (EX) normalizes sympathetic outflow. The mechanisms that regulate angiotensin converting enzyme (ACE) in the brain and the effect of EX on ACE are unknown. The aim of this study was to investigate the regulation of ACE and ACE2, a homologue of ACE, in various areas of the brain with a pacing CHF‐EX model. Experiments were performed on four groups of New Zealand White rabbits: sham, sham + EX, CHF, and CHF + EX (n=5/group). Punches of the PVN, NTS, and RVLM (n=2/group) were also measured. Western blot analysis and double immunofluoresence were performed to measure and localize expression of ACE and ACE2. ACE expression in the cerebellum, medulla, and hypothalamus was significantly higher in CHF rabbits compared to sham (1.0±0.2 to 2.2±0.1 in the hypothalamus, P<0.05). EX normalized this upregulation compared to CHF (0.5±0.1 to 2.2±0.1 in the hypothalamus). ACE2 was significantly decreased in CHF. EX normalized ACE2 in the PVN, NTS, and RVLM (0.03±0.02 CHF to 0.8±0.3 CHF + EX in the RVLM). ACE was present in both endothelial cells and neurons. The results suggest that EX normalizes ACE and ACE2 in sympatho‐regulatory areas.

Source of support: PO‐1 HL62222
Exercise training improves the exercise pressor reflex dysfunction via ameliorating the skeletal muscle oxidative stress in chronic heart failure
Authors: Wang Han‐Jun , Pan Yan‐Xia , Wang Wei‐Zhong , Zucker Irving H. , Wang Wei
Published: June 2021

In chronic heart failure (CHF), overactive skeletal muscle exercise pressor reflex (EPR) contributes to the exaggerated cardiovascular activity. It has been shown that exercise training (EX) improves the cardiovascular reflexes via ameliorating oxidative stress in CHF. In the present study, we tested the hypothesis that 6–8 weeks EX improved EPR in CHF rats via the mechanism of superoxide. EPR was elicited by electrical stimulation of L4/L5 ventral roots in the decerebrate rats. Superoxide fluorescence in the triceps surae muscle was enhanced in CHF rats compared with sham rats, which was reduced in EX+CHF rats. As shown in table, the EPR response in CHF rats was higher than in sham rats, and the intra‐arterial infusion of a superoxide dismutase (SOD) mimetic (tempol) normalized the EPR in CHF rats. EX improved the augmented EPR in CHF rats, which was abolished by the intra‐arterial infusion of a SOD inhibitor (DETC). The data suggest that EX improves the EPR dysfunction in CHF probably by ameliorating the muscle oxidative stress.
Contrasting Autonomic and Cardiovascular Phenotypes in ASIC1a and ASIC2 deficient mice
Authors: Sabharwal Rasna , Wemmie John A , Abboud Francois M , Chapleau Mark W
Published: June 2021

Members of the acid‐sensing ion channel (ASIC) family are differentially expressed in subpopulations of sensory and central neurons and have been implicated in a variety of neuronal functions. We recently demonstrated that mice deficient in ASIC2, a subunit implicated in mechanotransduction, exhibit baroreflex and autonomic dysfunction, and hypertension (FASEB J 20:2006). The goals of the present study were to characterize autonomic and cardiovascular phenotypes in conscious ASIC1a−/− mice and compare the results to those obtained in ASIC2−/− mice. Arterial blood pressure (BP), heart rate (HR), and locomotor activity were measured by radio‐telemetry (DSI) in the mouse home cage. Mean BP and HR (24 hr avg.), cardiac sympathetic tone (HR response to propranolol), and sympathetic vasomotor tone (BP response to ganglionic blocker chlorisondamine) were all significantly higher in ASIC2−/− (n=5–7), but not different in ASIC1a−/− (n=7) vs. wild‐type (WT, n=7) mice (Table, *P<0.05).

In contrast to the cardiovascular and autonomic phenotypes, locomotor activity was decreased in both ASIC1a−/− and ASIC2−/− mice compared with WT. We conclude: 1) ASIC1a does not contribute to baseline BP, HR, or sympathetic tone under resting conditions in conscious mice, 2) ASIC2 exerts a powerful restraining influence on sympathetic activity and BP, and 3) the ASIC2−/− mouse is a model of chronic neurogenic hypertension. (AHA 0725771Z, HL14388)
Hemodynamic responses to electrical stimulation of the aortic depressor nerve in conscious l‐name hypertensive rats
Authors: Salgado Helio Cesar , Durand Marina Toledo , Salgado Maria Cristina , Castania Jaci Airton
Published: June 2021

The hemodynamic responses to electrical stimulation of the aortic depressor nerve (ADN) were evaluated in conscious normotensive and L‐NAME hypertensive Wistar rats. In addition, it was also examined the relative role played by parasympathetic activation and sympathetic withdrawal during ADN stimulation. Hypertensive rats were obtained by means of L‐NAME dissolved in drinking water, given during 14 days. The animals were anesthetized (thiopental) for catheterization of femoral artery and vein, and placement of bipolar electrodes for ADN stimulation. Electrical stimulation of the ADN was applied during 20s (1mA, 2ms, and 5, 15, 30, 60 and 90 Hz), before and after atropine or atenolol. Hypertensive rats exhibited higher mean arterial pressure (MAP) (154±4 vs104±3 mmHg) and heart rate (HR) (394±8 vs 354±7 bpm) as compared to normotensive rats. Atenolol or atropine did not affect the hypotensive response in both groups. Atenolol attenuated (44%) the bradycardic response in L‐NAME hypertensive rats, but did not affect in normotensive rats. Atropine attenuated the bradycardic response in L‐NAME hypertensive (78%) and normotensive rats (71%). The results demonstrate a preserved baroreflex‐mediated decrease in MAP and HR in conscious L‐NAME hypertensive rats, and indicate that the withdrawal of the sympathetic drive plays a greater role in the reflex bradycardia in this hypertensive model. Support: FAPESP, CNPq.
Evaluation of the cardiovascular actions of sympathetic ganglion endothelin B receptors
Authors: Parker Lindsay M , Li Melissa , Esfahanian Mohammad , Fink Gregory D , Kreulen David L
Published: June 2021

Endothelin B receptors (ETBR) are found on blood vessels, renal tubules and sympathetic neurons and are upregulated in hypertensive celiac ganglion (CG). Chronic activation of ETBR with the selective agonist sarafotoxin 6c (S6c) causes hypertension that is attenuated in rats with celiac ganglionectomy (CGX). Thus S6c may increase blood pressure (BP) by activating ETBR on both blood vessels and CG. We evaluated the importance of ganglionic ETBR using two transgenic (TG) strains of rats: TG(+/+) rats have the normal complement of wild type ETBR (WT‐ETBR), whereas TG(sl/sl) rats lack functional WT‐ETBR; both strains have 70 copies of an ETBR transgene in sympathetic neurons. S6c infusion raised BP modestly in TG(+/+) rats and CGX blocked this effect. S6c‐induced hypertension was completely absent in TG(sl/sl) rats with or without CG. Thus, the ETBR transgene does not appear to be functional in sl/sl rats. We compared total (TG+WT) ETBR mRNA and protein levels in CG of the TG strains to that from WKY. ETBR mRNA levels were significantly greater in both TG strains compared to WKY (p<0.05, n=3). However, ETBR protein expression in CG was higher in WKY than in either TG strain. Thus, the presence of the ETBR transgene in ganglia appears to interfere with the expression of WT‐ETBR. We conclude that hypertension mediated by ETBR depends on the presence of WT‐ETBR in both sympathetic ganglia and vasculature. (Supported by PO1HL79687)
Exercise‐Induced Late Preconditioning is not Triggered by Reactive Oxygen Species
Authors: Starnes Joseph W , Taylor Ryan P , Nelson Mathew J
Published: June 2021

We tested the hypothesis that administration of the free radical scavenger N‐2‐mercaptopropionyl glycine (MPG) during exercise will prevent development of protection against ischemia‐reperfusion (I‐R) injury. Male, 4‐mo‐old, F344 rats were divided into sedentary controls (SED, n=7); sedentary plus 100 mg/kg MPG injection (SED/MPG, n=12); exercised 2 days on a treadmill at 70% VO2max for 60 min (RUN, n=7); and exercised after MPG injection (RUN/MPG, n=10). Twenty‐four hrs after the second exercise bout, isolated perfused working hearts were subjected to 22.5 min global ischemia followed by 30 min normoxic reperfusion. Left ventricle was analyzed for several putative mediators of cardioprotection including: heat shock protein 70 (HSP70); inducible (iNOS), endothelial (eNOS), and neuronal nitric oxide synthase; manganese superoxide dismutase (MnSOD); and catalase. In sedentary animals MPG did not affect recovery from I‐R (SED vs. SED/MPG, p>0.05). Both exercise groups displayed improved recovery of myocardial pump function vs. both sedentary groups (p<0.05). No changes in the activities of MnSOD and catalase, or the expression of iNOS and eNOS, were observed in any exercise group (p>0.05), and expression of HSP70 increased only in RUN and RUN/MPG (p<0.05). We conclude that production of reactive oxygen species during acute exercise does not trigger late preconditioning against I‐R injury. NIH AG022220
The carotid baroreflex is reset following prolonged exercise in humans
Authors: Hart Emma Charlotte Jane , Rasmussen Peter , Secher Niels H , George Keith P , Cable Tim , Volianitis Stefanos
Published: June 2021

Alterations in the carotid baroreflex (CBR) control of arterial pressure may explain the reduction in arterial pressure and left ventricular (LV) function observed after prolonged exercise. We examined the CBR control of heart rate (HR) and mean arterial pressure (MAP), in addition to changes in LV function, pre‐ to post exercise. Seven males (age, 29 ± 7 yrs) completed 4 h of ergometer rowing at a workload 10–15% below lactate threshold. CBR control of HR and MAP was assessed before and after exercise via the rapid neck suction/pressure method. LV systolic function was assessed by echocardiography, where ejection fraction (EF), systolic pressure/volume ratio (SBP/ESV) and stroke volume (SV) were estimated. After exercise MAP was reduced (12 ± 3%) and HR was elevated (35 ± 5%; P<0.05), indicating a relocation of the CBR operating point. Furthermore, the CBR operating pressure was relocated to the left on the HR and MAP stimulus‐response curve (P<0.05) demonstrating that the CBR operated at a lower arterial pressure. Concomitantly, EF (22 ± 2%), SBP/ESV (32 ± 14%) and SV (25 ± 5%, P<0.05) were reduced after exercise. The reduced EF and SBP/ESV were associated with the decreased MAP operating point (r2=0.71 and r2=0.47, P<0.05). In conclusion, the CBR is reset to a lower prevailing arterial pressure after prolonged exercise, which may contribute to the reduction arterial pressure and LV function observed.
Pressure Dependence of Compression‐Induced Dilation
Authors: Jasperse Jeffrey L. , Clifford Philip S.
Published: June 2021

We have recently demonstrated that mechanical compression causes dilation of soleus feed arteries. The current study examined the pressure dependence of compression induced dilation. We hypothesized that the magnitude of dilation would be directly related to the compression pressure. Rat soleus feed arteries (n=10) were isolated, cannulated, mounted on micropipettes in a sealed vessel chamber, and pressurized to 68 mmHg. Luminal diameter was measured using an inverted microscope. An electronically controlled solenoid valve system permitted delivery of accurately timed pulses of extravascular pressure at 68, 100, 300, or 600 mmHg. Arteries dilated to single, 1‐sec pressure pulses at each pressure and the dilation was similar at all compression pressures (15±2%, 16±2%, 17±2%, and 18±2% dilation respectively). Multiple pressure pulses (30 1‐sec pulses with 1‐sec intervals) produced dilations of 27±3%, 40±5%, 46±6%, and 44±6% dilation respectively (p<0.05). These data indicate that the dilation to single compressions is not dependent on compression pressure. The dilation to multiple compressions is related to compression pressure, but plateaus at pressures above 300 mmHg. Since the arteries dilated to pressures within the range of those observed during locomotory exercise, we conclude that compression induced dilation could contribute to the rapid hyperemia observed at the onset of exercise.

Supported by Seaver Research Council and the National Heart, Lung, and Blood Institute
Muscle mechanosensitive reflex is suppressed in the conscious condition: effect of anesthesia
Authors: Nakamoto Tomoko , Matsukawa Kanji , Tsuchimochi Hirotsugu
Published: June 2021

To test the hypothesis that a muscle mechanosensitive reflex is suppressed in the conscious condition, we examined the effect of anesthesia on the cardiovascular responses to passive mechanical stretch of the triceps surae muscle in six conscious cats. The triceps surae muscle was manually stretched for 30s by extending the hip and knee joints and subsequently by dorsiflexing the ankle joint; the lateral gastrocnemius muscle was lengthened by 19 ± 3 mm. Heart rate (HR) and mean arterial blood pressure (MAP) did not change significantly during passive stretch of the muscle in the conscious condition. At 10–40 min after intravenously administering sodium pentobarbital (20–25 mg/kg), the identical passive stretch of the triceps surae muscle was able to induce the cardiovascular responses; HR and MAP were increased by 14 ± 1 beats/min and 14 ± 1 mmHg, respectively. In contrast, vigorous stretching skin on the triceps surae muscle elicited no significant changes in HR and MAP in the anesthetized condition, indicating that if cutaneous nociceptors and/or mechanoreceptors might be activated by the skin stretch, pain‐induced reflex cardiovascular responses would be inhibited by anesthesia. We conclude that passive mechanical stretch of skeletal muscle is capable of evoking the reflex cardiovascular response, which is suppressed in the conscious condition but exaggerated by anesthesia. This study was supported by a Grant‐in‐Aid for Scientific Research (B) from the Japan Society for the Promotion of Science.
Contribution of intramuscular TG and plasma FFA to the long chain acyl‐carnitine pool: effects of insulin in trained and untrained humans
Authors: Kanaley Jill , Sheehan Michael , Guo ZengKui , Jensen Michael D
Published: June 2021

This study examined the plasma FFA, intramuscular long chain acylcarnitine (LCAC) fatty acids and imTG stores during basal and hyperinsulinemic conditions.

Methods:

Endurance trained (TR, n=8) and sedentary individuals (SED, n=15) were studied under overnight postabsorptive and euglycemic hyperinsulinemic clamp (1.0mU·kg FFM−1·min−1) conditions. FFA kinetics were measured using a 5‐h infusion of [U‐13C]oleate, for labeling of imTG and LCAC pools. Muscle biopsies were performed to measure imTG and imLCAC enrichment at 1400 h after 5‐h of infusion.

imTG concentrations were greater in TR than SED individuals (P<0.01).

Table

SED
Trained

Saline**
Clamp
Saline**
Clamp

plasma oleate FFA (mpe)
0.210±0.018*
0.271±0.036*
0.189±0.026*
0.361±0.050*

imLCAC (mpe)
0.025±0.003
0.005±0.002
0.016±0.005
0.002±0.002

imTG (mpe)
0.018±0.003†
0.011±0.001†
0.024±0.004†
0.013±0.001†

P<0.001 saline vs. clamp; *P<0.000 between compartment (plasma vs. imLCAC vs. imTG); †P<0.000 imLCAC and imTG

Methods:

Endurance trained (TR, n=8) and sedentary individuals (SED, n=15) were studied under overnight postabsorptive and euglycemic hyperinsulinemic clamp (1.0mU·kg FFM−1·min−1) conditions. FFA kinetics were measured using a 5‐h infusion of [U‐13C]oleate, for labeling of imTG and LCAC pools. Muscle biopsies were performed to measure imTG and imLCAC enrichment at 1400 h after 5‐h of infusion.

Conclusion:

The imTG contributes more to the imLCAC pools than plasma FFA both in the basal and hyperinsulinemic state, and hyperinsulinemia suppressed oleate FFA entry into the imLCAC pools, more so in trained than untrained adults. Muscle fatty acid oxidation is largely via obligatory cycling of fatty acids through imTG or other intracellular complex lipid pools.
Mild cold and overfeeding adaptive thermogenesis: role of mitochondrial uncoupling
Authors: Wijers Sander L.J. , Schrauwen Patrick , Saris Wim H.M. , van Marken Lichtenbelt Wouter D
Published: June 2021

Mild cold exposure and overfeeding are known to elevate energy expenditure, so‐called adaptive thermogenesis. In adult humans, mitochondrial uncoupling in skeletal muscle tissue is a candidate mechanism.

We aim to investigate whether diet induced and cold induced adaptive thermogenesis in humans are related to mitochondrial uncoupling in skeletal muscle tissue. The metabolic response to mild cold and overfeeding in 13 lean adult male subjects was measured in a respiration chamber in a baseline condition, after three days of mild cold exposure, and after three days of overfeeding. After each respiration chamber measurement a muscle biopsy was taken, in which mitochondrial uncoupling was measured by high‐resolution respirometry. The uncoupling ratio was defined as state 4 respiration (substrates + ADP + ATP‐synthase blocker) divided by state 3 respiration (substrates + ADP).

Total daily energy expenditure increased after overfeeding with 6.9% (0.79 MJ/day) and after mild cold exposure with 5.7% (0.65 MJ/day). Uncoupling ratio increased with respectively 58 and 24%. Uncoupling ratio correlated significantly with total daily energy expenditure (R2=0.533, p=0.01).

This study for the first time shows that skeletal muscle has the intrinsic capacity for adaptive thermogenesis in humans via mitochondrial uncoupling.
NADPH oxidase is critical for up‐regulation of HIF‐1[alpha] by intermittent hypoxia
Authors: Yuan Guoxiang , Khan Shakil A , Nanduri Jayasri , Semenza Gregg L , Prabhakar Nanduri R
Published: June 2021

Intermittent hypoxia (IH) increases HIF‐1α expression and HRE activation in PC12 cells (Yuan et al. J. Biol. Chem. 2005). Reactive oxygen species (ROS)‐mediated signaling plays a critical for IH‐evoked cellular and systemic responses. In the present study we examined whether ROS play a role in HIF‐1α accumulation by IH, and if so to assess the source of ROS generation. Experiments were performed on PC12 cells exposed to either 20% O2 (normoxic control) or 60 cycles of IH (30 sec at 1.5% O2 followed by 5 min at 20% O2). ROS scavengers, MnTMPyP, and PEG‐catalase prevented IH‐induced HIF‐1α accumulation. IH increased ROS generation as evidenced by decreases in aconitase enzyme activity in both cytosolic and mitochondrial fractions. Apocynin, an inhibitor of NADPH oxidase prevented IH‐induced HIF‐1α accumulation and ROS generation. IH upregulated NADPH oxidase subunits including gp91phox, p47phox, p22phox, and p67phox and increased NADPH oxidase enzyme activity. These observations suggest that ROS generated by NADPH oxidase plays an important role in IH‐induced up‐regulation of HIF‐1α. (Supported by NIH‐HL‐25830).
Doxorubicin stimulates oxidant activity, weakness and fatigue in murine skeletal muscle
Authors: Ashley Laura A. , Ferreira Leonardo F. , Smith Jeff D. , Hardin Brian J. , Noel Teresa J. , St. Clair Daret K. , Reid Michael B.
Published: June 2021

Doxorubicin is the most commonly prescribed chemotherapeutic agent. Doxorubicin causes an increase in free radical production in the myocardium and cardiac muscle dysfunction. Patients receiving chemotherapy also experience weakness and fatigue leading us to hypothesize doxorubicin (1) increases muscle‐derived oxidants and (2) depresses diaphragm and limb muscle force in mice. Doxorubicin treatment (20μM, 1hr) in vitro increased oxidant activity in diaphragm muscle as measured using DCF fluorescence (n=3, p<0.04). We injected C57B6 male mice intraperitoneally with doxorubicin (20 mg/kg), a cumulative dose commonly used in cardiotoxicity studies. Diaphragm and extensor digitorum longus (EDL) were excised three days post injection, compared to controls, doxorubicin lowered maximal force in diaphragm by 62.2 ± 7.1% (mean ± SE; n=5, p<0.01) and EDL by 28.7 ± 7.9% (n=3, p<0.03). Doxorubicin depressed diaphragm twitch characteristics, including peak force (−37.7 ± 12.5%; n=5, p<0.01) and time to peak force (Doxorubicin 0.15 ± 0.02 N s−1, Control 0.29 ± 0.02 N s−1). At the end of the fatigue trial, force was lower in doxorubicin treated mice 26.9 ± 12.2% of control values. The stability of the muscle in vitro was not altered. Our data shows that doxorubicin causes skeletal muscle weakness and predisposes muscle to fatigue, which may be due to an increase in muscle‐derived oxidants.

Supported by: HL 59878 (MBR)
Genetic profiling of wheel running exercise‐induced adaptation of mouse soleus muscle
Authors: Snoo Maartje de , Kruitwagen Cas , Cornelissen Peter , Jeneson Jeroen , Everts Maria
Published: June 2021

We previously reported improved Ca2+ handling capacity in mouse soleus muscle after 6 weeks of voluntary wheel running exercise (de Snoo et al., Faseb J, 19(4), A132, (2005)). To further investigate these results and underlying mechanisms, mRNA profiling was performed on pooled mRNA from soleus muscles isolated from animals in the running wheel (RWE; n=3) and sedentary control (SED; n=3) groups (male C57Bl/6 mice, age 10 weeks). The Affymetrix mouse 430 2.0 Genechip array was used. Data were analyzed with LIMMA (linear models of microarray), an implication in R. The top‐50 of most likely up‐ and downregulated genes were selected and clustered according to their function. Confirmation was obtained by RT‐PCR in subset of these genes. We found that genes responding to RWE fell into three major groups: (1) Ca2+ signaling, (2) Krebs cycle, and (3) cell cycle. The combination of up‐ and downregulated genes in group 1 (e.g. MARCKS↓, PLB↓, FK506BP↑) would enhance Ca2+ handling. Genes in group 2 (e.g. PDK4↓, Glut1↑) enhanced glucose metabolism and substrate oxidation (Krebs cycle). Genes in group (3) improved muscle differenti ation (e.g. MyoD↑, cyclinD1↓)
Subcontracture Depolarization Alters Doublet Potentiation in Isolated Skeletal Muscle
Authors: Barnes William Stephen
Published: June 2021

The addition of two closely spaced stimulating impulses (doublets) at the beginning of a low‐frequency train of stimuli has been shown to increase isometrictension and maximize tension‐time integral (TTI). The purpose of this investigation was to determine the combined effects of subcontracture K+‐induced depolarization, itself a powerful potentiating influence, and doublet potentiation on peak muscle tension development. Sartorii muscles (60–70 mg) from small male R. pipiens were dissected free and mounted vertically in water‐jacketed (20°C) chambers containing oxygenated (100%) amphibian Ringer's solution (pH 7.2) containing 2.5 mM KCl (R+KCl2.5). After equilibrium was achieved, muscles were exposed serially to single and paired (ISI = 20ms) supramaximal square wave pulses (1 ms) via field stimulation. Peak tension (PT) in response to paired stimulation (PTD, g/g) was greater when compared to single pulse stimulation (PTS, g/g): 202.2 ± 18.4 v. 104.8 ± 6.7, respectively. Following complete recovery, the stimulation protocol was repeated after substituting Ringer's containing 15 mM KCl (R+KCl15) for R+KCl2.5. PTS increased from 101.0 ± 4.9 (R+KCl2.5) to 152.7 ± 14.9 (R+KCl15). PTD increased from 101.0 ± 4.9 (R+KCl2.5) to 184.5 ± 18.61 (R+KCl15). Corresponding values for the TTI (g·s) mimicked results for PT. Subcontracture K+‐induced depolarization lessened the effect of subsequent paired stimulation: (+ 92.9% (R+KCl2.5) v. + 82.7% (R+KCl15)). These results suggest that alterations in extracellular environment may mitigate the potentiating effects of paired stimulation and alter it's efficacy as a motor control strategy.