bluuebunny/crossref_metadata_2025 huggingface dataset:
PP2B upregulation mediates increased NO production independent of NOS3 phosphorylation in the renal medullary thick ascending limb during diabetes mellitus
Authors: Foster Jan M , Carmines Pamela K , Pollock Jennifer S
Published: June 2021

The Ser/Thr phosphatase PP2B is known to regulate NO synthase 3 (NOS3) activity. Diabetes increases renal medullary NOS3 activity and reduces its phosphorylation (Thr495) in the medullary thick ascending limb (mTAL). We hypothesized that diabetes upregulates PP2B in the mTAL, which stimulates NO production by dephosphorylating NOS3. Three weeks after induction of diabetes (streptozotocin; STZ), immunoblotting revealed increased PP2B‐Aβ (catalytic subunit) in mTAL suspensions from STZ vs Sham rats (3.2±0.6 vs 1.7±0.1 RDU, P<0.05) and a trend for increased PP2B‐B (regulatory subunit; 0.47±0.12 vs 0.24±0.02 RDU, P=0.07). NO production by STZ mTALs was greater than Sham (977±161 vs 397±116 pmol/30min/mg prot., P<0.05) and reduced by CsA (551±67 pmol/30min/mg prot.; P<0.05 vs untreated). Although CsA decreased PP2B activity in STZ mTALs (64±14 vs 21±6 nmol PO4/mg prot., P<0.05), it did not alter NOS3 phosphorylation at Thr495, Ser1177, or Ser633 in Sham or STZ mTALs. NOS3 phosphorylation at Ser114 and Ser617 was not apparent in mTALs by immunostaining, although evident in other renal cells. We conclude that PP2B upregulation accelerates NO production by the mTAL during diabetes, but this does not involve PP2B‐mediated changes in NOS3 phosphorylation. We speculate that the PP2B‐dependent NO production may involve phosphorylation of another protein that, in turn, regulates NOS activity. (Supported by the AHA)
Functional genomics of diabetic nephropathy in a newly‐characterized rat model
Authors: McMahon K. Wyatt , Zanescu Dora I , Sehli Sharmila , Prabhakar Sharma
Published: June 2021

Diabetic nephropathy (DN) is the most common cause of end stage renal disease in the United States and approximately 40% of all diabetics eventually develop the disorder. A meta‐analysis of publicly available microarray data was performed to find genes that were consistently misexpressed in DN. From this list, those genes that also segregate with a predisposition for DN in humans were identified. Thus, a list of genes that are misexpressed both in patients and mouse models of DN, as well as linked to DN susceptibility, was prepared. This list of candidate genes, likely important for the onset and progression of DN, included vascular endothelial growth factor (VEGF), protein kinase B (Akt/Pkb), phospholipase Cγ (PLCγ), and interestingly PLCε, which is mutated in a familial form of nephrotic syndrome. These analyses, together with our recent observation of increased urinary VEGF in very early DN, suggest a central role for the VEGF pathway in DN onset and progression. The role of these genes is currently being investigated in ZSF rats, a newly‐characterized rat model for DN (Prabhakar et al., JASN, 2007). The identification of genes involved in DN pathogenesis could lead to better diagnostics and therapies for DN.

Supported by the Larry and Jane Woirhaye Foundation for renal research.
A proton‐independent function of ASIC in Ciona intestinalis
Authors: Coric Tatjana , Passamaneck Yale J. , Zhang Ping , Di Gregorio Anna , Canessa Cecilia M.
Published: June 2021

Acid‐Sensitive Ion Channels (ASICs) are neuronal‐specific voltage‐insensitive sodium channels gated by extracellular protons. ASIC functions in mammals include nociception, mechanosensation and modulation of synaptic transmission. However, the answer to what role protons play in mediating the effects of ASIC remains elusive. In this work we have examined ASIC from the ascidian Ciona intestinalis: a simple chordate organism with larval nervous system highly similar to the one of higher vertebrates. We show that Ciona has a single ASIC gene that gives rise to two spliced forms. CiASIC is expressed in most neurons of the larva, but absent in the adult nervous system. Despite sequence similarity with mammalian ASICs, CiASIC is proton‐insensitive when examined in heterologous systems or in larval neurons; the latter rules out the possibility that proton‐sensitivity is conferred by particular factors present only in Ciona neurons. Downregulation of CiASIC transcript by dsRNA disrupted the regular pattern of larval swimming, implying that proton‐independent mechanisms mediate the effects of ASIC in vivo. Together the data identify ASIC as a channel distinctive of chordate nervous system, and show that protons are not essential for ASIC function.

This work was supported by NIH RO1DK054062.06A1 and AHA GIA grants to CC, NIH/NICHD R01HD050704 to ADG and APS Postdoctoral Fellowship in Physiological Genomics to TC.
Using the nematode C. elegans to assay the effectiveness of novel anti‐nociceptive compounds targeting transient receptor potential vanilloid 1 (TRPV1) channels
Authors: Hamilton Kadine L. , Chapp Andrew D. , French Larry G. , Estevez Ana Y.
Published: June 2021

TRPV1 channels are expressed in nociceptive neurons and are involved in sensing noxious thermal and chemical stimuli. Because of their roles in the pain pathway, these channels have emerged as important therapeutic targets for the development of new analgesics. Capsaicin, the pungent ingredient in hot chili peppers, is a known agonist of TRPV1. Transgenic C. elegans nematodes expressing rat TRPV1 in sensory neurons exhibit a reversal response upon exposure to capsaicin. Pre‐incubation of the worms with capsazepine, a selective TRPV1 antagonist, potently blocks their response to capsaicin. Thus, we are using capsaicin‐induced reversal behavior as an assay to test the effectiveness of novel TRPV1 ligands. Data on the effectiveness of N‐cyanoguanidine analogs of capsaicin, capsazepine, SB 366791 and AMG 9810 will be presented.
Renal lipid accumulation and mitochondrial function in leptin deficient and melanocortin‐4 receptor deficient obese mice
Authors: Brandon Elizabeth , do Carmo Jussara M. , da Silva Alexandre A. , Hosler Jonathan P. , Hall John E.
Published: June 2021

We used two genetic mouse models of obesity to test the hypotheses that increased levels of leptin may protect against renal lipid accumulation and mitochondrial dysfunction in obesity. Studies of 26–30 week old leptin deficient obese mice (ob−/−) and melanocortin 4 receptor deficient obese mice (MC4R−/−), which have high leptin levels (26.2 ± 3.4 ng/mL leptin), revealed higher levels of lipid accumulation in the glomeruli and cortical tubular epithelia of ob−/− mice by oil red‐O staining. Ob−/− mice also had increased renal triglycerides (2.3 ± 0.03 v. 0.9 ± 0.2 mg TG/g kidney, n=3), albumin excretion (61.7 ± 8.4 v. 15.6 ± 1.6 μg/24 hrs, n=4), and a 47% increase in hyalinization of glomerular arterioles, compared to MC4R−/− mice. In addition, mitochondria from ob−/− kidneys had lower rates of oxygen consumption (67.6 ± 1.12 v. 89.2 ± 1.07 nmol O2/min/mg) and respiratory control ratios (2.5 ± 0.26 v. 3.6 ± 0.23). These data suggest that high levels of leptin may protect against renal lipid accumulation and lipotoxicity in obesity. Although the mechanisms for this protection are still unclear, increased mitochondrial oxidation of fatty acids could play a role, since leptin deficient mice have reduced mitochondrial O2 consumption compared to obese mice with high leptin levels. (PO1 HL51971and F32 DK 080591).
Plasma adiponectin concentration is strongly associated with VLDL‐TG catabolism in postmenopausal women
Authors: Lapointe Annie , Tchernof André , Lamarche Benoit , Piché Marie‐Ève , Weisnagel John , Bergeron Jean , Lemieux Simone
Published: June 2021

Objective: To investigate associations between plasma adiponectin concentration and VLDL‐TG secretion and catabolism in 30 postmenopausal women.

Methods: Plasma adiponectin concentration was measured by ELISA. Insulin sensitivity was assessed by a 2‐h euglycemic‐hyperinsulinemic clamp. Fasting plasma glucose (FPG) and 2‐hour plasma glucose (2hPG) were measured during an OGTT. The VLDL‐TG total secretion rate (TSR) and the VLDL‐TG fractional catabolic rate (FCR) were measured by electron impact ionization gas chromatography‐mass spectrometry. The MIDA approach was used to measure VLDL‐TG TSR following a 10h‐infusion of [1‐13C] acetate and the calculation of VLDL‐TG FCR was based on the monoexponential decrease of TG‐[2H5] glycerol values obtained following the administration of a 2H5‐glycerol bolus.

Results: Plasma adiponectin concentration was negatively associated with VLDL‐TG TSR (r=−0.42; p<0.02) and positively with VLDL‐TG FCR (r=0.60; p<0.0005). This latter association remained significant after statistical adjustments for insulin sensitivity, HDL‐C, TG, FPG and 2hPG. In a multivariate model, plasma adiponectin level was the best predictor of VLDL‐TG FCR.

Conclusion: Elevated plasma adiponectin concentration is associated with favourable VLDL‐TG metabolism. This study was supported by the Canadian Institutes of Health Research and the Heart and Stroke Foundation of Canada.
Glucagon‐like peptide‐1 receptor is a regulator of glucose clearance in oxidative tissues independent of insulin
Authors: Ayala Julio , Bracy Deanna , Wasserman David , Drucker Daniel
Published: June 2021

Activation of the glucagon‐like peptide‐1 receptor (GLP‐1R) stimulates insulin secretion. GLP‐1R also plays a role in the control of glucose homeostasis via mechanisms that are independent of this incretin action. Exercise was used to unmask glucoregulatory properties of GLP‐1R related to increased insulin‐independent glucose flux. Wild type (WT) and GLP‐1R knockout (KO) mice (n=7–8) were catheterized in the carotid artery (sampling) and jugular vein (infusion). 7 days later, 5h fasted mice underwent treadmill exercise (30 min @ 16.6 m/min) or remained sedentary. Fasting glucose (mg/dL) was higher in KO than WT (233±6 vs 198±9) and rose with exercise in KO but not WT (263±16 vs 201±11). Tissue glucose clearance (Kg μl/gtissue/min) was determined using 2[3H]deoxyglucose. Sedentary heart Kg was higher in KO than WT (84±17 vs 48±11) and did not increase further with exercise (ΔKg 6±14 vs 76±19). Sedentary diaphragm Kg was equal in WT and KO and increased with exercise in KO but not WT (ΔKg 29±10 vs 2±5). Insulin levels were equal in both groups. Oxygen consumption (VO2, ml/kg/min) was equal in WT and KO at rest but was higher in KO during moderate (75% VO2max 103±3 vs 121±4) and maximal (VO2max 132±4 vs 141±2) exercise. In summary, deletion of GLP‐1R leads to a) elevated sedentary cardiac glucose uptake to levels observed in exercised WT mice and b) increased diaphragm glucose uptake and higher VO2 during exercise. Thus exercise studies reveal the insulin‐independent glucoregulatory properties of GLP‐1R. Supported by DK50277.
Acute hyperglycemia decreases renal sympathetic nerve activity in conscious rats
Authors: Huber Domitila Augusta , Fazan Rubens , Salgado Helio Cesar
Published: June 2021

The effect of hyperglycemia on arterial pressure (AP), renal sympathetic nerve (RSN) activity and baroreflex control of RSN activity was investigated in conscious rats. The rats were submitted to surgical procedures under tribromoethanol and the experiments were conducted 6 to 8 hours after the recovery of anesthesia. Intravenous bolus (0.25 mL) administration of glucose 50% (n=10), or mannitol 30% (n=7), followed by continuous infusion (2 mL/h) during 30 min was carried out. RSN activity was evaluated during spontaneous activity (% basal) and by means of pressure‐nerve activity curve normalized as maximum activity (% max) as well. The infusion of glucose increased blood glucose (127±4 to 319±8 mg/dL) and plasma osmolarity (300±1 to 317±2 mOsm/kgH2O). The infusion of Mannitol increased the plasma osmolarity (301±2 to 313±3 mOsm/kgH2O) without affecting blood glucose (123±4 to 124±5 mg/dL). Glucose did not affect basal AP (108±1 to 106±2 mmHg) but decreased RSN activity (24±5 %). In addition, glucose reduced the range (103±2 to 82±6 % max) and gain (−3.43±0.25 to −2.59±0.30 % max/mmHg) of RSN activity, obtained by means of pressure‐nerve activity curve. Mannitol did not affect any parameter studied. In conclusion, the present study demonstrate, in conscious rats, that acute hyperglycemia promote reduction of spontaneous RSN activity and baroreflex control of RSN as well. Financial Support: CAPES, FAPESP, CNPq.
Non‐NMDA receptor activation in the Nucleus Tractus Solitarius (NTS) increases ventilatory drive
Authors: Carr J. Austin , Fu Zhenxing , Powell Frank L.
Published: June 2021

Previous studies showed an important role for N‐methyl‐D‐aspartate (NMDA) receptors but not non‐NMDA receptors in the NTS in the hypoxic ventilatory response (HVR) of awake rats. We hypothesized that plasticity of non‐NMDA receptors in the NTS is necessary for ventilatory acclimatization to hypoxia, which increases normoxic ventilatory drive and the acute HVR. To test this, we microinjected artificial CSF or NBQX in the NTS (4 mM in 50 nL, bilaterally) of normoxic control rats (CON, n=7) and chronically hypoxic rats (CH, n= 3, PIO2 = 70 Torr for 7 days) and used barometric pressure plethysmography to measure ventilation. Microinjection sites were identified by colloidal gold microinjections after the experiment. Ventilation (ml/(kg min) was (mean + sem):

Non‐NMDA receptor blockade significantly (P<0.05) decreased ventilation under all conditions with no significant interactions between drug and treatment or inspired O2. Ventilatory changes resulted from significant effects on breathing frequency but not tidal volume. Hence, non‐NMDA receptors in the NTS are important for ventilatory drive under normoxic and hypoxic conditions but they are not solely responsible for ventilatory acclimatization to hypoxia.

Supported by R01‐HL081823 (FLP) and The American Physiological Society (JAC).
Modulation of muscle mechanosensitive reflex in humans: comparison of the cardiovascular responses to passive cycling between the awake and sleep conditions
Authors: Matsukawa Kanji , Kashima Eri , Tsuchimochi Hirotsugu , Nakamoto Tomoko , Endo Kana , Sadamoto Tomoko
Published: June 2021

We have recently reported that mechanical stretch of skeletal muscle in cats is capable of evoking the reflex cardiovascular responses, which are suppressed in the conscious condition but exaggerated by anesthesia. To test the hypothesis that the reflex cardiovascular changes during passive limb movement in humans are also suppressed in the awake condition, the cardiovascular responses to supine passive leg cycling were compared between the awake and sleep conditions in 9 subjects. In the awake condition, heart rate (HR), stroke volume (SV), cardiac output (CO) increased slightly in response to passive cycling for 30 s at 60 rpm and total peripheral resistance (TPR) decreased slightly. Mean arterial blood pressure (MAP) did not change during passive cycling. In contrast, the increases in HR and CO and the decrease in TPR during passive cycling were largely enhanced during sleep in the daytime, whereas the changes in SV and MAP were not altered. Furthermore, to study the effect of overnight sleep on the muscle mechanoreflex, the HR response to passive cycling were repeatedly examined at various non‐REM and REM sleep. The increase in HR during passive cycling was augmented during non‐REM sleep, depending on the sleep depth. It was interesting that the increase in HR was peaked during REM sleep. It is concluded that a muscle mechanoreflex induced during passive cycling is suppressed in the awake condition but exaggerated by non‐REM and REM sleep in humans.
Voluntary exercise training attenuates the cardiovascular and brain responses to severe hemorrhage in conscious rats
Authors: Ahlgren Joslyn K. , Hayward Linda F.
Published: June 2021

Exercise training has been shown to be beneficial for various cardiovascular pathologies. This study was performed to test the hypothesis that chronic exercise is also beneficial to cardiovascular function during severe hemorrhage (HEM). Adult male rats were pair‐housed in cages with running wheels (n=6; exercise [EX] group) or without (n=6; sedentary [SED] group). After 6 weeks, all rats underwent a 15‐min HEM during which 30% of the estimated total blood volume was removed. Mean arterial pressure (MAP) and heart rate (HR) were recorded. 90 min post‐HEM, animals were anesthetized and transcardially perfused and brains processed for c‐Fos immunoreactivity. No statistical difference in HR was seen between groups during HEM. In contrast, MAP in SED rats dropped to 47% of baseline following HEM, while EX rats dropped only 25% (P<0.01). Several subnuclei within the lateral parabrachial nucleus (LPBN), a region known to modulate the response to HEM, displayed significantly less Fos‐positive cells in EX compared to SED animals. These data demonstrate that voluntarily active rats are better able to defend blood pressure during HEM, attenuating the severity of the cardiovascular decline associated with hypotensive shock. Furthermore, the benefits provided by exercise may have a central component involving LPBN area neurons. AHA‐53426 (LFH) & 65810 (JKA)
Exercise pressor reflex dysfunction in hypertension: a role for nitric oxide synthase (NOS) within the nucleus tractus solitarius (NTS)
Authors: Leal Anna K , Newcomb Richard , Squiers Jack , Smith Scott A
Published: June 2021

An overactive exercise pressor reflex (EPR; a circulatory reflex originating in skeletal muscle) mediates, in part, the exaggerated cardiovascular response to exercise in hypertension. Evidence suggests that decreases in the production of nitric oxide (NO) within the NTS of the medulla oblongata contribute to this EPR dysfunction. The mechanism by which NO production is reduced in the NTS remains unclear. NO is produced by the oxidation of l‐arginine by NOS. Thus, we hypothesized that reductions in NO production are due to the decreased expression of the neuronal (nNOS), endothelial (eNOS) and inducible (iNOS) isoforms of NOS within the NTS. Male, 20 week old, normotensive Wistar Kyoto (WKY; n=3) and spontaneously hypertensive (SHR; n=3) rats were anesthetized and transcardially perfused with 4% paraformaldehyde. Medullary brainstem tissue was harvested and interrogated for NOS protein expression using standard immunohistochemical techniques. Analyses determined that nNOS, eNOS and iNOS expression was markedly reduced in the NTS of SHR as compared to WKY. Although not direct evidence, these findings support the contention that decreases in NO production in hypertension may be due to reductions in NOS expression within the NTS. As such, decreases in NOS protein expression within the NTS may contribute significantly to the development of EPR dysfunction in hypertension. Supported by AHA SDG‐0735355N.
Postexercise cardiac hemodynamics in endurance‐trained men
Authors: Lynn Brenna Meaghan , Barrett‐O’Keefe Zachary , Lorenzo Santiago , Halliwill John R.
Published: June 2021

Initial observations in moderately fit endurance‐trained men have shown decreases in cardiac output with no change in systemic vascular conductance during postexercise hypotension, in contrast to other populations. As inadequate hydration could have been a cause for the differences, we decided to study a larger group of highly fit endurance‐trained men and to see if cardiac output is maintained when hydration status is standardized. We hypothesized that cardiac output would be increased postexercise under these circumstances. We studied 18 highly fit endurance‐trained men (63.8 ± 9.2 mL·kg−1·min−1 VO2peak). Mean arterial pressure, cardiac output, heart rate, and body weight were measured before and 40 min after 60 min of exercise at 60% VO2peak during which 15ml·kg−1 of water was consumed. Vascular conductance was calculated as flow/pressure. Following exercise, mean arterial pressure decreased (Δ −2.8 ± 1.3%; P = 0.034) but heart rate (Δ 24.2 ± 3.7%; P < 0.001), cardiac output (Δ9.6 ± 3.2%; P = 0.013), and systemic vascular conductance (Δ12.8 ± 3.2%; P = 0.001) increased. However, despite standardizing hydration, body weight still decreased (Δ −1.1 ± 0.1%; P < 0.001). Thus, partial maintenance of hydration status may be sufficient to prevent postexercise reductions in cardiac output in highly fit endurance‐trained men. Funding by AHA 555623Z and The Evonuk Foundation.
POTENTIAL BENEFIT FROM A H1‐ RECEPTOR ANTAGONIST ON POSTEXERCISE SYNCOPE IN THE HEAT
Authors: McCord Jennifer Lockwood , Pellinger Thomas K , Lynn Brenna M , Halliwill John R
Published: June 2021

H1‐receptors mediate the first 30 minutes of postexercise hypotension. Immediately after exercise, syncope can occur due to an exaggerated form of postexercise hypotension. Therefore, we hypothesized that orthostatic hypotension occurring immediately after exercise would be attenuated with a H1‐receptor antagonist. We studied 15 endurance exercise trained men and women in an environmental chamber set at 35 °C and 30% humidity. Subjects were studied in the supine position prior to a 45 minute bout of treadmill running at 50% of VO2max. Immediately after exercise, measurements were taken in the supine position before the subjects were moved into a 60° head‐up tilt. Measurements included arterial pressure and brachial blood flow on a control and a H1‐receptor antagonist (blockade) day. Mean arterial pressure was reduced 1 minute into the tilt compared to preexercise values on the control day (76.2 ± 0.5 vs 74.2 ± 0.5 mmHg; P < 0.05). This reduction was not seen on the blockade day (75.2 ± 0.3 vs 75.0 ± 0.5 mmHg; P > 0.41). There were no differences in brachial vascular conductance (calculated as flow/pressure) in response to the head‐up tilt between the study days (P > 0.23). When comparing the length of the head‐up tilt between study days, the blockade lengthened the mean tilt time by 94 seconds (P = 0.098). These data suggest that a H1‐receptor antagonist could potentially benefit postexercise syncope in a hot environment.
Chronic sustained and intermittent hypoxia reduce expression of ATP‐sensitive K+ (K‐ATP) channel subunits in the caudal NTS
Authors: Carreno Flavia R. , Zhang Weirong , Cunningham Tom , Mifflin Steve
Published: June 2021

Tissue hypoxia induces a K‐ATP channel mediated outward current in many brainstem neurons which is considered to serve a neuroprotective function. K‐ATP channels are made up by potassium channel subunits (Kir6.1 or Kir6.2) and sulphonylurea receptor subunits. The K‐ATP channel pore is formed by Kir6 subunits. The impact of chronic exposure to either normoxia (NORM), sustained hypoxia (SH; 7 days at 10% FIO2) or intermittent hypoxia (IH; alternating 3 min periods of 21% O2 with 10% O2 for 8 hr/day, 7 days) on the expression of the K‐ATP channel subunits was examined in NTS dissected from 1mm thick sections caudal to calamus. Tissue was homogenized by sonication in modified RIPA buffer plus protease inhibitors and 5μg of the total lysate was subjected to Western Blot analysis for both Kir6.1 and Kir6.2. Densitometric analysis showed that in the caudal NTS the ratio of Kir6.1:actin was .34 ± .06 in NORM rats (n=4) and this ratio was reduced to .13 ± .01 in SH rats (n=3, p<.05) and to .08 ± .01 in IH rats (n=4, p<.01). The ratio of Kir6.2:actin was .38 ± .15 in NORM rats (n=4) and this ratio was reduced to .20 ± .01 in SH rats (n=3, p<.01) and to .13 ± .01 in IH rats (n=4, p<.001). These results suggest that the reduced K‐ATP current observed during tissue hypoxia in 2nd order chemoreceptor neurons in NTS of SH rats (Zhang & Mifflin, presented at this meeting) could be the result of a reduced expression of K‐ATP channels. Supported by HL41894 and HL62579.
ACTIVATION OF NADPH‐OXIDASE BY 5‐HT MEDIATES SENSORY LTF OF THE CAROTID BODY BY CHRONIC INTERMITTENT HYPOXIA
Authors: Peng Ying‐Jie , Yuan Guaoxiang , Kumar Ganesh , Deneris Evan , Prabhakar Nanduri R
Published: June 2021

We previously reported that exogenous 5‐HT evokes sensory long‐term facilitation (sLTF) of the carotid body via activation of NADPH oxidase (Peng et al. J.Physiol. 576: 2006). In the present study, we examined the role 5‐HT and NADPH oxidase in chronic intermittent hypoxia (CIH) evoked sLTF of the carotid body. Experiments were performed on anaesthetized adult rats and mice exposed to normoxia or CIH. Sensory activity was recorded from in vivo and/or ex vivo carotid bodies. Acute hypoxia facilitated 5‐HT release from CIH but not from the control rat carotid bodies. CIH evoked sLTF in wild type but not in Pet‐1−/−mice with impaired 5‐HT expression in carotid body glomus cells. Ketanserin, a 5‐HT2 receptor antagonist prevented CIH‐evoked sLTF. CIH activated protein kinase C (PKC) and NADPH oxidase in the carotid bodies, and ketanserin prevented these effects. Bis‐1 (1μM, a PKC inhibitor), as well as DPI (3μM, a NADPH oxidase inhibitor) attenuated or abolished CIH‐evoked sLTF in rats. The results demonstrate that a) CIH facilities 5‐HT release from the carotid body and b) CIH‐induced sLTF requires PKC dependent activation of NADPH oxidase by 5‐HT2 receptors (Supported by NIH‐HL‐25830).
Adiponectin is Expressed in Skeletal Muscle and Influences Muscle Phenotype and Function
Authors: Krause Matthew Paul , Liu Ying , Vu Vivian , Chan Lawrence , Riddell Michael C , Sweeney Gary , Hawke Thomas J
Published: June 2021

Adiponectin (Ad) plays a vital role in various diseases and generally mediates anti‐diabetic and anti‐inflammatory effects. It was originally thought that Ad expression was limited to adipocytes, however, recent evidence has demonstrated its expression in other cells/tissues such as cardiac muscle. We present evidence that Ad is expressed in L6 myotubes and mouse soleus, as assessed by RT‐PCR and Western blot analysis. Furthermore, Ad was detected within the muscle fibers of wild‐type (WT) mouse tibialis anterior sections via IHC. Subsequently, we employed the Ad‐null (KO) mouse to determine the effect of Ad on muscle phenotype and function. Body mass increased significantly in KO mice (+5.5 ± 2.9% relative to WT) with no change in muscle mass. Epidydymal fat mass appeared to increase in KO mice (+35.1 ± 22.8%) vs. WT mice but was not statistically significant (p=0.16). Intramuscular triglyceride content was significantly greater in KO mice (+75.1 ± 25.1%) than WT. Muscle fiber type composition did not change, though there was a trend for an increase in muscle fiber area in KO mice (type IIA +13.6 ± 3.2%, IIB +25.5 ± 5.7%, IID +12.3 ± 12.7%) vs. WT. Using in situ electrical muscle stimulation, peak tetanic force was lower in KO mice (−47.5 ± 6.0) than WT with no change in muscle fatigue rates. Future studies will assess if muscle‐derived and circulating Ad elicit differential effects on skeletal muscle phenotype and function.
Effects of combined strength and endurance training on the expression of leptin receptors in human skeletal muscle
Authors: Olmedillas Hugo , Fuentes Teresa , Delgado‐Guerra Safira , Guadalupe‐Grau Amelia , Bernales Oscar , Dorado Cecilia , Santana Alfredo , Guerra Borja , Calbet Jose Al
Published: June 2021

Purpose. To determine if a sustained reduction in of plasma leptin concentration causes an alteration in the protein expression level of its receptors in human skeletal muscle.

Methods. Fifteen males ((mean ± SD), age: 35 ±5.1 years; height: 176 ±3.6 cm; body mass: 79.2 ± 7.4 kg and percentage of body fat 22.6 ± 5.4%) participated in a 12‐week weightlifting training program. Pre and post‐training muscle biopsies were obtained from the m. vastus lateralis to determine the expression of the leptin receptor (OB‐R) protein isoforms by Western blot (J Appl Physiol. 2007 102:1786–92). Body composition was determined by DXA.

Results. Training resulted in lower percentage of body fat was (from 22.5 ± 5.4 to 21.1 ±3.7 %, P = 0.06) and leptin concentration (from 9.0±7.7 to 5.7±3.7 ng/mL, P<0.05). The changes in leptin serum concentration correlated with the changes in the percentage of body fat mass (r = 0.66, p<0.05). However, non‐significant changes were observed in the expression level of the 170KDa isoform of the LR (0.77±0.2 vs. 0.85±0.5, P = 0.499) nor in the 128KDa (0.50±0.57 vs. 0.68±0.72, P=0.372) and 98KDa isoforms (1.75±1.27 vs. 1.76±2.08, P= 0.984) in skeletal muscle.

Conclusion. Skeletal muscle OB‐R expression is not modified by strength training, despite a reduction in fat mass and serum leptin concentration. These findings are compatible with increased leptin sensitivity after strength training.
Divergent myofibrillar protein synthesis and signalling in skeletal muscle in response to light and heavy resistance exercise loading
Authors: Holm Lars , van Hall Gerrit , Rose Adam J , Doessing Simon , Richter Erik A , Kjaer Michael
Published: June 2021

Objective. By standardizing exercise volume (intensity times repetitions), we investigated the isolated effect of concentric muscle contraction intensity on the anabolic response in the working skeletal muscle. One leg performed a light resistance exercise (LE) load at 15.5% of 1 repetition maximum (1RM) and the other leg a heavy resistance exercise (HE) load at 70% of 1RM.

Methods. In the overnight fasted state a primed, continuous infusion of 1‐13C‐leucine was initiated. Before exercise intervention with LE and HE intensities resting biopsies were obtained from vastus lateralis (VL) muscle. At 0:30, 3:00, and 5:30h post exercise biopsies were taken bilaterally from VL.

Results. LE resulted in a moderate elevation in FSR (0.11±0.01%/h, P<.05) in the early post exercise phase (0:30‐3h), and decreased thereafter towards basal values. The HE resulted in a marked increase in FSR only in the late phase (3–5:30h) after exercise (0.13±0.02%/h) compared to rest (P<.01), early HE (P<.05), and late LE (P<.10). The s6kinase(t421/s424) was markedly hyper‐phosphorylated (h‐phos) in HE at 0:30h compared with rest and LE (P<.001). The 4E‐BP1(t37/46) was h‐phos weakly in LE at 0:30h but markedly (P<.01) h‐phos following HE at 3:00h compared with rest and 0:30h.

Discussion. The temporal changes in myofibrillar FSR following LE and HE seem to be more related to the h‐phos of 4E‐BP1(t37/46) than to the s6kinase(t421/s424).
Endothelial cell proliferation is dependent on Nox4‐containing NADPH oxidases whereas Nox2 is anti‐apoptotic
Authors: Selemidis Stavros , Peshavariya Hitesh , Jiang Fan , Dusting Gregory J , Sobey Christopher G , Drummond Grant R
Published: June 2021

Endothelial cells produce reactive oxygen species (ROS) via NADPH oxidase complexes, which mediate signaling for proliferation, migration and cell death. We examined the roles of the Nox2‐ and Nox4‐containing NADPH oxidases in proliferation and survival of human microvascular endothelial cells (HMEC‐1). Superoxide and ROS production as well as Nox4 protein expression, but not Nox2 protein, were higher in proliferating than in quiescent HMEC‐1. Incubation of proliferating HMEC‐1 with diphenyleneiodinium, tiron (ROS scavenger), or Nox2‐ or Nox4‐specific siRNA, reduced ROS production and cell number. During proliferation, phosphorylation of ERK1/2 and Akt was increased. Inhibition of Nox4‐derived ROS production reduced both ERK1/2 and Akt phosphorylation, whereas inhibition of Nox2 only reduced the Akt phosphorylation. Furthermore, suppression of Nox2 but not Nox4 caused activation of caspase 3/7. In endothelial cells, Nox4‐derived ROS promote ERK1/2 and Akt signaling and proliferation, whereas Nox2 activity increases Akt and caspase 3/7 and prevents apoptosis. Isoform‐selective modulation of NADPH oxidase could be a useful therapeutic approach for either blocking or stimulating angiogenesis, as required for regenerative repair of ischemic or damaged tissues.
Oscillatory and pulsatile shear stress differentially regulate mitochondrial redox state via JNK activation
Authors: Ai Lisong , Hamilton Ryan T , Khalsa Bhavraj , Darbandi Farhad , Bressler Steven , Hsiai Tzung K
Published: June 2021

Oscillatory shear stress (OSS) induces oxidative stress in regions of arterial bifurcations or curvatures, whereas pulsatile shear stress (PSS) confers an opposite effect in the straight regions. Shear stress is known to activate JNK that plays an important role in up‐regulation of adhesion molecules. It remains unknown whether OSS and PSS differentially induce mitochondrial reactive oxygen species (ROS) via JNK in BAEC. Confluent BAEC were subjected to the flow conditions that simulated carotid bifurcation: (1) Control under static conditions (2)PSS; and (3)OSS. In response to OSS, JNK‐2 phosphorylation peaked at 30 minutes. In response to PSS, JNK‐2 phosphorylation peaked at 3 hours. Both OSS‐ and PSS‐induced JNK activation was significantly attenuated in the presence of a JNK inhibitor. Confocal microscope revealed that phosphorylated JNK‐2 colocalized with mitochondria in response to OSS at 30 minutes and to PSS at 2 hours. An increased mitochondrial O2− production was demonstrated. This merged intensity was significantly attenuated in the presence of a JNK inhibitor. We demonstrate that PSS and OSS differentially modulate mitochondrial ROS generation via JNK activation and its colocalization with mitochondria. Shear stress‐induced JNK may play a role in the metabolic regulation of mitochondria with relevance to endothelial dysfunction.
Electron‐dense vesicles in the endothelium of human extremity veins
Authors: Gõsi Gergely , Raffai Gábor , Fehér Erzsébet , Acsády György , Monos Emil
Published: June 2021

Secretory type electron‐dense vesicles containing vasoactive agents (endothelin, PDGF) were found in the endothelium of rat saphenous vein in high frequency, that decreases after long‐term orthostatic stress (Raffai et al. J Vasc Res 42(2), 157–64 (2005)). The purpose of this study was to identify these structures and to measure their quantity in human extremity veins that might play a role in gravitational adaptation via altering vascular tone, vascular permeability, and growth. Veins were collected from twelve patients, who underwent vascular surgery (upper/lower extremity n=5/7). Digitalized electron micrographs were used to measure vesicular density. Considering all vein samples, the microvesicular area (relative to the cross‐sectional area of the endothelial cells) was 4.5±1.36%. As our preliminary observations show, the amount of electron‐dense vesicles in the samples of the upper extremity was 2.30±0.88%, while that of the lower extremity was 6.07±2.11% (n.s.). Similarly to our observations, a substantial amount of secretory type microvesicles can be found also in the endothelium of human extremities. The difference, seen as a tendency, in vesicle content of upper and lower extremities suggests their possible role in orthostatic adaptation. There might be dependence between vesicle content and the orthostatic lifestyle. (Research grants: OTKA T‐042670/2003, ETT 128/2006, and TP‐163/2005)
Peroxiredoxin 1 is Upregulated by Laminar Shear Stress via Nrf2 Transcription Factor
Authors: Mowbray Amy Leigh , Chen Xi‐Lin , Kang Sang Won , Rhee Sue Goo , Jo Hanjoong
Published: June 2021

Endothelial cells (EC) exposed to oscillatory shear stress (OS) experience oxidative stress as a signature of atherosclerosis. Conversely, unidirectional laminar shear stress (LS) reduces reactive oxygen species (ROS) production and subsequent inflammatory responses. LS induces many protective genes via a shear‐sensitive antioxidant response element (ARE). We showed that the antioxidant enzyme peroxiredoxin 1 (PRX 1) is upregulated intracellularly by chronic LS, compared to OS and static (ST) culture. In addition, PRX 1‐knockdown attenuated LS‐dependent ROS reduction in EC. We hypothesize that shear‐dependent PRX 1 expression is regulated by ARE through the transcription factor nuclear factor (erythroid‐derived 2)‐related factor 2 (Nrf2). Quantitative, real‐time PCR indicated that chronic LS increased PRX 1 mRNA, compared to OS and ST. Immunoblots also showed increased PRX 1 protein expression in response to Nrf2 adenovirus infection of EC. These results indicate that shear‐dependent PRX 1 expression is regulated at the transcriptional level, and Nrf2 may play an important role in this mechanism. Through this pathway, PRX 1 acts as a mechanosensitive antioxidant implementing anti‐inflammatory and anti‐atherogenic effects of shear stress.

Supported by NIH HL71014, HL75209, and HL70531 (HJ), Korean Ministry of Science & Technology FPR05C2‐510 (SWK), and AHA pre‐doctoral fellowship (ALM).
Lipid Rafts‐Mediated Clustering and Activation of CD38 during M1‐Receptor Stimulation in Bovine Coronary Arterial Myocytes
Authors: Jia Sujie , Jin Si , Yi Fan , Zhang Fan , Xia Min , Li Pin‐Lan
Published: June 2021

CD38 is an ADP ribosylcyclase that mediates intracellular Ca2+ signaling via cADP‐ribose (cADPR). The activation mechanisms of CD38 remain unknown. The present study tested a hypothesis that lipid rafts (LRs) contribute to clustering and activation of CD38 in response to M1 receptor stimulation in bovine coronary arterial myocytes (CAMs). By confocal microscopy, oxotremorine (Oxo), a M1‐agonist, was found to increase LRs clustering on the membrane of CAMs with an aggregation of CD38 and LRs components, acid sphingomyelinase and ceramide in LR clusters. At 80 μM, Oxo increased LRs clustering by 78.8%. This effect was abolished by LR disruptors, methyl‐ß‐cyclodextrin (MCD) and filipin, respectively. Using FRET technique, about 17% energy transfer rate (vs. 5% of control) between CD38 and LRs component, GM1 was detected, further confirming the proximity of both molecules. In the presence of MCD or filipin, there were no FRET signals detected. By membrane microdomains flotation, CD38 was significantly increased in LR microdomains in CAMs treated by Oxo, which was blocked by MCD or filipin. HPLC analysis also demonstrated that MCD and filipin substantially attenuated Oxo‐induced production of cADPR via CD38 in CAMs. These results suggest that LRs clustering is crucial for Oxo‐induced activation of CD38 in CAMs which may mediate cADPR signaling response to M1‐receptor activation (Supported by NIH Grants HL‐57244, HL‐75316, and DK54927).
Conditional Overexpression of Cardiac‐Specific PI3Kα Improves Basal Myocardial Function
Authors: Yano Naohiro , Zhao Ting C , Tseng Andy , Padbury James F , Tseng Yi‐Tang
Published: June 2021

We have shown that PI3K signaling is highly regulated during cardiac development with the highest levels found during the fetal‐neonatal transition period and the lowest in adult. Transgenic models have established overexpression of the PI3K signaling results in increased organ size. The effects on proliferation, however, have not been carefully examined in vivo. To study the effects of PI3K on cardiomyocyte proliferation, we have engineered a tet‐off conditional transgenic mouse model to control the overexpression of cardiac‐specific PI3Kα. Newborn and adult transgenic mice were allowed to overexpress PI3Kα for two weeks and cell proliferation and basal myocardial functions were assessed, respectively. Basal cardiac function was assessed with the Langendorff perfusion system. Cell proliferation was measured with Ki‐67/troponin T double‐labeling immunohistochemistry.

Overexpression of cardiac PI3Kα, Akt and p70S6K was confirmed and was associated with an increase in cyclin D1 but not cyclin D3 levels. Adult transgenic mice had better myocardial performance including LV developed pressure, LV dP/dt max and LV dP/dt min. Heart rate and heart/body weight ratio were not changed. Overexpression of PI3Kα was also associated with an increase in cardiomyocyte proliferation in newborns. Hence, cardiac PI3K signaling can be conditionally overexpressed in postnatal stages when there is normally very limited PI3K activity. The new transgenic model can be used to investigate the mechanisms underlying regulation of cardiomyocyte proliferation during development and cardiac remodeling following injury. (NIH 1 P20 RR018728)
Effects of Spinal Cord Stimulation with different frequencies on blood flow in the rat hind paw
Authors: gao jie , wu mingyuan , qin chao , farber jay p , Linderoth Bengt , foreman robert d
Published: June 2021

Spinal cord stimulation (SCS) at a frequency of ∼50 Hz is used to improve peripheral blood flow. However, it is unclear whether SCS at higher frequencies induces further increases vasodilation and enhances clinical efficacy. This study investigated effects of SCS at different frequencies on peripheral vasodilation. SCS was setup at the lumbar 2–3 spinal cord segments in rats. Cutaneous blood flows from both ipsilateral and contralateral hind foot pads were recorded with laser Doppler flow perfusion monitors. SCS at frequencies of 50, 200 or 500 Hz was applied through the ball electrode at 30%, 60% and 90% of motor threshold (MT). Resiniferatoxin (RTX), an ultra potent analog of capsaicin, was applied to block the transient receptor potential vanilloid receptor (TRPV1); Furthermore, calcitonin gene related peptide (CGRP) 8–37, a CGRP‐1 receptor blocker, was used to elucidate mechanisms of SCS vasodilation at a high frequency. SCS applied with the three frequencies produced similar MT. SCS at the frequency of 500 Hz induced a higher increase of cutaneous blood flow and decreased vascular resistance compared to that with the frequency of 50 and 200 Hz (P<0.05). RTX (2 μg/Kg. i.v.) as well as CGRP8–37 (2.37 mg/Kg. i.v.) significantly reduced SCS‐induced vasodilation at this high frequency (500 Hz) (P<0.05). In conclusion, SCS at a high frequency significantly increased SCS‐induced vasodilation effect without influencing the MT. Furthermore, effects of SCS at high frequency seem to be predominantly mediated via activation of TRPV1 containing fibers including a release of CGRP.
Inter‐relationships between Noninvasive Measures of Peripheral Vascular Reactivity
Authors: Dhindsa Mandeep Singh , Sommerlad Shawn M. , DeVan Allison E. , Barnes Jill N. , Sugawara Jun , Tanaka Hirofumi
Published: June 2021

The clinical importance of peripheral vascular reactivity as an early marker of atherosclerosis has been well established. The relation between different methods employed to provide measurements of peripheral vascular reactivity are unclear as each technique evaluates different physiological aspects related to micro‐ and macro‐vascular reactive hyperemia. To address this, a total of 40 apparently healthy normotensive adults (age 19–68 yrs, 28 males) underwent 5‐minutes of suprasystolic forearm cuff‐induced ischemia followed by post‐ischemic measurements. Macrovascular reactivity measures included flow‐mediated dilatation (FMD) of brachial artery, and brachial to radial pulse wave velocity (ΔPWV; via Doppler flowmeters). Microvascular reactivity measures included reactive hyperemia index (RHI) assessed by fingertip arterial tonometry (RHI; via EndoPAT, Itamar) and fingertip temperature rebound (TR; via VENDYS, Endothelix). FMD was significantly and positively associated with RHI (r=0.54) and TR (r=0.44). There was a modest positive association between TR and RHI (r=0.29). ΔPWV was not significantly associated to any other measures. We concluded that different measures of peripheral micro‐ and macro‐vascular reactivity were not well correlated. These results suggest that a multi‐faceted approach may be necessary for the comprehensive assessment of peripheral vascular function.
Hypertension in male ETB‐receptor deficient rats is not mediated by splanchnic sympathetic nerves
Authors: Li Melissa Wei , Gariepy Cheryl E. , Fink Gregory Dale
Published: June 2021

Mean arterial pressure (MAP) is higher in rats lacking functional ETB receptors (ETBR) except in adrenergic tissues (transgenic dopamine‐β‐hydroxylase‐ETBR spotting lethal (sl/sl) rats), compared to transgenic controls (+/+). Plasma endothelin (ET‐1) levels are higher in sl/sl rats vs. +/+, so hypertension (HTN) in sl/sl could be mediated by stimulation of vascular ETA receptors. However, ET‐1 also can activate ETBRs in sympathetic neurons and there is evidence for increased sympathetic vasoconstriction in sl/sl. Here we tested the hypothesis that sympathetic activity to the splanchnic organs is critical for HTN in sl/sl. Sympathetic innervation to the splanchnic region was removed by surgical excision of the celiac and superior mesenteric ganglia (celiac ganglionectomy; CGX) in male +/+ and sl/sl (n=6–7/group). In rats on normal salt diet, MAP (radiotelemetry) and venous plasma ET‐1 concentrations (quantitative sandwich enzyme immunoassay) were measured three weeks after CGX. CGX significantly lowered MAP in +/+ (114 ± 1 vs.101 ± 1 mmHg), while it had no effect in sl/sl (124 ± 2 vs.120 ± 1). Plasma ET‐1 levels decreased significantly after CGX in +/+ (3.6 ± 0.1 vs. 2.0 ± 0.1 pg/ml), but did not change in sl/sl (5.2 ± 0.3 vs. 4.9 ± 0.4). Therefore, the data indicate that the splanchnic sympathetic nerves contribute to normal blood pressure regulation in +/+, but are not necessary for HTN in sl/sl rats (NIHP01HL70687).
Pro‐hypertensive role for leukotriene B4 receptors in the spontaneously hypertensive rat
Authors: Hendy Emma Bernice , Waki Hidefumi , Paton Julian F. R.
Published: June 2021

cDNA microarray analysis of the nucleus tractus solitarii (NTS) has indicated leukotriene B4 12‐hydroxydehydrogenase (LTB4 12‐HD) is down regulated in the spontaneous hypertensive rat (SHR) compared to its normotensive control (Wistar‐Kyoto or WKY; Waki et al. 2004; J.Physiol. 560P, PC6). As LTB4 12‐HD breaks down LTB4, we propose that it is up regulated in the NTS of the SHR, promoting vascular inflammation. To investigate the role of LTB4 receptors in the maintenance of the hypertension in the SHR, we have assessed whether systemic antagonism of LTB4 receptors has an anti‐hypertensive effect. In radio‐telemetered SHR, chronic oral administration of a LTB4 inhibitor (CP‐105696) was given in their drinking water (1 mg/ml in 1.25% DMSO). In all cases, a significant hypotensive effect of −13.2 ± 1.4 mmHg (P<0.05, n=5) was seen 11 ± 1.6 days after administration. In contrast, inhibiting LTB4 receptors in WKY rats was ineffective (n=5). When vehicle (1.25% DMSO) alone was added to drinking water there was no change in arterial pressure (n=2). These data indicate that LTB4 receptor activation contributes to the hypertension in the SHR. Our finding is consistent with the notion that the hypertension in the SHR is dependent, in part, on inflammation of the vasculature, which may include that within NTS and other brain regions controlling vasomotor tone. British Heart Foundation funded research.
Inhibition of the Janus (JAK)/Signal Transducers of Activated Transcription (STAT) Pathway prevents the Development of Angiotensin II‐induced Hypertension
Authors: Banes‐Berceli Amy K.L. , Brands Michael W , Labazi Hicham , Springfield Vanessa
Published: June 2021

Angiotensin II (ANG II) is a major physiologic and pathophysiologic controller of arterial pressure, but the molecular mechanisms underlying that effect are not completely understood. ANG II activates the JAK/STAT pathway in vitro and in vivo in models of type I diabetes but whether this activation occurs in hypertension and is required for ANG II‐mediated increases in blood pressure are unknown. Therefore we tested the hypothesis that inhibition of the JAK/STAT pathway would attenuate ANG II‐induced hypertension. We utilized male Sprague‐Dawley rats, chronically catheterized for 24 hr/day iv infusion and mean arterial pressure (MAP) measurements. All rats were maintained on a high‐salt intake (~12 mEq/day), and after control measurements ANG II (10 ng/kg/min, iv) was infused with or without the JAK2 inhibitor AG490 (10 ng/kg/min, iv). After 5 days of ANG II infusion the rats receiving AG490 had significantly lower MAP than the ANG II alone rats (106 + 6.1 vs 144 + 4.9 mm Hg, respectively). When AG490 was stopped during continued ANG II infusion, MAP rose within 1 day to match MAP in the ANG II alone group (158+ 2.1 vs 168 + 6.9, respectively). Western blot analysis revealed that JAK2 activation was significantly inhibited in the ANG II/AG490‐treated rats. From these data we conclude that activation of the JAK/STAT pathway is critical for the development of ANG II‐induced hypertension. (AHA SDG ABB, HL‐56259, HL‐74167 MWB).