bluuebunny/crossref_metadata_2025 huggingface dataset:
Role of A1 noradrenergic neurons in the pressor response induced by hypertonic saline (HS) infusion in unanesthetized rats
Authors: Pedrino Gustavo R , Korin Willian S , Cravo Sergio L
Published: June 2021

Several findings suggest that A1 noradrenergic neurons in the caudal ventrolateral medulla (CVLM) contribute to body fluid homeostasis. The present study sought to determine the effects of lesions of these neurons on the cardiovascular responses induced by HS infusion in awake rats. All experiments were performed in Wistar rats (280–350g). Lesion of A1 neurons were performed by microinjections of anti‐DβH–saporin (6.3 ng in 60 nl; SAP‐rats, N=8) into the CVLM, whereas sham rats received microinjections of free saporin (1.3 ng in 60 nl, N=6). Two weeks later the rats were instrumented for mean arterial pressure (MAP) and heart rare (HR) recordings. In the following day, rats were submitted to iv infusion of HS (3M NaCl, 0.18 ml/100g bw, 1 min). In sham rats, HS induced a sustained (>90min) increase of MAP (38±3 mmHg, 10 min) and a reduction in HR (−42±12 bpm, 10 min). In SAP‐rats, we observed a significant reduction in the changes in the MAP (15±3 mmHg) and a decrease of HR (−50±13 bpm). Hypernatremia induced by HS was similar in sham (146±1.4 mM) and SAP‐rats (146±0.9 mM). Blood hemoglobin concentration remains unchanged after HS in sham rats (100±0.3%). However, in SAP‐rats, we observed a sustained increased on this parameter (108±2%). Extension of A1 neurons lesion was confirmed by immunocytochemistry. These results suggest that A1 neurons mediate pressor response to acute changes in circulating volume. Supported by CAPES.
Splenic neural modulation of mesenteric vascular tone
Authors: Hamza Shereen M. , Kaufman Susan Elaine
Published: June 2021

Portal hypertension impedes outflow from the splanchnic vascular beds. We hypothesized that the increase in splenic intravascular pressure may initiate a neural reflex to increase mesenteric arterial tone and reduce blood flow into the plethoric mesenteric vascular bed. Splenic venous pressure was selectively increased by partial splenic vein occlusion (6.9±0.5 to 22.5±0.3 mmHg, n=47). During the 3 minutes following occlusion, mesenteric vascular conductance fell (−0.01±0.002 mmHg/mL·min‐1; p<0.05 n=7), an effect which was unchanged by renal denervation (−0.01±0.009 mmHg/mL·min‐1; n=6) and exacerbated by mesenteric denervation (−0.02±0.004 mmHg/mL·min‐1; p<0.05, n=7). The fall in conductance was abolished by splenic denervation (−0.002±0.002 mmHg/mL·min‐1; p<0.05 n=6). Similarly, the significant increase in mesenteric efferent nerve activity observed during splenic venous occlusion (22.2±2.8 to 27.9±3.8 spikes/sec, p<0.05 n=13) was absent after splenic denervation (32.4±2.4 to 31.2±1.6 spikes/sec, n=7). Although the spleen is thus integral to initiating the reflex, the accompanying increase in mesenteric nerve activity is not wholly responsible for increasing mesenteric arterial tone. (HSFC,CIHR).
Neuron‐targeted expression of ACE2 in the central nervous system prevents angiotensin‐II‐mediated hypertension
Authors: FENG Yumei , Xia Huijing , Bindom Sharell M , Lazartigues Eric
Published: June 2021

We previously showed that Angiotensin Converting Enzyme type 2 (ACE2) is a member of the brain renin‐angiotensin system. To elucidate the physiological importance of central ACE2, we developed a new transgenic mouse model (Syn‐ACE2) with neuron‐targeted ACE2 overexpression in the brain. Using immunohistochemistry we observed ACE2 overexpression in brain nuclei involved in blood pressure (BP) regulation, such as the RVLM, SFO and NTS, suggesting a local role for ACE2. To test this hypothesis, Syn‐ACE2 mice and littermates (n=6/group) were infused with AngII (osmotic pump: 600 ng/kg.min) for 14 days. Baseline BP (telemetry: 112±3 vs. 114±4 mmHg) and baroreflex gain (sequence method: 1.8±0.2 vs. 1.5±0.2 msec/mmHg) were similar in Syn‐ACE2 and control mice, respectively while the LF/HF ratio (BP variability) was significantly lower in transgenic mice (10±2 vs. 19±2; P<0.01). AngII infusion resulted in hypertension in littermates (135±3 mmHg) while Syn‐ACE2 mice remained normotensive (101±5 mmHg; P<0.01). At this time, baroreflex gain (2.3±0.1 vs. 2.2±0.1 msec/mmHg) and LF/HF ratio (17±2 vs. 15±2) were not significantly different between Syn‐ACE2 and littermates, respectively. Our data show that ACE2 overexpression improves autonomic function in Syn‐ACE2 mice, thus protecting them from AngII‐mediated hypertension, and support a role for ACE2 in the central regulation of BP. (NIH NS052479 and P20RR018766)
Testosterone stimulates peroxynitrite production in human coronary artery smooth muscle
Authors: Puttabyatappa Yashoda , White Richard
Published: June 2021

The increased incidence of cardiovascular disease in men compared with premenopausal women suggests an unfavorable effect of male sex hormone testosterone (T2) on the cardiovascular system. However, clinical and epidemiological studies report a controversial relationship between T2 and cardiovascular disease. Testosterone induces vasodilation, and this effect is thought to be mediated via nitric oxide. We now demonstrate that testosterone produces both nitric oxide (NO) and superoxide in human coronary artery smooth muscle cells (HCASMC). T2 stimulated NO‐dependent fluorescence in HCASMC, and this effect was attenuated by NOS inhibitors. T2‐stimulated superoxide production (measured by fluorescence and chemiluminescence) was inhibited by apocynin and sepiapterin. NO and superoxide react together to form peroxynitrite, and we have detected T2‐stimulated peroxynitrite production in HCASMC by employing the fluorescent dye DCF. Subsequent in vitro studies on porcine coronary artery support our findings. In conclusion, we hypothesize that testosterone stimulates production of peroxynitrite in vascular smooth muscle, and that peroxynitrite may mediate some of the effects of T2. (supported by HL080402)
Testosterone Activates mTOR/S6K1 Pathway Through Intracellular Calcium and ERK in Cardiomyocytes
Authors: Altamirano Francisco , Oyarce Cesar , Silva Patricio , Estrada Manuel
Published: June 2021

Testosterone has anabolic effects on heart, but the molecular mechanisms are unknown. The mammalian target of rapamycin (mTOR) is a nutrient sensor that regulates protein synthesis and it is a convergence point for several pro‐hypertrophy stimuli. We investigated testosterone effects on mTOR in neonatal ventricular cardiomyocytes. Testosterone increased both mTOR phosphorylation and its downstream targets S6K1 and 4E‐BP1, effects blocked by rapamycin and mTOR siRNA. The hormone increased both ERK1/2 and Akt phosphorylation, which were inhibited with PD98059 and Akt‐inhibitor‐X, respectively. Testosterone‐induced S6K1 phosphorylation was only blocked by ERK1/2 inhibition. Intracellular calcium chelator BAPTA‐AM or parvalbumin protein overexpression inhibited ERK1/2, S6K1 and 4E‐BP1 activation. Stimulation of cardiomyocytes in Ca2+‐free medium or inhibition of Ryanodine receptor did not modify ERK or S6K1 activation. However, inositol 1,4,5‐trisphosphate (IP3) pathway inhibitors (U‐73122, 2‐APB and Xestospongin C) blocked the activation of ERK and mTOR pathway. In addition, pertussis toxin inhibited ERK1/2 and S6K1 phosphorylation. Testosterone activation of mTOR pathway in cardiomyocytes could be important to understand the anabolic effects of this hormone.

Fondecyt 1060077, FONDAP 15010006
Rapid cardiac dysfunction caused by inducible cardiac specific leptin receptor deletion
Authors: Hall Michael Edward , Hall John E. , Smith Grant , Stec David
Published: June 2021

Leptin has been hypothesized to play a role in protecting the heart against lipotoxicity in obesity, and disruption of leptin signaling has been associated with cardiac lipid accumulation and dysfunction. However, the importance of leptin's direct effects on cardiac function is unclear. Using the Cre‐loxP recombinase system in C57B/J6 mice, we created an inducible, cardiac‐specific leptin receptor deficient model and assessed cardiac function with serial high resolution echocardiography (EC). Baseline data were obtained on 5 transgenic (TG) mice and 3 age matched (16–18 weeks) controls prior to induction of cardiac leptin receptor knockout with tamoxifen. Baseline ejection fraction (EF) and fractional shortening (FS) were not different between the groups and averaged 69 ± 5 and 39 ± 4% respectively. Seven days after initiation of the knockout, TG mice had a mean EF of 12 ± 1 and FS of 5 ± 0.5%, compared to 68 ± 3 and 38 ± 2%, respectively, in control mice. Heart weight/body weight ratios were higher in TG (7.69 mg/g) compared to control mice (5.45 mg/g). Acute cardiac specific deletion of the leptin receptor results in severe LV systolic dysfunction and rapid heart failure. The mechanism by which this occurs is unclear and could be related to altered cardiac metabolism, mitochondrial dysfunction, or other toxic effects of sudden cardiac leptin receptor deletion in adult mice (PO1 HL51971).
Impaired functional vasodilation in obese Zucker rats: role of insulin resistance and inflammation
Authors: Xiang Lusha
Published: June 2021

Obesity is characterized by insulin resistance (IR) and chronic inflammation, which may alter cyclooxygenase‐2 (COX‐2) activity and thus arachidonic acid (AA) metabolism. The obese Zucker rat (OZR) is a model of IR with increased inflammatory markers. We have shown an impaired functional vasodilation (FD) in the OZR due to altered arachidonic acid (AA) metabolism and increased thromboxane receptor (TP) activation. We hypothesize that IR alters AA metabolism via stimulation of COX‐2, resulting in impaired FD. Male lean (LZR) and OZR were treated with rosiglitazone (ROZ, 5mg/kg/day, rat chow) for 2 wks. The arteriolar diameters were measured in spinotrapezius muscle following muscle stimulation without and with the selective COX‐2 inhibitor NS‐398 or the TP antagonist SQ 29548 (SQ). Compared to LZR, untreated OZR exhibited an impaired oral glucose tolerance test and a two fold COX‐2 expression, both of which were normalized by ROZ treatment. The impaired FD in OZR was improved by 88% after acute treatment with NS‐398 and by 50% after chronic treatment with ROZ. SQ improved the vasodilatory responses by 58% in untreated OZR with no effect in lean or ROZ treated OZR. These results suggest that the increased TP activation and impaired FD in OZR is due to IR‐stimulated increase in COX‐2 activity. (Supported by AHA 55841, 53952, HL 51971)
The slingshot family of phosphatases mediates cofilin activation during PDGF‐induced migration in vascular smooth muscle cells
Authors: Torres Rebecca Ann , Drake Doulas , Weber David S
Published: June 2021

Platelet‐derived growth factor (PDGF) is a potent regulator of vascular smooth muscle cell (VSMC) migration. While a variety of signaling mechanisms have been reported during VSMC migration, ultimately the regulation of cell motility is via the actin cytoskeleton. We hypothesized that cofilin, a protein that facilitates actin depolymerization, is a key mediator of PDGF‐induced VSMC migration. In cultured rat aortic VSMCs, protein regulation was determined by immunoblotting with phosphospecific antibodies and migration was quantified using a modified Boyden chamber assay. Cofilin activity is increased by dephosphorylation at ser3, a process balanced by the dual regulation of LIM‐Kinase and the Slingshot family of phosphatases (SSH). Immunoblotting studies confirmed that PDGF increased cofilin activity in a time and dose dependent fashion. Relative to non‐treated control VSMCs, PDGF significantly increased VSMC migration (3.3 ± 0.4 fold), whereas in VSMCs treated with cofilin siRNA, PDGF‐induced migration is virtually eliminated (0.8 ± 0.1). The knockdown of SSH1 expression by siRNA resulted in attenuated PDGF‐induced activity of cofilin (∼80% inhibition) and a significant reduction in PDGF‐induced VSMC migration (4.4 ± 1.5; control vs 1.1 ± 0.7; siSSH1). Thus, these studies demonstrate that VSMC migration following PDGF stimulation is mediated by the SSH1 regulation of cofilin activity.

AHA 0330019N
SM22α Modulates Vascular Smooth Muscle Cells Phenotype through Bundling F‐actin
Authors: Han Mei , Dong Li‐hua , Shi Jian‐hong , Zheng Bin , Wen Jin‐kun , Cheng Yun‐hui
Published: June 2021

SM22α is a specific marker of contractile smooth muscle cells (SMCs). The recent study did demonstrate a role for SM22α in inhibiting the phenotypic modulation of SMCs from contractile to synthetic cells during atherogenesis. The investigators have showed that SM22α decorated contractile filament bundles in SMCs, and bound to and/or gel actin in vitro. However, it has been found no SM22α actin interaction in vitro or in vivo. These contradictory results suggest that the function and mechanism of SM22α in the actin cytoskeleton reorganization are unknown. We found that overexpression of SM22α induced F‐actin boundling, and forcedly expressed SM22α was incorporated into actin stress fibers. Coimmunoprecipitation and immunofluorescence showed that SM22α directly interacted and colocalized with F‐actin, participating in cytoskeleton reorganization in vascular SMCs that exhibit a differentiated phenotype. Co‐sedimentation assay demonstrated that SM22α facilitates actin filaments assemble into bundles. The blockade of SM22α expression by antisense SM22α transcripts made actin filaments dispersive and slim, and resulted in reduction of F‐/G‐actin ratio, consequently disappearance of cell contraction. This study first demonstrates that SM22α bundles F‐actin via a direct binding to F‐actin, participating in cytoskeleton reorganization in vascular SMCs that exhibit a differentiated phenotype.
The angiogenic action of tadalafil in zebrafish caudal fin regeneration
Authors: Tong Edmund Y. , Judkins Kyle M. , Greene‐Colozzi April E. , Chen Julia L. , Lee Joseph A. , Grimaldi Michael J. , Manos Philip D. , Saksa Erik V.
Published: June 2021

Tadalafil, better known as Cialis, is a phosphodiesterase E2 inhibitor which causes vasodilation. Many vasodilators have been shown to stimulate blood vessel growth. We used a zebrafish caudal fin regeneration model to investigate the angiogenic action of tadalafil. The three most ventral caudal fin bones of the zebrafish, Danio rerio, were amputated above the first branching point after being anesthetized with 0.2% Tricaine. After submerging five groups of zebrafish for 6o minutes in five increasing concentrations of taladifil (100 nM to 800 nM), they were placed in tanks containing regular water. Six days post amputation, each fish was anesthetized and its newly developed vascular bed in the regenerated tail fin region was examined under a microscope at 40X magnification. A 15 second video clip was also recorded. The area of each vascular bed was then quantified using our newly developed motion‐based angiogenesis analysis. As compared to the controls, zebrafish exposed to tadalafil showed significantly greater blood vessel growth. Statistically significant dose‐related responses were also demonstrated. In conclusion, tadalafil stimulates angiogenesis during caudal fin regeneration in zebrafish.
Differential regulation of angiogenic factors by radiation in rat brain
Authors: Lee Won Hee , Sonntag William E. , Lee Yong Woo
Published: June 2021

It has been hypothesized that radiation‐induced brain injury is a consequence of rarefaction of cerebral microvasculature. In the present study, we investigated the molecular regulatory mechanisms of radiation‐induced vessel rarefaction in the brain. Four month old F344×BN rats received either whole brain irradiation with a single dose of 10 Gy γ‐rays or sham‐irradiation, and were maintained for 4, 8, and 24 h following irradiation. The expression levels of a variety of angiogenic factors such as angiopoietin‐1 (Ang‐1), angiopoietin‐2 (Ang‐2), endothelial receptor tyrosine kinase (Tie‐2), and vascular endothelial growth factor (VEGF) were analyzed by quantitative real‐time RT‐PCR. A significant down‐regulation of Ang‐1 and Tie‐2 was observed in hippocampal and cortical regions isolated from irradiated animals. In addition, irradiation significantly attenuated the mRNA and protein expression levels of VEGF. In contrast, Ang‐2 expression was significantly and dramatically up‐regulated in the irradiated brain, demonstrating that irradiation markedly reduces the ratio of Ang‐1/Ang‐2 and increases the ratio of Ang‐2/VEGF. These data provide the first evidence that vascular plasticity may be impaired in irradiated animals though differential regulation of Ang‐1, Tie‐2, Ang‐2 and VEGF in the brain, and this may contribute to cerebrovascular rarefaction. (This work was supported by NINDS R01NS056218)
MDMA‐mediated cardiac toxicity is associated with increased oxidative stress
Authors: Shenouda Sylvia K. , Lord Kevin C , McIlwain Elizabeth , Lucchesi Pamela A. , Varner Kurt J.
Published: June 2021

Although MDMA (ecstasy) is considered to be a “safe” recreational drug, its use is associated with cardiovascular and cardiac events in humans and cardiac inflammation in rats. We tested the hypothesis that repeated administration of MDMA would alter cardiac structure and decrease left ventricular (LV) function. Male Sprague‐Dawley rats were treated with MDMA (n=8) or saline (n=8) (2xd for 4 d), followed by a 10 day drug‐free period (“binge”). After 4 MDMA binges, pressure‐volume (p‐v) and echocardiographic analysis revealed eccentric LV dilation (186 ±11 vs. 228±12μl) and diastolic dysfunction, indicated by increased end diastolic p‐v relationship (0.03±0.01 vs. 0.06±0.01). Proteomic analysis showed that MDMA treated hearts had higher levels of nitrated mitochondrial (Cyt c reductase, ATP synthase) and contractile proteins (troponin‐T, tropomyosin). Antioxidant enzyme levels in the LV were unchanged. While GSH/GSSG ratio was unchanged, glutathione reductase activity was decreased in the MDMA group. Electrically stimulated LV myocytes from MDMA rats had decreased contractility (SL shortening %) (8.3±0.4 vs. 6.7±0.4) and impaired relaxation compared to control at 0.5 Hz. Intracellular Ca2+ levels were the same in both groups. In conclusion, binge MDMA elicited eccentric LV dilation and dysfunction accompanied by oxidative stress. Support AHA 0615646B, NIH P20‐RR018766. MDMA courtesy of NIDA.
Regulation of iloprost and isoproterenol induced‐cAMP increases in rabbit erythrocytes (RBCs) requires different phosphodiesterase (PDE) activity
Authors: Adderley Shaquria , Sridharan Meera , Bowles Elizabeth , Stephenson Alan , Sprague Randy
Published: June 2021

Activation of the heterotrimeric G protein, Gs and adenylyl cyclase (AC) results in increases in cAMP in RBCs. Levels of cAMP are the product of synthesis via AC and hydrolysis by PDEs. Both iloprost (ILO), a PGI2 analog, and isoproterenol (ISO), a β‐adrenergic agonist, stimulate receptor‐mediated increases in cAMP. However, the PDEs associated with these pathways have not been characterized. Incubation of RBCs with ILO (1μM) or ISO (1μM) induced 141±16% and 172±34% increases in cAMP, respectively that were augmented by pretreatment with the non‐selective PDE inhibitor, IBMX (10μM). Selective inhibitors of PDE1 (vinpocetine), PDE2 (EHNA) and PDE4 (rolipram, ROL) had no effect on the ILO‐induced increases in cAMP, whereas, selective PDE3 inhibitors, cilostazol (CILO, 10μM) and milrinone (20μM), potentiated the ILO‐induced cAMP increases by 41±4% (n=7, P<0.01) and 22±5% (n=6, P<0.01), respectively. None of the PDE inhibitors alone had an effect on ISO‐induced cAMP increases. However, when the activity of protein kinase A, an activator of PDE4, was inhibited with H89 (10 μM), EHNA (10 μM) and ROL (10 μM) increased cAMP levels by 79±34% (n=8, P<0.01) and 116± 46% (n=8, P<0.01), respectively. These results suggest that in rabbit RBCs, ILO‐induced increases in cAMP are regulated by PDE3 while those associated with ISO are regulated by the activities of PDEs 2 and 4. Supported by NIH grant HL‐64180 and an ADA grant.
PAR‐2 receptor‐induced intracellular signalling in isolated intact human eccrine sweat gland cells and primary tissue culture cells
Authors: Bovell Douglas L. , Corbett Alistair D. , Lang Roland
Published: June 2021

PAR‐2 receptors can evoke intracellular signalling and anion transport in a cell line derived from human sweat glands, however, it is unknown whether PAR‐2 receptors are present and play a role in intact human sweat gland secretory cells. PAR‐2 receptors were investigated in isolated sweat glands and tissue culture cells derived from primary explants. Skin samples were obtained by informed consent and ethics approval from volunteers for immunohistochemistry and sweat gland isolation for; Western blot analysis; calcium imaging or explanted for tissue culture. Immunohistochemistry and Western blot using an anti human PAR‐2 antibody demonstrated the presence of these receptors in sweat gland secretory cells. Calcium imaging experiments using trypsin and the agonist peptide SLIGKV showed that both could increase intracellular calcium levels in intact glands and in the primary sweat gland cultures. Results also demonstrated that the effects of the agonist peptide were reversible upon washout, but not trypsin, and that the reverse peptide VKGILS failed to initiate changes in intracellular calcium. The results of this study demonstrate that PAR‐2 receptors are present in isolated human sweat gland secretory cells and in primary cultured cells and that these receptors are functionally active and can induce increases in intracellular calcium.

The study was supported by Glasgow Caledonian University.
Protein kinase C epsilon translocates to the Receptor for Activated C‐Kinase upon adenosine A1 receptor stimulation of the rat myocardium
Authors: Dobson James G. , Shea Lynne G. , Komatsu Satoshi , Ikebe Mitsuo , Fenton Richard A.
Published: June 2021

Protein kinase C epsilon (PKCε) in the heart plays an important role in adenosine‐induced anti‐adrenergic effects mediated by the adenosine A1 receptor (A1R). PKCε translocates to the transverse tubules (TT) of myocytes with A1R stimulation (AJP 287:H1721, 2004). Since the Receptor for Activated C‐Kinase (RACK2) resides in the TT we investigated whether PKCε translocates to RACK2 with A1R stimulation of rat ventricular myocytes and perfused hearts using the A1R agonist chlorocyclopentyladenosine (CCPA). Myocytes were exposed to primary rabbit PKCε and mouse RACK2 antibodies with secondaries conjugated to Texas red and Cy5, respectively. A scanning confocal microscope showed that CCPA caused PKCε to co‐localize with RACK2. RACK2 was immunoprecipitated (IP) from extracts of hearts perfused with ZM‐241385 (1 μM, ZM), an adenosine A2AR antagonist, and stimulated with either CCPA, phenylisoproplyadenosine (PIA) or phorbol 12‐myristate 13‐acetate (Pb) at 1 μM. As assessed by Western blotting CCPA, PIA and Pb increased the PKCε that co‐IP with RACK2 by 258%, 37% and >1000%, respectively. The adenosine A1R antagonist dipropylcyclopentylxanthine (DPCPX) prevented both the CCPA‐induced translocation of PKCε to, and co‐IP of PKCε with, RACK2. These results indicate that A1R stimulation of the heart is associated with PKCε translocation and association with RACK2 in transverse tubules. (HL‐066045 & 084160)
Inhibition of Gi/o‐proteins prevents hypoxia‐induced impairment of beta2‐adrenergic signalling in primary rat alveolar epithelial cells
Authors: Baloglu Emel , Ke Alberto , Bartsch Peter , Mairbaurl Heimo
Published: June 2021

Hypoxia inhibits lung alveolar reabsorption, whereas β2‐adrenergics stimulate. Hypoxia inhibits β2‐adrenergic signaling in a variety of tissues, but effects on alveolar epithelium are not known. To test this, primary rat alveolar type II cells were isolated, cultured to confluence, and incubated in normoxia or hypoxia (3% O2) for 24 hours. cAMP production was measured as an indicator of receptor function and adenylyl cyclase (AC) activity. Maximal terbutalin stimulated cAMP production was decreased by ∼30% in hypoxia; potency of terbutaline‐stimulation was not affected. However, forskolin‐stimulated AC was not inhibited by hypoxia. Receptor density assessed by ICYP binding in absence and presence of β2‐specific antagonist ICI‐118.551 (100μM) was decreased in hypoxia (−25%). To identify possible contribution of G proteins (Gs, Gi/o) to decrease cAMP production in hypoxia, AII cells were incubated with pertussis toxin (PTX; 0.5ng/ml) for 2 hours in normoxia or hypoxia before stimulation with terbutaline. PTX increased terbutalin‐stimulated cAMP production of hypoxic AII cells to normoxic control values, but did not further increase cAMP production in normoxia. PTX did not affect terbutalin potency. Our data suggest that hypoxia‐induced decrease in β2‐adrenergic signaling results mainly from an increased activity of Gi/o proteins.
Nitric oxide synthesis in neutrophil precursor cells and effect of nitric oxide modulators on their maturation
Authors: Kumar Sachin , Gupta Kanishka , Barthwal Manoj Kumar , Dikshit Madhu
Published: June 2021

Neutrophil maturation in bone marrow and their release in circulation have been shown to be modulated by nitric oxide (NO). Present study was undertaken to further assess the role of NO in neutrophil maturation in vitro. Neutrophil precursor cells were isolated from Sprague Dawley rat (200–250 g) bone marrow by Percoll density gradient in myeloblasts/promyelocytes rich band 3, myelocytes/metamyelocytes rich band 2 and band cells/segmented Neutrophils rich band 1, purity was confirmed by Giemsa staining/CD markers, NADPH oxidase and myeloperoxidase activity.

Intracellular NO level (assessed by DAF‐2DA), total nitrite (measured by Griess reagent) content, NOS activity (using 3H‐L‐arginine) and nNOS protein (Western blotting) were highest in band 1 and lowest in band 3. We also investigated effect of NO donor (SNP 1μM‐1mM) and NOS inhibitor (L‐NAME 1mM) on cell cycle by using Propidium iodide and BrdU staining during their culture for 24 hrs. SNP treated cells exhibited an increase in apoptosis, while viable cells showed a marginal increase in the S phase in band 2 and band 3. NOS inhibitor (L‐NAME) however increased the survival of cells without any modulation of cell cycle. Results obtained indicate that NO content augment with neutrophil maturation and seems to play an important role in the maturation of neutrophils.

The work was supported by CSIR and DBT India.
Effects of endurance training on energy substrate partitioning during exercise in postmenopausal women
Authors: Zarins Zinta Arija , Fattor Jill A. , Wallis Gareth A. , Mau Tamara L. , Faghihnia Nastaran , Horning Michael A. , Brooks George A.
Published: June 2021

PURPOSE: To examine the effects of endurance training on changes in energy substrate partitioning during exercise in sedentary, but otherwise healthy, postmenopausal women. METHODS: Ten postmenopausal women (aged 55±1 yr) completed 12 weeks of endurance exercise training [5 days/wk, 1 h/day, 65% peak oxygen consumption (VO2peak)]. Whole‐body energy substrate partitioning was determined by indirect calorimetry during 60 min cycle ergometer exercise in one pre‐training exercise trial [65% VO2peak, PRE] and two post‐training exercise trials [65% pre‐training VO2peak (ABT) and 65% post‐training VO2peak (RLT)]. RESULTS: Endurance training increased VO2peak by ∼16% (1.69±0.08 to 1.95±0.08 l/min, P<0.01). Compared to PRE (0.96±0.02), respiratory exchange ratio was reduced during exercise at ABT (0.93±0.01, P=0.01 vs. PRE), but not RLT (0.95±0.01). Accordingly, endurance training reduced the %energy from carbohydrate and increased the % energy from lipid during exercise at ABT (both P<0.05 vs. PRE), but not RLT. CONCLUSION: In sedentary, healthy postmenopausal women, endurance training increased peak aerobic capacity and lipid utilization during endurance exercise at a given absolute workload.

Supported by NIH grant AR42906.
Wnt3a promotes β‐catenin signaling and myotube formation during myogenic differentiation
Authors: Langen Ramon , van der Velden Jos , Kelders Marco , Laeremans Hilde , Schols Annemie
Published: June 2021

Satellite cells are muscle precursor cells required for postnatal skeletal muscle growth and regeneration. Glycogen Synthase Kinase 3beta (GSK‐3beta) has been implicated in negative regulation of skeletal muscle growth and muscle differentiation. Two distinct cellular pools of GSK‐3beta are selectively inhibited by IGF‐I/Akt‐or canonical Wnt signaling, respectively. Since canonical Wnt signaling and subsequent stabilization of beta‐catenin is involved in embryonic muscle formation, the effects of Wnt3a on differentiating myoblasts were investigated, and compared to those resulting from GSK‐3beta inactivation by LiCl or IGF‐I.

Wnt3a promoted C2C12 myoblast fusion, and induced beta‐catenin signaling in a time and dose dependent fashion. In addition, beta‐catenin‐dependent transcriptional activation was also induced after GSK‐3beta inhibition using LiCl, but not by IGF‐I. Furthermore, stimulation of muscle gene expression was only observed in response to LiCl but not Wnt3a, which uncouples the promoting effects of Wnt3a on myoblast fusion from muscle specific gene expression.

These data suggest that myoblast fusion and muscle specific gene expression are controlled by distinct cellular pools of GSK‐3beta, and provide evidence for a role of GSK‐3beta as a central regulator of muscle differentiation.

This work was supported by a research grant of the Netherlands Astma Foundation (NAF 3.2.02.63).
Neuregulin minimizes protein degradation induced by dexamethasone
Authors: Argadine Heather , Hellyer Nathan , Mantilla Carlos , Bailey Jeffrey , Sieck Gary
Published: June 2021

Neuregulin (NRG) is part of the epidermal growth factor family of proteins. Previous studies have shown that NRG activates PI3K/Akt pathways, leading to regulation of protein synthesis via downstream regulation of translation initiators. The principal translation initiator involved in protein synthesis is probably eIF4E, and the principal transcription factor regulating protein degradation is FOXO. We hypothesized that NRG signaling involves activation of the PI3K/Akt pathway, thus leading to an increase in expression of eIF4E and a decrease in expression of FOXO. An increase in eIF4E expression increases protein synthesis, and a decrease in FOXO expression reduces protein degradation. In this study we used dexamethasone, a synthetic glucocorticoid, to induce atrophy and cause an imbalance between protein synthesis and degradation in L6 myotubes. As expected, we found that after 48 hours of treatment with 10–100nM dexamethasone, eIF4E expression decreased and FOXO expression increased in a dose‐dependent manner. We found that treatment with 200 ng/ml NRG increased eIF4E expression. When L6 cells were treated with both NRG and dexamethasone for 48 hours, NRG blunted the catabolic effects of dexamethasone, thus increasing eIF4E expression and decreasing FOXO expression. These results indicate that NRG promotes protein synthesis and prevents protein degradation.

Supported by NIH grant AR51173.
Expression of genes regulating protein synthesis in young and old human muscle following resistance exercise and essential amino acid ingestion
Authors: Drummond Micah J , Miyazaki Mitsunori , Fry Christopher S , Dreyer Hans C , Pennings Bart , Dhanani Shaneen , Volpi Elena , Esser Karyn A , Rasmussen Blake B
Published: June 2021

Muscle growth requires an increase in both transcription and translation which results in enhanced muscle protein synthesis. The purpose of this study was to determine whether genes associated with the myogenic program and muscle protein synthesis are differentially expressed in old and young human skeletal muscle after a bout of resistance exercise and essential amino acid (EAA) ingestion. Twelve subjects (6 young, 6 old) completed 8 sets of leg extension exercise (70% 1RM) and muscle biopsies were obtained before and at 1, 3, and 6h post‐exercise. Subjects consumed a 20g leucine‐enriched EAA solution at 1h post‐exercise. RNA was isolated from biopsies and mRNA expression was determined using qRT‐PCR. At baseline, mRNA expression of Myogenin, TSC1/2, and Rheb were greater in old than young (P<0.05). Exercise resulted in increased levels of MyoD mRNA and decreased REDD1 and REDD2 mRNA in both groups (P<0.05). Myogenin mRNA increased at 3 and 6h and IGF‐1 mRNA increased at 6h only in young (P<0.05) suggesting that some genes are more responsive in young muscle following exercise. We conclude that reduced REDD1 and REDD2 mRNA levels are consistent with prolonged elevation of mTOR/S6K1 signaling and protein synthesis following resistance exercise and EAA ingestion in both young and old human muscle. Our data provides new insights into regulation of genes important for transcription and translation in young and old muscle.

Funded by NIH grant # P30 AG024832, R01 AR049877, and R01 AR045617
Plasticity of aged skeletal muscle: chronic contractile activity‐induced adaptations in muscle and mitochondrial function
Authors: Ljubicic Vladimir , Adhihetty Peter J. , Joseph Anna‐Maria , Huang Julianna H. , Saleem Ayesha , Ugoccioni Giulia , Menzies Keir J. , Hood David A.
Published: June 2021

The intent of the present study was to investigate the adaptive potential of skeletal muscle, as well as subsarcolemmal (SS) and intermyofibrillar (IMF) mitochondria in young (6 mo) and senescent (36 mo) animals in response to a standard regimen of chronic contractile activity (CCA). The TA and EDL muscles of 6 and 36 mo F344XBN rats were chronically stimulated (10 Hz, 3 h/day, 7 days) to induce CCA. The contralateral limb served as a non‐stimulated control. Subsequent to CCA, acute stimulation (1 Hz, 5 min) of the TA muscle in situ revealed greater fatigue resistance in both 6 and 36 mo animals. However, the improvement in endurance was 50% greater in the young, compared to the old animals. The CCA‐mediated increase in Hsp70 was also greater in the young animals. The expression of the longevity protein SIRT1 was increased 40–50% with CCA in both age groups. CCA also induced similar 30% elevations in COX enzyme activity in young and old animals. This may be due to the CCA‐elicited increases in mitochondrial biogenesis regulatory proteins PGC‐1α and Tfam that were also comparable (40–70%) between groups. CCA reduced mitochondrial ROS production by 40–80% in both age groups, whereas mitochondrial VO2 in the presence of succinate was increased with CCA only in the 6 mo group. Thus, muscle and mitochondria from senescent animals readily adapt to CCA, however the magnitude of the changes are less compared to young animals.
Brainstem sites for regulation of gill ventilation mapped by microinjection of DLH in the pre‐metamorphic bullfrog (Lithobates catesbeiana)
Authors: Hedrick Michael S. , Chen Anna K.
Published: June 2021

This study examined the role of ionotropic excitatory amino acid (EAA) receptors for regulating gill‐related respiratory rhythm in the bullfrog brainstem. Our objective was to ‘map’ the sites responsible for generating rhythmic gill burst activity by microinjection of the EAA agonist DL‐Homocysteic Acid (DLH). We used an in vitro brainstem preparation from pre‐metamorphic tadpoles (N=8; T‐K stages 8–16) that was superfused with oxygenated artificial CSF (20–22 °C) and generated spontaneous respiratory motor output measured by suction electrodes attached to cranial nerves (CN) V, X and XII. Pressure injections of small volumes (2–4 nL) of DLH in glass micropipettes were made in 7 locations from the rostral margin of CN V to the caudal margin of CN XII, lateral to the midline and at depths from 100 to 400 microns. DLH produced both inhibitory and excitatory responses. Inhibitory responses were largely confined to an area delineated by the extent of CN VII whereas excitatory responses, consisting of increases in respiratory frequency and burst amplitude, occurred in areas defined by the caudal margin of CN VIII to the rostral margin of CN XII and at most depths examined. These results indicate that EAAs have a widespread influence on gill burst activity in pre‐metamorphic bullfrogs that is not confined to a localized area of the brainstem. Supported by NIH SO6 GM48135.
Do pacemakers contribute to respiratory rhythm generation in the developing bullfrog brainstem?
Authors: Hedrick Michael S. , Chen Anna K. , Garcia Ryan
Published: June 2021

We tested the hypothesis that respiratory rhythm generation relies on pacemaker‐like activity in the bullfrog, Lithobates catesbeiana. We used in vitro brainstem preparations from pre‐metamorphic and post‐metamorphic tadpoles, superfused with oxygenated artificial CSF (20–22 °C), that generated spontaneous respiratory motor output. The potential role of pacemakers was tested with riluzole (RIL; 5–20 μM), a blocker of persistent Na+ currents, and flufenamic acid (FFA; 50 μM), a blocker of Ca2+‐activated cation currents. Both currents are hypothesized to be required for pacemaker activity in mammalian inspiratory neurons. RIL produced irreversible reductions in gill and lung respiratory frequency and burst amplitude in both pre‐ and post‐metamorphic brainstems, particularly at higher concentrations. FFA significantly reduced gill burst frequency and had no effect on lung burst frequency in pre‐metamorphic brainstems; in post‐metamorphic brainstems FFA had no effect on buccal burst frequency, but reversibly inhibited lung burst frequency. These data support the hypothesis that pacemaker‐like currents contribute to respiratory rhythm generation in the amphibian brainstem, although the specific role of these currents remains unclear. Supported by NIH SO6GM48135.
Depression of respiratory rate by intravenous opioids is not due to direct opioid effects on neurons within the preBotzinger Complex (pBC) region
Authors: Mustapic Sanda , Sanchez Antonio , Stucke Astrid G. , Dogas Zoran , Stuth Eckehard A. , Zuperku Edward J.
Published: June 2021

Clinical doses of i.v. mu‐opioids profoundly depress breathing rate. Mu‐opioid receptors (MORs) on pBC neurons, the putative kernel of rhythmogenesis, are potential targets. The purpose of this study was to determine the contribution of pBC MORs to respiratory depression by i.v. remifentanil (remi), a short‐acting, selective MOR agonist. Multi‐barrel micropipettes were used to record pBC neuronal activity and picoeject naloxone (NAL) in decerebrate, vagotomized, paralyzed and ventilated dogs during isocapnic hyperoxia. Remi was infused i.v. at a rate (0.2–0.4 mcg/kg/min) that produced a marked steady‐state bradypnea as determined from the phrenic neurogram (PNG). In study 1, NAL (5 mM) was picoejected onto single pBC neurons to determine the level of direct remi depression. In study 2, bilateral injections of NAL (100 nl each), 3 each side, centered in the pBC and 1‐mm rostral and caudal were used to locally block MORs. The results show that antagonism of MOR in the pBC region had no effect on neuronal and PNG patterns, but i.v. NAL (0.3 ml; 5 mM) completely reversed bradypnea. We conclude that opioids at plasma concentrations used for profound clinical analgesia depress breathing via MORs outside of the pBC region, which does not exclude that effects may be mediated indirectly via the pBC. Support: VA Med. Res. Funds & NIH R01 GM59234.
Estimation of the in vivo rate of tissue uptake for the organic osmolyte trimethylamine oxide in the winter skate (Leucoraja ocellata)
Authors: Treberg Jason Robert , Driedzic William R.
Published: June 2021

Elasmobranch fishes rely on nitrogenous organic osmolytes to maintain body fluids slightly hyperosmotic to the surrounding seawater. Methylamines are an important part of this osmoregulatory strategy with trimethylamine oxide (TMAO) being a significant osmolyte. The skate (Leucoraja ocellata) lacks significant TMAO synthesis, relying on low whole animal losses (≤ 1.2 % day−1) to maintain TMAO during periods of food deprivation [Treberg and Driedzic, 2006, AJP 291: R1790‐R1798]. TMAO is accumulated intracellularly, creating a substantial concentration gradient (> 50 mM). Herein we revisit TMAO handling in skates and provide the first quantitative estimates of TMAO uptake in tissues based on the exchange of radiolabeled tracer between the plasma and tissue compartments. Muscle TMAO uptake is very low (0.6 μmol g−1 day−1), indicating a long residency time once compartmentalized within muscle cells. The uptake of TMAO differs greatly amongst tissues; visceral tissues (liver and intestine) having rates an order of magnitude greater than muscle. Assuming sodium‐dependent concentrating mechanism, similar to other osmolyte systems, accumulation of TMAO is likely a small fraction of overall metabolic rate, but could constitute a significant part of the osmoregulatory energetic demand in skates. Presentation funded by NSERC and CIHR.
Dysregulation of SUMOylation During Hibernation
Authors: Cotter David G. , van Breukelen Frank
Published: June 2021

Hibernation is a dynamic process wherein animals oscillate between states of torpor and interbout arousal (IBA). IBA is characterized by a return to euthermy from the low temperatures and depressed metabolic rates of torpor. During late torpor (LT), the golden‐mantled ground squirrel, Spermophilus lateralis, must survive temperatures as low as −2º C at only 1% of active metabolic rates. As a result, cells may lose the ability to regulate key processes such as SUMOylation, which is involved in protein stabilization and transcriptional regulation. It has recently been shown that SUMO‐conjugate levels increase significantly during torpor. Here we present evidence that the observed increase in SUMO‐conjugate levels results from an inability to regulate SUMOylation at the low temperatures of torpor. We show that SUMOylation occurs at >20% of maximum rates at 0º C and that SUMOylation rates are not significantly different between LT, IBA, or summer active (SA) lysates at temperatures spanning 0º C to 37º C. We propose that the observed increase in SUMO‐conjugate levels during torpor is due to an inability to regulate SUMOylation combined with reduced de‐SUMOylation rates, rather than an increase in SUMOylation rates. We are currently working to identify targets of SUMOylation during hibernation, and relate these findings to their relevant cellular processes.
TG(mREN2)27 Females Show Differences in the Development of Systemic and Pulmonary Hypertension Compared to Ren2 Males
Authors: Schneider Rebecca Irene , Bosanquet James P , Habibi Javad , Whaley‐Connell Adam T , Sowers James R , Dellsperger Kevin C , DeMarco Vincent G
Published: June 2021

The Ren2 rat expresses the mouse renin gene in extra‐renal tissues leading to elevated tissue Ang II. In the male Ren2, reactive oxygen species (ROS) has been implicated in the development of pulmonary and systemic hypertension, as well as lung & cardiac remodeling. We hypothesized that a diminished hypertension in females is due to lower ROS in the heart and lung. Right ventricular systolic pressure (RVSP) & mean arterial pressure (MAP) of anesthetized 9 week old male & female Ren‐2 and Sprague Dawley (SD) rats, as well as indices of right & left ventricle hypertrophy (RVH & LVH) and ROS were analyzed and compared. RVSP & MAP were elevated in Ren2 males, but not in Ren2 females, compared to SD rats. Ren2 males exhibited moderate LVH, while Ren2 females exhibit mild LV hypertrophy. Neither Ren2 gender exhibited RVH when compared to SD. ROS was increased in the lung and LV of Ren2 males compared to SD. Interestingly, only ROS in LV of Ren2 females was elevated compared to SD females and this elevation was similar to that seen in Ren2 males. Ren2 females at this age do not exhibit the profound systemic and pulmonary hypertension found in age match Ren2 males. However, the increase of ROS and the appearance of mild LV hypertrophy in females suggests that Ren2 females have a protective, as yet unidentified, mechanism against an overactive RAS, which, without would lead to profound systemic and pulmonary hypertension. Support provided by NIH.
Paradoxical activation of eNOS by NADPH Oxidase 5
Authors: Malik Pulkit , Fulton David
Published: June 2021

Activity of endothelial nitric oxide synthase (eNOS), a key determinant of cardiovascular is modulated by several factors, including reactive oxygen species (ROS). The goal of the current study was to assess the direct effects of intracellular ROS on endothelial function and the activity of eNOS. To achieve this, we utilized Nox5, a unique member of the NADPH oxidase family that can function as a single gene product. We transiently co‐expressed eNOS and Nox5 in COS‐7 cells and measured the amount of NO produced. Contrary to our original hypothesis, we found increased amounts of NO produced in cells expressing Nox5. In endothelial cells transduced with an adenovirus expressing Nox5, we observed dose‐dependent increases in NO production in cells expressing Nox5 using both NO‐specific chemiluminescence and a co‐culture cGMP bioassay. To establish the functional significance of this observation in blood vessels, the endothelium of mouse aorta was transduced with control or Nox5 adenoviruses. In precontracted blood vessels, acute exposure to SOD induced significant vascular relaxation in vessels exposed to Nox5 versus control and unmasked the ability of Nox5 to activate eNOS. To identify the underlying mechanism we next assessed whether Nox5 modifies eNOS phosphorylation, the intra‐versus extra‐cellular effects of Nox5 and the ability of Nox5 to activate a calcium‐independent eNOS mutant.
Nitric Oxide ‐ Red Blood Cells Interactions under Oxidative Stress
Authors: Deonikar Prabhakar , Kavdia Mahendra
Published: June 2021

Role of endothelium‐derived nitric oxide (NO) as a potent vasodilator has been well established. Homeostasis nitric oxide in vasculature is maintained by its production by endothelial nitric oxide synthase and consumption by red blood cells and smooth muscle cells. Endothelial dysfunction observed in pathophysiological conditions such as diabetes is characterized with increase in oxidative stress due to excessive production of reactive oxygen species like superoxide which contribute to the reduced nitric oxide bioavailability for vasodilation. In this study, we investigated the effect of oxidative stress on NO‐RBC interactions. For this purpose, a novel stirred bioreactor with controlled headspace NO concentration was designed. Oxidative stress conditions were generated using xanthine oxidase‐hypoxanthine system. Gaseous NO was reacted with RBC and superoxide for 10 min and samples were drawn every 2 min. Nitrite and total nitrogen species (NOx) concentrations in the samples were measured by chemiluminescence assay and hemoglobin derivatives were measure by spectrophotometric assay. Nitrite and total NOx concentrations for NO‐RBC interactions in presence of superoxide were lesser than that of NO‐RBC interaction in absence of superoxide. These results are important step towards understanding of endothelial cell dysfunction related bioavailability of NO.