bluuebunny/crossref_metadata_2025 huggingface dataset:
Plasma membrane depolarization‐induced ERK activation: role in regulating paracellular permeability of the proximal tubular epithelium
Authors: Waheed Faiza , Speight Pam , Kapus Andras , Szaszi Katalin
Published: June 2021

Both physiological and pathological conditions, including apical ion transport, hypoxia, oxidative stress, and ATP depletion can alter the membrane potential of epithelial cells. We have previously shown that membrane depolarization induces Rho‐Rho kinase (ROK) mediated phosphorylation of myosin light chain (MLC) in proximal tubule epithelial cells and elevates paracellular permeability. Here we show that the permeability increase was absent in cells stably expressing a non‐phosphorylatable, dominant negative MLC. Depolarization, elicited by high extracellular [K+],– or ouabain, an inhibitor of the Na+/K+ ATPase, also activated the Ras‐Raf‐MEK‐ERK pathway. ERK activation was fast, reversible and independent of intracellular Ca2+. Immunofluorescent staining revealed that ERK translocates to the cell periphery. The MEK inhibitor PD98059 reduced the depolarization‐induced rise in paracellular permeability. Interestingly, PD98059 also mitigated the depolarization‐induced MLC phosphorylation, raising the possibility that ERK affects paracellular permeability by contributing to increased contractility.

In summary, our data suggest that cell contractility, regulated by the Rho‐ROK and the Ras‐ERK pathway might play a key role in epithelial barrier dysfunction caused by sustained depolarization.

Support: NSERC, Banting Foundation and Kidney Foundation of Canada.
Regulation of epithelial barrier through cell‐cell adhesion‐induced remodeling of N‐glycans of the Na,K‐ATPase beta1 subunit
Authors: Vagin Olga , Tokhtaeva Elmira , Yakubov Iskandar , Shevchenho Eugenia , Sachs George
Published: June 2021

Recent studies suggested the role of N‐glycans linked to the Na,K‐ATPase β1 subunit in cell‐cell adhesion in epithelia. The aim of this study was to test whether the structure of N‐glycans affects intercellular adhesion. The complexity of N‐glycans of the Na,K‐ATPase β1 subunit gradually decreases upon the formation of a tight monolayer from dispersed MDCK cells. This correlates with a cell‐cell adhesion‐induced increase in expression of GnT‐III (stops N‐glycan branching) and a decrease in expression of GnTs IVC and V (promote N‐glycan branching) as detected by real‐time quantitative PCR. Increased N‐glycan branching due to partial silencing of the gene encoding GnT‐III results in a 2‐fold increase in the paracellular permeability of the MDCK cell monolayer. However, reducing N‐glycan branching by swainsonine tightens and stabilizes cell‐cell junctions as detected by a 3‐fold decrease in the paracellular permeability and a 2‐ to 3‐fold increase in the resistance of the adherens junction proteins to extraction by non‐ionic detergent. Mutagenic removal of N‐glycosylation sites from the Na,K‐ATPase β1‐subunit impairs cell‐cell adhesion and decreases the tightening and stabilizing effects of swainsonine on cell‐cell junctions. These results suggest epithelial cells can regulate tightness of intercellular junctions via remodeling of N‐glycans, including those linked to the Na,K‐ATPase β1‐subunit.
Could differential expression of Apolipoprotein E (Apo E) in the alveolar epithelium be responsible for redox regulation of ENaC in type 1 and type 2 cells?
Authors: Helms My N , Self Julie L , Eaton Douglas C
Published: June 2021

We studied redox regulation of ENaC in the alveolar epithelium by patch clamping type1 and type2 cells in situ from a lung slice preparation. We found that type1 and type2 cells respond to nitric oxide (NO) donors differently. In 18 patch clamp recordings of type2 cells accessed in situ, 15μM NO‐donor, significantly decreased ENaC NPo from (control) 1.38±.26 to 0.82±.16 (p<0.05). Nitric oxide, however, does not alter lung ENaC activity in type1 cells; whether 15μM or 500μM NO‐donor was applied to type1 cells, ENaC activity did not significantly change from control NPo values from .63±.12; n=15. Using a cell culture model of the alveolar epithelium, we found that type1 cells have elevated levels of O2− compared to type2 cells, which suggests that differences in the cellular response to NO may be due to the oxidative state of pneumocytes. Apolipoprotein E, a 34 kDa plasma protein involved in the transport of cholesterol and other lipids, has been shown to be specifically expressed in type 1 cells (and not type 2 cells), and is up‐regulated in hyperoxic lung (Chen et al. 2006). Because of their known role in lipid transport, future studies are aimed at determining the effects of Apo E on lung ENaC under oxidative stress using single channel patch clamp analysis.

This work was supported by NIH grants HL071621‐01 and DK37963‐16 awarded to DCE and the Parker B. Francis Fellowship to MNH.
Fluorescein transport in isolated renal proximal tubules in experimental diabetic mice
Authors: Lungkaphin Anusorn , Chatsudthipong Varanuj
Published: June 2021

The renal clearance of organic anions compounds is mediated by organic anion transporters (OATs). Alterations of anion transport function could occur in pathological conditions such as diabetes. However, the role of the renal OATs under this condition has not been assessed. This study examined the transport function of renal OATs in diabetic mice induced by streptozotocin (200 mg/kg BW). The study was performed following 14, 21, 28 and 42 days of diabetic condition to investigate the duration of diabetes on the transport activity of the renal OATs. Fluorescein (FL) was used as a maker of anion transport by OAT1 and 3. Its accumulation in epithelial cells of isolated renal proximal tubules (RPT) was measured using confocal microscopy. We have used quantitative image analysis to follow transport of the organic anion FL from bath (blood side) to lumen in intact mice RPT. It was found that FL accumulation was not significance different from control group (n = 9) and the percentage of FL intensity were 96% and 105% of control, respectively. However, the significance reduction of cellular accumulation of FL was observed in longer period of diabetes, being 62% and 55% of control in 28 (n=5) and 42 (n=3) days of diabetes, respectively. Thus we hypothesize that diabetic condition induced tubular injury and reduced the expression of OAT1 and 3 that affected the transport function of these transporters. However, the mechanisms causing alterations in function and expression of renal OATs need to be further investigated. This work is supported by the Thailand Research Fund grants MRG4980200 and the Faculty of Medicine Endowment Fund, Chiang Mai University.
Molecular‐Detailed Modeling of Erythrocyte Membrane
Authors: Zhu Qiang , Vera Carlos , Asaro Robert , Sche Paul , Sung L. Amy
Published: June 2021

To investigate the correlation between the molecular design of the protein skeleton/lipid bilayer membrane of erythrocyte and its mechanical response, we have developed a molecular‐based model which incorporates the architecture of the junctional complex, a basic unit within the actin‐spectrin skeleton, together with the lipid bilayer. This model features a Fourier‐Space‐Brownian‐Dynamics model of the bilayer, a Brownian‐Dynamics model of the actin protofilament, and an experimentally based force‐elongation model of the spectrin. It thus enables us to predict the nano‐mechanics of single or multiple units of the protein network, the lipid bilayer, and the effect of their interactions. The present work is focused on the attitude of the actin protofilament at the equilibrium states coupled with the elevations of the lipid bilayer through their primary linkage at the suspension complex, as well as mechanical properties (i.e. shear modulus) of the system during large in‐plane deformations. Bifurcation in actin orientations has been predicted, although its occurrence depends on the exact manner of actin‐spectrin linkage. Thermal fluctuations at bifurcation may lead to mode‐switching, which may affect the network and the physiological performance of the membrane. These simulations suggest the importance of 3‐D molecular junctions and the lipid bilayer/protein network coupling on membrane mechanics.
Characterization of clock gene expression and their putative bioactive profiles on development in miniature pig
Authors: Lee Youngjeon Lee , Won Jinyoung , Lee Sang‐kil , Park Sookyeong , Hong Yonggeun
Published: June 2021

The aim of this study was to address the differential expression of circadian clock genes and their bioactivity profiles on growth in developmental stages of miniature pig (mini‐pig). In growing stages of piglets of either sex on day 0 (isolated from maternal by C‐section), day 1, 5 as well as parturient subjects were sacrificed. Most of all, differences exist in specific clock‐related gene expression between 0‐, 1‐ and 5‐day‐old neonatal and parturient mini‐pig of either sex, especially in brain. We confirmed the differential expression of clock‐related genes in heart and kidney excluding brain at 0‐, 1‐, 5‐day‐old of male mini‐pig. It is giving us the striking message that not only differential expression of clock genes in varies tissues but also dependency of these gene expression on the distinction of sex in neonatal subjects. Additionally, expression of clock, bmal1 and per1 were predominant in kidney compared to brain and heart but not shown any changing their expression on developmental stages. Taken together, therefore, these findings revealed a tissue‐specific differential expression of clock genes at developmental stages that modulates of development of mini‐pigs, suggesting differential physiological function at different developmental stage, especially neonatal periods in perspective donor for xeno‐transplantation. [Supported by BioGreen21 Program (Code#20070401034006), Republic of Korea].
IMPACT OF HYDROGEN SULFIDE (H2S) ON CAT CAROTID BODY (CB) FUNCTION
Authors: Fitzgerald Robert Schaefer , Shirahata Machiko , Kostuk Erik , Olson Ken
Published: June 2021

Current reports identify acetylcholine (ACh) and ATP as two excitatory transmitters in the cat and rat CBs. This study explored whether H2S could function as a neuromodulator, by measuring the release of ACh and ATP in response to the presence of H2S. The response to a KATP channel agonist, pinacidil, was also tested because in vascular tissues H2S is thought to act on that channel. In vascular tissue H2S is reported to mimic the effects of hypoxia. Harvested cat CBs were prepared for incubation in Krebs Ringer bicarbonate solutions (KRB) at 37°C using standard procedures consonant with the regulations of the Institutional Animal Care & Use Committee. ACh was measured with HPLC‐ECD methods; ATP, detected by a bioluminescence reaction. Positive immunohistochemical signals for Kir6.1 and SUR1 subunits of the KATP channel appeared in glomus cells (GC). The release of ACh from 2 cat CBs incubated in KRB made 100 μM Na2S (precursor of H2S) and bubbled with 30% O2/5%CO2 for 6 min significantly dropped to 60% of the control; the reduction in ATP was small and not significant. ACh release in response to KRB made 100 μM pinacidil and bubbled with 30% O2/5%CO2 for 6 min was significantly reduced to 42% of the control; ATP was reduced to 65% of the control. The data suggest the presence of the KATP channel in GCs; ACh and ATP release is affected by Na2S.

NHLBI:HL 05712–14, HL 72293.
Perinatal hyperoxia alters the expression of neurotrophic factors and their receptors in the rat carotid body
Authors: Dmitrieff Elizabeth F. , Dunmire Kyle B. , Piro Samantha E. , Bavis Ryan W.
Published: June 2021

Perinatal hyperoxia attenuates the hypoxic ventilatory response in rats by altering development of the carotid body and its afferent neurons. We hypothesized that these changes in carotid body growth and innervation involve changes in the expression of neurotrophic factors or their receptors. Rats were born and raised in hyperoxia (60% O2) or normoxia (21% O2) until studied at 1–14 days of age (i.e., P1–P14). Carotid body volume was 30% smaller in hyperoxia‐reared rats than their normoxia‐reared counterparts by P4 (P=0.02) and showed little growth thereafter. In contrast, carotid bodies of normoxia‐reared rats more than doubled in size between P4 and P14. We measured mRNA expression for brain‐derived neurotrophic factor (BDNF) and its receptor tyrosine kinase B (Trk B), and glial cell line‐derived neurotrophic factor (GDNF) and its receptor Ret in carotid bodies at P3 via quantitative RT‐PCR. Ret expression was reduced ~30% in hyperoxia‐treated rats (P<0.01), but no differences were detected for BDNF, TrkB, or GDNF. In addition, BDNF and GDNF protein expression levels were measured at P3 via ELISA. BDNF was reduced by ~70% in the carotid bodies of hyperoxia‐treated rats (P=0.02). GDNF appeared to decrease, depending on normalization methods. These data suggest a role for neurotrophic factors in hyperoxia‐induced respiratory plasticity. Supported by NIH grant P20 RR‐016463 (Maine INBRE).
Prediction of cardiorespiratory fitness without exercise testing in low back pain patients
Authors: Ivan Duque , Frederico Arango , Alain Duvallet
Published: June 2021

Cardiorrespiratory fitness is a capacity to perform, through body adaptations and responses, dynamic activities of moderate to high intensity using large muscle groups, during long time. It determines an important part of possibilities to perform activities of daily living and is closely related with health and quality of life. The measurement of the maximal oxygen uptake (VO2max) as a parameter of this fitness is useful for educating about the physical conditioning and in prescription and control of fitness programs in sick and healthy subjects.

The aim of the study was to develop a regression equation to predict VO2max based on non‐exercise data in patients with chronic low back pain (CLBP). All patients (N=70) achieved a maximal graded exercise test with measured VO2max (30.8 ml.kg−1.min−1 +/− 7.7). The regression model included as non‐exercise data, the patient's sex, body mass index (BMI) and intensity of sports activities (IPAS). Multiple linear regression generated the following formula (R2 = 38.3, SEE = 6.08 ml.kg−1.min−1): VO2max (ml.kg−1.min−1) = 35.337 − 0.475 * BMI + 0.155 * IPAS + 7.976 * sex (women = 0 and men =1)

The Durbin Watson statistic demonstrated inexistence of problems with serial autocorrelation (D–W = 1.86). The K–S normality test demonstrated that the errors are distributed normally. This study provides a relatively precise non‐exercise regression model to predict VO2max in CLBP patients.
A model of clinical inactivity with hypercortisolemia and hypocaloric diet induces peripheral insulin resistance and increases intramuscular fat
Authors: Cree Melanie G. , Ferrando Arny , Newcomer Brad R. , Aarsland Asle , Wolfe Robert R. , Paddon‐Jones Douglas
Published: June 2021

Many clinical conditions necessitate extended periods of bed rest and are characterized by a hypocaloric diet, hypercortisolemia and the rapid development of insulin resistance. Reduced insulin sensitivity has been linked to increased intracellular fat accumulation in the muscle and liver. However, it is unclear if tissue lipids increase during inactivity with a hypocaloric diet. To mimic a common clinical paradigm, we studied healthy volunteers before and after 14 days of bed rest with induced hypercortisolemia (20–24mg/dL) via oral hydrocortisone sodium succinate administration and hypocaloric diet (80% estimated daily requirement). Insulin sensitivity across the leg was assessed via blood sampling performed in association with a locally induced, hyperinsulinemic‐euglycemic clamp (0.15 mU/min/100ml leg). Intramuscular and liver fat were measured with magnetic resonance spectroscopy. Following bed rest, there was a decrease in insulin sensitivity across the leg (Pre: 5.82±0.19; Post: 4.21±0.33 mg/kg.leg/min; P=0.04) and increase in Intramuscular triglycerides (Pre: 0.13±0.02; Post: 0.18±0.02) (signal area of fat/signal area of standard; P=0.02). Liver triglycerides did not change (Pre: 0.10±0.02; Post: 0.11±0.02). Femoral glycerol and free fatty acid concentrations decreased with increasing insulin concentrations, but did not reach statistical significance following bed rest. This study demonstrates that insulin resistance is induced and intramuscular triglycerides increase during a model of clinical inactivity, despite a hypo‐caloric diet and the absence of detectable changes in plasma lipids or hepatic fat.
A Role for Forkhead Transcription Factors (FOXOs) in Disuse Atrophy of the Human Diaphragm (DIA) Associated with Mechanical Ventilation (MV)
Authors: Budak Murat Timur , Nguyen Taitan , Zhu Jianliang , Rothenberg Pamela , Shrager Joseph , Levine Sanford
Published: June 2021

We (Levine et al, NEJM, in press) have demonstrated that the combination of complete DIA inactivity and MV (i.e. DIA inact + MV) elicits marked atrophy of human DIA myofibers due to increased proteolysis. To assess the role of FOXOs 1 and 3 in effecting this atrophy, we biopsied the costal DIA of 10 brain dead organ donors prior to organ harvest (i.e., case subjects) and compared them to intra‐operative biopsies obtained from the DIA of 10 patients undergoing lung surgery (i.e., controls). Cases exhibited DIA inact + MV for much longer periods of time than controls (i.e., 18–69 hours versus 2–3 hours). We then isolated RNA from each DIA biopsy and we carried out real‐time reverse transcriptase PCR to assess the relative gene expressions of FOXOs 1 and 3, Atrogin‐1, and MuRF‐1 genes. In comparison to controls, case diaphragms exhibited the following fold increases in relative transcript number: FOXO 1, 5.6 (p<0.001); FOXO 3, 2.0 (p<0.04); Atrogin‐1, 2.6 (p<0.05), and MuRF‐1, 4.9 (p<0.01). These results are consistent with the concept that (a) case diaphragms increase transcription of FOXOs 1 and 3 that bind to the promoter region of the ubiquitin ligase genes (i.e., Atrogin‐1 and MuRF‐1); (b) increased mRNA expression of these latter genes increases the rate of proteolysis via the ubiquitin‐proteasome pathway; and (c) thereby accounts for some portion of the myofiber atrophy noted in case DIA. Supported by NIH Grant R01‐HL‐078834.
Stimulated disruption of Cav‐1/eNOS association plays a vital role in adaptation of H9c2 cells against high glucose concentration via Akt /HSP90 /eNOS activation.
Authors: Samuel Samson Mathews , Penumathsa Suresh Varma , Sudhakaran Perumana R , Maulik Nilanjana
Published: June 2021

Hyperglycemia is linked to reduced eNOS activity and Nitric Oxide (NO) production but the role of heat shock protein (HSP‐90)/Akt/Caveolin‐1 (Cav‐1) in this regulatory mechanism is unknown. In this study we examined the mechanism involved in the regulation of eNOS on high glucose (HG‐25mM) treatment in H9c2 (cardiac myoblast) cells. We examined the levels of Cav‐1, eNOS, p‐eNOS, p‐Akt and HSP‐90 in control (5mM) and HG treated cells at 24h, 48h and 72h in the sucrose gradient (5–40%) fractions to observe the status of these proteins in caveolar rich raft fractions and in the cytosol. We have observed increased expression of HSP‐90, p‐eNOS and p‐Akt at 24h and 48h of HG treatment. No significant alteration in Cav‐1 was observed at 24h of HG treatment however there was an increase in Cav‐1 levels at 48h. When the treatment was increased to 72h the HSP‐90, p‐eNOS, p‐Akt levels decreased while Cav‐1 levels increased and significant association of Cav‐1/eNOS was observed. These results were validated by immunoprecipitation assay at all time points to observe the association of Cav‐1/eNOS and HSP‐90/eNOS. Thus this study showed that earlier time points of HG treatment stimulate an adaptive mechanism by disruption Cav‐1/eNOS association and by phosphorylation of Akt and eNOS and increased expression of HSP‐90 which were reversely regulated on prolonged treatment resulting in reduced eNOS activation and NO production.
Effects of AGE on cardiac fibroblast (CF) signaling pathways and functions
Authors: Yamazaki Katrina Go , Villarreal Francisco
Published: June 2021

Long standing diabetes can result in the development of a cardiomyopathy which is accompanied by myocardial fibrosis. Chronic hyperglicemia accelerates the crosslinking between glucose and proteins or lipids and leads to the formation of advanced glycation end‐products (AGE). AGE can alter cell functions by binding to their receptors (RAGE). We assessed the effects of AGE on CF protein and collagen synthesis, matrix metalloproteinase (MMP) activity, cell proliferation and NFkB activity. RAGE levels were also determined. We cultured CF in low glucose media. CF were treated with either normal bovine serum albumin (BSA, 50 or 100 mg/ml) or glycated AGE‐BSA at equal concentrations. Results obtained after 48 h of treatment indicated that CF treated with AGE‐BSA significantly increased protein (~50%) and collagen synthesis (25>50%) in a dose dependent manner. AGE‐BSA also increased cell proliferation by 100%. AGE‐BSA Tx yielded a tendency to increase NFkB 3–4 fold. No effects on MMP were noted. Concentration dependent increases in RAGE protein levels occurred with Tx. Results suggest that diabetic patients activation of RAGE in CF may promote the development of adverse cardiac remodeling and tissue fibrosis. Supported by HL‐43617, 67922 and MD‐000220
Short Term Effect of Bariatric Surgery Induced Weight Loss on Cardiac Autonomic Function
Authors: Kim Areum , Fisher James P. , Young Colin N. , Nelson Jasmine , Scott James S. , Thyfault John P. , Fadel Paul J.
Published: June 2021

A primary metabolic alteration with obesity is insulin resistance (IR), which has been shown to be associated with impaired cardiac autonomic function. Considering recent work indicating marked improvements in IR as little as three weeks following bariatric surgery, we sought to determine whether such improvements in IR were accompanied by favorable alterations in cardiac autonomic control. Six patients (42±3 yr) were studied before and one month following Roux‐en‐Y gastric bypass surgery. Beat‐to‐beat heart rate and blood pressure (BP) were measured to assess heart rate and BP variability (power spectral analysis) and estimate spontaneous cardiac baroreflex sensitivity (transfer function gain, 0.04–0.15 Hz). Insulin resistance was estimated with the homeostasis model assessment (HOMA) index. Following surgery body mass index (53±3 pre vs. 47±3 post kg/m2), blood glucose, and HOMA (7.2±1.4 pre vs. 5.1±0.9 post) were all significantly reduced (P<0.05 pre vs. post). In contrast, low and high frequency heart rate variability, low frequency BP variability, and cardiac baroreflex sensitivity (6.3±1.4 pre vs. 7.4±2.1 post ms/mmHg; P=0.43) were unchanged. These preliminary findings suggest that despite marked reductions in body weight and improvements in insulin resistance one month following bariatric surgery, cardiac autonomic function and baroreflex sensitivity are unaltered.
Female gender is associated with decreased oxidative stress and increased insulin‐stimulated glucose uptake in skeletal muscle in TGR(mRen‐2)27 rats
Authors: Lastra Guido , Manrique Camila , Rehmer Nathan , Morris Mathew , Appesh Lama , Nusair Maein , Whaley‐Connell Adam , Ferrario Carlos , Sowers James R
Published: June 2021

The transgenic Ren‐2 rat is a model of enhanced Renin Angiotensin Aldosterone System activity, which exhibits insulin resistance, hypertension, and microalbuminuria. We investigated gender differences in the production of Reactive Oxygen Species (ROS) and NAPDH oxidase activity in 8 to 11 weeks old Ren 2 rats and age‐matched SD rats. ROS production was determined by photon emission in the presence of lucigenin and NADPH. Results are presented as mean ± SEM, and unpaired t test and ANOVA were used as appropriate. SBP was similar between males and females (170.5 ± 16.21 vs 180.4 ± 8.61 mmHg). In insulin‐sensitive soleus muscle, NAPDH oxidase activity was higher in Ren 2 males compared to females (6.378 ± 0.41 vs 4.98 ± 0.27 mOD/min/mg p <0.05), while it did not differ between SD males and females (4.1 ± 0.24 vs 4.22 ± 0.47 mOD/min/mg p = 0.83). No significant differences in NAPDH oxidase activity were found in aorta, kidney or left ventricle. ROS production in soleus muscle was lower in Ren‐2 females compared to males (102.69 ± 4.48 RLU/s/mg vs 170.69 ± 11.33, respectively. p=0.025). Insulin stimulated glucose uptake was higher in Ren‐2 female compared to males (0.655 ± 0.057 vs 0.195 ± 0.0531 nmol/mg) Our findings suggest that female gender may be protective against oxidative stress and leads to increased insulin sensitivity in skeletal muscle in the Ren‐2 rat model.

Funded by NIH 1R01‐HL073101‐02, VA Merit System 0018.
Constitutive activation of the mammalian target of rapamycin complex 1 (mTORC1) is associated with skeletal muscle insulin resistance
Authors: Reynolds Thomas Henry , Phelps Charlie , Berk Eli , Miller Andrew
Published: June 2021

The present study assessed mTORC1 activity in isolated rat skeletal muscles and in skeletal muscles of Ob/Ob (OB) mice. Epitrochlearis muscles isolated from rats were incubated with insulin (750 uU/ml) and/or amino acids (2.5X MEM). Amino acids produced a significant reduction in the ability of insulin to promote protein kinase B (PKB) phosphorylation, a process that was reversed by rapamycin (100nM). Amino acids did not inhibit the ability of insulin to promote the phosphorylation of PRAS40 on Thr‐246, a site whose phosphorylation enhances mTORC1 activity. In vivo insulin action was assessed in muscles of OB and wild type (WT) mice by intraperitoneal injections of insulin (0.25 U/Kg) or saline. In the basal state, phosphorylation of S6K on Thr‐389 and PRAS40 on Thr‐246 were increased significantly in muscles of OB compared to WT mice. In the insulin‐stimulated state, phosphorylation of S6K on Thr‐389 and PRAS40 on Thr‐246 were similar in muscles of OB compared to WT mice. We also assessed whether or not 5 days of rapamycin treatment (0.75 mg/Kg/day) could improve the insulin resistance of OB mice. Rapamycin treatment exacerbated the insulin resistance of Ob mice, indicating that some mTORC1 activity is necessary for normal in vivo glucose disposal. These results indicate that mTORC1 signaling is associated with insulin resistance, but inhibiting mTORC1 activity with rapamycin does not improve in vivo insulin action.
Mitochondrial coupling regulates responses of vascular endothelial cells to oxidative stress.
Authors: Brzezinska Anna K. , Bosnjak Zeljko J , Warltier David C
Published: June 2021

Failure to normalize generation of reactive oxygen species (ROS) is the most important factor in development of cardiovascular disease. Uncoupling of oxidative phosphorylation (OXPHOS) reduces ROS generation. We hypothesized that in human coronary artery endothelial cells (HCAECs) uncoupling of mitochondria reduces ability to generate ROS. Mitochondria were uncoupled using FCCP (100 nM), a chemical uncoupler. Oxidative stress was induced by doxorubicin (DOX, 5 μM, 1 hr) a redox cycling drug. HCAECs were treated with FCCP (10 min) or FCCP followed by DOX. The levels of ATP, LDH release and MTT assay were monitored 1‐ and 24 hours after treatment. FCCP alone had no effects on total ATP level, LDH release or cells viability. Treatment with DOX caused 20% (n=3, P<0.05) reduction in viability of HCAECs. Treatment with FCCP prior to the addition of DOX maintained cells viability at the control levels (n=3, NS). Treatment with DOX increased expression of Cu, Zn‐SOD and Mn‐SOD (2‐fold vs. control). Treatment with FCCP prior to addition of DOX maintained expression of Cu, Zn‐SOD and Mn‐SOD at the control levels. Functional importance of uncoupling was tested in the whole‐cell mode of the patch‐clamp. The addition of FCCP depolarized HCAECs by 40 mV (n=10, P<0.05). The dialysis of cells with a re‐coupling agent GDP (500 mM) caused a repolarization of HCAECs. Taken together, oxidative stress related changes of vascular function could be normalized by a short‐term, mild uncoupling of mitochondrial OXPHOS. The ability to control the level and time of mitochondrial coupling could be a way of delaying or preventing a vascular damage.
CaMK activation during exercise is required for histone hyper‐acetylation and MEF2A binding at the MEF2 site on the Glu4 gene.
Authors: Ojuka Edward Otim , Smith James A
Published: June 2021

Regular exercise protects against type II diabetes in part by increasing GLUT4 content. Previous work from our laboratory has established that GLUT4 expression is mediated by CaMK‐dependent binding of MEF2A to its cis element on the GLUT4 promoter. To further investigate how MEF2A binding to the GLUT4 gene is regulated, we injected rats with KN93 to inhibit CaMK II activation and measured; 1) MEF2A binding to, and histone H3 acetylation of, the MEF2 site on the GLUT4 gene by ChIP assay, 2) nuclear HDAC5 and GLUT4 contents by Western blot and 3) GLUT4 mRNA by RT‐PCR after exercise. Exercise increased CaMK II activity ∼50%, H3 acetylation and MEF2A binding to DNA ∼2‐fold and GLUT4 expression significantly. All the above increases by exercise were attenuated by KN93. Our data show that CaMK II activity modulates chromatin structure to increase MEF2A binding to the GLUT4 gene during exercise and suggests that drugs that mimic these effects might be worthy targets for controlling diabetes.
Chronic heme promotes sustained elevations in CO excretion, anorexia, attenuated physical activity and circulatory collapse in rats
Authors: Johnson Robert A , McGloin Kayla E , Johnson Fruzsina K , Nicholson Susannah E , Stewart Ronald M
Published: June 2021

Carbon monoxide (CO) is generated from the degradation of heme, in a reaction that is catalyzed by heme oxygenase, a heat shock protein (HSP32). Loss of appetite, confusion, lethargy, and systemic organ failure are all amongst the well documented adverse effects of chronic, subhypoxic, exogenous CO poisoning, and heme treatment has been shown to accelerate hemorrhage induced circulatory collapse. Accordingly, we wanted to determine if chronic elevations in endogenous CO, from heme alone, would affect appetite, activity and survival. Towards this end, rats (N=24) were treated with heme (15 or 45μmol/Kg heme as heme‐lysinate, IP q24h) for up to four weeks. Heme treatments produced up to a 3‐fold increase in expired CO, which was accompanied by a transient reduction in food intake, cessation of weight gain and a >95% decrease in wheel running activity. While both heme doses produced these adverse effects, heme administration of 45μmol/Kg per day also led to circulatory collapse and premature deaths in otherwise healthy unbled rats (50% at 14 days). Concurrent provision of high zinc diet (0.5%) ‐which promotes formation of ZnPP, a heme oxygenase inhibitor‐ conferred marked improvement against heme treatments. We conclude that heme drives heme oxygenase activity to increase endogenously formed CO and produce adverse effects which can promote circulatory collapse.

Jointly supported by NIH/DOD grant HL64577 (RAJ) and National Trauma Institute
Autonomic compensation to central hypovolemia monitored with heart period variability
Authors: Convertino Victor A. , Rickards Caroline A. , Ryan Kathy L. , Cooke William H.
Published: June 2021

A controversy exists as to whether components of heart rate variability may serve as indices of autonomic control of the circulation (JAP 101:676–682, 2006). We tested the hypothesis that frequency‐domain components of R‐R interval variability track autonomic function during progressive reductions in central blood volume induced by lower body negative pressure (LBNP) in humans. Subjects (n=33; 23 M/10 F) were instrumented to record ECG and muscle sympathetic nerve activity (MSNA; peroneal nerve) during a progressive LBNP protocol to cardiovascular collapse (i.e., presyncope). Although MSNA recordings were lost in many subjects during LBNP, MSNA was maintained through cardiovascular collapse in 12 subjects. Heart period variability was assessed by calculating the power spectral density in the high frequency (RRIHF; 015–0.4 Hz) and low frequency (RRILF; 0.04–0.15 Hz) domains with a fast Fourier transform. RRILF and RRIHF correlated poorly with MSNA at baseline (R2 ≤0.01). LBNP caused a progressive reduction in R‐R interval and a progressive increase in MSNA. LBNP produced a progressive reduction (p<0.001) in RRIHF but failed to elicit distinguishable (p=0.15) alterations in RRILF. As a result, RRIHF demonstrated a high, inverse amalgamated correlation with MSNA (R2 = 0.98), but a low amalgamated correlation existed between MSNA and RRILF (R2 = 0.09). Our data show that RRILF is not an accurate index of sympathetic compensation to progressive reductions in central blood volume. Conversely, reductions in RRIHF may serve as an accurate, non‐invasive index of elevated sympathetic activity.
Interindividual variability in heart rate variability (HRV) and complexity (HRC) measurements
Authors: Ryan Kathy L , Rickards Caroline A , Muniz Gary W , Moralez Gilbert , Convertino Victor A
Published: June 2021

Indices of HRV and HRC have been suggested as potential tools for assisting triage decisions in trauma patients (Aviat Space Environ Med 77:1107, 2007; J Trauma 63:512, 2007). Determination of intersubject variability in these measurements in normal individuals has not been performed. We hypothesized that common indices of HRV and HRC are not highly variable in healthy human subjects, thereby allowing the establishment of “normal” values. ECG was collected for 5 min in 31 supine, resting subjects. Time domain variables included RRISD, RMSSD, pNN50, and SD1 and SD2 from Poincaré plot analysis. Frequency domain variables (LF, 0.04–0.15 Hz; HF, 0.15–0.4Hz) were calculated using Fourier transform. HRC analyses included entropy (SampEn), fractal nature (FD‐L and FD‐DA), stationarity (StatAv), symbol dynamics (SymDyn) and detrended fluctuation analysis (DFA). Data are below:

Against expectations, many parameters demonstrated high interindividual variability. Since application of clinical vital signs depends on comparison to a “normal” value, a single measurement of HRV or HRC may not be beneficial for determining patient status. However, tracking changes in continuously‐measured HRV or HRC parameters over time might be more useful.
Reciprocal interactions between bradykinin and thromboxane A2 during stimulation of ischemically sensitive cardiac spinal afferent
Authors: Fu Liang‐Wu , Longhurst John
Published: June 2021

We have demonstrated that the ischemic metabolites thromboxane A2 (TxA2) and bradykinin (BK) contribute to activation of cardiac afferents during ischemia. The present study tested the hypothesis that there are reciprocal interactions between these two metabolites in stimulation of ischemically sensitive cardiac afferents. Nerve activity of single cardiac afferent units was recorded from the left sympathetic chain or rami communicantes (T2 – T5) of anesthetized cats. Twenty two ischemically sensitive afferents (CV= 0.46 – 4.34 m/s, 5 Aδ17 C‐fibers) were identified. In the first protocol, a repeat injection (2.5 μg) of TxA2 into left atrium (LA) 4 min after administration of BK (1 μg, LA) induced a significantly larger cardiac afferents’ response than the first TxA2 response (0.55±0.17 to 1.54±0.41 vs.0.64±0.13 to 2.28±0.36 imp/s, first vs. second injection, n=5). In a control group, repeated TxA2 (2.5 μg, LA) evoked consistent responses in four other ischemically sensitive afferents. Secondly, we observed that administration of TxA2 (2.5 μg) and BK (1 μg, LA) together appeared to cause a response that was approximately additive of the individual responses. Finally, blockade of the TxA2 receptors with BM13177 attenuated the responses of four cardiac afferent to BK (1 μg, LA) by 47%. In contrast, repeated BK (1 μg, LA) induced a consistent discharge activity in three other afferents. These preliminary data suggest that BK and TxA2 reciprocally enhance the responses of ischemically sensitive cardiac afferent endings to the other metabolite (Supported by NIH HL66217).
Novel neurotransmitters of the human intrinsic cardiac nervous system
Authors: Hoover Donald B. , Isaacs Emily R. , Jaques Frederic , Hoard Jennifer L. , Pagé Pierre , Armour J. Andrew
Published: June 2021

This study was done to anatomically identify novel neurotransmitters localized to human intrinsic cardiac ganglia (ICG). Samples were collected from 8 patients during cardiothoracic surgery and processed for immunofluorescent detection of specific neuronal markers. Colocalization of markers was evaluated by confocal microscopy. All intrinsic cardiac neuronal somata showed immunoreactivity for the cholinergic marker choline acetyltransferase (ChAT). These somata were bordered by cholinergic varicosities, evident as punctate immunostaining for high affinity choline transporter. Moderate to intense staining for neuronal nitric oxide synthase (nNOS) was likewise associated with all ganglion neurons as well as some proximal axons and nerve fiber bundles connected to ICG. Many calcitonin gene‐related peptide (CGRP)‐immunoreactive nerve fibers were identified in ICG, often surrounding cholinergic/nitrergic neuronal somata. Fewer nerve fibers displayed substance P (SP)‐immunoreactivity, but all SP was colocalized with CGRP. These findings suggest that NO may play a prominent role in cholinergic transmission within the human heart, CGRP being another key neuromodulator in the human intrinsic cardiac nervous system. Identification of such novel mediators may lead to new paradigms for cardiac neuromodulation. Supported by the AHA SE Affiliate and the Canadian Institutes for Health Research.
Transient Increase in P2Y2 Receptor Expression after Spinal Cord Injury in Rat Model
Authors: Rodríguez Ana E. , Torrado Aranza , Figueroa Johnny D. , González Fernando A. , Miranda Jorge D.
Published: June 2021

Spinal cord injury (SCI) increases inhibitory molecules release and reactive astrocytes proliferation at injury site producing nonpermissive environment for axonal regeneration. Reactive astrogliosis mechanism is unknown, but the release of nucleotides has been linked to this hypertrophic state. Our goal is to investigate the spatio‐temporal profile of P2Y2 after SCI. Molecular biology and immunohistochemical (IHC) studies were used to evaluate the expression and role of these receptors in rats injured at the T‐10 level using the NYU impactor device. P2Y2 gene temporal profile using standardized RT‐PCR showed a two‐fold increase after 4–7 days post‐injury (DPI) and returned to basal levels by 14 DPI. Double labeling IMH localized P2Y2 in cells bodies, axons, macrophages, oligodendrocytes and astrocytes. Also, levels of P2Y2 immunoreactivity were increased after injury in astrocytes. Therefore, the gene profile of P2Y2 after SCI showed that the levels of this mRNA and protein increased after trauma, suggesting a role in the establishment of the restrictive environment for axonal regeneration after SCI.

Support by NIH‐MRISP 2R2MH48190‐14, NIH‐SNRP NS39405, MBRS‐SCORE S06‐GM008224, MBRS‐RISE GM‐68138, PR‐EPSCOR EPS‐9874782
NADPH oxidase activity is necessary for phrenic motor facilitation induced by 5HT2B receptor activation
Authors: MacFarlane Peter M , Mitchell Gordon S
Published: June 2021

Acute intermittent hypoxia (AIH) elicits phrenic long‐term facilitation (pLTF), a form of respiratory plasticity that requires activation of spinal 5HT2 receptors (Mahamed and Mitchell, 2006). AIH‐induced pLTF requires NADPH oxidase‐derived ROS formation (MacFarlane et al., FASEB J., 2007). Spinal 5‐HT receptor activation in vivo is sufficient to induce phrenic motor facilitation by an NADPH oxidase‐dependent mechanism, but the specific 5‐HT receptor subtypes involved are not known. In this study, we tested 1) whether spinal 5‐HT2B receptor activation is sufficient to elicit phrenic motor facilitation (without hypoxia), and 2) whether phrenic motor facilitation following 5‐HT2B receptor activation requires spinal NADPH oxidase activity. In anesthetized, paralyzed and ventilated rats with constant arterial PO2 and PCO2, episodic intrathecal (i.t.) injections of 5‐HT (1mM) at C4 (5μl, 3 injections, 5min intervals) or the selective 5‐HT2B receptor agonist BW723C86 (1mM) both elicited significant phrenic motor facilitation at 60min post‐injections. Pre‐treatment with a single i.t. injection of apocynin (600μM, 12μl, i.t.), an NADPH oxidase inhibitor, blocked phrenic motor facilitation induced by both 5‐HT and BW723C86. Thus, episodic spinal 5‐HT2B receptor activation is sufficient to elicit phrenic motor facilitation in vivo by a mechanism that requires spinal NADPH oxidase activity. These findings sugest that 5‐HT2B receptors contribute to serotonin‐dependent phrenic motor facilitation (including AIH‐induced pLTF) by an NADPH oxidase dependent mechanism.
NMDA receptor activation is necessary for A2a‐induced phrenic motor facilitation
Authors: John Golder Francis
Published: June 2021

Adenosine 2a receptor agonists elicit persistent increases in phrenic motor output by transactivating the TrkB receptor near phrenic motoneurons. Our working model proposes that A2a‐induced TrkB activation strengthens glutamatergic synapses onto phrenic motoneurons. Activation of the glutamate NMDA receptors have been implicated in other forms of phrenic motor plasticity. Thus, we hypothesized that NMDA receptor activation also is necessary for A2a‐induced phrenic motor facilitation. Adult male Sprague Dawley rats were anesthetized with urethane, mechanically ventilated, neuromuscularly paralyzed, and bilaterally vagotomized. An catheter was positioned with the tip overlying the C4 spinal segment for intrathecal drug administration. Phrenic motor output was recorded before, during, and after intrathecal drug administration. Baseline phrenic motor output was established at 3 mmHg above an individual's apneic threshold. MK801, an NMDA receptor antagonist, was injected intrathecally (dose range 0.1, 1.0, 10.0, and 100 μM) 30 min before intrathecal administration of the A2a receptor agonist CGS 21680 (50 μM). MK801 dose‐dependently blocked A2a‐induced phrenic motor facilitation. The dose‐response curve was classically sigmoidal with an IC50 of 0.8 μM for MK801. These data demonstrate that NMDA receptor activation is necessary for A2a‐induced phrenic motor facilitation.
Is microglial activation necessary for phrenic long‐term facilitation following acute intermittent hypoxia?
Authors: Vinit Stephane , Mitchell Gordon S.
Published: June 2021

Microglial activation is critical for some forms of neuroplasticity in the spinal dorsal horn, such as the induction of chronic pain (Coull et al., 2005). Microglia regulate neuronal function in multiple ways, including the production of growth/trophic factors such as Brain Derivated Neurotrophic Factor (BDNF). BDNF is also critical for respiratory plasticity in the phrenic motor nucleus, such as phrenic long‐term facilitation (pLTF) following acute intermittent hypoxia (Baker‐Herman et al., 2004). Thus, we wondered if microglial activation plays a role in the induction and/or maintenance of pLTF. Since the antibiotic minocycline prevents microglial activation, we tested the hypothesis that minocycline would block pLTF following acute intermittent hypoxia in anesthetized rats. In anesthetized (urethane), paralyzed, vagotomized and ventilated rats, integrated phrenic nerve activity was measured in rats: 1) pretreated with minocycline (3 hrs before protocol; 10 mg i.v.) or 2) pretreated with a vehicle injection. 60 min post‐intermittent hypoxia, similar pLTF was observed in both sham and minocycline treated rats (~45% baseline in both groups). Thus, our data provide no evidence for microglial involvement in pLTF following acute intermittent hypoxia, although these findings do not rule out microglial involvement in longer‐lasting forms of respiratory plasticity.

Supported by NIH HL69064 and HL80209.
Role of eNOS in the Regulation of Substrate Metabolism during Exercise
Authors: Lee‐Young Robert , Bracy Deanna , James Freyja , Ayala Julio , Wasserman David
Published: June 2021

Endothelial nitric oxide synthase (eNOS) participates in the regulation of a blood flow and muscle mitochondrial function. The functional importance of eNOS was determined in mice with full (−/−) or partial (+/−) deletion of eNOS and wild‐type (+/+) littermates using treadmill exercise to amplify cardiovascular and metabolic responses. Five days after catheterization, 5h fasted mice performed a maximum of 30min of exercise (16m/min). At 5min, 2‐[14C]‐deoxyglucose and [3H]‐bromopalmitic acid were injected into the jugular vein to determine tissue‐specific glucose (Rg) and fatty acid (Rf) uptake. At the end of exercise microspheres were injected into the carotid artery to determine % cardiac output to the gastrocnemius (%QG). Running time was impaired in eNOS−/− mice (21±2 vs. 29±1 and 30±0min for eNOS+/− and eNOS+/+), as was the increase in %QG (2±1 vs. 3±1 and 6±1 fold). Muscle and liver glycogenolysis was greatest in eNOS−/− mice, as were plasma lactate levels (11±1 vs. 4±1 and 3±0mM). Despite elevated glycogenolysis eNOS−/− mice were hypoglycemic at the end of exercise (4±1 vs. 11±1 and 14±1mM) due to accelerated muscle glucose uptake (gastrocnemius Rg = 92±31 vs. 23±7 and 20±6μmol/100mg/min). Rf (4.4±1.4 vs. 2.8 ±0.6 and 2.5±0.4 μmol/100mg/min) was also accelerated. In conclusion, eNOS regulates %QG in a dose dependent manner with ~25% of QG being eNOS‐independent. Ablation of eNOS markedly increases Rg and Rf. The four‐fold increase in plasma lactate observed in eNOS−/− mice suggests that a high percentage of the substrate consumed by muscle does not undergo mitochondrial oxidation.
Investigation of the Potential for Intravascular Bubbles in Repetitively Diving Dolphins
Authors: Houser Dorian , Dankiewicz Lois , Stockard Torre , Ponganis Paul
Published: June 2021

Nitrogen (N2) bubbles resulting from behavioral responses to SONAR exposure have been postulated as a mechanism contributing to beaked whale strandings. To test whether N2 uptake during repetitive breath hold diving is sufficient to produce vascular bubbles in odontocetes, two bottlenose dolphins (Tursiops truncatus) were trained to perform sequences of 10–12 dives to 30, 50, 70 or 100 m. A ∼1 minute surface interval occurred between dives. A ∼90‐s wait on a biteplate placed at depth was performed on each dive to maximize time at depth. Performed dive sequences were predicted to produce muscle N2 saturations in excess of 500% upon surfacing from the final dive. Following the dive sequence, a 1 to 5 min, ultrasound bubble inspection of the portal or innominate vein was conducted. On several trials, serial voluntary blood samples were taken from the fluke and used to assess blood N2 partial pressures (PN2) via a Van Slyke technique. No vascular bubbles were observed in any of the post‐dive ultrasound inspections. Post‐dive blood PN2 values were not significantly elevated above those of a dolphin at rest. Ultrasound results suggest that for bouts of repetitive, prolonged dives up to 100‐m depth, the accumulation of N2 is insufficient to generate asymptomatic intravascular bubbles in bottlenose dolphins. (This research was funded by the U.S. Office of Naval Research and the International Association of Oil and Gas Producers.)