bluuebunny/crossref_metadata_2025 huggingface dataset:
Neuroendocrine peptides neuropeptide‐Y (NPY) and peptide‐YY (PYY) suppress Cl secretion and K secretion in guinea pig distal colon through action at Y2‐receptors
Authors: Halm Susan T , Zhang Jin , Halm Dan R
Published: June 2021

Electrogenic Cl and K secretion in isolated mucosa from guinea pig distal colon measured as short‐circuit current (Isc) and transepithelial conductance (Gt) were stimulated by epinephrine (epi), prostaglandin‐E2 (PGE2) and carbachol (CCh). NPY and PYY inhibited by 60% Cl secretion activated by either PGE2 or PGE2+CCh with EC50's of 16nM and 6nM, respectively. Neither peptide markedly inhibited the transient component of the PGE2+CCh response. Immunoreactivity (IR) for NPY was present in enteric ganglia and in proximity with crypts. Basolateral membranes of colonic crypt and surface epithelial cells had distinct IR for neuropeptide‐Y receptors Y1 and Y2. Receptor expression was supported further by immuno‐blot showing bands at molecular weights consistent with monomer and oligomer. Sub‐type selective antagonists BVD10 (Y1) and BIIE0246 (Y2) indicated that secretory suppression occurred through Y2 receptors. The Y2 selective peptide PYY(3–36) also suppressed Cl secretion with a EC50 of 9nM. BIIE0246 addition increased Isc and Gt during PGE2 or PGE2+CCh activation consistent with the presence of in vitro released PYY or NPY. PYY addition increased the epi EC50 to 18nM compared with BIIE0246 treated tissues, 4nM. Thus, PYY or NPY suppressed Cl secretory capacity and desensitized the adrenergic K secretory response, providing a negative feedback for secretory activation. [supported by NIH, DK65845]
Decreased eNOS activity induced by prenatal hypoxia results from abnormal interactions between eNOS and its regulatory proteins in adult sheep pulmonary arteries
Authors: Liu Jie , Gao Yuansheng , Negash Sewite , Tian Jia , Longo Lawrence D , Raj J. Usha
Published: June 2021

We previously reported that prenatal hypoxia resulted in persistent abnormalities in endothelium‐dependent relaxation responses of pulmonary arteries in adult sheep associated with unchanged eNOS protein expression but decreased eNOS activity (Faseb J. 21:A1203, 2007). To further elucidate the mechanisms responsible for the impairment of eNOS activity induced by prenatal hypoxia, we examined the regulatory proteins of eNOS and their interactions with eNOS by immunoprecipitation and immunoblotting analysis. Our results demonstrated that protein expression of caveolin–1, calmodulin and heat shock protein 90 did not change in prenatally hypoxic pulmonary arteries compared to controls. However, the interaction between eNOS and caveolin–1 was significantly increased, indicating that eNOS was tightly coupled with caveolin–1. The interactions of eNOS with calmodulin and heat shock protein 90 were greatly decreased, indicating that eNOS was dissociated from calmodulin and heat shock protein 90. Furthermore, phosphorylation of Ser 1177 and dephosphorylation of Thr495 of eNOS were significantly decreased in prenatally hypoxic pulmonary arteries, indicating that activation of eNOS was decreased. In conclusion, prenatal hypoxia impairs normal coupling between eNOS and its regulatory proteins and results in decreased eNOS activity. (Grants: NHLBI # HL 059435 and HL 075187).
Platelet activating factor (PAF) receptor expression is increased in lungs of neonatal rats with hypoxia‐induced pulmonary hypertension
Authors: Ibe Basil O. , Abdallah May F. , Nwafor‐Anene Victoria , Raj J. Usha
Published: June 2021

Endogenous PAF contributes to high vasomotor tone in the fetal pulmonary circulation and has been implicated in the pathogenesis of chronic‐hypoxia (CH)‐induced pulmonary hypertension (PH). We studied the effect of CH on neonatal rat (pup) body weight, right ventricle/left ventricle + septum ratio (RV/LV+S), and lung PAF receptor (PAF‐r) protein expression by Western blotting. Pups were placed in an air‐tight chamber ventilated with 13% oxygen (hypoxia) or room air (normoxia) from 1d to 22d of age. Three groups were studied (12 pups in each group): Group1, pups in Hypoxia; Group2, pups in hypoxia given 5mg/kg WEB 2170 daily, a PAF‐r antagonist, (Hypoxia +WEB); Group3, pups in Normoxia. We found that: a) Pups in Hypoxia gained 50% less weight than pups in Normoxia; WEB treatment in Hypoxia did not improve weight gain; b) RV/LV+S in Hypoxia group was highest at 0.79; 2‐fold higher than in WEB + Hypoxia and Normoxia groups; c) PAF‐r protein expression in lungs of pups in Hypoxia was >3‐fold higher than in WEB+Hypoxia and Normoxia groups. Our findings show that chronic hypoxia induces PAF‐r expression in lungs and suggest that increased PAF‐r expression may be responsible for the right ventricular hypertrophy and PH. Inhibition of PAF effects with a PAF‐r antagonist, WEB, though it did not improve weight gain of the pups in hypoxia, prevented RV hypertrophy in the pups. HL‐077819.
A novel constitutively active isoform of cGMP‐dependent protein kinase I (PKGI) is localized in the nucleus of vascular smooth muscle cells (VSMC)
Authors: Roberts Jesse David , Sugiura Takahiro , Nakanishi Hidehiko
Published: June 2021

Objective: Pediatric pulmonary vascular disease (PVD) is associated with excessive VSMC proliferation. Because recent studies suggest that nitric oxide attenuates PVD and VSMC proliferation by stimulating PKGI activity, the mechanisms affecting PKGI's nuclear localization were investigated.

Methods: PKGI was mapped to intracellular compartments using immunologic and biochemical methods and its catalytic activity was determined using a PKGI‐specific peptide substrate. The domain structure of nuclear PKGI was analyzed using antibodies and detailed using Edman‐based protein sequencing.

Results: PKGI immunoreactivity was detected in the nuclei of VSMC, where its activity was cGMP‐independent. Immunoblotting revealed that the nuclear PKGI isoform (PKGIγ) is 18 kDa smaller than the cytosolic one and lacks a NH2‐terminal dimerization domain, which tethers it to cytosolic proteins. The PKGI dimerization domain was observed in the perinuclear region where it colocalized primarily with the endoplasmic reticulum. Sequencing of immunopurified endogenous nuclear PKGIγ confirmed the absence of a dimerization domain and revealed that its putative scissile amino acids are not encoded by DNA located near typical RNA splice donor‐acceptor guidance nucleotides.

Conclusions: A novel constitutively active isoform of PKGI that appears to result from PKGI proteolytic processing is localized in the nuclei of VSMC.
Acute regulation of angiotensin II pressor response by heme oxygenase
Authors: Jackson Keith E , Jackson Debra W
Published: June 2021

Angiotensin II (AngII) induction is a well established model of hypertension with associated enhanced oxidative stress and increase in heme oxygenase activity. Vascular and renal tissues express heme oxygenase, which metabolizes heme to form biliverdin, free iron, and carbon monoxide (CO). CO has been demonstrated to relax vascular smooth muscle and to inhibit nitric oxide synthase. The current study examines the potential of the heme‐heme oxygenase carbon monoxide system to regulate acute AngII mediated increases in blood pressure. In vivo studies were conducted on Inactin anesthetized male Sprague Dawley rats (250–300g) on normal and high Zinc (1mM) diets. A dose response to AngII (5, 10, 20, 25ng/Kg, IV) was conducted to maintain a sustained elevation in blood pressure for an hour and half experimental period. Hemodynamic function was examined before and after acute administration of an inhibitor of CO production, zinc protoporphyrin‐IX (ZnPP; 30μmol/Kg, IV), a precursor for CO production, delta‐aminolevulinic acid (DALA; 40μmol/Kg, IV) or vehicle during AngII co‐infusion. A dose of 5ng/Kg AngII was observed to produce a 30mmHg sustained increase in blood pressure. In AngII hypertensive animals, ZnPP co‐infusion significantly attenuated the AngII increase in blood pressure. DALA or vehicle co‐infusion with AngII had no significant effects on the AngII mediated increase in blood pressure. In animals fed a high Zinc diet for 2 weeks, AngII infusion did not significantly increase blood pressure. This study demonstrates that an inhibitor of CO production can significantly attenuate the AngII mediated increase in blood pressure, while a precursor of CO production had no significant effects.

Support: NIH F32 HL76001
EFFECTS OF VITAMIN C CHRONIC TREATMENT ON BLOOD PRESSURE, SYMPATHETIC ACTIVITY AND GENE EXPRESSION IN THE RVLM IN RENOVASCULAR HYPERTENSION
Authors: Barbosa Oliveira‐Sales Elizabeth , Emy Nishi Erika , Aparecida Boim Mirian , Sterman Dolnikoff Miriam , Toledo Bergamaschi Cássia , Ribeiro Campos Ruy
Published: June 2021

Recently we showed that acute Vitamin C (Vit C) administration (intravenous or directly into the RVLM) caused a significant decrease in mean arterial pressure (MAP) and renal sympathetic nerve activity (RSNA) in 2K‐1C, but not in control rats. Moreover, we found that mRNA expression of NAD(P)H oxidase subunits (p47phox and gp91phox) was greater in 2K‐1C compared to the control group in the RVLM and CuZnSOD expression was similar in both groups. However, the chronic effects of Vitamin C (Vit C) in these parameters are unknown. Therefore, here we treated rats with Vit C (150 mg/kg/day) by gavage for 7 days and MAP, RSNA and mRNA expression of NAD(P)H oxidase subunits (p47phox and gp91phox) and CuZnSOD in the RVLM in the 2K‐1C rats was analyzed. The mRNA expression was analyzed by real‐time PCR. The Vit C resulted in a higher fall in MAP in 2K‐1C than in control group (2K‐1C, 22 ± 7%/C, 11 ± 3%) followed of a significant increase in RSNA (41± 2%) only in control group. The NAD(P)H oxidase subunits expression reduced significantly in both groups treated with Vit C in RVLM. In contrast, CuZnSOD expression was unchanged in all groups. The results support the idea that an increase in oxidative stress within the RVLM probably plays a major role in maintaining hypertension and sympathetic drive in 2K‐1C.

Supported by FAPESP.
Muscle interstitial norepinephrine and nucleotides during exercise and arterial ATP infusion
Authors: Mortensen Stefan P , González‐Alonso José , Saltin Bengt , Hellsten Ylva
Published: June 2021

Plasma ATP has been suggested to play a role in the skeletal muscle hyperaemia during muscle contractions, while interstitial ATP may contribute to the increase sympathetic nerve activity, by stimulating muscle afferents. We measured leg hemodynamics and muscle interstitial NE, ATP, ADP, AMP and adenosine during intra‐arterial ATP infusion (0.42±0.04 and 2.26±0.52 μmol min−1;mean±SEM) and one‐legged knee extensor exercise (18±1 and 37±1W) in 7 healthy, male subjects (24±2 years, 76±3 kg). ATP infusion increased leg blood flow (LBF) from 0.4±0.0 l min−1at baseline to 2.8±0.3 (0.42 μmol ATP min−1) and 4.5±0.4 (2.26 μmol ATP min−1) l min−1, while LBF during exercise increased from 0.4±0.1 at rest to 3.6±0.3 (18W) and 4.9±0.5 (37W) l min−1. Interstitial [NE] remained unchanged during the low ATP infusion (0.5±0.2 and 0.7±0.1 μM at baseline and ATP infusion, respectively), but increased to 2.0±0.5μM during the high ATP infusion rate, while [ATP], [ADP], [AMP] and [ADO] remained unchanged during both ATP infusions. Exercise increased interstitial [NE], [ATP], [ADP], [AMP] and [ADO] from 0.5±0.0, 2.0±0.7, 0.06±0.01, 0.06±0.01, 0.2±0.0 and 0.1±0.0 μM, respectively, to 2.9±0.7, 5.61±1.25, 4.80±1.30, 4.1±1.7 and 0.8±0.0 μM, respectively (P<0.05). These results indicate that ATP in plasma exerts its vasodilatory effects directly on the endothelium as arterially infused ATP and its breakdown products do not appear to reach the interstitium. Furthermore, the relationship between interstitial NE and ATP suggests that ATP may play a role in the increased sympathetic nerve activity observed during exercise.

Supported by Lundbeck Foundation
Anatomical and Physiological Characteristic of the Human Venous Valve
Authors: Janssen Herbert F , Buescher Clifford D , Janssen Ryan P , Skeen Nicholas P , Wijeratne Nilmini S , Hoo Karlene A
Published: June 2021

It has long been recognized that the venous valves play a significant role in the normal function of the vascular system. In addition to their beneficial role, they also serve as the major site for deep venous thrombosis (DVT) formation. We are attempting to better understand the physiological properties of the venous valve by measuring cross‐sectional slices through latex casting made from cadaver veins. A length of vein harvested from a fresh cadaver was filled with room temperature vulcanizing (RTV) silicone that was then allowed to cure. The tissue was stripped away leaving the underlying mold the vein's interior. Serial cross‐sections of the mold were made and photographed using a Cannon 10D equipped with an EF 75–300mm lens. These images were analyzed digitally using MatLab® Image Analysis toolbox (Natik, MA). The analysis provided interior vein measurements of a segment of vein wall including the length of the valve pocket. Additional images captured photographically were used to estimate the dimensions of the valve leaflets and identify their site of attachment on the vein. Using this information, an in silico 3‐D model of the vein segment was reconstructed using Inventor© software. These measurement techniques have provided a detailed evaluate the valve's normal anatomy and has allowed us to better understand and simulate the problems that develop after valve leaflet damage occurs in pathological conditions.
Local and distant manifestations of the venoarteriolar reflex
Authors: Kurtev Angel Dimitrov
Published: June 2021

The venoarteriolar reflex (VAR) is considered a good example of local vasomotor control depending on both myogenic and neural mechanisms. The presence of a neural component suggests generalized effects in‐so‐far as the sensory input may reach systemic vasomotor regulatory centers. The objective of the present study was to find out whether such distant effects are really present. In order to do this we compared the changes in the blood flow of the contralateral arm while inducing the VAR in the ipsilateral one. The VAR was elicited by a local orthostatic stress – lowering of the ipsilateral arm 40 cm below heart level. During the procedure the subject was in supine position with the other arm at heart level. Index finger photoplethysmography from both arms was simultaneously recorded before, during, and after the orthostatic stimulation using PPG100C transducer coupled to a TSD200 amplifier (Biopac Systems Inc.). Precordial single channel ECG was also recorded. The VAR was evaluated by the changes in the amplitude of the photoplethysmography.

The results showed the existence of contralateral VAR although less pronounced and more transient than the ipsilateral one. It is concluded that the local orthostatic stress does evoke more widespread changes affecting distant vascular beds.

The present study was supported by the Research Committee of Saba University School of Medicine.
cGMP regulates mechanosensitive TRPV4 channels in lung endothelial cells
Authors: Yin Jun , Wu Songwei , Neye Nils , Kuppe Hermann , Kuebler Wolfgang M
Published: June 2021

Previously, we and others have demonstrated that endothelial Ca2+ influx via activated TRPV4 channels promotes hydrostatic edema formation. Here, we used real‐time microscopy and patch clamp analyses to study the regulation of this mechanosensitive channel by NO/cGMP pathway. In vivo, we tested the ability of sildenafil which prevents the degradation of cGMP by inhibition of phosphodiesterase 5 to attenuate hydrostatic edema in a rat model of left anterior descending coronary artery (LAD) occlusion.

In isolated perfused rat lungs, elevation of left atrial pressure from 5 to 15 cmH2O induced an increase in endothelial Ca2+ concentration ([Ca2+]i) from 105±6 to 173±10 nM. This [Ca2+]i response was blocked by TRPV4 inhibitor ruthenium red (RuR; 1 μM), NO donor GSNO (250μM), or cGMP analog 8Br‐cGMP (100 μM) (n=5 each, p<0.05). Activation of TRPV4 by 4αPDD (10 μM) induced an [Ca2+]i increase from 103±4 to 172±20 nM which was again blocked by RuR and 8Br‐cGMP (p<0.05). In pulmonary microvascular endothelial cells, 4αPDD activated an inward current that reversed near +20 mV and was blocked by 8Br‐cGMP. In vivo, sildenafil (1 mg/kg bw) reduced lung wet/dry weight ratio and improved arterial pO2 after LAD occlusion.

Our data demonstrate that cGMP attenuates Ca2+ influx via TRPV4 in lung microvascular endothelial cells. This regulatory pathway may present a new therapeutic target for the treatment of hydrostatic lung edema.
Epithelial Sodium Channels Interact with Anionic Phospholipids
Authors: Chou Chu‐Fang , Liang You‐You , Ma He‐Ping
Published: June 2021

We and others have previously shown that the epithelial sodium channel (ENaC) is stimulated by anionic phospholipids (APs) such as phosphatidylinositol‐4,5‐bisphosphate (PI(4,5)P2) and phosphatidylinositol‐3,4,5‐trisphosphate (PI(3,4,5)P3). Although functional studies suggest that β‐ and γ‐ENaC interact with PI(4,5)P2 and PI(3,4,5)P3, respectively, however, whether ENaC selectively binds to PI(4,5)P2 and PI(3,4,5)P3 over other APs has not been carefully examined. By using the newly developed PIP Strips™ and different epithelial cell models, we found that the endogenous α‐ENaCs in human H441 lung epithelial cells and in mouse cortical collecting duct mpkCCDc14 cells bind to APs with a slight preference to APs containing less phosphoric residue. The endogenous β‐ and γ‐ENaCs also bind to APs with a similar binding pattern. To complement the possible inefficiency of some ENaC antibodies, we respectively expressed fluorescent protein‐tagged rat α‐, β‐, and γ‐ENaCs either in HEK293 cells or in mpkCCDc14 cells and detected each ENaC subunit with antibody against the attached fluorescent protein. Consistent with the data by using antibodies against ENaCs, all three subunits of the expressed rat ENaCs bind to APs, especially to those with less phosphoric residue rather than PI(4,5)P2 and PI(3,4,5)P3. These data suggest that none of ENaC subunit selectively binds to PI(4,5)P2 or PI(3,4,5)P3.
Differential Interaction of Sulfhydryl‐Reactive Reagents with hOAT1 and hOAT3: Cysteine 440 of hOAT1 is Accessible from the Hydrophilic Pore
Authors: Astorga Bethzaida O , Wright Stephen H , Pelis Ryan M
Published: June 2021

The organic anion transporters 1 (OAT1) and 3 (OAT3) are important for the renal elimination of organic anions. To better understand their structures the accessibility of native cysteines to membrane‐impermeant sulfhydryl‐reactive reagents was examined. When stably expressed in Chinese hamster ovary cells transport of paminohippurate by OAT1 was more sensitive to HgCl2 (IC50, 89 ± 22.4 μM vs 621 ± 111.1 μM) and several methanethiosulfonate reagents than OAT3 (estrone sulfate transport). Maleimide‐PEO2‐biotin and MTSEA‐biotin interacted preferentially with OAT1 in surface biotinylation experiments, further suggesting that cysteines of OAT1 are more accessible than those of OAT3. To determine if any of the 13 cysteines are accessible in OAT1, several cysteines located in helices that comprise the hydrophilic pore (site of substrate/protein interaction) were targeted for site‐directed mutagenesis, resulting in five mutants of OAT1 (C189A, C206S, C221A, C341A and C440A). All of the mutants transported p‐aminohippurate and glutarate. Interaction of maleimide‐PEO2‐biotin and MTSEA‐biotin with OAT1 was only reduced in the C440A mutant (~40–70%). Transport by the C440A mutant, but not the other mutants, was less sensitive to HgCl2 (IC50, 529 ± 126.9 μM) than wild‐type OAT1 (IC50, 101 ± 15.1 μM). These data suggest that C440 of OAT1 is exposed to the aqueous milieu of the pore. (DK58251, DK075242 and DK074022).
Transport of mutagenic vital dye ethidium bromide by the human organic cation transporters hOCT2 and hOCT3
Authors: Lee Wing‐Kee , Ciarimboli Giuliano , Koepsell Hermann , Thevenod Frank
Published: June 2021

Ethidium bromide (EtBr) and propidium iodide (PI) are widely used in molecular biology as DNA and also as necrosis‐markers. EtBr is not only mutagenic, it may also be carcinogenic, teratogenic or very toxic when inhaled or ingested and therefore poses a major safety problem. Since EtBr strongly stained nuclei in HEK293 cells stably overexpressing human organic cation transporter 2 (hOCT2) with no morphological signs of cell damage or death, vital dye accumulation by HEK293 cells overexpressing known hOCT isoforms was determined by fluorescence microscopy and quantified using a fluorescence microplate reader. All values are mean±SEM. Significant time and concentration‐dependent accumulation of EtBr by hOCT2 (Km=3.4±1.3 μM, n=4) and hOCT3 (Km=2.5±0.4 μM, n=6), but not by hOCT1 cells or HEK293 cells, was observed at 37°C. Conversely, PI was not taken up by any hOCT isoform. The organic cations cimetidine and quinine but not choline competed accumulation of 2.5 μM (IC50: 5.7 mM and 61 μM, respectively) and 12.5 μM (IC50: 10.6 mM and 152 μM, respectively) EtBr in hOCT2 and hOCT3 (IC50 for 12.5 μM EtBr: 1.4 mM and 59 μM, respectively) cells after 5 min. The data indicate that EtBr is transported by hOCT2 and hOCT3. Moreover, acute EtBr intoxication could be prevented by applying high concentrations of less harmful hOCT2 and hOCT3 substrates, such as quinine or cimetidine.

Funded by DFG TH 345/8‐1 and TH 345/10‐1
Suppression of influenza A proliferation by inhibiting Myosin Light Chain phosphorylation
Authors: Haidari Mehran , Ali Muzammil , Zhang Wei , Madjid Mohammad , Casscells Samuel W
Published: June 2021

Emergence of resistance to the drugs against influenza and inadequate flu vaccination coverage in human are cause for concern. Cytoskeleton plays crucial role in endocytosis of viruses by cells. We sought to determine if inhibition of Myosin Light Chian (MLC) phosphorylation blocks influenza virus proliferation. Our studies indicate that infection of cells with influenza increases the function of RhoA and Rho kinase proteins, accompanied by an increase in MLC‐phosphorylation and assembly of actin stress fibers. The inhibition of MLC phosphorylation via exposure of cells to Rho Kinase, MLC‐kinase, and protein kinase C inhibitors as well as statins resulted in suppression of influenza virus. In addition, Chelation of intracellular calcium or inhibition of calmodulin blocked the virus proliferation. Exposure of cells to all of the above mentioned inhibitors led to a reduction in MLC phosphorylation and actin stress fiber formation. In contrast, exposure of the cells to a protein kinase C agonist (Phorbol Myristate Acetate) led to induction of MLC phosphorylation and facilitation of influenza proliferation. Mechanism for inhibition of virus proliferation by all of the afore mentioned inhibitors including statins was blocking of influenza virus entry to the nucleus of host cell. Our studies for the first time provide compelling evidence for the role of MLC phosphorylation in proliferation of influenza virus.
A study of cell damage caused by bubble motion in a circular‐lumen endothelialized model of a microvessel
Authors: Wong Zheng Zheng , Fowlkes J. Brian , Bull Joseph L.
Published: June 2021

Gas embolotherapy, being researched as a potential therapy for cancer, involves selective, acoustic vaporization of liquid perfluorocarbon droplets into gas bubbles near a tumor to achieve occlusion of blood flow and “starvation” of the tumor. It is hypothesized that collateral endothelial cell damage is caused by the motion of these bubbles within the vicinity of the tumor, which might be desirable in assisting the tumor death. A microchannel with circular lumen was fabricated by curing a liquid elastomer Polydimethylsiloxane in a negative mold positioned on a device comprising a thin wire tightened between two fixed posts. The circular lumen has no sharp corners that might affect bubble properties. Endothelialized with HUVEC cells, the 80‐micron microchannel served as a model of a large arteriole. Air bubbles between 200–500 microns in length were moved through the model at rates of 0.1–2 cm/s. Cell viability was assessed with calcein AM and ethidium homodimer‐1 via fluorescent microscopy. The results show that bubble motion can cause significant damage to endothelial cells. Cells damaged by the bubble motion detached from the wall. The steep pressure gradient at the bubble front would be the most likely cause of cell damage. Ongoing work will aim to quantify the cell damage due to bubble size and speed. This work was supported by grants from NIH (R01EB006476 and EB003541) and a scholarship from A*STAR (Singapore).
BMAL1 and CLOCK proteins directly bind to the core enhancer of the Myod1 promoter
Authors: zhang xiping , McCarthy John J , Esser Karyn A
Published: June 2021

The molecular clock regulating circadian rhythms is a network of transcriptional‐translational feedback loops that drive rhythmic expression of genes over a 24‐h period. BMAL1 and CLOCK are two of the main transcription factors that regulate the molecular clock mechanism. Myod1 (myogenic differentiation 1) was identified as having a circadian pattern of expression in adult skeletal muscle. The primary objective of this study was to determine whether CLOCK and BMAL1 bind the Myod1 promoter in a temporal manner. Mice were entrained for 2 weeks in 12 hour light /12 hour dark and then hindlimb muscles were collected every 4 hours for 24 hours. The chromatin immuno‐precipitation (ChIP) assay with nuclear extracts from mouse muscle was performed. We tested binding of BMAL1, CLOCK, and RNA Polymerase to the core enhancer (CE) and distal regulatory region (DRR) of the Myod1 promoter. Results from these studies determined that BMAL1 and CLOCK did bind to the Myod1 promoter but only to the upstream core enhancer CE and not the DRR. Binding of BMAL1 and CLOCK at the CE was also found to oscillate over the course of the circadian cycle. We conclude that Myod1 is a direct clock controlled gene with BMAL1 and CLOCK regulating Myod1 mRNA levels via direct binding to the CE element of the promoter.

This research was supported by National Institute of Health grants KAE (AR045617) and JJM (AR053641).
Very low prevalence of acute mountain sickness in teenagers after rapid ascent to 3454 m
Authors: Bloch Jonathan , Duplain Herve , Rimoldi Stefano , Stuber Thomas , Kriemler Susi , Sartori Claudio , Scherrer Urs
Published: June 2021

Acute mountain sickness (AMS) is a frequent and debilitating complication of high‐altitude exposure. After a rapid ascent to tourist destinations located above 3000 m, as many as 60% of an adult population are affected. Circumstantial evidence suggests that children and teenagers may be even more susceptible to AMS than adults, but direct evidence is lacking. We, therefore, performed serial assessments of AMS (Lake Louise Score, LLS) in 48 teenagers (mean±SD age, 13.7±0.3 yrs; 20 girls and 28 boys) 6, 24, 48 hrs after arrival at the Jungfraujoch high‐altitude research station (3454 m) reached after a 2 hr ascent by train. The major new finding was that 6 hrs after arrival, AMS (LLS≥3) was present in only 22% of the subjects (mean score 4.1±0.4, range 3–7). The incidence of AMS was similar in both genders and steadily declined during the stay (17% at 24 hrs, 8% at 48 hrs). None of the subjects needed pharmacological treatment other than paracetamol. In conclusion, in teenagers the incidence of AMS after rapid arrival at an altitude where major high‐altitude tourist destinations are located world‐wide, is surprisingly low, benign, and resolves rapidly. These findings challenge the notion that teenagers are more susceptible to AMS than adults. They suggest that for the vast majority of teenagers, travel to these altitudes is safe and does not necessitate pharmacological prophylaxis for AMS.

Grant support: Swiss National Science Foundation.
Carotid body of transgenic mice with nicotinic ACh receptor a3 subunit deletion
Authors: Wei Edward , Lieu TinaMarie , Balbir Alex , Fujii Koichi , Zadzilka Nicole , Shirahata Machiko
Published: June 2021

The carotid body (CB) detects the arterial partial pressure of O2 and CO2. Previous studies suggested that nicotinic ACh receptors (nAChRs) play a role in these processes. We investigated if deletion of the nAChR α3 subunit influences gene expression, morphology and function of the CB in 2 week old mice. Real‐time PCR analysis was performed on CB from wild type (WT), heterozygous (HET), and knockout (KO) mice. Cholinergic traits (enzymes for ACh metabolism and receptors) were well preserved in all mice, except for the α3 subunit in KO mice. Dopaminergic traits and BK channels were down regulated in KO mice. Morphometry studies were performed in plastic embedded and serial‐sectioned CBs. The volumes and cell morphology between WT and HET CBs were similar. CB function was assessed by the ventilatory response to short exposures of 100% O2 and 15% O2 using whole body plethysmography. Both WT and HET have similar ventilatory responses to hyperoxia and hypoxia, hypoventilating in response to hyperoxia and hyperventilating in response to hypoxia. The results suggest that the α3 subunit is linked to the expression of BK channels and dopaminergic traits in the CB. Similarities between the phenotypes of WT and HET mice may be due to compensation occurring in the HET.

Supported by HL72293. Transgenic mice were originally provided by Dr. De Biasi, Baylor College of Medicine.
Effect of low calorie cranberry juice on the human glycemic response of type 2 diabetics.
Authors: Wilson Ted , Meyers Sara L
Published: June 2021

Diabetics have a high incidence of urinary tract infection and coronary heart disease, both of which may be positively affected by cranberry juice (CBJ) consumption. Euglycemia is a primary goal of nutritional therapy for type 2 diabetics, and fruit juice consumption can be problematic. Type two diabetic (n=14) responses to unsweetened low calorie CBJ (19 Cal/240 ml), normal calorie CBJ (120 Cal/240 ml), isocaloric low calorie sugar water, or isocaloric normal calorie sugar water were examined. Blood glucose peaked 30 minutes postprandially when normal calorie CBJ and control plasma glucose values were 242.7 ± 29.8 and 240.3 ± 35.8 (mg/dl) respectively and significantly greater than the low calorie treatment peak values of 152.4 ± 8.0 and 160.7 ± 10.1 for CBJ and control, respectively. The differences between the treatments remained significant 60 minutes, but not 120 minutes postprandially. Plasma insulin values 60 minute postprandially for normal calorie CBJ and control treatments were 30.84 ± 6.89 and 34.22 ± 8.48 (μU/ml) respectively and significantly greater than the low calorie treatment values of 15.68182 ± 4.585398 and 14.12857 ± 3.661446 for CBJ and control, respectively, but not 120 minutes postprandially. This study suggests that persons with type 2 diabetes can consume low calorie CBJ in a manner that promotes euglycemic control.
Regulation of vascular insulin signaling by protein tyrosine phosphatase 1B (PTP1B) in mouse models of obesity
Authors: Ketsawatsomkron Pimonrat , de Chantemele Eric Belin , Muta Kenjiro , Fulton David , Stepp David , Marrero Mario
Published: June 2021

Insulin resistance is a major risk factor in obese individuals, and PTP1B is a crucial negative regulator of insulin signaling in metabolic tissues but the role of PTP1B in the development of vascular insulin resistance is unknown. Previously, we have found that PTP1B impairs the insulin‐induced activation of anti‐mitogenic pathways (Akt) while enhancing insulin‐induced activation of mitogenic pathways (p42/44 MAPK) in cultured vascular smooth muscle cells. In this study we tested the hypothesis that deletion of PTP1B improves insulin‐induced activation of Akt while decreasing activation of p42/44 MAPK in the aorta of mouse models of obesity. On a high fat (35.5%) diet, PTP1B knockout (KO) mice were protected from weight gain and had improved insulin‐induced activation of Akt compared to those observed in aortae from wild type (WT) mice on the same diet. Activation of p42/44 MAPK was not statistically different between WT and PTP‐1B KO mice. In contrast, deletion of PTP1B did not correct weight gain in leptin receptor mutant (db/db) mice and did not improve the insulin‐induced activation of Akt in aortae of db/db mice. Moreover, insulin‐induced activation of p42/44 MAPK in aortae was not different in all groups. These data suggest that, in the absence of a functional leptin receptor, PTP1B does not contribute to vascular insulin resistance as assessed by activation of Akt. (NIH Grants HL58139 and DK616874)
The analysis of avian plasma for the glycation of serum albumin
Authors: Anthony Claire M. , Braun Eldon J.
Published: June 2021

Plasma glucose concentrations (Pglu) in humans are closely regulated at about 80–100mg%. If the Pglu levels reach 150mg% or greater for 4–6 weeks, the symptoms of Type 2 diabetes will begin to appear; peripheral vascular disease and/or retinal damage. These symptoms are the result of the glycation of serum albumin and a subsequent series of reactions that lead to formation of advanced glycation end‐products (AGEs). The AGEs bind to vascular tissues leading to pathologies that appear as symptoms of Type 2 diabetes. In contrast, to humans, the Pglu of birds ranges from 350–750mg%. The striking circumstance is that birds do not suffer the pathologies associated with Type 2 diabetes. In an attempt to gain an understanding of this discrepancy, we have begun experiments to analyze the interaction between glucose and the serum albumin of birds (experimental animal: Mourning doves). In particular, the analysis of this reaction under in vitro and in vivo conditions to determine the potential for avian albumin to become glycated and the amount of albumin normally occurring in one avian species.
Chronic exposure to glucagon impairs net liver glucose uptake by activating the gluconeogenic pathway
Authors: Mulligan Kimberly X , Chen Sheng‐Song , Santomango Tammy , Lacy D Brooks , Williams Phil , McGuinness Owen P
Published: June 2021

Elevated glucagon levels and impaired liver glucose uptake are commonly observed in stressed and diabetic patients. We observed that if dogs are placed on continuous (5 days) nutritional support the liver adapts to become a major consumer of glucose. The majority of the glucose is not stored as glycogen, but is released as lactate (i.e reverse Cori Cycle). We have recently reported (AJP 292: E928, 2007) that if glucagon (2.4 ng/kg/min) is chronically infused during nutritional support, it impairs net glucose uptake by the liver (5.1±0.4 vs. −1.0±0.4 mg/kg/min). This decrease was due to a rise in hepatic glucose production rather than to suppression of glucose uptake. In the present study we assessed mRNA expression in liver tissue (Affymetrix Canine genome 2.0 array). PEPCK and fructose‐1,6‐bisphosphatase‐1 expression were increased 11 fold and 3 fold, while the expression of glucokinase (GK) and acetyl‐CoA carboxylase (ACC) were decreased by 4.8 and 2.4 fold. CREB‐3 mRNA was increased 3 fold (positive regulator of PEPCK) and AtF‐3 decreased 5.4 fold (negative regulator of PEPCK). We are performing both rt‐PCR and westerns to confirm these observations. We also observed that the ratio of AMP/ATP (a negative regulator of ACC) increased 1.8 fold, while the total nucleotide pool did not change. In summary, the long term inhibition of liver glucose uptake by glucagon is mediated by the upregulation of gluconeogenesis. While many of the glucagon‐mediated effects can be explained by activation of the cyclic‐AMP pathway, changes in AMP may also contribute to these effects. (Support DK43748 and ADA)
Acute activation of the mammalian target of rapamycin complex 1 does not impair in vivo insulin action in mice
Authors: Berk Eli Zachary , Reynolds Thomas Henry
Published: June 2021

Recent cell culture studies have shown that the mammalian target of rapamycin complex 1 (mTORC1) plays a role in the development of insulin resistance. However, the ability of mTORC1 to produce whole‐body insulin resistance is not established. The purpose of the present study was to determine the effect of acutely activating mTORC1 with leucine on in vivo insulin action. C57BL/6 male mice (n = 20) received intraperitoneal injections of leucine (1.35 g/Kg body weight) or an equivalent volume of saline. Mice were subjected to an intraperitoneal insulin‐assisted glucose tolerance test (IAGTT) that involved injections of glucose (1 mg/Kg) and insulin (0.15 U/Kg) at 15 and 20 minutes following leucine or saline administration, respectively. Blood glucose was assessed by sampling tail vein blood (~ 5 ul) at baseline and 15, 30, 45, and 60 minutes following the injection of glucose. The blood glucose values were used to calculate the area under the IAGTT curve. The area under the IAGTT curve was similar in leucine treated mice compared to controls (7805 ± 675 vs. 7513 ± 462, p = 0.753), indicating that insulin sensitivity was not altered. In skeletal muscles of mice injected with leucine, mTORC1 signaling was elevated compared to mice that received saline. These results suggest that acute treatment with leucine appears to activate mTOR signaling but does not produce insulin resistance in mice.
Insulin signaling in Goto‐Kakizaki rat model of type II diabetes
Authors: Kelly Aisha Imani , Portik‐Dobos Vera , Mezzetti Erin , Ergul Adviye
Published: June 2021

Aberrant insulin signaling is a hallmark of insulin resistance. Decreased phosphorylation of insulin receptor substrate (IRS‐1) proteins and Akt, in particular, results in reduced glucose uptake by the tissues thereby contributing to the insulin resistant state. We have previously shown that insulin‐mediated aortic relaxation is reduced in Goto‐Kakizaki (GK) rat model of Type II diabetes. This study tested the hypothesis that blunted insulin signaling contributes to impaired vascular function and glucose metabolism in this lean model of diabetes. In GK rats, blood glucose was higher (168 ± 5 vs. 108 ± 3 mg/dl, p<0.05) and hyperinsulinemic euglycemic clamp studies showed decreased glucose utilization indicative of insulin resistance. Assessment of total and phosphorylated (p) IRS‐1 and Akt expression in aorta, liver, and skeletal muscle showed that IRS‐1 activation (pIRS‐1/total IRS‐1) was significantly decreased in all tissues as compared to Wistar controls (n= 4–5, p<0.05). Similarly, Akt activation (pAkt/total Akt) was reduced in the aorta and the liver (p<0.05). Akt activation in skeletal muscle, however, was significantly increased in GKs (p<0.05), suggesting a possible compensatory mechanism to promote peripheral glucose utilization.
Mechanisms of hyperglycemia‐ and hyperinsulinemia‐induced adiponectin resistance in cardiomyocytes
Authors: Park Min , Palanivel Rengasamy , Sweeney Gary
Published: June 2021

Current literature has indicated that low adiponectin levels in obesity and type2 diabetes are a potential risk factor for heart failure, yet the mechanisms responsible for this remain unclear. Since alterations in myocardial substratae metabolism precede cardiac dysfuntion, in the present study we investigated the effect of globular (gAd) and full‐length multimeric adiponectin forms (fAd) on glucose and fatty acid uptake and metabolism. Differentiated H9c2 rat cardiomyocytes were pre‐treated with high glucose (HG: 25mM, 24h) or high insulin (HI: 100nM, 24h) followed by treatment with gAd (5μg/ml) or fAd (10μg/ml) for up to 2hr. Both gAd and fAd forms of adiponectin stimulated glucose and fatty acid uptake and oxidation, but there was no increase in glycogen synthesis and lactate production. The increase in glucose and fatty acid uptake was attenuated by HI and HG. We observed down regulation of adiponectin receptor1 (AdipoR1) and AdipoR2 mRNA expression by HI and HG in H9C2 cells. Recruitment of GFP‐APPL1 to plasma membrane was induced by adiponectin, although HI and HG did not alter APPL1 expression. The phosphorylation level of AMPK and ACC was stimulated by gAd and fAd, and this was significantly attenuated in HG and HI conditions. In summary, our results indicate that both hyperglycemia and hyperinsulinemia significantly diminish gAd and fAd functions on cardiomyocyte substrate metabolism, likely via decreasing receptor expression, ultimately leading to ‘adiponectin resistance’ in cardiomyocytes.
Knock down of TRPC3 decreases Ca2+ influx and insulin‐mediated glucose uptake in adult skeletal muscle
Authors: Lanner Johanna T , Bruton Joseph D , Assefaw‐Redda Yohannes , Zhang Shi‐Jin , Zhang Zhi‐Bin , Zhang Shi‐Li , Katz Abram , Westerblad Håkan
Published: June 2021

The involvement of Ca2+ in insulin‐mediated glucose uptake in skeletal muscle is uncertain. Here we study the possible role of Ca2+ influx via canonical transient receptor potential 3 (TRPC3) channels in insulin‐mediated glucose uptake. Experiments were performed on adult skeletal mouse muscle fibers. Ca2+ influx and glucose uptake were measured with fluorescent indicators and confocal microscopy. TRPC3 protein expression was knocked down using a novel technique where functionalized carbon nanotubes were used to transfect cells with small interfering RNA. The interaction between TRPC3 and the glucose transporter 4 (GLUT4) was studied with immunoprecipitation and immunofluorescence staining. Knock down of TRPC3 resulted in ∼ 80% decrease in insulin‐mediated glucose uptake. TRPC3 can be activated by diacylglycerol (DAG) and knock down of TRPC3 inhibited the DAG‐induced Ca2+ influx. TRPC3 and GLUT4 co‐immunoprecipitated and showed co‐localization in the proximity of the t‐tubular system, which is the major site of insulin‐mediated glucose transport. In conclusion, TRPC3 interacts functionally and physically with GLUT4 and Ca2+ influx through TRPC3 has a large impact on insulin‐mediated glucose uptake.
Microcirculatory effects of red blood cells with different hemoglobin oxygen affinity in hemorrhagic shock
Authors: Villela Nivaldo R. , Cabrales Pedro , Tsai Amy G. , Intaglietta Marcos
Published: June 2021

Autoregulation can be considered to be a balance between blood flow and O2 extraction. Local regulatory mechanisms prevent excessive local O2 supply to tissue upstream arteriolar vasoconstriction. Effects of resuscitation with red blood cells (RBCs) with low and high P50 (partial pressure of O2 at 50% Hb saturation) were studied with the hamster window chamber in a model of 1 h shock. Introduction of inositol hexaphosphate (IHP) and 5‐hydroxymethyl‐2‐furfural (5HMF) into the RBCs by electroporation decreased and increased the P50 to 10 and 50 mmHg respectively (normal p50 = 32 mmHg). The animals were resuscitated with 25% of blood volume infusion using either the low and high P50 RBCs. Systemic parameters, microvascular hemodynamics, and functional capillary density (FCD, the number of capillaries perfused per unit area), were measured up to 90 min after resuscitation. RBCs with high P50 (reduced Hb‐O2 affinity) reduced arteriolar diameter and flow, and increased the oxygen extraction. RBCs with low P50 (increased Hb‐O2 affinity) had higher levels of systemic O2 delivery and tissue pO2. In summary, resuscitation with RBCs with increased Hb‐O2 affinity maintained adequate microhemodynamic conditions and tissue PO2 while RBCs with reduced Hb‐O2 affinity produced arteriolar vasoconstriction followed by higher O2 extraction and lower tissue PO2.

Supported in part by Bolsista CNPq, HL076182 and HL076182.
O‐GlcNAc agonist treatment improves survival, reduces inflammation and organ damage 24 hours after trauma‐hemorrhage in rats
Authors: Nöt Laszlo G , Brocks Charlye A , Marchase Richard B , Chatham John C
Published: June 2021

We have shown that O‐GlcNAc (N‐acetyl‐glucosamine) agonists improve survival and reduce inflammatory response 2 hours after trauma‐hemorrhage (T‐H). The aim of the study was to evaluate the effects of O‐GlcNAc levels on survival, inflammation and organ damage 24 hours after T‐H. Fasted male rats were subjected to either sham surgery (SH) or (T‐H) and during the resuscitation phase received glucosamine (0.64g/kg, GN) or O‐(2‐Acetamido‐2‐deoxy‐D‐glucopyranosylidene)amino N‐phenyl Carbamate, (7mg/kg , PUGNAc) to increase O‐GlcNAc levels, or mannitol as control (CON). Survival was followed up for 24 hours. Both GN and PUGNAc increased 24 hours survival compared to controls (CON: 53%, GN: 85%, PUGNAc: 86%, logrank test, p<0.05). PUGNAc attenuated the T‐H induced increase in serum IL‐6 (SH: 8±6, CON: 181±36, PUGNAc: 42±22 pg/mL, p<0.05), ALT (SH: 89±19, CON: 297±56, PUGNAc: 125±21 IU, p<0.05), AST (SH: 503±218, CON: 1661±215, PUGNAc: 897±155 IU, p<0.05) and LDH (SH: 129±26, CON: 1499±311, PUGNAc: 357±99 IU, p<0.05); however, GN had no effect on serum parameters. PUGNAc but not GN maintained O‐GlcNAc levels in liver and lung and also attenuated the enhanced NF‐κB DNA binding and iNOS expression in the liver. These results demonstrate that increasing O‐GlcNAc levels with GN and PUGNAc improves 24 hr survival after T‐H; the different effects of GN and PUGNAc on tissue injury require further studies. NIH grant HL076165.
How many heart beats are enough? A systematic assessment of data length reduction on measures of heart rate variability and complexity
Authors: Rickards Caroline A. , Ryan Kathy L. , Muniz Gary W. , Moralez Gilbert , Convertino Victor A.
Published: June 2021

Indices of heart rate variability (HRV) and complexity have become popular for the assessment of autonomic function and the presence of various disease states. Standard recommendations for R‐R interval (RRI) analysis include a minimum of 256 beats for HRV and 800 beats for heart rate complexity. However, the sensitivity and reliability of these indices to reduced data length have not been fully delineated. We therefore systematically determined minimum data length requirements for a variety of common HRV and complexity indices. ECG was recorded in resting, supine, healthy human subjects (n=19). Measurements were made from the same ectopy‐free ECG recording using 900, 800, 700, 600, 500, 400, 300, 256, 200, 150 and 100 heart beats by methodical truncation of the data set. Time domain variables assessed included RRISD, RMSSD, pNN50, and the Poincaré plot descriptors of SD1, SD2 and SD2/SD1. Frequency domain (LF, 0.04–0.15; HF, 0.15–0.4 Hz) variables were calculated via fast Fourier transform. Complexity analyses included entropy (ApEn and SampEn), fractal nature (FD‐L and FD‐DA), stationarity (StatAv), symbol dynamics (SymDyn) and detrended fluctuation analysis (DFA). Time and frequency domain variables were not significantly altered until the data were truncated to at least 150 beats. With the exception of ApEn and DFA, all complexity indices were unchanged until reaching 100 beats; ApEn and DFA were decreased (p≤0.034) at 600 and 250 beats. These data indicate that measurements of commonly used indices of HRV and complexity, with the exception of ApEn and DFA, are robust in data sets containing at least 150 heart beats.
Analysis of Cardiac Norepinephrine (NE) Transporter (NET) mRNA after removal of the stellate ganglia.
Authors: Esfahanian Mohammad , Wehrwein E A , Yoshimoto M , Osborn J W , Novotny M , Babankova D , Swain G M , Kreulen D L
Published: June 2021

NET is an important protein in the regulation of catecholamines, particularly NE, and its dysfunction has been linked to certain cardiovascular diseases. To evaluate the contribution of the sympathetic innervation from the stellate ganglia as a source of NET in the heart, we compared by qPCR NET mRNA in the heart in control rats to rats after removal of stellate ganglia. NET mRNA was present in heart homogenate, with atria expressing more NET mRNA than ventricles (p<0.05, n=4–6). Adult, male rats underwent bilateral stellate ganglionectomy (SGX) or sham surgery and hearts were assayed by chamber two weeks later. To confirm successful denervation, NE content was measured by HPLC and was reduced uniformly by 85.96% in all heart chambers (p<0.001, n=4–5). Although NE was depleted, there was a significant increase in NET mRNA (p<0.05, n=5) in the left atria while no differences were seen in any other chambers (p>0.05, n=4–6). The presence of NET mRNA after SGX suggests that NET mRNA is present in non‐stellate sources and is regionally increased by removal of the sympathetic innervation. P01HL79687, AHA predoctoral fellowship (EW).