bluuebunny/crossref_metadata_2025 huggingface dataset:
Microvascular oxygen transport in obese ZDF rats: an early model of type II diabetes
Authors: Benlamri Amina , Milkovich Stephanie , Burger Dylan , Feng Qingping , Goldman Daniel , Ellis Christopher
Published: June 2021

Sprague et al. (Diabetes 2006) reported impaired ATP release from human red blood cells (RBC) with type II diabetes that may contribute to impaired regulation of O2 delivery. Our goal was to quantify O2 transport in skeletal muscle of obese ZDF‐370 rats. Using dual wavelength intravital video microscopy we measured hemodynamics (RBC velocity, RBC supply rate, hematocrit) and RBC oxygen saturation in capillaries of extensor digitorum longus muscle as well as capillary density, glucose, TNF‐ α levels, arterial blood gases, arterial hemoglobin and hematocrit levels. Results reported as mean±SD. Two groups, 7 lean ZDF‐380 rats (179±13 g) and 8 obese ZDF‐370 rats (240±10 g), were studied at 7 weeks. Both glucose and TNF‐ α levels were elevated in the obese group compared to lean (glucose: 12.3±5.1 vs 6.8±0.6 mmol/L; TNF‐α: 112±43 vs 19±9 pg/mol). There were no significant differences in total capillary density. RBC supply rate was significantly decreased in obese rats (7.2±1.1 vs 9.0±1.8 RBC/s; p < 0.05); other hemodynamic parameters were unchanged (obese vs lean: velocity 130±26 vs 157±29 μm/sRBC, Hct 14.6±3.3 vs 15.3±1.6 %). There was a trend for decreased O2 saturation in the obese group (54±16% vs 64±5%; p = 0.19). The lower RBC supply rate and O2 saturations suggest the start of impaired regulation of O2 transport in this early diabetic model.

Supported by NIH grant (HL‐089125) and a CIHR operating grant to Ellis & Goldman.
Involvement of T‐type calcium channels in the vasodilation of mouse cremaster arterioles
Authors: Crowe William E. , Sarelius Ingrid H.
Published: June 2021

Increases in endothelial cell (EC) calcium (Ca) have been implicated in the activation of Ca‐dependent K channels, EDHF and eNOS, and hence local and remote vasodilations. In the mouse cremaster, store‐independent increases in Ca precede the spreading dilation invoked by ACh but not by adenosine. Since T‐type Ca channels are implicated in vasodilation, we tested the ability of the T‐type channel inhibitors, Ni (10μM) and amiloride (100μM) to block the ACh‐induced Ca increase. Arteriolar ECs in cremaster m. of anesthetized [Nembutal, 65 mg/kg ip] mice were loaded with fluo‐4; blood perfusion was then restored. The control ACh‐induced Ca response (peak: 8.7±3.2s; 1.6±0.1 F/Fo, n=6) preceded the dilation (peak: 12.3±3.3s; 9.8±2.1μm). In two vessels, a secondary Ca decrease was also observed (trough: 47s; 0.58 F/Fo). However, in the presence of either Ni (n=4) or amiloride (n=6), the initial Ca peak was limited to 30% of vessels (peak: 15±4s, 1.8±0.2 F/Fo) whereas in 50% of vessels a decrease in Ca (trough: 22±7s, 0.65±0.08 F/Fo) occurred following the dilation (peak: 17±5s, 9.9±2.5μm). In 20% of vessels both the Ca response and the dilation were inhibited. We conclude that some of the EC Ca entry occurs through T‐type channels but that the blocking of EC Ca entry does not preclude receptor‐mediated dilation in the majority of vessels. [Supported by: HL18208, HL75186, HL 76414]
Restoration of Vascular Relaxation in Cerebral Arteries of Congenic Dahl Rats Receiving the Brown Norway (BN) Renin Gene
Authors: Durand Matthew J. , Moreno‐Quinn Carol , Lombard Julian H.
Published: June 2021

The Dahl Salt Sensitive (SS) rat exhibits impaired regulation of the renin‐angiotensin system (RAS), chronically low levels of angiotensin II, and impaired vascular relaxation in response to vasodilator stimuli. This impaired vascular relaxation is likely due to chronic exposure of the animals to low levels of ANG II. The present study investigates responses to vasodilator stimuli in two different congenic strains in which a portion of the BN chromosome 13 was inserted onto the SS genetic background. Congenic strain SS.BN‐(D13rat88‐D13rat105) contains a 2.9 Mbp portion of chromosome 13 which excludes the BN renin gene, and would not be expected to restore normal regulation of the RAS. Congenic strain SS.BN‐(D13hmgc41‐D13hmgc23) contains a 2.0 Mbp adjacent region that includes the BN renin gene, which should restore normal ANG II levels in animals fed low salt (LS) diet. Males from each congenic strain were fed a LS diet (0.4% NaCl) after weaning. At the age of 10–14 weeks, middle cerebral arteries (MCA) were isolated and cannulated and dilator responses to acetylcholine (10−10M–10−5M) and reduced PO2 were assessed using television microscopy. MCA from the congenic strain containing the BN allele for renin dilated in response to acetylcholine and reduced PO2 while the MCA from the congenic strain containing the SS renin allele did not. These results suggest that the presence of a normally functioning renin gene from the BN rat in the SS genetic background restores normal vasodilator responses in the MCA. [NIH #HL‐29587; #HL‐65289, and #HL‐72920].
Why are Smooth Muscle Responses Unable to Conduct Along Skeletal Muscle Arteries?
Authors: Tran Cam Ha , Vigmond Edward J , Welsh Donald G
Published: June 2021

Electrical responses initiated in endothelial but not smooth muscle cells conduct robustly along skeletal muscle resistance arteries. The origin of this dichotomy is uncertain and was the focus of this investigation. Computational models based on physical and electrical properties of vascular cells provide two rationalizations for the inability of smooth muscle responses to conduct. First, given the differential orientation of vascular cells and the known variability in coupling resistance, it is conceivable that a resistance artery is inherently designed to limit the conduction of smooth muscle‐initiated responses. This view is consistent with the inability of a high K+ solution, discretely applied to stimulate smooth muscle, to elicit a conducted response. Second, given the passive nature of an artery's electrical properties, some smooth muscle agonists may fail to elicit the local electrical response required for conduction. This was confirmed by focally applying phenylephrine and monitoring vasomotor and electrical responses in hamster retractor muscle feed arteries. Further experiments revealed no evidence that the electrical feedback mechanisms in smooth muscle or endothelial cells limit the conduction of smooth muscle responses. In closing, this study highlights that the inability of smooth muscle responses to conduct is due to the inherent biophysical properties of resistance arteries. Supported by the Heart and Stroke Foundation of Canada.
The relationship between cochlear blood flow disorder and NF‐kB activation by loud sound situation
Authors: Yamamoto Hiroshi , Shi Xiaorui , Omelchenko Irina , Nuttall Alfred
Published: June 2021

Nuclear factor‐kappa B (NF‐κB) plays an essential role in the regulation of genes involved in various biological processes, such as inflammation, immune responses, neuronal development, and cellular apoptosis. NF‐κB activation can be achieved through two main pathways. The canonical (classical) pathway is known to be involved in the response of various cell types to pathogen associated molecules as well as proinflammatory cytokines, such as tumor necrosis factor alpha (TNF‐α) and interleukin‐1 (IL‐1). The noncanonical (alternative) is activated by a different specific set of stimuli, including B‐cell activating factor (BAFF), lymphotoxinβ, and CD40L.

In this study, using immunohistochemistry and immunoblot analysis, we determined whether one or both NF‐κB pathways, canonical (classical) or noncanonical (alternative), involved in loud‐sound induced cellular damages mechanism in guinea pig cochlea. In control (non‐sound stimulated) condition, we found that there was no detectable phosphoralated P65 in canonical pathway or P100 in the noncanonical pathway in both organ of Corti and cochlear lateral wall. However, NF‐κB activation related P65 phosphorylation was detected in the inner ear immediately following loud‐sound exposure at 120dB SPL/3hr for a day. There was no phosphorylation of p100 at any time periods following loud sound stimulation. These results suggest that loud‐sound stress activates NF‐κB dependent on P65 (canonical) pathway but not P100 (non‐canonical) pathway in the response to loud‐sound induced cellular damage. Supported by NIH/NIDCD R01 DC 00105, 008888 and 005983.
Role of epidermal growth factor receptor on angiotensin II‐induced hypertrophy and remodeling in cerebral arterioles
Authors: Chan Siu Lung , Baumbach Gary
Published: June 2021

We examined the effect of epidermal growth factor receptor (EGF‐R) inhibition on structural alternation in cerebral arterioles induced by angiotensin II (Ang II). C57BL/6J mice (8–10 weeks old) were infused with Ang II (1000 ng/kg/day) via osmotic minipumps for 28 days. AG1478 (12 mg/kg), a specific EGF‐R tyrosine kinase inhibitor, was given intraperitoneally once a day. Systolic arterial pressure (SAP) was measured by a tail‐cuff method. At the end of the treatment, pressure and diameter of cerebral arterioles were measured through an open cranial window in anesthetized mice. Chronic infusion of Ang II increased SAP (139±7 vs control of 115±5 mmHg), reduced external diameter of maximally dilated (by EDTA) cerebral arterioles (ED, 56±6 vs 66±3 μm at 50 mmHg intravascular pressure), and increased vessel wall cross sectional area (CSA, 527±31 vs 356±49 μm2) and immunohistochemical staining density of EGF‐R (19±2 vs 1±1 %). Thus, Ang II raised blood pressure, transactivated EGF‐R, promoted hypertrophy and remodeling. Concurrent treatment of AG1478 with Ang II did not attenuate increases in SAP (140±2 mmHg) or remodeling (ED: 57±3 μm), but prevented hypertrophy (CSA: 352±19 μm2) and diminished EGF‐R staining density (9±1 %). These findings suggest that increased activity of EGF‐R via transactivation by Ang II type 1 receptors may play an important role in Ang II‐induced hypertrophy, but may not in remodeling.
Acute hyperglycemia impairs NO‐mediated endothelial function of coronary arterioles by reducing L‐arginine availability independent of superoxide
Authors: Hein Travis W. , Xu Xin , Kuo Lih
Published: June 2021

Diabetes is associated with oxidative stress leading to impaired coronary flow regulation. However, the impact of acute elevation of glucose on coronary arteriolar function remains unclear. Herein, we examined the direct influence of high glucose on endothelium‐dependent nitric oxide (NO)‐mediated dilation of coronary arterioles and the role of superoxide in the high glucose‐mediated effect. Porcine coronary arterioles were isolated and pressurized without flow for in vitro study. In the presence of normal intraluminal physiological level of glucose (5 mM), coronary arterioles dilated dose‐dependently to endothelium‐dependent NO‐mediated agonist serotonin. In another group of vessels, the vasodilation to serotonin, but not to sodium nitroprusside, was reduced by an acute (90 min) elevation of intraluminal glucose in a concentration‐dependent manner (25 and 50 mM). Subsequent exposure of vessels to excess of NO precursor L‐arginine (3 mM), but not to superoxide dismutase mimetic TEMPOL, restored the normal dilation to serotonin. Our data show that acute exposure to high glucose can specifically impair endothelium‐dependent NO‐mediated dilation of coronary arterioles. It appears that reduction in L‐arginine availability, rather than increase in superoxide production, contributes to the endothelial dysfunction induced by an acute elevation of glucose. Support: HL‐71761 to LK.
Increased Coronary Reactivity and Endothelial Dysfunction in Young Cardiomyopathic Hamsters.
Authors: Ramos Jose A , Crespo Maria J , Santacana Guido E , Altieri Pablo I , Escobales Nelson
Published: June 2021

Previous data by our group indicated the presence of vascular dysfunction in the aorta of 2‐month‐old cardiomyopathic (CM) hamsters (Bio‐T02 strain). To determine the status of coronary resistance (CR) in these animals, isolated hearts from control (CT) and CM of 1, 2, and 6 months of age were studied using a Langendorff setup. At 1, 2, and 6 months of age, CR (mmHg × mL / min × g, RU) in CT was 1.11 ± 0.39, 1.79 ± 0.31, and 5.67 ± 2 RU, respectively. These values were similar to those of CM at their respective age groups. Infusion of thromboxane (THX, 0.1 μM) in CT, increased CR by 2.02, 3.58, and 3.78‐fold at 1, 2, and 6 months of age (P≤0.05), respectively. In CM, THX increased CR by 2.51, 4.70, and 5.16‐fold in these age groups (P≤0.05). Bradykinin (1 μM) inhibited the THX‐induced contraction in CT by 91±8%, 57±11%, and 48±7%, whereas in CM this parameter was reduced by 76±3%, 31±10%, and 48±7% at 1, 2, and 6 months of age, respectively (P≤0.05). SNP abolished the THX‐induced contraction to values not different from basal levels in all groups. These findings indicate that the coronary circulation of CM is hyperreactive to THX, an effect that increases as age progresses. The reduced bradykinin induced relaxation in CT and CM indicates the development of endothelial dysfunction, an effect that peaks (≈50%) at 2 months of age in CM. We conclude that coronary dysfunction is present in young CM. Supported by NIH 2S06‐GM 08224.
Endothelium‐dependent contractions in the carotid artery of young adult spontaneously hypertensive rats are mediated by cyclooxygenase and markedly augmented by nitric oxide inhibition
Authors: Denniss Steven G , Rush James WE
Published: June 2021

We have recently shown that ACh‐stimulated vasomotor activity in isolated PE‐precontracted common carotid artery (CCA) rings from young adult spontaneously hypertensive rats (SHR) involves a dose‐dependent relaxation phase from −9 to −7 LogM followed by a ‘re’‐contraction phase through to −4 LogM, whereas in normotensive Wistar Kyoto rats (WKY) only a robust relaxation phase existed. The objective of this study was to test the hypothesis that [ACh] >−7 LogM stimulates contractions in quiescent CCA from SHR but not WKY. Reference KCl contractions in CCA were similar between strains and across all drug conditions (0.9−1.0g). [ACh] from −9 to −4 did not elicit vasomotor activity in WKY CCA, nor did [ACh] elicit vasomotor activity in SHR CCA from −9 to −7; however, [ACh] >−7 stimulated a dose‐dependent contraction in SHR CCA reaching a plateau at −4 (0.16±0.03g). Endothelium denudation completely abolished these ACh‐stimulated contractions as did cyclooxygenase (COX) inhibition, whereas blocking endothelial nitric oxide (NO) synthase markedly increased the amplitude (0.61±0.08g) but not the threshold or sensitivity of the contractile response. These data demonstrate that only [ACh] >−7 LogM can stimulate contractions in CCA from young adult SHR (but not WKY) that are endothelium‐dependent, COX‐mediated, and powerfully modulated by the availability of NO.

Research Support: HSFO and NSERC
THE INTERACTION OF VASODILATOR‐STIMULATED PHOSPHOPROTEIN (VASP) WITH IP3R AND TRPC CHANNELS IN MADIN‐DARBY CANINE KIDNEY (MDCK) CELLS
Authors: Settles Deann Christine , Huss Andrew C , Sansom Steven C
Published: June 2021

Several dedifferentiating disorders of tissues are the result of aberrantly elevated [Ca2+]i leading to increased transcription. Although the cGMP‐activated protein kinase (PKG) pathway attenuates increases in [Ca2+]i and dedifferentiation, the mechanism is not understood. We recently found in mesangial cells that when VASP, an actin associated focal adhesion protein, is phosphorylated at Ser239 by PKG, it associates with TRPC4 channels. This was interesting because Homer, a scaffold protein, interacts with IP3R and TRP channels at the Ena/VASP Homology I (EVHI) domain. Because both VASP and Homer contain the EVH1 domains, we hypothesized that VASP forms a similar interaction with TRPC and IP3R in MDCK cells, which are model epithelia for studying diseases such as polycystic kidney disease (PKD). We identified with RT‐PCR and immunocytochemistry the presence of TRPC1 and TRPC4 in MDCK cells. We also demonstrated interaction between VASP and TRPC1 with immunocytochemistry and an interaction between VASP with IP3R using immunocytochemistry and Co‐IP. Moreover, upon PKG phosphorylation of VASP at Ser239, VASP no longer interacted with TRPC1, but instead interacted with TRPC4 on the cell membrane. These results suggest that PKG controls Ca2+ entry in MDCK cells by phosphorylating VASP at Ser239. This may be part of the mechanism by which PKG attenuates the elevated [Ca2+]i in some dedifferentiating diseases.
Molecular mechanism of ROMK channel endocytosis
Authors: Fang Liang , Kim Bo‐young , Wade James B. , Welling Paul A.
Published: June 2021

The apical surface density of ROMK channels in the renal CCD is down regulated by clathrin‐dependent endocytosis upon dietary potassium restriction. Aberrant stimulation of ROMK endocytosis may contribute to hyperkalemia in pseudohypoaldosteronism type II. ROMK contains an “NPXY‐type” internalization motif but the endocytotic machinery that recognizes the signal has been a mystery. Here we show that a member of a new class of clathrin adaptors, ARH, controls this activity. We found ARH is predominantly expressed in the distal nephron, where it co‐localizes with ROMK in subapical and perinuclear vesicles. As measured by cell surface biotinylation or antibody binding, ARH expression led to a dramatic attenuation of ROMK surface expression. A close relative of ROMK, Kir2.1, does not contain an “NPXY” type signal and is not affected by ARH. Swapping the C‐terminal domain of ROMK, containing the NPXF motif, onto the Kir2.1 channel conferred ARH‐dependent internalization. Alanine replacement of the NPXF motif increased cell surface expression and rendered the chimeric channel resistant to ARH. A “pull‐down” approach with recombinant ARH and GST‐ROMKC proteins revealed that ARH directly interacts with the ROMK C‐terminus in a NPNF signal dependent manner. In conclusion, ARH marks ROMK channels for endocytosis through direct interaction with the NPXF internalization signal. NIH RO1 DK63049 and DK54231.
Nephrogenic diabetes insipidus (DI) in a mouse model of renal insufficiency associated with developmental mis‐expression of Six2
Authors: Somponpun Suwit J , Fogelgren Ben , Kuroyama Mari , Drumhiller Wallace , Wong Brittany , Lozanoff Scott
Published: June 2021

Six2 is a developmental transcription factor that influences renal glomerular formation. We reported a radiation‐induced six2 mutant mouse called Br that displays glomerular dysmorphogenesis. This study was carried out to characterize the renal insufficiency associated with six2 mis‐expression in a Br/+ adult. Wildtype (WT) and mutant mice (Br) were separated into four groups: two received 2% NaCl for five days (WT‐S, Br‐S), the others had water (WT‐NS, Br‐NS). Urine composition, plasma osmolality (pOsm) and sodium (pNa+), and weight change were recorded while immuno‐analysis was undertaken for vasopressin (VP) and cFos in supraoptic neurons (SON). Compared to WT, salt‐loaded heterozygous Br mice: 1) failed to concentrate their urine (WT‐NS: 1640, WT‐S: 2587, Br‐NS: 1605, Br‐S: 1192 mOsm/kg, p<0.01) 2) had a significant increase in pOsm and pNa+ (325.4, 332.5, 344.8, 384.2 mOsm/kg, p<0.01; and 131.9, 136.9, 134.2, 145.6 mmol/L, p<0.01) 3) incurred weight loss (33.5, 31.5, 25.5, 19.4 g, p<0.01) and 4) had an increased fluid intake (2.1, 2.0, 2.2, 2.7 ml/10g, p<0.05). Interestingly, no change in VP or cFos expression to increased pNa+ was seen in Br‐S animals. Results suggest that developmental mis‐expression of six2 results in a defective kidney in adult animals with reduced urine concentrating capacity and polydipsia, consistent with nephrogenic DI. An uncoupling of VP response to increased pNa+ in SON of Br‐S mice warrants further study. Supported by R01 DK064752 (SL)
AT1, NMDA, and GABAA receptors in the median preoptic nucleus mediate greater vascular responses in Brown‐Norway/Mcw rats compared to Dahl salt‐sensitive/Mcw rats
Authors: Schwartz Julie A , Knuepfer Mark M
Published: June 2021

We have shown that GABAA, NMDA, and AT1 receptors in the hypothalamic median preoptic nucleus (MnPO) contribute to hemodynamic response variability to cocaine in Sprague‐Dawley rats. These receptors may also contribute to differences in response pattern in two unrelated strains of rats. We named Dahl salt‐sensitive/Mcw (DS) rats cardiac responders since they exhibit an increase in arterial pressure (AP) following cocaine that was due entirely to an increase in cardiac output (CO). We called Brown‐Norway/Mcw (BN) rats vascular responders since they exhibit a similar, sustained increase in AP following cocaine that was due entirely to an increase in systemic vascular resistance (SVR). Microinjection of either the GABAA agonist muscimol (80 pmol/100 nl) or the NMDA receptor antagonist MK‐801 (dizocilpine, 20 nmol) into the MnPO attenuated the decrease in CO and the increase in SVR in BN after cocaine without affecting the responses in DS. Injection of the AT1 receptor antagonist losartan (20 ng) into the MnPO prevented hemodynamic response differences to cocaine in DS and BN. Microinjection of l‐NAME (4 nmol) into the MnPO did not alter responses to cocaine in DS or BN. These results suggest that MnPO GABAA, NMDA, and AT1 receptors participate in producing the vascular response to cocaine in BN. Supported by USPHS DA017371, GM008306, and a predoctoral fellowship from the American Heart Association, Heartland Affiliate.
Oxytocin enhances glutamatergic afferent transmission and produces an inward current in second order medial solitary tract neurons
Authors: Peters James Henry , Andresen Michael
Published: June 2021

Cranial visceral afferents contact neurons within the solitary tract nucleus (NTS) and evoke essential homeostatic reflexes. Descending projections from the paraventricular nucleus (PVN) of the hypothalamus release oxytocin to modulate visceral afferent communication with NTS. The mechanisms through which oxytocin acts are poorly understood. In horizontal brainstem slices, remote electrical shocks to afferent axons in the solitary tract (ST) activated time invariant ST synaptic responses (jitter of < 200 μs) and identified second order NTS neurons. Blocking conducted transmission with tetrodotoxin (TTX), glutamate mediated mEPSC were isolated with the GABAA receptor antagonist gabazine (SR‐95531). Oxytocin concentration dependently (1 – 1000 nM) increased the frequency of mEPSCs but had no effect on the event kinetics – observations consistent with a presynaptic action at afferent terminals. The oxytocin receptor antagonist (d(CH2)51,Tyr(Me)2,Thr4,Orn8,des‐Gly‐NH29‐Vasotocin) blocked this effect. In addition, oxytocin evoked an inward current – observations consistent with postsynaptic modulation of ion channels. Taken together these findings demonstrate oxytocin released from PVN projections may enhance visceral afferent transmission within NTS via both pre‐ and postsynaptic sites of action.

Supported by: HL‐088894 (JHP) and HL‐041119 (MCA)
Chronic Hypoxia (CHx) Suppresses the Chemosensitive Response of Individual Nucleus Tractus Solitarius (NTS) Neurons from Adult Rats
Authors: Nichols Nicole Louise , Wilkinson Katie A. , Powell Frank L. , Putnam Robert W.
Published: June 2021

CHx alters the ventilatory response to hypoxia which is mediated in part by changes in NTS neurons. We hypothesized that the response of NTS neurons to hypercapnia is also increased by CHx. Rats were exposed to hypobaric hypoxia (10–11%) for 7–14 days, and we simultaneously measured the intracellular pH (pHi) and firing rate (FR) of NTS neurons in slices using whole cell patch and fluorescence imaging microscopy techniques. CHx did not affect basal pHi (∼7.30) or FR of NTS neurons (∼1 Hz). Synaptic block (SNB: 0.2 mM Ca2+, 11.4 mM Mg2+) increased the FR of neurons from control (1.4 ± 0.2 to 2.8 ± 0.5 Hz) and CHx rats (1.0 ± 0.2 to 3.0 ± 0.6 Hz). pHi responses to HA (hypercapnia acidosis: 15% CO2; pHo 7.0) were unchanged in neurons from CHx rats. The percentage of NTS neurons activated by HA decreased from 46% (control rats) to 34% (CHx rats). The chemosensitivity index of NTS neurons activated by HA was unchanged by CHx (166 ± 11% for controls and 160 ± 6% by CHx). The percentage of hypercapnia‐inhibited NTS neurons from CHx rats increased markedly (28%) compared to normal (13%). The HA responses of NTS neurons are intrinsic since they were unaffected by SNB. CHx causes neuronal plasticity resulting in suppression of the HA response in individual NTS neurons from adult rats. This is the first example of plasticity at the cellular level in individual chemosensitive neuronal responses to HA.

[Supported by NIH grants RO1 HL566683 and RO1 HL081823.]
Chronic hypoxia alters norepinephrine (NE) modulation of GABAergic inputs to second‐order neurons of peripheral chemoreceptors in the NTS
Authors: Zhang Weirong , Mifflin Steve
Published: June 2021

We have demonstrated that NE inhibits glutamatergic inputs to NTS receiving peripheral chemoreceptor inputs. The NE inhibition is mediated by presynaptic α‐adrenoreceptors (ARs), but it α2‐AR that is responsible for the enhanced NE inhibition following chronic hypoxia (CH). We investigated the role of NE in the modulation of GABAergic inputs to NTS second‐order neurons of peripheral chemoreceptors following CH (7 days at 10% FIO2). Whole‐cell recordings of NTS second‐order neurons identified by DiA labeling of carotid bodies were obtained in a brainstem slice. NE at 10μM reduced frequency of spontaneous release of GABA (mIPSCs) in the presence of TTX. NE inhibition effect was mediated by α‐ARs since α‐AR antagonist yohimbine abolished NE inhibition of mIPSC frequency (98±4% of control, n=5). CH significantly attenuated NE inhibition of mIPSC frequency (10μM NE, 27±4% vs 43±4% in normoxia, n=6 each, p<0.05). CH attenuated α2‐AR agonist clonidine‐mediated inhibition of mIPSC frequency (3μM, 15±4% vs 26±1% in normoxia, n=4 each, p<0.05). Neither NE nor α2‐AR agonist/antagonist significantly altered mIPSC amplitude. These results suggest CH attenuates NE presynaptic α2‐AR‐mediated inhibition of GABAergic inputs to NTS neurons receiving peripheral chemoreceptor afferent inputs. The attenuated NE inhibition of GABAergic inputs could play a role in central adaptations to CH.
Activation of opioid μ‐receptors in the medullary raphe region (MRR) attenuates the ventilatory response to hypoxia in anesthetized rats
Authors: Zhang Zhenxiong , Xu Fadi , Zhang Cancan , Liang Xiaomin
Published: June 2021

Caudal (cMRR) rather than medial and rostral MRR (mMRR and cMRR) μ‐receptors (μ‐Rs) were important in attenuating hypercapnic ventilation (VE) in anesthetized rats. Because opioids attenuate the peripheral chemoreceptor‐mediated early VE response to hypoxia partially via acting on peripheral μ‐Rs, and the MRR is also involved in modulating hypoxic VE, we asked whether local μ‐Rs modulate the early VE response to hypoxia. Cardiorespiratory activities and their responses to 10% O2 of 1 min were recorded in anesthetized and spontaneously breathing rats before and after: (1) intravenous administration of DAMGO, a μ‐R agonist, alone or coupled with a previous local injection of CTAP, a μ‐R antagonist; and (2) local microinjection of DAMGO. Systemic DAMGO markedly lowered VE and its response to hypoxia by 21% and 30%, respecitvely. VE was reduced only after microinjection of DAMGO into the cMRR, while the hypoxic VE was attenuated by 51% and 27% following microinjection made in the cMRR and mMRR, respectively, and unchanged by rMRR microinjection. Moreover, systemic DAMGO‐induced attenuation of the hypoxic VE was improved by 58% after microinjection of CTAP into both the cMRR and mMRR. Arterial blood pressure and its response to hypoxia were diminished by intravenous rather than MRR local injection of DAMGO. We conclude that activation of μ‐Rs in the cMRR and mMRR can attenuate hypoxic VE (Supported by NIH 74183).
Severe Hypoxia Enhances Lymphocyte Apoptosis Induced by Oxidative Stress during Moderate Exercise
Authors: Lin Chia‐Te , Wang Jong‐shyan
Published: June 2021

This study investigated how exercise combined with hypoxia affects lymphocyte apoptosis induced by oxidative stress and its underlying mechanisms. Eight sedentary men engaged six conditions in a normobaric hypoxia chamber for 1 hour: two normoxic exercise [i.e., strenuous exercise (SE, up to VO2max) and moderate exercise (ME, 50%VO2max) in 21% O2], two hypoxic exercise (i.e., ME in 12% and 15% O2), and two hypoxic conditions (i.e., 12% and 15% O2). Our results demonstrated that SE enhanced H2O2‐induced phosphatidylserine (PS) exposure of lymphocyte, which enhancement was accompanied by diminished mitochondrial transmembrane potential (MTP) as well as increased active caspase 3, 8, and 9 levels. Although ME combined with 12% O2 intervention also promoted PS exposure and caspase 3, 8, and 9 activation induced by H2O2, there was not significant change in H2O2‐induced lymphocyte PS exposure and caspases activation following ME combined with 15% O2 intervention. Therefore, we conclude that exposure of 12% O2, but not 15% O2, enhances lymphocyte apoptosis induced by H2O2 at performed ME, possible by promoting activation of caspases in lymphocyte.
Rate of Protein Synthesis Under Hypometabolic Conditions: The Down and Up and Down
Authors: Fähling Michael , Steege Andreas , Mrowka Ralf , Martinka Peter , Perlewitz Andrea , Persson Pontus Börje , Thiele Bernd Joachim
Published: June 2021

The first striking cellular response to hypoxia (1% oxygen) is the suppression of energy consuming processes, including gene expression. mRNA translation is of high energy cost and consequently undergoes a rapid inhibition following hypoxia. This translational arrest is mediated by several kinases which phosphorylate, e.g., translation elongation factors. The evaluation of reporter gene assays reveals a time dependent, multi‐phase response in the hypoxic adaptation of overall gene expression: We found that the short term suppression of protein synthesis rate under hypoxia can be rescued up to 18 h, but then comes down again under prolonged conditions. In the “rescue”‐phase, polysomal gradient analyses indicate a dissociation of polysomes, which seems to be compensated by a general shift of mRNAs into polysomal fractions. Under long term hypoxia the inhibition of gene expression is accompanied by the lack of small RNA species, e.g., tRNAs. Nevertheless, specific mRNAs can circumvent these inhibitory processes, showing an increased gene expression rate against the overall trend. RNA binding proteins such as nucleolin seems to be crucial for this selection. The findings indicate new possible pathways, with respect to short term or prolonged hypoxia, which assure a constant or even elevated gene expression rate under conditions of metabolic rate depression.
Nucleosome Assembly Protein 1 (NAP‐1) determines the progenitor status of endothelial cells
Authors: Alvarez Diego F. , Yoder Mervin C. , Parra‐Bonilla Glenda , Alexeyev Mikhail , Stevens Troy
Published: June 2021

Pulmonary microvascular endothelial cells (PMVECs) are enriched with a population of progenitors that enhance their growth potential. Since Nucleosome Assembly Protein‐1 (NAP‐1) expression increases endothelial cell proliferation, we sought to determine whether NAP‐1 is a key molecular determinant of an endothelial progenitor cell. PMVECs were subjected to the single cell clonogenic assay; 20% of the population was fully differentiated (i.e. did not proliferate), 30% exhibited low proliferative potential (LPP), and 50% exhibited high proliferative potential (HPP). Re‐seeding of single cells in the clonogenic assay revealed that HPPs, but not LPPs, reconstituted the entire hierarchy of growth potentials, indicating that HPPs possess the ability to self‐renew, an evidence of their intrinsic progenitor behavior. Western blot analysis demonstrated that NAP‐1 expression was increased in HPPs compared to LPPs. Stable NAP‐1 overexpression in LPPs conferred a progenitor behavior to this phenotype, where 40% of the LPP population exhibited HPP potential, with a capacity to self‐renew. Morphological and adhesive properties were similar in HPPs, LPPs, and NAP‐1‐LPP overexpressing cells, indicating that NAP‐1 regulates progenitor activity without affecting endothelial specification. Thus, NAP‐1 determines the progenitor behavior of endothelial cells. HL66299
Role of STIM1 in Regulation of Store‐Operated Ca2+ Entry in PC12 cells
Authors: Prakash Y S , Pabelick Christina M , Thompson Michael A
Published: June 2021

Store operated calcium entry (SOCE) is important for regulation of intracellular Ca2+ ([Ca2+]i) in neurons. Recent studies in other cell type suggest that stromal interaction molecule (STIM1) triggers SOCE. We examined the role of STIM1 in the sensitivity of SOCE to temperature, pH and glutamate in differentiated, fura‐2 loaded rat pheochromocytoma (PC12) cells, a model for neuronal cells. In STIM1 siRNA‐transfected vs. control cells, SOCE was triggered by introduction of extracellular Ca2+ following endoplasmic reticulum depletion via cyclopiazonic acid in zero Ca2+. Cells were washed and exposed to altered pH (6.0–8.0), altered temperature (34–40oC) or different concentrations of glutamate. In controls, SOCE was progressively inhibited by decreased pH or temperature, while alkalosis and increased temperature stimulated Ca2+ influx. Increasing glutamate concentrations progressively inhibited SOCE. siRNA inhibition of STIM1 substantially blunted both temperature sensitivity (especially increased temperature) and pH sensitivity (especially acidic pH) of SOCE. These data show that SOCE in PC12 cells is susceptible to acidosis, temperature, and agonist stimulation: factors that can adversely affect neuronal function under pathological conditions. In this regard, the SOCE triggering protein STIM1 may play a role in such susceptibility.

This work has been supported by NIH grants UL1RR024150‐01(CR to CMP, CR20 and Early Career Development Award to YSP), and the Department of Anesthesiology (CMP, YSP), Mayo Clinic College of Medicine, Rochester, MN
Differential calcium signaling and Kv1.3 trafficking to the immunological synapse in systemic lupus erythematosus
Authors: Nicolaou Stella A , Neumeier Lisa , Lee Susan Molleran , Ragupathy Manoj , Duncan Heather J , Kant Shashi K , Mongey Anne Barbara , Filipovich Alexandra H , Conforti Laura
Published: June 2021

Systemic lupus erythematosus (SLE) T cells have several activation signaling anomalies including defective Ca2+ response and increased NF‐AT nuclear translocation. The duration of the Ca2+ signal is critical in the activation of specific transcription factors and a sustained Ca2+ response activates NF‐AT. Yet, the distribution of Ca2+ responses in SLE T cells is not known. Furthermore, the mechanisms responsible for Ca2+ alterations are not fully understood. Kv1.3 channels control Ca2+ homeostasis in T cells. We reported a defect in Kv1.3 trafficking to the immunological synapse (IS) of SLE T cells that might contribute to the Ca2+ defect. Here, we compared single T cell quantitative Ca2+ responses upon formation of the IS in SLE, normal and rheumatoid arthritis (RA) donors. Also, we correlated cytosolic Ca2+ and Kv1.3 trafficking in the IS by two‐photon microscopy. We found that sustained Ca2+ responses are prevalent in SLE, but not in normal and RA T cells. The ratio of sustained over transient responses in SLE T cells was 3‐fold that of normal and RA T cells. Further, we observed that in normal T cells termination of Ca2+ influx is accompanied by Kv1.3 permanence in the IS, while Kv1.3 premature exit from the IS correlates with sustained Ca2+ responses in SLE T cells. Thus, we propose that the altered distribution in Ca2+ signaling in SLE T cells depends on the Kv1.3 channel trafficking defect. (NIH CA95286 and AHA 0615213B)
Carbon monoxide targets the pore‐forming BK alpha subunit in vascular smooth muscle Ca2+‐activated large‐conductance K+ channels
Authors: Essin Kirill , Schleifenbaum Johanna , Luft Friedrich C. , Gollasch Maik
Published: June 2021

Carbon monoxide (CO) induces vasodilation by activating arterial smooth muscle Ca2+‐activated large‐conductance potassium (BK) channels. BK channels are composed of the pore‐forming BK alpha and auxiliary BK beta1 subunits that confer an increased sensitivity for changes in membrane potential and calcium to BK channels. To elucidate the mechanism of BK channel activation, we performed whole‐cell perforated‐patch clamp experiments in fresh tibial artery vascular smooth muscle cells (VSMC) from BK beta1 gene‐deficient (−/−) and wild‐type (+/+) mice. BK currents were measured at physiological K+ gradients. Application of hemin (3 to 10 μmol/L), a substrate of heme oxygenase, induced a 4‐fold increase in current amplitude that was blocked by iberiotoxin (100 nmol/L) in +/+ cells. The effect was similar to that observed in cells from −/− mice. We next tested the vasodilator effects of hemin. Hemin (10 μmol/L) induced similar relaxations (~ 30%) of the serotonin (300 nmol/L) preconstricted VSMC from −/− and +/+ mice. This vasorelaxation was largely inhibited by iberiotoxin (100 nmol/L). This study pinpoints the BK alpha subunit as the molecule that senses CO, which results in myocyte BK channel activation and thereby endothelial‐independent relaxation. Our notion agrees with previous studies suggesting that CO modifies the binding of reduced heme to the heme‐binding domain of the channel alpha subunit C terminus.
The central nervous system contributes to vascular T cell infiltration during angiotensin II‐induced hypertension
Authors: Marvar Paul J , Guzik Tomasz J , Gordon Frank J , Harrison David G
Published: June 2021

Recently we have found that T cells play an important role in angiotensin II (ANG II) induced hypertension. We propose that ANG II can directly activate T cells as well as indirectly via increased sympathetic outflow. The purpose of this study was to determine the role of the central nervous system in T cell activation and vascular infiltration. We produced anteroventral third ventricle (AV3V) brain lesions in mice, which has been previously shown to prevent the central actions of ANG II. Following 2 weeks of ANG II infusion, the blood pressure of AV3V lesioned mice was significantly reduced compared to the sham group (AV3V: 118 ± 3.78 mmHg, n=5; Sham: 167.80 ± 8.85 mmHg, n=6). However there was no difference in the percent of circulating CD4+ cells expressing the early surface activation marker CD69 (Sham: 8.8 ± 0.55; AV3V: 7.5 ± 0.71). FACS analysis of collagenase digested aortas revealed that AV3V lesioning significantly reduced the total number of leukocytes (CD45+) and CD3+ cells infiltrating the aorta. In summary, following ANG II infusion, lesioning of the AV3V region prevents the increase in blood pressure and aortic T cell infiltration. We conclude that ANG II directly stimulates T cell activation but vascular infiltration of these cells is centrally mediated. These aortic infiltrated T cells then likely secrete cytokines that stimulate NADPH oxidase activity leading to vascular dysfunction.
BLOOD PRESSURE INCREASE AFTER SUCROSE FEEDING IS SODIUM‐INDEPENDENT
Authors: Freitas Raphael Ribeiro de Aquino , Oliveira‐Sales Elizabeth Barbosa , Boim Mirian Aparecida , Bergamaschi Cássia Toledo , Donilkoff Miriam Sterman , Campos Ruy Ribeiro
Published: June 2021

Dysregulation of sodium and/or water reabsorption by the kidney may contribute to hypertension. As chronic sucrose feeding reduces food intake and in consequence of this, the sodium (Na) intake. The objective of the present study was to investigate the role of kidney sodium and water channels on blood pressure increase in sucrose‐fed rats (SC). Wistar rats (150–180g) received, for 30 days, food ad libitum and sucrose solution (20%) (SC =7) or tap water (control group, C =8). The food ingestion was quantified using a metabolic cage and 1 mg of Na was given by gavage for another group of 5 sucrose fed‐rats (SS), also for 30 days (once a day) in order to normalize the sodium ingestion between SS and C. Mean arterial pressure (MAP) was recorded by a Power Lab system. The mRNA expression levels of epithelial sodium channel (ENaC) and water channel protein (AQP2) were analyzed in the cortical kidney tissue by Real Time RT‐PCR technique. MAP increases (P<0.05) in SC (128±1 mmHg) compared to C (102±4 mmHg) rats; MAP was 132±3 mmHg, in SS rats. Compared to C rats the ENaC and AQP2 expression levels were increased (P<0.05) in SC rats (ENaC: SC=1.95±0,19; C=1.03±0,07; AQP2: SC=2.12±0,19, C=1.07±0,10 arbitrary units). However, in SS group the high expression levels of ENaC and AQP2 were reversed (ENaC: 0,74±0,6, AQP2: 0,99±0,12 arbitrary units) but not MAP. Taken altogether, the results presented here indicate that in chronic sucrose fed‐rats the increased MAP is Na‐independent.

Supported by FAPESP, CNPq
Therapeutic aspirin therapy attenuates reflex cutaneous vasodilation in middle‐aged human skin
Authors: Holowatz Lacy A. , Kenney W. Larry
Published: June 2021

Full expression of reflex cutaneous vasodilation (VD) is dependent on cyclooxygenase‐ (COX) and nitric oxide synthase‐ (NOS) dependent mechanisms. Low‐dose therapeutic aspirin (ASA) therapy is utilized clinically in healthy populations to inhibit platelet aggregation for stroke prevention, however chronic COX inhibition may attenuate COX‐dependent VD mechanisms. We hypothesized that chronic COX inhibition with daily low‐dose ASA therapy would attenuated reflex VD in healthy humans. Two microdialysis (MD) fibers were placed in forearm skin of 4 middle‐aged (ASA: 57±3 years) normotensive humans taking daily low‐dose ASA therapy (81mg) and 4 unmedicated age‐matched control (no ASA: 56±2 years) subjects to serve as control (C: Ringers) and NOS inhibited (NOS‐I: 10mM L‐NAME) sites. VD was induced by using a water‐perfused suit to increase core temperature 0.8°C. Red cell flux was measured by laser‐Doppler over each site. Cutaneous vascular conductance was calculated (CVC=flux/MAP) and normalized to maximal CVC (28mM SNP). Cutaneous VD during heating was attenuated with daily low‐dose ASA treatment (ASA: 19±3 vs. no ASA: 49±5%CVCmax, p<0.01). NOS‐I decreased CVC in the no ASA group (21±4%CVCmax; p<0.05 vs. C), but failed to decrease CVC in the daily ASA treatment group (13±4%CVCmax). Daily low‐dose ASA therapy attenuates reflex cutaneous VD in middle‐aged humans though COX‐ and NOS‐dependent VD mechanisms.
UT‐A1 urea transporter activation does not involve furin‐dependent cleavage
Authors: Huang Haidong , Yang Yuan , Sands Jeff M , Chen Guangping
Published: June 2021

The serine protease furin is involved in the activation of a number of proteins. The urea transporter UT‐A1, located in the kidney inner medullary collecting duct (IMCD), is important for urine concentrating ability. UT‐A1's amino acid sequence revealed a consensus furin cleavage site (RSKR) in its N‐terminal region. Here, we report that UT‐A1 does not process furin cleavage in furin‐manipulated CHO cells, either in the cytosol or in the biotinylated cell surface pool. This result was further confirmed by in vitro furin digestion of 35S labeled UT‐A1 or a 126 N‐terminal fragment prepared from rabbit reticulocyte lysate translation system. As a positive control, the alpha subunit of the epithelial sodium channel (ENaC) was cleaved twice in vitro by furin. Functionally, mutation of the two key arginine residues in the furin consensus site (R78A, R81A) did not affect UT‐A1 urea transport activity in oocytes and protein cell surface expression. Our results indicate that UT‐A1 maturation and activation does not involve furin‐dependent proteolytic cleavage, and that the consensus furin site (R‐X‐R/K‐R) is required but may not be sufficient for furin digestion.
Mirthful laughter, as adjunct therapy in diabetic care, attenuates catecholamines, inflammatory cytokines, C‐RP, and myocardial infarction occurrence
Authors: Berk Lee S , Tan Linda , Tan Stanley
Published: June 2021

Objectives: Catecholamines are implicated in causing arrhythmias, hypertension, and MI. Inflammatory cytokines TNF‐α, IFN‐γ and IL‐6 contribute to the acceleration of atherosclerosis. Attenuation of catecholamines and inflammatory cytokines may provide cardiovascular protection. We hypothesized that mirthful laughter, by diminishing catecholamines production, can attenuate inflammatory cytokines, and provide cardiovascular protection.

Methods: We divided 20 high risk diabetic patients with hypertension and hyperlipidemia into 2 matched groups and followed them for one year in our Metabolic Syndrome Clinic. The experimental group was asked to view self‐selected humor for 30 minutes daily as an adjunct to standard diabetes (glipizide, TZD, metformin), hypertension (ACE inhibitor or ARB) and hyperlipidemia (statin) therapies. Plasma fractionated catecholamines levels, lipid panel, TNF‐α, IFN‐γ, IL‐6, and CRP were monitored and assayed every 2 months.

Results: Patients in the laughter group had lower EP and NEP levels by 2 months, increased HDL cholesterol, decreased TNF‐α, IFN‐γ, IL‐6 and CRP levels by 4 months, and a lower incidence of MI (1/10) than the control group (3/10).

Conclusions: Addition of mirthful laughter may be an effective cardiovascular preventive adjunct in diabetes mellitus and metabolic syndrome care. These effects may contribute to lower MI occurrence.
Dietary induced insulin resistance is independent of mammalian target of rapamycin complex 1 (mTORC1) activity in skeletal muscle of mice
Authors: Phelps Charlie , Berk Eli Zachary , Reynolds Thomas Henry
Published: June 2021

The purpose of this study was to determine the effects of insulin resistance on mTORC1 activity in skeletal muscle, the primary tissue for insulin‐mediated glucose disposal. Male C57BL/6 mice were fed a normal chow diet (CON) or a high fat diet (HFD) for four months and then insulin sensitivity was assessed by glucose tolerance (GT) and insulin tolerance (IT) testing. In mice fed a HFD the total area under the GT curve was similar to CON mice, but the area under the IT curve was significantly greater (p = 0.015) than CON mice, indicating the presence of peripheral insulin resistance. Basal and insulin‐stimulated mTORC1 activity was assessed in quadriceps muscle extracts by phospho‐specific immuno‐blotting with a pThr‐389 S6K antibody and a pThr‐246 PRAS40 antibody. There were no significant differences in pThr‐389 S6K immunoreactivity in muscles from the CON and HFD animals in the basal state or in response to insulin. Insulin significantly increased pThr‐246 PRAS40 immunoreactivity in muscles from CON and HFD animals compared to saline (p = 0.0001), but there was no significant effect of the HFD on pThr‐246 PRAS40 immunoreactivity in the basal or insulin‐stimulated state. These findings indicate that the insulin resistance observed after four months of a HFD is independent of mTORC1 activity.
Antidiabetic effect of Ecklonia cava extract
Authors: Kim Euikyung , Kang Changkeun , Lee Hyunkyung , Cha Meejin , Sohn Euntae
Published: June 2021

Diabetes mellitus is a metabolic disorder and characterized by high blood sugar (hyperglycemia). The symptoms of diabetes mellitus include excessive urine production, increased fluid intake and increased appetite. In the present study, we examined antidiabetic potential of methanol extracts of various brown algae collected near Korean ocean. Among them, we focused our research on Ecklonia cava (E. cava), which shows a relatively high level of polyphenol contents in its extract. We found that E. cava methanol extract (EME) showed a strong radical scavenging activity from DPPH assay. From cell‐based glucose uptake assay, we observed that EME enhanced glucose uptake in C2C12 cells in the presence or absence of insulin comparing with untreated respective controls. In streptozotocin‐induced type 1 diabetes mellitus SD rats, the high blood glucose level was significantly decreased to normal range by the treatment with EME (300 mg/kg). Furthermore, we could observe significant decreases in food intake and water intake of diabetic rats treated with EME. In conclusion, the present data suggest that E. cava methanol extract has a therapeutic potential in diabetes. 