bluuebunny/crossref_metadata_2025 huggingface dataset:
Hypoxia interacts with NFAT signaling in rat pulmonary arterial smooth muscle cells (PASMCs)
Authors: Cao Yuan‐Ning , Yang Xiao‐Ru , Tse Chung‐Ming , Sham James S.K.
Published: June 2021

Nuclear factors of activated T‐cells (NFATs) are calcium sensitive transcription factors regulated by calcineurin. They play critical roles in inflammation and cell proliferation. Recent studies showed that calcineurin/NFAT pathway is involved in the pathogenesis of pulmonary hypertension. Here we exposed rats to hypoxia for 3 weeks with daily injection of the calcineurin inhibitor cyclosporin (CsA, 35 mg/kg/day). Right ventricular systolic pressure (CsA treated: 34.3±2.0 mmHg; untreated: 53.9±2.6 mmHg, p<0.05), and right heart hypertrophy were significantly reduced in CsA treated rats. Real‐time RT‐PCR showed that NFATc3 mRNA was predominantly expressed in PA of rats and mice. It was 3‐5 times the amount of other NFAT isoforms, except that it was similar to NFATc1 in mouse PA. NFATc3 and NFATc1 proteins were also detected by western blot. NFATc3 nuclear translocation was examined by expressing hNFATc3‐EGFP fusion protein in rat PASMCs. Confocal images showed that nuclear accumulation of hNFATc3‐EGFP occurred within 5 min and peaked at about 15‐20 min after PASMCs were stimulated by ionomycin and PMA. NFAT‐dependent transcription activity, monitored with a 4xNFAT‐luciferase reporter, was increased 4 folds by ionomycin+PMA. Moreover, the stimulatory effect of ionomycin+PMA was further potentiated in PASMCs exposed to 24 hr hypoxia (2% O2). Since many agonists stimulate PLC to activate IP3‐induced Ca2+ release and PKC similar to ionomycin+PMA, this observation suggests that hypoxia may interact synergistically with agonists to enhance NFAT‐dependent gene transcription in PASMCs.
Lung neprilysin activity and expression are decreased in humans with COPD and pulmonary vascular remodeling
Authors: Wick Marilee J, , Barr Erica J. , Wehling Carol A. , Cool Carlyne D. , Zamora Marty , Miller York E. , Hersh Louis B. , Voelkel Norbert F. , Dempsey Edward C.
Published: June 2021

Pulmonary vascular remodeling and pulmonary hypertension (PHTN) are common in chronic obstructive pulmonary disease (COPD). Hypoxia, cigarette smoke, oxidant stress, and other factors may contribute to COPD, and may also decrease the activity or expression of the cell surface peptidase, Neprilysin (NEP). Recent studies with mice actually suggest that NEP may protect the lung vasculature from hypoxic PHTN, but NEP's importance in human pulmonary vascular disease is unknown. We hypothesized that NEP activity and/or expression is selectively reduced in humans with COPD and pulmonary vascular remodeling. Fixed sections were scored for pulmonary vascular remodeling in a blinded fashion. NEP activity and protein expression in lung lysates were reduced by 70% in COPD samples (n=14) vs control. In contrast, levels of dipeptidyl peptidase IV, another relevant peptidase, did not change. In order to understand the mechanisms responsible for these changes in NEP, human pulmonary artery SMCs were exposed to hypoxia, cigarette smoke extract, or ROS. Large decreases were measured in NEP activity and protein levels, while more modest decreases in NEP mRNA were noted, suggesting several levels of control. We conclude that decreased NEP may predispose to pulmonary vascular remodeling in COPD, and therapeutic strategies to increase NEP in the lung may prevent or attenuate some pulmonary vascular disorders.
NAP1‐L1 determines progenitor status of pulmonary endothelium
Authors: Alvarez Diego F. , Parra‐Bonilla Glenda , Alexeyev Mikhail , King Judy
Published: June 2021

Pulmonary MicroVascular Endothelial Cells (PMVECs) are enriched with progenitors that display enhanced proliferative and self‐renewal capacity. Since expression of Nucleosome Assembly Protein 1 Like 1 (NAP1‐L1) regulates cell proliferation, we sought to determine whether NAP1‐L1 determines the progenitor status of PMVECs. PMVECs subjected to the single cell clonogenic assay revealed that 20% of the population were fully differentiated (do not proliferate), 30% exhibited low proliferative potential (LPP) and 50% exhibited high proliferative potential (HPP). Re‐seeding cells in the clonogenic assay revealed that HPPs, but not LPPs, reconstituted the hierarchy of growth potentials, an indication of the HPPs self‐renewal capacity, and thus confirming their progenitor status. While NAP1‐L1 expression was increased in HPPs compared to LPPs, NAP1‐L1 overexpression in LPPs confered them with a progenitor status where 40% of the cells exhibited HPP‐ and self‐renewal capability. Immunohistochemical analysis in lungs from rats infected with Pseudomonas aeruginosa revealed that within injured, but not normal lung areas, there were endothelial cells that retained the proliferative marker Brdu and expressed NAP1‐L1. We conclude that NAP1‐L1 determines the progenitor status of pulmonary endothelium and associates in situ within vessels containing highly replicative endothelial cells. AHA 0835134N
Arsenic alters P2 receptor‐dependent Ca2+ signaling in human airway epithelial cells
Authors: Boitano Scott , Sherwood Cara , Liguori Andrew E. , Olsen Colin E. , Burgess Jefferey L. , Lantz R. Clark
Published: June 2021

Arsenic can affect the lung via inhalation or ingestion and has been shown to limit wound healing of lung epithelial cells in vitro. We examined the effects of acute exposure of low‐dose arsenic (24 hrs; 0, 60, and 290 ppb) on Ca2+ signaling, a component of wound recognition and repair. Arsenic reduced the number of cells responding to ATP‐dependent Ca2+ signaling emanating from local wounds (1‐2 cells) in a dose‐dependent manner. Examination of Ca2+ dynamics in cells adjacent to wounded cells showed that loss of Ca2+ signaling after 60 ppb exposure was a consequence of increased recovery of [Ca2+]i to resting values whereas loss of Ca2+ signaling after 290 ppb exposure was primarily due to a reduction in peak [Ca2+]i change. In Ca2+‐free solutions, [Ca2+]i changes were not significantly different between 0 and 60 ppb arsenic exposure, suggesting loss of ATP‐dependent Ca2+ entry at this dose. Activation of P2X channels resulted in a dose dependent‐loss of Ca2+ influx following arsenic exposure. Examination of P2Y and P2X receptor expression by real time RT‐PCR showed dose‐dependent reduction of P2Y2 and P2X4 mRNA expression in response to arsenic exposure. We propose that low‐dose arsenic can alter the reformation of the airway epithelial barrier by restricting proper wound repair in part through the alteration in paracrine P2 receptor Ca2+ signaling in lung epithelial cells. NIH P30‐ES006694, T32‐HL007249; EPA R832095
L‐NAME increases and apocynin decreases dihydroethidium fluorescence in endothelial cells in hyperglycemia
Authors: Rogers Steven Clay , Kavdia Mahendra
Published: June 2021

Diabetes is linked to numerous vascular pathologies as either a causative mediator or consequential product of a disease. The condition of diabetes is a slow progression of glucose imbalance in the body, which contributes to oxidative stress and endothelial dysfunction. We studied the superoxide levels in human umbilical vein endothelial cells (HUVEC's) under normal and hyperglycemic conditions. Superoxide concentrations were measured by dihydorethidium (DHE) fluorescence detection using a microplate reader and further analyzed using fluorescence microscopy. We also tested the impact of inhibitors and antioxidants such as L‐NAME (300 µM), apocynin (100 µM), and superoxide dismutase (100 units/ml). Cells incubated in hyperglycemic conditions showed an increase in fluorescence intensity over that of the normal glucose control. Treatment of apocynin and SOD reduced the fluorescence in hyperglycemic endothelial cells, whereas the treatment of L‐NAME caused an increase in fluorescence. A combined treatment of L‐NAME and apocynin had a fluorescence increase which was in between the respective individual treatments. The data shows that both NADPH oxidase and nitric oxide synthase can be an important source for oxidative stress in hyperglycemic conditions. Supported by: AHA 0530050N & NIH R01 HL084337.
The role of NO and rho kinase in contractions to serotonin in diabetic mice
Authors: Lamping Kathy G , Harrod Jeremy , Nuno Daniel W
Published: June 2021

Contraction to serotonin (5HT) is increased and relaxation to acetylcholine (ACH) is reduced in arteries from leptin receptor diabetic mice (DB) mice compared to non‐diabetics (ND). To determine the magnitude of the nitric oxide (NO) and rho kinase components in the vascular dysfunction in DB mice, responses of aorta from ND and DB to 5HT, ACH and nitroprusside (SNP) were measured before and after inhibition of NO synthase with nitro‐L‐arginine (LNA, 10μM) and rho kinase with H1152 (1μM). In ND aorta, 5HT produced concentration dependent contractions that were reduced by H1152. Contractions of aorta from DB mice to 5HT were increased compared to ND and also inhibited by H1152. Although LNA increased contraction to 5HT in ND mice, the inhibition by H1152 was similar. LNA had no effect on the contractions of aorta from DB mice to 5HT or the inhibition by H1152. LNA reduced whereas H1152 increased the relaxation to ACH in ND mice. Relaxation of aorta from ND to ACH was not affected by H1152 in the presence of LNA. Relaxation to ACH in DB mice was reduced approximately 50% compared to ND and LNA and H1152 had no effect on the relaxation. H1152 had no effect on relaxation to the NO‐independent dilator SNP in aorta from ND. We conclude that inhibition of rho kinase only increases relaxation to ACH in the presence of NO. Although NO modulates contractions to 5HT, it does not appear to modulate rho kinase‐mediated contractions to 5HT in mouse aorta.
Empirical study of transfecting early growth response gene‐1 DNA enzyme to prevent intimal hyperlasia of autogenous vein graft in rats
Authors: Wang Shu‐Qing , Liu Cheng‐Wei , Lv Da‐Ming , Kang Yu‐Ming , Wang Shu‐Qiu , Wei Xiao‐Dong , Qin Wen‐Bo
Published: June 2021

To detect the inhibitory action of early growth response gene‐1 DNA enzyme (EDRz) to vascular smooth muscle cell (VSMC) proliferation and intimal hyperplasia, with liposome as carrer, and to confirm the effect of gene therapy on stenosis and occlusion after vein transplantation. Construct early growth response gene‐1 DNA enzyme, autogenous vein graft model was established in 90 Sprage‐Dawley rats. After surgery, rats were randomly divided into at 1, 2, 6, 24 hours, 3, 7, 14, 28, 42 days groups. At 1h after graft, early growth response gene‐1 DNA enzyme was mainly located in media, adventitia and partial endothelial cells of vein graft. From 2h to 24h it was located in media of vein graft, while it was mainly located intimal of vein graft at 7d. There were not early growth response gene‐1 DNA enzyme in vein grafts at the late phase. We found that Egr‐1 protein mainly located in the medial VSMCs, monocytes and endothelium cells of vein graft at the early phase, and at the late phase no Egr‐1 protein was found in medial and neointimal VSMCs. The positive cells rate: at 2h, 6h, 24h, 3d is 15.3±4.2%, 9.7±2.4%, 6.4±1.8%, 2.3±0.2%. Conclusions: Early growth response gene‐1 DNA enzyme can reduce the expression of Egr‐1 in autogenous vein graft, it can effectively restrain VSMC proliferation and intimal hyperplasia, and then prevent vascular stenosis and occlusion after vein graft.
Decreased alpha 2B adrenergic receptor expression increases vascular but decreases renal eNOS expression.
Authors: Paredes Daniel A. , Duling Laura , Kanagy Nancy
Published: June 2021

Previous studies in mesenteric arteries from mice null for the a2B adrenergic receptor gene (a2B‐AR) suggest decreased a2B‐AR expression is associated with increased endothelial nitric oxide synthase (eNOS) expression in mesenteric arteries. Other studies have shown heterozygous a2B‐AR knockouts (HET) are less susceptible to renal hypertension than wildtype (WT) mice suggesting the HET genotype can be protective from some types of cardiovascular disease. Therefore we hypothesized a2B‐AR inhibit the expression of eNOS in other organs including the aorta, heart and kidney. Opposite to our prediction, western blots demonstrated a tendency for decreased eNOS protein expression in heart, aorta and renal medulla from HET mice and significantly lower expression in renal cortex (WT=1.0±0.1 vs HET=0.54±0.1 densitometry units, p<0.05). The renal medulla (WT=1.21±0.08 vs HET=0.84±0.05, p<0.05) and renal cortex (WT=1.06±0.05 vs HET=0.91±0.06, p<0.05) from HET mice also had decreased eNOS mRNA expression compared to WT. These results suggest α2B‐AR do not directly regulate eNOS expression but indirect effects of lower α2B‐AR expression lead to increased eNOS in small arteries and decreased eNOS in other tissues. The significance of decreased eNOS expression in the kidney on the altered blood pressure regulation in these mice remains to be investigated.
Luteolin improves vasorelaxation in aorta exposed to high glucose via NOS‐NO pathway
Authors: Wang Hui‐ping , Chen Fang‐xia , Qian Ling‐bo , Xia Qiang
Published: June 2021

Luteolin (Lut, 3',4',5,7‐tetrahydroxyflavone), the main flavonoid of Flos Chrysanthemi which is a popular traditional Chinese medicinal herb, has been demonstrated to attenuate myocardial ischemia‐reperfusion injury and cause vasorelaxation. The objective of the present study was to explore the effect of luteolin (Lut) on endothelium‐dependent vasorelaxation in isolated rat aortic rings exposed to high glucose, and its underlying mechanism. Male Sprague‐Dawley rats were killed by cervical dislocation. The thoracic aorta was rapidly dissected out and the effect of Lut on tension of aortic rings pretreated with 44 mmol/L glucose for 4 h was measured isometrically on the organ bath system. We found that pretreatment with high glucose (44 mmol/L glucose for 4 h) decreased acetylcholine (ACh)‐induced endothelium‐dependent vasorelaxation. However, high mannitol (33 mmol/L mannitol + 11 mmol/L glucose) had no effect on vasorelaxation. Lut (0.7~89.6 μmol/L) evoked a concentration‐dependent relaxation in aortic rings pre‐contracted by phenylephrine (PE), and the pD2 value was 5.21±0.04. EC50 concentration of Lut markedly attenuated the inhibition of vasorelaxation induced by high glucose, which was significantly weakened by pretreatment with L‐NAME (0.1 mmol/L) for 30 min, but not by indomethacin (0.01 mmol/L). The results indicate that the decrease of endothelium‐dependent vasorelaxation in rat aortic rings exposed to high glucose was not related to high osmolarity, whereas Lut markedly attenuated this decrease of vasorelaxation which may be mediated by maintaining the activity of NOS‐NO pathway.
Temporal profile of focal cerebral ischemic damage in type 2 diabetes
Authors: Li Weiguo , Elgebaly Mostafa M , Mezzetti Erin M , Hutchinson Jimmie R , Fagan Susan C , Ergul Adviye
Published: June 2021

While 90% clinical patients have type 2 diabetes, most experimental studies have focused on the effect of streptozotocin‐induced type 1 diabetes on cerebral ischemic injury. We have previously shown smaller infarcts in a lean type 2 diabetes model, Goto‐Kakizaki (GK) rats, after permanent and transient cerebral ischemia. However, the effect of extended period of type 2 diabetes on cerebral perfusion and stroke outcome remains unknown. Accordingly, the cerebral blood perfusion was measured with scanning laser Doppler imaging system and the infarct size and edema were evaluated after permanent middle cerebral artery occlusion induced ischemia in 10 and 18 wk old control Wistar and diabetic GK rats. Both 10 and 18 wk GK rats showed lower baseline cerebral perfusion (2.75 ± 0.19 and 2.76 ± 0.28 pixels/mm2, n=6‐9, p<0.01) compared to those obtained in Wistar rats. There was no difference in infarct size between young and old Wistars, while both groups of GK rats showed smaller infarct (10 wk GK, 29.07 ± 7.33% and 18 wk GK, 12.25 ± 1.38, p<0.05 vs. 10 or 18 wk Wistar). Both GK groups had increased edema when compared to controls. These results suggest that mild‐to‐moderate increases in blood glucose provide neuroprotection early in the disease process but effects of the severity and longer duration of diabetes on ischemic reperfusion injury remain to be determined.
Inhibition of TRPC3 by PKG contributes to NO‐mediated vasorelaxation
Authors: Chen Jie , Crossland Randy F. , Noorani Muzamil M.Z. , Marrelli Sean P.
Published: June 2021

Nitric oxide (NO) inhibits heterologously expressed TRPC3 channels via PKG‐dependent phosphorylation. We examined whether NO inhibition of TRPC3 occurs in freshly isolated smooth muscle cells (SMC) and, if so, whether TRPC3 channel inhibition contributes to NO‐mediated vasorelaxation. We used rat carotid artery (CA) as our model tissue.

Whole cell patch clamp of CA SMC demonstrated a non‐selective cation current that was activated by extracellular uridine triphosphate (UTP; 60 µM) and completely inhibited by La3+ (100 µM). The UTP‐stimulated current (IUTP) was also inhibited by anti‐TRPC3 antibody in the pipette solution, demonstrating that TRPC3 is the channel of IUTP. Application of an NO‐donor (MAHMA NONOate) or 8Br‐cGMP caused inhibition of IUTP. Pre‐incubation with a PKG inhibitor (KT5823) prevented inhibition by 8Br‐cGMP, demonstrating the essential role for PKG. Next, intact CA segments (in an artery myograph) were contracted with UTP (100 µM) in the presence or absence of La3+ (100 µM) and then relaxed with SNP, an NO donor. Relaxation to SNP was significantly less in the presence of La3+.

In summary, we demonstrate that rat CA SMC expresses functional TRPC3 channels and that these channels are inhibited by the NO/cGMP/PKG signaling pathway. Furthermore, our data suggests that NO inhibition of TRPC3 contributes to NO‐mediated vasorelaxation. Supported by: AHA 0665100Y and NIH HL088435 (SPM)
ATP released from sympathetic nerve endings in skeletal muscle arterioles
Authors: Kluess Heidi A , Stone Audrey J , Evanson Kirk W
Published: June 2021

The purpose of this study was to determine the feasibility of measuring ATP released from sympathetic nerve endings in arterioles. Using F344 rats (2‐3 months old), the gastrocnemius 1A arterioles from the red gastrocnemius muscle were dissected and cannulated with glass pipettes in a tissue bath. ATP probes were connected to a potentiometer, polarized, zeroed, and a normal ATP curve generated (6.25µM to 50 µM ATP). The ATP and null probes were then positioned in the tissue bath such that the tips of the probes were submerged in the physiological saline solution and the tips were touching the arteriole in similar anatomical locations. The arterioles were then field stimulated at 60Hz for 2000 impulses at 9mA using a constant current stimulator. The normal curve produced 7.19mA at 50 µM ATP, 6.09mV at 25 µM ATP, 3.83mA at 12.5 µM. Following linear curve fitting, the slope of the line was 7.15x10−2 mA/uM ATP and an intercept of 3.22mA. Field stimulation at 60Hz produced 3.44mA that corresponded to 3.01 µM of ATP released. These data suggest that it is possible to measure ATP released from the sympathetic nerve endings from arterioles using potentiometry.
Cyclic GMP‐dependent protein kinase (PKG) regulates smooth muscle gene expression : Possible involvement of Elk‐1 sumoylation
Authors: Choi ChungSik , Sellak Hassan , Brown Felricia M. , Lincoln Thomas M.
Published: June 2021

Although the regulation of smooth muscle cell (SMC) gene expression by cGMP‐dependent protein kinase (PKG) is now recognized, the mechanisms underling these effects are not fully understood. In this study, we report that PKG expression increased the level of a high molecular weight Elk‐1 as determined by western blot using a phospho‐Elk‐1 antibody. PKG expression also enhanced myocardin‐induced SM22 promoter activity in cultured SMC. With this background, we examined the hypothesis that PKG may modulate Elk‐1 activity through posttranslational modification by sumoylation, thus contributing to the expression of SMC specific genes. Using immunoprecipitation with sumo‐1 antibody and subsequent immunoblot with phospho‐Elk‐1 antibody, we identified the higher molecular weight species as sumoylated Elk‐1. PKG expression in SMC increased Elk‐1 sumoylation two‐fold compared to the control cells. The sumoylation of Elk‐1 was confirmed by the transfection of dominant negative form of the sumo‐conjugating enzyme, DN‐Ubc9. Furthermore, siRNA‐mediated suppression of PKG confirmed the role of the kinase in the up‐regulation of sumoylated phospho‐Elk‐1. Overexpression of Elk‐1, sumo‐1, and PKG in COS‐7 cells confirmed again that PKG increased the sumoylation of Elk‐1. Transfection of combined vectors demonstrated that PKG inhibited Elk‐1 binding and activity on the myocardin‐induced SMC specific gene promoter activity as measured by ChIP assay and reporter gene expression, respectively. Taken together, these data suggest that PKG increased sumoylation of Elk‐1, thereby increasing myocardin‐SRF activity on SMC‐specific gene expression.
Voluntary wheel running abolishes vascular inflammation and restores endothelial function in old mice
Authors: Connell Melanie L , Durrant Jessica R , Russell Molly J , Donato Anthony J , Seals Douglas R , Lesniewski Lisa A
Published: June 2021

Aging is associated with impaired endothelial dependent dilation (EDD) and vascular inflammation. Aerobic exercise improves EDD in older humans and rodents, but it's unknown if this is associated with reduced vascular inflammation. Inflammatory cytokines were measured using multiplex ELISA in aortas of young (YC, 4‐6 mo) and old (OC, 29‐34 mo) cage‐control and old voluntary wheel running (OVR, 1.1±0.6 km/d, 12 wk) B6D2F1 mice (N=4‐6/group). EDD to acetylcholine was measured in isolated femoral arteries (N=6‐8/group). Interleukin‐1β (IL‐1β: 3.7±0.9 vs 1.6±0.5 pg/ml, P<0.05), interferon γ (IFNγ: 45.1±8.4 vs 15.0±5.0 pg/ml, P<0.05) and tumor necrosis factor a (TNF a: 2.6±0.5 vs 1.2±0.4 pg/ml, P<0.05) were higher and maximal EDD was lower (85±4% vs 97±3%, P<0.01) in OC vs. YC. In OVR, IL‐1β (1.1±0.7 pg/ml), IFNγ (26.5±2.0 pg/ml), TNF a (1.2±0.3 pg/ml) and EDD (95±2%) were restored to levels similar to or better than YC. To determine if the absence of vascular inflammation in OVR is associated with reduced activation of the pro‐inflammatory transcription factor, nuclear factor κ B (NFκB), we measured aortic protein expression of the inhibitor of NFκB, IκBa (western blot). IκBa was 117% greater in aortas of OVR compared with OC mice 1.3±0.3 vs 0.6±0.2 AUs, P<0.05). This is the first evidence that habitual exercise may restore the loss of EDD with aging by abolishing vascular inflammation, possibly via inhibition of NFκB.

NIH AG013038, AG006537, AG015897, AG029337, AG000279
No evidence of pre‐synaptic ATP‐sensitive potassium (KATP) channel modulation of excitatory inputs to second order chemoreceptor neurons during hypoxia in the nucleus of solitary tract (NTS)
Authors: Zhang Weirong , Mifflin Steve
Published: June 2021

Activation of KATP channels is a neuroprotective mechanism in response to brain tissue hypoxia/ischemia. Three min of anoxia induces an outward current mediated by KATP channel in second‐order chemoreceptor NTS neurons. It is still unknown whether there are pre‐synaptic KATP channels modulating glutamate release in the NTS. Whole‐cell recordings of NTS second‐order chemoreceptor neurons identified following DiA labeling of the carotid bodies were obtained in a brainstem slice. We investigated the effect of anoxia (95% N2/5% CO2) vs normoxia (95% O2/5% CO2) on spontaneous pre‐synaptic glutamate release by recording mEPSCs in DiA labeled NTS neurons. Cs‐based pipette solution was used to block neuronal potassium channels. Three min anoxia did not significantly alter the frequency (95±4% of control) or amplitude (100±2% of control, n=7) of mEPSCs. The ineffectiveness of anoxia on mEPSCs was not altered by the KATP channel blocker tolbutamide (100 μM, 96±7% of control frequency and 97±2% of control amplitude, n=4). The KATP channel opener diazoxide (200 μM) did not significantly alter mEPSC frequency (96±3% of control), and amplitude (93±2% of control, n=3). Our data do not support pre‐synaptic KATP channel modulation of glutamate release in the NTS during acute tissue hypoxia. This work is supported by AHA BGIA 0865269F and NIH HL41894.
Apnea is exacerbated following exposure to intermittent hypoxia and is mitigated following administration of an antioxidant cocktail
Authors: Yokhana Sanar , Lee Dorothy S , Badr M Safwan , Mateika Jason H
Published: June 2021

Purpose

To determine whether exposure to intermittent hypoxia (IH) exacerbates apnea severity and whether administration of an antioxidant cocktail (AOX) mitigates this response.

Methods

Two studies were completed. Ten participants with OSA completed study 1 while 5 participants completed study 2. Study 1 required two visits while study 2 required 3 visits. For both studies, a baseline polysomnogram with no intervention was completed initially. During the remaining visits of both studies, participants were exposed to IH {12‐4 minute episodes of hypoxia (PETO2‐ 50 mmHg, PETCO2 ‐ 3 mmHg above baseline)} immediately followed by a sleep study. During visits 2 and 3 of study 2 participants were administered either an AOX or Placebo (PLB) in a randomized blinded fashion. Breathing events were scored during the initial 3 hours of sleep.

Results

Study 1 ‐ The apnea/hypopnea index (AHI) after exposure to IH was greater compared to baseline (43.3 ± 5.3 vs. 34.6 ± 3.2 events/hr, p = 0.04). Study 2 ‐ Similar results were observed when the AHI during PLB was compared to baseline (43.4 ± 6.2 vs. 27.6 ± 7.8 events/hr, p = 0.01). In contrast, the AHI during the PLB trial was greater compared to the AHI following AOX administration (43.4 ± 6.2 vs. 31.8 ± 5.7 events/hr, p = 0.04).

Conclusions

Exposure to IH may lead to an increase in the AHI while administration of an antioxidant cocktail may mitigate the increase.

Funding

NIH (HL‐085537)
dAIH restores phrenic long‐term facilitation contralateral to cervical spinal injury
Authors: Vinit Stéphane , Mitchell Gordon S
Published: June 2021

After C2 hemisection (C2HS), acute intermittent hypoxia (AIH) elicits phrenic long‐term facilitation (pLTF) ipsilateral (Golder and Mitchell, 2005), but not contralateral to injury (Doperalski and Fuller, 2006). Repetitive (daily) exposure to AIH (dAIH, 10‐5 min episodes of 11% O2, 5 min normoxic intervals, 7 days) nearly completely (>80%) restores breathing capacity 2 weeks post‐C2HS (Barr et al., 2007). Since dAIH also increases growth/trophic factor expression, including Brain Derived Neurotrophic Factor (BDNF) which is critical for AIH‐induced phrenic long‐term facilitation (pLTF), we tested the hypothesis that dAIH restores AIH‐induced pLTF contralateral to C2HS (15 days post‐injury). In urethane anesthetized, paralyzed, vagotomized and ventilated rats, integrated phrenic nerve activity was measured contralateral to C2HS following pretreatment with: 1) dAIH beginning 7 days post‐injury; and 2) normoxia (sham). Following AIH, pLTF was not observed in untreated rats contralateral to C2HS (7% ± 4% of hypoxic activity, 60 min post‐AIH; n=6; n.s.). In contrast, robust pLTF was observed contralateral to injury in dAIH treated rats (33% ± 11% % of hypoxic activity, 60 min post‐AIH; n=8; p<0.05). Thus, dAIH restores the capacity for AIH‐induced pLTF contralateral to C2HS. Repetitive AIH may be a useful treatment to induce functional recovery following cervical spinal injury.

Supported by NIH HL69064.
Repetitive Hypercapnia and Hypercapnic Hypoxia Slows Disease Progression in a Rodent Model of Amyotrophic Lateral Sclerosis (ALS)
Authors: Nashold Lisa J , Schumacher Bryan T , Mitchell Gordon S
Published: June 2021

Rats over‐expressing a mutated form of superoxide dismutase (SOD1G93A) exhibit progressive motor neuron degeneration and paralysis, and are a common model of familial ALS. We previously made the chance observation that weekly exposure to graded hypercapnia (5% and 7% CO2; 20 min) followed by hypercapnic hypoxia (7% CO2 in 10% O2 20 min) apparently delayed disease progression. In this uncontrolled comparison, treated rats were 16 days older at a defined end stage (>20% decrease in body mass) vs untreated littermates (p<0.05). Here we studied this protocol under controlled conditions. Eleven mutant SOD1G93A rats were paired with a mutant littermate of the same sex; within each pair, 1 rat was treated 3 times per week with the hypercapnia/hypoxia protocol and the other was exposed to air (sham). Exposures were performed from 82 days of age until one rat within the pair reached end‐stage. Rats were perfused to enable counts of surviving motor neurons (analysis underway). Decreases in body mass slowed in treated rats on the day of sacrifice (untreated: 19% +/‐2%, treated: 10% +/‐2%; p <0.05). In 7 of 11 pairs, untreated rats reached end‐stage first (p=0.06); treated rats tended to be older (164+/‐3; n = 4) vs untreated rats at end‐stage (149+/‐6; n = 7; p=0.11). Repetitive hypercapnia may slow disease progression and increase survival in SOD1G93A rats. Further studies to test this hypothesis are underway.

NIH NS057778 and RR023916
Anatomical Study of Serotonergic Nuclei Involvement in Cardiovascular Compensation following Hypotensive Hemorrhage
Authors: Glasgow Jaimee , Kung Ling‐Hsuan , Vantrease Jaime , Scrogin Karie
Published: June 2021

Lesion of serotonin (5‐HT) neurons in the hindbrain attenuates sympathetic compensation (SC) after hypotensive hemorrhage (HH) in awake rats. To determine the 5‐HT nuclei involved in SC, 5‐HT cells activated by HH were identified. Three days before the experiment, rats were implanted with vascular catheters under pentobarbital (65 mg/kg, i.p.) and randomized to 3 groups subjected to arterial blood withdrawal (21 ml/kg/10 min, n=7), hypotension (hydralazine, 10 mg/kg, i.v., n=8), or no treatment (n=7). After 90 min, rats were anesthetized (pentobarbital, 65 mg/kg, iv), perfused, and their brains processed for 5‐HT‐ and c‐Fos immunoreactivity. Larger numbers of double‐labeled cells were observed in the lateral extension of the B3 nucleus (P<0.05), the subependymal region lateral to B3 (P<0.05), and the raphe obscurus (P<0.01) in rats subjected to HH. In preliminary studies, adeno‐associated viral‐mediated knockdown of tryptophan hydroxylase 2 in the mid raphe obscurus attenuated SC (‐16% vs. +72% Δ baseline, 15 min after HH), while knockdown in more rostral regions did not. 5‐HT neurons in the brainstem, particularly in the raphe obscurus, likely contribute to SC following HH. Supported by HL 72354.
Chronic femoral artery occlusion augments exercise pressor reflex in decerebrated rats
Authors: Tsuchimochi Hirotsugu , McCord Jennifer L , Hayes Shawn G , Koba Satoshi , Kaufman Marc P
Published: June 2021

Although the exercise pressor reflex evoked by static muscle contraction has been reported to be potentiated by acute ischemia of skeletal muscles, the effect of chronic muscle ischemia on the exercise pressor reflex remains unclear. We, therefore, determined if ligation of the femoral artery 72 hours prior to static contraction augmented the exercise pressor reflex in male rats. 72 hours after ligation of one femoral artery, rats were decerebrated at midcollicular level. The pressor responses to static muscle contraction were compared between the side ligated previously for 72 hours and the contralateral side. The pressor response to contraction of the side whose femoral artery was ligated 72 hours previously averaged 28 ± 8 mmHg whereas the pressor response to contraction of the contralateral freely perfused side averaged 10 ± 8 mmHg (n=4, P<0.05). Likewise the pressor response to contraction on the side whose femoral artery was ligated for 72 hours previously averaged 28 ± 8 mmHg, whereas the pressor response to contraction on the contralateral side whose femoral artery was ligated for only 3 minutes averaged 8 ± 3 mmHg (n=4, P<0.05). We conclude that chronic femoral arterial occlusion augments exercise pressor reflex. Supported by NIH grant HL30710.
Lack of handling stress‐induced hyperthermia in orexin neuron‐ablated mice
Authors: Kuwaki Tomoyuki , Zhang Wei , Sakurai Takeshi
Published: June 2021

We have previously shown that multiple outputs during defense response against stressors (increases in blood pressure, heart rate, respiratory frequency and tidal volume, redistribution of blood flow from viscera to skeletal muscles, and stress‐induced analgesia) are attenuated in orexin‐deficient mice (Am J Physiol 290: R1654, 2006, Autonom Neurosci 142:11, 2008). In the present study, we examined whether the same is true for stress‐induced hyperthermia or not. We used prepro‐orexin knockout mice (ORX‐KO), orexin neuron‐ablated mice (ORX‐AB), and their controls. Increases in handling stress‐induced rectal temperature were significantly attenuated in ORX‐AB but not in ORX‐KO. Increases in uncoupling protein‐1, a major contributor in non‐shivering thermogenesis in the brown adipose tissue, were also blunted in ORX‐AB. Because orexinergic neurons contain not only orexin but also glutamate, dynorphin, and galanin, one of these substances should mediate stress‐induced hyperthermia.
Aerobic exercise training increases maximal in vivo cholinergic responsiveness but not sensitivity of eccrine sweat glands
Authors: Wilson Thad E. , Monahan Kevin D. , Kearney Matthew , Fogelman Amy , Sauder Charity L. , Ray Chester A.
Published: June 2021

Aerobic exercise training increases maximal sweat capacity and sweat responses at a given internal temperature. However, the mechanisms underlying these effects are unknown. The purpose of this study was to determine if aerobic exercise training improves in vivo cholinergic sensitivity and responsiveness of eccrine sweat glands. Eleven previously sedentary subjects were tested before and after 8 weeks of aerobic training consisting of 4 bouts of exercise per week that elicited an ~20% increase in peak oxygen uptake. In vivo cholinergic sensitivity and responsiveness were determined by dose‐response curves of acetylcholine (ACh) to sweat secretion rate on two forearm sites. ACh was administered via intradermal microdialysis while measuring cutaneous water loss (capacitance hygrometry) directly above the microdialysis membrane. Exercise training did not alter the number of cholinergic activated sweat glands or the dose‐response derived ED50 (2.0±0.1 to 2.1±0.1 ‐logM dose of ACh; P>0.05). However, the maximal response to the highest dose of ACh was greater after exercise training (0.84±0.11 to 1.16±0.21 mg/cm2/min, P<0.05). These data indicate that 8 weeks of aerobic exercise training does not increase in vivo cholinergic sensitivity as indicated by the ED50 but does increase maximal in vivo cholinergic responsiveness of human eccrine sweat glands.

Support: HL77670, AG24420, M01 RR10732, C06 RR016499
Dorsal spinal cord stimulation obtunds the capacity of intrathoracic extracardiac neurons to transduce myocardial ischemia
Authors: Ardell Jeffrey L. , Cardinal René , Vermeulen Michel , Armour J. Andrew
Published: June 2021

Populations of intrathoracic extracardiac neurons transduce myocardial ischemia, thereby contributing to sympathetic control of regional cardiac indices during such pathology. Our objective was to determine whether electrical neuromodulation using spinal cord stimulation (SCS) modulates such local reflex control.

Methods

In 10 anesthetized canines, middle cervical ganglion neurons were identified that transduce the ventricular milieu. Their capacity to transduce a global (rapid ventricular pacing) versus regional (transient regional ischemia) ventricular stress was tested before and after SCS (50 Hz, 0.2 msec duration at 90% motor threshold) applied to the dorsal, cranial thoracic spinal cord. Rapid ventricular pacing and transient myocardial ischemia both activated cardiac related middle cervical ganglion neurons. SCS obtunded their capacity to reflexly respond to the regional ventricular ischemia, but not rapid ventricular pacing.

Conclusions

Spinal cord inputs to the intrathoracic extracardiac nervous system obtund the latter's capacity to transduce regional ventricular ischemia, but not global cardiac stress. Given the substantial body of literature indicating the adverse consequences of excessive adrenergic neuronal excitation on cardiac function, these data delineate the intrathoracic extracardiac nervous system as a potential target for neuromodulation therapy in minimizing such effects. (Supported by HL71830)
Crystallographic and genetic studies of archaeal RNA polymerase
Authors: Murakami Katsu
Published: June 2021

The transcription apparatus in Archaea can be described as a simplified version of its eukaryotic RNA polymerase (RNAP) II (Pol II) counterpart. We reported the first crystal structure of the archaeal RNAP from Sulfolobus solfataricus at 3.4 ? resolution, completing the suite of multi‐subunit RNAP structures from all three domains of life (Hirata et al, Nature 2008). We also reported the high resolution (at 1.76 Å) crystal structure of the D/L subcomplex of archaeal RNAP and provide the first experimental evidence of any RNAP possessing an iron‐sulfur (Fe‐S) cluster, which plays a structural role in a key subunit of RNAP assembly. Our analysis of RNAP structures provides insight into the derivation of the cellular RNAPs from their common ancestor. In addition, the striking structural similarity between archaeal RNAP and eukaryotic Pol II highlights the simpler archaeal RNAP as an ideal model system for dissecting the molecular basis of eukaryotic transcription.

All archaeal genomes encode RNAP subunits E and F (encoded by rpoE and rpoF) that share a common ancestry with the Pol II subunits Rpb7 and Rpb4. By gene replacement, we have isolated the ΔrpoF Thermococcus kodakarensis cells that grew at the same rate as wild‐type at 70 °C, but much slower and to lower cell densities at 85 °C. RNAP preparations purified from ΔrpoF cells lacked subunit F and also subunit E and a transcription factor TFE that co‐purifies with RNAP from wild‐type cells, but in vitro, this mutant RNAP exhibited no discernible differences from wild‐type RNAP in promoter‐dependent transcription, abortive transcript synthesis, transcript elongation or termination (Hirata et al, Mol. Microbiol. 2008).
Changes of heat shock protein 70, intracellular Ca2+, NO and free radicals in epilepsy rat brain tissues affected by garoderma lucidum spores
Authors: Wang Shu‐Qiu , Kang Yu‐Ming , Liu Lei , Ma Xiao‐Ru , Wang Xian‐Gang , Chen Ting‐Yu , Zhang Dong‐Dong , Liang Yan‐Feng , Zhang Bo
Published: June 2021

To investigate the effects of garoderma lucidum spores on the change of heat shock protein 70 (HSP70), intracellular Ca2+, NO and NOS content in epilepsy rats brain tissues. Thirty male Wistar rats at the age of 10 weeks were divided into garoderma lucidum spores group, epilepsy model group and blank control group. The result showed that NOS, NO and intracellular Ca2+ in brain tissue of garoderma lucidum spores group were significantly lower than in epilepsy model group. The number of HSP70 positive cells was more in garoderma lucidum spores group, while there was no expression in blank contrast group.

Conclusion

By effectively reducing the Ca2+ overload of neuron cell, reducing the formation and harm of NO and free radicals, and enhancing the expression of HSP70 in brain tissue, garoderma lucidum spores can weaken the damage to nerve system caused by continuous epileptic seizure.
Serotonin Regulation by Astrocytes
Authors: Watson Cheryl , Bates Jonatha , Franczak Rod
Published: June 2021

The concentration of serotonin in synaptic spaces is regulated by serotonergic neurons through a cycle of serotonin synthesis and vesicular packaging via vesicular monoamine transporter (VMAT), release, reuptake via serotonin reuptake transporter (SERT), degradation via monoamine oxidase (MAO), and repackaging in vesicles. Astrocytes are also known to contribute to the regulation of synaptic serotonin concentration through uptake of serotonin via SERT, and the subsequent degradation by MAO. Our question was: can astrocytes also package serotonin into vesicles and release it in response to a physiological signal?

Astrocytes were dissociated from 1‐3 day old rat pups and cultured. Astrocytes were then exposed to 0.3 µM‐ 100 uM serotonin for 5 hours. Immunofluorescent experiments on serotonin loaded, GFAP positive astrocytes, show an uptake of serotonin into discrete clusters which co‐localize with the VMAT protein. This localization may be due to packaging in vesicles, which is suggested by the presence of VMAT. Western blot and RT‐PCR experiments show the presence of VMAT message and protein in astrocytes. During exocytosis assays serotonin is released from astrocytes into the surrounding media, in response to a treatment with 100 uM ATP. Serotonin uptake can be inhibited by SSRI's such as fluoxetine and citalopram. These results suggest that astrocytes may be capable of packaging serotonin after reuptake, and releasing it in response to a physiological signal.
EXERCISE‐INDUCED PLASTICITY IN THE RAT HIPPOCAMPUS, MOTOR CORTEX AND BRAIN STEM
Authors: Ferreira Ana FB , Real Caroline , Alves Adilson S , Britto Luiz RG , Filosa Jessica A
Published: June 2021

Increasing evidence shows that physical exercise is neuroprotective and enhances brain function by improving memory, cognitive functions, neuroplasticity, and learning. The aim of this study was to observe the plastic effects physical exercise has on regions of the CNS related to cognitive and motor functions by evaluating the expression of structural proteins. A group of 40 adult male Wistar rats were subjected to an exercise protocol on an adapted treadmill. Following a 2 day adaptation period, the exercised group ran for 40 min/day, at 0.6km/h for 3 (3D), 7 (7D) or 15 (15D) days. At the end of the exercise protocol, brains were subjected to immunoblotting analysis to evaluate the expression of neurofilaments (NF) in the hippocampus, motor cortex and brain stem. In the hippocampus, immunoblot analysis showed increased expression of NF68 (p<0.005) following 3D exercise. In the motor cortex, however, the levels of both NF68 and NF160 decreased (p<0.05) after 3D exercised and increased at 15D for NF68 (p<0.05). The brain stem showed increased expression of all NFs at 15D (p<0.05). These results suggest that physical exercise alters the expression of structural neuronal proteins in brain regions associated with motor control, cognition, memory and learning. Our current ongoing studies focuses on the functional role astrocytes have on exercise‐induced structural neuronal changes.

Support: FAPESP, CNPq, CAPES
Ambient glutamate levels under astrocytic control enhance excitability of supraoptic nucleus (SON) neurons. An in vitro and in vivo study
Authors: Fleming Tiffany Marie , Scott Victoria , Brown Colin H. , Stern Javier E
Published: June 2021

In addition to mediating conventional quantal synaptic transmission, glutamate (GLU) mediates a persistent, tonic form of excitation (GLUtonic) mediated by NMDA receptors. Here, we aimed to determine 1) if ambient GLU tonically activates NMDARs in SON neurons 2) the role of astrocytes in regulating GLUtonic 3) the functional relevance of GLUtonic and 4) if GLUtonic is altered in dehydration (48DH). In electrophysiological recordings obtained from hypothalamic slices, the NMDA receptor antagonist D‐AP5 blocked a tonic inward current of 54.0. ±2.4 pA, a proportion of which (35.3%) was sensitive to the NR2B subunit selective blocker ifenprodil. Blockade of the glial glutamate transporter GLT‐1 by dihydrokainate (DHK) increased GLUtonic (23.3 pA ± 2.0 pA, n = 7, P < 0.01) but effects were blunted in 48DH rats (13.5 pA ± 1.0 pA, n = 5). In single‐unit extracellular recordings obtained from SON neurons in vivo, firing rate was higher in 48DH rats (10.5 ± 2.1 Hz, n = 12, P<0.01) than in controls (4.2 ± 0.6 Hz, n = 11). Microdialysis administration of DHK increased SON neuron firing rate (by 121.7 ± 31.3%, n = 7, P < 0.02) in controls, but not in 48DH rats (4.3 ± 7.3%, n = 4). Our results support that by controlling ambient GLU levels, SON astrocytes regulate GLUtonic and SON neuronal activity, a function that is diminished during dehydration. Ongoing experiments assess the effects of GLUtonic on SON somatodendritic VP and OT release.

Supported by NIH RO1HL68725 , NIH R01HL090948‐01, the Otago School of Medical Sciences Bequest Fund, NSF EAPSI.
Characterization of the Cardiovascular Responses to Intrathecal Administration of Angiotensin II (Ang II) in Conscious Rats
Authors: Veitenheimer Britta , Osborn John
Published: June 2021

Currently, little is known about spinal neurotransmitters and receptors regulating sympathetic nerve activity (SNA) and therefore arterial pressure (AP) in conscious animals. Ang II is important in brain pathways regulating AP, but little is known about its role at the level of the spinal cord. Previous studies have reported AT1 receptors in the intermediolateral cell column (IML), the location of sympathetic preganglionic cell bodies. In addition, Ang II has been found in many neurons in the PVN ‐ a brain region that projects to the IML. However, to our knowledge, the AP and heart rate (HR) responses to intrathecally (i.t.) administered Ang II in conscious rats has not been reported. The present study was conducted to measure the AP and HR responses to several doses of i.t. administered AngII in conscious rats. At least 5 days prior to experiments, male Sprague Dawely rats were instrumented for telemetric measurement of AP and i.t. administration of three doses of Ang II: 1μg, 10μg and 100μg. Peak increases in AP occurred within ~4 minutes, reaching levels of 12+3, 22+5, and 56+5 mm Hg respectively. Corresponding changes in HR were 38+15, 17+30, and ‐27+20 BPM. These data support the hypothesis that i.t. Ang II increases AP in conscious rats and suggest a possible role for spinally released Ang II in the regulation of SNA and AP in conscious rats. Supported by NIH Grant HL064178.
Effects of gender and age on hypothalamic redox signaling and adrenomedullary catecholamine biosynthesis
Authors: Tawil Rabih , Erdos Benedek , Woods Mary E , Cudykier Idan , Vierck Charles J , Yezierski Robert P , Tumer Nihal
Published: June 2021

Increased sympathetic nervous system (SNS) activity plays a central role in age‐related cardiovascular diseases. SNS activity is regulated in part by hypothalamic redox signaling and catecholamine biosynthesis. The aim of this study was to examine whether age affects these regulatory pathways differently in males and females. Hypothalamic and adrenomedullary protein levels were measured from young (5mo) and old (25mo) male and female F344xBN rats. NADPH oxidase p47 subunits were significantly higher in the young female (YF) compared with young male (YM) rats, and while p47 levels declined in both old male (OM) and old female (OF) rats, the reduction was smaller in OF. NADPH oxidase p67 subunit levels were similar in YM and YF, but declined more in OM than in OF. Hypothalamic CuZnSOD levels were lower in YF than in YM, and these levels further decreased with age in only males. In contrast, catalase expression was higher in YF compared with YM, and decreased more in OM than in OF. Hypothalamic tyrosine hydroxylase (TH), the rate‐limiting enzyme in catecholamine biosynthesis, was significantly higher in YM vs. YF, and decreased significantly in OM but not in OF rats. Adrenomedullary TH, an indicator of SNS activity, was similar in YF and YM, and only increased with age in males. These data show that there is a significant gender difference in how age affects central redox signaling and SNS activity.