bluuebunny/crossref_metadata_2025 huggingface dataset:
Novel Rho Kinase Inhibitors with Bronchodilator and Anti‐inflammatory Properties Suitable for Treatment of Respiratory Diseases
Authors: Navratil Tomas , Wells Grace , Fulcher Emilee , Sorensen Scott , Bodnar Wanda , Crean Chris , Lampe John , Peterson Ward
Published: June 2021

Novel Rho kinase (ROCK) inhibitors were tested for attenuation of airway hyperreactivity (AHR) and pulmonary inflammation. Two distinct profiles of molecules were explored: orally bioavailable molecules, represented by INS117758, suitable for oral product development; and orally nonbioavailable molecules, represented by INS117509, suitable for development as an inhaled product with limited systemic exposure. INS117758 and INS117509 potently inhibit ROCK2 (Ki values of 3.8 and 3.3 nM, respectively). While INS117758 is orally bioavailable in rats following 10 mg/kg p.o. dosing (Cmax= 5.6 μM, Tmax = 0.83 hours, T1/2 = 1.55 hours), INS117509 was not detectable in rat plasma following p.o. dosing. INS117758 and INS117509 relaxed carbachol‐precontracted tracheal rings (242 nM and 103 nM IC50s, respectively). Dosing of INS117758 p.o. at 5 mg/kg and INS117509 intratracheally (i.t.) at 1.5 mg/kg fully reversed AHR and reduced eosinophilia in OVA‐sensitized mice and neutrophilia in LPS‐exposed mice. In conclusion, Inspire novel ROCK inhibitors are suitable for treatment of respiratory diseases with AHR and pulmonary inflammation via oral route of delivery as well as inhaled route of delivery with limited systemic exposure.

Funded by Inspire Pharmaceuticals.
Cytotoxic effects of manganese nanoparticles using different solvent system in astrocytes and neuronal cultured cell
Authors: Ali Syed F , Boulton M C , Braydish‐Stolle L K , Murdock R C , Jiang H , Rongzhu L , Miltatovic D , Aschner M , Schlager J J , Hussain S M
Published: June 2021

Recently we have shown that nanoparticles such as Mn, Ag and Cu can be neurotoxic by generating ROS and depleting dopamine concentration in PC‐12 cells. We synthesized Mn nanoparticles (Mn‐NP) using different solvents. These NP were subsequently characterized using DLS, Laser Doppler Velocimetry and TEM. Mn‐NP when reduced with different solvents produced semi‐spherical, poly‐dispersed particles with an average size of 97.6 nm in tea, 32 nm in isopropyl alcohol, and 23 nm in caffeine. In the present study, we evaluated the cytotoxicity of Mn‐NP using astrocytes and neuronal cultured cell. Mn‐NP (5‐40 ug/ml) produced a concentration‐dependent decrease in MTT, and increased LDH release from cultured astrocytes and neuronal cultured cells. Mn‐NP also produced a time‐dependent increase in the expression of nuclear factor erythroid 2‐related factor 2 (Nrf2). Morphological evaluation showed a time‐dependent effect of Mn‐NP on cell morphology. The most interesting effect was found in cells treated with Mn‐NP in tea. Eight hrs post‐treatment, astrocytes appeared deformed and increased in size by 24 hrs. However, with other media, the effects were not as pronounced. Similar effects were found in neuronal cultures, where Mn‐NP in tea produced the most pronounced dysmorphic effects. These data suggest Mn‐NP effectively produce neurotoxicity by affecting astrocytic and neuronal functions, most likely by generating oxidative stress and neurotoxicity.
ABC Transporter Structure and Mechanism
Authors: Rees Douglas Charles
Published: June 2021

ATP Binding Cassette (ABC) transporters constitute a ubiquitous superfamily of integral membrane proteins responsible for the ATP powered membrane translocation of a wide variety of substrates. The highly conserved ABC domains defining the superfamily provide the nucleotide powered engine that drives transport. In contrast, the transmembrane domains creating the translocation pathway are more variable, with three distinct folds currently recognized. Structural analyses of three bacterial importers, the BtuCD vitamin B12 importer and homologous HI1470/1, and the high affinity methionine MetNI importer, will be discussed within the mechanistic framework of the alternating access model. The interconversion of outward and inward facing conformations of the translocation pathway is coupled to the switching between open and closed interfaces of the ABC subunits that are associated with distinct nucleotide states. As with MetNI, additional domains may be present that can regulate transport activity. An important goal is to develop quantitative models that detail the kinetic and molecular mechanisms by which ABC transporters utilize the binding and hydrolysis of ATP to power substrate translocation.
Ethanol intoxication increases hepatic mitochondrial protein acetylation but not through sirtuin inhibition
Authors: Picklo Matthew J. , Lee Susan , Denu John M
Published: June 2021

Chronic ethanol intoxication induces a myriad of physiologic and biochemical changes in the liver. Recent work indicates that ethanol intoxication alters hepatic metabolism by promoting protein lysyl acetylation while inhibiting de‐acetylation. In this work, we tested the hypothesis that chronic ethanol intoxication (six weeks) in rats increases the lysyl acetylation of mitochondrial proteins in the liver. Six weeks of ethanol intoxication significantly increased the acetylation of several proteins in liver mitochondria. On the other hand, protein acetylation in heart mitochondria was not altered. Although mitochondrial protein acetylation is regulated by the mitochondrial de‐acetylase, Sirt3, mitochondrial content of Sirt3 was not altered by ethanol exposure. Mitochondrial levels of the sirtuin metabolite, O‐acetyl‐ADP‐ribose, were 3‐fold higher in ethanol‐treated rats than control rats, suggesting that sirtuin activity was not inhibited. These data suggest that chronic ethanol intoxication increased protein acetylation in hepatic mitochondria but not through inhibition of the sirtuin pathway. Funding : The Wisconsin Partnership Fund to JMD and SL and NIH grant R21 AA15145‐01 to MJP.
Enhanced heme oxygenase‐CO signaling in the rostral ventrolateral medulla of SHR
Authors: Li Guichu , Abdel‐Rahman Abdel A.
Published: June 2021

Heme oxygenase (HO)‐derived carbon monoxide (CO) is implicated in central control of blood pressure and baroreflex sensitivity. In the few reported studies that pharmacologically evaluated the activity of brainstem HO‐CO pathway, hemin was microinjected into the nucleus tractus solitarius of anesthetized normotensive rats. Whether HO‐CO signaling is upregulated (as defense mechanism) or downregulated (underlying factor for hypertension) in the rostral ventrolateral medulla of the SHR is not known. Therefore, we investigated in conscious SHRs, and their normotensive controls (WKY), the consequences of CO generation within the rostral ventrolateral medulla (RVLM) on BP, and neuronal norepinephrine (index of sympathetic activity). Hemin (1nmol) was microinjected into the RVLM and in vivo electrochemistry was used for measurement of NE changes as in our studies ( J Cardiovasc Pharmacol 45, 1‐11). Hemin microinjection elicited significantly (P<0.05) greater reductions in BP and NE in SHRs than in WKY rats; the durations of these responses were also greater in SHRs. The findings suggest exaggerated HO‐CO signaling in the RVLM of SHR, a brainstem area that controls sympathetic outflow. Such enhancement of HO‐CO signaling seems to serve as a defense mechanism to oppose further increases in BP in the SHR. The HO isoform (HO‐1 or HO‐2) implicated in these neurobiological effects remains to be elucidated.
Nrf2‐disrupted mice exhibits heightened liver injury induced by lithocholic acid
Authors: Tan Hendrick Kah Poh , Wood Geoffrey A , Yang Mingdong , Ito Shinya
Published: June 2021

Bile acid toxicity contributes to the pathogenesis of cholestasis. Lithocholic acid (LCA), the most toxic bile acid, induces cholestatic liver injury in rodents. On cultured hepatocytes we showed that LCA could activate the oxidative stress‐responsive Nrf2, triggering adaptive responses to mitigate resultant cell injury. Using Nrf2 disrupted (Nrf2‐/‐) mice, the in vivo role of Nrf2 in LCA‐induced hepatotoxicity was determined. Mice were injected (i.p.) with 125 mg/kg LCA. At 8 hours and 4 days of LCA treatment, hepatic expression of Nrf2‐target, cytoprotective genes including thioredoxin reductase 1, glutamate cysteine ligase subunits, glutathione S‐transferases, NAD(P)H: quinone oxidoreductase 1 and multidrug resistance‐associated proteins 3 and 4 was significantly enhanced in the wildtype, as compared to the Nrf2‐/‐ mice. Sex dimorphic influence in induction of Nrf2‐target genes was noticed. The blunted response in cytoprotective gene induction in Nrf2‐/‐ mice was accompanied by a more severe liver injury as evidenced by presence of multifocal liver necrosis, inflamed bile ducts, and significant elevated liver injury markers alanine aminotransferase and alkaline phosphatase. In conclusion, Nrf2 plays a crucial protective role against LCA‐induced liver injury by regulating cytoprotective genes which promotes cell survival upon toxic bile acid insults. Funded by CIHR
Renin from activated mast cells mediates the systemic inflammation of alveolar hypoxia
Authors: Chao Jie , Blanco V Gustavo , Wood John G. , Gonzalez Norberto C.
Published: June 2021

Alveolar hypoxia (FIO2 = 0.1) produces widespread systemic inflammation in rats. Recent data suggests that a mediator(s) released from alveolar macrophages (AM) activates mast cells (MC) and initiates an inflammatory cascade that includes activation of the local renin‐angiotensin system (RAS).

Objective

To determine whether MC degranulation leads to local RAS activation, and whether this is due to renin released from MC.

Methods

Intravital microscopy was used to study the rat mesentery microcirculation. Immunocytochemistry was used to detect the presence of renin in primary cultured AM, peritoneal macrophages, and peritoneal MC.

Results

1. Topical Angiotensin II (Ang II) caused leukocyte‐endothelial adherence (LEA) but not MC degranulation in the mesentery. 2. The MC secretagogue C4880 produced MC degranulation and LEA; Ang II receptor blockade inhibited C4880‐induced LEA but not MC degranulation. These combined observations suggest that RAS activation is downstream of MC degranulation. 3. Anti‐renin antibody detected the presence of renin in peritoneal MC, but not in peritoneal macrophages or AM.

Conclusions

The results support the hypothesis that renin released from MC activates the local RAS during alveolar hypoxia. Previous data indicate that MC activation, in turn, is the result of a circulating mediator released from AM during alveolar hypoxia. Supported by NIH HL 39443‐18 and AHA 0815652G
Lenti‐viral mediated overexpression of ACE2 or Angiotensin‐(1‐7) prevents bleomycin‐induced pulmonary fibrosis
Authors: Shenoy Vinayak , Ferreira Anderson J , Qi Yanfei , Dooies Autumn , Raizada Mohan K , Katovich Michael J
Published: June 2021

An activated pulmonary renin angiotensin system (RAS), comprising of ACE‐AngII‐AT1R has been implicated in the pathogenesis of idiopathic pulmonary fibrosis. The recent discovery of a counter‐regulatory arm of the RAS (ACE2‐Ang‐(1‐7)‐Mas), has led us to examine the role of this vasoprotective axis on pulmonary fibrosis with the use of lentiviral vector mediated gene transfer. Lentiviral particles carrying either the ACE2 or Ang‐(1‐7) fusion protein were administered intratracheally into the lungs of 5 weeks old male Sprague‐Dawley rats. Pulmonary fibrosis was induced by intratracheal administration of bleomycin (2.5mg/kg), following 2 weeks of gene transfer. Systemic blood pressure, body weights, right ventricular systolic pressure (RVSP) and right ventricular hypertrophy (RVH) were examined after two weeks of bleomycin administration. Bleomycin administration resulted in significant reduction in body weights, elevated RVSP, RVH and increased fibrotic lesions in the lungs. All these pathophysiological parameters were prevented in lenti‐ACE2 or lenti‐Ang‐(1‐7) treated rats. No significant change in the systemic blood pressure was observed among the different study groups. Collectively, these observations demonstrate that ACE2 or Ang‐(1‐7) gene transfer shifts the balance of the RAS from the vasoconstrictive‐proliferative axis (ACE‐AngII‐AT1R), towards vasoprotective axis (ACE2‐Ang‐(1‐7)‐Mas), resulting in attenuation of bleomycin‐induced pulmonary fibrosis.
Function of Klf4‐positive and Nanog‐positive Resident and Non‐Resident Progenitor Cells in Adult Lung
Authors: Cowan Colleen , Malik Asrar B , Wary Kishore
Published: June 2021

Given that regeneration and repair of tissues following injury require trafficking and differentiation of both resident and non‐resident progenitor stem cells, we tested the hypothesis that adult lungs harbor a limited subset of progenitor adult stem cells. To address this hypothesis, we used adult mice as a source for several tissues including the heart and the lungs. To identify and characterize presence of "candidate" primitive undifferentiated (progenitor) cells in adult tissues, we used Oct3/4, Klf4, Sox2, and Nanog transcription factors as markers. We used RT‐PCR, western blotting and confocal microscopy to determine the expression of these four transcription factors. RT‐PCR and western blotting experiment showed presence of a subset of Klf4+ and Nanog+ primitive cells. The number of Klf4+ and Nanog+ progenitor cells increased in the mice lungs that were challenged with bacterial endotoxin Lipopolysaccharide (LPS, 7.5 mg/kg body weight). These data indicate that adult tissues including lungs harbor a subset of Klf4 and Nanog expressing resident and non‐resident progenitor cells. These findings raise the intriguing possibility of previously unrealized existence of Nanog and Klf4 expressing adult progenitor cells that appear to respond to critical factors, and may contribute to regeneration and repair of tissue post‐injuries. Thus, further investigation into the function of Nanog and Klf4 in vivo may provide insights into the molecular mechanisms involved in the regeneration and repair of tissues in adult.
Radical gain and loss of CEACAM/PSG family genes in vertebrates
Authors: Hsu Sheau Yu Teddy , Chang Chia Lin
Published: June 2021

In placental mammals, a family of recently evolved tumor suppressor genes, carcinoembryonic antigen cell adhesion molecules (CEACAMs), and related pregnancy‐specific glycoproteins (PSGs) are critical to the regulation of tumorigenesis, fetal‐maternal interaction, and pathogen transmission. Despite having important roles in many basic physiological processes, these genes were believed to be "eutherian‐specific"; how they evolved remains a mystery. Based on syntenic mapping analyses, we reveal that, surprisingly, a large number of diverging CEACAM orthologs is present from teleosts to marsupial mammals. In addition, based on the deduced ancestral genotypes, we infer that the CEACAM gene family evolved from a couple of ancestral genes in the common ancestor of teleosts and tetrapods. Taken together, contrary to the conventional view that CEACAMs are placental mammal‐specific, our study reveals that CEACAM family genes represent an independent branch of the immunoglobulin superfamily that evolved early in vertebrates. Furthermore, while the observed accelerated gene gain of CEACAMs in therian mammals could be partly attributed to the presence of repetitive sequences and a lower genetic constraint associated with cell surface receptors, we propose an alternative evolution model to account for the radical changes in the CEACAM gene inventory.

The abstract was partly supported by the Taiwan National Health Research Institute grant HMRPG380011 and the NIDDK grant DK70652.
The effect of short‐term, low‐salinity acclimation on gill NHE, AE1 and HAT expression in the longhorn sculpin, Myoxocephalus octodecemspinosus
Authors: Hyndman Kelly Anne , Edwards Susan L , Kratochvilova Hana , Claiborne James B , Evans David H
Published: June 2021

We previously reported that the marine, longhorn sculpin, Myoxocephalus octodecemspinosus, can acclimate to 20% SW for days with no change in plasma osmolality, ion concentrations, or hematocrit level. Sculpin, however, lose ions during acclimation to 10% SW for 24 and 72 h and this reaches a lethal level after 6 days. During these acclimations, the sculpin is incapable of down‐regulating gill NKCC or CFTR, suggesting that they cannot turn off their gill ion secreting mechanisms. The counterpart to this is whether they can upregulate the transporters necessary for ion uptake during acclimation to low salinity water, such as the Na+/H+ exchangers (NHE), anion exchanger 1 (AE1) or H+‐ATPase (HAT), and thus maintain proper ion and water balance. We determined that sculpin upregulate gill NHE3 mRNA and NHE2c, while significantly down regulating AE1 during acclimation to 20% SW, and have undetectable levels of NHE2b during acclimation to 10% SW. There were no significant changes in HAT or NHE8. These transporters were immunolocalized in the sculpin gill, and we determined that during acclimation to 10 or 20% SW they are distributed in different aspects of the ion transporting cells of the gill. We conclude that longhorn sculpin are incapable of properly regulating gill ion transporters; thus, this is a physiological barrier from living in low‐salinity (below 10% SW) or fresh water.
Primary culture of isolated cardiac myocytes from the bullfrog, Rana catesbeiana
Authors: Duerr Jeffrey M , Luft Silvianne
Published: June 2021

Comparative vertebrate studies into physiological responses to adverse conditions can yield great insight into the design of robust systems and provide fundamental knowledge of stress adaptations. The classically studied cellular stress response is the mitogen‐activated protein kinase (MAPK) pathway. Prior investigations in our laboratory indicated a differential response of MAPK family activation to progressive dehydration in cardiac tissue from two species of anuran amphibians: Rana catesbeiana and Bufo marinus. Western blot analysis indicated that pERK increased by three‐fold in Bufo and 2‐fold in Rana. pJNK levels remained unchanged in Bufo whereas in Rana increased by 50%. pp38 did not exhibit any change in phosphorylation levels over the range of dehydration examined. To more specifically delineate signaling pathways activated in response to dehydrational stress (hypoxia, hyperosmolality) in anuran cardiac tissue, we adapted a variety of previously described methodologies to develop a reliable technique for isolating and retaining in primary culture cardiac myocytes from Rana catesbeiana. Isolated myocytes exhibited mostly a spindle‐like morphology with characteristic banding patterns and occasional spontaneous contractility when viewed under phase‐contrast microscopy. Myocytes were also immunostained with a fluorescent monoclonal antibody against the SR ryanodine receptor.
Hypoxia modifies the expression of clock genes in zebrafish larvae
Authors: Egg Margit , Pelster Bernd
Published: June 2021

Hypoxia is known to stimulate a wide range of responses at the organismic and the cellular level. The hypoxia inducible factor (Hif) appears to be the master key controlling the expression of a vast number of genes in order to coordinate acclimation to hypoxic conditions. Using real time PCR hypoxia induced expression levels of various Hif isoforms and of genes downstream in the Hif signalling pathway were assessed under different levels of hypoxia. Under hypoxia the circadian expression patterns of oxygen regulated genes were modified, suggesting a connection between circadian and hypoxic signalling pathways. Subsequent analysis of the clock genes period and clock revealed a down regulation of period expression starting 17h after the onset of hypoxia. During long term hypoxia (between 9 and 15 dpf) both genes were upregulated. It is hypothesized that the modification of the expression of clock genes contributes to achieve a general metabolic down regulation under hypoxic conditions.
Role of MLCP in relaxation of rat cavernosal smooth muscle
Authors: Leipheimer Robert E , Jackson Erin M , Toepfer James R
Published: June 2021

Calyculin A, a direct inhibitor of myosin light chain phosphatase (MLCP), was used to investigate the role of MLCP in mediating nitric oxide (NO)‐induced relaxation in rat corpus cavernosal smooth muscle. Isometric tension of rat cavernosal tissue was recorded and analyzed during contraction and relaxation in vitro. Strips of corpus cavernosum were placed in water‐jacketed tissue chambers filled with physiological buffered saline solution at 37oC and attached to force transducers. The buffer was continuously bubbled with 95% O2, 5% CO2 to maintain oxygenation and pH. After a 1 hour equilibration period, tissue samples were contracted by the addition of norepinephrine (10−4M) followed twenty minutes later by Calyculin A (10−6M) or dimethyl sulfoxide (DMSO, 10−5M) to experimental or control groups, respectively. Tissues were relaxed by adding the NO donor, sodium nitroprusside (SNP, 10−3M). Data were collected using the PolyVIEW data acquisition and analysis software system (Astro Med, Inc.). Results demonstrated that Calyculin A significantly inhibited SNP‐induced relaxation in corpus cavernosum (p < 0.001). Furthermore, the data suggest that MLCP plays a critical role in mediating the cellular mechanisms that regulate relaxation of corpus cavernosal tissue.

Supported by the Department of Biological Sciences, YSU.
Ethanol affects the respiratory control network through more than GABA receptors
Authors: Brundage Cord M , Iceman Kimberly E , Taylor Barbara E
Published: June 2021

Central nervous system networks that drive oscillatory activity rely on inhibitory amino acids like GABA for rhythm generation. Application of GABA to an intact but isolated ventilatory‐control network elicits an overall reduction in respiratory rhythm. In isolated bullfrog tadpole brainstem preparations the reduction in respiratory nerve output is both a decline in lung burst frequency and an abolition of gill‐associated neuroventilation. Ethanol is a GABA‐mimetic, and the ventilatory reduction exhibited during acute ethanol exposure has been associated with GABA‐meditated pathways. Chronic ethanol exposure has been found to attenuate responses to subsequent exposure to GABA and ethanol in the tadpole brainstem preparation. Despite these correlations between GABA and ethanol, preliminary experiments using acute ethanol application failed to reduce tadpole gill‐associated neuroventilation concomitant with a clear decrease in lung burst frequency. This suggests the effect of ethanol on respiratory rhythm generation is not exclusive to GABA‐mediated neurotransmission or, alternatively, that the role of GABA in the amphibian respiratory control network is more complex than previously speculated.
Sleep fragmentation attenuates the hypercapnic ventilatory response via adenosine A1 receptors in awake rats
Authors: Liu Chun , Cao Ying , Ling Liming
Published: June 2021

As a primary feature of obstructive sleep apnea (OSA), sleep fragmentation (SF) impairs respiratory long‐term facilitation and poikilocapnic hypoxic ventilatory responses. This study investigated the effect of SF on hypercapnic ventilatory responses (HCVR) and its underlying neurochemical mechanism in conscious adult male Sprague‐Dawley rats. SF was achieved by periodic, forced locomotion in a rotating drum (30s rotation/90s stop for 24h). Ventilation during baseline and hypercapnia (FiCO2=0.06) was measured using plethysmography. At ~1h after SF, resting ventilation (43.2±2.4 vs. 47.6±1.8 ml/100g/min) and arterial blood gases (PaCO2: 40.7±0.7 vs. 40.0±0.9; PaO2: 103.8±1.4 vs. 101.6±1.8 mmHg) were not significantly changed, but the HCVR (control: 172.8±17.5% above baseline) was attenuated (129.5±9.6%, p<0.05). This attenuated HCVR then approached spontaneously to the control level ~4h after SF (168.9±12.1%). This HCVR attenuation was also reversed (184.0±17.5%) by systemic injection of the adenosine A1 receptor antagonist 8‐CPT (2.5 mg/kg) shortly after SF, while 8‐CPT had little effect on HCVR in control rats (169.9±11.8%). These findings suggest that: 1) 24h SF causes an attenuated HCVR, which lasts only for several hours; 2) This attenuation requires activation of A1 receptors; and 3) SF of OSA may exacerbate OSA via impaired respiratory chemoreflexes. (Supported by: NIH HL64912)
Contribution of muscle afferent activation to the anterior cerebral artery blood velocity response to calf exercise in humans
Authors: Vianna Lauro C , Fisher James P
Published: June 2021

It has been proposed that the activation of mechanically sensitive muscle afferents (muscle mechanoreflex) is important for exercise‐induced increases in regional cerebral blood flow. To examine this possibility, and to assess how regional cerebral blood flow is influenced by activation of metabolically sensitive muscle afferents (muscle metaboreflex) and central signals arising from higher centers (central command), 12 subjects performed voluntary (Vol Ex) and electrically stimulated (Stim Ex) rhythmic calf exercise at 35% of maximum voluntary contraction, followed by post exercise muscle ischemia (PEMI). Anterior cerebral artery blood velocity (ACAVmean), mean arterial pressure (MAP), and end‐tidal PCO2 were measured throughout. Exercise induced increases in MAP were similar during Vol Ex (Δ6±2 mmHg) and Stim Ex (Δ7±2 mmHg; P>0.05). ACAVmean was increased from rest during Vol Ex (Δ15±1%; P<0.05), but remained unchanged during Stim Ex (P>0.05). MAP remained elevated during PEMI following both Vol Ex and Stim Ex (P<0.05 vs. rest), while ACAVmean was not different from rest (P>0.05). End‐tidal PCO2 was unchanged from rest throughout. These data suggest that selective activation of skeletal muscle afferents (i.e. Stim Ex or PEMI) does not increase ACAVmean, and that increases in ACAVmean during volitional contraction of an exercising calf muscle are dependent upon the presence of central command.
Cutaneous vasoconstriction during 30 minutes of progressive cold exposure is increased by hypoxia
Authors: Simmons Grant H , Minson Christopher T , Halliwill John R
Published: June 2021

The goal of this study was to test the effect of hypoxia on cutaneous vasoconstriction during prolonged cold exposure. Twelve subjects had 2 microdialysis fibers placed in the ventral forearm and were immersed to the sternum in a bathtub with arms positioned out of the water at heart level. One fiber was designated as control and the other received 5mM yohimbine + 1mM propranolol (to block adrenergic receptors). Skin blood flow was assessed at each site (laser‐Doppler flowmetry), divided by mean arterial pressure to calculate cutaneous vascular conductance (CVC), and scaled to baseline. Cold exposure was induced by a progressive reduction in water temperature from 36°C to 23°C over 30 minutes on 2 randomized study days (normoxia and poikilocapnic hypoxia [SaO2 = 80%]). During normoxia, cold stress reduced CVC by 44 ± 4 and 13 ± 7 % baseline in control and yohimbine sites (both P < 0.05 vs. pre‐cooling). During hypoxia, cold stress reduced CVC by 67 ± 7 and 35 ± 11 % normoxic baseline in control and yohimbine sites (both P < 0.05 vs. pre‐cooling). Hypoxia increased the vasoconstrictor response at both sites (both P < 0.05 vs. normoxia). We conclude that hypoxia increases cutaneous vasoconstriction during prolonged cold exposure, and this effect is at least partially mediated through non‐noradrenergic mechanisms. Supported by the Evonuk and ACSM Foundations
Enhanced heat loss despite blunted renal sympathoexcitation in diabetic rats during heat stress
Authors: Leite Laura Hora Rios , Zheng Hong , Coimbra Candido Celso , Patel Kaushik
Published: June 2021

In the present study we investigated the effect of hyperthermia on the regulation of sympathetic activity and blood flow redistribution in diabetic rats. Renal sympathetic nerve activity (RSNA), mean arterial pressure (MAP), heart rate (HR), body (Tb) and tail (Ttail) temperatures were recorded in α‐chloralose‐ and urethane‐anesthetized control and streptozotocin (STZ)‐induced diabetic rats (n=6/group) during heat stress. Heat stress was induced by a heating pad with increases in temperature from 37 to 43°C during 30 min. This heat stimulus resulted in blunted RSNA, MAP and HR in diabetic rats compared with controls. The highest values were attained at the end of heat stress (ΔRSNA: 46.4 ± 10.0% vs. 155.6 ± 22.1%; MAP: 91 ± 5mmHg vs. 114 ± 5mmHg; HR: 395 ± 11bpm vs. 460 ± 11bpm; Diabetic vs Control, p< 0.05), suggesting a decreased renal vasoconstriction. Diabetic animals also showed lower Tb during the first 13 min of heat stress and decreased heat storage rate (HSR: 15.9 ± 1.6cal.min−1 Diabetic vs. 22.0 ± 0.9cal.min−1 Control; p< 0.01). These results may be due to improved heat dissipation shown by diabetic rats as indicated by a decreased ΔTb until tail vasodilation (0.3 ± 0.1°C Diabetic vs. 0.7 ± 0.1 °C Control; p< 0.02). In conclusion, although diabetic rats have a decreased RSNA response (renal vasoconstriction), the tail vasodilation was sufficient to decrease Tb and HSR to heat stress.

Supported by: CNPq , NIH (HL‐62222)
Isolated effects of elevated local temperature on sweating in humans
Authors: Wingo Jonathan E , Low David A , Keller David M , Brothers R Matthew , Shibasaki Manabu , Crandall Craig G
Published: June 2021

This study tested the hypothesis that elevated local skin temperature (Tsk), independent of skin blood flow (SkBF) and nitric oxide (NO), augments sweating. Two microdialysis membranes were placed in posterior forearm skin in 7 subjects. Each site was instrumented for the measurement of local sweat rate and SkBF, and the control of Tsk. After insertion trauma subsided, an NO synthase inhibitor (L‐NAME) was administered at each site, while Tsk was clamped at 34 °C. Then, one site was locally heated to 40 °C. After ~20 min, progressively increasing doses of methacholine (MCh) were administered (6 min/dose) to stimulate sweating. Despite 40 °C local heating at one site, cutaneous vascular conductance was not different between sites prior to and across all doses of MCh (P=0.844). Non‐linear regression analysis revealed no difference (P=0.63) between sites in the effective drug concentration causing 50% of the peak sweating response (log [M] EC50 = ‐2.59±0.56 and ‐2.73±0.87 for the 34 °C and 40 °C sites, respectively). Likewise, no main effect of site or interaction between sites and drug dose for absolute sweat rate (P=0.59 for main effect and P=0.58 for interaction) or percent peak sweat rate (P=0.07 for main effect and P=0.30 for interaction) were detected. These results indicate that elevated local skin temperature to 40 °C, independent of SkBF and NO, does not modulate MCh‐induced sweating.

Supported by NIH grants 61388 & 84072.
Spinal cord stimulation (SCS) stabilizes intrinsic cardiac neurons to reduce neurally induced atrial fibrillation
Authors: Gibbons David D. , Richer Louis‐Philippe , Southerland E. Marie , Vinet Alain , Kus Teresa , Armour J. Andrew , Ardell Jeffrey L.
Published: June 2021

Objective

To determine the contribution of discrete neuronal inputs to the intrinsic cardiac nervous system for targeted reduction of the atrial arrhythmogenic substrate.

Methods

In 15 anesthetized dogs, trains of electrical stimuli (1 mA, 1ms) were delivered during the atrial refractory period to mediastinal nerves associated with the superior vena cava to induce atrial tachyarrhythmias. Neuronal activity was recorded in the right atrial ganglionated plexus (RAGP). For SCS, the dorsal aspect of the T1‐T3 spinal cord was stimulated electrically (50 Hz; 0.2 ms; 90% of motor threshold).

Results

Mediastinal nerve stimulation caused an abrupt increase in RAGP neuronal activity (~5 fold) that was maintained for the duration of transient neurally‐induced atrial fibrillation. SCS reduced basal neuronal activity and mitigated any mediastinal nerve‐induced increases in RAGP neuronal activity. Prior to SCS, mediastinal nerve stimulation evoked atrial fibrillation/atrial flutter (AF/Afl) from 95% of sites. After SCS, atrial rhythms induced by mediastinal nerve stimulation were 38% AF/Afl, 56% bradycardia/sinus rhythm, and 6% sinus tachycardia. SCS likewise reduced dispersion in early pacemaker breakthrough loci secondary to mediastinal nerve stimulation.

Conclusions

Neuromodulation with dorsal cranial thoracic SCS stabilizes elements within the intrinsic cardiac nervous system to reduce the atrial arrhythmogenic substrate to its excessive neural inputs. (Supported by HL71830)
Intracellular signaling mechanisms underlying the activity and internalization of the neuronal norepinephrine transporter by endothelin‐3 in the rat posterior hypothalamus
Authors: Hope Sandra , Bianciotti Liliana , Vatta Marcelo
Published: June 2021

Brain norepinephrine (NE) is involved in relevant physiological functions regulated by the hypothalamus. We previously reported that endothelins (ET) participate in the short term regulation of NE uptake in the anterior and posterior hypothalamus by changing the kinetic and internalization of the neuronal NE transporter (NET). In the present work we studied the mechanisms involved in the regulation of NET by ET‐3 in the rat posterior hypothalamic region (PHR). Experiments were performed in vitro and neuronal NE uptake and nisoxetine binding were used as indexes of activity and internalization of NET, respectively. Results showed that ET‐3 reduced neuronal NE uptake through the activation of a non conventional ET receptor involving PKC, PKA and PKG pathways. Furthermore ET‐3 enhanced NET internalization. However incubation with inhibitors of PKC (GF‐109203x), PKA (H‐89) or PKG (KT5823) inhibited ET‐3 response on NET internalization. Present findings suggest that the effect of ET‐3 on NET in the rat PHR is mediated by a non‐conventional ET receptor coupled to various intracellular pathways including PKC, PKA and PKG.

(UBACyT B079, CONICET‐PIP 5929, ANPCyT PICT‐38333)
Striatal interneurons in dissociated culture
Authors: Schock Sarah C , Jolin‐Dahel Kheira , Staines William , Arbuthnott Gordon , Garcia‐Munoz Marianela
Published: June 2021

There are 4 interneuron types in the striatum. Three contain GABA and either parvalbumin, calretinin or NOS/NPY/somatostatin. The fourth is cholinergic. Most studies characterizing the striatum use rat organotypic cultures, in part because cholinergic interneurons don't survive in late embryonic dissociated striatal cultures. Our aim was to produce a dissociated striatal culture containing cholinergic interneurons and to characterize them after 35 days in vitro. Cholinergic neurons from E12.5 CD‐1 mice were combined with striatal neurons and cortical neurons from E14.5 CD‐1 mice using cryopreservation technical support from QBM Cell Science.

We evaluated the characteristics of striatal interneurons after 35 days in vitro in cultures of striatal neurons alone or in corticostriatal cocultures. Inclusion of cortical neurons increased dendritic branching and axon terminal outgrowth of the interneurons compared to striatal cultures alone. Cortex from GFP transgenic mice enabled identification of cortical versus striatal neurons. Interneurons showed extensive GABAergic and glutamatergic innervation. Since we are able to obtain cultures with and without the cholinergic neurons we now can study the contribution of the giant aspiny neurons to the network properties of the striatum.

Funded in collaboration with OISTpc.
Enhanced NMDA receptor function in the hypothalamic PVN contributes to support of sympathetic nerve discharge and blood pressure in Ang II + Salt hypertensive rats
Authors: Calderon Alfredo S , Dong Ying , Andrade Mary Ann , Shi Peng , Chen Qing‐Hui , Toney Glenn M
Published: June 2021

Rats consuming a high salt diet and treated with angiotensin II (ANG II) develop neurogenic hypertension that involves activation of sympathetic nerve discharge (SND). Here, we investigated control of renal SND and mean arterial pressure (MAP) by NMDA receptors in the hypothalamic PVN. Control (0.4 % NaCl diet, no ANG II, n=6) and ANG II + Salt hypertensive rats (2.0% NaCl, 150 ng ANG II/kg/min, s.c., n=4) were anesthetized (urethane/chloralose) and MAP and renal SND responses to PVN microinjection of the NMDA receptor antagonist AP5 (3.0 nmol in 50 nl) were recorded. Baseline MAP was higher by ~12 mmHg in ANG II + Salt rats compared to controls. MAP was unchanged by AP5 in control rats, but decreased by 13 ± 2 mmHg (P<0.05) in the ANG II+ Salt group. Whereas renal SND was unchanged by AP5 in control rats, it was reduced from baseline by 13 ± 4 % in ANG II + Salt rats. These data suggest that maintenance of renal SND and MAP in rats with ANG II + Salt hypertension involves an enhanced role for NMDA receptor activation in the PVN. Whether common adaptive mechanisms underlie this enhancement of NMDA receptor function and that reported to occur in other sympathoexcitatory states (e.g., water deprivation, heart failure, intermittent hypoxia) requires further investigation. Support: HL076312 (NCDC)
Pre‐ and Postsynaptic Effects of Adenosine on Spinally Projecting Paraventricular Neurons
Authors: Li De‐Pei , Pan Hui‐Lin
Published: June 2021

Abstract

Adenosine produces depressor effects in various brain areas. However, the cellular mechanisms underlying the depressor effect of adenosine remain unclear. The presympathetic neurons in hypothalamic paraventricular nucleus (PVN) play an important role in the regulation of arterial blood pressure and sympathetic outflow through projections to spinal cord and brainstem. In this study, we performed whole‐cell patch‐clamp recordings on retrogradely labeled PVN neurons projecting to the intermediolateral cell column of the spinal cord in rats. Adenosine (10‐100 μM) dose‐dependently decreased the firing activity with a significant hyperpolarization in 12 of 26 neurons tested. Blockade of A1 receptors with DPCPX or intracellular dialysis of GDP‐β‐s eliminated the inhibitory effects of adenosine. Also, blocking ATP‐dependent K+ (KATP) channel with glibenclamide (50 μM) or tolbutamide (200 μM) abolished the effect of adenosine on the firing activity of PVN neurons. Furthermore, glibenclamide or tolbutamide significantly decreased the outward K+ current elicited by adenosine. In addition, adenosine decreased the frequency of glutamatergic and GABAergic postsynaptic currents in labeled PVN neurons. Collectively, these data suggested that adenosine inhibits the excitability of PVN presympathetic neurons mainly through postsynaptic A1 receptors and opening of KATP channels.
AT1 receptors in the paraventricular nucleus (PVN) are required for sympathoexcitation due to angiotensin II in the subfornical organ of nonpregnant and pregnant rats.
Authors: Kvochina Lyudmyla I , Phaup Jeffery G. , Hasser Eileen M. , Heesch Cheryl M.
Published: June 2021

Activation of AT1 receptors in the subfornical organ (SFO) by circulating angiotensin A II (A II) increases mean arterial pressure (MAP) through a pathway which includes the PVN. A II levels are increased and CNS actions of A II may support sympathetic nerve activity in pregnancy. The role of the PVN, and AT1 receptors in the PVN, in responses to microinjection of A II into the SFO was tested in nonpregnant (NP) and term pregnant (P) rats. In 7 NP and 8 P sinoaortic denervated (SAD) inactin anesthetized rats, baseline mean arterial pressure (MAP) was lower (P= 96 ±5; NP= 120 ±8 mmHg) and heart rate was higher (P =374 ±11; NP =330 ±10 bpm) in P rats. The increase in MAP in response to A II (20 µM, 50 nl) in the SFO was greater in P compared to NP rats (ΔMAP: P = +22 ±3; NP = +14 ±2 mmHg). Bilateral microinjection of the GABAA receptor agonist, muscimol (1.5 mM, 100 nl), into the PVN decreased MAP (ΔMAP: P= ‐43±7 mmHg; NP= ‐46±4 mmHg) and LSNA (ΔLSNA: P= ‐34±13%; NP= ‐37±3%) and eliminated responses to activation of the SFO in both groups. In 3P and 2NP rats AT1 receptor blockade (L158, 809, 20 mM, 100 nl) in the PVN also eliminated responses to activation of the SFO with A II. Thus, AT1 receptors in the PVN are required for sympathoexcitation mediated by SFO A II in both NP and P rats. (NIH HL 36245).
Glucagon‐like peptide‐1 mitigates myocardial ischemia‐reperfusion injury, and is associated with differential chemokine expression
Authors: Dokken Betsy B. , Davis‐Gorman Grace , La Bonte Laura R. , McDonagh Paul F.
Published: June 2021

After myocardial infarction, restoration of blood flow is critical to survival, however, reperfusion causes an inflammatory response that exacerbates the injury. Glucagon‐like Peptide‐1 (GLP‐1) has protective effects, but it's mechanism is poorly understood. Earlier, we found that postconditioning with GLP‐1 decreased ischemia‐reperfusion injury (IRI) after experimental myocardial ischemia in vivo, which was associated with decreased neutrophil accumulation. In this study, Sprague Dawley rats underwent a 30 minute coronary occlusion. Recombinant human GLP‐1 (30 pmol/kg/min) or vehicle was then infused for 2 hours of reperfusion. To determine the mechanism by which GLP‐1 mitigates neutrophil‐mediated IRI, we analyzed the expression of chemokines in plasma taken from animals before ischemia and at R120 using a rat cytokine array kit (RayBiotech, Inc). We found that two chemokines were differentially expressed. β‐NGF almost doubled in response to IR; GLP‐1 treatment abolished this increase (pre‐ischemia 16.6 ± 0.5; IR 30.9 ± 0.9; IR + GLP‐1 17.5 ± 1.3, p< 0.05, n=5). MCP‐1 decreased in response to IR; GLP‐1 treatment attenuated the decrease (pre‐ischemia 61.4 ± 4.8; IR 27.3 ± 2.1; IR + GLP‐1 54.6 ± 6.2, p< 0.05, n=5). GLP‐1 postconditioning may mitigate myocardial IRI in part by regulating the expression of chemokines. Supported by the Arizona Sarver Heart Center and NIH HL 58859.
Radiation combined injury enhances iNOS expression and cytokine concentrations, delays wound closure, and alters ileal villi histology
Authors: Kiang Juliann G , Jiao Wan , Cary Lynnette H , Mog Steven R , Pellmar Terry C , Elliott Thomas B , Ledney G David
Published: June 2021

Radiation combined injury (CI) results in mortality greater than that following radiation injury (RI) or skin‐wound trauma. The molecular mechanism for this increase is not clear. In this study, mice were given 9.75 Gy r‐radiation followed by a 15% total body skin surface wound. On 1d, 3d, and 7d after RI or CI, sections of ileum and skin were removed and histologically examined. Also, iNOS in ileum and skin and serum cytokines were determined. RI but not wounding vastly shortened and widened villi 3 d post‐RI, whereas CI resulted in smaller alterations in villus. However, the serosal layer was thinner after CI than after RI. The skin in CI mice was less cellular with a smaller healing bud than in wounded mice resulting in significantly delayed wound closure times after CI from about 14 d to 28 d. CI enhanced the levels of iNOS in both tissues that were upregulated by increases in transcriptional factors KLF6, NF‐kB, and NF‐IL6. The latter was related to a burst in cytokine concentrations including IL‐1b, ‐6, ‐8, ‐9, ‐10, ‐13, and c‐GSF. Bacteria were isolated from heart blood and liver, indicating translocation from intestinal tract and wound site may have induced increases in these cytokines. The data are consistent with the idea that increases in iNOS and cytokines by CI may contribute to the CI‐induced mortality. Agents that inhibit iNOS and the cytokine cascade may ameliorate CI‐induced sequelae including death.
Association of NRF2 and NQO1 Polymorphisms and Susceptibility to Emphysema
Authors: Marzec Jacqui M , Hertzberg Andrew , Yoon Hyoung‐Kyu , Kleeberger Steve
Published: June 2021

Rationale

Emphysema is defined as airway enlargement due to loss of alveolar structure and diminished elasticity, and is mainly caused by cigarette smoking and air pollution. NRF2 activates antioxidants such as NQO1 in response to oxidative stress, and deficiency increases susceptibility to cigarette smoke‐induced emphysema in mice. The objective of this study was to determine whether functional polymorphisms in NRF2 and NQO1 associate with emphysema susceptibility.

Methods

Emphysema cases and controls were recruited for study. Regions of the NRF2 promoter and NQO1 promoter were amplified from genomic DNA and sequenced.

Results

Individuals homozygous for the NRF2 rs35652124 G allele were at increased risk for development of emphysema compared to those with wildtype alleles [OR 2.514; CI: 1.173, 5.389]. Individuals homozygous for the NQO1 rs689455 C allele were at decreased risk for emphysema compared to those with wildtype alleles [OR 0.299; CI: 0.124, 0.722].

Conclusions

In this translational investigation, we found that the NRF2 rs35652124 and the NQO1 rs689455 mutations confer differential risk of emphysema. Previous investigations demonstrated similar risk effects of NRF2 and NQO1 promoter SNPs for susceptibility to acute lung injury. These investigations suggest that NRF2 and NQO1 are pivotal determinants of susceptibility to oxidative stress‐induced lung diseases.
Correlations between peripheral vascular function, inflammation and depression in human subjects
Authors: Stapleton Phoebe A. , d'Audiffret Alexandre C. , Frisbee Stephanie J. , Goodwill Adam G. , James Milinda E. , Frisbee Jefferson C.
Published: June 2021

In addition to traditional risk factors for peripheral vasculopathy, including altered lipid profiles and chronic inflammation, ongoing study has suggested that psychological health, and specifically personal depression, may represent a profound contributor to poor vascular outcomes. The present study interrogated relationships between established risk factors for peripheral vascular disease, indices of personal depression, and vascular/endothelial function in human subjects recruited through a vascular surgery clinic at WVU HSC. During clinic visits, patients completed a depression screener, received baseline evaluations of health, provided venous blood samples and had brachial artery hyperemia evaluated following alleviation of 2 and 5 minute occlusions via ultrasound imaging. Results indicated that reactive hyperemia was reduced in patients in proportion to plasma TNF‐α and IL‐1β, (IL‐1β > TNF‐α). Additionally, depression severity was correlated both inflammation markers, as well as with impairments to reactive hyperemia. However, there was no evidence that self‐reported depression was an exacerbating influence on RH independent from that for inflammation. These results suggest that depression may not represent a causative factor contributing to poor vascular outcome, but may reflect a pathology arising from establishment of vascular dysfunction. (NIH R01 DK64668, AHA EIA 0740129N)
Characterization of a stable cell line expressing human Na+/taurocholate cotransporting polypeptide for high throughput screening
Authors: Roth Megan , Alnouti Yazen , Hagenbuch Bruno
Published: June 2021

Human Na+/taurocholate cotransporting polypeptide (NTCP) is selectively expressed on the basolateral membrane of hepatocytes and mediates Na+‐dependent uptake of conjugated bile acids. It has also been shown to transport non‐bile acid substrates, including some statins. To examine the substrate specificity of NTCP using high‐throughput screening, we established a stable Chinese Hamster Ovary cell line that transports taurocholate with Km and Vmax values of 18.4 μM and 6.9 nmoles/mg/min, respectively. To screen for interactions we determined uptake on 96‐well plates in the absence and presence of xenobiotics that affect hepatocytes. Potential substrates were tested directly using either radiolabels or LC‐MS/MS analysis. Numerous strong inhibitors were identified, including troglitazone and rosiglitazone (IC50s of 3.5 μM and 0.93 μM, respectively), perfluorodecanoic acid (IC50 = 8 μM), fluvastatin (IC50 = 40 μM) and carbenoxolone (IC50 = 9 μM). Neither glitazone was a substrate of NTCP. Unexpectedly, pravastatin, which did not inhibit NTCP‐mediated transport, was a substrate for NTCP, indicating the potential presence of multiple binding sites. These results show that our cell line can be used for high‐throughput screening to identify xenobiotics that may interfere with enterohepatic circulation of bile acids, or are potential substrates of NTCP.