bluuebunny/crossref_metadata_2025 huggingface dataset:
Teasing a(PAR)t biased‐agonism: Protease‐activated Receptor‐1 at signaling crossroads in endothelial barrier function
Authors: Soh Unice JK , Trejo JoAnn
Published: June 2021

Protease‐activated G protein‐coupled receptors (PARs) elicit cellular responses to extracellular proteases. The coagulant protease thrombin contributes to inflammatory responses associated with vascular injury and thrombotic disease, whereas the anti‐coagulant protease activated protein C (APC) diminishes thrombin generation, promotes cytoprotective and anti‐inflammatory signaling in endothelial cells. Both thrombin and APC signal through PAR1 but elicit opposite effects on endothelial barrier function. Activation of PAR1 by thrombin stimulates RhoA and disassembly of adherens junctions. Remarkably, incubation with APC stimulates activation of Rac, and not RhoA, and ablates thrombin‐induced endothelial barrier permeability. The mechanism by which APC protects endothelial barrier from thrombin signaling remains poorly understood. We examined PAR1 distribution with cytoprotective and inflammatory signaling mediators in caveolin‐enriched microdomains using density gradient fractionation. We found that β‐arrestins co‐sediment with PAR1 and are essential for APC‐stimulated Rac activation and endothelial barrier protection. Interestingly, APC induces translocation of Disheveled, a signaling scaffold that may function to distinguish differential regulation of endothelial barrier permeability. Supported by NIH R01 HL073328, UC TRDRP and AHA Postdoctoral Fellowship Award.
Organization of NADPH‐Cytochrome P450 Reductase and CYP1A2 in the Endoplasmic Reticulum – Microdomain localization affects monooxygenase function
Authors: Brignac‐Huber Lauren Michelle , Reed James , Backes Wayne L
Published: June 2021

Cytochrome P450 is found in the endoplasmic reticulum (ER), with its catalytic function requiring interactions with NADPH‐cytochrome P450 reductase (CPR). The goals of this study were to examine how the P450 system proteins are organized in the membrane and to determine if they are distributed in detergent‐resistant lipid microdomains (DRM). Liver microsomes from untreated rabbits were treated with 1% Brij 98, and DRMs were isolated via sucrose gradient centrifugation. Lipid analysis showed that DRM fractions were enriched in cholesterol and sphingomyelin, similar to that found at the plasma membrane DRMs. Approximately 73% of CYP1A2 and 68% of CPR resided in DRM fractions as compared to only 33% of total ER proteins. Cholesterol depletion experiments suggest that these DRMs are cholesterol‐dependent. CYP1A2 function was studied in three purified lipid vesicles consisting of 1) phosphatidylcholine (V‐PC), 2) lipids with composition similar to ER lipids (V‐ER) and 3) lipids with composition similar to the DRMs (V‐DRM). Association between CPR and CYP1A2 were measured and V‐ER and V‐DRM vesicles produced lower apparent Km values compared to V‐PC without any significant change in Vmax. These findings suggest that CYP1A2 and CPR reside in ER‐DRMs and that the unique lipid components of these domains enhance CYP1A2 substrate metabolism through more efficient CPR‐CYP1A2 binding. Supported by NIEHS ES004344
PXR Prevents Cholesterol Gallstone Disease by Affecting Bile Acid Biosynthesis and Transport
Authors: He Jinhan , Nishida Shigeru , Makishima Makoto , Xie Wen
Published: June 2021

Cholesterol gallstone disease (CGD), one of the most prevalent and costly digestive diseases, is resulted from biochemical imbalance of lipids and bile salts in the gallbladder bile. In this report, we showed that the previously known “xenobiotic receptor” pregnane X receptor (PXR) plays an important role in preventing CGD. Loss of PXR sensitized mice to lithogenic diet‐induced CGD, which was associated with decreased biliary concentrations of bile salts and phospholipids, leading to an increased cholesterol saturation index (CSI) and formation of cholesterol crystals. The decreased bile acid pool size in lithogenic diet‐fed PXR−/− mice was associated with a major suppression of Cyp7a1, the ratelimiting enzyme of cholesterol catabolism and bile acid formation. In contrast, treatment of C57L mice with the PXR agonist pregnenolone‐16alpha‐carbonitrile (PCN) prevented cholesterol precipitation by increasing biliary bile salt concentration and reducing CSI. The benefit of PXR in preventing CGD was achieved by its coordinate regulation of the biosynthesis and transport of bile salts in the liver and intestine. The current findings demonstrate an important role of PXR in maintaining biliary bile acid homeostasis, and establish PXR as a potential therapeutic target for cholesterol gallstone disease.
Meclizine is an agonist of human pregnane X receptor
Authors: Lau Aik Jiang , Yang Guixiang , Rajaraman Ganesh , Baucom Christie C. , Chang Thomas K. H.
Published: June 2021

Pregnane X receptor (PXR) is a receptor that regulates the transcription of genes involved in various functions, including drug metabolism and transport. Conflicting data exist as to whether meclizine, which is a histamine H1 antagonist, is an activator of human PXR (hPXR). Therefore, we conducted a detailed, systematic investigation to assess the effect of meclizine on hPXR activity by performing a cell‐based reporter gene assay, a time‐resolved fluorescence resonance energy transfer competitive ligand‐binding assay, a mammalian two‐hybrid assay to assess coactivator recruitment, and a hPXR target gene expression assay. Given the pronounced species differences in PXR activation, we also compared the effect of meclizine on rat PXR (rPXR) and hPXR function. In transfected HepG2 cells, meclizine activated hPXR to a greater extent than rPXR. The drug bound to hPXR ligand‐binding domain and recruited steroid receptor coactivator‐1 to the receptor. Consistent with its hPXR agonism, meclizine increased hPXR target gene (CYP3A4) mRNA expression in cultured human hepatocytes. However, it did not increase but decreased testosterone 6β‐hydroxylation, suggesting inhibition of CYP3A catalytic activity. In conclusion, our principal findings indicate species differences in hPXR and rPXR activation by meclizine and its mechanism of hPXR activation involves receptor agonism.[Supported by CIHR and MSFHR]
Induction of hepatic CYP enzymes by a subtoxic dose of acetaminophen in rats
Authors: Kim Sun J , Kwon Do Y , Nam Yea I , Kim Young C
Published: June 2021

Acetaminophen (APAP) is activated by CYP enzymes to N‐acetyl‐benzoquinoneimine, which reacts with cellular macromolecules to initiate liver damage. Alteration of CYP enzyme system by a toxic dose of APAP is well known; however, its potential adverse effect at a lower dose has hardly been explored. Rats were treated with APAP (500 mg/kg, ip) 18 h before a challenge with CCl4. APAP treatment only did not alter serum AST, ALT, or SDH activities. But a prior dose of APAP increased the CCl4 hepatotoxicity significantly. In rats treated with APAP 18 h before, hepatic p‐nitrophenol hydroxylase, p‐nitroanisole O‐demethylase, and aminopyrine N‐demethylase activities were increased significantly. Protein levels of CYP2E1, 3A, 1A1/2, and NADPH CYP reductase were increased, but mRNA expressions of the CYP enzymes were reduced. Ubiquitinated protein expression was suppressed by a prior dose of APAP. The immunoprecipitated CYP proteins were analyzed using a specific Ab against ubiquitin. The ubiquitin‐conjugated proteins in the immunoprecipitated CYP enzymes were decreased, implying that protein stabilization would play an essential role in the induction of CYP enzymes by APAP. These results suggest that a subtoxic dose of APAP may induce CYP enzymes and their metabolizing capacities, thereby influencing the metabolic conversion of xenobiotics that are substrates for the same enzyme systems.
Competitive inhibition of [14C]‐TEA uptake in A549 cell monolayers by formoterol and salbutamol
Authors: Salomon Johanna Jessica , Hosoya Ken‐ichi , Ehrhardt Carsten
Published: June 2021

Formoterol (For) and salbutamol (Sal), two β2‐receptor agonistic drugs, are positively charged at physiological pH. Here, we investigated, if organic cation/carnitine transporters (OCT/Ns) are involved in the translocation of the drugs in human alveolar epithelial cells (A549). A549 cells were grown to confluent monolayers for 5 days. Investigation of OCT/N‐mediated transport was performed using [14C]‐tetraethylammonium (TEA, 10 μM), [3H]‐acetylcarnitine (5.5 nM, Ac‐Car) and the β2‐agonists (both at 500 μM) in KRB. A549 monolayers were incubated for 30 min (TEA) or 20 min (Ac‐Car) at 37°C. Competitive inhibition studies were also performed in which TEA uptake (0.25 – 2 mM) was studied in the presence and absence of For and Sal for 30 min. The β2‐agonists showed pronounced inhibition of TEA uptake into A549 monolayers. TEA uptake was decreased to 15.4% (For) and 54.5% (Sal) of control, respectively. Neither β2‐receptor agonist had a significant effect on Ac‐Car uptake. Eadie‐Hofstee analysis of TEA uptake revealed linearity and Km values of 858.2 μM and 1050.6 μM in the presence of For and Sal, respectively, which was a 6.5‐fold (For) and 4.5‐fold (Sal) increase compared to control values. No significant change in Vmax was observed. Ki values were 115 μM (For) and 271 μM (Sal). β2‐agonists competitively inhibited TEA uptake via OCTs, but no interaction between the drugs and OCTNs was found.
Pre‐systemic fexofenadine drug‐drug interactions at OATP1A2
Authors: Flynn Colleen Ann , Hagenbuch Bruno , Reed Gregory A.
Published: June 2021

Fexofenadine (Fex) disposition is controlled by transporters, however there is uncertainty regarding the roles of specific transporters in Fex absorption. We previously tested a probe drug cocktail containing Fex, buspirone (Busp), caffeine, dextromethorphan (DM) and losartan (Los) for the measurement of transporter and CYP activities. A pre‐systemic Fex‐probe drug interaction was observed with this cocktail, decreasing Fex Cmax and AUC. We have characterized Fex transport and Fex‐probe drug interactions by the two major intestinal OATP uptake transporters in transfected mammalian cell models. We found that Fex is transported by OATP1A2 (Km=32 μM), but that Fex is not transported by OATP2B1. Additional studies demonstrated that Los, DM, and Busp each inhibited transport of Fex by OATP1A2. The efficient transport of Fex by OATP1A2 suggests that this transporter is a major determinant of the pharmacokinetics of oral Fex. Moreover, the inhibition of OATP1A2‐dependent transport of Fex by three of the probe drugs included in our cocktail is consistent with the observed decrease in Fex bioavailability. The replication of the drug‐drug interaction from the clinical study in these well‐defined model systems provides additional support for the conclusion that OATP1A2 is the major uptake transporter responsible for Fex absorption and suggests that Fex may serve as an effective probe drug for this transporter. Supported by NIH grants AT002907, RR021940, and ES007079.
An Inhalation Exposure System for the Oil Dispersant COREXIT® EC9500A
Authors: Goldsmith William Travis , McKinney Walter , Jackson Mark , Law Brandon , Bledsoe Toni , Siegel Paul , Frazer David
Published: June 2021

An automated whole body inhalation exposure system capable of exposing 12 rats was designed to examine the potential health effects of the oil dispersant (COREXIT® EC9500A [CE]) used during the Deepwater Horizon oil spill. A computer‐controlled syringe pump injected the CE into an atomizer where droplets and vapor were formed and mixed with diluent air. The aerosolized CE was passed into a customized exposure chamber where a calibrated light scattering instrument estimated the real‐time particle mass concentration of the aerosol in the chamber. A custom software GUI was developed to record and control parameters during the exposures. Software feedback loops controlled the chamber aerosol concentration and pressure throughout each exposure. Particle size of the dispersant aerosol was measured and shown to have a number peak at 271 nm and a mass peak at 961 nm. The method used to determine the chamber concentration was a modification of the acidified methylene blue spectrophotometric assay for anionic surfactants. Filter samples were extracted in 5 mL water/filter using sonication for 30 min and diluted 1/10 for analyses. CE filter content was extrapolated from a standard plot using the original CE (4–130 nL/mL water) material and adjusted for density. Five automated 5 hour animal exposures were performed that produced controlled and consistent CE concentrations (27.1 ± 2.9 mg/m3).
Acute effects of COREXIT® EC9500A on cardiovascular function
Authors: Krajnak Kristine , Kan Hong , Roberts Jenny R , Goldsmith William Travis , Frazer David , Castranova Vincent
Published: June 2021

The goal of these studies was to characterize cardiovascular responses after an acute inhalation exposure to COREXIT® EC9500A, the oil dispersant used in the Deepwater Horizon oil spill response. Male Sprague Dawley rats underwent a single 5 h exposure to COREXIT® EC9500A (N= 5 exposures, average exposure level 27.12 mg/m3) or air. On days 1 and 7 following the exposure, rats were implanted with indwelling catheters and changes in heart rate and blood pressure were assessed in response to increasing levels of adrenoreceptor agonists. Separate groups of rats were euthanized at the same time points, ventral tail arteries were dissected, and vascular tone along with dose‐dependent responses to vasoconstricting and dilating factors were assessed in vitro. Agonist‐induced dose‐dependent increases in heart rate and blood pressure where greater in COREXIT® EC9500A than air exposed rats 1 but not 7 days after the exposure. COREXIT® EC9500A exposure also induced an increase in basal tone and a reduced responsiveness of tail arteries to acetylcholine‐mediated vasodilation 1 day following the exposure. These findings demonstrate that an acute exposure to COREXIT® EC9500A had transient effects on cardiovascular and peripheral vascular function.
Neuronal synaptic and cytoskeletal protein aberration following acute inhalation exposure to the oil dispersant COREXIT® EC9500A
Authors: SRIRAM KRISHNAN , JEFFERSON AMY M , LIN GARY X , GOLDSMITH WILLIAM T , JACKSON MARK , FRAZER DAVID G , ROBINSON VICTOR A , CASTRANOVA VINCENT
Published: June 2021

Consequent to the Gulf oil spill, there is an emergent concern about the short‐ and long‐term adverse health effects of exposure to oil, weathered‐oil products and dispersants among the workforce employed to contain and clean‐up the spill. Workers are at risk for exposure primarily via inhalation of volatile or non‐volatile aerosolized components. These inhaled fractions can potentially translocate to the brain via olfactory or systemic circulation causing nervous system abnormalities. To determine if crude oil and/or dispersant pose a neurological risk, we have initiated investigations to evaluate their effects in a rodent model. Male Sprague‐Dawley rats were exposed by whole‐body inhalation exposure to the dispersant, COREXIT® EC9500A (~27 mg/m3 × 5h/day × 1d), and various indices of neural dysfunction were evaluated in discrete brain areas, at 1 or 7d post‐exposure. Exposure to the dispersant did not elicit neuroinflammation or oxidative stress in any of the brain areas examined. However, up‐regulation of L‐type voltage‐gated calcium channel, as well as, altered expression of synaptic and cytoskeletal proteins were observed in the striatum and hippocampus. Whether these molecular aberrations will persist and cause long‐term neurological deficits remains to be ascertained.
EFFECT OF PROPOLIS WITH TOPICAL ADMINISTRATION ON ACID‐INDUCED COLITIS IN RATS
Authors: Ozturk Haluk , Surer Ilhami , Surer Hatice , Demirbag Suzi , Guven Ahmet , Atabek Cuneyt , Caliskan Bahadir
Published: June 2021

PURPOSE

Propolis, which is a specific product of honeybee hive exhibits potential antioxidant and anti‐inflammatory properties. This study was designed to determine the effects of propolis on acid‐induced experimental colitis model in rats.

METHODS

The experimental acid induced colitis was performed in 56 Wistar‐Albino rats according to Mann and Demers. Then rats were divided into two equal groups (Sl‐saline irrigation and Pl‐propolis irrigation). After preliminary saline irrigation in all rats either propolis (1.5 ml–100 mg/kg) or saline enema (1.5 ml) was administered by rectal route after acetic acid induction/In both groups irrigation procedure was performed in every 12 hours until colonic resection time. At 3rd – 7th – 10th and 14th post operative days rats were killed and distal colonic segments were harvested to determine the histopathological changes and tissue levels of malondialdehyde (MDA).

RESULTS

Severity of the mucosal injury in Pl group was significantly lower when compared with the Sl group. Additionally MDA levels in Sl group was higher statistically significant than Pl group.

COCLUSION

Topical administration of propolis as a rectal enema has significantly improved rectal mucosal restoration rate and both histopathological parameters and MDA levels on acid‐induced colitis model.
Critical role of Paneth cell‐derived IL‐17A in hepatic ischemia and reperfusion injury
Authors: Lee H Thomas , Kim Mihwa , D'Agati Vivette D , Park Sang Won
Published: June 2021

Hepatic ischemia and reperfusion (IR) injury is a major clinical problem with frequent extra‐hepatic complications including intestinal dysfunction and acute kidney injury (AKI). We aimed to determine the mechanisms of hepatic IR‐induced systemic complications. Mice subjected to 60 min of hepatic IR not only developed severe hepatic injury but also developed significant AKI and small intestinal injury. Hepatic IR induced small intestinal Paneth cell degranulation and increased IL‐17A levels in portal plasma and small intestine. IL‐17A neutralizing antibody treatment or genetic deletion of either IL‐17A or IL‐17A receptors significantly protected against hepatic IR‐induced acute liver, kidney and intestinal injury. Leukocyte IL‐17A does not contribute to organ injury as wild type splenocytes infused into IL‐17A deficient failed to exacerbate liver and kidney injury after hepatic IR. Laser capture micro‐dissection of Paneth cells showed significant IL‐17A mRNA induction after hepatic IR. Finally, wild type mice treated with dithizone to deplete Paneth cells or Paneth cell deficient (Sox9flox/flox;VilCre) mice were protected against hepatic, renal and intestinal injury following liver IR. Taken together, the results show that Paneth cell derived IL‐17A plays a critical role in hepatic IR injury and extra‐hepatic organ dysfunction. Modulation of Paneth cell dysregulation may have therapeutic implications by reducing systemic complications arising from hepatic IR.

Supported by Department of Anesthesiology, Columbia University.
Skeletal muscle troponin I as a biomarker of skeletal muscle injury
Authors: Knapton Alan , Peters David , Rosen Elliot , Lu Quynh‐Ahn , Estis John , Agee Sara , Todd John , Herman Eugene
Published: June 2021

Many classes of drugs induce SM injury and creatine kinase (CK) has been used as a biomarker to detect this injury. The present study utilized an ultrasensitive immunoassay (Errena) to determine if slow or fast‐twitch troponin I (ssTnI, fsTnI) would be more useful biomarkers. Sprague‐Dawley (SD) rats (N=27, ♀, 12 wks) were dosed (po) with Simvastatin (SVS) (80 mg/kg) or water (n=15) once a day for up to 14 days. Tissues and blood samples were collected 24 hrs after final dosing. Fifteen SD dosed for 6‐11days were euthanized early (ED) due to excessive weight loss. SVS SM injury was detected in 9 ED animals (primarily in the psoas muscle (9/9)and multiple muscles in 3/9). All 15 had elevated levels of fsTnI. The 9 SD with muscle injury (lesion score>1.0) had higher mean levels (4691 pg/ml) compared to (651 pg/ml) in the remaining 6 ED animals and 129 pg/ml in15 control SD. SVS SM injury was absent in the Soleus muscle and ssTnI increased in only 1 of 15 ED. The levels of CK were not consistent. SVS SM injury was present in 5 of 12 SD that were treated for 14D (TS). Lesions were consistently present in the psoas muscle (5/5) and in multiple muscles in 3/5 TS rats. Serum levels of fsTnI were elevated 5 SD with lesions (8,317 pg/ml) and were normal in 7 unaffected TS SD (147 pg/ml). Microscopic SVS SM injury was absent in the Soleus muscle and ssTnI levels frrm all 12 SD were normal (156 pg/ml). CK levels increased from 199 IU (no lesions) to 526 IU (psoas lesions). These results suggest ssTnI and fsTnI are usefull biomarkers to identify drug‐affected SM fiber types and may offer advantages over routine microscopic evaluation or CK.
Mechanisms of Glucosamine‐Mediated Relaxing Responses in Isolated Rat Aortic Rings
Authors: Hilgers Rob , Oparil Suzanne
Published: June 2021

Glucosamine (GlcN) has been shown to block capacitative Ca2+ entry, also referred to as store‐operated Ca2+ entry (SOCE), in rat cardiomyocytes in response to phenylephrine (PHE). We hypothesized that GlcN would suppress SOCE in smooth muscle cells and lower intracellular [Ca2+] in PHE‐contracted rat aortic rings leading to vasorelaxation. In the wire‐myograph, relaxing responses to GlcN (0.01 – 30 mM) were assessed in PHE‐contracted rat aortic rings. GlcN relaxed endothelium‐denuded (−E) aortic rings with a sensitivity (pEC50) of 2.73 ± 0.09. The cell impermeable and osmotic control L‐ glucose (L‐Glc; 0.01 – 30 mM) did not result in arterial relaxations. Inhibition of the sarcoendoplasmic reticulum (ER) Ca2+‐ATPase (SERCA) results in Ca2+ depletion in the ER, which activates SOCE. The selective SERCA inhibitors thapsigargin (10 μM) and cyclopiazonic acid (50 μM) both significantly suppressed relaxations to GlcN in −E aortic rings. In Ca2+ depleted –E aortic rings, re‐titration of CaCl2 (0.01 – 5 mM) resulted in a lower sensitivity to CaCl2 in the presence of 5 mM GlcN compared to L‐Glc. Pre‐incubation with the SOCE inhibitor SKF96365 (10 μM) reduced sensitivity to CaCl2to comparable levels for both GlcN and L‐Glc. The present findings demonstrate that GlcN acts via an endothelium‐independent and cGMP‐dependent mechanism that inhibits SOCE to lower smooth muscle cell [Ca2+]i with subsequent vasorelaxation.
Free radical signaling in the subfornical organ (SFO) is required for the neurometabolic flow coupling dysfunction induced by angiotensin‐II (AngII) hypertension (HTN)
Authors: Iadecola Costantino , Capone Carmen , Faraco Giuseppe , Cao Xian , Davisson Robin L , Wang Gang
Published: June 2021

HTN disrupts cerebral blood vessels and causes stroke and dementia. In particular, HTN alters the coupling of cerebral blood flow (CBF) with brain metabolism. In AngII “slow pressor” HTN, reactive oxygen species (ROS) in the SFO mediate the blood pressure (BP) elevation. We studied whether ROS in the SFO are also required for the cerebrovascular dysfunction. Neocortical CBF was monitored by a laser‐Doppler probe through a cranial window in anesthetized mice (n=5/group). The CBF rise induced by neural activity (whisker stimulation; WS) was tested after infusion of AngII (600ng/kg/min) or vehicle (V) for 14dd. Ang II elevated mean BP from 70±2 to 92±2 mmHg, increased ROS in the SFO (2.3±0.3 folds; p<0.05), and attenuated the CBF response to WS (V: 23±2; AngII: 12±1±%)(p<0.05). Comparable HTN by phenylephrine (PE) did not alter the CBF response (V: 22±1; PE:23±1%). Adenoviral gene transfer of the ROS scavenger CuZn‐superoxide dismutase to the SFO attenuated the AngII‐induced ROS increase in the SFO (1.1±0.5 folds; p>0.05), prevented the HTN (V: 73±4; AngII: 74±2mmHg) and blocked the CBF dysfunction (V: 21±3; AngII:22±1%). We conclude that the dysfunction in flow‐metabolism coupling induced by slow pressor AngII HTN cannot be attributed to the BP elevation, but requires ROS signaling in the SFO. The effect is likely to involve neurohumoral mechanisms linking the SFO to neurovascular coupling (Supported by HL96571).
PLC gamma‐1 is Required for IP3‐mediated Activation of TRPM4 and Pressure‐Induced Depolarization and Vasoconstriction in Cerebral Arteries
Authors: Garcia Zarine , Earley Scott
Published: June 2021

In cerebral artery smooth muscle cells (SMCs), the melastatin transient receptor potential (TRP) channel TRPM4 is activated by intracellular Ca2+ release from inositol trisphosphate (IP3) receptors located on the sarcoplasmic reticulum. IP3 is a product of phospolipase C (PLC), an enzyme which cleaves the membrane phospholipid phosphatidylinositol 4,5‐biphosphate (PIP2) into IP3 and diacylglycerol (DAG). Multiple isoforms of PLC are present in vascular smooth muscle and can be classified into one of three families – β, γ, or δ. This study tested the hypothesis that PLC activity is required for TRPM4 activation, and investigated the particular PLC isoform responsible. Patch clamp electrophysiology recordings from freshly isolated cerebral myocytes revealed that the PLC inhibitor U‐73122 decreased the total open probability (NPo) of transient inward cation currents (TICCs) associated with TRPM4 by ~75%, while its inactive analog, U‐73343, had no effect. In addition, siRNA‐mediated knockdown of the PLCγ1 isoform decreased the TICC NPo. Following knockdown of PLCγ1, we also found that pressure‐induced SMC depolarization and myogenic vasoconstriction of cerebral arteries were greatly diminished compared with controls. These data suggest that PLCγ1 generates the IP3 required for TRPM4 activation in cerebral myocytes, and pressure‐induced SMC depolarization and vasoconstriction. RO1HL091905
Age‐related differences in cerebrovascular reactivity in response to COX inhibition
Authors: Barnes Jill N , Hines Casey N , Nicholson Wayne T , Schmidt John E , Katusic Zvonimir S , Joyner Michael J
Published: June 2021

Reduced cerebral blood flow and reactivity may explain age‐related changes in cognitive function. We examined cerebrovascular reactivity, and the potential role of prostaglandins, in young and old healthy humans. Middle cerebral artery velocity (MCAv) responses to stepped hypercapnia were measured in young (26±2y; 6M/5F) and old (66±2y, 3M/4F) adults before (PRE) and 90 min after (POST) oral indomethacin (1.2mg/kg). To account for any changes in blood pressure, cerebrovascular conductance index (CVCi) was calculated as the MCAv divided by mean arterial pressure. Cerebrovascular reactivity slopes were calculated using linear regression between either MCA velocity or CVCi and end‐tidal CO2. There were no differences between young and old adults in height, weight, BMI, or brachial blood pressures. Young adults demonstrated greater MCAv (53.7±6.7 vs. 32.0±3.3), MCAv reactivity (1.62±0.21 vs. 0.94±0.10), and CVCi (0.66±0.08 vs. 0.39±0.04) compared with old adults at PRE (P<0.05 for all). In both young and old adults, MCAv, MCAv reactivity, CVCi, and CVCi reactivity decreased between PRE and POST measurements. Furthermore, any age‐related differences in these cerebrovascular variables were abolished by indomethacin. These results suggest that cerebral blood flow and cerebrovascular reactivity is attenuated in aging adults and that this reduction may be mediated by prostaglandins.

NIH: AG16574‐11PP2
Contribution of IKCa Channels to the Control of Coronary Blood Flow
Authors: Berwick Zachary C , Kurian Michelle M , Kohr Meredith C , Moberly Steven P , Tune Johnathan D
Published: June 2021

The purpose of this investigation was to determine the role of intermediate conductance calcium‐activated potassium channels (IKCa) in regulating coronary arteriolar resistance at rest, in response to changes in coronary perfusion pressure, and during cardiac ischemia. Experiments were conducted in open‐chest anesthetized dogs in the presence of IKCa channel inhibitor, TRAM‐34 (1 μg/min, ic), or the nitric oxide (NO) synthase inhibitor, L‐NAME (150 μg/min, ic). Administration of the SKCa and IKCa channel agonist NS309 dose‐dependently increased coronary blood flow and inhibition of IKCa channels with TRAM‐34 attenuated this response by ~ 90%. The increase in coronary blood flow with NS309 was also decreased ~100% by L‐NAME. In addition, TRAM‐34 diminished the autoregulatory capability of the coronary circulation at pressures ranging from 60 – 120 mmHg. However, inhibition of IKCa channels did not affect coronary reactive hyperemia. Our data reveal that IKCa channels are functionally expressed in the coronary vasculature and that activation of these channels produces marked coronary vasodilation in vivo, primarily via increases in endothelial NO production. In addition, IKCa channels modestly regulate vascular resistance in response to alterations in coronary perfusion pressure but do not modulate coronary vasodilation in response to cardiac ischemia. Support 10PRE4230035, HL092245
Subendocardial Coronary Reserve as a Mechanism for the Preserved Cardiac Function in Rats vs Mice with Chronic Pressure Overload
Authors: Gao Shumin , Tian Yimin , Ho David , Lin Lin , Park Misun , Gelpi Ricardo , Vatner Dorothy E. , Vatner Stephen F.
Published: June 2021

We compared the effects of chronic (4 wks) pressure overload induced by aortic banding in rats and mice. As expected, LV function, as assessed by LV ejection fraction (EF), measured echocardiographically, declined in the mouse model (from 72±0.3% to 61±1%, p<0.01) accompanied by increased LV systolic wall stress (102±5.4 vs. 58±1.8 dyn/cm2, p<0.01); the LV/aortic pressure gradient was 98±3mmHg. In the rat, the pressure gradient was greater (145±7mmHg, p<0.05), but LVEF actually increased (from 73±1% to 78±1%, p<0.05) and LV wall stress did not increase (68±5.6 dyn/cm2). Reduced coronary reserve is one mechanism responsible for the reduced LV function with LV overload and hypertrophy (H). Coronary reserve, the increase in coronary flow velocity with adenosine measured echocardiographically, was similar in sham rats and mice (2.22 vs 2.44), and was reduced, p<0.05, in LVH mice (1.76), but preserved in LVH rats (2.21). One potential mechanism for the preserved coronary reserve is angiogenesis. Capillary density, measured by the endothelial cell marker (isolectin), was similar in sham rats and mice, but higher, p<0.05, in rats than mice with LVH (497 vs 407 units/mm2). Thus, the rat model appears unique in tolerating an unusually severe pressure gradient without compromising LV function or LV wall stress, which may be attributed to the preservation of coronary reserve through enhanced angiogenesis.
NADPH oxidase upregulates intermediate‐conductance Ca2+ ‐ activated K+ channels (KCa3.1) in coronary smooth muscle
Authors: Gole Hope , Bowles Douglas
Published: June 2021

Oxidative stress is known to play a role in cardiovascular disease including atherosclerosis. While several ROS may be involved, NADPH oxidase appears to play a key role. Studies have shown VSMC phenotypic modulation is critical to the development of atherosclerotic plaques. KCa3.1 is one mechanism by which increased VSMC proliferation and migration occurs. Our objective was to determine if NADPH oxidase plays a role in KCa3.1 upregulation. In coronary SMCs, bFGF increased superoxide (O2.‐) production; increased KCa3.1 mRNA levels ~2.5 fold, protein expression ~2.5 fold, and channel activity ~4.0 fold; and increased AP‐1 activity ~2.0 fold. Apo inhibited the bFGF increase in O2.‐, KCa3.1 message and activity, and AP‐1 activity. Coronary smooth muscle expressed all four cardiovascular Nox isoforms (Nox1,2,4, and 5) with Nox4 being the predominant isoform. bFGF decreased Nox1,2, and 4 message, while Apo increased message of all four isoforms. Nox2 and Nox4 siRNA did not affect KCa3.1 message response to bFGF or Apo. Our findings provide novel evidence that NADPH oxidase contributes to VSMC phenotypic modulation through AP‐1 transcriptional upregulation of KCa3.1.
Hibiscus sabdariffa extracts on vascular smooth muscle cell proliferation
Authors: Wu Chieh‐Hsi
Published: June 2021

Hibiscus sabdariffa Linne (HSL) has been found to be a great antioxidant. Since many antioxidants have been indicated in preventing restenosis, HSL is therefore elected to elucidate its effect on balloon injury‐induced neointimal formation. Extractions of HSL at different temperatures were tested on neointimal formation. Our results showed that room temperature extracts of HSL significantly inhibited neointimal formation. This in vivo results were supported by our in vitro study showing that cell proliferation, migration, apoptosis and oxidation were all suppressed by room temperature extracts of HSL on A10 vessel smooth muscle cells. The cell cycle was found to arrest at G0/G1 phase. The organic extracts were more potent to inhibit cell migration as compared to the water extracts. Similar results were found in organic extracts to significantly suppress the protein expression level of PCNA, Raf and FAK. Our results also showed that organic extracts of HSL significantly increased the protein expression level of caspase‐3. Taken together the above results, the room temperature organic extracts of HSL may possess potential preventive effects on PTCA‐induced restenosis.
A functional channel in kidney epithelial cells exhibit characteristics of gentamicin uptake‐related TRPV4 channel
Authors: Jiang Zhi‐Gen , Wang Tian , Karasawa Takatoshi , Yang Yuqin
Published: June 2021

Aminoglycosides (AGs) are important antibiotics but toxic to cochlear hair cells. Using kidney epithelial cell‐line cultures, we found that fluorescent gentamicin (GTTR) can enters MDCK and KPT2 cells independently of endocytosis and the uptake is enhanced by low [Ca2+]o and by exogenous expression of TRPV4 in KPT2 cells (KPT2‐TRPV4 cells), suggesting an important role for TRPV4 in gentamicin transmembrane transport. In this study, using whole‐cell and patch‐clamp recording techniques, we identified a channel in MDCK and KPT2‐TRPV4 cells that exhibited characteristics of TRPV4: (1) In physiological pipette and bath solutions, cell‐attached and outside‐out patches from MDCK and KPT2‐TRPV4 cells frequently displayed unitary current activity with a reversal potential near zero mV and a single‐channel conductance of ~100 pS for outward current and 30 – 60 pS for inward current. The unitary activity (Po) was enhanced by low extracellular Ca2+ and suppressed by Gd3+, La3+ and Ruthenium Red. (2) Hypotonic media (225 mOsmol), 100 μM ATP and 1 μM 4α‐phorbol 12,13‐didecanoate (4αPDD), a selective TRPV4 activator, stimulated this unitary activity. (4) The spontaneous or 4αPDD‐elicited unitary current was not sensitive to chloride channel blocker niflumic acid, flufenamic acid (FFA) or N‐phenylanthranilic acid. (3) In whole‐cell recordings, 4αPDD caused a net current with an I/V curve reversing its polarity near zero mV. (5) Control cells with empty vector (KPT2‐pBabe) showed very rare spontaneous NSC unitary currents. 4αPDD (2 μM) failed to elicit the unitary activity or whole‐cell I/V change in the KPT2‐pBabe cells. We conclude that MDCK and KPT2‐TRPV4 cells express functional TRPV4 channels; these data further substantiates the role of TRPV4 as a route for cellular gentamicin uptake and/or clearance. Funded by NIDCD R01 04716 (ZGJ), R03 DC009501 (T.K.)
Basolateral trafficking of KCa3.1 in a polarized epithelium
Authors: Winter Barclay J , Balut Corina M , Butterworth Michael B , Gao Yajuan , Devor Daniel C , Hamilton Kirk L
Published: June 2021

In this study, we examined the trafficking of the intermediate‐conductance, Ca2+‐activated K+ channel (KCa3.1) using novel biotin‐ligase acceptor peptide (BLAP) – BirA‐KDEL immunofluorescence (IF) technology. Fischer rat thyroid (FRT) cells were transfected with KCa3.1‐BLAP and BirA‐KDEL and grown on filters as a monolayer. To define the membrane location of KCa3.1‐BLAP, we labeled cell monolayers with streptavidin‐Alexa 546 on either the apical or basolateral side of the monolayers and incubated cells for 0 or 2 hr. Only basolateral labeling was evident demonstrating that KCa3.1‐BLAP was targeted to the basolateral membrane. Using IF, we determined which part of KCa3.1 was responsible for the basolateral trafficking of the channel. Various C‐terminal constructs of KCa3.1 were engineered to the neural growth factor receptor (NGFR) that is an apically targeted receptor. WT‐NGFR trafficked to the apical membrane while NGFR‐137‐KCa3.1 (entire C‐terminus of KCa3.1) and NGFR‐C137‐D370STOP‐KCa3.1 trafficked to the basolateral membrane, however, NGFR‐C59‐KCa3.1 (last 59 amino acids of KCa3.1) trafficked to the apical membrane. Our results demonstrate, for the first time in a polarized epithelium, that KCa3.1 is trafficked to the basolateral membrane and that the amino acids between the end of S6 and D370 of KCa3.1 are required for basolateral targeting of this channel. This work was funded by Lotteries Health and Department of Physiology (KLH) and NIH (DCD).
Role of c‐Src tyrosine kinase in airway smooth muscle contraction
Authors: Humber Brent , Tazzeo Tracy , Janssen Luke
Published: June 2021

One component of asthma is airway hyperactivity whereby the airway smooth muscle (ASM) cells abnormally constrict (to a greater degree) in the presence of an excitatory stimulus. It is not known whether serotonin (5‐HT) and acetylcholine (ACh) differentially impact ASM contraction via c‐Src phosphorylation (activation).

This study investigated the contractile responses to ACh and 5‐HT in the presence of a non‐selective tyrosine kinase inhibitor (genistein) and Src‐family kinase inhibitors (PP1 and PP2). We also examined whether these contractile agonists influenced c‐Src phosphorylation. Bovine tracheal ASM segments (0.5cm × 3cm) were dissected, mounted vertically in an organ bath and the isometric changes in tension were measured. Segments were flash frozen and subjected to western blotting. PP1 and PP2 significantly reduced the contractile response of 5‐HT by 73% and 70% respectively, but did not change the response to ACh. Genistein completely abolished responses to 5‐HT but did not affect responses to ACh. Western blotting data suggests increased c‐Src phosphorylation in response to both 5‐HT and ACh.

These data suggest c‐Src tyrosine kinase activation is uniquely sensitive to 5‐HT (relative to ACh) and might be a key molecular player in ASM contraction when ASM cells are stimulated by the 5‐HT G‐protein coupled receptor.

Research Support: CIHR
Membrane fusion protein annexin A7 and lamellar body protein ABCA3 co‐localize at the cell surface in secretagogue‐stimulated alveolar type II cells
Authors: Chander Avinash , Vasa Pavan Kumar , Gerelsaikhan Tudevdagva
Published: June 2021

Lung surfactant secretion requires fusion of lamellar bodies (LB) with the plasma membrane in type II (T2) cells. Annexin A7 (A7) contributes to such membrane fusion since 1) it promotes surfactant secretion in permeabilized T2 cells and 2) secretagogues increase its association with the LB and plasma membrane in T2 cells. In this study, we hypothesized that secretagogues would increase membrane insertion of A7 to facilitate its fusion promoting activity. As a result, A7 may be expressed at the cell surface. Isolated rat T2 cells were cultured for 20–22h on glass cover slips and stimulated for 2h without or with 100nM phorbol myristate acetate (PMA). The cells were fixed in 2% paraformaldehyde, (PFA, non‐permeabilized) or in ice‐cold methanol (permeabilized) and immuno‐stained for A7 and LB specific protein, ABCA3 (P180). With methanol fixation, control cells showed P180 and A7 staining at the cell surface as well as in the cell interior without significant co‐localization of two proteins. The PMA‐stimulated cells showed increased co‐localization of two proteins. With PFA fixation, control cells showed that A7 and some P180 staining at the cell surface without significant co‐localization of the two proteins. Very little staining was seen inside the cells. In PMA‐stimulated cells, increased staining and co‐localization of the two proteins were observed at the cell surface. Our results suggest that increased secretion is associated with increased insertion of A7 and P180 into the plasma membrane leading to exposure of these two proteins at the cells surface, possibly at the secretion site.
Niemann‐Pick type C1 (NPC1) and cholesterol transport in type II pneumocytes
Authors: Bates Sandra R. , Yu Kevin J. , Huang Shaohui , Tao Jian‐Qin
Published: June 2021

Although cholesterol comprises 10% of the total lipid content of lung surfactant, few studies address the regulation of surfactant cholesterol levels. Surfactant is synthesized by type II cells and stored in specialized organelles, lamellar bodies (LBs), prior to secretion into the alveolar space. LBs are considered to be part of the late endosome/lysosome compartment, the same location as NPC1, a transmembrane protein shown to modulate intracellular cholesterol transport. Deficiency of NPC1 results in abnormal lysosomal accumulation of cholesterol and other lipids. Thus, we examined the role of NPC1 in pneumocytes. Using a Western blot protocol and equal protein loading, NPC1 was found in the intact rat lung, enriched (1.6‐fold) in primary cultures of type II cells and greatly enriched (11.6‐fold) in LBs isolated from the lung. By immunocytochemistry of type II cells, NPC1 colocalized with ABCA3 on the limiting membrane of LBs. U‐18666A (UA) is a compound that inhibits cholesterol trafficking from the late endosomes to the endoplasmic reticulum, thus mimicking NPC1 deficiency. UA treatment of isolated type II cells resulted in a 50% increase in free cholesterol content and the accumulation of cholesterol (filipin‐stain) in LB‐like structures. The data are consistent with a role for NPC1 in removal of cholesterol during the maturation of LBs, thereby regulating the surfactant cholesterol content. [HL 19737]
Rac mediates actin remodeling and permeability during alveolar epithelial stretch
Authors: DiPaolo Brian Christopher , Margulies Susan S.
Published: June 2021

Alveolar epithelial cells (AECs) maintain integrity of the blood‐gas barrier via gasket‐like transcellular tight junction proteins bound to the actin cytoskeleton. AEC monolayers stretched biaxially undergo rapid actin cytoskeletal remodeling. We hypothesize that Rac1 and downstream effectors LIMK½, cofilin, Akt, and SSH1L are activated in stretched AECs. Furthermore we hypothesize that Rac1 pathway inhibition will attenuate actin remodeling and preserve paracellular permeability during AEC stretch. Primary AEC monolayers were stretched biaxially to create a change in surface area of 37% at ¼ Hz for 10 or 60 min. Additional monolayers were pretreated with Rac1 pathway inhibitors wortmannin, EHT‐1864, or IPA‐3 and stretched. Unstretched treated and untreated AECs served as two control groups. Without treatments, biaxial stretch rapidly increased Rac1, Akt, LIMK½, and cofilin activity, and resulted in rapid actin remodeling and increased monolayer permeability. Compared to stretched vehicle control monolayers, Rac1 pathway inhibitors reduced Rac1, Akt, LIMK½, and cofilin activity, attenuated actin remodeling, and preserved monolayer permeability in stretched monolayers. This study provides a foundation for developing treatment strategies aimed at reducing cellular injury during high magnitude biaxial deformation. NIH R01 HL057204
Acute Respiratory Distress Syndrome (ARDS): Are corticosteroids harmful in lung injury
Authors: Chupin Cecile , Lavoie Jacynthe , Privé Anik , Brochiero Emmanuelle , Berthiaume Yves
Published: June 2021

ARDS is a rapidly evolving lung injury that is linked with permeability oedema and excessive inflammation, leading to a severe oxygenation defect. Albeit there has been significant progress in survival, ARDS mortality remains ~ 40 %. Glucocorticoids therapy has been proposed to decrease the inflammatory response, but its usage remains controversial.

The objective of this study was to evaluate the impact of dexamethasone treatment (dex; 0.5 mg/kg/d) on the in vivo evolution of bleomycin‐induced (bleo; 4 U/kg) lung injury in NMRI mouse. Treatment with dex did not decrease the mortality, the weight loss, the wet/dry ratio, the severity of epithelial injuries and the inflammatory infiltrating of neutrophils induced by bleo. To better evaluate the lack of response to dex, the impact of dex on the repair process of primary epithelial cells was studied. Our results show that dex inhibits the closure of mechanical wound. This effect of dex is not related to a default of proliferation, or an activation of apoptosis. This inhibitory effect of dex on wound closure is associated with an increase in cell adhesion suggesting that its effect could be related a modulation of the cytoskeleton.

Our results indicate that dex does not decrease the severity of lung injury model and might slow its recovery by preventing proper repair of the alveolar epithelium.

Supported by RSR‐FRSQ, FCFK, IRSC.
Motility of alveolar mitochondria
Authors: Brandenberger Christina , Bhattacharya Jahar
Published: June 2021

Mitochondria are the major source of cellular ATP. As studied best in axons, mitochondrial motility regulates spatial ATP distribution within the cell. However little is known regarding regulation of mitochondrial motility in non‐neuronal cells. Here, we addressed this issue in intact alveoli of mouse lungs. Isolated, perfused lungs were held at constant pulmonary artery, left atrial and alveolar pressures of 10, 2 and 5 cm H2O, respectively. We visualized alveoli by confocal microscopy. To record mitochondrial density, we microinfused alveoli with the mitochondria‐specific dye, MitoTracker Green (MTG). Spontaneous, time‐dependent fluctuations of mitochondrial density were evident at several alveolar locations, suggesting that mitochondria were motile. To quantify the motility, we photobleached MTG‐labeled regions (3–6 μm) to 23±3% of the initial fluorescence. Then we determined time dependent fluorescence recovery after photobleaching (FRAP). Fluorescence recovery was 43±4 % of the initial in 3 min (P<0.05; n=3), indicating repopulation of the photobleached region by fluorescent mitochondria from adjoining regions. Our findings are the first evidence that alveolar epithelial mitochondria are motile. The motility might enable equitable delivery of ATP to different alveolar regions. (Swiss National Foundation, HL57556, HL36024)
Branching pattern of the single renal artery. Study on corrosion casts
Authors: MATUSZ Petru L. , BORDEI Petru , SAPTE Elena , ILIESCU Dan , BULBUC Ionut
Published: June 2021

Terminologia Anatomica (1998) homologated the forking of the renal artery (RA) in an anterior and a posterior branch. The purpose of this paper is to analyze the variability of branching of the trunk of the single RA on a number of 200 corrosion casts (100 from the right and 100 from left). The corrosion casts were realized by injection of the vasculo‐ductal systems with plastic mass, followed by the corrosion of the renal parenchyma. In 71% of the cases the RA gives rise at two branches; in 25% of the cases – it gives rise at three branches, and in 4% of the cases it gives rise at four branches. In case of bifurcation, the branches of the RA were: anterior and posterior (in 61% of cases); anterior and inferior (6.5%); anterior and superior (35%). In case of triforking, the branches of the RA were: anterior‐superior, anterior‐inferior, and posterior (10%); superior, anterior and inferior (6%); anterior, posterior and superior (4%); anterior, posterior and inferior (5%). In case of forking in four, the branches of the RA were: superior, anterior, inferior and posterior (1.5%); superior, anterior‐superior, anterior‐inferior and posterior (2%). The knowledge of these morphological aspects, is very important when performing partial kidney resections surgical techniques. (Supported by Grant CNMP 42097/2008).