bluuebunny/crossref_metadata_2025 huggingface dataset:
PAR1 inhibition does not enhance the cardioprotective effect of postconditioning
Authors: Jiang Rong , Rutledge Caleb , Shi Weiwei , Guyton Robert A , Vinten‐Johansen Jakob
Published: June 2021

Postconditioning (postcon) protects the heart from ischemia reperfusion (IR) injury. Protease activated receptor1 (PAR1) inhibition has protective effects after IR. Therefore, we hypothesize that if postcon and pharmacological PAR1 inhibition target the same pathway, and the combination of postcon and PAR1 inhibition may not have additive protective effect.

Methods

In anesthetized open‐chest rats, the left coronary artery (LCA) was occluded for 30 min and reperfused for 3 hours. Rats were randomly assigned to1) Control: saline; 2) PAR1 antagonist (BMS 226001) 1mg/Kg was administered 5 min before R; 3) Postcon with 3 cycles of 10 s R and 10 s re‐occlusion and 4) BMS 1 mg/Kg plus postcon.

Results

Area at risk (36%–39%) was comparable among 4 groups. Compared to control, infarct size (TTC staining) was significantly less in BMS, postcon and the combination groups. The combination of BMS and postcon did not further significantly reduce infarct size compared to either intervention alone.

Conclusions

Inhibition of PAR1 and postcon alone at reperfusion are cardioprotective in vivo; however, concomitant PAR1 inhibition does not enhance the cardioprotection of postcon.

Group
N
AAR/LV (%)
An/AAR (%)

Control
7
37.9 ± 2.4
57.0 ± 2.1

BMS
8
38.1 ± 2.5
42.5 ± 2.5*

Postcon
7
39.3 ± 2.1
39.1 ± 2.3*

BMS + Postcon
7
35.6 ± 1.4
39.9 ± 3.0*

Values are expressed as mean ± S.E.M.

P < 0.05 vs. values in control.
Hypoxia reduces cerebrovascular myogenic reactivity in diabetes
Authors: Kelly‐Cobbs Aisha Imani , Ergul Adviye
Published: June 2021

Diabetes increases the risk for acute ischemic stroke and poor stroke outcome. The outcome of cerebral ischemia is mainly determined by vessel diameter which is regulated by myogenic, endothelial, and neuronal factors. However, how diabetes affects myogenic reactivity and tone of cerebral vessels, especially under hypoxic conditions, is not known. We hypothesized that myogenic tone is increased in diabetes and that ischemia reduces cerebrovascular myogenic reactivity, thereby making the affected brain more prone to edema and bleeding. Lumen diameter (LD) and wall thickness (WT) of middle cerebral arteries (MCAs) from Wistar (n=6–8) and type 2 diabetic Goto‐Kakizaki (GK) rats (n=6–8) were analyzed under normoxic and hypoxic conditions and myogenic tone was calculated. In vitro hypoxia was achieved by exposing the isolated vessels to 95% N2/5%CO2 in 1 mM glucose to mimic oxygen‐glucose deprivation. The autoregulatory capacity of vessels in all groups was retained across the normal range. Under normoxic conditions, active LD was smaller in diabetes and myogenic tone was higher across the pressure range. Ischemia reduced myogenic tone in both groups but this effect was more significant in diabetic animals, potentially making them more likely to bleed after ischemia‐reperfusion injury. Hypoxia increased WT in Wistars alone, possibly making them less likely to hemorrhage and more prone to larger cerebral infarct.
Effect of Nitric Oxide Synthase and growth conditions on hydrogen peroxide production in cultured endothelial cells during shear stress
Authors: Zinkevich Natalya S , Gutterman David D
Published: June 2021

Endothelial cells release nitric oxide (NO) and reactive oxygen species (ROS) during acute shear stress (SS). We hypothesized that chronic SS attenuates the production of H2O2 during acute SS.

Human microvascular endothelial cells (HMVEC) were exposed to one hour of laminar SS, either with or without a preceding 24 hour SS exposure (chronic SS). Cells were studied either in full serum media (5%) or after serum‐starving (to increase ROS) overnight (0.5% serum). 2′,7′‐dichlorofluorescein (DCF) assessed H2O2 formation with flow cytometry (N=3–7).

Acute SS increased H2O2 release compare to static conditions (4.4±0.5 fold increase (F.I.) for 0.5% serum and 1.7±0.2 F.I for 5% serum (p<0.05)). Presence of L‐Nitro‐Arginine Methyl Ester (L‐NAME; nitric oxide synthase inhibitor) during acute shear reduced production of H2O2 in serum starved cells (2.7±0.2 F.I. vs. 4.4±0.5 F.I. (p<0.05)), but not in full serum. Acute SS after chronic SS reduced H2O2 in cells grown in low (1.9±0.8 F.I.) and full serum (0.9±0.1 F.I.). This reduction in H2O2 was reversed by L‐NAME in full serum (2.2±0.5 F.I. vs. 0.9±0.1 F.I. (p<0.05)), but not in low serum.

Preconditioning with 24 hrs of SS inhibits generation of H2O2 during acute SS. We conclude that NOS modulates H2O2 production differentially in high and low serum conditions.
The Putative TRPM4 Blocker 9‐Phenanthrol Causes Smooth Muscle Cell Hyperpolarization and Arterial Dilation
Authors: Garcia Zarine Isabel , Gonzales Albert Louis , Earley Scott
Published: June 2021

The contractile state of vascular smooth muscle cells is regulated by small changes in membrane potential that gate voltage‐dependent calcium channels. The melastatin transient receptor potential (TRP) channel TRPM4 is a critical mediator of pressure‐induced membrane depolarization and arterial constriction. A recent study shows that the tricyclic compound 9‐phenanthrol (9‐phen) inhibits TRPM4, but not the related channel TRPM5. The current study investigated the effects of 9‐phen on pressure‐induced smooth muscle depolarization and arterial constriction. Patch‐clamp electrophysiology revealed that 9‐phen (30 μM) had no effect on maximum evoked currents in HEK 293 cells expressing TRPC3. In freshly isolated cerebral arterial myocytes, 9‐phen decreased transient inward cation currents (TICCs) associated with TRPM4. Using intracellular microelectrodes to record smooth muscle membrane potential in isolated cerebral arteries pressurized to 70 mmHg, we found that 9‐phen reversibly hyperpolarized the membrane to from ~−40 mV to ~−70 mV. Lastly, we found myogenic tone was reversibly abolished when vessels were exposed to 9‐phen. These data suggest that TRPM4 is a major depolarizing factor in smooth muscle membrane depolarization and arterial constriction in response to intraluminal pressure. RO1HL091905(SE); F31HL094145(AG).
Disruption of Caffeine‐Induced Constriction in Isolated Cerebral Arteries Following Treatment with siRNA Against STIM 1
Authors: Gonzales Albert Louis , Earley Scott
Published: June 2021

The regulation of cytosolic free Ca2+ is crucial for controlling smooth muscle contraction. Ca2+ release from sarcoplasmic reticulum (SR) stores contribute to the activation of Ca2+ sensitive ion channels located proximally in the plasma membrane. Regulation of these channels governs membrane potential and the opening of voltage‐gated Ca2+ channels, ultimately leading to muscle contraction. The stromal‐interacting molecule (STIM) 1 acts as the Ca2+‐sensing protein, and is important for the refilling of SR Ca2+ stores. In the current study, we use RNAi technology to further characterize the role of STIM1 in caffeine‐induced SR Ca2+‐depletion and arterial constriction. Treatment of intact cerebral arteries with STIM1 siRNA decreased mRNA expression by ~70% compared with controls. Using ratiometric Ca2+ imaging, we found that caffeine‐induced increases in cytosolic Ca2+ levels were attenuated in STIM1 siRNA‐treated vessels. Furthermore, using simultaneous video dimensional analysis we found that transient caffeine‐induced constriction of arteries treated with STIM1 siRNA was blunted compared with controls. These data suggest that STIM1 plays a central role in regulating SR Ca2+ stores responsible for smooth muscle contraction and the maintenance of arterial tone. RO1HL091905 (SE); F31HL094145‐01 (AG)
Increases in Vascular Smooth Muscle Stiffness with Aging
Authors: Qiu Hongyu , Zhu Yi , Gansner Meredith , Depre Christophe , Peppas Athanasios , Resuello Ranillo , Natividad Filipinas , Hunter William , Meininger Gerald , Genin Guy , Elson Elliot , Vatner Dorothy E. , Vatner Stephen F.
Published: June 2021

It is generally assumed that the increased aortic stiffness during aging is due to changes in the extracellular matrix (ECM). Our hypothesis was that the mechanical properties of vascular smooth muscle cells (VSMCs) contribute to the increased vascular stiffness with aging. Young (YM, 6.4±0.1 yr) and old male (OM, 25±0.4 yr) monkeys (Macaca fascicularis) (n=7/group) were studied. The mechanical properties of the VSMCs were measured in vitro using a VSMC‐ECM interaction model. Using the reconstituted tissue model with cultured aortic VSMC embedded in collagen, the tissue force response was measured with a force transducer and strain by a micrometer. The total stiffness of the VSMC construct in OM was higher (46.7±6.1 kPa, p<0.01) vs that in YM (27.4±4.9 kPa). Then cytochalasin D (2 μM) was used to disrupt the actin cytoskeleton of VSMC and eliminate the “active” force induced by VSMC, leaving the ECM component. The stiffness of ECM was greater (p<0.05) in OM (36.1±4.9 kPa) than in YM (20.6±3.9 kPa), and VSMC stiffness calculated from the difference between the total and the ECM stiffness, was also significantly higher (p<0.01) in OM (10.5±1.2 kPa) vs YM (6.8±1.0 kPa). Therefore, aortic VSMCs from OMs not only produce higher active forces compared to YMs but also remodel collagen more extensively to produce a stiffer ECM, which contributes to increased vascular stiffness with aging.
Mitochondria control NF‐κB‐dependent functional CaV1.2 expression in cerebral artery smooth muscle cells
Authors: Narayanan Damodaran , Xi Qi , Pfeffer Lawrence , Jaggar Jonathan
Published: June 2021

In myocytes of resistance‐size cerebral arteries, we investigated Ca2+ signals that regulate mitochondrial Ca2+ concentration ([Ca2+]mito) and tested the hypothesis that [Ca2+]mito controls ion channel transcription, thereby regulating contractility. Endothelin‐1 (ET‐1), a vasoconstrictor, activated Ca2+ waves, elevated global intracellular Ca2+ concentration ([Ca2+]i), elevated [Ca2+]mito, caused mitochondrial depolarization, and stimulated ROS generation in arterial myocytes. Thapsigargin, a sarcoplasmic reticulum (SR) Ca2+ ATPase inhibitor and xestospongin C (XeC), an IP3 receptor (IP3R) inhibitor, blocked ET‐1‐induced [Ca2+]mito elevation, mitochondrial depolarization, and ROS elevation. ET‐1 stimulated the generation of mitochondria‐derived ROS which subsequently stimulated an additional small rise in NAD(P)H oxidase‐derived ROS. ET‐1‐induced IP3R‐mediated mitochondrial ROS stimulated nuclear translocation of the NF‐κB p50 subunit and NF‐κB‐dependent luciferase activity. NF‐κB p105 knockdown reduced basal CaV1.2 channel expression and attenuated pressure (myogenic tone)‐ and depolarization‐induced, CaV1.2 channel‐dependent vasoconstriction. ET‐1 elevated CaV1.2 channel expression, myogenic tone, and depolarization‐induced vasoconstriction, and these effects were blocked by XeC, bortezomib, a proteasome inhibitor, and NF‐κB p105 knockdown. These data indicate that in arterial myocytes, IP3R‐mediated SR Ca2+ release controls [Ca2+]mito, which regulates mitochondrial ROS generation. Mitochondrial ROS regulate NF‐κB activity, which controls functional CaV1.2 channel expression. Thus, myocyte mitochondria control arterial contractility by regulating NF‐κB‐dependent functional CaV1.2 channel expression. AHA GSE 09PRE2220266, NIH R01077678.
Neural activity in hippocampal slices from hibernating Syrian hamsters (Mesocricetus auratus) is more tolerant to anoxia than slices from nonhibernating hamsters
Authors: Tan Jennifer S , Singh Rajpreet , Taylor Kelleen C , Hamilton Jock S , Horowitz John M , Horowitz Barbara A
Published: June 2021

During arousal from hibernation, when sympathetic signals fully activate brown adipose tissue to generate heat and shivering in muscle adds to oxygen consumption, the brain may, for brief periods, become anoxic. Whether hippocampal neural networks in the hibernating hamster (HH) exhibit greater hypoxic tolerance than do those in the euthermic hamster (EH) is not known. We tested the hypothesis that CA1 pyramidal cell responses in HH hippocampal slices are more tolerant to anoxia than those in EH slices. The HH group was housed in a 6°C cold room on a 8:16 light:dark cycle, and the EH in a 23°C room on a 14:10 L:D cycle. Population spike amplitude (PSA) in both HH and EU groups were measured at 30°C bath temperature each minute throughout the experiment. After a 15 min. control period in oxygenated (95%O2/5%CO2) buffer, oxygen was replaced with nitrogen (95%N2/5%CO2) for 15 min. Slices were then perfused with oxygenated buffer for a 30 min. recovery period. Three minutes after nitrogen exposure, PSA in HH slices dropped 5.0 ± 4.4 % (mean ± SE) from control, compared to a larger drop (20.3 ± 5.3%) in EH slices. Five minutes after nitrogen exposure, PSA in HH slices had dropped 32.2 ± 5.7% (mean ± SE) from control, versus 58.3 ± 8.0% in EH slices. Differences are significant (P<0.05), consistent with our hypothesis that hypoxic tolerance (and thus, neuroprotection) is enhanced in preparation for and/or during the hibernating state.
Brain MCP‐1 is elevated in a mouse model of cancer‐related fatigue
Authors: Gordon Benjamin Thomas , Murphy Angela , McClellan Jamie L. , Carmichael Martin , Davis Mark
Published: June 2021

Cancer‐related fatigue (CRF) is associated with elevated pro‐inflammatory cytokines in the central nervous system (CNS). Previous data from our laboratory show an increase in brain IL‐1β in the ApcMin/+ mouse model of colon cancer that is associated with reduced voluntary wheel running activity. These effects occur at a time of imperceptible illness in this mouse model of CRF. MCP‐1 has been identified as an important chemokine involved in tissue macrophage recruitment and inflammation. However little is known about the CNS role of brain MCP‐1. The purpose of this study was to examine brain MCP‐1 gene expression in the ApcMin/+ mouse model of colon cancer. ApcMin/+ mice (n=11) or wildtype mice (n=8) were sacrificed at 20 weeks of age. The brain was grossly dissected (brain stem, cerebellum, cortex, frontal lobe, hippocampus, hypothalamus and mid brain) and analyzed for MCP‐1 mRNA using real time PCR. Statistical analysis of MCP‐1 was performed using a one‐way ANOVA with Student‐Newman‐Keuls post hoc analysis (P≤0.05). Gene expression of MCP‐1 was significantly increased in the cerebellum (4‐fold) and the hippocampus (3‐fold) (P<0.05) of the ApcMin/+ mouse. MCP‐1 was also increased in the cortex, frontal lobe, hypothalamus and mid brain but these did not reach statistical significance. These data suggest a potential role for brain MCP‐1in CRF in the ApcMin/+ mouse model of colon cancer.
Influences of Alcohol Consumption on Transient Focal Cerebral Ischemia
Authors: Sun Hong , Xiong Wanfen , Zhao Honggang , Sharpe Glenda M , Arrick Denise M , Mayhan William G
Published: June 2021

Ischemic stroke is a leading cause of mortality and long‐term disability. Alcohol is one of the most abused drugs. We examined the dose‐dependent influences of chronic alcohol consumption on transient focal cerebral ischemia. Sprague‐Dawley rats were fed liquid diets with or without alcohol (1%, 3%, 5%, and 6.4% v/v) for 8 weeks. Transient focal cerebral ischemia was induced by a 2‐hour middle cerebral artery occlusion (MCAO). Ischemic area was determined by contrast‐enhanced perfusion‐weighted imaging at the end of MCAO. Ischemic brain damage was evaluated on days 1, 3, and 14. Gene expression profile in peri‐infarct area was measured at 24 hours of reperfusion by DNA microarray. There was no difference in ischemic area between nonalcohol‐fed and alcohol‐fed rats. However, total infarct volume was reduced by 45% in 1% alcohol‐fed rats and increased by 32% in 6.4% alcohol‐fed rats at 24 hours of reperfusion. The protective effect of 1% alcohol consumption and detrimental effect of 6.4% alcohol consumption on transient focal cerebral ischemia could be seen at later time points. 3% and 5% alcohol did not significantly altered total infarct volume. In addition, ischemia/reperfusion induced an upregulation in inflammatory genes. The magnitude of upregulation in intergrin β2 and MMP‐9 was less in 1% alcohol‐fed rats but greater in 6.4% alcohol‐fed rats compared to nonalcohol‐fed rats. Our findings suggest that disparate effects of low and high dose alcohol consumption on ischemic brain damage may be related to an altered inflammatory response.
The aryl hydrocarbon receptor (AhR) upregulates P‐glycoprotein (P‐gp) at the blood‐brain barrier (BBB)
Authors: Miller David S , Wang Xueqian
Published: June 2021

Widespread, persistent organic pollutants, e.g., polychlorinated biphenyls and 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin (TCDD), activate AhR causing it to translocate to the cell nucleus where it induces transcription of target genes, e.g., cytochrome P450 1A1 and 1B1 and phase II drug metabolizing enzymes. AhR's ability to modulate expression of xenobiotic transport proteins is largely unexplored. We show here that exposing isolated rat brain capillaries to the AhR ligands, β‐naphthoflavone (βNF, 0.5–5μM) or TCDD (0.05–0.5nM), increased P‐gp‐mediated transport and P‐gp protein expression 2–3 fold. These effects were abolished by actinomycin D or cycloheximide or by the AhR antagonists, resveratrol and αNF. Dosing rats with TCDD (1–5μg/kg, ip) increased P‐gp mediated transport and protein expression in brain capillaries assayed ex vivo. This is the first evidence that AhR ligands upregulate P‐gp mediated transport in any tissue and that AhR can alter drug transport at the BBB. Thus, environmental toxicants that are AhR ligands selectively tighten the BBB, providing increased protection, but at the expense of reduced penetration of therapeutic drugs. Supported by the Intramural Research Program, NIH/NIEHS.
beta‐Amyloid is removed from the brain CSF by a non‐diffusion process
Authors: Thompson Corbin , Crossgrove Janelle S.
Published: June 2021

Alzheimer's disease (AD), the most commonly diagnosed neurodegenerative condition in the United States, is thought to be caused by the accumulation of beta‐amyloid (Aβ) plaques within the brain. Therefore, any mechanism which removes these toxic Aβ peptides from brain tissue may help in the treatment or prevention of AD. We hypothesize that Aβ peptides are removed from the brain and CSF by the choroid plexus, a major component of the blood‐CSF barrier, through a concentration‐dependent export mechanism and not simply by diffusion. To test this hypothesis, an artificial CSF containing Aβ1–40 (10 or 1000 ng/mL) and a reference compound (Evans blue) was perfused into the lateral brain ventricles of adult male Long Evans rats. Outflow CSF samples were collected from the cisterna magna and analyzed for Aβ and Evans blue levels by ELISA and spectrophotometry, respectively. The percent of Aβ remaining in cisternal CSF at steady‐state was less than the percent of Evans blue, suggesting a non‐diffusion‐mediated process to remove Aβ peptides from CSF.
Elevated Blood Pressure and Abnormal Evolution of Pregnancy in ACE2‐Deficient Female Mice
Authors: Pimentel Renata Vieira , Santos Robson Augusto Souza , Todiras Mihail , Velloso Elizabeth Portugal , Braga Janaína Félix , Penninger Josef , Bader Michael , Alenina Natalia
Published: June 2021

Angiotensin‐converting enzyme 2 (ACE2) generates Ang‐(1–7) which interacts with Mas. We have recently observed that pregnant Mas‐deficient− mice presented a preeclampsia‐like state. However, it is not clear whether this effect is caused by a reduction of the activity of the ACE2/Ang‐(1–7)/Mas axis or other Mas‐dependent actions. Here we evaluated the phenotypic alterations induced by ACE2 deficiency during pregnancy in C57Bl/6 mice (ACE2−/−). Twenty weeks old ACE2−/− and WT female mice were used. Blood pressure was measured by telemetry before, during pregnancy and 4 days after delivery. Female ACE2−/− mice presented increased MAP before pregnancy (106±1 in ACE2−/− vs. 99±2 mmHg in WT). During pregnancy, ACE2−/− MAP remained constant with only small fluctuations. However, after delivery, MAP decreased in WT, but not in ACE2−/− KO (108.7±1 mmHg in ACE2−/− vs. 97±1 in WT). The ACE2−/− mice also presented a significant reduction in body weight after delivery and the ACE2−/− pups showed fetal growth restriction (1.33±0.03 g in ACE2−/− vs.1.49±0.01 g in WT). More interesting, ACE2−/− mothers had a significant number of dead pups presenting developmental abnormalities. These data show, for the first time, that ACE2 plays a critical role in fetal development. In addition, our data further support an important role of the ACE2/Ang‐(1–7)/Mas axis in the hemodynamic adjustment during pregnancy. Support: CAPES – PROBRAL
Role of Angiotensin II Receptor (AT1aR) in Thick Ascending Limb of Henle's Loop and Distal Tubules
Authors: Li Huiping , Davis Deborah R. , Sigmund Curt D.
Published: June 2021

The intrarenal renin‐angiotensin system (RAS) plays an important role in the regulation of renal hemodynamics and tubular transport, mainly mediated by AT1aR. However the role of AT1aR in the thick ascending limb of Henle's loop (TAL) and distal convoluted tubules (DCT) remains unknown. To examine this, we generated a transgenic mouse expressing a constitutively active form of HA‐tagged AT1aR (N111G) under the control of the human uromodulin promoter. Tissue‐ and cell‐specific expression was confirmed using quantitative real‐time PCR and immunohistochemistry. There was no difference in baseline blood pressure of transgenic (Tg) and wild type (WT) littermates assessed by radiotelemetry and tail cuff. However, baseline urine osmolality (Os) and sodium concentration [Na] in Tg were significant decreased (Os: 800±90 vs 1100±90 mOsM, P<0.05; [Na]: 48±6 vs 65±9 mM, P<0.05). Preliminary studies revealed increased localization of sodium potassium chloride co‐transporter (NKCC2) to the apical membrane. Expression of the urea transporter (UT‐A) was also increased in Tg. Water loading and deprivation revealed no serious impairment in the urine concentrating and diluting capacity of the Tg mice. We are currently examining the responses to high salt diet and Ang‐II infusion. Our data suggest that AT1aR in the TAL and DCT, like other nephron segments, may play a role in regulating sodium reabsorption.
Identification of key residues in WW domains of ubiquitin‐protein ligase Nedd4‐2 that bind to calcium channel TRPV6
Authors: Zhang Wei , Na Tao , Peng Ji‐Bin
Published: June 2021

We previously observed that ubiquitin‐protein ligase Nedd4‐2 mediated degradation of calcium channel TRPV6 in Xenopus oocytes. The goal of this study was to identify the domains and key residues in Nedd4‐2 that interact with TRPV6 as an association between TRPV6 and Nedd4‐2 was demonstrated by Co‐IP upon co‐expression in oocytes. As revealed by far‐western approach, TRPV6 interacted with WW1 and WW2 domains, but not with WW3, WW4, C2 or HECT domain of Nedd4‐2. To identify the residues in WW1 and WW2 domains responsible for the interactions, 5 residues in WW1 that are conserved in WW1‐2 but not in WW3‐4 domains were mutated to their corresponding residues in WW3‐4. Only D204H mutation abolished the binding of WW1 to TRPV6. Similarly, the corresponding D376H mutation in WW2 also impaired its binding to TRPV6. On the other hand, the H539D mutation in the corresponding position of WW4 enabled its interaction to TRPV6. However, the 2 sites appeared not necessary for the association between TRPV6 and Nedd4‐2 as assessed by Co‐IP. Interestingly, both TRPV6 ubiquitination and degradation were increased by Nedd4‐2 with D204H and D376H mutations. This suggests that the binding between TRPV6 and WW1‐2 domains may prevent the ubiquitination of TRPV6 by Nedd4‐2. Nedd4‐2 co‐localized with TRPV6 in rat intestinal tract as observed by immunostaining with antibodies against TRPV6 and Nedd4‐2. Our results provide evidence that Nedd4‐2 plays a role in regulating TRPV6 stability under physiological conditions.
Voltage‐dependent gating underlies the PHA1 phenotype caused by mutation of a conserved HG motif in ENaC
Authors: Kucher Volodymyr , Boiko Nina , Pochynyuk Oleh , Stockand James D.
Published: June 2021

Autosomal recessive pseudohypoaldosteronism type 1 (PHA1) is caused by loss of ENaC function leading to inappropriate renal salt wasting. ENaC is a member of the ENaC/Deg superfamily. All members of this superfamily contain a conserved HG motif in their NH2‐terminus. Mutation of the Gly in this motif decreases ENaC activity causing PHA1 in humans. Here, we tested the role of the conserved HG motif to ENaC function. Targeted substitution of either H or G dramatically decreases ENaC Po by causing marked voltage‐dependent gating with Po increasing at hyperpolarizing potentials but being lower at all potentials compared to wild type channels. Mutant ENaC has transient activation and deactivation, not present in wild type channels, associated with hyperpolarization and depolarization. We recently reported that targeted substitution of a conserved Trp at the base of TM1 in ENaC unmasks a similar voltage‐sensitivity associated with decreases in Po, and activation at hyperpolarizing potentials. M2 proton channels gate through formation and breaking of a ring‐like structure beneath the intracellular mouth of the channel pore dependent on transient interaction between a transmembrane Trp and cytosolic His residue. Interrupting such interaction between Trp‐His may underlie the abnormal voltage‐sensitive gating reported here for PHA1 mutations of ENaC.
Phospho‐specific antibodies to Ser126 and Ser874 of the apical Na‐K‐2Cl cotransporter (NKCC2) demonstrate vasopressin‐regulated phosphorylation in the renal thick ascending limb (TAL)
Authors: Gunaratne Ruwan , Pisitkun T , Braucht DWW , Xie L , Rinschen MM , Hoffert JD , Chou C‐L , Knepper MA
Published: June 2021

Renal countercurrent multiplication is controlled in part through the action of vasopressin to regulate NaCl absorption in the TAL. Our recent phosphoproteomic analysis in rat has identified two predominant phosphorylation sites in the apical cotransporter NKCC2, viz Ser126 and Ser874. To investigate effects of vasopressin, we made phospho‐specific antibodies to both sites. Both antibodies specifically recognized the phosphorylated forms of NKCC2. Confocal immunofluorescence in rat showed TAL‐specific labeling, strongest in the cortical TAL. Immunoblotting of rat medullary TAL suspensions or homogenates from intact rat kidneys showed that vasopressin increases phosphorylation at both Ser126 (>8‐fold) and Ser874 (>2‐fold). Increases at both sites were blocked by H89 (10 μM). In vitro assays revealed that purified protein kinase A can phosphorylate synthetic NKCC2 peptides at either Ser126 or Ser874. However, in the same assay, AMP kinase produced little or no phosphorylation at either site. In addition, we re‐examined two previously demonstrated phosphorylation sites, viz Thr96 and Thr101, with the R5 double‐phospho‐antibody. Immunoblotting with ‘cross‐preadsorbed’ R5 antibodies showed that vasopressin increases phospho‐Thr96 (2‐fold) but does not increase phospho‐Thr101. These studies demonstrate regulated phosphorylation of NKCC2 at three sites in response to vasopressin.
Akt/PKB‐sensitive proximal tubular glucose and phosphate transport
Authors: Föller Michael , Kempe Daniela S. , Siraskar Gulab , Fröhlich Henning , Ackermann Teresa F. , Siraskar Balasaheb , Völkl Harald , Judenhofer Martin , Pichler Bernd , Leavens Karla F. , Birnbaum Morris J. , Pearce David , Lang Florian
Published: June 2021

Akt/PKB stimulates the glucose carrier GLUT4. Here we explored whether Akt directly regulates Na+‐coupled transport of glucose (by SGLT1) or phosphate (by NaPiIIa). Immunohistochemistry showed that Akt2 is expressed in proximal renal tubules. According to dual electrode voltage clamp experiments the coexpression of Akt increased the glucose‐induced current in SGLT1‐expressing and the phosphate‐induced current in NapiIIa‐expressing Xenopus oocytes. Renal transport was further studied using Akt2 deficient (akt2−/−) and wild type mice (akt2+/+). akt2−/− mice suffered from glucosuria which was partially due to increased plasma glucose levels but was not observed in akt2+/+ mice made similarly hyperglycemic as akt2−/− mice by fructose feeding pointing to defective renal tubular glucose reabsorption. Glucose‐induced depolarization was significantly smaller in isolated perfused renal tubules from akt2−/− than from akt2+/+ mice. In addition, the phosphate clearance was higher in akt2−/− than in akt2+/+ mice despite a significantly decreased plasma PTH and enhanced 1,25‐dihydroxyvitamin D3 concentration and a tendency of lower plasma phosphate. Bone density was significantly reduced in akt2−/− mice. In conclusion, Akt plays a role in the regulation of renal glucose and phosphate transport contributing to the maintenance of phosphate balance and adequate mineralization of bone.
Pendrin regulates ENaC abundance and function by modulating luminal HCO3− concentration
Authors: Pech Vladimir , Pham Tuyen D. , Hong Seongun M. , Spencer Kathryn B. , Duke Billy J. , Walp Erik , Kim Young H. , Sutliff Roy L. , Eaton Douglas C. , Wall Susan M.
Published: June 2021

Pendrin regulates abundance and function of the epithelial Na+ channel, ENaC, in kidney. Since pendrin mediates HCO3− secretion we hypothesized that ENaC is regulated by changes in luminal HCO3− concentration. Thus mice received either aldosterone with a NaCl‐replete diet containing NaHCO3 to increase pendrin‐dependent HCO3− secretion or aldosterone, diet and acetazolamide to increase luminal HCO3− concentration through a pendrin‐independent mechanism. ENaC abundance and function were lower in pendrin null relative to wild type mice when given aldosterone alone. However, when acetazolamide was added ENaC abundance and function were similar in wild type and pendrin null mice. To determine if HCO3− concentration has a direct effect on ENaC abundance and function, amiloride‐sensitive current and ENaC subunit abundance were measured in mpkCCD cells, a mouse principal cell line. ENaC‐mediated current and subunit abundance rose with increased HCO3− concentration on the apical or basolateral side achieved by substitution with either Cl− or methanesulfonate. However, ENaC was more sensitive to changes in HCO3− concentration on the basolateral than the apical side. We conclude that pendrin modulates ENaC abundance and function, at least in part, by raising luminal HCO3−concentration and/or pH. Extracellular HCO3− concentration likely modulates ENaC function through an intracellular signaling event.
Gastric H+/K+ATPase beta subunit in the colonic H+/K+ATPase knockout mice
Authors: Wei Guojun , Higashi Sakura , Nakamura Suguru
Published: June 2021

Three H+K+ATPase subunits (HKAalpha1; gastric, HKAalpha2; colonic, HKAbeta) are expressed in the renal collecting duct. It is known that the HKAalpha1 (gastric) plays an important role under normal conditions, whereas the HKAalpha2 (colonic) is induced in potassium depletion and mediates increased HCO3− in the maintenance of metabolic alkalosis. The beta‐subunit is essential for the functional expression of both the HKAalpha1 and HKAalpha2. The purpose of the current experiments was to examine the role of the beta‐subunit of HKA in wild type (WT) and in selective disruption of the HKAalpha 2 gene (KO) mice under normal and K+ depletion conditions. Mice were placed on a potassium‐deficient diet for 2 weeks. The mouse kidney medulla was dissected and lysed with a lysis buffer (pH 8). Cell lysate was incubated with monoclonal antibody (HKA beta subunit, 34 kDa) overnight under agitation at 4°C. The amount of HKA beta subunit was quantified by a modified Bradford protein assay. At a wavelength of 540 nm, the HKA beta subunit protein absorbance was 0.052 ± 0.018 (n=20) in WT mice with a potassium replete diet. In WT mice with a potassium‐deficient diet, the HKA beta subunit absorbance was 0.065 ± 0.028 (n=20, p<0.01). A similar pattern of the HKA beta subunit absorbance has been observed in KO mice with a potassium replete diet vs. a potassium‐deficient diet. Results suggested that the HKA beta‐subunit is stimulated by hypokalemia in the renal collecting ducts. The HKA beta‐subunit is likely to associate with the HKAalpha1 in the HKAalpha2 KO mice under normal and K+ depletion conditions..
Acid–induced currents in freshly isolated cerebral artery smooth muscle cells are predominately conducted by ASIC1b
Authors: Chung Wen‐Shuo , Swenson Alyssa , Farley Jerry M. , Drummond Heather A.
Published: June 2021

Our laboratory has demonstrated the presence of acid‐sensing ion channels (ASIC)‐like currents in freshly isolated cerebral vascular smooth muscle cells (VSMC). In these cells, extracellular H+ (pH≤6.5) induces an amiloride‐sensitive inward Na+ current. However, the ASIC subunits mediating this response are unknown. In this study, Western blotting and immunostaining demonstrated robust expression of ASIC1b, although other subunits were also detected to a lesser extent. Patchclamp was utilized to determine if ASIC1b may mediate H+‐gated currents; we evaluated responses to psalmotoxin 1 (PcTX1, 100nM), a known inhibitor and activator of ASIC1a and ASIC1b channels, respectively. In voltage‐clamped VSMCs, PcTX1 inhibited H+‐induced (pH 6.0) inward current in only 4 of 44 VSMCs examined. PcTX1 enhanced currents in 34/44 VSMCs by 12.0±2.7 pA/pF (p<0.001) and increased the mean desensitization constant of the initial decline phase of peak current (2.2±0.3 to 4.7±0.9 s; p<0.05; n=15). Taken together, these findings suggest that ASIC1b conducts most H+ gated currents in freshly isolated VSMC.
Small Molecule Inhibitors of Acid Sensing Ion Channel‐1
Authors: Qadri Yawar J , Song Yuhua H , Fuller Catherine M , Benos Dale J
Published: June 2021

Amiloride is known to interact with Acid Sensing Ion Channel (ASIC) proteins. However, the structural basis for this interaction has not been fully elucidated. Years of functional tests on chimeric and mutated channel proteins has implicated many residues and domains in the pore region and extracellular loops. This work uses computational and experimental techniques to examine the interaction of amiloride with ASIC‐1. Using small molecule docking software, amiloride and related molecules were docked to models of homomeric human ASIC‐1, generating potential interaction sites and predicting the affinity of analogs for the channel. These predictions were experimentally tested using whole‐cell patch clamp in two model cell lines. Two drugs previously classified as inhibitors of sodium transporters were predicted to and verified to inhibit hASIC‐1. Finding that sodium competes with amiloride and that the tested inhibitors exhibited voltage sensitivity, the potential docking sites were re‐examined for interactions in the putative conductive pathway for the channel. Using the aggregated data from the computational and functional experiments, putative interaction pockets for amiloride with hASIC‐1 are defined at the pore and cation binding sites in the extracellular loops. This work also shows the feasibility of virtual screening of drug libraries at these domains. This work was supported by NIH DK 037206.
Inhibition of Murine Macrophage Inwardly Rectifying Kir2.1 Channels by Gambogic Acid
Authors: O'Bryant Zaven , Colden Margaret
Published: June 2021

Cs+‐sensitive K+ inward rectifier (Kir) ionic currents in macrophages are modulated during VCAM‐1/VLA‐4 interactions affecting macrophage function that may possibly contribute to atherogenesis (Colden‐Stanfield, 2009). While heavy metals, Cs+ and Ba2+, are known to nonspecifically block the Kir family of channels, specific blockade of the Kir2.1 subspecies by gambogic acid (GA) has only been recently demonstrated (Zaks‐Makhina et al., 2009). To determine the effect of GA on Kir currents in murine macrophages, we recorded patch‐clamp whole‐cell currents and resting membrane potential (RMP) in bone marrow‐derived macrophages (BMDMs) following a 3‐h pretreatment with GA or its vehicle, DMSO. In the absence of exogenous GA 97% of BMDMs (32 of 33 cells) possessed Kir currents that were elicited during hyperpolarization and an RMP of −57 mV. When the cells were incubated with GA (500 nM) for 3 h, Kir currents were completely inhibited (19 of 19 cells). RMP of cells was significantly depolarized to −11 mV compared to control cells. Exposure to DMSO (0.0005%) for 3 h did not alter Kir currents. As a comparison, exogenous Cs+ (10 mM for 30 min) completely blocked Kir currents but only depolarized the membrane to −46 mV. This data and the lack of necrotic or programmed cell death in the presence of GA indicate that Kir2.1 channels underlie Kir whole‐cell currents in BMDMs. This research was supported by NHLBI K01 HL076628.
Human muscular variations: comparative, evolutionary and developmental perspectives
Authors: Diogo Rui , Wood Bernard
Published: June 2021

The anatomy of primates and other mammals has been the subject of numerous comparative studies. However, the vast majority of these studies deal with skeletal structures. The lack of detailed comparative works on the head, neck, pectoral and forelimb musculature of primates and their closest living relatives, i.e. tree‐shrews and colugos, creates difficulties in the discussion of subjects such as the evolution of primates, the origin of modern humans, and the peculiar muscular variations of both human and non‐human mammals. The main aim of the present work is to discuss human muscular variations in a broader comparative, evolutionary and developmental framework, based on our own dissections of numerous vertebrate specimens and on an extensive review of the literature. One of topics that will be explored in the talk is the relationship between ontogeny, genetics, and the so‐called (somewhat controversially) ‘muscular atavisms’.
A cAMP‐activated Cl− channel in the apical membrane of chick (Gallus gallus) proximal tubule cells
Authors: Laverty Gary , Reddy Varudhini , Árnason Sighvatur S
Published: June 2021

In primary culture, filter‐grown monolayers from chick renal proximal tubules express a parathyroid hormone‐activated transport current under epithelial voltage clamp conditions. Data suggest that this positive current response is due to Cl− ion transport and is mediated by a cystic fibrosis transmembrane regulator (CFTR)‐like Cl− channel on the apical membrane. To further examine this transport pathway we permeabilized the basolateral membrane of proximal tubule monolayers in Ussing chambers. After establishing either basolateral to apical (Bl > Ap) or apical to basolateral (Ap > Bl) chloride gradients by glutamate substitution, nystatin (300 μg/ml) was added to the basolateral reservoirs. Subsequent stimulation with 1 μM forskolin caused a short circuit current (ΔISC) response of 7.67 ± 0.99 and − 19.50 ± 3.54 μA/cm2 in the Bl > Ap and Ap > Bl monolayers, respectively (means ± SEM, N = 6). Basolateral addition of 100 μM bumetanide, a blocker of the sodium‐potassium‐2 chloride cotransporter, had no effect on these ISC responses, indicating complete basolateral permeabilization. In contrast, apical addition of 20 μM CFTR inhibitor 172 reduced the forskolin‐stimulated ISC by 38 and 48%, for the secretory and reabsorptive gradients, respectively. These data support the hypothesis that chick proximal tubule cells are capable of active, stimulated chloride secretion, mediated by a cAMP‐activated Cl− channel.
Saponin Selectively Permeabilizes the Sweat Duct Basolateral Membrane (BLM)
Authors: Reddy M.M.
Published: June 2021

Understanding the role of endogenous phosphatases and kinases in regulating CFTR in the native epithelial cells is hindered by our inability to add or delete large, putative regulatory proteins to and from native cells at will. Therefore, we investigated the ability of Saponin to selectively permeabilize the BLM to macromolecules. We measured the CFTR Cl‐ conductance (CFTR‐gCl) in the sweat ducts as previously described (Quinton, 1986) and permeabilized the BLM either with α‐toxin (1000 U/ml) or with Saponin (0.01%) in the basolateral bath. ATP (5mM), cAMP (0.1 mM), PP2A inhibitor Okadaic acid (10−8 M) was added to the cytoplasmic bath as needed. α‐toxin permeabilization of the BLM spontaneously inhibited CFTR‐gCl (3.5±2 mS/cm2, n=12) Such deactivation was prevented either by the application Okadaic acid (CFTR‐gCl= 38± 4 mS/cm2, n=5) or cAMP CFTR‐gCl=45±6 mS/cm2, n=12). In contrast, after permeabilizing the BLM with saponin, CFTR‐gCl failed to deactivate if ATP was present. These results seem to indicate that: a.) α‐toxin pores in the BLM are small enough to retain endogenous phosphatases in the cytosol that dephosphorylate and deactivate CFTR even while depleting cytosolic cAMP through α‐toxin pores, b.) Saponin appears to make larger pores in the BLM so that endogenous phosphatases are lost from the cytosol that prevents deactivation by dephosphorylation of CFTR‐gCl.

We thank Mr. K. Taylor for technical assistance. Supported by the NIH
Glucose stimulates BK channels in human podocytes
Authors: Huang Wen‐Tan , Ma He‐Ping
Published: June 2021

There is a significant loss of podocytes in diabetic patients. However, the mechanism remains unclear. Recent studies suggest that activation of a big conductance, Ca2+‐activated K+ (BK) channel induces apoptosis and that BK channel is expressed in podocytes. Therefore, we hypothesize that hyperglycemia may promote podocyte apoptosis by stimulating BK channels. In the present study, we used patch‐clamp techniques to determine whether glucose regulates BK channels in human podocytes by culturing the cells either in control or high glucose condition. The data demonstrated that BK channels were seen more often and more active in the podocytes cultured in high glucose condition than those cultured in control condition. Since glucose metabolism generates reactive oxygen species (ROS), confocal microscopy experiments were performed to evaluate intracellular ROS with a cell membrane‐permeable fluorescent dye. The data showed that intracellular ROS were significantly elevated in the podocytes cultured in the presence of high glucose. We also found that exogenous hydrogen peroxide elevated intracellular Ca2+ and stimulated BK channels in podocytes. These data suggest that high glucose stimulates BK channels in cultured human podocytes probably by causing oxidative stress and elevating intracellular Ca2+. This work is supported by Guangxi Scholarship to W.T.H. and NIH Grant R01‐DK‐067110 to H.P.M.
A Pseudomonas aeruginosa toxin (Cif) reduces plasma membrane CFTR by inactivating the deubiquitinating enzyme USP10
Authors: Bomberger Jennifer , Ye Siying , O'Toole George , Stanton Bruce
Published: June 2021

P. aeruginosa is an opportunistic pathogen critical to the pathogenesis of nosocomial infections, chronic obstructive pulmonary disease, ventilator‐associated pneumonia, cystic fibrosis, and bronchiectasis. Cif, a bacterial toxin secreted by clinical isolates of P. aeruginosa, reduces CFTR‐mediated chloride secretion by decreasing the endocytic recycling of CFTR. The aim of the current study was to investigate the mechanism by which Cif reduces the endocytic recycling of CFTR. Experiments in polarized human airway epithelial cells using Optiprep gradients revealed that Cif redirected CFTR from recycling endosomes to lysosomes for degradation, without affecting CFTR endocytosis. Cif redirected CFTR from recycling endosomes to lysosomes by increasing the amount of ubiquitinated CFTR (Ub‐CFTR). Employing proteomics and a deubiquitinating enzyme (DUB) activity assay, we observed that USP10 deubiquitinates CFTR, and that the Ras‐GAP SH3 domain binding protein‐1 (G3BP1) inactivates USP10. Moreover, Cif, by stabilizing USP10‐G3BP1 interaction, inactivates USP10 and thus, enhances the ubiquitination of CFTR, which is then directed to lysosomes for degradation. siRNA knockdown of G3BP1 can block the Cif effect on CFTR trafficking. These data suggest that chronic infection of P. aeruginosa will reduce mucociliary clearance, and thereby severely compromising the immune defenses of the lung.
Inhibition of Na+ transport in mouse lung exposed to chlorine (Cl2)
Authors: Lazrak Ahmed , Cain Laura , Doran Stephen , Matalon Sadis
Published: June 2021

Lung fluid balance is finely tuned by vectorial transport of ions across the epithelium. Active sodium reabsorption generates the driving force needed for fluid clearance. In the inflammatory disease of the lung, high levels of Hypochlorus acid (HOCl) are generated by stimulated neutrophils. HOCl causes extensive damage through its reaction with cellular substances such as thiols and amines. In addition to the physiological sources, HOCl can be generated by inhalation of Cl2 known to induce lung edema. Herein; we investigated the effect of HOCl on ENaC in mouse lungs exposed to 400 ppm Cl2, one hour and 24 hours later using the single channel recording on live lung slices. The control alveolar cells exhibited two Na+ conductances, highly selective 4–6 pS and moderately selective 14–16 pS. Both were amiloride sensitive. One hour after exposure to Cl2, the Po of both channels was significantly reduced from 0.25 ± 0.05 to 0.06 ± 0.01 (p<0.001) and 0.12 ± 0.07 to 0.02 ± 0.005 (p<0.001) for highly and moderately selective channels, respectively. At 24 hours post exposure to Cl2 the Po of both channels recovered to 50% of its initial value in addition a new non selective conductance of 25 pS was observed. In experiments where animals were exposed to Cl2 and injected with a cocktail of antioxidants the recovery was faster, both pre‐existing channels recovered by 90% of their initial values. The non selective conductance (25 pS) was less active. Cl2 gas inhalation inhibits ENaC which may lead to alveolar edema formation. This injury was prevented by post administration of antioxidants.

This Research was supported by NIH U01 ES015675 and U54 ES017218 grant to Sadis Matalon.
Regulation of Plasma Membrane Expression of TRPV4 Channel by GSK1016790A and Hypotonic Stress in HeLa and M‐1 Cells
Authors: Jin Min , James Jose , O'Neil Roger G.
Published: June 2021

TRPV4 (Transient Receptor Potential Vanilloid 4) channels are activated by a wide range of stimuli, including hypotonic stress, phorbol ester 4α‐PDD, non‐noxious heat and mechanical stress. GSK1016790A (GSK101, GlaxoSmithKline), a potent and specific activator of TRPV4 was evaluated in HeLa cells transiently transfected with TRPV4 (HeLa‐TRPV4) and M‐1 (mouse cortical collecting duct) cells expressing TRPV4 endogenously. GSK101 (10 nM) causes a TRPV4 specific calcium influx in both over‐expressed and endogenous cell models for TRPV4, which can be inhibited by Ruthenium Red (RR). Hypotonic stress (225 mOsm/kg) causes a calcium influx in HeLa‐TRPV4 and M‐1 cells, but only partially responds to RR inhibition. Plasma membrane proteins were extracted and Western blotting was utilized to study the expression of TRPV4 over a time course of 30 minutes under GSK101 and hypotonic stress treatment. GSK101 causes a steady down‐regulation of TRPV4 plasma membrane expression over 30 minutes, while the hypotonic stress causes a more complex change in TRPV4 plasma membrane expression. Fluorescence confocal microscopy shows co‐localization of TRPV4 with microtubules and actin, suggesting likely trafficking and endocytosis of TRPV4 channel. Immunocytochemistry also shows co‐localization of TRPV4 with large‐conductance calcium‐activated potassium channels (BK), indicating a potential macro‐complex of these channels are probably involved in intracellular calcium and volume regulation. It is concluded that GSK101 specifically activates TRPV4 in HeLa‐TRPV4 cells and M‐1 cells, which likely involves regulation of endocytosis of TRPV4 from the plasma membrane. Hypotonic stress activates TRPV4 in a more complex pattern of regulation that may involve recycling.

Supported by NIH DK70950.