bluuebunny/crossref_metadata_2025 huggingface dataset:
Rosetta modeling of the inner KCa3.1 pore, a hotspot for small molecule modulation
Authors: Jenkins David Paul , Wulff Heike , Yarov‐Yarovoy Vladimir
Published: June 2021

The intermediate‐conductance KCa3.1 and the related small‐conductance KCa2 channels constitute potential pharmacological targets for immunosuppression, fibroproliferative disorders, hypertension and various neurological diseases. However, there currently is no available crystal structure or molecular model of these medically relevant channels that could be used for structure‐assisted drug design. Using the Rosetta modeling method we constructed a model of the KCa3.1 pore and energy minimized it in the presence of the KCa3.1 blocker TRAM‐34. The lowest energy confirmation of the KCa3.1 complex with TRAM‐34 identified Thr250 and Val275 as interaction sites, two residues which had been previously demonstrated to completely abolish triarylmethane sensitivity when mutated to the corresponding residues in KCa2.3. The model further correctly identified the interaction of the benzothiazinone‐type KCa3.1 blocker NS6180 with Thr250 and Val275 in KCa3.1 and modeled binding of the negative KCa2 channel gating modulator NS8593 to a KCa3.1‐Thr250Ser‐ Val275Ala mutant channel, which is blocked more potently by NS8593 than the WT‐KCa2.3 channel. The Rosetta KCa3.1 model can be used to understand the molecular mechanism of action of KCa2/3 channel modulators and assist with drug design of blockers and gating modulators.

Supported by RO1 GM076063 (to H.W.) and UC Davis startup (to V.Y.).
β4‐subunit palmitoylation controls cell surface expression of large conductance calcium‐ and voltage‐ dependent potassium (BK) channels
Authors: Chen Lie , Bi Danlei , McClafferty Heather , Steeb Franziska , Tian Lijun , Shipston Mike
Published: June 2021

BK channels are involved in controlling neuronal excitability and thus mechanisms that regulate cell surface expression of the channels are important determinants of neuronal function. We have identified that the cell surface expression of BK channel α–subunit (α) is regulated by s‐acylation. The β4‐subunit (β4) is an accessory subunit of α predominantly expressed in neurones and using 3H‐palmitate incorporation and acyl‐RAC we revealed β4 is palmitoylated in mouse brain and when β4 is expressed in HEK293 (HEK) and N2a cells. Using quantitative imaging assays, cell surface expression of β4 in HEK or N2a cells was significant decreased (>; 50%) by mutation of a single cysteine to alanine (β4‐ mut) resulting in increased co‐localisation at the ER (Pearson's R value from 0.36 to 0.83). When we co‐expressed β4 with an N‐terminal Flag tagged α (ZERO), the cell surface expression of ZERO was significantly enhanced in both HEK (by 50 ± 5.7%) and N2a (by 90 ± 8.1%) cells that was associated with a decreased ER co‐localization of ZERO (R value from 0.80 to 0.48). However, this enhanced cell surface expression of ZERO was dependent upon the palmitoylation of β4. Co‐expression of ZERO with β4 –mut resulted in no significant enhancement of ZERO surface expression in either HEK or N2a cells. Therefore, β4 is a palmitoylated protein that regulates the trafficking of the BK channel to the cell surface in a palmitoylation dependent mechanism.
Palmitoylation of large conductance calcium‐ and voltage activated potassium (BK) channel β‐subunits
Authors: Bi Danlei , Chen Lie , McClafferty Heather , Tian Lijun , Shipston Mike
Published: June 2021

BK channels control diverse physiological functions including vascular tone and neuronal excitability. In these tissues, BK channel pore‐forming α‐subunits (α) typically assemble with regulatory β‐subunits (β). Though the properties of α are regulated by a range of post‐translational modifications (PTMs) like S‐acylation, whether β are also modified by this PTM is unknown. We predicted that both β1 and β4 were palmitoylated and confirmed it by 3H‐Palmitate incorporation and acyl‐RAC assays. We investigated the effect of palmitoylation of the β1 and β4 on the trafficking and activity of theα ZERO variant in HEK293 cells by quantitative immunofluorescence imaging and patch clamping. The cell surface expression of ZERO was significantly enhanced upon co‐expression of either β1 or β4 (>;100% by β1 and >;50% by β4). Depalmitoylation of β4, but not β1, by mutation of a single cysteine residue to alanine, significantly reduced co‐expressed ZERO variant surface expression. In macropatch recordings, co‐expression of wild‐type β1 and β4 significantly left‐shifted the V0.5max of the channels as expected at 10uM intracellular free calcium. However, depalmitoylation of either βdid not significantly affect the normal left‐shift of V0.5max. These data suggest that palmitoyaltion is crucial for trafficking of BKα upon β4, but not β1, but not their effect on V0.5max. The functional role of β1 pamitoylation is to be determined.

The work is supported by Wellcome Trust and The China Scholarship Council.
The Hv1 voltage‐gated proton channel regulates the pH of neutrophil but not macrophage phagosomes
Authors: Nunes Paula , El Chemaly Antoine , Jimaja Wedali , Castelbou Cyril , Demaurex Nicolas
Published: June 2021

Phagosomal acidification and reactive oxygen species (ROS) production are important for an effective immune response. Macrophage phagosomes acidify rapidly due to vATPase activity, whereas neutrophil phagosomes remain near‐neutral for >;30 min due to ROS‐dependent inhibition of vATPase activity. Hv1 proton channels sustain high level ROS production at the plasma membrane by extruding protons from the cytosol. Here we tested whether the phagosomal pH of neutrophils and macrophages is regulated by Hv1 channels. Immunostaining revealed that Hv1 was recruited to phagosomes of both neutrophils and macrophages. ROS production, measured with OxyBurst‐coupled targets, was reduced in neutrophil phagosomes of Hv1 knockout mice. Ratiometric imaging of fluorescein isothiocyanate‐coupled targets showed that macrophage phagosomes acidified rapidly and independently of Hv1. In contrast, neutrophil phagosomes remained neutral 30 min after target ingestion, while 80% of Hv1 knockout phagosomes were alkaline. The remaining 20% where highly acidic and this subpopulation of acidic phagosomes was eliminated by inhibition of vATPase. These data suggest that whereas Hv1 contributes little to macrophage phagosomal pH, in neutrophils Hv1 ablation deregulates phagosome pH, leading either to alkalinization or to early recruitment of vATPase.
Patch clamp screening of a7 nAChRs and the temperature dependence of allosteric modulation on nAChR and GABAA
Authors: Haedo Rodolfo Jorge
Published: June 2021

The nicotinic acetylcholine receptor (nAChR) is a member of the ligand‐gated ion channel superfamily which includes GABAA, 5HT3, NMDA and glycine receptors. Neuronal nAChR are found in many areas of the mammalian peripheral and central nervous systems including the autonomic ganglia, hippocampus and thalamus. Despite their relative scarcity of expression in the CNS, when activated, nAChR have been shown to have profound effects on cognitive performance, locomotor activity, cardiovascular function and pain perception, amongst others.

Additionally, the effect of PNU‐120596 was investigated at physiological temperatures (37 °C), where the current amplitude was significantly smaller, compared with using the same concentrations applied at room temperature. This supports the idea of a strong temperature dependence of the allosteric modulation by PNU‐102596 on nAChRs, also reported by others (Sitzia, Front. Pharmacol. 2011 2: 81). More importantly, we demonstrate for the first time an equal temperature dependence of GABAARs positive allosteric modulators, such as Diazepam, suggesting a general temperature dependence on positive allosteric modulation of ligand‐gated ion channels.
9‐Phenanthrol Inhibits Calcium Oscillations in HL‐1 Mouse Cardiomyocytes
Authors: Burt Rees , Graves Bridget M. , Li Chaunfu , Williams David L. , Fregoso Santiago P. , Hoover Donald B. , Wondergem Robert
Published: June 2021

Transient Receptor Potential Melastatin 4 (TRPM4) is functionally expressed throughout the heart and has been implicated as a calcium‐activated nonselective cation channel that mediates membrane depolarization. The functional significance of TRPM4 in regards to Ca2+ signaling and its effects on cellular excitability and pacemaker function remains inconclusive. We show by Fura2 Ca‐imaging that pharmacological inhibition of TRPM4 in HL‐1 mouse cardiac myocytes by 9‐phenathrol (10 μM) decreases Ca2+ oscillations followed by an overall increase in [Ca2+]i. The latter occurs also in HL‐1 cells in Ca‐free solution and after depletion of sarcoplasmic reticulum Ca2+ with thapsigargin (10 μM). Furthermore, by whole‐cell voltage clamp we show that 9‐ phenanthrol reversibly inhibits TRP‐like membrane current; by fluorescence immunohistochemistry we demonstrate that HL‐1 cells display punctate surface labeling with TRPM4 antibody. We conclude that 9‐phenanthrol inhibits TRPM4 ion channels in HL‐1 cells, which in turn decreases Ca2+ oscillations followed by a compensatory increase in [Ca2+]i from an intracellular store other than the sarcoplasmic reticulum. We speculate that the most likely source is the mitochondrion.

RB supported by APS summer undergrad fellowship.
ROS‐induced membrane lipid peroxidation impairs TRPV1 channels in the vasculature
Authors: Kmetz John G. , DelloStritto Daniel J. , Sinha Sayantani , Prudner Bethany , Damron Derek S. , Bratz Ian N.
Published: June 2021

A critical amount of reactive oxygen species (ROS) contributes to coronary blood flow (CBF) regulation; however, oxidative stress impairs CBF regulation. We have previously demonstrated TRPV1‐dependent coupling of CBF to metabolism is disrupted in diabetic cardiomyopathy (DCM). Accordingly, we hypothesized that basal levels of H2O2 stimulate TRPV1 whereas enhanced oxidative stress desensitizes TRPV1 via an H2O2 dependent post translational modification involving the lipid peroxidation product 4‐Hydroxynonenal (4‐HNE). Using contrast echocardiography, H2O2 dose‐dependently increased CBF in control and TRPV1−/− mice, yet was greater in controls compared to TRPV1−/−. Similarly, H2O2 potentiated capsaicin‐mediated CBF increases in controls. H2O2 caused robust dilation in control coronary microvessels (blunted in the presence of the TRPV1 inhibitor SB366791 and in TRPV1−/− vessels), suggesting H2O2 induced vasodilation occurs in part via TRPV1. H2O2 induced Ca2+ influx was TRPV1 dependent in HEK and TRPV1−/− endothelial cells (ECs) transfected with TRPV1 and further verified using Ca2+ imaging and electrophysiology. Lastly, ECs incubated with H2O2 (1hr) and 4‐ HNE (1hr) demonstrated increased 4‐HNE levels and 4‐HNE modified TRPV1 via western/co‐immunoprecipitation. These data suggest low levels of H2O2 can potentiate TRPV1 however, increased ROS concentrations as seen in DCM can lead to enhanced 4‐HNE levels which modulate TRPV1 and disrupt its signaling, resulting in tissue perfusion impairments.
TRPV4‐mediated Ca2+ influx promotes airway smooth muscle cell proliferation via NFATc3
Authors: Zhao Limin , Sullivan Michelle Nicole , Gonzales Albert Louis , Earley Scott
Published: June 2021

Bronchial asthma is a highly prevalent chronic respiratory disease with significant impact on human health. Airway remodeling due to proliferation of airway smooth muscle cells (ASMC) causes irreversible airflow obstruction during asthma, but the underlying molecular mechanisms are unclear. We hypothesized that Ca2+ influx via TRPV4 contributes to ASMC proliferation. We find that the selective TRPV4 agonist GSK1016790A elicited concentration‐dependent Ca2+ influx (EC50 = 18.1±2.4 nM) in primary rat ASMC that was blocked by the TRPV4 antagonist HC067047 (500 nM). GSK1016790A enhanced ASMC proliferation (EC50=31.0±7.5 nM) whereas HC067047 (IC50=220 ±46 nM) had the opposite effect. Involvement of the Nuclear Factor of Activated T cells (NFAT), a Ca2+‐activated transcription factor important for SMC proliferation, was examined. The calcineurin blocker cyclosporine (10 μM), and expression of the NFAT‐inhibiting peptide VIVIT attenuated GSK1016790A‐induced ASMC proliferation. In addition, GSK1016790A induced translocation of a cytoplasmic NFATc3‐GFP fusion protein to the nucleus of ASMC. Our findings show that Ca2+ influx via TRPV4 induces proliferation of ASMC by activating NFATc3. Supported by HL091905.
IL‐8 Inhibits cAMP‐stimulated Alveolar Epithelial Fluid Transport via a GRK2/PI3K‐dependent Mechanism
Authors: Wagener Brant M , Roux Jeremie , McNicholas Carmel M. , Carles Michel , Goolaerts Arnaud , Houseman Benjamin T. , Dickinson Dale A. , Iles Karen E. , Ware Lorraine B. , Matthay Michael A. , Pittet Jean‐Francois
Published: June 2021

Patients with acute lung injury (ALI) who retain maximal alveolar fluid clearance (AFC) have better clinical outcomes. Experimental and small clinical studies have shown that β2‐adrenergic receptor (β2AR) agonists enhance AFC via a cAMP–dependent mechanism. Two phase 3 clinical trials failed to show that β2AR agonists provide a survival advantage in ALI patients. We hypothesized that IL‐8, an important mediator of ALI, antagonizes the alveolar epithelial response to β2AR agonists. Short‐circuit current and whole‐cell patch‐clamping experiments revealed that IL‐8 or its rat analog CINC‐1 decreases by 50% β2AR agonist‐stimulated vectorial Cl− and net fluid transport across rat and human alveolar epithelial type II cells via a reduction in the cystic fibrosis transmembrane conductance regulator activity and biosynthesis. This reduction was mediated by heterologous β2AR desensitization and downregulation (50%) via the G‐protein coupled receptor kinase 2 (GRK2)/PI3K signaling pathway. Inhibition of CINC‐1 restored β2AR agonist‐stimulated AFC in an experimental model of ALI in rats. Finally, consistent with the experimental results, high pulmonary edema fluid levels of IL‐8 (>; 4,000 pg/ml) were associated with impaired AFC in ALI patients. These results demonstrate a novel role for IL‐8 in inhibiting β2AR agonist‐stimulated alveolar epithelial fluid transport via GRK2/PI3K‐dependent mechanisms.
Sustained Elevated Adenosine Causes Lung Endothelial Apoptosis and Emphysema via Nucleoside Transporter‐ Facilitated Mitochondrial Defects
Authors: Lu Qing , Newton Julie , Shamirian Paul , Hsiao Vivian , Sakhatskyy Pavlo , Curren Sean , Blackburn Michael R. , Rounds Sharon
Published: June 2021

Cigarette smoking (CS) and sustained elevated adenosine cause alveolar cell apoptosis and emphysema. Adenosine deaminase (ADA) activity is decreased in the lungs of patients with COPD. However, it is unknown whether CS increases lung adenosine levels and whether increased lung adenosine contributes to CS‐induced endothelial cell (EC) apoptosis and emphysema. In this study, we found that lung tissue adenosine levels were significantly elevated in AKR mice exposed to CS for 3 weeks; an effect associated with increased EC apoptosis and early emphysema. We used our previously established in vitro model of sustained adenosine exposure by incubating lung EC with adenosine and an ADA inhibitor for up to 48 hours. We demonstrated that sustained adenosine exposure caused lung EC apoptosis via nucleoside transporter‐facilitated, not receptor‐mediated, intracellular adenosine uptake and metabolism, subsequent mitochondrial oxidative stress‐mediated activation of p38 and JNK, and ultimately mitochondrial defects and activation of mitochondrial apoptotic pathway. Our results suggest that CS‐induced lung EC apoptosis and emphysema is due to sustained elevated adenosine. Inhibition of adenosine uptake and mitochondria oxidative stress may become new therapeutic targets in treatment of smoke‐induced lung diseases. Funded by HL64936 (Rounds), VA Merit Review (Rounds), ATS/PHA research grant (Lu)
Endoplasmic reticulum stress linked to cystic fibrosis cellular manifestations through PI3‐kinase p110alpha/histone deacetylase 6 signaling cascade
Authors: Rymut Sharon M , Corey Deborah A , Kelley Thomas J
Published: June 2021

Cystic fibrosis (CF) is an autosomal recessive disorder, characterized by dysfunctional cystic fibrosis transmembrane conductance regulator (CFTR) protein. Determining how the loss of CFTR function leads to cellular manifestations in CF is an ongoing challenge. Among these cellular manifestations are impaired endosomal transport, perinuclear cholesterol accumulation, and pro‐inflammatory signaling. CF epithelial cells and tissues exhibit endoplasmic reticulum (ER) stress independent of mutant CFTR folding marked by two‐fold increase in expression of GRP78. We have identified a novel signaling cascade linking ER stress to CF cellular manifestations through PI3‐kinase p110alpha (PI3KCA) modulation of histone deacetylase 6 (HDAC6) activity. Activation of this pathway leads to deacetylated microtubules resulting in impaired endosomal movement, cholesterol accumulation, and pro‐inflammatory and pro‐proliferative signaling cascades. Efficacy of the ER stress reliever 4‐phenylbutyrate and the PIK3CA inhibitor PIK‐75 in reversing these CF related phenotypes is demonstrated. This study directly links ER stress to microtubule modifications that result in a number of disease related cellular phenotypes. Supported by NIH/NHLBI grant HL104358 and a grant from the CFF.
Modulation of αENaC mRNA expression under different stresses: Implication of its 3′UTR.
Authors: Migneault Francis , Pascariu Mihai , Dagenais André , Berthiaume Yves
Published: June 2021

Active sodium transport by the epithelial Na+ channel (ENaC) is important for fluid movement across the alveolar epithelium in the lungs and for the resolution of pulmonary oedema in acute respiratory distress syndrome (ARDS). It was shown that cellular stress induced by lipopolysaccharides (LPS) or cycloheximide (Chx) downregulate αENaC mRNA via different molecular mechanisms. The purpose of this project was to investigate the importance of the αENaC 3′UTR in the mRNA modulation of ENaC. Alveolar epithelial cells were co‐transfected with 3′UTR mutants (V5/ENaC±3′UTR and Luc±3′UTR) inserted in pTRE‐tight vector along with the Tet‐Off vector. The cells were treated with Chx (1.0μM), LPS (15μg/mL) or Actinomycin D (5μg/mL) along with Dox (1μg/mL) to inhibit transcription. We found that 3′UTR deletion caused a ~60% decrease of luciferase activity and expression compared to the complete recombinant. The T1/2 of V5‐αENaC mRNA was 60 min and the deletion of 3′UTR reduced T1/2 to 37min. Chx reduced αENaC mRNA T1/2 to 33min whereas LPS had no effect. Actinomycin D, a transcription inhibitor, increased T1/2 >; 120min. These results suggest that the αENaC mRNA half‐life is shorter then previously estimated and that 3′UTR seems to be important for the stability of αENaC mRNA.

Supported by FRSQ, CFC and CIHR.
Rab38 targets to and regulates the sizes of a subset of larger and older LBs in the alveolar type II pneumocyte
Authors: Huang Shaohui , Zhang Zongqi , DeBolt Kristine M. , Mulugeta Surafel , Feinstein Sheldon I , Fisher Aron B , Zhang Linghui
Published: June 2021

Hermansky‐Pudlak syndrome associated pulmonary fibrosis is characterized by giant lamellar bodies (LBs) in degenerative alveolar type II (ATII) pneumocytes. We have previously shown a prominent giant LB pathology in the ATII cells isolated from the Rab38‐null Fawn‐hooded hypertension (FHH) rats. EGFP‐Rab38 reexpressed in cultured FHH ATII cells rescues the giant LB pathology while only being targeted to 30–40% LBs at any moment. The objective of this study is to characterize the Rab38‐ targeted LBs in the ATII pneumocytes. Using quantitative confocal microscopy, we found that EGFP‐Rab38 selectively targeted to a subset of larger LBs with higher endogenous ABCA3 contents, resulting in apparent inhibition of growth of the targeted LBs. Expressing ABCA3‐EGFP in cultured FHH ATII cells enhanced both LB biogenesis and growth in size. Importantly, newly synthesized ABCA3‐EGFP was associated with pre‐LB compartments and nascent LBs, while newly synthesized mRFP‐Rab38 was directly trafficked to larger existing LBs. After further cell culture, the ABCA3‐EGFP‐associated LBs grew substantially larger and were increasing targeted by mRFP‐Rab38, resulting in inhibition of growth of the targeted LBs. Our study reveals a LB biogenesis and growth process resolvable by the spatially and temporally differentiated Rab38 and ABCA3 trafficking, and establishes Rab38 as the first molecular marker for a subgroup of older and larger LBs in the ATII pneumocyte.
S100A12 Induce Pro‐inflammatory Response in Pulmonary Fibroblasts
Authors: Medina Maria Elena Monzon , Santos‐Falcon Nieves , Campos MIchael
Published: June 2021

Chronic obstructive pulmonary disease (COPD) is a progressive disease characterized by a chronic and irreversible airflow obstruction that is associated to structural changes of the airway wall (remodeling). The major sites of remodeling in COPD are the bronchioles. The tissue remodeling process starts with epithelial damage followed by an inflammatory response that propagates into the underlying tissues. S100A12 is a member of the S100 protein family that is elevated in numerous chronic inflammatory diseases and that triggers cellular activation in endothelial cells, and leukocytes through its binding to RAGE receptor. Systemic S100A12 is elevated in IPF and cystic fibrosis. In the present report to assess the role of S100A12/RAGE in the pathogenesis of COPD, S100A12 concentration was measure in bronchoalveolar lavages (BAL), from healthy and COPD patients by ELISA. The expression of S100A12 and RAGE were studied in primary cultures of pulmonary fibroblasts (Fb) and human bronchial epithelial (HBE) cells by qPCR and IF. Pro‐inflammatory IL‐6 and GRO‐α expression were assessed by qPCR in Fb and HBE cultures exposed to S100A12. We found that S100A12 was significantly increased in BAL samples of COPD when compared to healthy individuals. We also found that while both Fb and HBE cells express RAGE, only HBE produce S100A12. When cell cultures were exposed we found that 100A12 significant increase IL‐6 and GRO‐α mRNA expression in Fb. No significant differences were found in HBE cells compared to untreated controls. These results suggest a possible role of S100A12 released by bronchial epithelium in inducing inflammatory response in Fb.
FBXL12 regulates G1 arrest via degradation of CaMKI
Authors: Agassandian Marianna , Kaercher Leah , Snavely Courtney , Pilijala Roopa , Chen Beibei , Coon Tiffany , Mallampalli Rama
Published: June 2021

Cell cycle progression through its regulatory control by changes in intracellular Ca2+ levels at the G1/S transition controls the proliferation and viability of cells. Ca2+/CaM‐dependent kinase 1 (CaMKI) appears critical in regulating the assembly of the cyclin D1/cdk4 complex during G1 progression, but the molecular mechanisms are unknown. Here, we show that ectopic expression of a ubiquitin E3 ligase component, F‐box protein FBXL12, mediates CaMKI degradation via a proteasome‐directed pathway leading to disruption of cyclin D1/cdk4 complex formation and G1 arrest in lung epithelia. Cyclin D1/cdk4 assembly in the early G1 phase is also regulated via binding to p27, a protein that has been shown previously to be phosphorylated by AKT, PKC, ERK1/2 at Thr157 and Thr198. Our studies demonstrate that i) CaMKI also phosphorylates p27 to modulate its subcellular localization; ii) FBXL12‐induced CaMKI degradation attenuates p27 phosphorylation at these sites in early G1 and iii) activation of CaMKI during G1 transition followed by p27 phosphorylation appears to be upstream from other p27 phosphorylation events, an effect abrogated by FBXL12 overexpression in epithelia. Last, known inducers of G1 arrest significantly increased FBXL12 levels in cells. Thus, FBXL12 may be a previously uncharacterized, functional growth inhibitor regulating cell cycle progression that might be used for mechanism‐based therapy.
Endothelial caveolar modulation of SK3 channel activity
Authors: Lin Mike , Adelman John P , Maylie James
Published: June 2021

Small‐ and intermediate‐conductance Ca2+‐activated K+ channels (SK3/Kcnn3 and IK1/Kcnn4) are expressed in vascular endothelium. Their activities play important roles in regulating vascular tone through their modulation of [Ca2+]i required for the production of endothelium‐derived vasoactive agents. Activation of endothelial IK1 or SK3 channels hyperpolarizes endothelial cell membrane potential, increases Ca2+ influx, and leads to the release of vasoactive factors, thereby impacting blood pressure. To examine the distinct roles of IK1 and SK3 channels, we used electrophysiological recordings to investigate IK1 and SK3 channel trafficking in acutely dissociated endothelial cells from mouse aorta. The results show that SK3 channels undergo Ca2+‐dependent cycling between the plasma membrane and intracellular organelles; disrupting Ca2+‐dependent endothelial caveolae cycling abolishes SK3 channel trafficking. Moreover, transmitter induced changes in SK3 channel activity and surface expression modulate endothelial membrane potential. In contrast, IK1 channels do not undergo rapid trafficking and their activity remains unchanged when either exo‐ or endocytosis is block. Thus, modulation of SK3 surface expression may play an important role in regulating endothelial membrane potential in a Ca2+‐dependent manner.
Mechanisms of oxLDL induced endothelial stiffening and its role in capillary formation
Authors: Oh Myung‐Jin , Shentu Tzu‐Pin , Kohler Erin , Wary Kishore , Levitan Irena
Published: June 2021

Dyslipidemia is a major risk factor for the development of atherosclerosis which is initiated by endothelial injury but the mechanisms of dyslipidemia‐induced endothelial injury are still poorly understood. Our lab has shown that plasma dyslipidemia, oxidized low density lipoprotein (oxLDL), and 7‐ketocholesterol, leads to an increase in endothelial cell (EC) stiffness. We propose that endothelial stiffening plays a significant role in neovascularization of atherosclerotic plaques. Our goal is to understand the mechanism by which oxLDL induces EC stiffening and whether EC stiffening contributes to the formation of capillaries. We tested whether oxLDL‐induced EC stiffening is mediated by the Rho‐GTPases. We specifically targeted RhoA or its downstream effector Rho kinase (ROCK). We show that oxLDL induced EC stiffening is abrogated when inhibiting either RhoA or ROCK, and 7‐ketocholesterol induced EC stiffening is also abrogated by inhibition of RhoA. To test whether oxLDL is involved in capillary formation in vivo, we performed a matrigel plug assay and quantified the number of vessels formed from cells exposed to oxLDL. We show that oxLDL induces an increase in the number of vessels formed compared to normal vessel formation. We propose, therefore, that oxLDL induced endothelial stiffening is mediated by the RhoA/ROCK pathway, and that it plays an important role in dyslipidemic conditions in vivo.
Angiotensin II‐induced impairment of vasodilation in mouse mesenteric arteries: role of endothelial TRPV4 channels
Authors: Nishijima Yoshinori , Zheng Xiaodong , Lund Hayley , Mattson David , Zhang David X.
Published: June 2021

Up‐regulation of angiotensin II (Ang II) signaling causes endothelial dysfunction and altered production of endothelial relaxing factors. Endothelial TRPV4 is a Ca2+entry channel critically involved in endothelium‐mediated dilation in various species and vascular beds; however, the importance of this channel in disease remains largely unknown. This study investigated the contribution of TRPV4 to Ang II‐induced impairment of vasodilation in mice. The study was performed in TRPV4‐deficient (TRPV4−/−) and wild‐type (WT) mice implanted with an osmotic mini‐pump for continuous infusion of Ang II (600 ng/kg/min) for 14 days. After Ang II infusion, mean arterial pressure was similar in TRPV4−/− (143 ± 4 mmHg) and WT (137 ± 7 mmHg). In isolated mesenteric arteries from control WT mice, acetylcholine (ACh) elicited potent endothelium‐dependent dilation (dilation at 10−5 M, 92 ± 2%; n=9). The dilation was markedly reduced in Ang II‐treated WT mice (71 ± 5%; n=7). Ang II also reduced the dilation to TRPV4 agonist GSK1016790A (dilation at 10−8 M, 14 ± 5% vs. 77 ± 7% of control). Surprisingly, Ang II did not affect ACh‐induced dilation in TRPV4−/− mice (71 ± 3% vs. 69 ± 9% of control; n=9 and 5, respectively). In conclusion, Ang II reduces endothelium‐mediated dilation in mouse mesenteric arteries from normal but not TRPV4−/− mice. The precise role of TRPV4 in Ang II‐induced endothelial dysfunction remains to be determined.
Mesenteric vascular remodeling in different mouse strains
Authors: Familtseva Anastasia , Qipshidze Natia , Pushpakumar Sathnur Basappa , Tyagi Suresh C
Published: June 2021

Inflammatory Bowel Disease (IBD) is a category of inflammatory conditions of the colon and small intestine associated with oxidative stress (OS). Hyperhomocysteinemia (HHcy) is known as the cause of mesenteric vascular remodeling, inducing hypertension and acute abdomen pain. Patients with IBD often have HHcy compare to healthy people. We followed alterations of homocysteine (Hcy) level and mesenteric artery remodeling comparing four different strains of mice: C5BJ/L6 wild type (WT) as a control; cystathione beta synthase heterozygote knockout (CBS+/−) with increased level of Hcy; C3H, which is known as the most resistant to (OS) and double‐transgenic CBS/C3H mice. We suggested that Hcy level will be decreased in CBS/C3H compared to CBS, there will be amelioration of hemodynamics in mesenteric artery of CBS/C3H in comparison with CBS mice and attenuation of mesenteric vascular remodeling in CBS/C3H versus CBS. We used blood flow and mesenteric artery reactivity measurements, immunohistochemistry, electron microscopy of mitochondria, measurement of Dpr‐1 level (mediates mitochondrial fission during OS), measurement of Hcy level in plasma, Masson trichrome, PCR and Western blot (eNOS/iNOS, DDAH‐2, SAHH, CBS, CSE, 3MST, MTHFR, DNMT). Our preliminary data suggests that mesenteric vascular remodeling and HHcy are attenuated and hemodynamic of mesenteric artery is ameliorated in CBS/C3H versus CBS mice.
Effect of Relaxation Breathing on Glycemic Response in Type II Diabetic Subjects
Authors: Settell Megan Leigh , Gregor Angela L , Ackermann Kristin C , Segner Sara A , Anderson Melanie M , Anderson Ashley D , Wilson Ted
Published: June 2021

Background

Pharmacological treatments are often used to improve hyperglycemia in Type II diabetics. Stress is associated with development of insulin resistance and hyperglycemia. Non‐pharmacological interventions that reduce stress could improve glycemic response. This study determined if a standard relaxation breathing (RB) exercise could improve glycemic response in non‐insulin dependent Type II diabetics.

Methods

Subjects (n=9; 62.0 ± 9.3 yrs; BMI 31.0 ± 6.5) were randomized in a double‐cross over design. Following a 9 hour fast from food/medications and a 30 min acclimation period of control breathing (CB) or RB, baseline blood glucose was measured (0 min) prior to an oral glucose tolerance test drink (OGTT; 50g/240mL). Blood glucose was measured 30, 60, 120 min post OGTT. RB consisted of sequentially increasing exhalation durations (1, 2, …10 sec) repeated every 10 min for 20 min pre‐OGTT and every 10 min for 120 min post OGTT.

Results

Blood glucose at 0, 30, 60 and 120 min was 158 ± 41, 251 ± 66, 307 ± 58, and 231 ± 57 mg/dl for control, and 156 ± 43, 260 ± 67, 309 ± 54, and 214 ± 60 mg/dl for RB. Blood glucose AUC for control and RB was 30683 ± 2201 and 30394 ± 2191. No significant differences between groups were observed for blood glucose, AUC, heart rate, or %SpO2.

Conclusion

Acute implementation of RB does not appear to improve glycemic response in non‐insulin dependent Type II diabetics.
The mitochondrial Na+/Ca2+ exchanger‐NCLX is an integrating hub for glucose dependent Na+ and Ca2+ signaling in pancreatic β cells
Authors: Sekler Israel , Hershfinkel Michal , Nita Inlia I.
Published: June 2021

Although Na+ channels are abundantly expressed in β cells how Na+ fluxes are linked to glucose dependent Ca2+ signaling linked to pancreatic secretion is poorly underwood. Here we addressed the role of Na+ influx in regulating glucose dependent mitochondrial Ca2+ shuttling and cytosolic Ca2+ transients by fluorescently monitoring cytosolic and mitochondrial Na+ and Ca2+ transport in pancreatic b cells. Influx of Na+ via the TTX sensitive channels activated the mitochondrial Na+/Ca2+ exchanger NCLX, which in turn stimulated glucose dependent cytosolic Ca2+ influx. This effect was mimicked by pretreating cells with ouabain that by triggering cytosolic Na+accumulation eliminated the TTX dependent inhibition of mitochondrial and cytosolic Ca2+ signaling. Importantly knock down of NCLX expression by siNCLX eliminated the stimulatory effects of ouabain. Our results identify NCLX as the integrating hub for Na+ and Ca2+ signaling. Thus activation of the mitochondrial NCLX by Na+ influx accelerate mitochondrial Ca2+ shuttling and glucose dependent cytosolic Ca2+ rise in pancreatic β cells. Supported by a ISF grant to IS
Chlorine Gas Exposure on Human Bronchial Cells Decreases Mitochondrial Quality and Activates Autophagy
Authors: Jurkuvenaite Asta , Benavides Gloria A , Komarova Svetlana , Doran Stephen F , Darley‐Usmar Victor M , Matalon Sadis
Published: June 2021

Chlorine (Cl2) can cause significant morbidity and mortality when inhaled due to the formation of reactive intermediates. We hypothesized that mitochondria of lung epithelial cells are injured by Cl2 causing decreased bioenergetic capacity of these organelles and membrane depolarization. Human epithelial cells (H441) were exposed to Cl2 (100 ppm for 15 min) and returned to the incubator with air/5% CO2. Measurements of mitochondrial oxygen consumption rate (OCR) using extracellular flux analysis showed a significant reduction of maximal OCR and bioenergetic reserve capacity at 1h post exposure, the values returning to air controls 6 h later. Mitochondrial ROS production, assessed by MitoSOX fluorescence (both by fluorescent microscopy and FACS analysis) showed increased signals at 1 and 6 h post Cl2 exposure. Furthermore, mitochondrial membrane potential showed a 30% decrease when compared with controls at 1h but not 6 h post exposure, indicative of significant but reversible mitochondrial injury (TMRM assay). The recovery of mitochondrial membrane potential at 6h post exposure was accompanied by increased levels of autophagy protein LC3‐II which increased even more 24h post exposure. Taken together these data support the hypothesis that mitochondrial quality is decreased by Cl2 exposure and suggests that autophagy may play a regulatory role in this process. Supported by 5U01ES015676
Vasoactive agonists induce oscillatory changes in vascular smooth muscle cell stiffness and alter cytoskeletal architecture
Authors: Hong Zhongkui , Sun Zhe , Li Zhaohui , Trzeciakowski Jerome P. , Meininger Gerald A.
Published: June 2021

Vasoactive agonists induce coordinated and simultaneous changes in vascular smooth muscle cell (VSMC) stiffness and integrin adhesion. In this study we analyzed the dynamic effects of vasoactive agonists on VSMC stiffness and cortical cytoskeletal structure. Real‐time measurement of cell stiffness was obtained with atomic force microscopy (AFM) and analyzed with Eigen decomposition. Stiffness in VSMCs displayed temporal oscillations with three components at 0.001, 0.004, and 0.007 Hz frequency. The vasoconstrictor angiotensin II (ANG II, 10−6M) increased the amplitude of these oscillations (+100%) whereas adenosine (ADO, 10−4M), a vasodilator, had the opposite effects (−50%). To examine whether alterations in cortical cytoskeletal structure was part of these effects, AFM was used to image VSMC topography before and after treatment with ANG II or ADO. Topographical AFM images revealed the distribution and orientation of cytoskeletal stress fibers. Analysis with Matlab showed that ANG II increased the parallel alignment, density and thickness of the cortical stress fibers. In contrast, ADO showed reverse effects on the stress fibers. These findings demonstrate that the vasoactive agonists dynamically alter temporal behavior and structure of the VSMC cytoskeleton and may play an important role in contributing to the normal contractile behavior of VSMC. (1P01HL095486 to G.A.M. and AHA 0835676N to Z.S.)
N‐terminal region of cardiac myosin binding protein‐C impairs myofilament function
Authors: Sarkey Jason P. , Govindan Suresh , Chen Xin , Ge Ying , de Tombe Pieter P. , Sadayappan Sakthivel
Published: June 2021

Cardiac myosin binding protein‐C (cMyBP‐C) is a thick filament protein that plays a critical role in the regulation of sarcomere structure and contractility. Previously, we demonstrated that the N‐terminal fragment of cMyBP‐C (C0‐C1f), released during ischemia‐reperfusion (I‐R) injury, is cytotoxic and significantly impairs contractile function. However, the mechanisms in which C0‐C1f impairs cardiomyocyte contractile function remain unknown. The hypothesis of this study is that C0‐C1f depresses cardiac myofilament function. The molecular mass of C0‐C1f was analyzed by top‐down mass spectrometry. The observed molecular weights were 30974.72 Da and 31989.20 Da for mouse and human species respectively. Permeabilized left ventricular (LV) muscle strips were incubated with purified C0‐C1f. Myofilament function was characterized by measuring active tension‐[Ca2+] relationships to determine changes in maximum tension development (tmax) and Ca2+ sensitivity. Tension development and ATP consumption were simultaneously measured to determine tension‐cost over a range of [Ca2+]. C0‐C1f significantly reduced myofilament Ca2+ sensitivity and tmax, increased tension‐cost, and increased the rate of tension redevelopment (ktr). These data contribute to the understanding of contractile dysfunction following I‐R injury and provide a novel therapeutic target for the treatment of heart failure.
Coupling between stretch‐activated channels and ryanodine receptors in vacsular smooth muscle cells.
Authors: Savineau Jean‐Pierre , Gilbert Gillaume , Ducret Thomas , Marthan Roger , Quignard Jean‐François
Published: June 2021

Resistance arteries constrict in response to mechanical stress a phenomenon called “the myogenic response” that is inherent to vascular smooth muscle cells (SMC) properties. In the present study, we investigated the signaling pathway of this response both in physiological and pathological conditions (i.e. pulmonary hypertension using chronically hypoxic and monocrotaline‐treated rats). Stretching the membrane of pulmonary artery smooth muscle cells (PASMC) either via the pressure in the patch clamp pipette or a hypo osmotic shock, induced Ca2+ influx through stretch‐activated channels (SAC) which subsequently activated (1) BKCa channels, (2) RyR allowing an amplification of the calcium increase. Immunolabelling of RyR revealed that the three RyrR isoforms (Ryr‐1, RyR‐2, RyR‐3) were expressed in PASMC but with different subcellular segregation of calcium stores according the physiological status of vessels (control vs hypertensive). Indeed, calcium influx trough SAC was amplified by only subplasmalemnal RyR‐1 in PASMC from control rats while the other RyR subtypes were also involved in this amplification in PASMC from pulmonary hypertensive rats Altogether, these data suggest that SAC and RyR are necessary to this response and spatial organization of RyR and calcium stores in PASMC are important for cell signaling and response to stretch and thus in pulmonary hypertension.
Phenylephrine‐induced calcium oscillations in human mesenteric arterial smooth muscle
Authors: Tejerina Teresa , Navarro‐Dorado Jorge , Fameli Nicola , Garcia‐Alonso Mauricio , Ramajo Marta , Urraca Concha , Redondo Santiago , Medina Ursula , van Breemen Casey
Published: June 2021

Phenylephrine (PE)‐induced oscillatory fluctuations in intracellular Ca2+ concentration ([Ca2+]i) of vascular smooth muscle (SM) have been observed in every blood vessel isolated from a wide variety of mammals. Paradoxically, until recently similar observations in humans have proven elusive. In this study, we report for the first time observations of adrenergically‐stimulated [Ca2+]i oscillations in human mesenteric artery SM. We partially characterized these oscillations by using the inhibitory drugs nifedipine, 2‐aminoethoxydiphenyl borate (2‐APB), cyclopiazonic acid (CPA), and HA‐1077. PE and one of these drugs were added to different arterial segments, which had previously been loaded with Fluo‐4 AM in PSS and mounted on a myograph on the stage of a confocal microscope. We observed PE‐induced oscillations in [Ca2+]i, which initiated and maintained vasoconstriction. After PE contraction, all drugs provoked relaxation of the vessel segments, sometimes only partial, and reduced or inhibited oscillations, except CPA, which rarely caused relaxation. These preliminary results point to a potential involvement of the sarcoplasmic reticulum Ca2+ and IP3R in the maintenance of the observed oscillations.

FIS PI080920, RECAVA RD06/0014/1007
Stimulation of M3 but not M2 muscarinic receptor type inhibits large‐conductance Ca2+ activated K+ channel in rat detrusor smooth muscle
Authors: Parajuli Shankar Prasad , Petkov Georgi V
Published: June 2021

Large conductance Ca2+‐activated K+ (BK) channel is a major regulator of detrusor smooth muscle (DSM) contractility. Whether the regulation of BK channel function is mediated by muscarinic receptors (M‐Rs) remains unexplored. We studied the functional link between BK channels and M‐Rs in freshly isolated DSM cells using the perforated patch‐clamp technique. Carbachol (CCh) initially elicited large outward BK currents followed by inhibition of the amplitude and frequency of transient BK currents (TBKCs). CCh also inhibited the spontaneous transient hyperpolarizations and depolarized the cell membrane potential (MP). CCh inhibitory effects on the BK channels were suppressed by selectively blocking the M3‐Rs with 4‐DAMP, but not M2‐Rs with methoctramine. Blocking the IP3 receptors with xestospongin C attenuated CCh‐evoked initial effects on the large outward BK currents without altering the TBKCs suggesting an involvement of Ca2+ release from IP3 receptors in the initiation of the large outward BK currents. In the presence of thapsigargin, ryanodine, and nifedipine – CCh did not change the steady‐state BK currents suggesting requirement of intracellular Ca2+in the BK channel inhibition by cholinergic pathways. In conclusion, our results revealed that stimulation of M3‐Rs, but not M2‐Rs inhibits TBKCs, depolarized MP, and promotes DSM contractions. Supported by NIH DK08424 to GVP and AUA Fellowship to SPP.
A novel role for spontaneous endothelial cell calcium activity in the vascular myogenic response
Authors: Bagher Pooneh , Beleznai Timea , Kansui Yasuo , Mitchell Ray , Garland Christopher J. , Dora Kim A.
Published: June 2021

Although global increases in vascular smooth muscle cell (SMC) calcium are crucial for myogenic tone mainly in resistance arterioles, historically the myogenic response was thought to be independent of endothelial cell (EC) function. We have identified a novel role for ECs in the myogenic response, namely increases in localized spontaneous EC calcium event frequency at low intraluminal pressure activates transient receptor potential, vanilloid 4 channels (TRPV4) and subsequently intermediate conductance calcium‐activated K channels (IKCa), to activate an EC‐dependent vasodilation. Isolated rat cremaster arterioles were cannulated and pressurized (5–80mmHg) at ~34°C. Myogenic tone developed and EC function was assessed. Calcium events were imaged in Oregon Green® 488 BAPTA‐1 loaded ECs and/or SMCs using laser scanning confocal microscopy. Myogenic tone‐pressure response curves were performed in the presence of nitric oxide synthase (L‐NAME, 100 μM), KCa (TRAM‐34, 1 μM, and/or apamin 50 nM) and TRPV4 (RN1734, 30 μM) inhibitors. Calcium event frequency increased in ECs at low pressure versus high, with the reciprocal observed in SMCs. Inhibition of IKCa, TRPV4, but not SKCa at low pressures resulted in a loss of EC‐dependent dilation and a significant increase in myogenic tone. These data support a role for pressure‐dependent TRPV4‐mediated spontaneous EC calcium activity in the modulation of myogenic tone.
ASIC1a opposes exercise induced hyperemia and maximal exercise capacity
Authors: Drummond Heather , Gousset Monette , Abu‐Zaid Ahmed , Daklallah Mohammed , Weisman Jennifer , Murphey Beau , Hester Robert
Published: June 2021

Acid Sensing Ion Channel 1a (ASIC1a) is an extracellular proton‐gated, cation‐selective channel in neurons and vascular smooth muscle cells (VSMC). It is unknown if ASIC1a contributes to vascular tone under conditions associated with extracellular acidosis, such as strenuous exercise. During exercise, vessel recruitment and dilation, mediated by the accumulation of extracellular protons, contribute to increases in blood flow to the exercising muscles. To determine if ASIC1a contributes to vascular responses during exercise, we measured femoral vascular conductance (FVC) during a step‐wise electrical stimulation (0.5 – 5.0 Hz at 3V for 60 sec) of the left major hindlimb muscle groups in anesthetized ASIC1a+/+ and ASIC1a−/− mice. Baseline hemodynamic parameters were similar. FVC increased to a greater extent in ASIC1a−/− versus ASIC1a+/+ mice (0.44 ± 0.03 vs. 0.30 ± 0.04 ml×min−1×100g hindlimb mass−1, n=5 each, p=0.0009) with stimulation. The enhanced FVC was associated with a greater peak oxygen (O2) uptake in conscious ASIC1a−/− mice during exhaustive treadmill running (9563 ± 120 vs. 8836 ± 276 ml×kg−1×hr−1, n=6–7, p=0.0082). Differences in peak O2 uptake were not due to differences in absolute or relative lean body mass. Our findings suggest ASIC1a activity may oppose increases in vascular conductance during strenuous exercise thus limiting peak exercise capacity. Supported by NHLBI 51971.
Defective Nrf2‐Dependent Redox Signaling Contributes To Microvascular Dysfunction In Type 2 Diabetes
Authors: Velmurugan Gopal V , White Carl
Published: June 2021

In type‐2 diabetes, antioxidant depletion contributes to increased oxidative stress in the microvasculature. However, the mechanisms and physiological consequences are poorly understood. The objective of this study is to assess how oxidative stress contributes to functional changes in the diabetic microvasculature and to determine the effective role of transcription factor Nrf2. Myogenic tone was larger in diabetic (db/db) vessels and was directly correlated with elevated basal and pressure‐induced reactive oxygen species (ROS). Levels of Nrf2 and Nrf2‐regulated genes were downregulated in db/db vessels. Notably, expression of GCLC and GCLM, enzymes important for glutathione (GSH) synthesis and total GSH were significantly reduced. Normal myogenic function was restored to db/db arterioles by incubation with cell‐permeant GSH. Similarly, the db/db myogenic phenotype was recapitulated in the nondiabetic (db/m) vessels by GSH depletion. Treatment with the Nrf2‐activator sulforaphane increased GCLC, GCLM expression levels in db/db and dramatically lowered ROS and myogenic tone to db/m levels. In conclusion, depleted Nrf2 and its dependent genes compromises antioxidant capacity resulting in dysfunctional myogenic tone in diabetes. Defective Nrf2 signaling and its physiological consequences are rescued by sulforaphane, highlighting the compounds potential as a therapeutic tool.