Channels In Smooth Muscle Cells Research Articles

Enhanced isradipine sensitivity in vascular smooth muscle cells due to hypoxia-induced Cav1.2 splicing and RbFox1/Fox2 downregulation.

Calcium influx via the L-type voltage-gated Cav1.2 calcium channel in smooth muscle cells regulates vascular contraction. Calcium channel blockers (CCBs) are widely used to treat hypertension by inhibiting Cav1.2 channels. Using the vascular smooth muscle cell line, A7r5 and primary culture of cerebral vascular smooth muscle cells, we found that the expression and function of Cav1.2 channels are downregulated during hypoxia. Furthermore, hypoxia induces structural changes in Cav1.2 channels via alternative splicing. The expression of exon 9* is upregulated, whereas exon 33 is downregulated. Such structural alterations of Cav1.2 channels are caused by the decreased expression of RNA-binding proteins RNA-binding protein fox-1 homolog 1 and 2 (RbFox1 and RbFox2). Overexpression of RbFox1 and RbFox2 prevents hypoxia-induced exon 9* inclusion and exon 33 exclusion. Importantly, such structural alterations of the Cav1.2 channel partly contribute to the enhanced sensitivity of Cav1.2 to isradipine (a CCB) under hypoxia. Overexpression of RbFox1 and RbFox2 successfully reduces isradipine sensitivity in hypoxic smooth muscle cells. Our results suggest a new strategy to manage ischemic diseases such as stroke and myocardial infarction.

TMEM16A in smooth muscle cells acts as a pacemaker channel in the internal anal sphincter

Maintenance of fecal continence requires a continuous or basal tone of the internal anal sphincter (IAS). Paradoxically, the basal tone results largely from high-frequency rhythmic contractions of the IAS smooth muscle. However, the cellular and molecular mechanisms that initiate these contractions remain elusive. Here we show that the IAS contains multiple pacemakers. These pacemakers spontaneously generate propagating calcium waves that drive rhythmic contractions and establish the basal tone. These waves are myogenic and act independently of nerve, paracrine or autocrine signals. Using cell-specific gene knockout mice, we further found that TMEM16A Cl− channels in smooth muscle cells (but not in the interstitial cells of Cajal) are indispensable for pacemaking, rhythmic contractions, and basal tone. Our results identify TMEM16A in smooth muscle cells as a critical pacemaker channel that enables the IAS to contract rhythmically and continuously. This study provides cellular and molecular insights into fecal continence. R01HL139686 and R21HD097458. This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

TMEM16A in smooth muscle cells acts as a pacemaker channel in the internal anal sphincter.

Maintenance of fecal continence requires a continuous or basal tone of the internal anal sphincter (IAS). Paradoxically, the basal tone results largely from high-frequency rhythmic contractions of the IAS smooth muscle. However, the cellular and molecular mechanisms that initiate these contractions remain elusive. Here we show that the IAS contains multiple pacemakers. These pacemakers spontaneously generate propagating calcium waves that drive rhythmic contractions and establish the basal tone. These waves are myogenic and act independently of nerve, paracrine or autocrine signals. Using cell-specific gene knockout mice, we further found that TMEM16A Cl- channels in smooth muscle cells (but not in the interstitial cells of Cajal) are indispensable for pacemaking, rhythmic contractions, and basal tone. Our results identify TMEM16A in smooth muscle cells as a critical pacemaker channel that enables the IAS to contract rhythmically and continuously. This study provides cellular and molecular insights into fecal continence.

Kv2.1 Channels Prevent Vasomotion and Safeguard Myogenic Reactivity in Rat Small Superior Cerebellar Arteries.

Vascular smooth muscle voltage-gated potassium (Kv) channels have been proposed to contribute to myogenic autoregulation. Surprisingly, in initial experiments, we observed that the Kv2 channel inhibitor stromatoxin induced vasomotion without affecting myogenic tone. Thus, we tested the hypothesis that Kv2 channels contribute to myogenic autoregulation by fine-tuning the myogenic response. Expression of Kv2 channel mRNA was determined using real-time PCR and 'multiplex' single-cell RT-PCR. Potassium currents were measured using the patch-clamp technique. Contractile responses of intact arteries were studied using isobaric myography. Expression of Kv2.1 but not Kv2.2 channels was detected in intact rat superior cerebellar arteries and in single smooth muscle cells. Stromatoxin, a high-affinity inhibitor of Kv2 channels, reduced smooth muscle Kv currents by 61% at saturating concentrations (EC50 36 nmol/L). Further, stromatoxin (10-100 nmol/L) induced pronounced vasomotion in 48% of the vessels studied. In vessels not exhibiting vasomotion, stromatoxin did not affect myogenic reactivity. Notably, in vessels exhibiting stromatoxin-induced vasomotion, pressure increases evoked two effects: First, they facilitated the occurrence of random vasodilations and/or vasoconstrictions, disturbing the myogenic response (24% of the vessels). Second, they modified the vasomotion by decreasing its amplitude and increasing its frequency, thereby destabilizing myogenic tone (76% of the vessels). Our study demonstrates that (i) Kv2.1 channels are the predominantly expressed Kv channels in smooth muscle cells of rat superior cerebellar arteries, and (ii) Kv2.1 channels provide a novel type of negative feedback mechanism in myogenic autoregulation by preventing vasomotion and thereby safeguarding the myogenic response.

The Impact of T- and L-Type Calcium Channels Blockers on Pulmonary Microhemodynamics in Experimental Model of Pulmonary Thromboembolism

In acute experiments on isolated perfused rabbit’s Сhinchilla lungs changes of pulmonary microhemodynamics were studied in case of pulmonary embolization in the comparison group and after pretreatment with ethosuximide, mibefradil, chloroquine and nifedipine. In response to administration of T-type Ca2+-channel blockers ethosuximide and mibefradil, pulmonary artery pressure, precapillary and pulmonary vascular resistance decreased approximately to the same extent, postcapillary resistance did not change. After pretreatment with chloroquine pulmonary artery pressure, precapillary and pulmonary vascular resistance decreased more than in response to the administration of ethosuximide and mibefradil and the antagonist of L-type Ca2+-channels nifedipine; postcapillary resistance decreased. In the case of chloroquine administration combined with infusion of the KATP- channel blocker glibenclamide, most parameters of pulmonary microcirculation decreased almost to the same extent as in response to mibefradil administration, and postcapillary resistance did not change. After pretreatment with chloroquine in response to pulmonary embolization, pulmonary vascular resistance, pre- and postcapillary resistance increased less pronounced than with thromboembolism after pretreatment with ethosuximide, mibefradil and nifedipine. When modeling thromboembolism after pretreatment with chloroquine combined with KATP-channels blocker glibenclamide, the studied hemodynamics parameters increased to the same extent as after nifedipine pretreatment. Thus, chloroquine exhibits the properties of L- and T-type Ca2+-channels blocker, as well as an activator of KATP-channels, whereas ethosuximide has a blocking effect mainly on T-type Ca2+-channels of smooth muscle cells of pulmonary arterial vessels. Shifts of capillary filtration coefficient under these conditions depend more on changes of precapillary resistance than from the changes of permeability of endothelium of pulmonary vessels.

Calcium-dependent modulation of BKCa channel activity induced by plasmonic gold nanoparticles in pulmonary artery smooth muscle cells and hippocampal neurons.

Gold nanoparticles are widely used for biomedical applications, but the precise molecular mechanism of their interaction with cellular structures is still unclear. Assuming that intracellular calcium fluctuations associated with surface plasmon-induced calcium entry could modulate the activity of potassium channels, we studied the effect of 5nm gold nanoparticles on calcium-dependent potassium channels and associated calcium signaling in freshly isolated rat pulmonary artery smooth muscle cells and cultured hippocampal neurons. Outward potassium currents were recorded using patch-clamp techniques. Changes in intracellular calcium concentration were measured using the high affinity Ca2+ fluorescent indicator fluo-3 and laser confocal microscope. In pulmonary artery smooth muscle cells, plasmonic gold nanoparticles increased the amplitude of currents via large-conductance Ca2+ -activated potassium channels, which was potentiated by green laser irradiation near plasmon resonance wavelength (532 nm). Buffering of intracellular free calcium with ethylene glycol-bis-N,N,N',N'-tetraacetic acid (EGTA) abolished these effects. Furthermore, using confocal laser microscopy it was found that application of gold nanoparticles caused oscillations of intracellular calcium concentration that were decreasing in amplitude with time. In cultured hippocampal neurons gold nanoparticles inhibited the effect of EGTA slowing down the decline of the BKCa current while partially restoring the amplitude of the slow after hyperpolarizing currents. We conclude that fluctuations in intracellular calcium can modulate plasmonic gold nanoparticles-induced gating of BKCa channels in smooth muscle cells and neurons through an indirect mechanism, probably involving the interaction of plasmon resonance with calcium-permeable ion channels, which leads to a change in intracellular calcium level.

Dual effect of amitriptyline in the control of vascular tone: Direct blockade of calcium channel in smooth muscle cells and reduction of TLR4-dependent NO production in endothelial cells

Background and purposeAmitriptyline (AM) is a classical and typical tricyclic antidepressant drug. Despite its well-known effects on the nervous system, it has been described to work as a TLR4 antagonist and several clinical works suggested some unexpected cardiovascular effects. The role of amitriptyline on vascular tone is not clear, thus we hypothesized that amitriptyline has a double effect on vascular tone by both endothelial TLR4-dependent nitric oxide down-regulation and calcium channel blockade in smooth muscle cells. Experimental approachChanges in isometric tension were recorded on a wire myograph. NO production was evaluated by fluorescence microscopy and flow cytometry in the mouse aorta and EAhy926 cells using DAF fluorescence intensity. Calcium influx was evaluated in A7r5 cells by flow cytometry. Western blot was used to analyze eNOS and nNOS phosphorylation. Key resultsAM reduced PE-induced contraction by calcium influx diminution in smooth muscle cells (F/F0 = 225.6 ± 15.9 and 118.6 ± 17.6 to CT and AM, respectively). AM impaired Ach-dependent vasodilation (Emax = 95.8 ± 1.4; 78.1 ± 1.8; 60.4 ± 2.9 and −7.4 ± 1.0 for CT, 0.01, 0,1 and 1 μmol/L AM, respectively) through reduction of calcium influx and NO availability and TLR4 antagonism in a concentration-dependent manner. AM or TLR4 gene deletion significantly reduced NO production (Fluorescence = 9503 ± 871.7, 2561 ± 282, 4771 ± 728 and 1029 ± 103 to CT, AM, TLR4−/− and AM + TLR4−/−, respectively) by an increase in nNOSser852 and reduction in eNOSser1177 phosphorylation in endothelial cells. Conclusions and implicationsOur data show that amitriptyline impaired vascular function through two different mechanisms: blockade of TLR4 in endothelial cells and consequent decrease in NO production and calcium influx reduction in smooth muscle and endothelial cells. We also suggest, for the first time, nNOS activity reduction by AM in non-neuronal cells.

Docking-based analysis and modeling of the activity of bile acids and their synthetic analogues on large conductance Ca2+ activated K channels in smooth muscle cells.

The objective of this study was to perform docking-based analysis of bile acid binding on the protein complex of channels and to derive neural network that predicts the influence of bile acids and their synthetic analogues on the activity of BK(Ca) channels in smooth muscle cells based on descriptors for bile acids and their synthetic analogues and on their already published activities using patch-clamp techniques. Ligands for molecular docking were optimized using computer routine for minimization of energy by using the force field MMFF94 via Chem3D 15.0 and ligands and protein channel complex were prepared in AutoDockTools 1.5.6. AutoDock Vina 4.0 software was used for blind docking; processing and verification of the obtained results was performed via Discovery Studio 4.0. Neural network was derived using descriptors for bile acids and their synthetic analogues and their already published activities on calcium-activated K+ channels in smooth muscle cells (ChemDraw Professional 15.0, Dragon 6 software). Molecular docking was performed for: lithocholic acid, deoxycholic acid, 5β-cholanoic acid, 3β-hydroxi-5β-cholanoic acid, henodeoxycholic acid, ursocholic acid and α-muricholic acid. Neural network model Multiple layer perceptron is derived, having 0.9259 training performances and 0.3673 test performances, training error 0.0073 and test error 0,1607. Model was tested for henodeoxycholic, ursocholic and α-muricholic acid, and internal validation of the model is performed. Molecular docking suggested that the pharmacophore for maximizing the activity of BK(Ca) channels in the steroid skeleton of bile acids is the C3 quasi-axial α-OH group and the C24 carboxyl function. Derived neural network model successfully predicted activities of tested bile acids on Ca2+ activated K+ channels in smooth muscle cells.

Abstract 12772: Regulation of Blood Pressure by Smooth Muscle TRPV4 Channels Under Normal and Hypertensive Conditions

Introduction: Recent studies suggest that transient receptor potential vanilloid 4 (TRPV4) channel is a functionally important Ca 2+ influx pathway in arterial smooth muscle cells (SMCs). Hypothesis: We hypothesized that SMC TRPV4 (TRPV4 SMC ) channels regulate blood pressure under normal conditions and in hypertension. Results: Radiotelemetric recordings showed lower blood pressures in the SMC-specific TRPV4 -/- (TRPV4 SMC -/- ) mice than the control mice. Phenylephrine (PE, α1 adrenergic receptor or α1AR agonist)-induced constriction was blunted in small mesenteric arteries (MAs) from TRPV4 SMC -/- mice, whereas intraluminal pressure-induced constriction was increased. These data demonstrated that TRPV4 SMC channels contribute to α1AR-induced vasoconstriction but oppose pressure-induced vasoconstriction. Both PE and intraluminal pressure increased the activity of elementary Ca 2+ influx signals through TRPV4 SMC channels (TRPV4 SMC sparklets). TRPV4 agonist GSK1016790A (30 nM) activated the currents through large-conductance, Ca 2+ -sensitive K + (BK) channels in SMCs, whereas phenylephrine had no effect on BK channel currents. These data implied the presence of two functionally distinct pools of TRPV4 SMC channels: 1) α1AR-activated, constrictor TRPV4 SMC channels; and 2) pressure-activated, dilator TRPV4 SMC channels. Next, direct STORM imaging demonstrated that ~30% TRPV4 SMC channels co-localize with α1ARs and ~10% TRPV4 SMC channels co-localize with BK channels. Importantly, only ~3% TRPV4 SMC channels co-localized with both α1ARs and BK channels, supporting the concept of spatially distinct constrictor and dilator TRPV4 SMC channels. Angiotensin II (Ang II, 7-14 days) infusion resulted in a lower increase in blood pressure in TRPV4 SMC -/- mice than the control mice, demonstrating a crucial role of TRPV4 SMC channels in blood pressure elevation in hypertension. Moreover, Ang II infusion in C57BL6 mice increased α1AR-TRPV4 SMC signaling; reduced pressure-activation of TRPV4 SMC channels; and reduced TRPV4 SMC -BK channel signaling. Conclusions: Our results show that increased constrictor TRPV4 SMC channel activity and reduced dilator TRPV4 SMC channel activity combine to elevate blood pressure in hypertension.

LRRC8A is essential for volume-regulated anion channel in smooth muscle cells contributing to cerebrovascular remodeling during hypertension.

ObjectivesRecent studies revealed LRRC8A to be an essential component of volume‐regulated anion channel (VRAC), which regulates cellular volume homeostasis. However, evidence for the contribution of LRRC8A‐dependent VRAC activity in vascular smooth muscle cells (VSMCs) is still lacking, and the relevant functional role of LRRC8A in VSMCs remains unknown. The primary goal of this study was to elucidate the role of LRRC8A in VRAC activity in VSMCs and the functional role of LRRC8A in cerebrovascular remodeling during hypertension.Materials and MethodssiRNA‐mediated knockdown and adenovirus‐mediated overexpression of LRRC8A were used to elucidate the electrophysiological properties of LRRC8A in basilar smooth muscle cells (BASMCs). A smooth muscle–specific overexpressing transgenic mouse model was used to investigate the functional role of LRRC8A in cerebrovascular remodeling.ResultsLRRC8A is essential for volume‐regulated chloride current (I Cl, Vol) in BASMCs. Overexpression of LRRC8A induced a voltage‐dependent Cl− current independently of hypotonic stimulation. LRRC8A regulated BASMCs proliferation through activation of WNK1/PI3K‐p85/AKT axis. Smooth muscle‐specific upregulation of LRRC8A aggravated Angiotensin II‐induced cerebrovascular remodeling in mice.ConclusionsLRRC8A is an essential component of VRAC and is required for cell volume homeostasis during osmotic challenge in BASMCs. Smooth muscle specific overexpression of LRRC8A increases BASMCs proliferation and substantially aggravates basilar artery remodeling, revealing a potential therapeutic target for vascular remodeling in hypertension.

Nitric Oxide Signals Through IRAG to Inhibit TRPM4 Channels and Dilate Cerebral Arteries.

Nitric oxide (NO) relaxes vascular smooth muscle cells (SMCs) and dilates blood vessels by increasing intracellular levels of cyclic guanosine monophosphate (cGMP), which stimulates the activity of cGMP-dependent protein kinase (PKG). However, the vasodilator mechanisms downstream of PKG remain incompletely understood. Here, we found that transient receptor potential melastatin 4 (TRPM4) cation channels, which are activated by Ca2+ released from the sarcoplasmic reticulum (SR) through inositol triphosphate receptors (IP3Rs) under native conditions, are essential for SMC membrane depolarization and vasoconstriction. We hypothesized that signaling via the NO/cGMP/PKG pathway causes vasodilation by inhibiting TRPM4. We found that TRPM4 currents activated by stretching the plasma membrane or directly activating IP3Rs were suppressed by exogenous NO or a membrane-permeable cGMP analog, the latter of which also impaired IP3R-mediated release of Ca2+ from the SR. The effects of NO on TRPM4 activity were blocked by inhibition of soluble guanylyl cyclase or PKG. Notably, upon phosphorylation by PKG, IRAG (IP3R-associated PKG substrate) inhibited IP3R-mediated Ca2+ release, and knockdown of IRAG expression diminished NO-mediated inhibition of TRPM4 activity and vasodilation. Using superresolution microscopy, we found that IRAG, PKG, and IP3Rs form a nanoscale signaling complex on the SR of SMCs. We conclude that NO/cGMP/PKG signaling through IRAG inhibits IP3R-dependent activation of TRPM4 channels in SMCs to dilate arteries.Significance StatementNitric oxide is a gaseous vasodilator produced by endothelial cells that is essential for cardiovascular function. Although NO-mediated signaling pathways have been intensively studied, the mechanisms by which they relax SMCs to dilate blood vessels remain incompletely understood. In this study, we show that NO causes vasodilation by inhibiting the activity of Ca2+-dependent TRPM4 cation channels. Probing further, we found that NO does not act directly on TRPM4 but instead initiates a signaling cascade that inhibits its activation by blocking the release of Ca2+ from the SR. Thus, our findings reveal the essential molecular pathways of NO-induced vasodilation—a fundamental unresolved concept in cardiovascular physiology.

Clinical Importance of the Human Umbilical Artery Potassium Channels.

Potassium (K+) channels are usually predominant in the membranes of vascular smooth muscle cells (SMCs). These channels play an important role in regulating the membrane potential and vessel contractility—a role that depends on the vascular bed. Thus, the activity of K+ channels represents one of the main mechanisms regulating the vascular tone in physiological and pathophysiological conditions. Briefly, the activation of K+ channels in SMC leads to hyperpolarization and vasorelaxation, while its inhibition induces depolarization and consequent vascular contraction. Currently, there are four different types of K+ channels described in SMCs: voltage-dependent K+ (KV) channels, calcium-activated K+ (KCa) channels, inward rectifier K+ (Kir) channels, and 2-pore domain K+ (K2P) channels. Due to the fundamental role of K+ channels in excitable cells, these channels are promising therapeutic targets in clinical practice. Therefore, this review discusses the basic properties of the various types of K+ channels, including structure, cellular mechanisms that regulate their activity, and new advances in the development of activators and blockers of these channels. The vascular functions of these channels will be discussed with a focus on vascular SMCs of the human umbilical artery. Then, the clinical importance of K+ channels in the treatment and prevention of cardiovascular diseases during pregnancy, such as gestational hypertension and preeclampsia, will be explored.

PIP2: A critical regulator of vascular ion channels hiding in plain sight

The phosphoinositide, phosphatidylinositol 4,5-bisphosphate (PIP2), has long been established as a major contributor to intracellular signaling, primarily by virtue of its role as a substrate for phospholipase C (PLC). Signaling by Gq-protein-coupled receptors triggers PLC-mediated hydrolysis of PIP2 into inositol 1,4,5-trisphosphate and diacylglycerol, which are well known to modulate vascular ion channel activity. Often overlooked, however, is the role PIP2 itself plays in this regulation. Although numerous reports have demonstrated that PIP2 is critical for ion channel regulation, how it impacts vascular function has received scant attention. In this review, we focus on PIP2 as a regulator of ion channels in smooth muscle cells and endothelial cells-the two major classes of vascular cells. We further address the concerted effects of such regulation on vascular function and blood flow control. We close with a consideration of current knowledge regarding disruption of PIP2 regulation of vascular ion channels in disease.

Vasodilation activity of dipfluzine metabolites in isolated rat basilar arteries and their underlying mechanisms

Identifying the metabolites of a drug has become an indispensable task in the development of new drugs. Dipfluzine (Dip) is a promising candidate for the treatment of cerebral vascular diseases and has 5 metabolites (M1∼M5) in rat urine and liver microsomes, but their biological activity is still unknown. Because selective cerebral vasodilation is a main role of Dip, we investigated the vasodilation of Dip and its 5 metabolites in isolated Sprague-Dawley (SD) male rat basilar arteries preconstricted with high-K+ or 5-HT. The results showed that only M1 possessed concentration-dependent inhibitory activity on the vasoconstriction of arteries with or without the endothelium, and M1 has a more potent vasodilatory effect than Dip on both contraction models. Like Dip, the vasodilatory mechanisms of M1 may be not only related to receptor-operated and voltage-dependent calcium ion channels of smooth muscle cells but also to the release of NO and EDHF from endothelial cells and the opening of Ca2+-activated K+ channels and ATP-sensitive potassium ion channels. Unlike Dip, the vasodilation mechanism of M1 is also related to the opening of voltage-sensitive K+ channel. Together with more selectivity to non-VDCC than Dip, this may partially explain why M1 has stronger vasodilatory effects than Dip. The mechanisms of vasodilation of Dip and M1 may result from the combined action of these or other factors, especially blocking non-endothelium dependent non-VDCC and endothelium dependent IKCa channels. These results point to the possibility that M1 provides synergism for the clinical use of Dip, which may inform the synthesis of new drugs.

Extra Virgin Olive Oil Phenols Dilate the Rat Mesenteric Artery by Activation of BKCa2+ Channels in Smooth Muscle Cells

Accumulating evidence has shown the beneficial health effects of extra virgin olive oil (EVOO) consumption in reducing blood pressure and preventing the risk of developing hypertension. Some studies associate the hypotensive activity of EVOO to a minor component—the phenols. This study was designed to investigate the effects of EVOO phenols on the rat resistance mesenteric artery (MA) and to find out the possible vascular pathways involved. The experiments were carried out using a pressurized myograph, which allowed the effects of phenols on isolated MA to be tested under different conditions: (a) with endothelium removed; (b) with inhibition of nitric oxide synthase by Nω-Nitro-l-arginine methyl ester hydrochloride (l-NAME, 10−4 M) + Nω-Nitro-l-arginine (l-NNA, 10−4 M); (c) with inhibition of cyclooxygenase by indomethacin (10−5 M); (d) with inhibition of guanylate cyclase by 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ,10−5 M) or adenylate cyclase by 9-(Tetrahydro-2′-furyl)adenine (SQ, 10−5 M); (e) with depolarization by high potassium chloride (40 mM); and (f) with inhibition of the large conductance Ca2+–potassium channels (BKCa2+) with paxilline (10−5 M). EVOO phenols induce vasodilation of the endothelium, mediated by a direct effect on smooth muscle cells (SMC) by activation of BKCa2+ channels, an action by which phenols can regulate the vascular tone of the resistance artery. Phenols can be regarded as bioactive molecules that may contribute to the antihypertensive effects of EVOO.

Altered anoctamin-1 and tyrosine phosphorylation in congenital ureteropelvic junction obstruction

PurposeUreteropelvic junction (UPJ) obstruction is the most common cause of congenital hydronephrosis in children. The pathophysiology of UPJ obstruction and the exact mechanism of pelviureteral peristalsis are poorly understood. Anoctamin-1 (ANO1), a Ca2+-activated chloride channel, has been shown to play a key role in muscle wall contractions in the gastrointestinal tract. We designed this study to investigate the hypothesis that ANO1 is expressed in smooth muscle cells (SMCs) of the human UPJ and that tyrosine phosphorylation is altered in UPJ obstruction. Materials and methodsFresh frozen specimens of UPJ obstruction (n = 28) and control specimens from patients who underwent Wilms' tumor nephrectomy (n = 20) were prepared. Western blot (WB) was performed to evaluate levels of ANO1 protein expression and changes in tyrosine phosphorylation. In addition analysis of ANO1 and phalloidin using confocal-immunofluoresence-double staining and 3D reconstruction were carried out. ResultsOur WB results revealed increased tyrosine phosphorylation in UPJ obstruction samples compared to controls, and decreased ANO1 expression in UPJ obstruction. Confocal microscopy showed that ANO1 immunoreactivity was decreased in SMCs of UPJ obstruction compared to controls. ConclusionsWe provide evidence, for the first time, of the presence of ANO1 expression in the human UPJ. We speculate that altered tyrosine phosphorylation, observed in UPJ obstruction, may lead to a failure of transmission of peristaltic waves in UPJ obstruction by inhibiting Ca2+-activated chloride channels in SMCs.

Cobalt-Induced Hypercontraction is Mediated by Generationof Reactive Oxygen Species and Influx of Calcium in Isolated RatAorta

To investigate the mechanism of cobalt-mediated phenylephrine (PE)-induced contraction in endothelium-intact isolated Wistar rat aortic rings. Effect of dose-dependent concentrations of cobalt on PE-induced contraction was investigated in isolated Wistar rat aortic rings using an organ bath system. Aortic rings were pre-incubated with verapamil (1μM and 20μM), gadolinium, apocynin, indomethacin or N-G-nitro-L-arginine methyl ester (L-NAME) separately before incubation with cobalt. Endothelium-intact aortic rings were incubated with 800nM, 1μM, 10μM, 50μM cobalt; we observed 20%, 22%, 32% and 27% increased contractions respectively, while no effect was seen in tension recording on cobalt exposure. Incubation of endothelium-intact aortic rings with 100μM apocynin and 100μM L-NAME suggested the role of NADPH oxidase in generation of reactive oxygen species (ROS) and decrease in bioavailability of nitric oxide (NO) from eNOS on exposure to cobalt. Aortic rings pre-incubated with 1μM and 20μM verapamil suggested role of both L-type and T-type calcium channels in influx of extracellular calcium in smooth muscle cells. We observed no role of store-operated calcium channels (SOCC) in calcium influx due to cobalt exposure and cyclooxygenase in generation of prostanoids in isolated aortic rings. Cobalt caused rise of PE-induced contractions as a result of the endothelial generation of ROS, by decreasing bioavailability of NO. Generation of ROS may be responsible for causing the influx of extracellular calcium through L-type and T-type Ca2+ channels in smooth muscle cells.

Activation of adenosine A2A but not A2B receptors is involved in uridine adenosine tetraphosphate-induced porcine coronary smooth muscle relaxation

Activation of both adenosine A2A and A2B receptors (A2BR) contributes to coronary vasodilation. We previously demonstrated that uridine adenosine tetraphosphate (Up4A) is a novel vasodilator in the porcine coronary microcirculation, acting mainly on A2AR in smooth muscle cells (SMC). We further investigated whether activation of A2BR is involved in Up4A-mediated coronary SMC relaxation. Both A2AR and A2BR may stimulate H2O2 production leading to activation of KATP channels in SMCs, we also studied the involvement of H2O2 and KATP channels in Up4A-mediated effect. Coronary small arteries dissected from the apex of porcine hearts were mounted on wire myograph for Up4A concentration responses. Up4A-induced coronary SMC relaxation was attenuated by A2AR but not A2BR antagonism or non-selective P2R antagonism, despite greater endogenous A2BR expression vs. A2AR in both coronary small arteries and primary cultured coronary SMCs. Moreover, Up4A-induced coronary SMC relaxation was blunted by H2O2 catabolism. This effect was not altered by KATP channel blockade. Combination of H2O2 catabolism and A2AR antagonism attenuated Up4A-induced coronary SMC relaxation to the similar extent as A2AR antagonism alone. Collectively, Up4A-induced porcine coronary SMC relaxation is mediated by activation of A2AR-H2O2 pathway. This process does not involve A2BR, P2R or KATP channels.

EFFECT OF LEVOSIMENDAN, A CALCIUM SENSITIZER, ON RENAL HEMODYNAMICS IN L-NAME INDUCED HYPERTENSIVE RATS

Objective:Levosimendan acts preferentially by increasing the sensitivity of troponin C to calcium in myocardial cells. In addition, it also causes vasodilatation by opening the ATP-sensitive potassium channels in smooth muscle cells. We investigated the effect of levosimendan on renal hemodynamics a

BIN1 clusters CaV1.2 channels in vascular smooth muscle

Resistance vessel such as mesenteric arteries respond to increases in intravascular pressure by constricting. Ca2+ influx via voltage‐gated CaV1.2 channels in the smooth muscle surrounding these vessels is critical for the development of myogenic tone and thus the regulation of arterial dimeter and blood flow. Our group discovered that CaV1.2 channels have an inherent ability to form clusters and functionally couple in response to local elevations in [Ca2+]i. The consequence of these channels coming together is that they amplify Ca2+ influx during depolarization. However, the mechanisms dictating the arrangement of CaV1.2 channels in smooth muscle cells are unknown. BIN1, a member of the BAR (Bin1‐Amphiphysin‐Rvs) domain superfamily, has been implicated in the trafficking of CaV1.2 channels but has not yet been studied in smooth muscle. In this study, we test the hypothesis that BIN1 promotes the formation of CaV1.2 clusters enhancing overall Ca2+ influx into the vascular smooth muscle and shaping arterial diameter. We report that BIN1 is expressed and translated in arterial smooth muscle while ground state depletion (GSD) super‐resolution imaging suggest that smooth muscle specific down‐regulation of BIN1 diminishes CaV1.2 cluster formation. Using patch clamp electrophysiology and pressurized arteriography, we observed that loss of BIN1 substantially diminishes ICa and attenuates arterial tone. Overall, these results suggest that BIN1 is a critical component in shaping CaV1.2 clustering and plays a physiological role in vascular smooth muscle Ca2+ entry and arterial diameter.Support or Funding InformationThis work is supported by NIH R01 HL085870 (LFS), T32GM099608 (SO), and 18PRE33960249 (SO)This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.