Channels In Smooth Muscle Cells Research Articles

Hydrogen Sulfide as an allosteric modulator of ATP sensitive potassium channels in experimental colitis

The ATP sensitive potassium channel (KATP) in mouse colonic smooth muscle cell is a complex containing a pore forming subunit (Kir6.1) and a sulfonyl urea receptor subunit (SUR2B). We used whole‐cell voltage‐clamp techniques to study the alterations in these channels in smooth muscle cells in experimental colitis. Colitis was induced in BALB/C mice following an intracolonic administration of trinitrobenzene sulfonic acid. KATP currents were measured at Vh −60 mV in high K+ external solution. The dose‐response to levcromakalim (LEV), a KATP channel opener, was significantly shifted to the left in the inflamed smooth muscle cells. Both the affinity and maximal currents induced by LEV were enhanced in inflammation. The EC50 in control was 6489 nM (n=10) and 423.4 nM (n=8) in inflamed colon while the maximal currents were 9.3 ± 1.1 pA/pF (60 μM) in control and 47.7 ± 16.8 pA/pF (3 μM) following inflammation. Similar to LEV, KATP currents activated by sodium hydrogen sulfide (NaHS) (10–1000 μM) were significantly greater in inflamed compared to controls. In control cells, pretreatment with 100 μM NaHS shifted the EC50 for LEV‐induced currents from 6489 nM (n=10) to 180.9 nM (n=8). These data suggest that NaHS can act as an allosteric modulator for LEV‐induced KATP currents. Decreased colonic motility may result from enhanced KATP activation by increased release of H2S in colitis. Supported by NIH DK046367.

Cyp‐epoxygenases mediate adenosine A2A receptor induced vascular relaxation via KATP channels

A2A adenosine receptor (AR) causes aortic relaxation through generation of epoxyeicosatrienoic acids (EETs) formed via Cyp‐epoxygenases (Nayeem et al, AJP 2008). We hypothesize that A2AAR‐mediated aortic relaxation occurs via EETs through opening of KATP channels in smooth muscle cells. Organ bath experiments were done with isolated aortas from A2AKO and wild type (WT) mice. Pinacidil (KATP opener; 10−6M) caused higher relaxation in WT aorta compared to A2AKO (48.09±5.23% vs. 25.41±2.37%; p<0.05). Cyp‐epoxygenase inhibitor MSPPOH (10−5M) caused significant decrease (p<0.05) in pinacidil response in WT (48.09±5.23% vs. 24.93±5.34%) and A2AKO (25.41±2.37% vs. 14.66±3.88%) aorta. Adenosine analog NECA (10−5M) caused significant relaxation in WT aorta (19.65±3.25%) compared to contraction (13.94±6.57%) in A2AKO, that was lowered by KATP blocker glibenclamide in WT (0.17±6.43%) while higher contraction was observed in KO (28.47±5.1%). Relaxation to A2A agonist CGS 21680 (10−6M) in WT (19.02±2.75%) changed to contraction (23.66±6.31%; p<0.05) with glibenclamide. Mitochondrial KATP opener diazoxide had no effect on precontracted tissues, while 5‐HD (mitochondrial KATP blocker, 100uM) had no effect on CGS 21680 response in WT and A2AKO aorta. These data suggest adenosine mediated aortic relaxation occurs via generation of EETs through sarcolemmal KATP channels. (HL027339, HL09444, HL07180)

Modulation of H2S Metabolism by Statins: A New Aspect of Cardiovascular Pharmacology

Statins (3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors) are commonly used in the treatment of cardiovascular diseases. Statins reduce plasma low-density lipoproteins, inhibit inflammatory reaction, improve endothelial function, ameliorate oxidative stress, and reduce platelet activity. Consequently, statins markedly decrease the risk of acute cardiovascular events. H(2)S is synthesized in all layers of the vascular wall, including the endothelium, smooth muscle cells, and perivascular adipose tissue (PVAT). Recent studies demonstrate that PVAT-derived H(2)S decreases vascular tone by activating K(ATP) and/or KCNQ potassium channels in smooth muscle cells. Lipophilic atorvastatin, but not hydrophilic pravastatin, increases net H(2)S production in PVAT by inhibiting its mitochondrial oxidation, and augments the anticontractile effect of PVAT. Inhibition of H(2)S metabolism results from atorvastatin-induced decrease in coenzyme Q, which is a cofactor of H(2)S oxidation by sulfide:quinone oxidoreductase. In contrast to H(2)S, statins do not impair mitochondrial oxidation of organic substrates. Taking into account antiatherosclerotic and anti-inflammatory effect of H(2)S, the gas may mediate some of the beneficial effects of statins on the cardiovascular system. In addition, specific statins differ in their ability to enhance H(2)S signaling. Since both statins and H(2)S reduce ischemia-reperfusion injury, the possible effect of statins on H(2)S oxidation in other tissues such as the heart and the kidney needs to be examined. Inhibition of H(2)S metabolism may be a new therapeutic strategy to improve H(2)S signaling, especially in the mitochondrial compartment.

Diphosphate Regulation of Adenosine Triphosphate Sensitive Potassium Channel in Human Bladder Smooth Muscle Cells

To clarify the properties of adenosine triphosphate sensitive K+ channel in human detrusor smooth muscle we examined the effect of the representative nicotinic acid derivatives β-nicotinamide adenine dinucleotide, cyclic adenosine diphosphate ribose and nicotinic acid adenine dinucleotide phosphate (Sigma-Aldrich®) on human detrusor adenosine triphosphate sensitive K+ channels. Patch clamp procedures were done in human detrusor cells. Reverse transcriptase and real-time polymerase chain reaction were performed to clarify the subunit components of adenosine triphosphate sensitive K+ channels. The K+ channel opener levcromakalim induced a long lasting outward current that was inhibited by glibenclamide (Sigma-Aldrich) under the whole cell configuration. The single channel study revealed that the unitary conductance of the adenosine triphosphate sensitive K+ channel in the human detrusor was 11 pS and nucleotide diphosphates increased its open probability. Applying β-nicotinamide adenine dinucleotide also activated the adenosine triphosphate sensitive K+ channel but applying cyclic adenosine diphosphate ribose or nicotinic acid adenine dinucleotide phosphate had little effect on channel activation. Molecular studies indicated that Kir6.1 and SUR2B were the predominant components of the adenosine triphosphate sensitive K+ channel in the human detrusor. To our knowledge we report the first single channel study of the adenosine triphosphate sensitive K+ channel in the human detrusor. The properties of this channel, ie unitary conductance, adenosine triphosphate sensitivity and diphosphate activation, were consistent with those of other smooth muscle organs. β-Nicotinamide adenine dinucleotide has the potency to activate adenosine triphosphate sensitive K+ channels in the human detrusor. This channel likely has some role during ischemic conditions as well as physiological muscle motion leading to the activation of cell metabolism.

Hydrogen sulfide dilates cerebral arterioles by activating smooth muscle cell plasma membrane K ATP channels

Hydrogen sulfide (H2S) is a gaseous signaling molecule that appears to contribute to the regulation of vascular tone and blood pressure. Multiple potential mechanisms of vascular regulation by H2S exist. Here, we tested the hypothesis that H2S dilates piglet cerebral arterioles by activating ATP-sensitive K+ (KATP) channels in smooth muscle cells. Patch-clamp electrophysiology indicated that Na2S or NaHS, two different H2S donors, activate K+ currents in isolated cerebral arteriole smooth muscle cells. H2S-induced K+ current activation was partially reversed by glibenclamide, a selective KATP channel inhibitor. H2S donors dilated isolated pressurized (40 mmHg) piglet cerebral arterioles, and this vasodilation was partially reversed by glibenclamide. Vasoregulation by H2S was also studied in resistance-size cerebral arteries of mice with a targeted deletion in the gene encoding sulfonylurea receptor 2 (SUR2 null mice), a KATP channel subunit. H2S-induced vasodilation was smaller in pressurized cerebral arteries of SUR2 null mice than in wild-type controls. These data indicate that H2S dilates cerebral arterioles of newborn piglets and cerebral arteries of mice by activating arterial smooth muscle cell KATP channels and that SUR2 subunits are necessary for this functional effect.

Postsynaptic density 95 (PSD95) scaffolding of Shaker ‐type potassium channels in smooth muscle cells regulates the diameter of rat cerebral arteries

Postsynaptic density 95 (PSD95) is a 95 kDa scaffolding protein in the brain that clusters postsynaptic proteins including ion channels, receptors and other signaling partners. Voltage-gated, Shaker-type, K+ (KV1) channels are key binding partners of PSD95 scaffolds in postsynaptic neurons. KV1 channels are also found in cerebral vascular smooth muscle cells (cVSMCs) and mediate hyperpolarization and vasodilation of cerebral arteries. This study was designed to determine if PSD95 scaffolds KV1 channels in cVSMCs to promote vasodilation of cerebral arteries. PSD95 mRNA and protein was detected in cVSMCs by real-time PCR and Western blotting. Furthermore, PSD95 co-immunoprecipitated with the pore-forming α1.2 subunit of the KV1 channel. Antisense-mediated knockdown of PSD95 in rat cerebral arteries profoundly reduced KV1 channel expression and patch-clamped cVSMCs exhibited a loss of KV1 current. Loss of PSD95 also depolarized cVSMCs in pressurized cerebral arteries and induced a strong constriction associated with a loss of functional KV1 channels. Our findings provide initial evidence that PSD95 is expressed in cVSMCs, and KV1 channels are one of its important binding partners. PSD95 appears to function as a critical “dilator” scaffold in rat cerebral arteries by increasing the number of functional KV1 channels at the plasma membrane. Supported by NIH R01 HL097107 (SWR).

T‐type calcium channels and vascular function: the new kid on the block?

While L-type voltage-dependent calcium channels have long been considered the predominant source of calcium for myogenic constriction, recent studies of both cerebral and systemic circulations have provided evidence for the prominent expression of other members of the voltage-dependent calcium channel family, in particular the low voltage activated T-type channels. Although physiological studies have not supported the involvement of a classical low voltage activated, T-type channel in vascular function, evidence is accumulating that points to the involvement of a non-L-type, high voltage activated channel with sensitivity to T-type channel antagonists. We propose that this may arise due to expression of a T-type channel splice variant with unique biophysical characteristics resulting in a more depolarised profile. Expression of these channels in smooth muscle cells would broaden the voltage range over which sustained calcium influx occurs, while expression of T-type channels in endothelial cells could provide a feedback mechanism to prevent excessive vasoconstriction. Perturbation of this balance during pathophysiological conditions by upregulation of channel expression and endothelial dysfunction could contribute to vasospastic conditions and therapy-refractory hypertension.

H 2 O 2 Is the Transferrable Factor Mediating Flow-Induced Dilation in Human Coronary Arterioles

Endothelial derived hydrogen peroxide (H(2)O(2)) is a necessary component of the pathway regulating flow-mediated dilation (FMD) in human coronary arterioles (HCAs). However, H(2)O(2) has never been shown to be the endothelium-dependent transferrable hyperpolarization factor (EDHF) in response to shear stress. We examined the hypothesis that H(2)O(2) serves as the EDHF in HCAs to shear stress. Two HCAs were cannulated in series (a donor intact vessel upstream and endothelium-denuded detector vessel downstream). Diameter changes to flow were examined in the absence and presence of polyethylene glycol catalase (PEG-CAT). The open state probability of large conductance Ca(2+)-activated K(+) (BK(Ca)) channels in smooth muscle cells downstream from the perfusate from an endothelium-intact arteriole was examined by patch clamping. In some experiments, a cyanogen bromide-activated resin column bound with CAT was used to remove H(2)O(2) from the donor vessel. When flow proceeds from donor to detector, both vessels dilate (donor:68±7%; detector: 45±11%). With flow in the opposite direction, only the donor vessel dilates. PEG-CAT contacting only the detector vessel blocked FMD in that vessel (6±4%) but not in donor vessel (61±13%). Paxilline inhibited dilation of endothelium-denuded HCAs to H(2)O(2). Effluent from donor vessels elicited K(+) channel opening in an iberiotoxin- or PEG-CAT-sensitive fashion in cell-attached patches but had little effect on channel opening on inside-out patches. Vasodilation of detector vessels was diminished when exposed to effluent from CAT-column. Flow induced endothelial production of H(2)O(2), which acts as the transferrable EDHF activating BK(Ca) channels on the smooth muscle cells.

Hydrogen sulfide: synthesis and function in the adipose tissue

Apart from nitric oxide (NO) and carbon monoxide (CO), hydrogen sulfide (H 2 S) is the third gaseous mediator in mammals. H 2 S is synthesized from L-cysteine by cystathionine β-synthase (CBS), cystathionine γ-lyase (CSE), or by sequential action of alanine aminotransferase and 3-mercaptopyruvate sulfurtransferase. In the cardiovascular system, H 2 S is involved in the regulation of vascular tone and blood pressure, inhibits atherogenesis, and protects myocardium from ischemia-reperfusion injury. Recent studies indicate that H 2 S is synthesized also in the adipose tissue. Hydrogen sulfide produced in periadventitial adipose tissue (tunica adiposa) of the blood vessels induces vasodilation by activating K + channels in smooth muscle cells. On the other hand, H 2 S inhibits basal and insulin-stimulated glucose uptake in visceral adipose tissue, and may be involved in the pathogenesis of insulin resistance. H 2 S production in periadventitial adipose tissue is stimulated by vasoconstrictors and aortic banding-induced hypertension and downregulated by aging. H 2 S signaling in adipose tissue may be affected by pharmacotherapy. Lipid-soluble statins (3-hydroxy-3-methylglutarylcoenzyme A reductase inhibitors) increase H 2 S level in periadventitial adipose tissue and thus augment its anticontractile effect on the blood vessels. This effect of statins results from the depletion of ubiquinone - a component of mitochondrial respiratory chain - and the impairment of mitochondrial H 2 S oxidation. Adipobiology 2010; 2: 41-50.

Endothelial SKCa and IKCa Channels Regulate Brain Parenchymal Arteriolar Diameter and Cortical Cerebral Blood Flow

Calcium-sensitive potassium (K(Ca)) channels have been shown to modulate the diameter of cerebral pial arteries; however, little is known regarding their roles in controlling cerebral parenchymal arterioles (PAs). We explored the function and cellular distribution of small-conductance (SK(Ca)) and intermediate-conductance (IK(Ca)) K(Ca) channels and large-conductance K(Ca) (BK(Ca)) channels in endothelial cells (ECs) and smooth muscle cells (SMCs) of PAs. Both SK(Ca) and IK(Ca) channels conducted the outward current in isolated PA ECs (current densities, ~20 pA/pF and ~28 pA/pF at +40 mV, respectively), but these currents were not detected in PA SMCs. In contrast, BK(Ca) currents were prominent in PA SMCs (~154 pA/pF), but were undetectable in PA ECs. Pressurized PAs constricted to inhibition of SK(Ca) (~16%) and IK(Ca) (~16%) channels, but were only modestly affected by inhibition of BK(Ca) channels (~5%). Blockade of SK(Ca) and IK(Ca) channels decreased resting cortical cerebral blood flow (CBF) by ~15%. NS309 (6,7-dichloro-1H-indole-2,3-dione3-oxime), a SK(Ca)/IK(Ca) channel opener, hyperpolarized PA SMCs by ~27 mV, maximally dilated pressurized PAs, and increased CBF by ~40%. In conclusion, these data show that SK(Ca) and IK(Ca) channels in ECs profoundly modulate PA tone and CBF, whereas BK(Ca) channels in SMCs only modestly influence PA diameter.

Adaptation to Short-Term Stress Exposures Prevents Poststress Dysfunction of Calcium-Activated Potassium Channels in Coronary Vessels

Adaptation to short-term stress exposures prevents immobilization stress-induced decrease in functional activity of BK(Ca)channels in smooth muscle cells of coronary vessels developing against the background of NO overproduction and increase in the relative content of oxidized glutathione (change in redox state of the glutathione system). The protective antistress mechanisms of adaptation probably include prevention of NO overproduction by inducible NO synthase and maintenance of dependent glutathione redox state functional activity of BK(Ca)channels in smooth muscle cells of coronary vessels.

Canonical TRP channels and mechanotransduction: from physiology to disease states

Mechano-gated ion channels play a key physiological role in cardiac, arterial, and skeletal myocytes. For instance, opening of the non-selective stretch-activated cation channels in smooth muscle cells is involved in the pressure-dependent myogenic constriction of resistance arteries. These channels are also implicated in major pathologies, including cardiac hypertrophy or Duchenne muscular dystrophy. Seminal work in prokaryotes and invertebrates highlighted the role of transient receptor potential (TRP) channels in mechanosensory transduction. In mammals, recent findings have shown that the canonical TRPC1 and TRPC6 channels are key players in muscle mechanotransduction. In the present review, we will focus on the functional properties of TRPC1 and TRPC6 channels, on their mechano-gating, regulation by interacting cytoskeletal and scaffolding proteins, physiological role and implication in associated diseases.

Magnesium lithospermate B dilates mesenteric arteries by activating BKCa currents and contracts arteries by inhibiting KV currents

To examine the involvement of K(+) channels and endothelium in the vascular effects of magnesium lithospermate B (MLB), a hydrophilic active component of Salviae miltiorrhiza Radix. Isolated rat mesenteric artery rings were employed to investigate the effects of MLB on KCl- or norepinephrine-induced contractions. Conventional whole-cell patch-clamp technique was used to study the effects of MLB on K(+) currents in single isolated mesenteric artery myocytes. MLB produced a concentration-dependent relaxation in mesenteric artery rings precontracted by norepinephrine (1 micromol/L) with an EC(50) of 111.3 micromol/L. MLB-induced relaxation was reduced in denuded artery rings with an EC(50) of 224.4 micromol/L. MLB caused contractions in KCl-precontracted artery rings in the presence of N-nitro-L-arginine methyl ester (L-NAME) with a maximal value of 130.3%. The vasodilatory effect of MLB was inhibited by tetraethylammonium (TEA) in both intact and denuded artery rings. In single smooth muscle cells, MLB activated BK(Ca) currents (EC(50) 156.3 micromol/L) but inhibited K(V) currents (IC(50) 26.1 micromol/L) in a voltage- and concentration-dependent manner. MLB dilated arteries by activating BK(Ca) channels in smooth muscle cells and increasing NO release from endothelium, but it also contracted arteries precontracted with KCl in the presence of L-NAME.

Expression of KCa2.x and KCa3.1 channels in vascular smooth muscle cells from mouse portal vein

Calcium‐activated K+ channels are key regulators of smooth muscle membrane potential, and therefore are essential to smooth muscle function. Small and intermediate conductance Ca2+‐activated K+ channels (KCa2.x and KCa3.1) have been reported in vascular myocytes only during proliferation. Portal vein is an atypical blood vessel as it shows action potentials (AP). As KCa2.x channels have been shown to be involved in the control of AP in different cell types, we investigated the presence of KCa2.x and KCa3.1 channels in vascular smooth muscle cells from mouse portal vein (mPV). PCR experiments showed the presence of mRNA for all KCa2.x and KCa3.1 channels in smooth muscle cells from mPV. In Patch clamp experiments, freshly isolated myocytes were dialyzed with a pipette solution containing 1 μM free Ca2+ to stimulate KCa channels. To minimize the contribution of BKCa channels, paxilline (10 μM) was added in the pipette as well as in the superfusion solution. Exposure of myocytes to TRAM‐34 (10 μM), an inhibitor of KCa3.1 channels, induced a decrease of ≈ 65% in membrane currents. However, Apamin (300 nM), a KCa2.x channels blocker, had little if any effect on membrane currents. These data suggest that KCa2.x and KCa3.1 channels are expressed in mPV and may play an important role in the control of membrane potential. This study is financially supported by FRSQ and the Fondation de l'Institut de Cardiologie de Montréal.

Isoform-Selective Physical Coupling of TRPC3 Channels to IP3 Receptors in Smooth Muscle Cells Regulates Arterial Contractility

Many vasoconstrictors bind to phospholipase C (PLC)-coupled receptors on arterial smooth muscle cells, leading to an intracellular inositol 1,4,5-trisphophate (IP3) elevation and vasoconstriction. IP3-induced vasoconstriction can occur independently of intracellular Ca2+ release and via IP3 receptor (IP3R) and canonical transient receptor potential (TRPC) channel activation, but signaling mechanisms mediating this effect are unknown. Here, we studied the mechanisms by which IP3Rs stimulate TRPC channels in smooth muscle cells of resistance-size cerebral arteries. Immunofluorescence resonance energy transfer (immuno-FRET) microscopy in smooth muscle cells indicated that endogenous type 1 IP3Rs (IP3R1) are in close spatial proximity to TRPC3, but distant from TRPC6 or TRPM4 channels. Endothelin-1 (ET-1), a PLC-coupled receptor agonist, elevated the immuno-FRET signal between IP3R1 and TRPC3, but not between IP3R1 and TRPC6 or TRPM4. IP3R1 co-immunoprecipitated with TRPC3, but not with TRPC6. An antibody targeting TRPC3 channels and TRPC3 channel knockdown with short hairpin RNA inhibited IP3-induced nonselective cation current (ICat) activation, whereas an antibody to TRPC6 and TRPC6 channel knockdown had no effect. Biotinylation indicated that ET-1 did not alter total or plasma membrane-localized TRPC3. RT-PCR demonstrated that a calmodulin and IP3R binding (CIRB) domain is present on the C-terminus of both TRPC3 and TRPC6 channels. A CIRB domain peptide attenuated IP3- and ET-1-induced ICat activation. A peptide corresponding to the IP3R region that can interact with TRPC channels activated ICat. A HIV-1 TAT-conjugated CIRB domain peptide reduced IP3- and ET-1-induced vasoconstriction in pressurized arteries. These data indicate that IP3 stimulates direct coupling between IP3R1 and membrane-resident TRPC3 channels in arterial smooth muscle cells, leading to ICat activation and vasoconstriction. Data also indicate that close spatial proximity between IP3R1 and TRPC3 establishes this isoform-selective functional interaction.

Patho-, physiological roles of voltage-dependent K+ channels in pulmonary arterial smooth muscle cells

In this review, we demonstrate the basic properties, modulation of, and pathological changes in voltage-dependent K+ (Kv) channels that are expressed in pulmonary arterial smooth muscle cells (PASMCs). Pulmonary Kv channels are thought to play a crucial role in the maintenance of resting membrane potentials, and therefore the vascular tone of the pulmonary arteries. Although the molecular identity of pulmonary Kv channels is not clear, Kv1.1, Kv1.2, Kv1.5, Kv2.1, Kv9.3, and Kv3.1 subtypes are expressed in PASMCs. In addition, resistant PASMCs contain greater amount of Kv channels as compared to conduit PASMCs. This heterogenetic expression of Kv channels is consistent with regional differences in the contractile response to hypoxia. Similar to other K+ channels, pulmonary Kv channels can also be modulated by several vasoconstrictors concomitant with the activation of protein kinase C (PKC). Alterations in Kv channel function have several additional and interrelated consequences, including the regulation of cell proliferation and apoptosis, which ultimately lead to pulmonary vascular remodeling. Increased pulmonary vasoconstriction in pulmonary arterial hypertension is attributable to decreased expression and activity of Kv channels in smooth muscle cells. Kv channels play a central role in the maintenance of cellular homeostasis and ion channels, and consequential signaling cascades. Therefore, Kv channels are potential therapeutic targets for the treatment of pulmonary vascular disease.

TRPC channels in smooth muscle cells

Transient receptor potential canonical (TRPC) proteins constitute a family of seven (TRPC1-7) nonselective cation channels within the wider TRP superfamily. TRPC1, TRPC3, TRPC4, TRPC5 and TRPC6 channels are expressed in vascular smooth muscle cells from human vessels of all calibers and in smooth muscle from organs such as the uterus and the gastrointestinal tract. TRPC channels have recently emerged as important players in the control of smooth muscle function. This review will focus on the retrospective analysis of studies proposing contributions of TRPC channels to native calcium entry pathways in smooth muscle and to physiological and pathophysiological responses with emphasis on the vascular system.

The Guanylyl Cyclase Activator YC-1 Directly Inhibits the Voltage-Dependent K+ Channels in Rabbit Coronary Arterial Smooth Muscle Cells

We investigated the effects of YC-1, an activator of soluble guanylyl cyclase (sGC), on voltage-dependent K+ (Kv) channels in smooth muscle cells from freshly isolated rabbit coronary arteries by using the whole-cell patch clamp technique. YC-1 inhibited the Kv current in a dose-dependent fashion with an apparent Kd of 9.67 μ M. It accelerated the decay rate of Kv channel inactivation without altering the kinetics of current activation. The rate constants of association and dissociation for YC-1 were 0.36 ± 0.01 μ M −1·s−1 and 3.44 ± 0.22 s−1, respectively. YC-1 did not have a significant effect on the steady-state activation and inactivation curves. The recovery time constant from inactivation was decreased in the presence of YC-1, and application of train pulses (1 or 2 Hz) caused a progressive increase in the YC-1 blockade, indicating that YC-1–induced inhibition of Kv currents is use-dependent. Pretreatment with Bay 41-2272 (also a sGC activator), ODQ (a sGC inhibitor), or Rp-8-Br-PET-cGMPs (a protein kinase G inhibitor) did not affect the basal Kv current and also did not significantly alter the inhibitory effect of YC-1. From these results, we suggest that YC-1 directly inhibits the Kv current independently of sGC activation and in a state-, time-, and use-dependent fashion.

Propagation of Fast and Slow Intercellular Calcium Waves in Primary Cultured Smooth Muscle Cells

Tissue blood flow is controlled by the changes in the diameter of the arteries and arterioles due to the coordinated contraction and relaxation of smooth muscle cells (SMCs) within the vascular wall. The contractile state of SMCs is regulated primarily by the intracellular Ca2+ concentration ([Ca2+]i). The increase in [Ca2+]i in response to hormonal stimuli propagates from cell to cell along the vessel wall as a wave, and activates the process of contraction. The mechanism underlying this phenomenon, however, is not yet fully revealed.In this work, we study the onset and propagation of intercellular calcium waves through gap junctions in primary cultured vascular SMCs. For imaging intercellular Ca2+ waves, SMCs seeded along a collagen line and loaded with the fluorescent Ca2+ indicator Fluo-4 were locally stimulated mechanically or chemically. The stimulation evoked two distinct calcium waves: 1) a fast Ca2+ wave (several mm/s), and 2) a much slower Ca2+ wave (few tens of μm/s); both waves propagated to neighboring cells. The fast Ca2+ wave was caused by the propagation of membrane depolarization and subsequent Ca2+ influx through voltage operated channels. This fast wave facilitated the onset and propagation of a slow, but higher amplitude Ca2+ wave that started from the stimulated cell and propagated to neighboring cells. Our results suggest a possible mechanism for intercellular Ca2+ wave propagation through gap junction channels in SMCs.

Orai1 Channel And Ca2+-independent Phospholipase A2 Are The New Determinants Of Proliferation And Migration Of Vascular Smooth Muscle Cells

Ca2+ entry is known to play an important role in proliferation and migration of vascular smooth muscle cells (SMC), but the molecular mechanisms that mediate these processes are far from being understood. Here we introduce Ca2+-independent phospholipase A2β (iPLA2β) and Orai1-encoded plasma membrane channel (that mediate store-operated Ca2+ entry (SOCE) in non-excitable as well as excitable cells) as new molecular determinants of proliferation and migration of aortic SMC. Patch-clamp, Ca2+ imaging, immunocytochemistry and molecular approaches were used in this study. We demonstrated that molecular knock down of either iPLA2β or Orai1 (but not TRPC1) channel resulted in full inhibition of store-operated current and Ca2+ entry in primary aortic SMC. Transfection of SMC with siRNA to Orai1 impaired their proliferation: Orai1 knock down resulted in 69±4% decrease in the number of BrDU positive cells and 60±1% reduction in the rate of their proliferation. Similar effects were observed in iPLA2β-deficient cells (65±3% and 71±4% reductions, respectively). Interestingly, transfection of SMC with antisense to TRPC1 did not affect SOCE, but significantly reduced the rate of cell proliferation (by 48±4%). Orai1 and TRPC1 produced additive effects on proliferation, and knock down of both channels resulted in 76±8% reduction in the rate of SMC proliferation, consistent with different mechanisms for Orai1and TRPC1-mediated Ca2+ entry. Molecular knock down of iPLA2β, or Orai1 channel also impaired SMC ability to migrate in response to 20% FBS, high glucose, or SERCA inhibition. These results suggest that SOCE mediated by iPLA2β-dependent activation of Orai1 channel in SMC is required for their proliferation and migration, and may be involved in vascular SMC responses and angiogenesis triggered by different physiological and pathological conditions.