Kv Channel Current Research Articles

Stretch‐activation of angiotensin II type 1a receptors contributes to the myogenic response of mouse mesenteric and renal arteries (1067.8)

Wall stretch is a major stimulus for the myogenic response of small arteries to pressure. Recent findings suggest that G protein‐coupled receptors can elicit a stretch response. Our aim was to determine if angiotensin II type 1 receptors (AT1R) in vascular smooth muscle cells (VSMC) exert mechanosensitivity and identify the downstream ion channel mediators of myogenic vasoconstriction. We used mice deficient in AT1R signaling molecules and putative ion channel targets, namely AT1R, angiotensinogen, TRPC6 channels or subtypes of the KCNQ (Kv7) gene family (KCNQ3, 4 or 5). We identified a mechano‐sensing mechanism in mesenteric arteries and the renal circulation that relies on coupling of the AT1R subtype a (AT1aR) to a Gq/11‐protein as a critical event to accomplish the myogenic response. The mechano‐activation occurs after block of AT1R, and in the absence of angiotensinogen or TRPC6. Activation of AT1aR suppresses XE991‐sensitive Kv channel currents in VSMCs, blocking these channels enhances mesenteric and renal myogenic tone. Although KCNQ3, 4 and 5 are expressed in VSMCs, XE991‐sensitive K+ current and myogenic contractions persist in arteries deficient in these channels. Our results provide evidence that myogenic responses of mouse mesenteric and renal arteries rely on ligand‐independent mechano‐activation of AT1aR. This signal relies on an ion channel distinct from TRPC6 or KCNQ3, 4 or 5.Grant Funding Source: Supported by Deutsche Forschungsgemeinschaft (DFG)

Inhibition of insulin secretion from rat pancreatic islets by dexmedetomidine and medetomidine, two sedatives frequently used in clinical settings

The aim of this study was to determine whether dexmedetomidine (DEX) and medetomidine (MED), α2-adrenergic agonists clinically used as sedatives, influence insulin secretion from rat pancreatic islets. Islets were isolated from adult male Wistar rats after collagenase digestion. Static incubation was used to determine effects of DEX or MED on insulin secretion and ionic-channel currents of β-cells. Results indicate that both drugs dose-dependently inhibit insulin secretion, DEX more potently than MED. The inhibitory effects were attenuated by addition of yohimbine or by pretreatment of rats with pertussis toxin (PTX). 10 nM DEX decreased the current amplitude of voltage-dependent Ca2+ channels, but this did not occur when the N-type Ca2+ channel blocker ω-conotoxin was added. In the presence of tetraethylammonium, a classical voltage-gated K+ channel (Kv channel) blocker, the magnitude of inhibition of insulin secretion by MED was reduced. However, when tolbutamide, a specific blocker of the ATP-sensitive K+ channel (KATP channel), was present, the magnitude of MED inhibition of insulin secretion was not influenced, suggesting that Kv-channel activity alteration, but not that of KATP channels, is involved in MED-associated insulin secretory inhibition. The Kv-channel currents were increased during 1 nM MED exposure at membrane potentials ranging from -30 mV to -10 mV, where action potentials were generated in response to glucose stimulation. These results indicate that DEX and MED inhibit insulin secretion through an α2-adrenoceptor and PTX-sensitive GTP-binding protein pathway that eventually involves Kv channel activation and Ca2+ channel inhibition.

Hypoxia induces Kv channel current inhibition by increased NADPH oxidase-derived reactive oxygen species

There is current discussion whether reactive oxygen species are up- or downregulated in the pulmonary circulation during hypoxia, from which sources (i.e., mitochondria or NADPH oxidases) they are derived, and what the downstream targets of ROS are. We recently showed that the NADPH oxidase homolog NOX4 is upregulated in hypoxia-induced pulmonary hypertension in mice and contributes to the vascular remodeling in pulmonary hypertension. We here tested the hypothesis that NOX4 regulates Kv channels via an increased ROS formation after prolonged hypoxia. We showed that (1) NOX4 is upregulated in hypoxia-induced pulmonary hypertension in rats and isolated rat pulmonary arterial smooth muscle cells (PASMC) after 3days of hypoxia, and (2) that NOX4 is a major contributor to increased reactive oxygen species (ROS) after hypoxia. Our data indicate colocalization of Kv1.5 and NOX4 in isolated PASMC. The NADPH oxidase inhibitor and ROS scavenger apocynin as well as NOX4 siRNA reversed the hypoxia-induced decrease in Kv current density whereas the protein levels of the channels remain unaffected by siNOX4 treatment. Determination of cysteine oxidation revealed increased NOX4-mediated Kv1.5 channel oxidation. We conclude that sustained hypoxia decreases Kv channel currents by a direct effect of a NOX4-derived increase in ROS.

Ghrelin Attenuates cAMP-PKA Signaling to Evoke Insulinostatic Cascade in Islet β-Cells

OBJECTIVEGhrelin reportedly restricts insulin release in islet β-cells via the Gαi2 subtype of G-proteins and thereby regulates glucose homeostasis. This study explored whether ghrelin regulates cAMP signaling and whether this regulation induces insulinostatic cascade in islet β-cells.RESEARCH DESIGN AND METHODSInsulin release was measured in rat perfused pancreas and isolated islets and cAMP production in isolated islets. Cytosolic cAMP concentrations ([cAMP]i) were monitored in mouse MIN6 cells using evanescent-wave fluorescence imaging. In rat single β-cells, cytosolic protein kinase-A activity ([PKA]i) and Ca2+ concentration ([Ca2+]i) were measured by DR-II and fura-2 microfluorometry, respectively, and whole cell currents by patch-clamp technique.RESULTSGhrelin suppressed glucose (8.3 mmol/L)-induced insulin release in rat perfused pancreas and isolated islets, and these effects of ghrelin were blunted in the presence of cAMP analogs or adenylate cyclase inhibitor. Glucose-induced cAMP production in isolated islets was attenuated by ghrelin and enhanced by ghrelin receptor antagonist and anti-ghrelin antiserum, which counteract endogenous islet-derived ghrelin. Ghrelin inhibited the glucose-induced [cAMP]i elevation and [PKA]i activation in MIN6 and rat β-cells, respectively. Furthermore, ghrelin potentiated voltage-dependent K+ (Kv) channel currents without altering Ca2+ channel currents and attenuated glucose-induced [Ca2+]i increases in rat β-cells in a PKA-dependent manner.CONCLUSIONSGhrelin directly interacts with islet β-cells to attenuate glucose-induced cAMP production and PKA activation, which lead to activation of Kv channels and suppression of glucose-induced [Ca2+]i increase and insulin release.

Impaired Voltage Gated Potassium Channel Responses in a Fetal Lamb Model of Persistent Pulmonary Hypertension of the Newborn

We investigated the hypothesis that oxidative stress in persistent pulmonary hypertension of the newborn (PPHN) impairs voltage gated potassium (Kv) channel function. We induced PPHN in fetal lambs by prenatal ligation of ductus arteriosus; controls had sham ligation. We studied changes in the tone of pulmonary artery (PA) rings and Kv channel current of freshly isolated PA smooth muscle cells (PASMC) using standard techniques. 4-Aminopyridine (4-AP), a Kv channel antagonist, induced dose-dependent constriction of control PA rings; this response was attenuated in PPHN pulmonary arteries. Exogenous superoxide and peroxynitrite inhibited the response to 4-AP in control rings. Tiron, a superoxide scavenger, improved the response to 4-AP in PPHN rings. 4-AP inhibited the NOS-independent relaxation response to ATP in control PA rings. Relaxation response to ATP was blunted in PPHN rings and was improved by NOS antagonist, N-nitro-L-arginine methyl ester (L-NAME). 4-AP attenuated this response in L-NAME-treated PPHN rings. Exogenous superoxide suppressed 4-AP sensitive Kv current in control PASMC. Kv channel current was attenuated in cells from PPHN lambs and was restored by tiron. Oxidative stress impairs Kv channel function in PPHN. Superoxide scavengers may improve pulmonary vasodilation in PPHN in part by restoring Kv channel function.

Regulation of voltage-gated K + channels by glucose metabolism in pancreatic β-cells

Regulation of delayed rectifier-type K + channels (Kv-channels) by glucose was studied in rat pancreatic β-cells. The Kv-channel current was increased in amplitudes by increasing glucose concentration from 2.8 to 16.6 mM, while it was decreased by 2.8 mM glucose in a reversible manner (down-regulation) in both perforated and conventional whole-cell modes. The current was decreased by FCCP, intrapipette 0 mM ATP or AMPPNP. Glyceraldehyde, pyruvic acid, 2-ketoisocaproic acid, and 10 mM MgATP prevented the down-regulation induced by 2.8 mM or less glucose. The residual current after treatment with Kv2.1-specific blocker, guangxitoxin-1E, was unchanged by lowering or increasing glucose concentration. We conclude that glucose metabolism regulates Kv2.1 channels in rats β-cells via altering MgATP levels.

Sphingomyelinase dependent apoptosis following treatment of pancreatic beta-cells with amyloid peptides Aß1-42 or IAPP

Amyloid peptides interfere with survival of pancreatic beta-cells. In some cells apoptosis is paralleled by ceramide-dependent alterations of ion channel activity. The purpose of the present study was to elucidate the dependence of amyloid peptides Abeta(1-42) and islet amyloid polypeptide (IAPP)-induced cell death on ceramide formation and ion channel activity in murine pancreatic islet cells. As disclosed by TUNEL (terminal dUTP nick-end labelling) and cleaved caspase 3 staining, apoptotic cell death was induced by Abeta(1-42), IAPP and exogenously added C2-ceramide in islet cells from wild type mice. In islet cells from acid sphingomyelinase-deficient mice (ASMKO) Abeta(1-42) and IAPP but not exogenously added N-acetyl-D-sphingosine (C2-ceramide, 20 microM) failed to stimulate apoptosis. Immunofluorescent staining revealed a stimulatory effect of Abeta(1-42) on ceramide formation. According to patch clamp experiments, administration of Abeta(1-42) and IAPP significantly decreased outwardly rectifying whole cell currents in wild type but not in ASMKO islet cells. C2-ceramide but not inactive di-ceramide (20 microM) mimicked the inhibitory effect on Kv channel current. In conclusion, amyloid peptides induce apoptosis of pancreatic islet cells at least in part through activation of acid sphingomyelinase resulting in production of ceramide and subsequent inhibition of ion channel activity.

Mitochondria-dependent regulation of Kv currents in rat pulmonary artery smooth muscle cells

Voltage-gated K+ (Kv) channels are important in the regulation of pulmonary vascular function having both physiological and pathophysiological implications. The pulmonary vasculature is essential for reoxygenation of the blood, supplying oxygen for cellular respiration. Mitochondria have been proposed as the major oxygen-sensing organelles in the pulmonary vasculature. Using electrophysiological techniques and immunofluorescence, an interaction of the mitochondria with Kv channels was investigated. Inhibitors, blocking the mitochondrial electron transport chain at different complexes, were shown to have a dual effect on Kv currents in freshly isolated rat pulmonary arterial smooth muscle cells (PASMCs). These dual effects comprised an enhancement of Kv current in a negative potential range (manifested as a 5- to 14-mV shift in the Kv activation to more negative membrane voltages) with a decrease in current amplitude at positive potentials. Such effects were most prominent as a result of inhibition of Complex III by antimycin A. Investigation of the mechanism of antimycin A-mediated effects on Kv channel currents (IKv) revealed the presence of a mitochondria-mediated Mg2+ and ATP-dependent regulation of Kv channels in PASMCs, which exists in addition to that currently proposed to be caused by changes in intracellular reactive oxygen species.

Oxyhemoglobin-Induced Suppression of Voltage-Dependent K + Channels in Cerebral Arteries by Enhanced Tyrosine Kinase Activity

Cerebral vasospasm following aneurysmal subarachnoid hemorrhage (SAH) has devastating consequences. Oxyhemoglobin (oxyhb) has been implicated in SAH-induced cerebral vasospasm as it causes cerebral artery constriction and increases tyrosine kinase activity. Voltage-dependent, Ca(2+)-selective and K(+)-selective ion channels play an important role in the regulation of cerebral artery diameter and represent potential targets of oxyhb. Here we provide novel evidence that oxyhb selectively decreases 4-aminopyridine sensitive, voltage-dependent K(+) channel (K(v)) currents by approximately 30% in myocytes isolated from rabbit cerebral arteries but did not directly alter the activity of voltage-dependent Ca(2+) channels or large conductance Ca(2+)-activated (BK) channels. A combination of tyrosine kinase inhibitors (tyrphostin AG1478, tyrphostin A23, tyrphostin A25, genistein) abolished both oxyhb-induced suppression of K(v) channel currents and oxyhb-induced constriction of isolated cerebral arteries. The K(v) channel blocker 4-aminopyridine also inhibited oxyhb-induced cerebral artery constriction. The observed oxyhb-induced decrease in K(v) channel activity could represent either channel block, or a decrease in K(v) channel density on the plasma membrane. To explore whether oxyhb altered trafficking of K(v) channels to the plasma membrane, we used an antibody generated against an extracellular epitope of K(v)1.5 channels. In the presence of oxyhb, staining of K(v)1.5 on the plasma membrane surface was markedly reduced. Furthermore, oxyhb caused a loss of spatial distinction between staining with K(v)1.5 and the general anti-phosphotyrosine antibody PY-102. We propose that oxyhb-induced suppression of K(v) currents occurs via a mechanism involving enhanced tyrosine kinase activity and channel endocytosis. This novel mechanism may contribute to oxyhb-induced cerebral artery constriction following SAH.

Oxyhemoglobin‐induced suppression of KV channels by enhanced tyrosine kinase activity and channel endocytosis

Oxyhemoglobin (oxyhb) released following SAH has been implicated in cerebral vasospasm as it causes cerebral artery constriction and increases tyrosine kinase activity. Voltage-dependent, Ca2+-selective and K+-selective ion channels play an important role in the regulation of cerebral artery diameter and represent potential targets of oxyhb. Here, we demonstrate that acute exposure (10 min) of oxyhb selectively decreases 4-AP-sensitive, voltage-dependent K+ channel (KV) currents by ≈30 % in myocytes isolated from rabbit cerebral arteries, but did not directly alter the activity of voltage-dependent Ca2+ channels (VDCCs) or large conductance Ca2+-activated (BK) channels. A combination of tyrosine kinase inhibitors abolished the ability of oxyhb to decrease KV channel currents. To explore whether oxyhb altered trafficking of KV channels to the plasma membrane, we used an antibody generated against surface KV 1.5 channels. In the presence of oxyhb, staining of surface KV 1.5 was markedly reduced, while staining with the early endosomal marker, EEA-1, was enhanced. Further, oxyhb caused a loss of spatial distinction between staining with KV 1.5 and the general anti-phosphotyrosine antibody, PY-102. We propose that oxyhb-induced suppression of KV currents occurs via a mechanism involving tyrosine phosphorylation and channel endocytosis. This novel mechanism may contribute to oxyhb-induced cerebral artery constriction following SAH. Supported by the Totman Medical Research Trust Fund and the NIH (P20 RR16435 & R01 HL078983).

Gene therapy targeting survivin selectively induces pulmonary vascular apoptosis and reverses pulmonary arterial hypertension

Pulmonary arterial hypertension (PAH) is characterized by genetic and acquired abnormalities that suppress apoptosis and enhance cell proliferation in the vascular wall, including downregulation of the bone morphogenetic protein axis and voltage-gated K+ (Kv) channels. Survivin is an "inhibitor of apoptosis" protein, previously thought to be expressed primarily in cancer cells. We found that survivin was expressed in the pulmonary arteries (PAs) of 6 patients with PAH and rats with monocrotaline-induced PAH, but not in the PAs of 3 patients and rats without PAH. Gene therapy with inhalation of an adenovirus carrying a phosphorylation-deficient survivin mutant with dominant-negative properties reversed established monocrotaline-induced PAH and prolonged survival by 25%. The survivin mutant lowered pulmonary vascular resistance, RV hypertrophy, and PA medial hypertrophy. Both in vitro and in vivo, inhibition of survivin induced PA smooth muscle cell apoptosis, decreased proliferation, depolarized mitochondria, caused efflux of cytochrome c in the cytoplasm and translocation of apoptosis-inducing factor into the nucleus, and increased Kv channel current; the opposite effects were observed with gene transfer of WT survivin, both in vivo and in vitro. Inhibition of the inappropriate expression of survivin that accompanies human and experimental PAH is a novel therapeutic strategy that acts by inducing vascular mitochondria-dependent apoptosis.

Overexpression of human KCNA5 increases IK V and enhances apoptosis.

Apoptotic cell shrinkage, an early hallmark of apoptosis, is regulated by K+ efflux and K+ channel activity. Inhibited apoptosis and downregulated K+ channels in pulmonary artery smooth muscle cells (PASMC) have been implicated in development of pulmonary vascular medial hypertrophy and pulmonary hypertension. The objective of this study was to test the hypothesis that overexpression of KCNA5, which encodes a delayed-rectifier voltage-gated K+ (Kv) channel, increases K+ currents and enhances apoptosis. Transient transfection of KCNA5 caused 25- to 34-fold increase in KCNA5 channel protein level and 24- to 29-fold increase in Kv channel current (I(K(V))) at +60 mV in COS-7 and rat PASMC, respectively. In KCNA5-transfected COS-7 cells, staurosporine (ST)-mediated increases in caspase-3 activity and the percentage of cells undergoing apoptosis were both enhanced, whereas basal apoptosis (without ST stimulation) was unchanged compared with cells transfected with an empty vector. In rat PASMC, however, transfection of KCNA5 alone caused marked increase in basal apoptosis, in addition to enhancing ST-mediated apoptosis. Furthermore, ST-induced apoptotic cell shrinkage was significantly accelerated in COS-7 cells and rat PASMC transfected with KCNA5, and blockade of KCNA5 channels with 4-aminopyridine (4-AP) reduced K+ currents through KCNA5 channels and inhibited ST-induced apoptosis in KCNA5-transfected COS-7 cells. Overexpression of the human KCNA5 gene increases K+ currents (i.e., K+ efflux or loss), accelerates apoptotic volume decrease (AVD), increases caspase-3 activity, and induces apoptosis. Induction of apoptosis in PASMC by KCNA5 gene transfer may serve as an important strategy for preventing the progression of pulmonary vascular wall thickening and for treating patients with idiopathic pulmonary arterial hypertension (IPAH).

Role of Voltage-Gated K+(KV) Channels in Vascular Function

Voltage-dependent K+ (KV) channels represent the most diverse group of K+ channels ubiquitously expressed in vascular smooth muscles. The KV channels, together with other types of K+ conductances, such as Ca2+-activated (BKCa), ATP-sensitive (KATP), and inward rectifier, play an important role in the control of the cell membrane potential and regulation of the vascular contractility. Comparison of the expression of different KV channel isoforms obtained from RT-PCR studies showed that virtually all KV genes could be detected in vascular smooth muscle cells (VSMC). Based on the analysis of both mRNA and protein expressions, it is likely that KV1.1, KV1.2, KV1.3, KV1.5, KV1.6, KV2.1, and KV3.1b channel isoforms are mainly responsible for the delayed rectifier current characterized electrophysiologically in most VSMC types studied to date. It has been recently demonstrated by our research group and by others that functional expression of multiple KV channel α-subunits is not homogeneous and varies in different vascular beds of small and large arteries. Growing evidence suggests that in some small arteries, e.g., cerebral arteries and arterioles, the KV channels are activated at more negative membrane voltages than BKCa, thus making a greater contribution to the control of vascular tone. Our data also suggest that in some blood vessels, such as the rat aorta and mouse small mesenteric arteries, the KV channel current (identified mainly as passed through KV2.1 channels), but not BKCa, is the predominant conductance activated even under conditions where intracellular Ca2+ concentration is increased up to 200 nM. In addition, our data indicate that the KV2.1 channel current could also contribute to the regulation of the induced rhythmic activity in the rat aorta in vitro acting as a negative feedback mechanism for membrane depolarization. We and other experimenters also demonstrated that functional expression of KV channels is a dynamic process, which is altered under normal physiological conditions (e.g., during the development of the vessels), and in various pathological states (e.g., pulmonary hypertension developing during chronic hypoxia). Recent findings also suggest that activation of KV channels can also play a role in vascular apoptosis (causing loss of intracellular K+ and subsequent cell shrinking, one of the essential prerequisites of cellular apoptosis). To summarize, the KV channels are essential for normal vascular function, and their expression and properties are altered under abnormal conditions. Therefore, understanding of the molecular identity of native KV channels and their functional significance and elucidation of the mechanisms, which govern and control the expression of the KV channels in the vasculature, represent an important and challenging task and could also lead to the development of useful therapeutic strategies for the treatment of cardiovascular diseases.

Electrophysiologically distinct smooth muscle cell subtypes in rat conduit and resistance pulmonary arteries.

Pulmonary arteries (PAs), particularly those of the rat, demonstrate a prominent voltage-gated K+ (Kv) current (I(Kv)), which plays an important role in the regulation of the resting potential. No detailed characterization of electrophysiological and pharmacological properties of I(Kv), particularly in resistance PA myocytes (PAMs), has been performed. The aim of the present study was therefore to compare I(Kv) in rat conduit and resistance PAMs using the standard patch clamp technique. We found that 67% of conduit PAMs demonstrated a large, rapidly activating I(Kv) which was potently blocked by 4-aminopyridine (4-AP; IC50, 232 microM), but was almost insensitive to TEA (18% block at 20 mM). Thirty-three percent of cells exhibited a smaller, more slowly activating I(Kv) which was TEA sensitive (IC50, 2.6 mM) but relatively insensitive to 4-AP (37% block at 20 mM). These currents (termed I(Kv1) and I(Kv2), respectively) inactivated over different ranges of potential (V(0.5) = -20.2 vs. -39.1 mV, respectively). All resistance PAMs demonstrated a large, rapidly activating and TEA-insensitive K+ current resembling I(Kv1) (termed I(KvR)), but differing significantly from it with respect to 4-AP sensitivity (IC50, 352 microM), activation rate, and inactivation potential range (V(0.5), -27.4 mV). Thus, cells from conduit PAMs fall into two populations with respect to functional I(Kv) expression, while resistance arteries uniformly demonstrate a third type of I(Kv). Comparison of the properties of the native I(Kv) with those of cloned Kv channel currents suggest that I(Kv1) and I(KvR) are likely to be mediated by Kv1.5-containing homo/heteromultimers, while I(Kv2) involves a Kv2.1 alpha-subunit.

High glucose impairs voltage-gated K(+) channel current in rat small coronary arteries.

Hyperglycemia is associated with impaired endothelium-dependent dilation that is due to quenching of NO by superoxide (O(2)(. -)). In small coronary arteries (CAs), dilation depends more on smooth muscle hyperpolarization, such as that mediated by voltage-gated K(+) (Kv) channels. We determined whether high glucose enhances O(2)(.-) production and reduces microvascular Kv channel current and functional responses. CAs from Sprague-Dawley rats were incubated 24 hours in medium containing either normal glucose (NG, 5.5 mmol/L D-glucose), high glucose (HG, 23 mmol/L D-glucose), or L-glucose (LG, 5.5 mmol/L D-glucose and 17 mmol/L L-glucose). O(2)(.-) production was increased in HG arteries. Whole-cell patch clamping showed a reduction of 4-aminopyridine (4-AP)-sensitive current (Kv current) from smooth muscle cells of HG CAs versus NG CAs or versus LG CAs (peak density was 9.95+/-5.3 pA/pF for HG versus 27.8+/-6.8 pA/pF for NG and 28.5+/-5.2 pA/pF for LG; P<0.05). O(2)(.-) generation (xanthine+xanthine oxidase) decreased K(+) current density, with no further reduction by 4-AP. Partial restoration was observed with superoxide dismutase and catalase. Constriction to 3 mmol/L 4-AP was reduced in vessels exposed to HG (13+/-5%, P<0.05) versus NG (30+/-7%) or LG (34+/-4%). Responses to KCl and nifedipine were not different among groups. Superoxide dismutase and catalase increased contraction to 4-AP in HG CAs. This is the first direct evidence that exposure of CAs to HG impairs Kv channel activity. We speculate that this O(2)(.-)-induced impairment may reduce vasodilator responsiveness in the coronary circulation of subjects with coronary disease or its risk factors.

Differential expression of KV and KCa channels in vascular smooth muscle cells during 1-day culture.

Voltage-dependent delayed rectifier K+ (KV) channels and Ca(2+)-activated K+ (KCa) channels both play important roles in the regulation of the membrane potential and contractility of vascular smooth muscle cells (SMCs). The expression and function of these K+ channels in cultured vascular SMCs have been extensively studied. The long-term in vitro cell culture would change the properties of K+ channels in SMCs. However, whether the short-term culture could differentially affect the expression and function of KV and KCa channels was not clear. In the present study, both KV and KCa channel currents were identified in freshly dissociated SMCs and in 1-day-cultured SMCs from rat tail arteries. KCa currents were inhibited by iberiotoxin or tetraethylammonium (TEA), and amplified by the calcium ionophore A-23187. Kv currents were inhibited by 4-aminopyridine or beta-dendrotoxin. By using different pharmacological agents and manipulating the calcium concentrations in the recording solutions, it was revealed that in freshly dissociated SMCs the predominant component of total outward K+ currents is KCa current, and KV current a minor component. In contrast, KV current was found to be the predominant component of total outward K+ currents in SMCs primarily cultured with 10% fetal bovine serum at 37 degrees C for 24 h. Differential expression of KV and KCa channels in 1-day-cultured SMCs was thus demonstrated under our experimental conditions. Our results are important for interpreting the electrophysiological properties of vascular SMCs under different cell culture conditions and for understanding the relative contributions of KV and KCa channels to different cellular functions.

Hypoxic pulmonary vasoconstriction: role of voltage-gated potassium channels.

Activity of voltage-gated potassium (Kv) channels controls membrane potential, which subsequently regulates cytoplasmic free calcium concentration ([Ca2+]cyt) in pulmonary artery smooth muscle cells (PASMCs). Acute hypoxia inhibits Kv channel function in PASMCs, inducing membrane depolarization and a rise in [Ca2+ ]cyt that triggers vasoconstriction. Prolonged hypoxia inhibits expression of Kv channels and reduces Kv channel currents in PASMCs. The consequent membrane depolarization raises [Ca2+]cyt, thus stimulating PASMC proliferation. The present review discusses recent evidence for the involvement of Kv channels in initiation of hypoxic pulmonary vasoconstriction and in chronic hypoxia-induced pulmonary hypertension.

Inhibition of cytochrome P-450 reduces voltage-gated K+ currents in pulmonary arterial myocytes

Cytochrome P-450 (P-450) is a NADPH-requiring and O2-dependent monooxygenase system. It is present in lung and has been postulated to act as an O2 sensor in hypoxic pulmonary vasoconstriction. To determine whether P-450 is involved in the regulation of voltage-gated K+ (KV) channel activity in pulmonary artery (PA) myocytes, we used the whole cell patch-clamp technique to evaluate the effects of P-450 inhibitors on KV channel currents (IKV) and membrane potential (Em). Bath application of the P-450 inhibitors clotrimazole, miconazole, and 1-aminobenzotriazole (1-ABT) significantly and reversibly inhibited steady-state IKV (IKss) and depolarized PA cells bathed in either Ca(2+)-containing (1.8 mM) or Ca(2+)-free [0.5-1 mM ethylene glycol-bis(beta-aminoethyl ether)-N,N,N'N'-tetraacetic acid present] bath solution. Clotrimazole (1 microM), miconazole (10 microM), and 1-ABT (1 mM) reversibly reduced IKss, elicited by a test potential of +80 mV, by 40, 70, and 31%, respectively. Pretreatment of PA smooth muscle cells with 10 mM 4-aminopyridine (4-AP) prevented the subsequent inhibitory effect of clotrimazole on IKV. However, pretreatment of the cells with 1 mM tetraethylammonium negligibly altered the effects of miconazole on IKV and Em. In current-clamp (I = 0) measurements, clotrimazole depolarized PA myocytes by 9 and 11 mV during perfusion with Ca(2+)-containing and Ca(2+)-free bath solution, respectively. 1-ABT also caused a 9-mV depolarization in PA myocytes bathed in Ca(2+)-free solution. These effects are similar to those induced by hypoxia, reduced glutathione, and 4-AP. Clotrimazole also decreased IKV and depolarized mesenteric arterial myocytes. These data raise the possibility that the P-450 system, due to its influence on IKV and sensitivity to O2 tension and NADPH, may play a role in linking the regulation of pulmonary vascular tone to the alteration of cellular redox status through a common pathway of KV channel activity.