Chronic hypoxia decreases K(V) channel expression and function in pulmonary artery myocytes

Activity of voltage-gated K+ (KV) channels regulates membrane potential (E(m)) and cytosolic free Ca2+ concentration ([Ca2+](cyt)). A rise in ([Ca2+](cyt))in pulmonary artery (PA) smooth muscle cells (SMCs) triggers pulmonary vasoconstriction and stimulates PASMC proliferation. Chronic hypoxia (PO(2) 30-35 mmHg for 60-72 h) decreased mRNA expression of KV channel alpha-subunits (Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3) in PASMCs but not in mesenteric artery (MA) SMCs. Consistently, chronic hypoxia attenuated protein expression of Kv1.1, Kv1.5, and Kv2.1; reduced KV current [I(KV)]; caused E(m) depolarization; and increased ([Ca2+](cyt)) in PASMCs but negligibly affected KV channel expression, increased I(KV), and induced hyperpolarization in MASMCs. These results demonstrate that chronic hypoxia selectively downregulates KV channel expression, reduces I(KV), and induces E(m) depolarization in PASMCs. The subsequent rise in ([Ca2+](cyt)) plays a critical role in the development of pulmonary vasoconstriction and medial hypertrophy. The divergent effects of hypoxia on KV channel alpha-subunit mRNA expression in PASMCs and MASMCs may result from different mechanisms involved in the regulation of KV channel gene expression.     

Alterations in a redox oxygen sensing mechanism in chronic hypoxia.

The mechanism of acute hypoxic pulmonary vasoconstriction (HPV) may involve the inhibition of several voltage-gated K+ channels in pulmonary artery smooth muscle cells. Changes in PO2 can either be sensed directly by the channel(s) or be transmitted to the channel via a redox-based effector mechanism. In control lungs, hypoxia and rotenone acutely decrease production of activated oxygen species, inhibit K+ channels, and cause constriction. Two-day and 3-wk chronic hypoxia (CH) resulted in a decrease in basal activated oxygen species levels, an increase in reduced glutathione, and loss of HPV and rotenone-induced constriction. In contrast, 4-aminopyridine- and KCl-mediated constrictions were preserved. After 3-wk CH, pulmonary arterial smooth muscle cell membrane potential was depolarized, K+ channel density was reduced, and acute hypoxic inhibition of whole cell K+ current was lost. In addition, Kv1.5 and Kv2.1 channel protein was decreased. These data suggest that chronic reduction of the cytosol occurs before changes in K+ channel expression. HPV may be attenuated in CH because of an impaired redox sensor.     

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.     

Kvbeta1.2 subunit coexpression in HEK293 cells confers O2 sensitivity to kv4.2 but not to Shaker channels.

Voltage-gated K+ (KV) channels are protein complexes composed of ion-conducting integral membrane alpha subunits and cytoplasmic modulatory beta subunits. The differential expression and association of alpha and beta subunits seems to contribute significantly to the complexity and heterogeneity of KV channels in excitable cells, and their functional expression in heterologous systems provides a tool to study their regulation at a molecular level. Here, we have studied the effects of Kvbeta1.2 coexpression on the properties of Shaker and Kv4.2 KV channel alpha subunits, which encode rapidly inactivating A-type K+ currents, in transfected HEK293 cells. We found that Kvbeta1.2 functionally associates with these two alpha subunits, as well as with the endogenous KV channels of HEK293 cells, to modulate different properties of the heteromultimers. Kvbeta1.2 accelerates the rate of inactivation of the Shaker currents, as previously described, increases significantly the amplitude of the endogenous currents, and confers sensitivity to redox modulation and hypoxia to Kv4.2 channels. Upon association with Kvbeta1.2, Kv4.2 can be modified by DTT (1,4 dithiothreitol) and DTDP (2,2'-dithiodipyridine), which also modulate the low pO2 response of the Kv4.2+beta channels. However, the physiological reducing agent GSH (reduced glutathione) did not mimic the effects of DTT. Finally, hypoxic inhibition of Kv4.2+beta currents can be reverted by 70% in the presence of carbon monoxide and remains in cell-free patches, suggesting the presence of a hemoproteic O2 sensor in HEK293 cells and a membrane-delimited mechanism at the origin of hypoxic responses. We conclude that beta subunits can modulate different properties upon association with different KV channel subfamilies; of potential relevance to understanding the molecular basis of low pO2 sensitivity in native tissues is the here described acquisition of the ability of Kv4. 2+beta channels to respond to hypoxia.     

15-HETE对缺氧兔肺动脉平滑肌钾离子通道的影响

用肺动脉环和全细胞膜片钳技术研究15-羟化二十烷四烯酸(15-HETE)对缺氧兔肺动脉平滑肌钾离子通道的影响。新出生的幼兔分两组,一组放入吸氧分数为0．12的低氧舱内;另一组保持正常氧环境。9d后,称重、取肺动脉进行细胞培养并制作肺动脉环。分别加入4-氨基吡啶(4-aminopyridione,4-AP)、四乙胺(tetraethylammonium,TEA)、glyburide(GLYB)三种特异性钾离子通道阻断剂,观察15-HETE对兔肺动脉平滑肌钾离子通道的作用变化,同时采用全细胞膜片钳测定钾电流。结果显示：5mmol／L 4-AP阻断Kv通道后可以抑制15-HETE诱导的缺氧兔肺动脉收缩;TEA和GLYB分别阻断大电导型钙激活钾通道(BKCa)和KATP通道后并不影响15-HETE诱导的缺氧兔肺动脉收缩;15-HETE可降低兔肺动脉平滑肌细胞钾电流幅度。上述结果提示：缺氧兔肺动脉中,15-HETE阻断电压依赖钾通道(Kv通道),引起膜去极化,可能是缺氧性肺血管收缩的机制之一。     

O2 sensing in the human ductus arteriosus: redox-sensitive K+ channels are regulated by mitochondria-derived hydrogen peroxide

The ductus arteriosus (DA) is a fetal artery that allows blood ejected from the right ventricle to bypass the pulmonary circulation in utero. At birth, functional closure of the DA is initiated by an O2-induced, vasoconstrictor mechanism which, though modulated by endothelial-derived endothelin and prostaglandins, is intrinsic to the smooth muscle cell (DASMC) [Michelakis et al., Circ. Res. 91 (2002); pp. 478-486]. As pO2 increases, a mitochondrial O2-sensor (electron transport chain complexes I or III) is activated, which generates a diffusible redox mediator (H2O2). H2O2 inhibits voltage-gated K+ channels (Kv) in DASMC. The resulting membrane depolarization activates L-type Ca2+ channels, thereby promoting vasoconstriction. Conversely, inhibiting mitochondrial ETC complexes I or III mimics hypoxia, depolarizing mitochondria, and decreasing H2O2 levels. The resulting increase in K+ current hyperpolarizes the DASMC and relaxes the DA. We have developed two models for study of the DA's O2-sensor pathway, both characterized by decreased O2-constriction and Kv expression: (i) preterm rabbit DA, (ii) ionically-remodeled, human term DA. The O2-sensitive channels Kv1.5 and Kv2.1 are important to DA O2-constriction and overexpression of either channel enhances DA constriction in these models. Understanding this O2-sensing pathway offers therapeutic targets to modulate the tone and patency of human DA in vivo, thereby addressing a common form of congenital heart disease in preterm infants.     

Hypoxia regulates expression and activity of Kv1.3 channels in T lymphocytes: a possible role in T cell proliferation

T lymphocytes are exposed to hypoxia during their development and also when they migrate to hypoxic pathological sites such as tumors and wounds. Although hypoxia can affect T cell development and function, the mechanisms by which immune cells sense and respond to changes in O(2)-availability are poorly understood. K(+) channels encoded by the Kv1.3 subtype of the voltage-dependent Kv1 gene family are highly expressed in lymphocytes and are involved in the control of membrane potential and cell function. In this study, we investigate the sensitivity of Kv1.3 channels to hypoxia in freshly isolated human T lymphocytes and leukemic Jurkat T cells. Acute exposure to hypoxia (20 mmHg, 2 min) inhibits Kv1.3 currents in both cell types by 20%. Prolonged exposure to hypoxia (1% O(2) for 24 h) selectively decreases Kv1.3 protein levels in Jurkat T cells by 47%, but not Kvbeta2 and SK2 Ca-activated K(+) channel subunit levels. The decrease in Kv1.3 protein levels occurs with no change in Kv1.3 mRNA expression and is associated with a significant decrease in K(+) current density. A decrease in Kv1.3 polypeptide levels similar to that obtained during hypoxia is produced by Kv1.3 channel blockage. Our results indicate that hypoxia produces acute and long-term inhibition of Kv1.3 channels in T lymphocytes. This effect could account for the inhibition of lymphocyte proliferation during hypoxia. Indeed, we herein present evidence showing that hypoxia selectively inhibits TCR-mediated proliferation and that this inhibition is associated with a decrease in Kv1.3 proteins.     

Pathophysiology of voltage-gated K channels in vascular smooth muscle cells: Modulation by protein kinases

In this review, the pathological alteration and clinical relevance of voltage-gated K⁺ (Kv) channels and their specific regulation by protein kinase-dependent signaling in vascular smooth muscle cells are described, particularly focusing on the pulmonary vasculature. The physiological relevance, channel characteristics, pharmacological modulation, and expression of Kv channels vary between different arterial beds and between subdivisions of arteries within those vascular beds. Although detailed signaling cascades regulating Kv channels are not clearly elucidated, it is known that the Kv channels in vascular smooth muscle cells can be tightly regulated by protein kinases C (PKC) and A (PKA). Alterations in Kv channel expression and function has been noted in pathological and pathophysiological conditions including hypertension (pulmonary and systemic), in diabetes and in individuals subjected to prolonged hypoxia (high altitude living). Vascular Kv channels are potential therapeutic targets in diseases such as pulmonary arterial hypertension and, therefore, it is important to understand the specific pharmacological modulation of Kv channel isoforms in different vascular beds.     

Bcl-2 decreases voltage-gated K+ channel activity and enhances survival in vascular smooth muscle cells

Cell shrinkage is an incipient hallmark of apoptosis in a variety of cell types. The apoptotic volume decrease has been demonstrated to attribute, in part, to K+ efflux; blockade of plasmalemmal K+ channels inhibits the apoptotic volume decrease and attenuates apoptosis. Using combined approaches of gene transfection, single-cell PCR, patch clamp, and fluorescence microscopy, we examined whether overexpression of Bcl-2, an anti-apoptotic oncoprotein, inhibits apoptosis in pulmonary artery smooth muscle cells (PASMC) by diminishing the activity of voltage-gated K+ (Kv) channels. A human bcl-2 gene was infected into primary cultured rat PASMC using an adenoviral vector. Overexpression of Bcl-2 significantly decreased the amplitude and current density of Kv currents (I(Kv)). In contrast, the apoptosis inducer staurosporine (ST) enhanced I(Kv). In bcl-2-infected cells, however, the ST-induced increase in I(Kv) was completely abolished, and the ST-induced apoptosis was significantly inhibited compared with cells infected with an empty adenovirus (-bcl-2). Blockade of Kv channels in control cells (-bcl-2) by 4-aminopyridine also inhibited the ST-induced increase in I(Kv) and apoptosis. Furthermore, overexpression of Bcl-2 accelerated the inactivation of I(Kv) and downregulated the mRNA expression of the pore-forming Kv channel alpha-subunits (Kv1.1, Kv1.5, and Kv2.1). These results suggest that inhibition of Kv channel activity may serve as an additional mechanism involved in the Bcl-2-mediated anti-apoptotic effect on vascular smooth muscle cells.     

Effect of normoxia and hypoxia on K(+) current and resting membrane potential of fetal rabbit pulmonary artery smooth muscle

At birth, the increase in O(2) tension (pO(2)) is an important cause of the decrease in pulmonary vascular resistance. In adult animals there are impressive interspecies differences in the level of hypoxia required to elicit a pulmonary vasoconstrictor response and in the amplitude of the response. Hypoxic inhibition of some potassium (K(+)) channels in the membrane of pulmonary arterial smooth muscle cells (PASMCs) helps to initiate hypoxic pulmonary vasoconstriction. To determine the effect of the change in pO(2) on fetal rabbit PASMCs and to investigate possible species-dependent differences, we measured the current-voltage relationship and the resting membrane potential, in PASMCs from fetal resistance arteries using the amphotericin-perforated patch-clamp technique under hypoxic and normoxic conditions. Under hypoxic conditions, the K(+) current in PASMCs was small, and could be inhibited by 4-aminopyridine, iberiotoxin and glibenclamide, reflecting contributions by Kv, K(Ca) and K(ATP) channels. The average resting membrane potential was -44.3+/-1.3 mV (n=29) and could be depolarized by 4-AP (5 mM) and ITX (100 nM) but not by glibenclamide (10 microM). Changing from hypoxia, that mimicked fetal life, to normoxia dramatically increased the K(Ca) and consequently hyperpolarized (-9.3+/-1.7 mV; n=8) fetal rabbit PASMCs. Under normoxic conditions K(+) current was reduced by 4-AP with a significant change in resting membrane potential (11.1+/-1.7 mV; n=8). We conclude that resting membrane potential in fetal rabbit PASMCs under both hypoxic and normoxic conditions depends on both Kv and K(Ca) channels, in contrast to fetal lamb or porcine PASMCs. Potential species differences in the K(+) channels that control resting membrane potential must be taken into consideration in the interpretation of studies of neonatal pulmonary vascular reactivity to changes in O(2) tension.     

Acute hypoxia selectively inhibits KCNA5 channels in pulmonary artery smooth muscle cells

Acute hypoxia causes pulmonary vasoconstriction in part by inhibiting voltage-gated K⁺ (Kv) channel activity in pulmonary artery smooth muscle cells (PASMC). The hypoxia-mediated decrease in Kv currents [I_K(V)] is selective to PASMC; hypoxia has little effect on I_K(V) in mesenteric artery smooth muscle cells (MASMC). Functional Kv channels are homo- and/or heterotetramers of pore-forming -subunits and regulatory -subunits. KCNA5 is a Kv channel -subunit that forms functional Kv channels in PASMC and regulates resting membrane potential. We have shown that acute hypoxia selectively inhibits I_K(V) through KCNA5 channels in PASMC. Overexpression of the human KCNA5 gene increased I_K(V) and caused membrane hyperpolarization in HEK-293, COS-7, and rat MASMC and PASMC. Acute hypoxia did not affect I_K(V) in KCNA5-transfected HEK-293 and COS-7 cells. However, overexpression of KCNA5 in PASMC conferred its sensitivity to hypoxia. Reduction of PO₂ from 145 to 35 mmHg reduced I_K(V) by 40% in rat PASMC transfected with human KCNA5 but had no effect on I_K(V) in KCNA5-transfected rat MASMC (or HEK and COS cells). These results indicate that KCNA5 is an important Kv channel that regulates resting membrane potential and that acute hypoxia selectively reduces KCNA5 channel activity in PASMC relative to MASMC and other cell types. Because Kv channels (including KCNA5) are ubiquitously expressed in PASMC and MASMC, the observation from this study indicates that a hypoxia-sensitive mechanism essential for inhibiting KCNA5 channel activity is exclusively present in PASMC. The divergent effect of hypoxia on I_K(V) in PASMC and MASMC also may be due to different expression levels of KCNA5 channels. membrane potential; potassium channels; vascular smooth muscle     

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 (I(Kv)) revealed the presence of a mitochondria-mediated Mg(2+) 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.     

Expression and function of potassium channels in the human placental vasculature

In the placental vasculature, where oxygenation may be an important regulator of vascular reactivity, there is a paucity of data on the expression of potassium (K) channels, which are important mediators of vascular smooth muscle tone. We therefore addressed the expression and function of several K channel subtypes in human placentas. The expression of voltage-gated (Kv)2.1, KV9.3, large-conductance Ca2+-activated K channel (BKCa), inward-rectified K+ channel (KIR)6.1, and two-pore domain inwardly rectifying potassium channel-related acid-sensitive K channels (TASK)1 in chorionic plate arteries, veins, and placental homogenate was assessed by RT-PCR and Western blot analysis. Functional activity of K channels was assessed pharmacologically in small chorionic plate arteries and veins by wire myography using 4-aminopyridine, iberiotoxin, pinacidil, and anandamide. Experiments were performed at 20, 7, and 2% oxygen to assess the effect of oxygenation on the efficacy of K channel modulators. KV2.1, KV9.3, BKCa, KIR6.1, and TASK1 channels were all demonstrated to be expressed at the message level. KV2.1, BKCa, KIR6.1, and TASK1 were all demonstrated at the protein level. Pharmacological manipulation of voltage-gated and ATP-sensitive channels produced the most marked modifications in vascular tone, in both arteries and veins. We conclude that K channels play an important role in controlling placental vascular function.     

Graded response of K+ current,membrane potential,and [Ca2+]i to hypoxia in pulmonary arterial smooth muscle

Many studies indicate that hypoxic inhibition of some K+ channels in the membrane of the pulmonary arterial smooth muscle cells (PASMCs) plays a part in initiating hypoxic pulmonary vasoconstriction. The sensitivity of the K+ current (I(k)), resting membrane potential (E(m)), and intracellular Ca2+ concentration ([Ca2+]i) of PASMCs to different levels of hypoxia in these cells has not been explored fully. Reducing PO2 levels gradually inhibited steady-state I(k) of rat resistance PASMCs and depolarized the cell membrane. The block of I(k) by hypoxia was voltage dependent in that low O2 tensions (3 and 0% O2) inhibited I(k) more at 0 and -20 mV than at 50 mV. As expected, the hypoxia-sensitive I(k) was also 4-aminopyridine sensitive. Fura 2-loaded PASMCs showed a graded increase in [Ca2+]i as PO2 levels declined. This increase was reduced markedly by nifedipine and removal of extracellular Ca2+. We conclude that, as in the carotid body type I cells, PC-12 pheochromocytoma cells, and cortical neurons, increasing severity of hypoxia causes a proportional decrease in I(k) and E(m) and an increase of [Ca2+]i.     

Hypoxia sensitivity of a voltage-gated potassium current in porcine intrapulmonary vein smooth muscle cells

Hypoxia contracts the pulmonary vein, but the underlying cellular effectors remain unclear. Utilizing contractile studies and whole cell patch-clamp electrophysiology, we report for the first time a hypoxia-sensitive K(+) current in porcine pulmonary vein smooth muscle cells (PVSMC). Hypoxia induced a transient contractile response that was 56 ± 7% of the control response (80 mM KCl). This contraction required extracellular Ca(2+) and was sensitive to Ca(2+) channel blockade. Blockade of K(+) channels by tetraethylammonium chloride (TEA) or 4-aminopyridine (4-AP) reversibly inhibited the hypoxia-mediated contraction. Single-isolated PVSMC (typically 159.1 ± 2.3 μm long) had mean resting membrane potentials (RMP) of -36 ± 4 mV with a mean membrane capacitance of 108 ± 3.5 pF. Whole cell patch-clamp recordings identified a rapidly activating, partially inactivating K(+) current (I(KH)) that was hypoxia, TEA, and 4-AP sensitive. I(KH) was insensitive to Penitrem A or glyburide in PVSMC and had a time to peak of 14.4 ± 3.3 ms and recovered in 67 ms following inactivation at +80 mV. Peak window current was -32 mV, suggesting that I(KH) may contribute to PVSMC RMP. The molecular identity of the potassium channel is not clear. However, RT-PCR, using porcine pulmonary artery and vein samples, identified Kv(1.5), Kv(2.1), and BK, with all three being more abundant in the PV. Both artery and vein expressed STREX, a highly conserved and hypoxia-sensitive BK channel variant. Taken together, our data support the hypothesis that hypoxic inhibition of I(KH) would contribute to hypoxic-induced contraction in PVSMC.