Hypoxia-induced reactive oxygen species downregulate ETB receptor-mediated contraction of rat pulmonary arteries

Production of reactive oxygen species (ROS) may be increased during hypoxia in pulmonary arteries. In this study, the role of ROS in the effect of hypoxia on endothelin (ET) type B (ETB) receptor-mediated vasocontraction in lungs was determined. In rat intrapulmonary (approximately 0.63 mm ID) arteries, contraction induced by IRL-1620 (a selective ETB receptor agonist) was significantly attenuated after 4 h of hypoxia (30 mmHg Po2) compared with normoxic control (140 mmHg Po2). The effect was abolished by tiron, a scavenger of superoxide anions, but not by polyethylene glycol (PEG)-conjugated catalase, which scavenges H2O2. The hypoxic effect on ETB receptor-mediated vasoconstriction was also abolished by endothelium denudation but not by nitro-L-arginine and indomethacin. Exposure for 4 h to exogenous superoxide anions, but not H2O2, attenuated the vasoconstriction induced by IRL-1620. Confocal study showed that hypoxia increased ROS production in pulmonary arteries that were scavenged by PEG-conjugated SOD. In endothelium-intact pulmonary arteries, the ETB receptor protein was reduced after 4 h of exposure to hypoxia, exogenous superoxide anions, or ET-1. BQ-788, a selective ETB receptor antagonist, prevented these effects. ET-1 production was stimulated in endothelium-intact arteries after 4 h of exposure to hypoxia or exogenous superoxide anions. This effect was blunted by PEG-conjugated SOD. These results demonstrate that exposure to hypoxia attenuates ETB receptor-mediated contraction of rat pulmonary arteries. A hypoxia-induced production of superoxide anions may increase ET-1 release from the endothelium and result in downregulation of ETB receptors on smooth muscle.     

Oxygen-dependent signaling in pulmonary vascular smooth muscle

The pulmonary circulation constricts in response to acute hypoxia, which is reversible on reexposure to oxygen. On exposure to chronic hypoxia, in addition to vasoconstriction, the pulmonary vasculature undergoes remodeling, resulting in a sustained increase in pulmonary vascular resistance that is not immediately reversible. Hypoxic pulmonary vasoconstriction is physiological in the fetus, and there are many mechanisms by which the pulmonary vasculature relaxes at birth, principal among which is the acute increase in oxygen. Oxygen-induced signaling mechanisms, which result in pulmonary vascular relaxation at birth, and the mechanisms by which chronic hypoxia results in pulmonary vascular remodeling in the fetus and adult, are being investigated. Here, the roles of cGMP-dependent protein kinase in oxygen-mediated signaling in fetal pulmonary vascular smooth muscle and the effects of chronic hypoxia on ion channel activity and smooth muscle function such as contraction, growth, and gene expression were discussed.     

Increased hydrogen peroxide downregulates soluble guanylate cyclase in the lungs of lambs with persistent pulmonary hypertension of the newborn

Similar to infants born with persistent pulmonary hypertension of the newborn (PPHN), there is an increase in circulating endothelin-1 (ET-1) and decreased cGMP-mediated vasodilation in an ovine model of PPHN. These abnormalities lead to vasoconstriction and vascular remodeling. Our previous studies have demonstrated that reactive oxygen species (ROS) levels are increased in pulmonary arterial smooth muscle cells (PASMC) exposed to ET-1. Thus the initial objective of this study was to determine whether the development of pulmonary hypertension in utero is associated with elevated production of the ROS hydrogen peroxide (H(2)O(2)) and if this is associated with alterations in antioxidant capacity. Second we wished to determine whether chronic exposure of PASMC isolated from fetal lambs to H(2)O(2) would mimic the decrease in soluble guanylate cyclase expression observed in the ovine model of PPHN. Our results indicate that H(2)O(2) levels are significantly elevated in pulmonary arteries isolated from 136-day-old fetal PPHN lambs (P 0.05). In addition, we determined that catalase and glutathione peroxidase expression and activities remain unchanged. Also, we found that the overnight exposure of fetal PASMC to a H(2)O(2)-generating system resulted in significant decreases in soluble guanylate cyclase expression and nitric oxide (NO)-dependent cGMP generation (P 0.05). Finally, we demonstrated that the addition of the ROS scavenger catalase to isolated pulmonary arteries normalized the vasodilator responses to exogenous NO. As these scavengers had no effect on the vasodilator responses in pulmonary arteries isolated from age-matched control lambs this enhancement appears to be unique to PPHN. Overall our data suggest a role for H(2)O(2) in the abnormal vasodilation associated with the pulmonary arteries of PPHN lambs.     

ET－1，NO和缺氧对肺动脉平滑肌细胞钙内流及胶原合成的影响

肺血管平滑肌细胞是肺血管收缩反应的主要执行者,也是肺血管结构重建的重要参与者。本研究观察了内皮素１（ＥＴ１）,一氧化氮（ＮＯ）和缺氧对培养的新生小牛肺动肺平滑肌细胞（ＰＡＳＭＣ）钙内流以及胶原合成的影响。结果表明ＥＴ１和缺氧可促进ＰＡＳＭＣ的钙内流,ＮＯ供应剂硝普钠（ＳＭＰ）可抑制ＥＴＩ诱导的钙内流,其作用呈剂量依赖性,ＳＮＰ还可以剂量依赖地抑制ＰＡＳＭＣ的胶原合成,而缺氧可促进ＰＡＳＭＣ的胶原合成。     

Oxidant and redox signaling in vascular oxygen sensing mechanisms: basic concepts, current controversies, and potential importance of cytosolic NADPH

Vascular smooth muscle (VSM) derived from pulmonary arteries generally contract to hypoxia, whereas VSM from systemic arteries usually relax, indicating the presence of basic oxygen-sensing mechanisms in VSM that are adapted to the environment from which they are derived. This review considers how fundamental processes associated with the generation of reactive oxygen species (ROS) by oxidase enzymes, the metabolic control of cytosolic NADH, NADPH and glutathione redox systems, and mitochondrial function interact with signaling systems regulating vascular force in a manner that is potentially adapted to be involved in Po2 sensing. Evidence for opposing hypotheses of hypoxia, either decreasing or increasing mitochondrial ROS, is considered together with the Po2 dependence of ROS production by Nox oxidases as sensors potentially contributing to hypoxic pulmonary vasoconstriction. Processes through which ROS and NAD(P)H redox changes potentially control interactive signaling systems, including soluble guanylate cyclase, potassium channels, and intracellular calcium are discussed together with the data supporting their regulation by redox in responses to hypoxia. Evidence for hypothesized potential differences between systemic and pulmonary arteries originating from properties of mitochondrial ROS generation and the redox sensitivity of potassium channels is compared with a new hypothesis in which differences in the control of cytosolic NADPH redox by the pentose phosphate pathway results in increased NADPH and Nox oxidase-derived ROS in pulmonary arteries, whereas lower levels of glucose-6-phosphate dehydrogenase in coronary arteries may permit hypoxia to activate a vasodilator mechanism controlled by oxidation of cytosolic NADPH.     

Altered pulmonary vasoreactivity in the chronically hypoxic lung

Prolonged exposure to alveolar hypoxia induces physiological changes in the pulmonary vasculature that result in the development of pulmonary hypertension. A hallmark of hypoxic pulmonary hypertension is an increase in vasomotor tone. In vivo, pulmonary arterial smooth muscle cell contraction is influenced by vasoconstrictor and vasodilator factors secreted from the endothelium, lung parenchyma and in the circulation. During chronic hypoxia, production of vasoconstrictors such as endothelin-1 and angiotensin II is enhanced locally in the lung, while synthesis of vasodilators may be reduced. Altered reactivity to these vasoactive agonists is another physiological consequence of chronic exposure to hypoxia. Enhanced contraction in response to endothelin-1 and angiotensin II, as well as depressed vasodilation in response to endothelium-derived vasodilators, has been documented in models of hypoxic pulmonary hypertension. Chronic hypoxia may also have direct effects on pulmonary vascular smooth muscle cells, modulating receptor population, ion channel activity or signal transduction pathways. Following prolonged hypoxic exposure, pulmonary vascular smooth muscle exhibits alterations in K+ current, membrane depolarization, elevation in resting cytosolic calcium and changes in signal transduction pathways. These changes in the electrophysiological parameters of pulmonary vascular smooth muscle cells are likely associated with an increase in basal tone. Thus, hypoxia-induced modifications in pulmonary arterial myocyte function, changes in synthesis of vasoactive factors and altered vasoresponsiveness to these agents may shift the environment in the lung to one of contraction instead of relaxation, resulting in increased pulmonary vascular resistance and elevated pulmonary arterial pressure.     

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 K(v) 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 K(v)1.5 and NOX4 in isolated PASMC. The NADPH oxidase inhibitor and ROS scavenger apocynin as well as NOX4 siRNA reversed the hypoxia-induced decrease in K(v) current density whereas the protein levels of the channels remain unaffected by siNOX4 treatment. Determination of cysteine oxidation revealed increased NOX4-mediated K(v)1.5 channel oxidation. We conclude that sustained hypoxia decreases K(v) channel currents by a direct effect of a NOX4-derived increase in ROS.     

Effect of L-arginine on pulmonary artery smooth muscle cell apoptosis in rats with hypoxic pulmonary vascular structural remodeling

This study investigated the effect of L-arginine (L-Arg) on the apoptosis of pulmonary arterysmooth muscle cells (PASMC) in rats with hypoxic pulmonary vascular structural remodeling,and itsmechanisms.Seventeen Wistar rats were randomly divided into a control group (n=5),a hypoxia group(n=7),and a hypoxia L-Arg group (n=5).The morphologic changes of lung tissues were observed underoptical microscope.Using the terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling assay,the apoptosis of PASMC was examined.Fas expression in PASMC wasexamined using immunohistochemistry.The results showed that the percentage of muscularized artery insmall pulmonary vessels,and the relative medial thickness and relative medial area of the small and medianpulmonary muscularized arteries in the hypoxic group were all significantly increased.Pulmonary vascularstructural remodeling developed after hypoxia.Apoptotic smooth muscle cells of the small and median pul-monary arteries in the hypoxia group were significantly less than those in the control group.After 14 d ofhypoxia,Fas expression by smooth muscle cells of median and small pulmonary arteries was significantlyinhibited.L-Arg significantly inhibited hypoxic pulmonary vascular structural remodeling in association withan augmentation of apoptosis of smooth muscle cells as well as Fas expression in PASMC.These resultsshowed that L-Arg could play an important role in attenuating hypoxic pulmonary vascular structural remod-eling by upregulating Fas expression in PASMC,thus promoting the apoptosis of PASMC.     

Endothelin-1 decreases endothelial NOS expression and activity through ETA receptor-mediated generation of hydrogen peroxide

Similar to infants born with persistent pulmonary hypertension of the newborn (PPHN), there is an increase in circulating endothelin-1 (ET-1) and decreased endothelial nitric oxide synthase (eNOS) gene expression in an ovine model of PPHN. These abnormalities lead to vasoconstriction and vascular remodeling. Our previous studies have demonstrated that reactive oxygen species (ROS) levels are elevated in the pulmonary arteries from PPHN lambs and that ET-1 increases ROS production in pulmonary arterial smooth muscle cells (PASMC) in culture. Thus the objective of this study was to determine whether there was a feedback mechanism between the ET-1-mediated increase in ROS in fetal PASMC (FPASMC) and a decrease in eNOS gene expression in fetal pulmonary arterial endothelial cells (FPAEC). Our results indicate that ET-1 increased H2O2 levels in FPASMC in an endothelin A receptor-dependent fashion. This was observed in both FPASMC monoculture and in cocultures of FPASMC and FPAEC. Conversely, ET-1 decreased H2O2 levels in FPAEC monoculture in an endothelin B receptor-dependent fashion. Furthermore, ET-1 decreased eNOS promoter activity by 40% in FPAEC in coculture with FPASMC. Promoter activity was restored in the presence of catalase. In FPAEC in monoculture treated with 0-100 microM H2O2, 12 microM had no effect on eNOS promoter activity, but it increased eNOS protein levels by 50%. However, at 100 microM, H2O2 decreased eNOS promoter activity and protein levels in FPAEC by 79 and 40%, respectively. These data suggest a role for smooth muscle cell-derived H2O2 in ET-1-mediated downregulation of eNOS expression in children born with PPHN.     

Hypoxic pulmonary vasoconstriction: role of ion channels.

Acute hypoxia induces pulmonary vasoconstriction and chronic hypoxia causes structural changes of the pulmonary vasculature including arterial medial hypertrophy. Electro- and pharmacomechanical mechanisms are involved in regulating pulmonary vasomotor tone, whereas intracellular Ca(2+) serves as an important signal in regulating contraction and proliferation of pulmonary artery smooth muscle cells. Herein, we provide a basic overview of the cellular mechanisms involved in the development of hypoxic pulmonary vasoconstriction. Our discussion focuses on the roles of ion channels permeable to K(+) and Ca(2+), membrane potential, and cytoplasmic Ca(2+) in the development of acute hypoxic pulmonary vasoconstriction and chronic hypoxia-mediated pulmonary vascular remodeling.     

Hypoxic constriction and reactive oxygen species in porcine distal pulmonary arteries

To determine whether reactive oxygen species (ROS) play an essential role in hypoxic pulmonary vasoconstriction (HPV) and the cellular locus of ROS production and action during HPV, we measured internal diameter (ID) at constant transmural pressure, lucigenin-derived chemiluminescence (LDCL), and electron paramagnetic resonance (EPR) spin adduct spectra in small distal porcine pulmonary arteries, and dichlorofluorescein (DCF) fluorescence in myocytes isolated from these arteries. Hypoxia (4% O2) decreased ID, increased DCF fluorescence, tended to increase LDCL, and in some preparations produced EPR spectra consistent with hydroxyl and alkyl radicals. Superoxide dismutase (SOD, 150 U/ml) or SOD + catalase (CAT, 200 U/ml) did not alter ID during normoxia but reduced or abolished the constriction induced by hypoxia. SOD also blocked HPV in endothelium-denuded arteries after restoration of the response by exposure to 10-10 M endothelin-1. Confocal fluorescence microscopy demonstrated that labeled SOD and CAT entered pulmonary arterial myocytes. SOD, SOD + CAT, and CAT blocked the increase in DCF fluorescence induced by hypoxia, but SOD + CAT and CAT also caused a stable increase in fluorescence during normoxia, suggesting that CAT diminished efflux of DCF from cells or oxidized the dye directly. We conclude that HPV required increased concentrations of ROS produced by and acting on pulmonary arterial smooth muscle rather than endothelium.     

Neutral sphingomyelinase, NADPH oxidase and reactive oxygen species. Role in acute hypoxic pulmonary vasoconstriction

The molecular mechanisms underlying hypoxic pulmonary vasoconstriction (HPV) are not yet properly understood. Mitochondrial electron transport chain (ETC) and NADPH oxidase have been proposed as possible oxygen sensors, with derived reactive oxygen species (ROS) playing key roles in coupling the sensor(s) to the contractile machinery. We have recently reported that activation of neutral sphingomyelinase (nSMase) and protein kinase C ζ (PKCζ) participate in the signalling cascade of HPV. Herein, we studied the significance of nSMase in controlling ROS production rate in rat pulmonary artery (PA) smooth muscle cells and thereby HPV in rat PA. ROS production (analyzed by dichlorofluorescein and dihydroethidium fluorescence) was increased by hypoxia in endothelium-denuded PA segments and their inhibition prevented hypoxia-induced voltage-gated potassium channel (K(V) ) inhibition and pulmonary vasoconstriction. Consistently, H(2) O(2) , or its analogue t-BHP, decreased K(V) currents and induced a contractile response, mimicking the effects of hypoxia. Inhibitors of mitochondrial ETC (rotenone) and NADPH oxidase (apocynin) prevented hypoxia-induced ROS production, K(V) channel inhibition and vasoconstriction. Hypoxia induced p47(phox) phosphorylation and its interaction with caveolin-1. Inhibition of nSMase (GW4869) or PKCζ prevented p47(phox) phosphorylation and ROS production. The increase in ceramide induced by hypoxia (analyzed by immunocytochemistry) was inhibited by rotenone. Exogenous ceramide increased ROS production in a PKCζ sensitive manner. We propose an integrated signalling pathway for HPV which includes nSMase-PKCζ-NADPH oxidase as a necessary step required for ROS production and vasoconstriction.     

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     

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.     

AQP1促进肺动脉平滑肌细胞的增殖与迁移（应作者要求撤稿）

该文应作者要求已撤稿。肺动脉平滑肌细胞（PASMCs）的迁移和增殖是肺动脉重塑进而造成肺动脉高压的主要病理基础。水通道蛋白1（AQP1）具有促进上皮细胞、内皮细胞迁移的作用,但机制不清。由于AQP1也表达于血管平滑肌细胞,推测AQP1可能参与缺氧诱导的PASMCs增殖及迁移。通过PCR和免疫印迹分析,检测AQP的表达以及缺氧对AQP表达水平的影响,并通过细胞迁移以及增殖实验观察AQP1在缺氧诱导的PASMCs迁移与增殖中的作用。AQP1在PASMCs和主动脉平滑肌细胞（AoSMCs）均表达,但缺氧只增加PASMCs中AQP1的表达,以及促进PASMCs的迁移与增殖。敲除AQP1可抑制PASMCs的增殖以及缺氧诱导的细胞增殖和迁移。过表达AQP1促进PASMCs的增殖和迁移。缺氧促进β联蛋白在PASMCs内的表达。敲除β联蛋白后,抑制AdAQP1所介导的PASMCs迁移与增殖。这些结果表明,缺氧可促进AQP1在肺动脉内的表达,AQP1可通过β联蛋白对PASMCs的增殖和迁移进行调节。     

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.     

Heterogeneity of hypoxia-mediated decrease in I(K(V)) and increase in [Ca2+](cyt) in pulmonary artery smooth muscle cells

Hypoxic pulmonary vasoconstriction is caused by a rise in cytosolic Ca(2+) ([Ca(2+)](cyt)) in pulmonary artery smooth muscle cells (PASMC) via multiple mechanisms. PASMC consist of heterogeneous phenotypes defined by contractility, proliferation, and apoptosis as well as by differences in expression and function of various genes. In rat PASMC, hypoxia-mediated decrease in voltage-gated K(+) (Kv) currents (I(K(V))) and increase in [Ca(2+)](cyt) were not uniformly distributed in all PASMC tested. Acute hypoxia decreased I(K(V)) and increased [Ca(2+)](cyt) in approximately 46% and approximately 53% of PASMC, respectively. Using combined techniques of single-cell RT-PCR and patch clamp, we show here that mRNA expression level of Kv1.5 in hypoxia-sensitive PASMC (in which hypoxia reduced I(K(V))) was much greater than in hypoxia-insensitive cells (in which hypoxia negligibly affected I(K(V))). These results demonstrate that 1) different PASMC express different Kv channel alpha- and beta-subunits, and 2) the sensitivity of a PASMC to acute hypoxia partially depends on the expression level of Kv1.5 channels; hypoxia reduces whole-cell I(K(V)) only in PASMC that express high level of Kv1.5. In addition, the acute hypoxia-mediated changes in [Ca(2+)](cyt) also vary in different PASMC. Hypoxia increases [Ca(2+)](cyt) only in 34% of cells tested, and the different sensitivity of [Ca(2+)](cyt) to hypoxia was not related to the resting [Ca(2+)](cyt). An intrinsic mechanism within each individual cell may be involved in the heterogeneity of hypoxia-mediated effect on [Ca(2+)](cyt) in PASMC. These data suggest that the heterogeneity of PASMC may partially be related to different expression levels and functional sensitivity of Kv channels to hypoxia and to differences in intrinsic mechanisms involved in regulating [Ca(2+)](cyt).