Chronic hypoxia decreases KV channel expression and function in pulmonary artery myocytes

Oleksandr Platoshyn1,*, Ying Yu1,*, Vera A. Golovina2, Sharon S. McDaniel1, Stefanie Krick1, Li Li2, Jian-Ying Wang2, Lewis. J. Rubin1, and Jason X.-J. Yuan1

1 Division of Pulmonary and Critical Care Medicine, Department of Medicine, University of California, San Diego, California 92103-8382; and 2 Departments of Physiology and Surgery, University of Maryland School of Medicine, Baltimore, Maryland 21201


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Activity of voltage-gated K+ (KV) channels regulates membrane potential (Em) 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 (PO2 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 [IK(V)]; caused Em depolarization; and increased [Ca2+]cyt in PASMCs but negligibly affected KV channel expression, increased IK(V), and induced hyperpolarization in MASMCs. These results demonstrate that chronic hypoxia selectively downregulates KV channel expression, reduces IK(V), and induces Em 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.

voltage-gated potassium channel; membrane depolarization; increases in cytosolic free calcium; voltage-gated potassium current


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THE PULMONARY CIRCULATION BEARS many unique properties that are dramatically different from the systemic circulation. One of the differences is that hypoxia causes pulmonary vasoconstriction (26, 34) but systemic (e.g., cerebral, coronary, and renal) vasodilation (10, 35). Hypoxic pulmonary vasoconstriction (HPV) is an important physiological mechanism involved in maximizing the oxygenation of blood by directing blood flow away from hypoxic regions of the lung. In patients with hypoxic pulmonary diseases (e.g., chronic obstructive pulmonary disease) and residents of high-altitude areas, continuous alveolar hypoxia causes pulmonary hypertension that could lead to right heart failure and death. Persistent pulmonary vasoconstriction and medial thickening of the pulmonary arteries (PAs) due to smooth muscle growth greatly contribute to the elevated pulmonary vascular resistance and arterial pressure in these patients (42, 55).

Hypoxia causes contraction in endothelium-denuded PA rings and single PA smooth muscle cells (SMCs) but not in isolated mesenteric artery (MA) rings and single systemic (aortic and cerebral) artery SMCs (34, 35, 37, 44, 79, 83). These results suggest that HPV is an intrinsic property of pulmonary vascular smooth muscle and that the mechanism of HPV involves direct oxygen sensing by PASMCs. HPV is inhibited by Ca2+ channel blockers (39), and removal of extracellular Ca2+ abolishes HPV in isolated PA rings (79). These results suggest that Ca2+ influx through sarcolemmal voltage-dependent Ca2+ channels is involved in the development of HPV (26, 34). In PASMCs, acute hypoxia decreases voltage-gated K+ (KV) currents [IK(V)], induces membrane depolarization, augments Ca2+ influx through voltage-dependent Ca2+ channels, and increases cytosolic free Ca2+ concentration ([Ca2+]cyt) (48, 53, 54, 57, 65, 68, 78). Inhibition of KV channels by 4-aminopyridine also increases pulmonary arterial pressure in isolated perfused rat lungs, which mimics the hypoxia-induced pressor response (27).

In lungs from chronically hypoxic rats, the pressor response to acute hypoxia is impaired, whereas the pressor response to vasoconstrictor agonists (e.g., angiotensin II, prostaglandin F2alpha , and norepinephrine) is enhanced (40). These observations suggest that the reduced pressor response to acute hypoxia in chronically hypoxic rats may result from abnormalities in the mechanism that couples acute hypoxia to contraction of the pulmonary vascular smooth muscle. That is, chronic hypoxia-mediated pulmonary hypertension may share the same mechanisms that are responsible for acute HPV. Accordingly, a decrease in the activity of KV channels and the resultant membrane depolarization may also be involved in the development of chronic hypoxia-mediated pulmonary hypertension by mediating pulmonary vasoconstriction and vascular remodeling through increased [Ca2+]cyt in PASMCs.

Sweeney and Yuan (65) and Wang et al. (70) previously reported that chronic hypoxia (60-72 h) downregulates mRNA and protein expression of Kv1.2 and Kv1.5, two KV channel alpha -subunits that encode delayed rectifier KV channels. The rationale of this study was to examine further 1) whether hypoxia-mediated downregulation of KV channel alpha -subunit expression decreases KV channel activity and reduces whole cell IK(V) in PASMCs and 2) whether chronic hypoxia inhibits the expression and function of KV channels only in PA but not in systemic artery (MA) SMCs (i.e., whether hypoxia-induced downregulation of KV channel expression is selective to PASMCs). In addition, we also investigated whether the hypoxia-mediated changes in membrane potential (Em) differed between PASMCs and MASMCs.


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Cell preparation and culture. Primary cultured SMCs from PAs and MAs were prepared from Sprague-Dawley rats (125-250 g) (77). The isolated arterial rings were incubated in Hanks' balanced salt solution containing 1.5 mg/ml of collagenase (Worthington) for 20 min. After incubation, a thin layer of the adventitia was carefully stripped off with fine forceps and the endothelium was removed by gently scratching the intimal surface with a surgical blade. The remaining smooth muscle was then digested with 2.0 mg/ml of collagenase and 0.5 mg/ml of elastase (Sigma) for 45 min at 37°C. The cells were plated onto 25-mm coverslips (for patch-clamp and fluorescence microscopy experiments) or 10-cm petri dishes (for molecular biological experiments) in 10% fetal bovine serum (FBS)-DMEM and cultured in a 37°C, 5% CO2 humidified incubator for 3-5 days. Before each experiment, the cells were incubated in 0.3% FBS-DMEM for 12-24 h to stop cell growth.

The purity of PASMCs and MASMCs in the primary cultures was confirmed by the specific monoclonal antibody raised against smooth muscle alpha -actin (Boehringer Mannheim). Primary cultured cells were first stained with the membrane-permeable nucleic acid stain 4',6'-diamidino-2-phenylindole (DAPI, 5 µM; Molecular Probes) to estimate the total cell number in the cultures. All the DAPI-stained cells also cross-reacted with the SMC alpha -actin antibody, indicating that the cultures contained only SMCs.

Treatment with hypoxia. Quiescent (growth-arrested) PASMCs cultured in 0.3% FBS-DMEM were divided into two groups. One group of cells was incubated continuously in an incubator containing 5% CO2 in air as a normoxic control. The hypoxic group was incubated in an incubator equilibrated with a gas mixture composed of 3% O2, 5% CO2, and 92% N2.

Primary cultured PASMCs and MASMCs were incubated under normoxic and hypoxic conditions for 60-72 h before each experiment. For cells cultured in normoxia, PO2 in the culture medium was 140 ± 5 mmHg and was retained at this level until the cells were used for the measurement of IK(V), Em, KV channel mRNA level, and [Ca2+]cyt. For cells treated with hypoxia, PO2 in the culture medium reached 30-35 mmHg within 2 h after placement of the culture dishes in the incubator equilibrated with 3% O2, 5% CO2, and 92% N2. The PO2 level in the culture medium was automatically controlled by an O2 sensor and a controller incorporated in the incubator and could be stably maintained at a level close to ambient PO2 in the incubator for days. There were no significant changes in the pH values of the culture medium as well as in cell morphology during the 72-h incubation in the hypoxic incubator.

Electrophysiological measurements. Whole cell K+ currents (IK) were recorded with an Axopatch-1D amplifier and a DigiData 1200 interface (Axon Instruments) with patch-clamp techniques (25, 77). Patch pipettes (2-4 MOmega ) were made on a Sutter electrode puller with borosilicate glass tubes and fire polished on a Narishige microforge. Step-pulse protocols and data acquisition were performed with pCLAMP software. Currents were filtered at 1-2 kHz (-3 dB) and digitized at 2-4 kHz with an Axopatch-1D amplifier. All experiments were performed at room temperature (22-24°C). Em in single SMCs was measured in the current-clamp mode (I = 0) with whole cell patch-clamp techniques. In some experiments, Em was recorded with an intracellular electrode (50-100 MOmega ) filled with 3 M KCl. Data were acquired by an electrometer (Electro 705, World Precision) coupled to an IBM-compatible computer and analyzed with the DATAQ data acquisition software WINDAQ (Dataq Instruments).

Measurement of [Ca2+]cyt. Cells were loaded with fura 2-AM (3 µM) for 30 min at room temperature (24°C) under an atmosphere of 5% CO2 in air. The fura 2-loaded cells were then superfused with a standard bath solution for 20 min at 34°C to wash away extracellular dye and permit intracellular esterases to cleave the cytosolic fura 2-AM into active fura 2. Fura 2 fluorescence (510-nm emission; 360- and 380-nm excitation) images from the cells and background were obtained with a Gen III charge-coupled device camera (Stanford Photonics) coupled to a Carl Zeiss microscope. Image acquisition and analysis were performed with a MetaMorph imaging system (Universal Imaging). Video frames containing images of fura 2 fluorescence from cells as well as the corresponding background images (fluorescence from fields devoid of cells) were digitized at a resolution of 512 horizontal × 480 vertical pixels with an 8-bit gray scale. [Ca2+]cyt was calculated from fura 2 fluorescent emission excited at 360 and 380 nm with the ratio method (23).

Reagents and solutions. A coverslip containing the cells was positioned in the recording chamber (approx 0.75 ml) and superfused (2 ml/min) with the extracellular (bath) physiological salt solution (PSS) for recording IK or measuring [Ca2+]cyt. The PSS contained (in mM) 141 NaCl, 4.7 KCl, 1.8 CaCl2, 1.2 MgCl2, 10 HEPES, and 10 glucose (pH 7.4). In Ca2+-free PSS, CaCl2 was replaced by equimolar MgCl2 and 1 mM EGTA was added to chelate residual Ca2+. The internal (pipette) solution for recording IK(V) contained (in mM) 125 KCl, 4 MgCl2, 10 HEPES, 10 EGTA, and 5 Na2ATP (pH 7.2).

RT-PCR. Total RNA [ratio of optical density (OD) at 260 nm to OD at 280 nm > 1.7] prepared from primary cultured SMCs by the acid guanidinium thiocyanate-phenol-chloroform extraction method was reverse transcribed with the Superscript first-strand cDNA synthesis kit (GIBCO BRL). The sense and antisense primers were specifically designed from the coding regions of Kv1.1 (GenBank accession no. X12589), Kv1.5 (GenBank accession no. M27158), Kv2.1 (GenBank accession no. X16476), Kv4.3 (GenBank accession no. U42975), Kv9.3 (GenBank accession no. AF029056), Kvbeta 1.1 (GenBank accession no. X70662), Kvbeta 2 (GenBank accession no. X76724), and Kvbeta 3 (GenBank accession no. X76723; Table 1). The fidelity and specificity of the sense and antisense oligonucleotides were examined with the BLAST program.

                              
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Table 1.   Oligonucleotide sequences of the primers used for RT-PCR

The cDNA samples were amplified in a DNA thermal cycler (PerkinElmer), the PCR products were electrophoresed through a 1.5% agarose gel, and amplified cDNA bands were visualized by GelStar gel staining (FMC BioProducts). To quantify the PCR products, an invariant mRNA of beta -actin was used as an internal control. The OD values in the channel signals, measured by a Kodak electrophoresis documentation system, were normalized to the OD values in the beta -actin signals; the ratios are expressed as arbitrary units for quantitative comparison (70).

Western blot analysis. Primary cultured PASMCs were gently washed twice in cold PBS, scraped into 0.3 ml of lysis buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 0.1% SDS, 100 µg/ml of phenylmethylsulfonyl fluoride, and 30 µl/ml of aprotinin), and incubated for 30 min on ice. The lysates were then sonicated and centrifuged at 12,000 rpm for 10 min, and the insoluble fraction was discarded. The protein concentrations in the supernatant were determined by the bicinchoninic acid protein assay (Pierce, Rockford, IL) with bovine serum albumin (BSA) as a standard. Ten micrograms of protein were mixed and boiled in SDS-PAGE sample buffer for 5 min. The proteins fractionated by 10% SDS-PAGE were then transferred to nitrocellulose membranes by electroblotting in a Mini Trans-Blot cell transfer apparatus (Bio-Rad) under conditions recommended by the manufacturer. After incubation overnight at 4°C in a blocking buffer (0.1% Tween 20 in PBS) containing 5% nonfat dry milk powder, the membranes were incubated with the affinity-purified rabbit polyclonal antibodies specific for Kv1.1, Kv2.1 (Alomone Labs, Jerusalem, Israel), and Kv1.5 (Upstate Biotechnology, Lake Placid, NY). The monoclonal antibody specific for smooth muscle alpha -actin (Boehringer Mannheim) was used as a control. The membranes were then washed and incubated with anti-rabbit horseradish peroxidase-conjugated IgG for 90 min at room temperature. The bound antibody was detected with an enhanced chemiluminescence detection system (Amersham).

Statistical analysis. Data are expressed as means ± SE. Statistical analysis was performed with unpaired Student's t-test or ANOVA and post hoc tests (Student-Newman-Keuls) as indicated. Differences were considered to be significant when P < 0.05.


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Linear relationship between PCR products and cycle number. The quantity of PCR products for Kv1.5 and beta -actin correlated linearly with the changes in cycle number between 22 and 30 cycles, whereas 2 µg of total RNA and 1 µl of cDNA were used for RT-PCR (Fig. 1). Therefore, to appropriately quantify the mRNA levels of KV channels, we used 2 µg of total RNA for RT and 1 µl of cDNA and 25 cycles for PCR in this study. The amount of PCR products from KV channel alpha - and beta -subunits were normalized to the mRNA levels of beta -actin for comparison.


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Fig. 1.   Linear relationships between the quantity of PCR products for Kv1.5 and beta -actin and the changes in cycle number. RT-PCR products are displayed in agarose gels for Kv1.5 (267 bp; A, top) and beta -actin (244 bp; B, top). The cDNA samples were amplified for the indicated number of cycles. M, 100-bp DNA ladder. The data were normalized to the maximal values of the PCR products for Kv1.5 (A, bottom) and beta -actin (B, bottom) and are expressed as means ± SE; n = 6 samples/experiment. The linear regression lines were drawn through the data points between 22 and 30 cycles.

Divergent effects of chronic hypoxia on mRNA expression of KV channel alpha -subunits in PASMCs and MASMCs. Exposure to hypoxia for 72 h did not induce significant PASMC death and negligibly affected the total amount of proteins in culture (Fig. 2). However, chronic hypoxia (60 h) significantly attenuated the mRNA levels of 1) Kv1.1 and Kv1.5, two KV channel alpha -subunits belonging to the Shaker subfamily (82); 2) Kv2.1, a member of the Shab subfamily; 3) Kv4.3, a member of the Shal subfamily; and 4) Kv9.3, an electrically silent alpha -subunit that assembles with the Shab subfamily members to form functional heteromeric channels (28, 50) in PASMCs (Fig. 3). The inhibitory effects of hypoxia on all the KV channel alpha -subunits (Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3) were selective to PASMCs because hypoxia negligibly affected the mRNA expression of these channels in MASMCs (Fig. 3). The amplitude of the current through a heteromeric KV channel formed by the electrically silent alpha -subunit Kv9.3 and the functional alpha -subunit Kv2.1 was much greater than that through a homomeric KV channel formed by Kv2.1 (50). Thus inhibition of Kv9.3 expression would significantly reduce the heteromeric Kv2.1/Kv9.3 channels that are believed to be an O2-sensitive KV channel in rat PASMCs (28, 50).


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Fig. 2.   Effect of chronic exposure of pulmonary artery (PA) smooth muscle cells (SMCs) to hypoxia on cell viability and the amount of total protein. Summarized data show the number of viable cells (A), the total amount of protein/well (B), and the amount of protein/cell (C) in PASMCs incubated under normoxic and hypoxic conditions for 72 h. Values are means ± SE; n = 6 experiments.



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Fig. 3.   The mRNA levels of voltage-gated K+ (KV) channel alpha -subunits in PA and mesenteric artery (MA) SMCs incubated under normoxic (N) and hypoxic (H) conditions. A: PCR-amplified products displayed in agarose gels stained with ethidium bromide for Kv1.1 (594 bp), Kv1.5 (267 bp), Kv2.1 (269 bp), Kv4.3 (270 bp), Kv9.3 (568 bp), and beta -actin (244 bp) transcripts. M, 100-bp DNA ladder. B: data normalized to the amount of beta -actin in PASMCs (a) and MASMCs (b) before (Nor) and after (Hyp) treatment with hypoxia (PCR experiments were repeated 4 times independently). Values are means ± SE. ** P < 0.01 vs. Nor.

In contrast to the inhibitory effect on the pore-forming alpha -subunits (Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3) in PASMCs, chronic hypoxia negligibly affected the mRNA levels of the regulatory beta -subunits (Kvbeta 1.1, Kvbeta 2, and Kvbeta 3) in PASMCs and MASMCs (Fig. 4). As an internal control, hypoxia had little effect on mRNA expression of beta -actin (Figs. 3 and 4). These results suggest that hypoxia-induced downregulation of KV channel mRNA expression was selective to alpha -subunits in PASMCs.


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Fig. 4.   The mRNA levels of KV channel beta -subunits in PASMCs and MASMCs incubated under normoxic and hypoxic conditions. A: PCR-amplified products displayed in agarose gels for Kvbeta 1.1 (150 bp), Kvbeta 2 (141 bp), Kvbeta 3 (178 bp), and beta -actin (244 bp) transcripts. M, 100-bp DNA ladder. B: data normalized to the amount of beta -actin in PASMCs and MASMCs before and after treatment with hypoxia (PCR experiments were repeated 6 times independently). Values are means ± SE.

Divergent effects of chronic hypoxia on protein expression of KV channel alpha -subunits in PASMCs and MASMCs. A change in the number of functional KV channels occurs within hours because of the rapid turnover rate of channel mRNA and protein; the half-lives of Kv1.5 mRNA and protein, for example, are only 0.5 and 4 h, respectively (66). Thus the downregulation of KV channel mRNA expression in PASMCs during chronic hypoxia would correspondingly cause a reduction in KV channel protein expression. Indeed, chronic exposure to hypoxia for 72 h significantly and selectively downregulated the protein expression of Kv1.1, Kv1.5, and Kv2.1 in PASMCs (Fig. 5). Previous observations by Sweeney and Yuan (65) and Wang et al. (70) indicated that chronic hypoxia also downregulates the protein expression of Kv1.2 in rat PASMCs. Consistent with the inability to affect mRNA expression, chronic exposure to hypoxia negligibly affected the protein expression of Kv1.1, Kv1.5, and Kv2.1 in MASMCs (Fig. 5).


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Fig. 5.   The protein levels of KV channel alpha -subunits in PASMCs and MASMCs incubated under normoxic and hypoxic conditions. A: Western blot analyses of Kv1.1 (a), Kv1.5 (b), and Kv2.1 (c) channel proteins in PASMCs and MASMCs cultured in normoxia and hypoxia for 72 h. Nos. on left, molecular mass markers. The molecular masses of Kv1.1, Kv1.5, Kv2.1, and alpha -actin are ~86, 63, 100-130, and 42 kDa, respectively. B: summarized data showing the protein levels for Kv1.1 (a), Kv1.5 (b), and Kv2.1 (c) normalized to the amount of alpha -actin in PASMCs and MASMCs cultured during normoxia and hypoxia for 72 h. Values are means ± SE. ** P < 0.01 vs. Nor.

Hypoxia-induced decrease in IK(V) was selective to PASMCs. Whole cell IK(V) was isolated in cells superfused with a Ca2+-free bath solution (plus 1 mM EGTA) and dialyzed with a Ca2+-free (plus 10 mM EGTA) and ATP-containing pipette solution. Under these conditions, contributions of Ca2+-activated K+ currents and ATP-sensitive K+ currents to the whole cell outward currents were minimized (20, 47). Consistent with the molecular biological results, chronic hypoxia significantly decreased whole cell IK(V) in PASMCs (Fig. 6). The hypoxia-sensitive IK(V) was activated at a potential close to the resting Em value (approximately -40 mV) in PASMCs. Chronic hypoxia caused a 54% decrease in IK(V) at +80 mV and an 85% decrease at -40 mV in PASMCs (Fig. 4, B and C). Consistent with the inability of hypoxia to inhibit mRNA and protein expression of KV channel alpha -subunits, chronic hypoxia had no inhibitory effect on whole cell IK(V) in MASMCs (Fig. 7). In fact, hypoxia slightly, but insignificantly, increased the amplitude of the whole cell IK(V) from 7.7 ± 4.1 (n = 18 cells) to 13.3 ± 6.1 pA (P = 0.49; n = 25 cells) at -40 mV in MASMCs. These results indicate that hypoxia-mediated downregulation of KV channel alpha -subunit mRNA and protein expression decreased the number of functional KV channels and attenuated whole cell IK(V) in PASMCs but not in MASMCs.


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Fig. 6.   Whole cell voltage-gated K+ current [IK(V)] in PASMCs cultured in normoxia and hypoxia. A: representative currents elicited by depolarizing the cells to a series of test potentials ranging from -40 to +80 mV in 20-mV increments from a holding potential of -70 mV in PASMCs incubated under normoxic and hypoxic conditions for 60 h. B: composite current-voltage relationships (I-V curves) from PASMCs cultured in normoxia (n = 17) and hypoxia (n = 16). The I-V curve for hypoxic cells was significantly different from the curve for normoxic cells (P < 0.001 by Student-Newman-Keuls test). C: summarized data showing amplitudes of the K+ current (IK) elicited by a test potential of -40 mV in normoxic and hypoxic cells. Current amplitudes were measured at 250-290 ms during a 300-ms test pulse. Data are means ± SE; nos. in parentheses, no. of cells tested. * P < 0.05 vs. Nor.



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Fig. 7.   Whole cell IK(V) in MASMCs cultured in normoxia and hypoxia. A: representative currents elicited by depolarizing the cells to a series of test potentials ranging from -40 to +80 mV in 20-mV increments from a holding potential of -70 mV in cells incubated under normoxic and hypoxic conditions for 60 h. B: composite I-V curves from MASMCs cultured in normoxia (n = 18) and hypoxia (n = 25). C: summarized data showing amplitudes of the IK elicited by a test potential of -40 mV in normoxic and hypoxic cells. Current amplitudes were measured at 250-290 ms during a 300-ms test pulse. Data are means ± SE; nos. in parentheses, no. of cells tested.

Hypoxia-mediated attenuation of IK(V) induced membrane depolarization and increases in [Ca2+]cyt in PASMCs. The membrane input resistance of resting PASMCs is very high, usually on the order of 1-10 GOmega (47, 77). Therefore, a small change in outward IK(V) would be expected to cause a large change in Em. As shown in Fig. 6C, chronic hypoxia decreased the amplitude of IK(V) elicited by a test potential of -40 mV from 21.8 ± 6.9 to 3.2 ± 3.7 pA. The 18-pA reduction in IK(V) could be extrapolated to an 18-mV change in Em, given that the resting membrane input resistance in PASMCs is 1 GOmega . Indeed, chronic hypoxia caused a 15-mV depolarization in PASMCs (from -38 ± 1 to -23 ± 1 mV) but a 4-mV hyperpolarization in MASMCs (from -44.7 ± 2.1 to -49.5 ± 5.1 mV; P = 0.37; Fig. 8). These results indicate that the hypoxia-mediated decrease in whole cell IK(V) was sufficient to cause substantial membrane depolarization in PASMCs.


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Fig. 8.   Resting membrane potential (Em) in PASMCs and MASMCs incubated under normoxic and hypoxic conditions. A: resting Em measured by an intracellular electrode filled with 3 M KCl in PASMCs incubated in normoxia and hypoxia. B: summarized data showing resting Em measured with whole cell current-clamp techniques in PASMCs and MASMCs in normoxia and hypoxia. Data are means ± SE; nos. in parentheses, no. of cells tested. *** P < 0.001 vs. Nor.

Studies on the kinetics of the L-type voltage-gated Ca2+ channel and its relationship to [Ca2+]cyt demonstrated that prolonged membrane depolarization at a range of -35 to -20 mV (a voltage range where Ca2+ channel inactivation is incomplete while the channel activation begins) can open the Ca2+ channel sufficiently to cause a sustained increase in [Ca2+]cyt (17, 46). Indeed, chronic hypoxia significantly increased resting [Ca2+]cyt in PASMCs (Fig. 9) as a result of membrane depolarization due to decreased KV channel activity. The increased [Ca2+]cyt activates the contractile apparatus (actomyosin) in the cytosol and causes pulmonary vasoconstriction. Because of the minimal resistance of nuclear membrane to Ca2+, the sustained elevation in [Ca2+]cyt would rapidly increase the nuclear Ca2+ concentration ([Ca2+]) in PASMCs. In the cell cycle, Ca2+ is required for the transition from the resting state (G0) to DNA synthesis and mitosis (41). Thus a rise in [Ca2+]cyt also plays a critical role in stimulating PASMC proliferation (which leads to pulmonary vascular medial hypertrophy).


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Fig. 9.   Resting cytosolic free Ca2+ concentration ([Ca2+]cyt) in PASMCs incubated under normoxic and hypoxic conditions. A: fura-2 fluorescence (360-nm excitation) images (top) showing the cells in which [Ca2+]cyt was measured and pseudocolor images (bottom) showing resting [Ca2+]cyt in PASMCs cultured under normoxic and hypoxic conditions for 60 h. Bar, 10 µm. B: summarized data showing resting [Ca2+]cyt in normoxic (n = 125) and hypoxic (n = 95) PASMCs. Data are means ± SE. *** P < 0.001 vs. normoxia.


    DISCUSSION
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Acute hypoxia-induced pulmonary vasoconstriction is an important physiological mechanism for the matching of ventilation and perfusion in the lung to ensure maximal oxygenation of venous blood circulating through the lungs. Chronic exposure to hypoxia, however, causes pulmonary hypertension that is characterized pathologically by vasoconstriction and vascular remodeling (39, 40, 42, 55). Indeed, pulmonary hypertension induced by persistent alveolar hypoxia has been well implicated in patients with chronic obstructive pulmonary disease and residents living at high altitude (45, 59). Furthermore, chronic hypoxia-induced pulmonary vasoconstriction and pulmonary hypertension are important causes of high-altitude pulmonary edema (19, 29, 58). Thus understanding the cellular and molecular mechanisms involved in chronic hypoxia-mediated pulmonary vasoconstriction and vascular remodeling would greatly help in developing therapeutic approaches for patients with pulmonary hypertension and edema.

Chronic exposure to hypoxia (PO2 30-35 mmHg for 60-72 h) downregulated the mRNA and protein expression of the pore-forming KV channel alpha -subunits (including Kv1.1, Kv1.5, and Kv2.1) in PASMCs but not in MASMCs (Figs. 3 and 5). The inhibitory effect of hypoxia was specific to the alpha -subunits because hypoxia negligibly affected the mRNA expression of the cytoplasmic regulatory beta -subunits (including Kvbeta 1.1, Kvbeta 2, and Kvbeta 3; Fig. 4). Consistent with its inhibitory effect on KV channel expression, chronic hypoxia reduced IK(V) (Fig. 6) and depolarized PASMCs (Fig. 8) but slightly increased IK(V) and hyperpolarized MASMCs (Figs. 7 and 8). Similar results were also observed in freshly dissociated PASMCs isolated from chronically hypoxic animals (62, 63). The selectivity of the hypoxia-induced decrease in IK(V) (2, 53, 54, 65, 78) and downregulation of KV channel expression (65, 70; this study) in PASMCs (compared with MASMCs) suggests that hypoxia regulates the activity of KV channels via an intrinsic mechanism that exists uniquely in PASMCs.

The KV channel alpha - and beta -subunits identified in PASMCs (49, 50, 82), such as Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2, Kvbeta 1.1, Kvbeta 2, and Kvbeta 3, are also expressed in MASMCs (74). Qualitative differences in KV channel alpha -subunits between PASMCs and MASMCs have not been observed so far. The potential candidates of KV channel alpha -subunits (65) that could form O2-sensitive heteromeric or homomeric channels include Kv1.2 (8, 28, 70), Kv1.5 (3, 70), Kv2.1 (3, 28, 50), Kv3.1 (48), Kv4.2 (51), and Kv9.3 (28, 50). As cytoplasmic regulatory domains, KV channel beta -subunits (e.g., Kvbeta 1.1 and Kvbeta 1.2) have been demonstrated to be O2 sensors in the hypoxia- or redox-mediated regulation of KV channel activity (11, 38, 51, 56). PASMCs and MASMCs, while expressing homogeneous KV channel alpha - and beta -subunits, have divergent responses to hypoxia (65). Therefore, the "O2 sensor" in PASMCs may exist in the upstream regulation cascade of the KV channel gene promoter (6, 14, 18, 69). These observations also suggest that the mechanisms involved in the regulation of KV channel gene expression are different between PASMCs and systemic artery SMCs.

Multiple pathways may exist for hypoxia-induced KV channel inhibition to ensure the efficacy and sensitivity of HPV, a critical physiological mechanism involved in maximizing the oxygenation of blood. Acute hypoxia may inhibit KV channel function by 1) inhibiting oxidative phosphorylation (15, 80), 2) changing the redox status (2, 80), 3) altering a membrane-delimited O2-sensitive regulatory moiety that is adjacent or coupled to the channel protein (24, 33), 4) activating mitochondrial NAD(P)H oxidase and increasing superoxide production (6, 9, 36, 71), 5) inhibiting cytochrome P-450 to synthesize endothelium-derived hyperpolarizing metabolites (5, 32, 81), 6) affecting directly the KV channel protein (24, 33), and 7) activating protein kinase C (4, 12, 13). Chronic hypoxia may inhibit KV channel activity by directly or indirectly downregulating mRNA and protein expression of KV channel alpha -subunits. In addition to the mechanisms by which acute hypoxia decreases KV channel activity, the underlying mechanisms involved in chronic hypoxia-induced decrease of KV channel expression may include 1) inhibition of gene transcription, 2) decrease in mRNA (and/or protein) stability, 3) up- or downregulation of transcription factors (e.g., hypoxia-inducible factor-1, nuclear factor-kappa B, c-fos/c-jun, Kv1.5 repressor element binding factor, CCAAT enhancer binding protein, and FixL/FixJ) and signal transduction proteins (e.g., p53, p38, mitogen-activated protein kinase, tyrosine kinase, and protein kinase C) (4, 12, 13, 21, 22, 30, 43, 67, 72, 75) that can bind directly to KV channel gene promoters (e.g., Kv1.5 repressor element) and modulate KV channel gene transcription (14, 18, 69), and 4) induction of transcription factors that upregulate intermediate inhibitors (e.g., endothelin-1) of the KV channel genes (31, 61).

Under resting conditions, Em is controlled primarily by transmembrane K+ permeability and gradient. Thus the K+ permeability (or IK) is a key determinant of Em when the K+ gradient is kept unchanged (47). Transmembrane IK is carried by at least four types of K+ channels in vascular SMCs: 1) KV channels, 2) Ca2+-activated K+ channels, 3) ATP-sensitive or -inhibited K+ channels, and 4) inward rectifier K+ channels (47). All four types of K+ channels contribute to the regulation of Em.

It has been demonstrated that under resting conditions, K+ permeability through KV channels is responsible for determining Em in PASMCs (16, 20, 76). Thus Em is directly related to the whole cell IK(V) that is determined by IK(V) = N × iKv × Po, where N is the number of membrane KV channels, iKv is the unitary current of a KV channel, and Po is the steady-state open probability of a KV channel. When KV channel expression declines (N is decreased) and/or the KV channel closes (iKv or Po is decreased), Em becomes less negative (depolarized) due to decreased IK(V). When KV channel expression rises (N is increased) and/or KV channel opens (iKv or Po is increased), Em becomes more negative (hyperpolarized) due to increased IK(V).

The downregulation of KV channel alpha -subunits during chronic hypoxia would decrease the number of functional KV channels expressed in the plasma membrane and reduce whole cell IK(V) in PASMCs. The resultant membrane depolarization opens voltage-gated Ca2+ channels, augments Ca2+ influx, increases [Ca2+]cyt in PASMCs, and causes pulmonary vasoconstriction (17, 46, 76). In addition to triggering cell contraction, a rise in [Ca2+]cyt can activate the mitogen-activated protein kinase cascade that stimulates synthesis of the transcription factors required for cell proliferation (41). There are at least four Ca2+/calmodulin-sensitive steps in the cell cycle: transitions from 1) G0 (resting state) to G1 phase (the beginning of DNA synthesis), 2) G1 to S phase (an interphase during which replication of the nuclear DNA occurs), 3) G2 to M phase (mitosis), and 4) through the M phase (41). Ca2+ diffuses quickly between the cytosol and the nucleus (1); therefore, the hypoxia-induced increase in [Ca2+]cyt would rapidly increase nuclear [Ca2+] and promote cell proliferation (73) by moving quiescent cells into the cell cycle and by propelling the proliferating cells through mitosis (7, 41, 52, 60). Furthermore, maintaining a sufficient level of Ca2+ in the sarcoplasmic or endoplasmic reticulum is also critical for cell growth; indeed, depletion of the sarcoplasmic reticulum Ca2+ stores induces growth arrest (64).

In summary, a rise in [Ca2+]cyt in PASMCs is essential in the development and maintenance of pulmonary hypertension by mediating pulmonary vasoconstriction and stimulating pulmonary vascular smooth muscle proliferation (Fig. 10). The hypoxia-induced downregulation of KV channel mRNA expression and inhibition of KV channel function in PASMCs induces a membrane depolarization that opens the voltage-gated Ca2+ channels and enhances Ca2+ influx. The resultant increase in [Ca2+]cyt triggers pulmonary vasoconstriction and stimulates PASMC proliferation (leading to pulmonary vascular medial hypertrophy; Fig. 10). Abnormalities in the transcriptional regulation of KV channel alpha -subunit genes mediated by hypoxia-sensitive transcription factors may be involved in the decreased KV channel mRNA expression during hypoxia. The precise mechanisms responsible for the divergent effects of hypoxia on gene expression of KV channels in PASMCs and systemic artery (e.g., MA) SMCs may underlie the upstream regulation of the KV channel gene promoter.


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Fig. 10.   Schematic diagram illustrating the proposed mechanisms involved in hypoxia-mediated pulmonary hypertension. Chronic exposure to hypoxia downregulates gene expression of KV channel alpha -subunits and inhibits KV channel function by multiple mechanisms. The resultant decrease in whole cell IK(V) leads to membrane depolarization that opens voltage-gated Ca2+ channels, promotes Ca2+, and raises [Ca2+]cyt in PASMCs. Because of the high ratio of intracellularly stored Ca2+ concentration ([Ca2+]) in the sarcoplasmic reticulum (SR; [Ca2+]SR) to the [Ca2+]cyt and the minimal resistance of the nuclear membrane to Ca2+, a rise in [Ca2+]cyt subsequently increases [Ca2+]SR and nuclear [Ca2+] ([Ca2+]n). Increases in cytosolic, nuclear, and intracellularly stored [Ca2+] would 1) stimulate PASMC proliferation by activating cytosolic signal transduction proteins [e.g., cAMP response element binding protein (CREB)] and transcription factors (e.g., c-fos and c-jun, Ca2+-sensitive early responsive genes), 2) induce vasoconstriction by activating cytosolic calmodulin and contractile proteins, and 3) facilitate cell proliferation by enhancing SR or endoplasmic reticulum (ER) functions (e.g., protein sorting and processing and lipid synthesis). The pulmonary vasoconstriction and vascular remodeling increase pulmonary vascular resistance and ultimately cause pulmonary hypertension. PKC, protein kinase C; MAPK, mitogen-activated protein kinase; P-450, cytochrome P-450; NAD(P)H, NAD(P)H oxidase; HIF, hypoxia-inducible factor; NF-kappa B, nuclear factor-kappa B; ROS, reactive oxygen species; HPH, hypoxia-induced pulmonary hypertension.


    ACKNOWLEDGEMENTS

We thank Dr. J. Wang for his preliminary study on voltage-gated potassium channel expression and Dr. N. Kim and A. Limsuwan for technical assistance.


    FOOTNOTES

* O. Platoshyn and Y. Yu contributed equally to this work.

This work was supported by National Heart, Lung, and Blood Institute Grants HL-54043 and HL-64549 (to J. X.-J. Yuan); National Institute of Diabetes and Digestive and Kidney Diseases Grants DK-45314 and DK-57819 (to J.-Y. Wang); the American Heart Association Mid-Atlantic Affiliate (to V. Golovina); and a Merit Review Grant from the Department of Veterans Affairs (to J.-Y. Wang).

S. Krick is an Ambassadorial Scholar of Rotary International. J. X.-J. Yuan is an Established Investigator of the American Heart Association (974009N).

Address for reprint requests and other correspondence: J. X.-J. Yuan, UCSD Medical Center, 200 W. Arbor Dr., San Diego, CA 92103-8382 (E-mail: xiyuan{at}ucsd.edu).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

Received 8 May 2000; accepted in final form 18 October 2000.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS AND MATERIALS
RESULTS
DISCUSSION
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