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Published ahead of print on May 19, 2004, doi:10.1165/rcmb.2003-0386OC
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American Journal of Respiratory Cell and Molecular Biology. Vol. 31, pp. 337-343, 2004
© 2004 American Thoracic Society
DOI: 10.1165/rcmb.2003-0386OC

Subacute Hypoxia Decreases Voltage-Activated Potassium Channel Expression and Function in Pulmonary Artery Myocytes

Zhigang Hong, E. Kenneth Weir, Daniel P. Nelson and Andrea Olschewski

VA Medical Center, University of Minnesota, Minneapolis, Minnesota; and Department of Anesthesiology, Intensive Care Medicine and Pain Therapy, Justus-Liebig-University, Giessen, Germany

Address correspondence to: Andrea Olschewski, Department of Anesthesiology, Intensive Care Medicine and Pain Therapy, Justus-Liebig-University, D-35392 Giessen, Germany. E-mail: andrea.j.olschewski{at}physiologie.med.uni-giessen.de


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Chronic hypoxia results in both structural changes in the pulmonary artery and a sustained increase in pulmonary vascular tone. This study investigated the effects of subacute moderate hypoxia on expression and function of potassium (K+) channels in rat pulmonary artery myocytes (PASMCs). The rats were kept at 0.67 atmospheres for 6, 12, or 24 h. We found that the expression of mRNA for voltage-activated K+ channels (Kv)1.2, Kv1.5, and Kv2.1 is reduced after less than 24 h of this moderate hypoxia. K+ current (Ik) is significantly inhibited in PASMCs from rats hypoxic for 24 h, resting membrane potential is depolarized and cytosolic [Ca2+] is increased in these cells. In addition, antibodies to Kv1.2, Kv1.5, and Kv2.1 inhibit Ik, cause membrane depolarization and attenuate both hypoxia- and 4-AP–induced elevation in [Ca2+]i in PASMCs from normoxic rats but not from 24 h hypoxic rats. Subacute hypoxia does not completely remove the mRNA for Kv1.2, Kv1.5, and Kv2.1, but antibodies against these channels no longer alter Ik or cytosolic calcium, suggesting that subacute hypoxia may inactivate the channels as well as reduce expression. As the expression of mRNA for Kv1.2, Kv1.5, and Kv2.1 is sensitive to subacute hypoxia and decreased expression/function of these channels has physiologic effects on membrane potential and cytosolic calcium, it seems likely that these Kv channels may also be involved in the mechanism of high-altitude pulmonary edema and possibly in the signaling of chronic hypoxic pulmonary hypertension.

Abbreviations: 4-aminopiridine, 4-AP • ethylene glycol-bis(ß-aminoethyl ether) N,N,N',N'-tetraacetic acid, EGTA • resting membrane potential, Em • high-altitude pulmonary edema, HAPE • N-(2-hydroxyethyl)-piperazine-N'-(2-ethane sulfonic acid), HEPES • hypoxic pulmonary vasoconstriction, HPV • potassium current, Ik • inward-rectifier potassium channel, Kir • voltage-activated potassium channels, Kv • optical density, OD • pulmonary artery, PA • rat pulmonary artery myocytes, PASMCs • polymerase chain reaction, PCR • reverse transcriptase, RT


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
Potassium (K+) channels are tetrameric arrangements of structural protein subunits in the cell membrane, whose role in O2-sensing in different cell types has been investigated since 1,988 (1). In smooth muscle cells from resistance pulmonary arteries (PASMCs) it is now well established that one of the immediate cellular responses to hypoxia is inhibition of one or several K+ channels, leading to cell depolarization, opening of voltage-gated Ca2+ channels, and myocyte contraction (2–6). This hypoxic pulmonary vasoconstriction (HPV) is a physiologic response of small pulmonary arteries that diverts mixed venous blood away from hypoxic alveoli, thus optimizing the matching of perfusion and ventilation and reducing arterial hypoxemia. The potassium channels which control resting membrane potential in PASMCs appear to belong to the family of voltage-dependent K+ (Kv) channels. At the molecular level, Kv channels are homo- or heteromultimeric tetramers that are composed of two structurally distinct subunits: the pore forming {alpha}-subunits and the regulatory ß-subunits (7). The potential candidate Kv channel {alpha}-subunits that could form O2-sensitive channels in PASMCs are Kv1.2 (8, 9), Kv1.5 (8, 10), Kv2.1 (9–11), Kv3.1 (12), and Kv9.3 (9, 11).

Chronic hypoxia in obstructive lung disease, or during a prolonged stay at high altitude, results in both structural changes in the PA and a sustained increase in pulmonary vascular tone. Despite their pathophysiologic importance, the molecular and cellular mechanisms underlying these phenomena have not been fully elucidated. The increase in vascular tone relates, to membrane depolarization of PASMCs (13) and to an increase in intracellular calcium concentration ([Ca2+]i) of PASMCs (14). Electrophysiologic studies have revealed that chronic hypoxia induces a downregulation of Kv in PASMCs (8, 15–17). Moreover, it has been shown that exposure to chronic hypoxia downregulates mRNA and protein expression of Kv1.1, Kv1.2, Kv1.5, and Kv2.1, that constitute delayed-rectifier Kv channels in PASMCs (8, 18). Downregulation of Kv channels by chronic hypoxia could thus explain the observed depolarization of PASMCs (19). Although various studies have been performed to study the mechanisms of PASMC adaptation to chronic hypoxia, no information is available on how subacute hypoxia may affect Kv channel activity, membrane potential, and change in [Ca2+]i. The present study was undertaken to investigate the sensitivity to subacute (6, 12, and 24 h) moderate hypoxia of different Kv channels in PASMCs. Hypoxia of this duration and severity can cause high-altitude pulmonary edema (HAPE). Preliminary results of these studies have been published (20).


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
All animal studies were approved by the Institutional Animal Care and Use Committee of the Minneapolis VA Medical Center and conform to current National Institutes of Health and American Physiological Society guidelines for the use and care of laboratory animals.

Subacute Hypoxia
Adult male Sprague-Dawley rats (300–350 g) (Harlan, Indianapolis, IN) were randomly allocated into subacute (6–24 h) hypoxic and normoxic groups. The rats of the subacute hypoxic group were maintained in a hypobaric chamber at a simulated altitude of 3,000 m (0.67 atmospheres) for 6, 12, or 24 h. Control animals (normoxic group) were maintained the same length of time in room air. All studies were begun within 1 h of removal from the hypobaric chamber.

Reverse Transcriptase–Polymerase Chain Reaction
Reverse transcriptase (RT)-polymerase chain reaction (PCR) was used to investigate the effects of subacute hypoxia on expression of K+ channels in rat pulmonary arteries. The lungs were removed under anesthesia and immediately placed on ice. Resistance PAs were carefully isolated and the endothelial and adventitial tissue removed. Total RNA was extracted with the guanidinium thiocyanate-phenol-chloroform method (GIBCO BRL Kit, Rockville, MD). After homogenization, the samples were processed according to the reagent instructions. To eliminate residual contaminating genomic DNA, the RNA preparation was further treated with RQ1 RNase-free DNase (1 U/1µg RNA) for 30 min at 37°C, then repurified by phenol:chloroform extraction and ethanol precipitation. The RNA was dissolved in diethyl pyrocarbonate–treated water and stored at –70°C. Optical density (OD) was measured to determine the RNA concentration (ratio of OD at 260 nm to OD at 280 nm > 1.7).

One microgram of RNA was reverse-transcribed in first-strand cDNA synthesis reagent (20 µl; Clontech, Palo Alto, CA) with oligo (dT) 18 primer used for each sample. The final reaction was diluted to a volume of 100 µl, and then 5 µl of this RT reaction taken for each PCR. Oligonucleotide primers used to amplify Kv channel {alpha}-subunits (Kv1.2, Kv1.3, Kv1.5, Kv2.1, Kv3.1), Kv ß–subunits (ß1, ß2, ß3) and calcium-sensitive potassium channels (KCa) are shown in Table 1.


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  		TABLE 1. Oligonucleotide sequence of primers used for RT-PCR

 
The cDNA samples were amplified in an MJ Research (Waltham, MA) thermocycler. The number of PCR cycle for ß-actin was 20, for Kv1.2, Kv2.1, KCa was 30, for Kv1.3, Kv1.5, Kv3.1, Kvß1, Kv ß2, Kv ß3 was 35. The PCR products were electrophoresed through a 1.5% agarose gel, and amplified cDNA bands were visualized by Gel Dol 2,000 (Bio-Rad, Hercules, CA). An invariant mRNA of ß-actin was used as an internal control to quantify the PCR products. Densitometry was used in relative semiquantitative assessment of the RT-PCR product with NIH Image. The OD values in the channel signals were normalized to the OD values in the ß-actin signals; the ratios are expressed as arbitrary units for semi-quantitative comparison. ß-actin is not thought to be changed by hypoxia over 24 h (21, 22).

Cell Isolation
Adult rat smooth muscle cells from resistance PAs were freshly dissociated for patch-clamp and Ca2+ imaging studies every day. PASMCs were isolated using a method adapted from that described previously (23).

Several digestions were done each day to ensure cell viability. Gentle trituration produced a suspension of single cells, which was then aliquoted into a perfusion chamber on the stage of an inverted microscope (Diaphot 200; Nikon, Tokyo, Japan). After a brief period to allow partial adherence to the bottom of the recording chamber, cells were perfused via gravity with an extracellular solution (see SOLUTIONS) at a rate of 2 ml/min.

Electrophysiology
To investigate changes in K+ current (Ik) and resting membrane potential (Em) caused by subacute hypoxia and the effects of antibodies to Kv1.2, Kv1.5, and Kv2.1, the conventional, whole cell patch-clamp technique was used (24). It was decided to administer the antibodies intracellularly as this is the only way to rapidly deliver the antibodies and thus clearly show acute changes in Ik and Em (10, 25, 26). Voltage and current clamp measurements were performed as previously described (23). Patch pipettes were pulled from glass tubes (PG 150T; Warner Instruments Corp., Hamden, CA). The pipettes were fire-polished directly before the experiments and had a resistance of 2–3 M{Omega} when filled with a pipette solution. The patch-clamp amplifiers were Axopatch 200A and B (Axon Instruments, Foster City, CA) in all voltage- and current-clamp experiments. Offset potentials were nulled directly before formation of a seal. No leak subtraction was made. Leakage current was monitored using hyperpolarizing steps (–30 mV) from the holding potential, a procedure that did not activate ion channels, but allowed measurement of passive membrane properties and leak during the experiments. Cells expressing holding current at –70 mV of > 20 pA before or during the recordings were discarded. Smooth muscle cells were voltage-clamped at a holding potential of –70 mV. The standard protocol used to obtain current-voltage relationships consisted of 300 ms voltage-clamp pulses applied in 20-mV steps between –70 and +50 mV. Estimation of cell capacitance (in pF) was made from whole-cell capacitance compensation, with current density calculated by dividing each whole-cell amplitude by cell capacitance (pA/pF) and plotting this against the test potential. The effective corner frequency of the low-pass filter was 1 kHz. The frequency of digitization was at least twice that of the filter. For resting membrane potential, cells were held in current-clamp at their resting Em (without current injection).

For the antibody-blocking experiments, electrodes were dipped into a filtered antibody-free pipette solution and then back-filled with the pipette solution containing the antibodies of interest with a 1:125 dilution. Series resistance and leak were checked at the beginning and end of each membrane potential experiment to eliminate artifactual changes in potential.

The data were stored and analyzed with commercially available pCLAMP 8.0 software (Axon Instruments).

All experiments were performed at 30°C.

Measurement of Intracellular Ca2+
[Ca2+] was measured by dual-excitation ratiometric imaging, using fura-2 (27). Freshly dispersed cells were transferred to the experimental chamber (Molecular Probes, Eugene, OR) and exposed to the antibody mixture of Kv1.2, Kv1.5, Kv2.1, or saline (1:400) for 1 h to permit membrane permeabilization, and then loaded in Ca2+-free extracellular solution with 0.1 µM fura-2-AM and 0.8 µM pluronic acid for 15 min at room temperature (23). The plates were then washed with extracellular solution containing 2.0 mM Ca2+ and incubated at room temperature for a further 15 min. This loading method allows low concentrations of fura-2 to be quickly introduced into the cells without the potential effects on cell morphology that may occur from long exposures to high concentrations. Background fluorescence was recorded from each dish of cells and subtracted before calculation of the 340- to 380-nm ratio. Changes in [Ca2+]i were measured using a cooled CCD camera (Hamamatsu, Japan) with MetaFluor image capture and analysis software (Universal Imaging Corp., West Chester, PA). Individual exposure times were adjusted so that similar gray values were used for both wavelengths. Measurements were made every 5 s and [Ca2+]i was calculated according to the method of Grynkiewicz and colleagues (27). A dissociation constant of 220 nM was calculated from in vitro calibration. Maximal and minimal ratio values were determined at the end of each experiment by first treating the cells with 1 µM ionomycin (maximal ratio) and then chelating all free Ca2+ with 10 mM EGTA (minimal ratio). Any cells not responding to ionomycin were discarded, as were cells showing significant photobleaching.

Plates were perfused with a warmed experimental solution (30°C) bubbled with normoxic gas mixture. PASMCs received either 4-AP (5 mM) or hypoxic experimental solution for 4 min. In either case, this was followed by angiotensin-II (AII, 1 µg). Peak increases in [Ca2+]i were measured during each intervention and data are given as averaged peak values.

Solutions and Drugs
The extracellular or experimental solution contained (in mM): NaCl 115, KCl 5.4, MgCl2 1, CaCl2 2.0, NaHCO3 25, HEPES 10, glucose 10, (pH 7.4 with NaOH). The standard intracellular pipette solution contained (in mM): KCl 145, MgCl2 1, ATP 1, EGTA 0.1, HEPES 10 (pH was adjusted to 7.2 by KOH).

Experimental solutions were equilibrated with 21% O2, 5% CO2 and 74% N2 or 0% O2, 5% CO2, balance N2. These procedures produced PO2 values in the cell chamber of 140–160 mm Hg under normoxic and 11–18 mm Hg under hypoxic conditions. PCO2 was 36–42 mm Hg, and pH was 7.37 to 7.42 under these conditions.

Fura-2AM was obtained from Molecular Probes and the anti-Kv antibodies were obtained from Alomone Laboratories (Jerusalem, Israel). All other compounds were purchased from Sigma Chemical Co. (St. Louis, MO). Antibodies were prepared according to the manufacturer's instructions. All drugs were dissolved in experimental solution. pH of solutions containing drugs was tested and corrected to eliminate potential pH-induced effects.

Statistical Analysis
Numerical values are given as mean ± standard error of the mean (SEM) of n cells. In all figures the SEM is indicated when it exceeds the symbol size. Student's unpaired t tests were used to compare membrane potential recordings and changes in Ik. Intergroup differences in changes of mRNA expression of Kv channel, effects of acute hypoxia on Em and intracellular Ca2+ levels were assessed by a factorial ANOVA with post hoc analysis with Fisher's least significant difference test. P values < 0.05 were considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Subacute Hypoxia Decreases mRNA Expression of Kv Channel {alpha}-Subunits in PASMCs
Using RT-PCR, we investigated the expression of a subset of Kv channels that have been previously reported to be present in pulmonary arteries and involved in hypoxic vasoconstriction. Exposure to hypoxia for 24 h did not affect significantly the total amount of protein in resistance pulmonary arteries. However, subacute hypoxia of 24 h significantly attenuated the mRNA levels of Kv 1.2, Kv1.5, and Kv2.1 (Figure 1). Moreover, the mRNA levels of Kv1.2 and Kv2.1 were markedly decreased after only 6 h moderate hypoxia (Figure 2). Unlike Kv1.2, Kv1.5 and Kv2.1, the expression levels of Kv 1.3, Kv3.1, and Ca2+-activated K+ channels (KCa) mRNA did not show a significant difference between the two groups (Figure 1; normoxic versus subacute hypoxic group).



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  		Figure 1. Moderate subacute hypoxia decreases mRNA expression of Kv1.2, Kv 1.5, and Kv2.1 in PASMCs. (A) Representative gel shows mRNA expression of Kv1.2 (407 bp, lane 2), Kv1.3 (238 bp, lane 3), Kv1.5 (340 bp, lane 4), Kv2.1 (557 bp, lane 5), Kv 3.1 (414 bp, lane 6), KCa (312 bp, lane 7), and Kv ß-subunits (lanes 8–10) in PASMCs under normoxic conditions and after exposure to 24 h moderate hypoxia. Lane 1 shows the molecular weight marker used to indicate the size of the PCR fragments. (B) Effect of moderate 24 h hypoxia (striped bars) on mRNA levels of Kv {alpha}- and ß-subunits, and KCa in PASMCs. Data that were normalized to the amount of ß-actin are expressed as means ± SEM (experiments were repeated six times independently). **P < 0.01 versus normoxic controls (0 h, open bar).

 


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  		Figure 2. Effect of short-term (6 h [shaded bars], 12 h [striped bars], and 24 h [cross-hatched bars]) hypoxia on mRNA levels of Kv1.2, Kv1.5, and Kv2.1 {alpha}-subunits in PASMCs. (A) PCR amplified products are displayed in agarose gel for Kv1.2, Kv1.5, Kv2.1 and ß-actin transcripts. Lane 1 shows the molecular weight marker used to indicate the size of the PCR fragments. (B) Data were normalized to the amount of ß-actin in PASMCs in normoxia (0 h) or after 6, 12, and 24 h moderate hypoxia. Values are means ± SEM (experiments were repeated 6 times independently). *P < 0.05, **P < 0.01 versus normoxic controls (0 h, open bar).

 
In contrast to the inhibitory effect on the pore-forming {alpha}-subunits (Kv1.2, Kv1.5, and Kv2.1) in PASMCs, subacute hypoxia negligibly affected the mRNA levels of the regulatory ß-subunits (Kvß1, Kvß2, Kvß3). As an internal control, hypoxia had no effect on mRNA-expression of ß–actin (Figure 1). These results suggest that short-term hypoxia-induced downregulation of Kv channel mRNA expression was selective for {alpha}-subunits in PASMCs.

Ik Downregulation in Response to Hypoxia
To evaluate the effect of moderate subacute hypoxia and to test the contribution of particular Kv channels (Kv1.2, Kv1.5, and Kv2.1) to whole-cell outward Ik and resting membrane potential (Em), the whole-cell patch-clamp technique was used in PA smooth muscle cells. There was no significant change in cell capacitance after 24 h moderate hypoxia (13.03 ± 0.46 pF control, n = 38 versus 13.01 ± 0.61 pF subacute hypoxia, n = 41).

Consistent with the molecular biological results, 24 h of moderate hypoxia significantly decreased whole cell Iks recorded from PASMCs (Figure 3). The averaged current density–voltage relationship for Ik is shown in Figure 3A. The hypoxia-sensitive Ik was activated at a potential close to the resting membrane potential in these cells. Subacute hypoxia caused a 46% decrease in Ik of PASMCs at –30 mV and a 21% decrease at +50 mV as shown in Figures 3A and 3B. The average resting Em value in PASMCs, measured by using current-clamp technique under normoxic conditions, was –52.6 ± 0.8 mV (n = 52; Figure 4A). Resting membrane potentials recorded from SMCs from the subacute hypoxic rats were significantly depolarized compared with controls (–45.0 ± 1.0 mV, n = 49, P < 0.001).



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  		Figure 3. Subacute hypoxia inhibits whole-cell voltage-gated K+ current. (A) Average current density-voltage plot showing current (I) densities (in pA/pF) recorded from single PASMCs from normoxic rats and from PASMCs of the subacute hypoxia group. Currents were activated by 20-mV voltage steps from a holding potential of –70 mV to +50 mV. (B and C) Summarized data showing current density of PASMCs elicited by a test potential at –30 mV and +30 mV in normoxic and hypoxic cells. Values are mean ± SEM. Numbers of cells shown in parentheses. **P < 0.01 for difference from control.

 


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  		Figure 4. Modulation of resting membrane potential (Em) of PASMCs under normoxic and hypoxic conditions. (A) Summarized data showing Em in single PASMCs from normoxic rats and from PASMCs of the subacute hypoxia group. (B and C) Recording of Em for 1 and 8 min after onset of dialysis with vehicle, anti-Kir2.1/Kir2.3/Kir4.1 control-antibodies or anti-Kv1.2/Kv1.5/Kv2.1 antibodies in the patch pipette. B shows Em in control PASMCs, whereas C shows Em from the subacute hypoxia group. Values are mean ± SEM of Em measured with current-clamp (I = 0) in PA smooth muscle cells. ***P < 0.001 for difference from control. (D) Change in membrane potential. Values are mean ± 95% confidence interval (CI) of Em measured with current-clamp (I = 0). Numbers of cells shown in parentheses.

 
To further demonstrate that Kv1.2, Kv1.5, and Kv2.1 subunits are the molecular substrate for the Ik, beyond the resting membrane potential in PASMCs, we assayed the efficiency of the anti-Kv1.2/Kv1.5/Kv2.1 antibodies to block this outward current component. Whole-cell current-clamp experiments were performed with anti-Kv1.2/Kv1.5/Kv2.1 antibodies delivered to the cell by dialysis through the patch pipette. The first measurements were obtained upon breaking into the whole-cell configuration (1 min) in current-clamp described in MATERIALS AND METHODS. When the control (normoxic) PASMCs were dialyzed with anti- Kv1.2/Kv1.5/Kv2.1, a time-dependent irreversible depolarization was observed, indicating that the Kv1.2, Kv1.5, and Kv2.1 subunits form the channels that contribute to the Em in PASMCs (Figure 4B). The change in Em induced by antibody application usually reached steady-state within 6–8 min. Consistent with the reduction of mRNA levels of Kv 1.2, Kv1.5 and Kv2.1 in PASMCs of rats from the 24 h subacute hypoxic group, the dialysis of these antibodies did not change the Em in the PASMCs (Figure 4C). Control recordings were made either in the presence of albumin (vehicle control) or in the presence of anti–inward-rectifier K+ channels (Kir)-Kir2.1/Kir2.3/Kir4.1 antibodies in the pipette solution (antibody control), to exclude nonspecific effects.

The sensitivity of the Em of PASMCs to acute hypoxia was also examined in both normoxic and subacute hypoxic rats (Figure 5). Superfusion of PASMCs with experimental solution bubbled with 0% O2 caused a significant partial depolarization in normoxic and subacute hypoxic rats (17.8 ± 3.3 mV, n = 12 and 11.8 ± 3.2 mV, n = 9, respectively) that was reversible on return to normoxia (Figures 5A and 5B). The immunological blockade of K+ channels is a powerful tool with which to assay the role of particular channel subunits under a variety of physiologic conditions, e.g., hypoxia. In anti-Kv1.2/Kv1.5/Kv2.1–treated PASMCs from both groups, no additional effect on Em was seen after exposure to acute hypoxia (Figures 5A and 5B).



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  		Figure 5. Effect of acute hypoxia on Em of PASMCs from normoxic and sub-acute hypoxic rats. (A) Average Em of PASMCs from normoxic rats under normoxic conditions (control 1) and after 8 min exposure to acute hypoxia (left). Summarized data showing Em in anti-Kv1.2/Kv1.5/Kv2.1 antibody-treated cells (intracellularly) under normoxia, 1 min (control 2) and after 8 min acute hypoxia (right). (B) Average Em of PASMCs from subacute hypoxic rats under normoxic conditions (control 1) and after 8 min exposure to acute hypoxia (left). Summarized data showing Em in anti-Kv1.2/Kv1.5/Kv2.1 antibody-treated cells (intracellularly) under normoxia, one minute (control 2) and after 8 min acute hypoxia (right). Values are mean ± SEM of the change in Em measured with current-clamp (I = 0) in PASMCs. Numbers of cells shown in parentheses. *P < 0.05 and ***P < 0.001 for difference from control.

 
Measurement of [Ca2+]i and Effects of Anti-Kv1.2/Kv1.5/Kv2.1 Antibodies
Subacute hypoxia (24 h) caused an increase in intracellular [Ca2+]i in PASMCs. Average resting [Ca2+]i under normoxia was calculated to be 109 ± 4 nM (n = 18) in the control (normoxic) group. PASMCs from rats kept in the hypobaric chamber for 24 h showed a higher [Ca2+]i of 143 ± 3 nM (n = 23, P < 0.01). Elevated basal [Ca2+]i was seen in the anti-Kv1.2/Kv1.5/Kv2.1–treated cells of the control group (146 ± 11, n = 18, P < 0.01). In contrast, the [Ca2+]i did not differ among groups after 24 h hypoxia, with or without pretreatment of anti-Kv1.2/Kv1.5/Kv2.1 (150 ± 7 nM, n = 25; 143 ± 3 nM, n = 23, respectively).

Acute hypoxia and 4-AP caused a rapid increase in [Ca2+]i of PASMCs in the normoxic group. Anti-Kv1.2/Kv1.5/Kv2.1 attenuated both hypoxia- and 4-AP–induced elevation in [Ca2+]i significantly, without altering the response to AII (Figure 6). In contrast, acute hypoxia or exposure to 4-AP showed a significant but relatively small effect on [Ca2+]i in PASMCs from rats in the 24 h subacute hypoxia group without altering the response to AII (Figure 6). The incubation with anti-Kv1.2/Kv1.5/Kv2.1 antibodies did not change the effect of acute hypoxia or 4-AP on [Ca2+]i in these PASMCs (Figure 6).



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  		Figure 6. Modulation effect of anti-Kv1.2/Kv1.5/Kv2.1 antibodies on [Ca2+]i of control PASMCs. (A) Superfusion of control PASMCs with a bath solution bubbled with 0% O2 significantly increased [Ca2+]i (318 ± 17 nM, n = 18, P < 0.01). Preincubation with anti-Kv1.2/Kv1.5/Kv2.1 antibodies decreased the hypoxia-induced increase in [Ca2+]i. (160 ± 13 nM, n = 18, P < 0.01). Bath application of 5 mM 4-AP increased [Ca2+]i (211 ± 13 nM, n = 15, P < 0.01). Pretreating with anti-Kv1.2/Kv1.5/Kv2.1 antibodies attenuated the 4-AP induced increase in [Ca2+]i (145 ± 10 nM, n = 10, P < 0.01). Antibody did not interfere with the rise in [Ca2+]i caused by A-II. (B) Superfusion of PASMCs from the subacute hypoxia group with a hypoxic solution significantly increased [Ca2+]i (139 ± 12 nM, n = 23, P < 0.01). Preincubation with anti-Kv1.2/Kv1.5/Kv2.1 antibodies did not change the hypoxia-induced increase in [Ca2+]i (131 ± 13 nM, n = 25). Bath application of 5 mM 4-AP increased [Ca2+]i (111 ± 9 nM, n = 11, P < 0.01). Preincubation with anti-Kv1.2/Kv1.5/Kv2.1 antibodies did not change the 4-AP induced increase in [Ca2+]i (96 ± 8 nM, n = 14). Antibody did not interfere with the rise in [Ca2+]i caused by A-II. Values are mean ± SEM. *P < 0.01 for difference from the response to acute hypoxia of normoxic rats. #P < 0.01 compared with 4-AP response of normoxic rats.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The results from this study demonstrate that 24 h of hypoxia downregulates gene transcription of Kv 1.2, Kv1.5, and Kv2.1 channel {alpha}-subunits in PASMCs, but negligibly affects transcription of KCa and Kv channel ß-subunits. Moreover, the mRNA levels of Kv1.2 and Kv2.1 were markedly decreased after only 6 h moderate hypoxia. Similar findings were reported previously for chronic hypoxia (8, 17, 18), but data for PASMCs subjected to short-term hypoxic stimuli (24 h or less) are lacking. In contrast to previous investigations, we studied not only the regulation of Kv channels by subacute hypoxia, but also their effects on resting membrane potential and cytosolic [Ca2+] in freshly isolated PASMCs of rats kept under moderate hypoxic conditions. Consistent with the postulated role of Kv 1.2, Kv1.5, and Kv2.1 as hypoxia-sensitive K+ channels in PASMCs at least in rats, 24 h hypoxia reduces whole cell Kv currents, depolarizes PASMCs, and consequently increases cytosolic [Ca2+].

The activity of certain Kv channel subtypes and KCa channels has been shown to be O2 sensitive. Because K+ channels are important determinants of vascular tone control and the proliferative status of vascular smooth muscle cells, the role of K+ channels and membrane potential have been investigated in several animal models of chronic pulmonary hypertension. Significant alterations in the electrophysiologic parameters of freshly isolated PASMCs from chronically hypoxic animals have been observed as early as 2 d after exposure to decreased O2 (17). We wondered whether a shorter exposure to less marked hypoxia (altitude of 3,000 m) causes significant changes in electrophysiological properties of PASMCs. We found that as little as 24 h hypoxia significantly inhibits Kv current. Resting membrane potentials recorded from SMC from the subacute hypoxic rats are significantly depolarized compared with controls. Similar results have been observed in PASMCs from chronically hypoxic animals after 2–4 wk exposure to hypoxia (15, 16, 28). These findings differ somewhat from those reported by Peng and colleagues (29). In that earlier study, chronic hypoxia reduced the activity of KCa, whereas the delayed rectifier K+ current remained unaffected in cultured human main pulmonary arterial smooth muscle cells. This discrepancy may relate to differences in electrophysiologic properties of PASMCs from conduit and resistance pulmonary arteries (5), in vivo and in vitro exposure to hypoxia and to differences between cultured and freshly isolated cells.

It has been proposed that the observed reduction in K+ current amplitudes is as a result of decreased channel expression. The first evidence for a downregulation of Kv channels {alpha}-subunit (Kv1.2 and Kv1.5) in cultured rat PASMCs under chronic hypoxia was provided by Wang and coworkers (8). More recently it was reported that chronic hypoxia also decreases the mRNA expression of Kv1.1, Kv1.5, Kv2.1, Kv4.3, and Kv9.3 {alpha}-subunits in cultured rat PASMCs (18, 30). These results are similar to several reports in freshly isolated PASMCs from chronically hypoxic animals (13, 16, 17, 28). Consistent with the reduction of mRNA levels of Kv1.2, Kv1.5 and Kv2.1, as assessed by semiquantitative RT-PCR, in PASMCs of rats from the subacute hypoxic group, the acute dialysis of antibodies against these channels did not change the Em in these cells. Exposure to severe acute hypoxia (11–18 mm Hg PO2) significantly depolarized PASMCs from both groups, though the depolarization was less marked in PASMCs from subacute hypoxic rats. Thus, it appears that the changes observed in cellular electrophysiology of subacute hypoxic PASMCs do not reflect the loss of all hypoxia-sensitive K+ channels. There is still persistence of hypoxia-sensitive Ik and membrane depolarization under severe acute hypoxia. The residual depolarization may be the result of hypoxic inhibition of the expression or function of other 4-AP–sensitive K+ channels.

Reduction of the Kv channel activity and the consequent membrane depolarization appears to be involved in the development of chronic hypoxic pulmonary hypertension by mediating pulmonary vasoconstriction and vascular remodeling through increased resting [Ca2+]i in PASMCs. Exposure to subacute hypoxia causes an elevated [Ca2+]i, at least in part as a result of membrane depolarization due to decreased Kv channel activity, a finding consistent with observations in cultured PASMCs (18). To demonstrate the link between the effects of the antibodies on Ik, Em and the effects on tone, we studied the ability of acute hypoxia to raise [Ca2+]i in PASMCs from both control and the subacute hypoxic rats. Acute hypoxia caused still an increase in [Ca2+]i of PASMCs in both groups. Pretreatment with anti-Kv1.2/Kv1.5/Kv2.1 attenuated the hypoxia-induced elevation in [Ca2+]i significantly in controls, but did not change the effect of acute hypoxia on [Ca2+]i in PASMCs from subacute hypoxic rats. Some of this increase in [Ca2+]i may reflect release from the sarcoplasmic reticulum, unreleated to changes in Ik or Em.

The main findings of the present study are that PASMCs responded to short-term hypoxia (equivalent to an altitude of ~ 3,000 m) with a significant downregulation of Kv1.2, Kv1.5, and Kv2.1 mRNA expression, marked depolarization of the resting membrane potential and elevated [Ca2+]i levels. Persistent alveolar hypoxia often leads to the development of pulmonary hypertension associated with both structural and functional changes in the PA, including a decreased pressor response to subsequent acute hypoxic challenges. Our results suggest that the reduced pressor response to acute hypoxia in the subacute hypoxic rats may result from abnormalities in the pathway of hypoxia-sensing, related to inactivation and/or lack of expression of several Kv channels. In addition, such changes in K+ channel function occurring within 24 h might explain the pulmonary hypertension that occurs in HAPE (31) and indeed the mechanism of the increased pulmonary capillary pressure (31), if the same changes occur in K+ channels in the SMC of pulmonary veins (32).


   Acknowledgments
 
A.O. is supported by the Deutsche Forschungsgemeinschaft (DFG Ol 127/1-1 and SFB 547). E.K.W. is supported by VA Merit Review Funding and NIH (RO1-HL 65322-01A1).

Received in original form October 29, 2003

Received in final form April 26, 2004


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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