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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ABSTRACT |
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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 -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
-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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INTRODUCTION |
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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 F2, 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 -subunits that encode delayed
rectifier KV channels. The rationale of this study was to
examine further 1) whether hypoxia-mediated downregulation of KV channel
-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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METHODS AND MATERIALS |
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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 muscleTreatment 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 M) 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 M
) 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 (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), Kv1.1 (GenBank accession no. X70662), Kv
2 (GenBank
accession no. X76724), and Kv
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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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 -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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RESULTS |
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Linear relationship between PCR products and cycle number.
The quantity of PCR products for Kv1.5 and -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
- and
-subunits were normalized to the mRNA levels of
-actin for
comparison.
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Divergent effects of chronic hypoxia on mRNA expression of
KV channel -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
-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
-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
-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
-subunit Kv9.3 and the functional
-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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Divergent effects of chronic hypoxia on protein expression of
KV channel -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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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
-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
-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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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 G (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 G
. 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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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 -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
-subunits because
hypoxia negligibly affected the mRNA expression of the cytoplasmic
regulatory
-subunits (including Kv
1.1, Kv
2, and Kv
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 - and
-subunits identified in PASMCs
(49, 50, 82), such as Kv1.2, Kv1.4, Kv1.5, Kv2.1, Kv4.2,
Kv
1.1, Kv
2, and Kv
3, are also expressed in MASMCs
(74). Qualitative differences in KV channel
-subunits between PASMCs and MASMCs have not been observed so far.
The potential candidates of KV channel
-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
-subunits (e.g., Kv
1.1 and Kv
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
-
and
-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 -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-
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 -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 -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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ACKNOWLEDGEMENTS |
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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.
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FOOTNOTES |
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* 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.
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