Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21224
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ABSTRACT |
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We tested the
hypothesis that chronic hypoxia alters the regulation of
K+ channels in intrapulmonary
arterial smooth muscle cells (PASMCs). Charybdotoxin-insensitive,
4-aminopyridine-sensitive voltage-gated K+
(KV,CI) and
Ca2+-activated
K+
(KCa) currents were measured in
freshly isolated PASMCs from rats exposed to 21 or 10%
O2 for 17-21 days. In
chronically hypoxic PASMCs, KV,CI
current was reduced and KCa
current was enhanced. 4-Aminopyridine (10 mM) depolarized both normoxic
and chronically hypoxic PASMCs, whereas charybdotoxin (100 nM) had no
effect in either group. The inhibitory effect of endothelin (ET)-1
(107 M) on
KV,CI current was significantly
reduced in PASMCs from chronically hypoxic rats, whereas inhibition by
angiotensin (ANG) II (10
7
M) was enhanced. Neither ET-1 nor ANG II altered
KCa current in normoxic PASMCs;
however, both stimulated KCa
current at positive potentials in chronically hypoxic PASMCs. These
results suggest that although modulation of
KV,CI and
KCa channels by ET-1 and ANG II is
altered by chronic hypoxia, the role of these channels in the
regulation of resting membrane potential was not changed.
voltage-gated potassium current; calcium-activated potassium current; membrane potential
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INTRODUCTION |
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PROLONGED EXPOSURE to decreased alveolar oxygen
tension, as occurs with many pulmonary diseases, leads to sustained
pulmonary vasoconstriction followed by vascular remodeling and
pulmonary hypertension. The morphological changes associated with
chronic hypoxia include smooth muscle cell hypertrophy and hyperplasia, muscularization of precapillary arterioles, and increased deposition of
extracellular matrix components (25, 33, 40). Functional changes that
occur in the pulmonary vasculature as a consequence of chronic hypoxia
include membrane depolarization (48, 51) and altered vasoreactivity in
response to endothelin (ET)-1, serotonin, angiotensin (ANG) II,
norepinephrine, prostaglandin
F2, acetylcholine, bradykinin, and drugs that open K+
channels or inhibit Ca2+ channels
(1, 10, 15, 18, 23, 24, 37, 43, 54).
Through control of Ca2+ influx and cytosolic Ca2+ concentration ([Ca2+]i), membrane potential may play a vital role in regulating vascular caliber and the proliferative state of smooth muscle cells. In intrapulmonary arterial smooth muscle cells (PASMCs), the resting membrane potential appears to be regulated predominantly by specific subtypes of voltage-gated K+ (KV) channels, which are 4-aminopyridine (4-AP) sensitive and charybdotoxin (ChTX) insensitive (KV,CI) because 4-AP, but not ChTX, causes membrane depolarization and increased [Ca2+]i (3, 47, 56). In vivo exposure to chronic hypoxia attenuates KV-channel currents (48), causes membrane depolarization (48, 51), and elevates basal [Ca2+]i (46) in PASMCs, whereas in vitro exposure of PASMCs to hypoxia reduces Ca2+-activated K+ (KCa)-channel activity (35). Under normoxic conditions, inhibiting KCa channels with ChTX has no effect on membrane potential or [Ca2+]i (3, 47, 56); however, under conditions where the membrane potential is depolarized or [Ca2+]i is increased, KCa channels operate as a negative feedback mechanism to repolarize membrane potential and reduce [Ca2+]i (9, 56). It is possible, therefore, that the depolarization, elevated [Ca2+]i, and altered vascular reactivity associated with chronic hypoxia involve changes in the activity and regulation of K+ channels.
Circulating factors may also influence membrane potential, contraction
and proliferation of PASMCs during exposure to chronic hypoxia. ET-1,
the most potent vasoconstrictor known to date, is abundantly present in
the pulmonary vasculature. Both acute and chronic exposure to hypoxia
increases ET-1 gene expression, transcription, secretion, and plasma
ET-1 levels (12, 17, 22, 33). It was recently demonstrated that ET-1
caused membrane depolarization, inhibited
KV current, and increased
[Ca2+]i
in PASMCs under normoxic conditions (5, 44, 47). ET-1 also activates
KCa and
Ca2+-activated
Cl channels secondary to
intracellular Ca2+ release in
PASMCs (5, 44). ANG II production may also be affected by prolonged
exposure to hypoxia. ANG-converting enzyme (ACE) activity is increased
in the small pulmonary arteries of chronically hypoxic rats, and both
ACE inhibitors and ANG type 1 (AT1) receptor antagonists
attenuate the development of chronic hypoxic pulmonary hypertension
(27, 28, 32, 59). Contraction induced by ANG II is enhanced in arteries
from hypoxic lungs, and the signal transduction pathways involved in
ANG II-induced contraction have many similarities to those of ET-1,
including inhibition of KV
currents, depolarization, and increased
[Ca2+]i (11, 20, 49).
Exposure to chronic hypoxia appears to alter the electrophysiological characteristics of PASMCs, resulting in cells that exhibit membrane depolarization, reduction in KV- and/or KCa-channel activity, and increased resting [Ca2+]i (35, 46, 48, 51). Because K+-channel inhibition is common to the signal transduction pathways of both ET-1 and ANG II, two important mediators of pulmonary vascular tone, we hypothesized that in addition to altering the basal activity of the KV,CI and ChTX-sensitive, 4-AP-insensitive KCa channels and the contribution of these channels to the control of resting membrane potential, exposure to chronic hypoxia would also alter the modulation of these channels by ET-1 and ANG II. To test this hypothesis, we used whole cell patch-clamp techniques in PASMCs from rats exposed to 10% O2 for 17-21 days to determine the effect of chronic normobaric hypoxia on 1) basal KV,CI and KCa currents and membrane potential, 2) the effects of K+-channel antagonists on membrane potential, and 3) the effects of ET-1 and ANG II on KV,CI and KCa currents.
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METHODS |
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Chronic Hypoxia
Male Wistar rats (150-250 g) were placed in a hypoxic chamber and exposed to either normoxia or normobaric hypoxia for 17-21 days. The chamber was continuously flushed with either room air or a mixture of room air and N2 (10 ± 0.5% O2) to maintain a low CO2 concentration (<0.5%). Chamber O2 and CO2 concentrations were continuously monitored (OM-11 oxygen analyzer and LB-2 gas analyzer, Sensormedics, Anaheim, CA). The rats were exposed to room air for 10 min twice a week to clean the cages and replenish food and water supplies.Cell Preparation
The rats were injected with heparin, anesthetized with pentobarbital sodium (130 mg/kg ip), and exsanguinated. The heart and lungs were removed en block and transferred to a petri dish of physiological salt solution (PSS) containing (in mM) 130 NaCl, 5 KCl, 1.2 MgCl2, 1.5 CaCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with 5 M NaOH. The right ventricle of the heart was separated from the left ventricle and the septum, and the two portions were weighed. The method for obtaining single PASMCs has been previously described (47). Briefly, intrapulmonary arteries (300-800 µm OD) were isolated and cleaned of connective tissue. After the endothelium was disrupted by gently rubbing the luminal surface with a cotton swab, the arteries were allowed to recover for 30 min in cold (4°C) PSS followed by 20 min in reduced-Ca2+ PSS (20 µM CaCl2) at room temperature. The tissue was digested in reduced-Ca2+ PSS containing collagenase (type I; 1,750 U/ml), papain (9.5 U/ml), bovine serum albumin (2 mg/ml), and dithiothreitol (1 mM) at 37°C for 20 min. After digestion, single smooth muscle cells were dispersed by gentle trituration with a wide-bore transfer pipette in Ca2+-free PSS, and the cell suspension was transferred to the cell chamber for study.Electrophysiological Measurements
The myocytes were continuously superfused with PSS containing (in mM) 130 NaCl, 5 KCl, 1.2 MgCl2, 2 CaCl2, 10 HEPES, and 10 glucose, with pH adjusted to 7.4 with 5 M NaOH. Patch pipettes (tip resistance 3-5 MExperimental Protocols
Effect of chronic hypoxia on whole cell
K+ currents.
To characterize the K+ currents
present in the rat PASMCs, membrane currents were activated by
depolarizing pulses of 800 ms from a holding potential of 60 mV
to test potentials ranging from
50 to +40 mV in +10-mV step
increments. These current-voltage (I-V)
relationship measurements were made under control conditions 3-4
min after the cells were treated with ChTX (100 nM) to inhibit KCa channels and 3-4 min
after the subsequent addition of 4-AP (10 mM) to inhibit
KV,CI channels. In subsequent
experiments, we isolated KV,CI
currents by pretreating the cells with ChTX (100 nM) or
KCa currents by pretreating the
cells with 4-AP (10 mM). The effect of chronic hypoxia on
KV,CI and
KCa currents was determined by
comparison of the
I-V
relationships of peak KV,CI- and
KCa-current densities measured in
cells from normoxic and hypoxic animals.
Effect of chronic hypoxia and K+-channel antagonists on membrane potential. Membrane potential was measured in current-clamp mode with I = 0. The effect of K+-channel antagonists on membrane potential was evaluated by measuring membrane potential for 1 min before, 2 min during, and 2 min after exposure to either 4-AP (10 mM) or ChTX (100 nM).
Effect of chronic hypoxia on the response of
KV,CI and KCa
channels to ET-1 and ANG II.
The effect of ET-1 and ANG II on
K+ current was determined by
exposing PASMCs to agonists in the presence of 100 nM ChTX
(KV,CI currents) or 10 mM 4-AP
(KCa currents).
K+ currents were activated at 5-s
intervals by a 400-ms step depolarization to +20 mV from a holding
potential of 60 mV while ET-1
(10
7 M) or ANG II
(10
7 M) was applied and
until stable currents (<1% change in magnitude) were attained. The
effect of agonists on peak KV,CI
and KCa currents was then
determined by comparing the
I-V
curves for both K+-current
components before and 3-4 min after exposure to ET-1 or ANG II.
The effect of chronic hypoxia on the response to the agonists was
determined by comparison of the
I-V
relationships in cells from normoxic and chronically hypoxic animals.
Drugs and Chemicals
ET-1 and ChTX were obtained from American Peptides (Sunnyvale, CA). ANG II was obtained from Calbiochem (San Diego, CA). All other chemicals were obtained from Sigma (St. Louis, MO). Stock solutions of ANG II (10Data Analysis
The amplitude of the currents is expressed as current density obtained by normalizing peak current with cell capacitance. To separate KV,CI current into rapidly and slowly inactivating and noninactivating components, the time course of inactivation of KV,CI-current density at +40 mV was fit with a biexponential equation (2, 47, 56): I(t) = Ao + A1e(Significance was determined with Student's t-test (paired or unpaired as applicable) and two-way analysis of variance (ANOVA) with repeated measures with a Student-Newman-Keuls post hoc test. A P value < 0.05 was accepted as significant. Data are expressed as means ± SE; n is the number of cells tested.
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RESULTS |
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Effect of Chronic Hypoxia on Right Ventricular Weight
Chronic hypoxia-induced pulmonary hypertension was verified by the development of right ventricular (RV) hypertrophy. After 17-21 days of chronic hypoxia, RV weight was significantly increased to 0.31 ± 0.01 g (P < 0.01; n = 32) compared with 0.16 ± 0.01 g in normoxic rats (n = 31). The ratio of RV weight to left ventricular plus septal (LV+S) weight was also significantly greater in chronically hypoxic rats (0.38 ± 0.01) than in normoxic rats (0.21 ± 0.01); however, LV+S weights were not significantly different.Effect of Chronic Hypoxia on K+ Currents
Average cell capacitance in normoxic and chronically hypoxic rats was 16.1 ± 1.2 (n = 23) and 15.1 ± 0.7 pF (n = 32), respectively. We (47) previously demonstrated that in the presence of ATP, whole cell outward K+ currents in PASMCs were primarily composed of KV,CI and KCa currents. Similar to PASMCs from normoxic rats, depolarizing pulses to test potentials positive to
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PASMCs from rats exposed to chronic hypoxia showed a significant
reduction in KV,CI-current
density, which was apparent as a downward shift in the
I-V
relationship (Fig. 2). Peak
KV,CI-current density was reduced
from 4.1 ± 1.5 (n = 8) to 1.01 ± 0.17 pA/pF (P < 0.01;
n = 19) at 20 mV and
from 35.0 ± 9.5 to 10.8 ± 1.8 pA/pF
(P < 0.005) at +40 mV. The time
course of inactivation of the
KV,CI current at +40 mV was fit
with a biexponential equation to separate
KV,CI current into rapidly and
slowly inactivating and noninactivating components (46). All three
components of KV,CI-current
density (at +40 mV) were significantly reduced after exposure to
chronic hypoxia, with the rapidly inactivating component decreasing
from 9.74 ± 2.6 to 5.48 ± 1.4 pA/pF, the slowly inactivating component decreasing from 5.27 ± 0.8 to 2.91 ± 0.5 pA/pF, and the noninactivating component decreasing from 13.76 ± 1.1 to 5.11 ± 1.1 pA/pF (P < 0.05). The fast
and slow time constants of inactivation were 16.9 ± 4.1 and 174.4 ± 29.8 ms, respectively, in cells from normoxic rats and 9.87 ± 1.2 and 158.6 ± 49.6 ms, respectively, in cells from chronically
hypoxic rats. The fast time constant appeared to be smaller in PASMCs
from chronically hypoxic rats, although the difference did not achieve
significance (P = 0.1). There was no apparent difference in the threshold or voltage
dependence of activation of KV,CI
channels between PASMCs from normoxic and chronically hypoxic rats.
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In contrast to the reduction in KV,CI current observed with chronic hypoxia, KCa-current density was significantly enhanced at test potentials positive to 0 mV as evidenced by an upward shift in the I-V relationship (Fig. 2). At +60 mV, peak KCa-current density was increased from 18.1 ± 4.1 (n = 7) to 61.7 ± 21.5 pA/pF (P < 0.01; n = 13). There was no apparent difference in the threshold and the voltage dependence for KCa-current activation between PASMCs from normoxic and chronically hypoxic rats.
Role of KV,CI and KCa Channels in Membrane Potential Regulation in PASMCs After Chronic Hypoxia
The resting membrane potential in PASMCs from chronically hypoxic rats was significantly depolarized to
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Effect of ET-1 on KV,CI and KCa Currents
In PASMCs from normoxic rats, ET-1 (10
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In PASMCs from normoxic rats, ET-1
(107 M) had no significant
effect on KCa-current density as
indicated by the
I-V
relationships shown in Fig. 5. Peak
KCa-current density at +60 mV was
18.12 ± 4.1 and 17.7 ± 3.9 pA/pF (n = 7)
before and during, respectively, the application of ET-1. In
contrast, application of ET-1 to PASMCs from chronically hypoxic rats
increased peak KCa-current density at potentials positive to +30 mV but had no effect at lower test potentials (Fig. 5). At +60 mV, peak
KCa-current density was increased from 70.1 ± 33.9 to 87.9 ± 37.1 pA/pF
(P < 0.05;
n = 8).
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Effect of ANG II on KV,CI and KCa Currents
In PASMCs from normoxic rats, ANG II (10
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ANG II had no significant effect on
KCa-current density in PASMCs from
normoxic rats except at the highest potential tested (+60 mV; 8.1 ± 1.1 to 9.3 ± 1.5 pA/pF; n = 6). In
contrast, KCa current in PASMCs
from chronically hypoxic rats was significantly increased by the
addition of ANG II at all test potentials positive to +10 mV
(P < 0.01;
n = 9). At +60 mV,
KCa-current density was increased
50% (19.1 ± 4.1 to 28.7 ± 7.5 pA/pF; Fig.
7).
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DISCUSSION |
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In the present study, PASMCs from rats chronically exposed (17-21 days) to hypoxia (10% O2) exhibited depolarized resting membrane potential, reduced KV,CI current, and increased KCa current. Application of 4-AP to these PASMCs caused depolarization, whereas ChTX had no effect on the membrane potential, results similar to those observed in PASMCs from normoxic rats. We also found that after exposure to chronic hypoxia, ET-1 had no effect on KV,CI current and had a stimulatory effect on KCa current at highly positive test potentials. This is in contrast to the marked ET-1-induced inhibition of KV,CI current and the lack of effect of ET-1 on KCa current in PASMCs from normoxic rats. In PASMCs from normoxic rats, ANG II inhibited only the steady-state portion of the KV,CI current and had no direct effect on the KCa current. In contrast to ET-1, the inhibitory effect of ANG II on the KV,CI current was enhanced in PASMCs from chronically hypoxic rats, whereas the stimulatory effect of ANG II on the KCa current observed in PASMCs from chronically hypoxic rats resembled that measured in response to ET-1. These data suggest that hypoxia altered the baseline activity of the KV,CI and KCa channels and the modulation of these channels by both ET-1 and ANG II but did not alter the absolute contribution of these channels to the regulation of resting membrane potential.
The chronically hypoxic rat model of pulmonary hypertension has been widely studied (8, 12, 14, 16, 25, 33, 40, 48, 51). Significant alterations in the electrophysiological parameters of PASMCs have been observed as early as 1 wk after exposure to decreased O2 was begun and remained stable during prolonged exposure (51). In the present study, rats were exposed to hypoxia for 3 wk to ensure development of pulmonary hypertension and alterations in membrane ion transport mechanisms. Development of pulmonary hypertension as a result of exposure to chronic hypoxia was confirmed by the presence of significant RV hypertrophy, with RV weights and RV-to-LV+S ratios consistent with those previously reported (8, 15, 33). Significant changes in the electrophysiological parameters of PASMCs were also observed after exposure to chronic hypoxia. The 50-70% decrease in KV,CI-current density we observed was quantitatively similar to the reduction in KV,CI-current density previously reported in PASMCs from rats exposed to 10% O2 for 4 wk (48). Our results stand in contrast, however, to a recent study (35) in human smooth muscle cells from main pulmonary arteries that showed that KCa-channel activity was decreased after 4 wk of culture in 5-7% O2 without significant change in KV-channel activity. The discrepancies could be related to differences between cultured and freshly isolated cells, in vivo and in vitro exposure to hypoxia, animal species, or the size of the vessels from which the PASMCs were isolated.
The hypoxia-induced reduction in
KV,CI current did not appear to be
confined to a specific kinetic component of
KV,CI current (47) because the
amplitudes of the fast, slow, and sustained components were all
decreased. Several KV-channel
-subunits have been identified in PASMCs, including
KV1.1,
KV1.2,
KV1.3,
KV1.4, KV1.5,
KV.16, and
KV2.1 (4, 53, 58). Unfortunately,
antagonists for specific
KV-channel
-subunits are not
currently available; however, the channels corresponding to the
KV,CI current we examined in this
study are likely to consist of the
KV1.5 and
KV2.1
-subunits because they
were insensitive to ChTX, whereas the
KV channels of other
-subunits
identified in rat PASMCs can be blocked by ChTX (4).
The mechanism(s) by which chronic hypoxia alters K+ currents is unclear. The alterations in KCa- and KV,CI-current density were not likely to have been mediated by the same mechanism as the reversible changes in K+ currents observed during acute hypoxia (3, 7, 38, 39, 57) because the currents were recorded in PASMCs that had been exposed to normoxic conditions throughout the isolation procedure and experiments. Furthermore, the changes in K+-current density were not due to increased [Ca2+]i, which inhibits KV channels (20, 38) and stimulates KCa channels (5, 44) because the cells were buffered with 10 mM BAPTA, which theoretically restricts the diffusion radius of Ca2+ to <30 nm around an open Ca2+-permeating channel (31, 50) and experimentally inhibited the local Ca2+ signaling between L-type Ca2+ channels and ryanodine receptors in cardiac diadic junctions, which have a cleft distance of <15 nm (45). Instead, possibilities that may account for the hypoxia-induced changes in K+ current include a change in the expression of K+-channel proteins (53), upregulation of protein kinase C (PKC) (13, 21, 26, 55), or a change in PASMC phenotype (19). Future studies will be required to further elucidate these possibilities.
Exposure to chronic hypoxia caused a sustained depolarization in our
PASMCs, findings consistent with observations in rat main (51) and
intralobar (48) pulmonary arteries and in smooth muscle cells cultured
from human main pulmonary arteries (35). Under normoxic conditions,
KV,CI channels contribute
significantly to the regulation of resting membrane potential and
vascular tone because inhibition of
KV,CI, but not of
KCa, channels results in
depolarization and increased
[Ca2+]i
(3, 47, 56). The reduction in
KV,CI current observed after
prolonged hypoxia in this study and by others (48) may contribute to
the observed depolarization, leading to enhanced Ca2+ influx and sustained
vasoconstriction. Interestingly, despite a reduction in current
density, KV,CI channels still
appear to play a significant role in membrane potential regulation
because 4-AP caused a depolarization in PASMCs from chronically hypoxic rats similar in magnitude to that observed in PASMCs from normoxic rats. The similar magnitude of depolarization is likely due to the fact
that at the elevated resting membrane potential of 20 mV, the
KV,CI-current density of 1.01 ± 0.2 pA/pF is similar to the value of 1.5 ± 0.2 pA/pF observed
at the resting membrane potential of
40 mV in PASMCs from
normoxic rats. Furthermore, the fact that the membrane potential was
significantly more positive in PASMCs from chronically hypoxic than
from normoxic rats after complete blockade of
KV,CI channels suggests that ionic
conductances with positive equilibrium potentials (i.e.,
Na+ or
Ca2+) could be enhanced and/or
chloride conductance could be inhibited by chronic hypoxia. Elucidating
the role of other ions in membrane potential regulation during chronic
hypoxia requires future experiments. On the other hand, ChTX had no
effect on the membrane potential in PASMCs from either normoxic or
chronically hypoxic rats, indicating that
KCa channels did not play a more
prominent role in the regulation of membrane potential after exposure
to chronic hypoxia even though the
KCa-current density was enhanced.
This finding was not surprising because
KCa channels were only activated
at potentials more positive than the resting membrane potential. Under
conditions where PASMCs are stimulated, however, such as during
exposure to vasoactive agonists, the enhancement of
KCa current may provide an extra capacity for repolarizing the membrane potential.
The modulation of both KV,CI and KCa channels by ET-1 and ANG II enhanced the alterations in basal K+ currents induced by exposure to chronic hypoxia. Although ET-1 caused a marked inhibition of KV,CI channels in PASMCs from normoxic rats, the inhibitory effect of ET-1 on KV,CI currents was significantly attenuated in PASMCs from chronically hypoxic rats. The inhibition of KV,CI current induced by ANG II was much smaller than that observed in response to ET-1. This may be due to the fact that, in contrast to ET-1, which inhibits all components of the KV current in normoxic PASMCs (47), ANG II only inhibited the steady-state or noninactivating portion of the KV,CI current. Furthermore, we (47) previously demonstrated that concentration-dependent ET-1-induced inhibition of the KV current was attenuated by inhibitors of phospholipase C and PKC and was mimicked by activators of PKC. Although ANG II has been shown to cause PKC-dependent inhibition of KV channels in portal vein smooth muscle (11), the effect of ANG II on KV channels in renal smooth muscle is entirely dependent on intracellular Ca2+ release (20). If this is indeed the case in PASMCs, then buffering intracellular Ca2+ with BAPTA would reduce the ability of ANG II to modulate the KV,CI current.
The divergence in the effect of chronic hypoxia on the modulation of KV,CI current by ET-1 and ANG II may reflect differences by which these agonists regulate KV,CI channels or a difference in receptor distribution with hypoxia. AT1 receptors, the predominant receptor subtype in both normoxic and hypoxic lungs (59), are increased after exposure to chronic hypoxia. Both ETA and ETB receptors mediate contraction in rat lungs, and the distribution of these receptors changes with chronic hypoxia (22, 30). The difference in the signal transduction pathways coupled to these receptor subtypes has yet to be defined, and thus further experiments are required to determine whether changes in receptor density and subtype can account for the observed changes in K+-channel responsiveness to these agonists.
It is intriguing that both ET-1 and ANG II stimulated KCa currents after exposure to chronic hypoxia, whereas under normoxic conditions, neither had a direct effect on KCa channels. In the present study, [Ca2+]i was highly buffered with 10 mM BAPTA; thus the effect of ET-1 and ANG II on the KCa current was clearly different from that previously observed, which depended on Ca2+ release from the sarcoplasmic reticulum (20, 44). Although the stimulatory effects of ET-1 and ANG II on the KCa channels were small and observed only at voltages well above the resting membrane potential, it suggests that changes in the signal transduction pathways common to ET-1 and ANG II occurred during exposure to chronic hypoxia. In addition to the activation of PLC, ET-1 and ANG II can also activate phospholipases A and D (36, 42), the products of which are known to stimulate KCa channels (34, 41). Although enhanced agonist-induced activation of these phospholipases might contribute to the altered effect of ET-1 and ANG II on KCa channels after exposure to chronic hypoxia, the exact mechanism(s) underlying these changes as well as the physiological significance of this stimulatory effect remains to be elucidated.
Roles for both ET-1 and ANG II in mediating the pathogenesis of hypoxic pulmonary hypertension have been proposed based on several lines of evidence. First, chronic exposure to hypoxia increases ET-1 gene expression, transcription, secretion, and plasma ET-1 levels (12, 17, 22, 33) and upregulates ACE activity in small pulmonary arteries (27). Second, the pulmonary contractile response to ET-1 and ANG II is enhanced (1, 6, 15, 23, 52), ETA, ETB, and AT1 receptors are upregulated (22, 30, 59), and ET-1-induced vasodilation is lost (15) after exposure to chronic hypoxia, suggesting modifications in the ET-1- and ANG II-mediated regulation of pulmonary vascular tone. Finally, treatment with ET-1-receptor antagonists, ACE inhibitors, and AT1-receptor antagonists prevents (8, 12, 14, 16, 27, 28, 33, 59) and, in the case of ET-1, partially reverses (12, 14) pulmonary hypertension associated with chronic hypoxia, whereas prolonged infusion of ET-1 mimics the sequelae of pulmonary hypertension due to chronic hypoxia (29). Although the exact mechanisms by which ET-1 and ANG II may mediate the pathogenesis of hypoxic pulmonary hypertension remain to be elucidated, our findings suggest that hypoxia-induced elevations in ET-1 and ANG II levels may potentiate the effects of chronic hypoxia on K+ currents and membrane potential, thus promoting the development of pulmonary hypertension by elevating [Ca2+]i, inducing vasoconstriction and PASMC proliferation.
In summary, exposure to chronic hypoxia alters not only the baseline activity of KCa and KV,CI channels in rat PASMCs but also their modulation by ET-1 and ANG II. The major changes in ionic transport mechanisms could be due to the direct effects of chronic hypoxia on the pulmonary vasculature or may be secondary to hypoxia-induced physiological changes such as hypertension or vascular remodeling.
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ACKNOWLEDGEMENTS |
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This work was supported by the National Heart, Lung, and Blood Institute Grants HL-52652 (to J. S. K. Sham), HL-51912 (to J. T. Sylvester) and HL-09543 (to L. A. Shimoda) and an American Heart Association Established Investigator Award (to J. S. K. Sham).
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FOOTNOTES |
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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. §1734 solely to indicate this fact.
Address for reprint requests and other correspondence: J. S. K. Sham, Division of Pulmonary and Critical Care Medicine, 5501 Hopkins Bayview Circle, Baltimore, MD 21224 (E-mail: jsks{at}welchlink.welch.jhu.edu).
Received 6 July 1998; accepted in final form 13 April 1999.
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