Chronic hypoxia alters effects of endothelin and angiotensin on K+ currents in pulmonary arterial myocytes

Larissa A. Shimoda, J. T. Sylvester, and James S. K. Sham

Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21224


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 (10-7 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


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 F2alpha , 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.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 MOmega ) were pulled from glass capillary tubes, fire polished, and filled with an internal solution containing (in mM) 35 KCl, 90 potassium gluconate, 10 NaCl, 10 HEPES, and 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA), with pH adjusted to 7.2 with 5 M KOH. GTP (0.5 mM) was added to provide a substrate for the signal transduction pathways. Because [Ca2+]i may affect K+-channel activity (5, 20, 38), 3 mM Ca2+ was added to buffer the [Ca2+]i at a physiological level (~75 nM). MgATP (5 mM) was included to inhibit ATP-sensitive K+ currents and provide a substrate for energy-dependent processes. Whole cell currents were recorded with an Axopatch 200A amplifier (Axon Instruments) in voltage-clamp mode; membrane potential was recorded in current-clamp mode. Pipette potential and capacitance and access resistance were electronically compensated. Voltage-clamp protocols were applied with pClamp software (Axon Instruments). Data were filtered at 5 kHz, digitized with a Digidata 1200 analog-to-digital converter (Axon Instruments), and analyzed with pClamp software (Axon Instruments). Cell capacitance was calculated from the area under the capacitive current elicited by a 10-mV hyperpolarizing pulse from a holding potential of -70 mV. Whole cell current was normalized to cell capacitance and is expressed as picoamperes per picofarad. External solutions were changed with a rapid-exchange system with a multibarrel pipette connected to a common orifice positioned 100-200 µm from the myocyte studied. Complete solution exchange was achieved in <1 s. All experiments were conducted at room temperature (22-25°C).

Experimental 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 (10-2 M), ET-1 (10-5 M), and ChTX (10-4 M) were made up in distilled water, divided into aliquots, and kept frozen at -20°C until used. 4-AP was made up daily as a stock solution (10-1 M) in PSS, with the pH adjusted to 7.4 with HCl. On the day of experiment, the stock solutions were diluted as needed with PSS to the appropriate concentrations.

Data 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(-t/tau 1) + A2e(-t/tau 2), where I(t) is the current at time t, Ao is the steady-state current, A1 and A2 are the amplitudes of the exponentials, and tau 1 and tau 2 are the time constants.

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.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 -30 mV elicited outward K+ currents in PASMCs from chronically hypoxic rats (Fig. 1). ChTX inhibited a portion of the outward current. The residual KV,CI current had low noise, exhibited time-dependent activation and inactivation kinetics, and was almost completely abolished after the addition of 4-AP. These results indicate that the outward K+ current in PASMCs from chronically hypoxic rats was also composed primarily of a KCa and a KV,CI current. Therefore, in subsequent experiments, KV,CI currents were studied in the presence of 100 nM ChTX to inhibit KCa currents and KCa currents were examined in the presence of 10 mM 4-AP.


View larger version (30K):
[in this window]
[in a new window]
 
Fig. 1.   Characterization of K+ currents in pulmonary arterial smooth muscle cells (PASMCs) from chronically hypoxic animals. Traces represent K+ currents measured under control conditions and in presence of charybdotoxin (ChTX) before and after exposure to 4-aminopyridine (4-AP) and subtracted difference between currents measured under control conditions and in presence of ChTX, corresponding to ChTX-sensitive portion of current.

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.


View larger version (24K):
[in this window]
[in a new window]
 
Fig. 2.   Whole cell outward K+ currents recorded in PASMCs from normoxic and chronically hypoxic rats. A: ChTX-insensitive, 4-AP-sensitive voltage-gated K+ (KV,CI) current measured in presence of ChTX (100 nM). B: Ca2+-activated K+ (KCa) current measured in presence of 4-AP (10 mM). Mean current-voltage (I-V ) relationships (right) for peak KV,CI- and KCa-current densities were compared in cells from normoxic (n = 8 cells from 3 animals for KV,CI and 7 cells from 3 animals for KCa currents) and chronically hypoxic (n = 19 cells from 7 animals for KV,CI and 13 cells from 9 animals for KCa currents) animals.

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 -20.0 ± 1.8 mV (P < 0.001; n = 16) compared with -31.3 ± 1.1 (n = 21) in PASMCs from normoxic rats. Application of 4-AP (10 mM) depolarized PASMCs from chronically hypoxic rats from -19.6 ± 2.9 to -1.1 ± 5.7 mV (P < 0.05; n = 6; Fig. 3) compared with the 4-AP-induced depolarization from -32.1 ± 2.5 to -13.2 ± 2.1 mV in normoxic rats (n = 8). The change in membrane potential induced by 4-AP was not significantly different between PASMCs from normoxic and chronically hypoxic rats. In contrast, ChTX (100 nM) had no significant effect on the membrane potential in any of the cells tested (Fig. 3).


View larger version (22K):
[in this window]
[in a new window]
 
Fig. 3.   A and B: representative tracings of membrane potential (Em) in PASMCs from chronically hypoxic rats during exposure to 4-AP, a KV,CI-channel antagonist, or ChTX, a KCa-channel antagonist, respectively. C and D: average Em before and during exposure to 4-AP (n = 8 cells from 4 animals for normoxia and 6 cells from 3 animals for chronic hypoxia) and ChTX (n = 12 cells from 5 animals for normoxia and n = 6 cells from 3 animals for chronic hypoxia), respectively. * Significant difference from control value, P < 0.05. § Significant difference from normoxic value, P < 0.05.

Effect of ET-1 on KV,CI and KCa Currents

In PASMCs from normoxic rats, ET-1 (10-7 M) significantly reduced KV,CI-current density at test potentials positive to -30 mV, resulting in a significant downward shift in the I-V relationship (Fig. 4). At +40 mV, ET-1 reduced KV,CI-current density from 35.9 ± 9.5 to 19.8 ± 7.6 pA/pF (P < 0.001; n = 5). In contrast, the effect of ET-1 (10-7 M) on KV,CI-current density was abolished in PASMCs from chronically hypoxic rats (Fig. 4). At +40 mV, KV,CI-current density in the absence and presence of ET-1 (10-7 M) was 11.4 ± 2.5 and 9.7 ± 2.4 pA/pF (n = 11), respectively.


View larger version (39K):
[in this window]
[in a new window]
 
Fig. 4.   Effects of endothelin (ET)-1 on KV,CI current of PASMCs from normoxic (A) and chronically hypoxic (B) animals. Representative traces show KV,CI current in absence (top) and presence (middle) of ET-1. Bottom: mean I-V relationships obtained from 5 cells from 3 normoxic animals and 11 cells from 7 hypoxic animals. All measurements were done in presence of ChTX.

In PASMCs from normoxic rats, ET-1 (10-7 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).


View larger version (36K):
[in this window]
[in a new window]
 
Fig. 5.   Effects of ET-1 on KCa current in PASMCs from normoxic (A) and chronically hypoxic (B) rats. Representative current traces show KCa current in absence (top) and presence (middle) of ET-1. Bottom: mean I-V relationships from 7 cells from 3 normoxic rats and 8 cells from 7 hypoxic rats. All measurements were made in presence of 4-AP.

Effect of ANG II on KV,CI and KCa Currents

In PASMCs from normoxic rats, ANG II (10-7 M) had no significant effect on peak KV,CI-current density (34.6 ± 7.6 to 31.1 ± 5.9 pA/pF); however, the steady-state component of KV,CI-current density was reduced 21% at +40 mV from 23.9 ± 4.8 to 18.8 ± 3.9 pA/pF (P < 0.05; n = 7; Fig. 6). After exposure to chronic hypoxia, ANG II had an inhibitory effect on both the peak and steady-state components of the KV,CI current. In PASMCs from chronically hypoxic rats, peak KV,CI-current density was significantly reduced 18% (15.9 ± 2.7 to 13.0 ± 2.3 pA/pF) at +40 mV (n = 9), whereas the steady-state component was reduced 37% (8.1 ± 1.9 to 5.2 ± 1.2 pA/pF) at +40 mV.


View larger version (31K):
[in this window]
[in a new window]
 
Fig. 6.   Effects of ANG II on KV,CI current of PASMCs from normoxic (A) and chronically hypoxic (B) animals. Representative traces show KV,CI current in absence (top) and presence (middle) of ANG II. Bottom: mean I-V relationships obtained from 7 cells from 3 normoxic animals and 9 cells from 3 hypoxic animals. All measurements were done in presence of ChTX.

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).


View larger version (35K):
[in this window]
[in a new window]
 
Fig. 7.   Effects of ANG II on KCa current in PASMCs from normoxic (A) and chronically hypoxic (B) rats. Representative current traces show KCa current in absence (top) and presence (middle) of ANG II. Bottom: mean I-V relationships from 6 cells from 3 normoxic rats and 9 cells from 3 hypoxic rats. All measurements were made in presence of 4-AP.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

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 alpha -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 alpha -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 alpha -subunits because they were insensitive to ChTX, whereas the KV channels of other alpha -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.


    ACKNOWLEDGEMENTS

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).


    FOOTNOTES

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.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

1.   Adnot, S., B. Raffestin, S. Eddahibi, P. Braquet, and P. E. Chabrier. Loss of endothelium-dependent relaxant activity in the pulmonary circulation of rats exposed to chronic hypoxia. J. Clin. Invest. 87: 155-162, 1991[Medline].

2.   Aiello, E. A., O. Clement-Chomienne, D. P. Sontag, M. P. Walsh, and W. C. Cole. Protein kinase C inhibits delayed rectifier K+ current in rabbit vascular smooth muscle cells. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H109-H119, 1996[Abstract/Free Full Text].

3.   Archer, S. L., J. M. Huang, H. L. Reeve, V. Hampl, S. Tolarova, E. Michelakis, and E. K. Weir. Differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia. Circ. Res. 78: 431-442, 1996[Abstract/Free Full Text].

4.   Archer, S. L., E. Souil, A. T. Dinh-Xuan, B. Schremmer, J.-C. Mercier, A. El Yaagoubi, L. Nguyen-Huu, H. L. Reeve, and V. Hampl. Molecular identification of the role of voltage-gated K+ channels, KV1.5 and KV2.1, in hypoxic pulmonary vasoconstriction and control of resting membrane potential in rat pulmonary artery myocytes. J. Clin. Invest. 101: 2319-2330, 1998[Abstract/Free Full Text].

5.   Bakhramov, A., S. A. Hartley, K. J. Salter, and R. Z. Kozlowski. Contractile agonists preferentially activate Cl- over K+ currents in arterial myocytes. Biochem. Biophys. Res. Commun. 227: 168-175, 1996[Medline].

6.   Bialecki, R. A., C. S. Fisher, W. W. Murdoch, H. G. Barthlow, R. B. Stow, M. Mallamaci, and W. Rumsey. Hypoxic exposure time dependently modulates endothelin-induced contraction of pulmonary artery smooth muscle. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L552-L559, 1998[Abstract/Free Full Text].

7.   Bonnet, P., C. Vandier, C. Cheliakine, and D. Garnier. Hypoxia activates a potassium current in isolated smooth muscle cells from large pulmonary arteries of the rabbit. Exp. Physiol. 79: 597-600, 1994[Abstract].

8.   Bonvallet, S. T., M. R. Zamora, K. Hasunuma, K. Sato, N. Hanasato, D. Anderson, K. Sato, and T. J. Stelzner. BQ123, an ETA-receptor antagonist, attenuates hypoxic pulmonary hypertension in rats. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H1327-H1331, 1994[Abstract/Free Full Text].

9.   Brayden, J. E., and M. T. Nelson. Regulation of arterial tone by activation of calcium-dependent potassium channels. Science 256: 532-535, 1992[Medline].

10.   Carville, C., B. Raffestin, S. Eddahibi, Y. Blouquit, and S. Adnot. Loss of endothelium-dependent relaxation in proximal pulmonary arteries from rats exposed to chronic hypoxia: effects of in vivo and in vitro supplementation with L-arginine. J. Cardiovasc. Pharmacol. 22: 889-896, 1993[Medline].

11.   Clement-Chomienne, O., M. P. Walsh, and W. C. Cole. Angiotensin II activation of protein kinase C decreases delayed rectifier K+ current in rabbit vascular myocytes. J. Physiol. (Lond.) 495: 689-700, 1996[Abstract].

12.   Chen, S.-J., Y.-F. Chen, Q. C. Meng, J. Durand, V. S. DiCarlo, and S. Oparil. Endothelin-receptor antagonist bosentan prevents and reverses hypoxic pulmonary hypertension in rats. J. Appl. Physiol. 79: 2122-2131, 1995[Abstract/Free Full Text].

13.   Dempsey, E. C., D. B. Badesch, E. L. Dobyns, and K. R. Stenmark. Enhanced growth capacity of neonatal pulmonary arterial smooth muscle cells in vitro: dependence on cell size, time from birth, insulin-like growth factor I, and auto-activation of protein kinase C. J. Cell. Physiol. 160: 469-481, 1994[Medline].

14.   DiCarlo, V. S., S.-J. Chen, Q. C. Meng, J. Durand, M. Yano, Y.-F. Chen, and S. Oparil. ETA-receptor antagonist prevents and reverses chronic hypoxia-induced pulmonary hypertension in rat. Am. J. Physiol. 269 (Lung Cell. Mol. Physiol. 13): L690-L697, 1995[Abstract/Free Full Text].

15.   Eddahibi, S., B. Raffestin, P. Braquet, P. E. Chabrier, and S. Adnot. Pulmonary vascular reactivity to endothelin-1 in normal and chronically hypertensive rats. J. Cardiovasc. Pharmacol. 17: S358-S361, 1991[Medline].

16.   Eddahibi, S., B. Raffestin, M. Clozel, M. Levame, and S. Adnot. Protection from pulmonary hypertension with an orally active endothelin receptor antagonist in hypoxic rats. Am. J. Physiol. 268 (Heart Circ. Physiol. 37): H828-H835, 1995[Abstract/Free Full Text].

17.   Elton, T. S., S. Oparil, G. R. Traylor, P. H. Hicks, R. H. Yang, H. Jin, and Y.-F. Chen. Normobaric hypoxia stimulates endothelin-1 gene expression in the rat. Am. J. Physiol. 263 (Regulatory Integrative Comp. Physiol. 32): R1260-R1264, 1992[Abstract/Free Full Text].

18.   Emery, C. J., D. Bee, and G. R. Barer. Mechanical properties and reactivity of vessels in isolated perfused lungs of chronically hypoxic rats. Clin. Sci. 61: 569-580, 1981[Medline].

19.   Frid, M. G., A. A. Aldashev, E. C. Dempsey, and K. R. Stenmark. Smooth muscle cells isolated from discrete compartments of the mature vascular media exhibit unique phenotypes and distinct growth capabilities. Circ. Res. 81: 940-952, 1997[Abstract/Free Full Text].

20.   Gelband, C. H., and J. R. Hume. [Ca2+]i inhibition of K+ channels in canine renal artery. Novel mechanisms for agonist-induced membrane depolarization. Circ. Res. 77: 121-130, 1995[Abstract/Free Full Text].

21.   Goldberg, M., H. L. Zhang, and S. F. Steinberg. Hypoxia alters the subcellular distribution of protein kinase C isoforms in neonatal rat ventricular myocytes. J. Clin. Invest. 99: 55-61, 1997[Abstract/Free Full Text].

22.   Li, H. L., T. S. Elton, Y.-F. Chen, and S. Oparil. Increased endothelin receptor gene expression in hypoxic rat lung. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L552-L560, 1994.

23.   MacLean, M. R., K. M. McCulloch, and M. Baird. Effects of pulmonary hypertension on vasoconstrictor responses to endothelin-1 and sarafotoxin S6C on inherent tone in rat pulmonary arteries. J. Cardiovasc. Pharmacol. 26: 822-830, 1995[Medline].

24.   McMurtry, I. F., M. D. Petrum, and J. T. Reeves. Lungs from chronically hypoxic rats have decreased pressor response to acute hypoxia. Am. J. Physiol. 235 (Heart Circ. Physiol. 4): H104-H109, 1978[Abstract/Free Full Text].

25.   Meyrick, B. O., and E. A. Perkett. The sequence of cellular and hemodyanmic changes of chronic pulmonary hypertension induced by hypoxia and other stimuli. Am. Rev. Respir. Dis. 140: 1486-1489, 1989[Medline].

26.   Minamino, T., M. Kitakaze, K. Komamura, K. Node, H. Takeda, M. Inoue, M. Hori, and T. Kamada. Activation of protein kinase C increases adenosine production in the hypoxic canine coronary artery through the extracellular pathway. Arterioscler. Thromb. Vasc. Biol. 15: 2298-2304, 1995[Abstract/Free Full Text].

27.   Morrell, N. W., E. N. Atochina, K. G. Morris, S. M. Danilov, and K. R. Stenmark. Angiotensin converting enzyme expression is increased in small pulmonary arteries of rats with hypoxia-induced pulmonary hypertension. J. Clin. Invest. 96: 1823-1833, 1995[Medline].

28.   Morrell, N. W., K. G. Morris, and K. R. Stenmark. Role of angiotensin-converting enzyme and angiotensin II in development of hypoxic pulmonary hypertension. Am. J. Physiol. 269 (Heart Circ. Physiol. 38): H1186-H1194, 1995[Abstract/Free Full Text].

29.   Mortensen, L. H., C. M. Pawloski, N. L. Kanagy, and G. D. Fink. Chronic hypertension produced by infusion of endothelin in rats. Hypertension 15: 729-733, 1990[Abstract].

30.   Muramatsu, M., M. Oka, Y. Morio, S. Soma, H. Takahashi, and Y. Fukuchi. Chronic hypoxia augments endothelin-B receptor-mediated vasodilation in isolated perfused rat lungs. Am. J. Physiol. 276 (Lung Cell. Mol. Physiol. 20): L358-L364, 1999[Abstract/Free Full Text].

31.   Neher, E. Concentration profiles of intracellular calcium in the presence of a diffusible chelator. Exp. Brain Res. S14: 80-96, 1986.

32.   Nong, Z., J. M. Stassen, L. Moons, D. Collen, and S. Janssens. Inhibition of tissue angiotensin-converting enzyme with quinapril reduces hypoxic pulmonary hypertension and pulmonary vascular remodeling. Circulation 94: 1941-1947, 1996[Abstract/Free Full Text].

33.   Oparil, S., S.-J. Chen, Q. C. Meng, T. S. Elton, M. Yano, and Y.-F. Chen. Endothelin-A receptor antagonist prevents acute hypoxia-induced pulmonary hypertension in the rat. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L95-L100, 1995[Abstract/Free Full Text].

34.   Ordway, R. W., J. V. Walsh, and J. J. Singer. Arachidonic acid and other fatty acids directly activate potassium channels in smooth muscle cells. Science 244: 1176-1179, 1989[Medline].

35.   Peng, W., J. R. Hoidal, S. V. Karwande, and I. M. Farrukh. Effect of chronic hypoxia on K+ channels: regulation in human pulmonary vascular smooth muscle cells. Am. J. Physiol. 272 (Cell Physiol. 41): C1271-C1278, 1997[Abstract/Free Full Text].

36.   Plevin, R., N. A. Kellock, M. J. Wakelam, and R. Wadsworth. Regulation by hypoxia of endothelin-1-stimulated phospholipase D activity in sheep pulmonary artery cultured smooth muscle cells. Br. J. Pharmacol. 112: 311-315, 1994[Abstract].

37.   Porcelli, R. J., and M. J. Bergman. Effects of chronic hypoxia on pulmonary vascular responses to biogenic amines. J. Appl. Physiol. 55: 534-540, 1983[Abstract/Free Full Text].

38.   Post, J. M., C. H. Gelband, and J. R. Hume. [Ca2+]i inhibition of K+ channels in canine pulmonary artery. Circ. Res. 77: 131-139, 1995[Abstract/Free Full Text].

39.   Post, J. M., J. R. Hume, S. L. Archer, and E. K. Weir. Direct role for potassium channel inhibition in hypoxic pulmonary vasoconstriction. Am. J. Physiol. 262 (Cell Physiol. 31): C882-C890, 1992[Abstract/Free Full Text].

40.   Rabinovitch, M., W. Gamble, A. S. Nadas, O. S. Miettinen, and L. Reid. Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features. Am. J. Physiol. 236 (Heart Circ. Physiol. 5): H818-H827, 1979[Abstract/Free Full Text].

41.   Ren, J., E. Karpinski, and C. G. Benishin. The actions of prostaglandin E2 on potassium currents in rat tail artery vascular smooth muscle cells: regulation by protein kinase A and protein kinase C. J. Pharmacol. Exp. Ther. 277: 394-402, 1996[Abstract].

42.   Resink, T. J., T. Scott-Burden, and F. R. Buhler. Activation of phospholipase A2 by endothelin in cultured vascular smooth muscle cells. Biochem. Biophys. Res. Commun. 158: 279-286, 1989[Medline].

43.   Rodman, D. M. Chronic hypoxia selectively augments rat pulmonary artery Ca2+ and K+ channel-mediated relaxation. Am. J. Physiol. 263 (Lung Cell. Mol. Physiol. 7): L88-L94, 1992[Abstract/Free Full Text].

44.   Salter, K. J., J. L. Turner, S. Albarwani, L. H. Clapp, and R. Z. Kozlowski. Ca(2+)-activated Cl- and K+ channels and their modulation by endothelin-1 in rat pulmonary arterial smooth muscle cells. Exp. Physiol. 80: 815-824, 1995[Abstract].

45.   Sham, J. S. K. Ca2+ release-induced inactivation of Ca2+ current in rat ventricular myocytes: evidence for local Ca2+ signaling. J. Physiol. (Lond.) 500: 285-295, 1997[Abstract].

46.   Shimoda, L. A., G. M. Booth, T. H. Shimoda, J. Y. Sylvester, and J. S. K. Sham. Chronic hypoxia attenuates endothelin-1 (ET-1)-induced mobilization of intracellular Ca2+ in rat intrapulmonary arterial smooth muscle cells (PASMCs) (Abstract). FASEB J. 12: A232, 1998.

47.   Shimoda, L. A., J. T. Sylvester, and J. S. K. Sham. Inhibition of voltage-gated K+ current in rat intrapulmonary arterial myocytes by endothelin-1. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L842-L853, 1998[Abstract/Free Full Text].

48.   Smirnov, S. V., T. P. Robertson, J. P. T. Ward, and P. I. Aaronson. Chronic hypoxia is associated with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells. Am. J. Physiol. 266 (Heart Circ. Physiol. 35): H365-H370, 1994[Abstract/Free Full Text].

49.   Smith, J. B., L. Smith, E. R. Brown, D. Barnes, M. A. Sabir, J. S. Davis, and R. V. Farese. Angiotensin II rapidly increases phosphatidate-phosphoinositide synthesis and phosphoinositide hydrolysis and mobilizes intracellular calcium in cultured arterial muscle cells. Proc. Natl. Acad. Sci. USA 81: 7812-7816, 1984[Abstract].

50.   Stern, M. D. Buffering of calcium in the vicinity of a channel pore. Cell Calcium 13: 183-192, 1992[Medline].

51.   Suzuki, H., and B. M. Twarog. Membrane properties of smooth muscle cells in pulmonary hypertensive rats. Am. J. Physiol. 242 (Heart Circ. Physiol. 11): H907-H915, 1982[Abstract/Free Full Text].

52.   Tjen-A-Looi, S., R. Ekman, J. Osborn, and I. Keith. Pulmonary vascular pressure effects by endothelin-1 in normoxia and chronic hypoxia: a longitudinal study. Am. J. Physiol. 271 (Heart Circ. Physiol. 40): H2246-H2253, 1996[Abstract/Free Full Text].

53.   Wang, J., M. Juhaszova, L. J. Rubin, and X. J. Yuan. Hypoxia inhibits gene expression of voltage-gated K+ channel alpha subunits in pulmonary arterial smooth muscle cells. J. Clin. Invest. 100: 2347-2353, 1997[Abstract/Free Full Text].

54.   Wanstall, J. C., and S. R. O'Donnell. Responses to vasodilator drugs on pulmonary artery preparations from pulmonary hypertensive rats. Br. J. Pharmacol. 105: 152-158, 1992[Abstract].

55.   Yamaoka, Y., S. Shimohama, J. Kimura, R. Fukunaga, and T. Taniguchi. Changes in protein kinase C isozymes in the rat hippocampus following transient hypoxia. Neurosci. Lett. 154: 20-22, 1993[Medline].

56.   Yuan, X.-J. Voltage-gated K+ currents regulate resting membrane potential and [Ca2+]i in pulmonary arterial myocytes. Circ. Res. 77: 370-378, 1995[Abstract/Free Full Text].

57.   Yuan, X.-J., W. F. Goldman, M. L. Tod, L. J. Rubin, and M. P. Blaustein. Hypoxia reduces potassium currents in cultured rat pulmonary but not mesenteric arterial myocytes. Am. J. Physiol. 264 (Lung Cell. Mol. Physiol. 8): L116-L123, 1993[Abstract/Free Full Text].

58.   Yuan, X.-J., J. Wang, M. Juhaszova, V. A. Golovina, and L. J. Rubin. Molecular basis and function of voltage-gated K+ channels in pulmonary arterial smooth muscle cells. Am. J. Physiol. 274 (Lung Cell. Mol. Physiol. 18): L621-L635, 1998[Abstract/Free Full Text].

59.   Zhao, L., R. al-Tubuly, A. Sebkhi, A. A. Owji, D. J. Nunez, and M. R. Wilkins. Angiotensin II receptor expression and inhibition in the chronically hypoxic rat lung. Br. J. Pharmacol. 119: 1217-1222, 1996[Abstract].


Am J Physiol Lung Cell Mol Physiol 277(3):L431-L439
0002-9513/99 $5.00 Copyright © 1999 the American Physiological Society