ET-1 modulates KCa-channel activity and arterial tension in normoxic and hypoxic human pulmonary vasculature

Wei Peng, John R. Michael, John R. Hoidal, S. V. Karwande, and Imad S. Farrukh

Division of Respiratory, Critical Care, and Occupational Medicine, Department of Internal Medicine, The University of Utah Health Sciences Center, Salt Lake City, Utah 84132

    ABSTRACT
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Abstract
Introduction
Methods
Results
Discussion
References

The molecular mechanisms by which endothelin (ET)-1 induces pulmonary hypertension are poorly understood. We investigated the effects of ET-1 on outward K+ currents of normoxic and chronically hypoxic human pulmonary arterial (PA) smooth muscle cells (HPSMCs). In normoxic HPSMCs, ET-1 has dual effects. In intact cells, 5 nM ET-1 activates the large-conductance and Ca2+-activated K+ (KCa)-channel current [IK(Ca)] by increasing intracellular Ca2+ concentration, whereas it directly inhibits IK(Ca) in isolated membrane patches. At a higher concentration (10 nM), ET-1-induced IK(Ca) inhibition predominates. In hypoxic HPSMCs, ET-1 at 5 nM significantly reduces IK(Ca). The ETA-receptor antagonist BQ-123 reverses the ET-1-induced decrease in IK(Ca). Chronic BQ-123 treatment also prevents the hypoxia-induced decrease in IK(Ca). In PA rings obtained from human organ donors, ET-1 causes a concentration-dependent increase in tension. The ET-1-mediated increase in tension is reversed by a KCa-channel agonist. The increase in tension at the highest concentration studied (9 nM) was more pronounced in PA rings obtained from patients with chronic obstructive pulmonary disease. These results imply that an ET-1-induced decrease in IK(Ca) contributes to chronic hypoxia-induced pulmonary hypertension.

endothelin-1; calcium-activated potassium channel; pulmonary hypertension; chronic hypoxia; potassium currents; BQ-123; human lung

    INTRODUCTION
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Abstract
Introduction
Methods
Results
Discussion
References

ENDOTHELIN (ET)-1 is a potent vasoactive 21-amino acid peptide that was originally reported to be produced by endothelial cells (31). A recent study (33) demonstrated that ET-1 is also expressed and produced from rat pulmonary arterial (PA) smooth muscle cells (SMCs). ET-1 has been implicated in the pathogenesis of pulmonary hypertension. Compared with control subjects, patients with primary or secondary pulmonary hypertension have elevated circulating plasma ET-1 levels (28). Other studies (27, 33) reported an increase in intrapulmonary production of ET-1 in the fawn-hooded rat model for pulmonary hypertension. The role of ET-1 in the pathogenesis of pulmonary vascular disease is not certain. In particular, the role of ET-1 in the development of acute hypoxic pulmonary vasoconstriction is controversial. The ET-1-receptor antagonist BQ-123 is reported to block acute hypoxic pulmonary vasoconstriction (20). Other authors have noted that ET-1 causes pulmonary vasodilation in hypoxic rat lungs (10) and that pretreatment with BQ-123 does not affect the hypoxia-induced pulmonary vasoconstriction of isolated canine PA rings (6). Recent work suggested an association between ET-1 and the development of pulmonary hypertension after chronic hypoxia. Chronic infusion of BQ-123 into rats attenuates chronic hypoxia-induced pulmonary hypertension, right ventricular hypertrophy, and new muscle growth (3-5). Continuous treatment with BQ-123 beginning after 2 wk of hypoxia also significantly decreases the PA pressure and prevents the progressive worsening of the right ventricular hypertrophy that occurs during the third and fourth weeks of exposure to hypoxia (5). Thus an ETA-receptor antagonist both prevents and halts the progression of the physiological and morphological changes in rat pulmonary vasculature arising from chronic hypoxia. Another study (14) demonstrated that exposing rats to hypoxia for 48 h or 4 wk increases pulmonary vascular ET-1 mRNA levels as well as plasma, lung, and main PA homogenate ET-1 levels. In addition to vasoactive properties, ET-1 has a comitogenic effect on pulmonary vascular SMCs, suggesting that it may play a role in the vascular remodeling of chronic hypoxia (13, 18, 32). The mechanisms by which ET-1 contributes to the development of pulmonary hypertension in chronic hypoxia are not fully understood. The vasoactivity of ET-1, the pulmonary vasoconstriction after acute exposure to hypoxia, and the development of pulmonary hypertension after chronic hypoxia are all believed to be mediated, in part, via modulation of K+ channels (9, 22, 26). Salter et al. (26) reported in rat PA SMCs that ET-1 enhanced Ca2+- and voltage-activated outward K+ channels and induced the periodic oscillation of inward Cl- currents. The authors suggested that these currents are involved in the contractile responses of PA SMCs to ET-1 (26). The effects of ET-1 on K+-channel activity in human PA SMCs (HPSMCs) are unknown. In the present study, we investigated the effects of ET-1 on the outward K+ current (IKo) of HPSMCs that were grown under either normoxic or hypoxic conditions.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

HPSMCs

As previously described (22, 23), HPSMCs were cultured from explants of the main PA from eight control human donors in The University of Utah Transplant Program. Briefly, the arteries were separated from their adventitia and endothelium and then minced into l- to 3-mm2 pieces with sterile scalpel blades. The tissues were mounted in culture wells with 0.05 ml of chicken plasma (Sigma, St. Louis, MO) plus 0.05 ml of chick embryo extract (Sigma). The tissue, and eventually the HPSMCs, were then plated in SMC culture medium (SmGM; Clonetics, San Diego, CA) that was supplemented with 5% fetal bovine serum, 10% bovine calf serum (HyClone, Logan, UT), and human recombinant epidermal growth factor (0.1 µg/ml) plus insulin (5 µg/ml; Clonetics). Antimicrobial agents (25 µg of gentamicin and 25 µg of amphotericin B; Clonetics) were added to 500 ml of culture medium (SmGM-2 BulletKit, Clonetics). All experiments were performed predominantly in primary HPSMCs.

HPSMCs were grown from PA explants under either normoxic or hypoxic conditions. The chamber of the hypoxic incubator was infused with 5-7% O2-5% CO2-balance N2. This achieved a medium PO2 of 63 ± 2.7 mmHg (n = 4 explants). The O2 level was monitored by an O2/CO2 sensor incorporated in the tissue incubator (Forma Scientific, Mariette, OH). The duration of cell culture for both hypoxic and normoxic HPSMCs was 25-28 days.

Electrophysiological Measurements

Single cells were voltage clamped, and membrane currents were recorded with cell-attached, outside-out, and inside-out configurations of the patch-clamp technique (22). The effects of charybdotoxin (CTX) on IKo were determined in outside-out membrane patches, which allowed treatment washout in the same experimental run. All other membrane patches were performed in the inside-out configuration. Inside-out patches are excised membrane patches in which the intracellular side of the membrane is facing the bathing solution and the extracellular side is facing the pipette solution. Voltage-clamp potentials were applied to membrane patches, and membrane currents were recorded with an Axopatch 200A amplifier (Axon Instruments, Foster City, CA). The current data were filtered at 1 kHz and digitized at 5 kHz with a Digidata 1200 interface (Axon Instruments). Data were acquired and analyzed with pClamp software (Axon Instruments). Membrane potential (Em) is reported with the extracellular surface as the reference or ground. Outward currents were recorded as upward deflections.

Data analysis. In HPSMCs, the large-conductance component of IKo was identified by our laboratory as the Ca2+-activated K+ (KCa) channel and the small-conductance component as the delayed rectifier K+ (Kdr) channel (23). In this study, the KCa channel was identified by amplitude, sensitivity to CTX, and lack of inhibition with the use of 4-aminopyridine (4-AP) (23). The activity of K+ channels during single-channel recordings was computed by scanning the channel events at the 50-percentile threshold. A frequency-amplitude histogram was obtained by digitizing the current records, and an all-points histogram was derived. The histograms were fitted by double Gaussian continuous lines. The open probability (Po) values were calculated by measuring the area under each fitted peak. Po was used for channel activity when one peak was identified in the histogram. For patches containing multiple (N) large-conductance channels, N was determined as the maximal number of channels observed in conditions of a high level of Po. Mean values for the state of channel Po and mean open times were obtained from a 5- to 15-min steady-state recording, and channel Po is expressed as NPo. Average channel activity or NPo in membrane patches was determined as follows: NPo = (<LIM><OP>∑</OP></LIM>Nj=1tjj)/T, where T is the duration of recording and tj is the time spent with j = 1, 2, 3, ..., N channels open.

Solutions and Chemicals

For cell-attached, outside-out, and inside-out experiments, we used the following bath and pipette solution (in mM): 130 potassium gluconate, 10 KCl, 2 MgCl2, 3 EGTA, 10 HEPES, and 2.29 CaCl2, and pH was adjusted to 7.4. The estimated free Ca2+ concentration ([Ca2+]) was 300 nM as calculated by Fabiato's (7) computer program. For other modifications of the bath or pipette solution, see RESULTS. ET-1 and the ETA-receptor antagonists BQ-123 and JKC-301 were obtained from Alexis Biochemicals (San Diego, CA). The selective KCa-channel inhibitor CTX was obtained from Alomone Laboratories. 4-AP, an inhibitor of the Kdr channel; the cGMP analog 8-bromo-cGMP; EGTA; and the naturally occurring steroid and KCa-channel opener dehydroepiandrosterone (DHEA) (8) were obtained from Sigma.

Isolated Human PA Ring Preparations

The arteries were dissected from their adventitia and cut into rings (4-6 mm in width). The arterial rings were mounted on wire hooks. One hook was fixed to the bottom of a tissue chamber, and the other hook was connected to a force displacement transducer (Grass FT03). The arterial rings were vertically centered with a Multi-Axis stage manipulator (Newport, Irvine, CA) that held the force displacement transducer. Changes in isometric force were recorded on a Gould recorder. Baseline tension was adjusted to 1 g baseline tension/0.5 g tissue with the Z-axis actuator (SM-50, Newport) of the manipulator. This baseline tension was determined based on preliminary experiments indicating that 1 g of tension applied to an average arterial ring of 0.5 g produced a near-maximal response to 60 mM KCl. The arterial rings were allowed to equilibrate for 1 h, and the bath solution was then washed out. The vessels were incubated in 50 ml of a physiological salt solution that was gassed with 5% CO2-21% O2-balance N2. The composition of the physiological salt solution (in mM) was 137 NaCl, 4.7 KCl, 2.5 CaCl2, 1.3 KH2PO4, 1.2 MgSO4, 11 dextrose, and 18 NaHCO3. The temperature was maintained at 37°C with an external heat exchanger (Lauda M3, Baxter Scientific). The endothelium of the PA rings was removed by gently rubbing the lumen of the vessel. Removal of the endothelium was confirmed physiologically by the lack of dilatation to acetylcholine (2 µM) in arterial rings precontracted with norepinephrine (2 × 10-8 M) (23).

Statistical Analysis

All data are expressed as means ± SE. Statistical analyses were performed with two-way ANOVA (Scheffé's F-test significant at 95%; StatView, Abacus Concepts). Differences were considered to be significant when P < 0.05.

    RESULTS
Top
Abstract
Introduction
Methods
Results
Discussion
References

Normoxia

Effect of ET-1 during normoxia on K+ channels in HPSMCs. Experiments were done on intact normoxic HPSMCs in the cell-attached configuration at an Em of +30 mV. The bath and pipette solutions contained 300 µM and 300 nM Ca2+, respectively. Using these Ca2+ concentrations, we observed no significant cell contraction. During normoxia, ET-1 (5 nM) significantly increased K+-channel activity (NPo = 0.0117 ± 0.0005 for control cells vs. 0.0690 ± 0.0013 for ET-1; P < 0.0001, n = 6 cells/group; Fig. 1). The effect of ET-1 was completely blocked by pretreating the cells with the ETA-receptor blocker BQ-123 [1 µM; NPo = 0.0124 ± 0.0008 for BQ-123+ET-1 vs. 0.0117 ± 0.0005 for control cells; not significant (NS); n = 6 cells] (4, 12). Treating the cells with BQ-123 alone had no significant effect on baseline K+-channel activity (NPo = 0.0117 ± 0.0005 for control cells vs. 0.0128 ± 0.0007 for BQ-123; NS; n = 6 cells/group; Fig. 1A). A higher concentration of ET-1 (10 nM) caused a sustained decrease in K+-channel activity (NPo = 0.0117 ± 0.0005 for control cells vs. 0.0035 ± 0.0004 for 10 nM ET-1; P < 0.0005; n = 8 cells/group). To further elucidate the dual effects of ET-1 on K+-channel activity, in a second group of experiments, we studied the concentration-dependent effects of ET-1 (0.1, 0.5, 1.0, 3.0, 5.0, 7.0, and 10 nM) on K+-channel activity of HPSMCs. Experiments were done on intact normoxic HPSMCs in the cell-attached configuration at an Em of +50 mV. During normoxia, ET-1 caused a concentration-dependent increase in K+-channel activity over the range of 0.1-5 nM (Fig. 1B). A higher concentration of ET-1 (7.0 nM) caused a reversal in K+-channel activity and a sustained inhibition at 10 nM (Fig. 1B).


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Fig. 1.   A: single-channel recordings in cell-attached configuration in human pulmonary arterial smooth muscle cells (HPSMCs) under normoxic conditions. Experiments were performed at a membrane potential (Em) of +30 mV. HPSMCs were perfused with a bath solution containing 300 µM Ca2+ concentration ([Ca2+]), and estimated [Ca2+] in pipette solution was 300 nM. O, open; C, closed. a: control cells. Endothelin (ET)-1 (5 nM) caused a significant increase in outward K+ current (IKo; P < 0.0001; b). ET-1-induced increase in IKo was blocked by pretreating HPSMCs with ETA-receptor antagonist BQ-123 (1 µM; d). Treating normoxic HPSMCs with BQ-123 alone has no significant effect on baseline K+-channel activity (c). B: effect of ET-1 concentration ([ET-1]) on IKo activity [expressed as no. of channels (N) × open probability (Po; NPo)] in normoxic HPSMCs. Experiments were performed in cell-attached configuration at Em of +50 mV. Increasing [ET-1] from 0.1 to 5.0 nM caused an ~5-fold increase in normalized (%control) K+-channel activity (n = 5 cells/point). Further increases in [ET-1] reversed increase in K+-channel activity, and at 10 nM, ET-1 caused a sustained decrease in K+-channel activity.

Identification of the K+ channel affected by ET-1. In the cell-attached configuration at an Em of +30 mV, the ET-1-induced increase in IKo was blocked by CTX (100 nM), the selective inhibitor of the KCa channel, but was unaffected by pretreating the cells with 4-AP, an inhibitor of the Kdr channel (NPo = 0.0675 ± 0.0005 for ET-1 vs. 0.0006 ± 0.0003 for ET-1+CTX; P < 0.0001; n = 8 cells/group). ET-1 had no effect on the Kdr channel, the low-conductance, 4-AP-sensitive, and CTX-insensitive K+ channel (NPo for Kdr channel = 0.0061 ± 0.0003 for control cells vs. 0.0062 ± 0.0004 for ET-1; NS; n = 6 cells/group). These findings indicate that during normoxia 5 nM ET-1 increased IKo by augmenting KCa-channel activity.

Role of Ca2+ flux in the increase in KCa-channel activity produced by 5 nM ET-1. To further elucidate the mechanism by which ET-1 induced KCa-channel modulation during normoxia, we studied the effect of depleting intracellular [Ca2+] ([Ca2+]i) or blocking extracellular Ca2+ entry. The experiments were performed in intact HPSMCs, and single-channel recordings were obtained in a cell-attached configuration at an Em of +30 mV. The estimated [Ca2+] in the bath solution was 300 µM. To determine the effect of intracellular Ca2+ (Ca2+i) depletion, HPSMCs were treated with thapsigargin (1 µM) before the addition of ET-1. Thapsigargin released stored Ca2+i, leading to an initial increase in [Ca2+]i followed by a decrease due to the inhibition of Ca2+ reuptake by the storage sites. In HPSMCs, thapsigargin caused a transient increase in KCa channels (P < 0.0001 at 1 min; Fig. 2B) followed by a sustained decrease in KCa-channel activity (NPo = 0.0042 ± 0.0002 after 10 min of thapsigargin treatment vs. 0.0072 ± 0.0002 for control cells; P < 0.0001; n = 6 cells; Fig. 2C). The transient increase in KCa-channel activity was most likely due to release of stored Ca2+i and secondary activation of the KCa-channel component of IKo (2). The addition of ET-1 to thapsigargin-treated HPSMCs eliminated KCa-channel activity (Fig. 2D). These results suggest that the ET-1-induced increase in KCa channels is mediated, in part, via release of Ca2+i and that the sustained effect of ET-1 on KCa channels is inhibitory.


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Fig. 2.   Treating normoxic HPSMCs with 1 µM thapsigargin caused a transient increase in Ca2+-activated K+ (KCa)-channel current [IK(Ca); B] followed by a significant decrease in IK(Ca) below baseline level (P < 0.0001; C). A: control cells in a cell-attached configuration at Em of +30 mV. In thapsigargin-treated HPSMCs, ET-1 eliminated IK(Ca) (n = 6 cells; D). This suggests that ET-1-induced increase in IK(Ca) of normoxic HPSMCs is mediated, in part, via release of stored intracellular [Ca2+] and that the sustained effect of ET-1 on IK(Ca) is inhibitory.

In another group of experiments, we tested the effect of extracellular Ca2+ entry blockade with either verapamil (10 µM) or NiCl2 (500 µM). Experiments were performed in a cell-attached configuration at an Em of +30 mV. Both verapamil and NiCl2 partially reduced the ET-1-mediated increase in KCa-channel activity (NPo = 0.0400 ± 0.002 and 0.0390 ± 0.002 for ET-1+ verapamil and ET-1+NiCl2, respectively, vs. NPo = 0.0650 ± 0.001 for ET-1 alone; P < 0.001; n = 8 cells/group). Thus we conclude that in normoxic HPSMCs the ET-1-induced increase in KCa-channel activity at 5 nM is predominantly mediated by an increase in [Ca2+]i arising from both the release of stored Ca2+i and Ca2+ entry.

Effect of ET-1 on KCa channels in HPSMC membrane patches. To further characterize the effects of ET-1 on KCa channels during normoxia, we examined the direct effect on KCa channels in excised HPSMC membrane patches. Membrane patches were studied in an inside-out configuration with a symmetrical transmembrane K+ concentration (intracellular K+ concentration/extracellular K+ concentration = 140/140 mM) and the Em electrically depolarized to +50 mV. Applying ET-1 (5 nM) to the inner surface of the membrane reduced KCa-channel activity (NPo = 0.1690 ± 0.009 for control patches vs. 0.0680 ± 0.007 for ET-1; P < 0.0001; n = 6 patches; Fig. 3). Treating the membrane patches with the ETA-receptor antagonist BQ-123 (1 µM) reversed the inhibitory effect of ET-1 on IKo (NPo = 0.1640 ± 0.008 for ET-1+BQ-123 vs. 0.1690 ± 0.009 for control patches; NS; n = 6 patches; Fig. 3). BQ-123 had no effect on baseline IKo (data not shown). This indicates that the ETA receptor mediates the ET-1-induced inhibition of KCa channels. In the presence of ET-1, the BQ-123-induced increase in IKo was blocked by CTX but not by 4-AP, demonstrating an effect on KCa channels (Fig. 3).


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Fig. 3.   Effects of ET-1 on KCa-channel activity in inside-out normoxic HPSMC membrane patches (A-C, E) at Em of +50 mV and symmetrical transmembrane K+ concentration (intracellular K+ concentration/extracellular K+ concentration = 140/140 mM). A: control patches. Treating membrane patches with ET-1 (5 nM) significantly reduced KCa-channel activity (P < 0.0001; B). Effect of ET-1 was blocked by ETA-receptor antagonist BQ-123 (C). BQ-123 had no significant effect on baseline KCa-channel activity (data not shown). In presence of ET-1, BQ-123-induced increase in KCa-channel activity was blocked by 100 nM charybdotoxin (CTX; outside-out patches; D) but was not affected by 2 mM 4-aminopyridine (4-AP; E). In outside-out patches, activity of IK(Ca) after treatment washout was not significantly different from baseline control (data not shown).

These observations suggest that in normoxic HPSMCs ET-1 has the following dual effects on KCa-channel activity: first, an indirect activator role due to its ability to increase Ca2+i, and second, a direct inhibitory effect that was observed in isolated membrane patches and after depletion of Ca2+ stores in intact HPSMCs.

Chronic Hypoxia

Effect of ET-1 on K+ channels in chronically hypoxic HPSMCs. In these experiments, we studied the effect of ET-1 on K+ channels in intact chronically hypoxic HPSMCs (25-28 days of exposure). Experiments were performed in a cell-attached configuration at an Em of +30 mV and under reduced O2 tension. Reduced O2 tension was maintained by bubbling the perfusate solution with 5% CO2-balance N2. Chronic exposure to hypoxia decreased baseline HPSMC K+-channel activity (NPo = 0.0035 ± 0.0002 for chronic hypoxia vs. 0.0117 ± 0.0005 for control normoxia; P < 0.001; n = 6 cells; Fig. 4A). Exposing hypoxic HPSMCs to 5 nM ET-1 further decreased the large-conductance K+-channel activity (NPo = 0.0006 ± 0.0001 for ET-1 vs. 0.0035 ± 0.0002 for hypoxia; P < 0.0001; n = 6 cells/group). Acute BQ-123 (1 µM) treatment of chronically hypoxic HPSMCs increased the activity of baseline K+ channels (0.0110 ± 0.0007 for BQ-123 vs. 0.0035 ± 0.0002 for hypoxia; P < 0.0001; n = 6 cells) and reversed the inhibitory effect of ET-1 on the large-conductance K+-channel component of IKo (Fig. 4A).


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Fig. 4.   A: single-channel recordings of effects of chronic hypoxia on ET-1-induced modulation of IKo in intact HPSMCs in cell-attached configuration. Experiments were performed at Em of +30 mV. HPSMCs were perfused with physiological solution bubbled with 5% CO2-95% N2, and estimated [Ca2+] in pipette solution was 300 nM. a: control cells. ET-1 (5 nM) caused a significant decrease in IKo (P < 0.001; b). Treating chronically hypoxic HPSMCs with BQ-123 (1 µM) alone (c) caused a significant increase in baseline IKo (P < 0.0001) and reversed the effects of ET-1 (d). B: experiments were done in inside-out (a-c, e) and outside-out (d) hypoxic HPSMC membrane patches at Em of +50 mV and transmembrane symmetrical K+ concentration (intracellular K+ concentration/extracellular K+ concentration = 140/140 mM). a: control cells. Treating HPSMC membrane patches with ET-1 further decreased KCa-channel activity (b). Treating chronically hypoxic HPSMC membrane patches with BQ-123 increased baseline KCa-channel activity (P < 0.0001) and prevented ET-1-induced KCa-channel inhibition (c). BQ-123-induced increase in KCa-channel activity was blocked by 100 nM CTX (d) but was not affected by 2 mM 4-AP (e). In outside-out patches, activity of KCa channels after treatment washout was not different from baseline control values (data not shown). C: frequency-amplitude graphs of IK(Ca) recorded in HPSMCs during normoxia, chronic hypoxia, and chronic hypoxia+BQ-123. Histograms were fitted by double Gaussian continuous lines. Chronic hypoxia decreased KCa-channel Po from 0.140 for control normoxia to 0.009. BQ-123 reversed the effect of chronic hypoxia on KCa-channel activity, increasing Po to 0.135.

Mechanism of ET-induced inhibition of IKo during chronic hypoxia. EFFECT OF ETA-receptor blockade. Experiments were performed on membrane patches in the inside-out configuration with a symmetrical transmembrane K+ concentration (intracellular K+ concentration/extracellular K+ concentration = 140/140 mM) and the Em electrically depolarized to +50 mV. Applying ET-1 (5 nM) to the inner surface of the membrane caused a significant decrease in activity of the large-conductance K+ channels (NPo = 0.0065 ± 0.0004 for hypoxia vs. 0.0005 ± 0.0001 for ET-1; P < 0.0001; n = 6 patches). Treating the membrane patches with the ETA-receptor antagonist BQ-123 (1 µM) by itself caused a significant increase in IKo activity (NPo = 0.1260 ± 0.0050 for BQ-123 vs. 0.0065 ± 0.0004 for hypoxia; P < 0.0001; n = 6 patches; Fig. 4B) and reversed the effect of ET-1 on IKo (NPo = 0.1130 ± 0.008 for ET-1+BQ-123 vs. 0.0005 ± 0.0001 for ET-1 alone; P < 0.0001; n = 6 patches; Fig. 4, B and C).

To further test whether the effect of ET-1 on IKo was mediated via ETA receptors, we studied the effect of an additional ETA-receptor antagonist, JKC-301 (30), on the ET-1-induced inhibition of IKo in isolated membrane patches. The effect of ET-1 (5 nM) on IKo was also reversed with JKC-301 (1 µM; NPo = 0.1197 ± 0.006 for ET-1+JKC-301 vs. 0.0005 ± 0.00006 for ET-1 alone; P < 0.0001; n = 6 patches; Fig. 5).


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Fig. 5.   Single-channel recordings in membrane patches of chronically hypoxic HPSMCs in inside-out (A-C, E) and outside-out (D) configurations at Em of +50 mV. A: control patches. B: control patches treated with ET-1. JKC-301 (1 µM) reversed the effect of ET-1 on KCa-channel activity (C). JKC-301-induced increase in IKo activity was sensitive to CTX (100 nM; D) but not to 4-AP (2 mM; E). In outside-out patches, activity of KCa channels after treatment washout was not different from baseline control activity (data not shown).

IDENTIFICATION OF THE K+ CHANNEL AFFECTED BY ET-1. To further characterize the effect of ET-1 on IKo in chronically hypoxic HPSMCs, we investigated the differential effects of ET-1 on the major K+ channels. We tested the effect of the specific KCa-channel blocker CTX (100 nM) on the increase in IKo activity produced by ETA-receptor antagonists. Experiments were done in inside-out membrane patches of chronically hypoxic HPSMCs. In the presence of ET-1 (5 nM), the BQ-123- and JKC-301-induced increases in IKo activity were blocked by CTX (100 nM) but not by 4-AP (Figs. 4B and 5). The results of these experiments further support the thesis that ET-1 modulates KCa-channel current [IK(Ca)] and that the direct effects of ET-1 on K+ channels in HPSMCs are mediated via ETA receptors.

EFFECT OF KCA AGONISTS ON THE ET-1-INDUCED INHIBITION OF IK(CA). We next studied the effect of KCa-channel activators (cGMP and DHEA) on the ET-1-induced decrease in IK(Ca). Both cGMP (1, 19, 21) and DHEA (8) have been shown to increase KCa-channel activity in pulmonary SMCs. Experiments were performed in inside-out membrane patches at an Em of +50 mV. Both 8-bromo-cGMP (n = 6 patches) and DHEA (n = 6 patches) reversed the effect of ET-1 on IK(Ca) (Fig. 6). The cGMP- and DHEA-mediated increases in IKo were blocked by treating the cells with 100 nM CTX but not with 2 mM 4-AP. These experimental results further support the observation that ET-1 selectively decreases KCa-channel activity in hypoxic HPSMCs.


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Fig. 6.   Single-channel recordings in inside-out (a-c, e) and outside-out (d) configurations at Em of +50 mV. Experiments were performed at high Em to partially compensate for effect of chronic hypoxia on KCa-channel activity. In outside-out patches, activity of KCa channels after treatment washout was not different from baseline control activity (data not shown). Treating hypoxic membrane patches (control patches; a) with ET-1 (5 nM; b) caused a significant decrease in KCa-channel activity. A: effect of ET-1 on KCa channels was reversed by 1 mM cGMP analog 8-bromo-cGMP (c). In presence of ET-1, CTX (100 nM) blocked effect of cGMP on KCa activity (d). Treating membrane patches with 2 mM 4-AP blocked delayed rectifier K+ current [IK(dr)] and had no effect on cGMP-mediated increase in IK(Ca) (e). B: dehydroepiandrosterone (DHEA; 50 mM) reversed ET-1-induced decrease in IK(Ca) (c). Effect of DHEA was blocked with 100 nM CTX (d) and not with 2 mM 4-AP (e).

EFFECT OF ET-1 ON THE SENSITIVITY OF KCA CHANNELS TO [CA2+]I. Peng et al. (22) previously demonstrated in HPSMCs that chronic hypoxia reduces the sensitivity of KCa channels to [Ca2+]i. To further investigate the effect of ET-1 on KCa channels after chronic hypoxia, we studied the effect of changing [Ca2+]i on the ET-1-induced KCa-channel modulation in isolated membrane patches from chronically hypoxic HPSMCs.

Experiments were performed on chronically hypoxic HPSMC membrane patches in the inside-out configuration at an Em of +20 mV. We varied the perfusate free [Ca2+] or [Ca2+]i from 0.1 to 10 µM. ET-1 significantly decreased KCa-channel activity at all [Ca2+]i values (P < 0.0001; n = 10 patches; Fig. 7). The linear fit of ln[NPo /(1 - NPo)] vs. pCa demonstrated that ET-1 caused an e-fold decrease in KCa-channel activity at all [Ca2+]i values over the range of 0.1-10 µM (Fig. 7). ET-1 did not affect the conductance of KCa channels (control hypoxic HPSMCs: 12 ± 0.02 pA; chronic hypoxia+ET-1: 12 ± 0.06 pA; NS; n = 10 patches/group). The results of these experiments demonstrate that ET-1 further reduces the sensitivity of KCa channels to [Ca2+]i in chronically hypoxic HPSMCs. Treating the membrane patches with 1 µM BQ-123 reverses the effects of ET-1 on KCa-channel sensitivity to [Ca2+]i (Fig. 7).


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Fig. 7.   Logarithmic plot of NPo /(1 - NPo) vs. [Ca2+] in bath solution [log10[Ca2+]i (p[Ca2+]i)] for inside-out membrane patches from chronically hypoxic HPSMCs (n = 6 patches/point), hypoxic HPSMCs treated with ET-1 (n = 6 patches/point), and hypoxic HPSMCs treated with ET-1+BQ-123 (n = 6 patches/point). Experiments were done at Em of +20 mV. Treating hypoxic HPSMC membrane patches with ET-1 significantly decreased KCa-channel activity at all [Ca2+]i values in range of 0.1-10 µM. BQ-123 reversed effects of ET-1 on KCa-channel activity.

Effect of chronic BQ-123 treatment on chronic hypoxia-induced inhibition of KCa channels. In these experiments, we tested the effect of chronic hypoxia on IKo of HPSMCs after chronic BQ-123 (1 µM) treatment. The BQ-123 concentration was chosen on the basis of evidence that it reduces chronic hypoxia-induced pulmonary hypertension in rats (4). BQ-123 (1 µM) was added to the culture medium of HPSMCs that were exposed to chronic hypoxia. This treatment was maintained throughout the period of exposure to low O2 tension. Experiments were performed on inside-out membrane patches of chronically hypoxic HPSMCs at an Em of +50 mV. Chronic BQ-123 treatment prevented the hypoxia-induced decrease in KCa channels (NPo = 0.1255 ± 0.0086 for hypoxia+BQ-123 vs. 0.0089 ± 0.0006 for hypoxia alone; P < 0.001; n = 6 patches; Fig. 8). The increase in IKo activity caused by chronic BQ-123 treatment was blocked by CTX but not by 4-AP (Fig. 8).


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Fig. 8.   HPSMCs were chronically treated with BQ-123 (1 µM added to cell culture medium) for duration of chronic hypoxia exposure. Medium that contained BQ-123 was changed 2 times/wk. A: control cells in normoxia. B: control cells in hypoxia. BQ-123 reversed effect of chronic hypoxia on IK(Ca) (single-channel recordings in inside-out configuration at Em of +50 mV; C). Effect of BQ-123 on IKo was completely blocked by CTX (100 nM; outside-out configuration; D) but was not affected by 4-AP (E). In outside-out patches, activity of KCa channels after treatment washout was not different from baseline control activity (data not shown).

Physiological Experiments

Effect of ET-1 on vascular tone in human PA rings. The concentration effect of ET-1 on human pulmonary vascular tension was examined in PA rings obtained from either the main PA or a fourth-generation intrapulmonary artery (intraPA) from human donors (n = 4 for each group) and from the main PA of chronically hypoxic patients with end-stage lung disease [chronic obstructive pulmonary disease (COPD)-PA; n = 3]. In PAs and intraPAs, ET-1 (2.5-9.0 nM) caused a concentration-dependent increase in arterial tension normalized for arterial ring mass [change in grams of arterial tension per gram of arterial ring mass (Delta Gtension/Gmass)], and the increase in arterial tension was sustained for at least 2 h. The ET-1-induced increase in arterial tension at the highest concentration studied was more pronounced in COPD-PAs than in PAs or intraPAs (Delta Gtension/Gmass = 7.4 ± 1.3 for COPD-PA vs. 3.3 ± 0.2 for PA and 3.5 ± 0.7 for intraPA; P < 0.01; Fig. 9).


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Fig. 9.   A: ET-1 causes concentration-dependent increases in arterial tension in both main pulmonary artery (Main PA) and intrapulmonary 4th-generation arterial rings (IntraPA). Gtension/Gmass, g arterial tension/g arterial ring mass. There was no difference in increases in arterial tension induced by [ET-1] of 2.5-9 nM. At 9 nM, ET-1-induced increase in arterial tension was more prominent in PA rings obtained from Main PA of chronically hypoxic lungs [chronic obstructive pulmonary disease (COPD)-PA; n = 3; dagger  P < 0.01]. B: effect of DHEA on ET-1-induced increase in arterial tension. Experiment was performed on an arterial ring obtained from Main PA of a patient with end-stage COPD. ET-1 (9 nM) caused a sustained increase in arterial tension. Oscillation in arterial tension was observed in arterial rings of 1 of 3 patients. DHEA reversed 80% of ET-1-induced increase in arterial tension. Vertical scale bar, Gtension/Gmass; horizontal scale bar, 5 min.

Effect of KCa-channel agonists on ET-1-induced pulmonary vasoconstriction. To determine the potential importance of KCa-channel inhibition in ET-1-induced pulmonary vasoconstriction, we tested the ability of a KCa-channel opener to reverse ET-1-mediated pulmonary vasoconstriction. Farrukh et al. (8) previously reported that the naturally produced hormone DHEA selectively activates KCa channels in isolated ferret PA MSCs and HPSMCs. In these experiments, we tested the effect of DHEA (0.2 mM) on the ET-1-induced increase in arterial tension. DHEA reversed 80% of the ET-1-induced increase in the tension of arterial rings obtained from main PAs (Fig. 9B) and 63% of the vasoconstriction observed in intraPA rings.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The present study demonstrates that, in normoxic HPSMCs, ET-1 has a dual effect on KCa channels. ET-1 (0.1-5 nM) causes a concentration-dependent increase in K+-channel activity. At 5 nM, ET-1 causes both an indirect increase in KCa-channel activity by increasing [Ca2+]i and a direct inhibition of the KCa channel. At a higher ET-1 concentration (10 nM), the inhibitory effect on KCa channels predominates. In chronically hypoxic HPSMCs, ET-1 causes an inhibitory effect in intact cells and in isolated membrane patches of HPSMCs. Treatment of normoxic and hypoxic HPSMC membrane patches with the ETA-receptor antagonist BQ-123 reverses the acute effect of ET-1 on KCa channels. Chronic treatment of hypoxic HPSMCs with BQ-123 prevents the chronic hypoxia-induced decrease in KCa-channel activity. In isolated human PA rings, ET-1 causes a concentration-dependent increase in tension. The increase in arterial tension was more pronounced in arterial rings from PAs obtained from end-stage COPD patients who were exposed in vivo to chronic hypoxic. The ET-1-induced increase in arterial tension was significantly reduced by a KCa-channel agonist.

ET-1-Induced Modulation of IKo Activity During Normoxia

During normoxia, ET-1 has a dual effect on IKo. At 5 nM, it causes a concentration-dependent increase in IK(Ca), the large-conductance and CTX-sensitive component of IKo. ET-1 has no effect on IK(dr), the low-conductance, 4-AP-sensitive, and CTX-insensitive component of IKo. A higher ET-1 concentration (10 nM) also can directly inhibit KCa channels. This dual action of ET-1 on KCa channels was reported in porcine coronary arterial SMCs. Using a cell-attached patch-clamp technique, Hu et al. (11) reported that ET-1 at 1-10 nM activated KCa channels of porcine coronary SMCs but caused a sustained inhibition at concentrations > 10 nM.

The low-concentration ET-1-induced increase in KCa-channel activity is most likely mediated by an increase in [Ca2+]i. ET-1 has been reported to cause an increase in [Ca2+]i of vascular SMCs (15). Increased [Ca2+]i is known to activate KCa channels, which act as a negative inhibitory mechanism after acute and chronic agonist stimulation (19, 22). This ET-1-induced activation of KCa channels was blocked in thapsigargin-treated HPSMCs. Thapsigargin releases stored Ca2+i, leading to an initial increase in [Ca2+]i followed by a sustained decrease due to inhibition of Ca2+ reuptake by the storage sites. ET-1 caused near-complete blockade of KCa channels in thapsigargin-treated HPSMCs. This suggests that ET-1 has a direct inhibitory effect on KCa channels. In isolated normoxic HPSMC membrane patches, ET-1 significantly decreases KCa-channel activity. Our observations are different from the those of Salter et al. (26). These authors reported in rat small PA SMCs that ET-1 (0.8-16 nM) activated a Ca2+- and voltage-dependent IKo. The ET-1-induced increase in IKo was mediated by intracellular release of Ca2+. In intact HPSMCs, we observed that 1-5 nM but not 10 nM ET-1 increased IK(Ca). The concentration effect of ET-1 on IKo is likely species and/or arterial-size dependent.

The ET-1-induced modulation of KCa channels is mediated by ETA receptors. The ETA-receptor antagonist BQ-123 blocks the ET-1-mediated changes in KCa channels without affecting baseline channel activity. This is consistent with previously reported observations (4, 5, 29) that infusion of BQ-123 had no effect on resting PA pressure or pulmonary vascular resistance. This also suggests that ETA receptors do not play a significant role in regulating resting pulmonary vascular tone. In isolated normoxic and endothelium-denuded human PA rings, we observed that ET-1 caused concentration-dependent increases in arterial tension. The mechanisms by which ET-1 causes pulmonary vasoconstriction are complex. Mann et al. (16) and others (25) have reported that ET-1 may cause pulmonary vasoconstriction by stimulating Ca2+ entry, activating protein kinase C, and increasing the generation of cyclooxygenase products. Our experimental results demonstrate that ET-1 can also inhibit KCa-channel activity. The mechanism by which ET-1 inhibits KCa channels is unclear. In porcine coronary SMCs, it has been reported that ET-1 inhibits KCa channels via a protein kinase C-independent mechanism (17). The observed inhibitory effect of ET-1 on KCa-channel activity deprives the vascular SMCs of an effective mechanism to counteract the ET-1-induced pathways that lead to pulmonary vasoconstriction. In our physiological experiments, treating PA rings with DHEA reversed the vasoconstrictive effect of ET-1. DHEA causes pulmonary vasodilatation, in part, by activating KCa channels (8). Our results emphasize that KCa channels play an important role in regulating pulmonary vascular tone during agonist stimulation and that one mechanism by which ET-1 induces pulmonary vasoconstriction is by inhibiting KCa-channel activity.

ET-1 Effects on IKo Activity After Chronic Hypoxia

In HPSMCs chronically exposed to hypoxia, ET-1 reduces KCa-channel activity in both intact cells and isolated membrane patches. Chronic hypoxia eliminates the initial ET-1-induced increase in KCa-channel activity. The effect of ET-1 on KCa-channel activity was blocked by two different ETA-receptor antagonists, BQ-123 (4, 12) and JKC-301 (30). This strongly suggests that the ET-1-induced decrease in KCa-channel activity is mediated by ETA receptors. The inhibitory effect of ET-1 on KCa-channel activity was also reversed by treating the hypoxic HPSMCs with either a cGMP analog or DHEA. Both compounds have been shown to increase KCa-channel activity in vascular SMCs (1, 8, 19, 21). The reduction in KCa-channel activity produced by ET-1 is likely mediated by a decrease in channel sensitivity to [Ca2+]i. In inside-out membrane patches, ET-1 causes an e-fold decrease in KCa-channel activity at all [Ca2+]i values in the range of 100 nM to 10 µM. Additionally, chronic treatment of HPSMCs with the ETA-receptor antagonist BQ-123 reverses the effect of chronic hypoxia on KCa-channel activity.

It is unclear how BQ-123 prevents the chronic hypoxia-induced decrease in KCa-channel activity. Chronic infusion of BQ-123 into rats exposed to 2 wk of hypobaric hypoxia attenuates the development of pulmonary hypertension despite elevated ET-1 plasma levels (4). In a recent study, DiCarlo et al. (5) reported in rats that chronic blockade of ETA receptors with BQ-123 significantly reversed the chronic hypoxia-induced pulmonary hypertension. Pulmonary vascular SMCs express ET receptors and produce ET-1 (32, 33). In addition, the production of ET-1 by the main PA and by distal sites of the pulmonary vasculature is augmented after exposure to chronic hypoxia (14, 24). Exposing rats to 4 wk of hypoxia increases lung tissue ETA- and ETB-receptor mRNAs (14). In our physiological experiments, the ET-1-induced increase in human PA ring tension at the highest concentration studied (9 nM) was more pronounced in PAs obtained from three COPD patients. Thus it is possible that exposing HPSMCs to chronic hypoxia causes an increased production of pulmonary vascular ET-1, a change in ETA and/or ETB receptors, or a change in KCa-channel sensitivity to ET-1. These chronic hypoxia-mediated changes could result in a more potent inhibition of KCa-channel activity by ET-1. Thus we speculate that treating chronically hypoxic HPSMCs with BQ-123 prevents the inhibition of KCa channels that results from enhanced ET-1 and/or ETA effect(s).

Our observations agree with the reported finding that chronic treatment of hypoxic rats with ETA-receptor antagonists attenuates the development of pulmonary hypertension (5). Furthermore, our observations provide a molecular mechanism by which ET-1 may contribute to the pathogenesis of hypoxic pulmonary vasoconstriction. The ability of ETA-receptor blockers to reverse the effects of chronic hypoxia on KCa-channel activity provides an explanation for why these drugs are so effective in preventing and reversing the pulmonary vascular effects of chronic hypoxia. KCa channels have large unitary conductances; thus opening a few KCa channels has a significant impact on Em (19, 21, 23). The ability of BQ-123 to open KCa channels and to reverse the effect of chronic hypoxia on KCa-channel activity may provide a novel therapeutic agent for pathological conditions, such as chronic hypoxia, where the pulmonary vascular membrane potential is depolarized.

In summary, ET-1 has a direct inhibitory effect on KCa-channel activity in normoxic and hypoxic HPSMCs. The ETA-receptor antagonists BQ-123 and JKC-301 block and reverse the inhibitory effect of ET-1 on KCa-channel activity. Chronic treatment of HPSMCs with BQ-123 also prevents the inhibitory effect of chronic hypoxia on KCa-channel activity.

    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: I. S. Farrukh, Division of Respiratory, Critical Care, and Occupational Pulmonary Medicine, Dept. of Internal Medicine, Univ. of Utah Health Sciences Center, Salt Lake City, UT 84132.

Received 4 March 1998; accepted in final form 8 June 1998.

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Abstract
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Methods
Results
Discussion
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