Inhibition of voltage-gated K+ current in rat intrapulmonary arterial myocytes by endothelin-1

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

Although endothelin (ET)-1 is an important regulator of pulmonary vascular tone, little is known about the mechanisms by which ET-1 causes contraction in this tissue. Using the whole cell patch-clamp technique in rat intrapulmonary arterial smooth muscle cells, we found that ET-1 and the voltage-dependent K+ (KV)-channel antagonist 4-aminopyridine, but not the Ca2+-activated K+-channel antagonist charybdotoxin (ChTX), caused membrane depolarization. In the presence of 100 nM ChTX, ET-1 (10-10 to 10-7 M) caused a concentration-dependent inhibition of K+ current (56.2 ± 3.8% at 10-7 M) and increased the rate of current inactivation. These effects of ET-1 on K+ current were markedly reduced by inhibitors of protein kinase C (staurosporine and GF 109203X) and phospholipase C (U-73122) or under Ca2+-free conditions and were mimicked by activators of protein kinase C (phorbol 12-myristate 13-actetate and 1,2-dioctanoyl-sn-glycerol). These data suggest that ET-1 modulated pulmonary vascular reactivity by depolarizing pulmonary arterial smooth muscle, due in part to the inhibition of KV current that occurred via activation of the phospholipase C-protein kinase C signal transduction pathway.

potassium ion; protein kinase C; membrane potential; patch clamp

    INTRODUCTION
Top
Abstract
Introduction
Methods
Results
Discussion
References

ENDOTHELIN (ET)-1, a 21-amino acid peptide secreted by the vascular endothelium, causes profound constriction in vascular tissues from many different species and is the most potent vasoconstrictor known to date (49). For example, at concentrations between 10-10 and 10-8 M (EC50 = 3 × 10-9 M), ET-1 constricts isolated pulmonary arteries (6) and causes long-lasting increases in vascular resistance in isolated perfused lungs (26). In addition to its vasoconstrictive properties, ET-1 has potent mitogenic effects on pulmonary vascular smooth muscle cells (53). Synthesis and release of ET-1 from pulmonary arterial endothelial cells can be induced by numerous stimuli, including hypoxia (23), thrombin (36) and tumor necrosis factor-alpha (13), and both ETA- and ETB-receptor mRNA and binding sites are present in pulmonary vascular smooth muscle (11). Recent evidence demonstrates that ET-1-receptor antagonists can prevent acute hypoxic pulmonary vasoconstriction in rats (8, 37), dogs (48), and pigs (16) as well as block or partially reverse (8) pulmonary hypertension and vascular remodeling associated with prolonged exposure to decreased oxygen tensions. These findings indicate that ET-1 is an important modulator of pulmonary vascular tone, which contributes to pulmonary hypertension secondary to hypoxia.

Previous work (27, 30) in systemic vessels suggests that ET-1 causes membrane depolarization and increases intracellular Ca2+ concentration ([Ca2+]i), but the mechanisms of these effects have not been completely elucidated. Binding of ET-1 to G protein-coupled ETA receptors activates the phospholipase C (PLC) cascade (27, 30), initiating the conversion of phosphoinositol to inositol trisphosphate, which evokes Ca2+ release from inositol trisphosphate-gated intracellular stores, and 1,2-diacylglycerol, which activates protein kinase C (PKC). Once activated, PKC can exert a number of significant effects in vascular smooth muscle, including stimulation of Na+/H+ exchange (39), induction of smooth muscle cell proliferation (53), and modulation of ion channels. Electrophysiological studies in systemic myocytes demonstrate that ET-1 activates L-type Ca2+, receptor-operated Ca2+, Cl-, and nonselective ion channels (14, 20, 46); inhibits ATP-sensitive K+ (KATP) channels (33); and either increases or decreases activity of Ca2+-activated K+ (KCa) channels depending on concentration (19, 32). It is not clear whether these effects are due to direct coupling between channels and ET receptors or are secondary to other cellular events such as increased intracellular Ca2+, depolarization, or PKC activation. Thus modulation of ion-channel activity by ET-1 in systemic vascular smooth muscle cells, as well as the physiological relevance and role of these actions, requires further elucidation.

In isolated perfused lungs, the PKC inhibitors staurosporine and calphostin C abolished the pressor effect of ET-1 (5), suggesting involvement of PKC activation. The vasoconstrictor effect of ET-1 appears to be mediated by intracellular Ca2+ release (5) and extracellular Ca2+ influx via L-type Ca2+ channels (18, 29) or other Ca2+ entry pathways (24). Electrophysiological studies in isolated pulmonary arterial smooth muscle cells (PASMCs) showed that ET-1 caused depolarizing oscillations in membrane potential in association with ETA receptor-dependent activation of Cl- and KCa currents secondary to intracellular Ca2+ release (4, 42, 43), as well as ETB- and G protein-dependent inhibition of voltage-gated delayed rectifier K+ (KV) current (42). Moreover, a recent study (1) in portal arterial myocytes indicated that PKC activation with phorbol esters can inhibit KV channels. Because the KV channel is thought to play a major role in regulating the resting membrane potential in PASMCs (50), inhibition of KV channels by ET-1 may cause membrane depolarization, contributing to the activation of L-type Ca2+ channels and increased intracellular Ca2+ levels via Ca2+ influx.

On the basis of this information, we hypothesized that in PASMCs ET-1 causes membrane depolarization by inhibiting KV-channel activity via PLC- and PKC-dependent pathways, leading to smooth muscle cell contraction. To test this hypothesis, we used whole cell patch-clamp techniques in PASMCs and isolated arterial ring segments to determine whether 1) ET-1 and antagonists of K+ channels cause membrane depolarization, 2) ET-1 inhibits KV currents, 3) the effects of ET-1 on KV current are blocked by PLC and PKC inhibitors and are mimicked by PKC activators, and 4) voltage-gated Ca2+-channel antagonists block ET-1-induced vasoconstriction.

    METHODS
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Abstract
Introduction
Methods
Results
Discussion
References

Cell Preparation

Single PASMCs were obtained with the method of Smirnov et al. (45). Briefly, male Wistar rats (150-250 g) were injected with heparin and anesthetized with pentobarbital sodium (130 mg/kg ip). The rats were exsanguinated, and the heart and lungs were removed en bloc 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. Intrapulmonary arteries (300- to 800-µm OD) were isolated and cleaned of connective tissue. 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 at 37°C for 20 min 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). After digestion, single smooth muscle cells were dispersed by gentle trituration with a wide-bore transfer pipette in Ca2+-free PSS. The cell suspension was then transferred to the cell chamber for study. A total of 92 cells from 44 animals were used in this study. Cells used for experiments were either relaxed or partially contracted and exhibited spindle-shaped morphology; round and fully contracted cells were discarded. Smooth muscle cell identity was verified by alpha -actin staining.

Electrophysiological Measurements

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 with a tip resistance of 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, and 10 HEPES, with pH adjusted to 7.2 with 5 M KOH. GTP (0.5 mM) was added to provide substrate for the signal transduction pathways, and MgATP (5 mM) was included to inhibit KATP currents and provide the substrate for energy-dependent processes. In some of the experiments measuring membrane potential and in all of the experiments measuring K+ currents, 10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA) and 3 mM Ca2+ were added to the pipette solution to provide a strong Ca2+-buffering capacity and clamp [Ca2+]i at a physiological level of ~75 nM. Membrane currents were recorded with an Axopatch 200A amplifier (Axon Instruments) and the whole cell configuration in voltage-clamp mode. Pipette potential and capacitance and access resistance were compensated for electronically. 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 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).

Isolated Arterial Ring Segments

Intrapulmonary arteries (300- to 800-µm OD) were obtained as described in Cell Preparation, cleaned of connective tissue, and cut into ring segments 4 mm in length. The arterial rings were mounted on stainless steel wires for isometric tension recording in organ baths containing a modified Krebs solution containing (in mM) 118 NaCl, 4.7 KCl, 0.57 MgSO4, 1.18 KH2PO4, 25 NaHCO3, 10 glucose, and 2.5 CaCl2. The solution was gassed with 16% O2-5% CO2 to maintain a pH of 7.4. Force was recorded with a force-displacement transducer (model FT03, Grass, West Warwick, RI) attached to a micrometer. The arteries were adjusted to a resting tension of 2 g in 0.5-g steps over a period of 40 min. The arteries were exposed to 80 mM KCl to establish viability and maximum contraction and to phenylephrine (3 × 10 -7 M) followed by acetylcholine (10-6 M) to verify endothelium integrity.

Experimental Protocols

Effects of ET-1 and antagonists of K+ channels on membrane potential. Membrane potential measurements were made under current-clamp mode, with the current equal to zero. The effect of ET-1 (10-8 M), 4-aminopyridine (4-AP; 10 mM) or charybdotoxin (ChTX; 100 nM) on membrane potential was determined by recording the membrane potential for 1 min before, 2 min during, and 2 min after exposure to either ET-1 or the K+-channel antagonists.

Characterization of K+ currents. 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 measurements were made under control conditions 3-4 min after the cells were treated with ChTX (100 nM) and 3-4 min after the subsequent addition of 4-AP (10 mM). Whole cell currents remaining after treatment with ChTX were further characterized by analyzing the time course of current inactivation as described in Characterization of Whole Cell K+ Currents.

Effect of ET-1 on KV currents. To study the effect of ET-1 on KV currents, all experiments were conducted in the presence of 100 nM ChTX. To examine the time course of the ET-1 effect, KV 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 (10-9 M) was applied. Once stable currents were attained, ET-1 was washed out. To determine the effects of ET-1 on the current-voltage (I-V) relationship of peak KV currents, membrane currents were measured during depolarizing pulses (800 ms in duration) from a holding potential of -60 mV to test potentials ranging from -50 to +40 mV before and 3-4 min after exposure to increasing concentrations of ET-1 (10-10 to 10-7 M).

Role of PLC and/or PKC in ET-1 effect on KV current. The involvement of the PLC and/or PKC signaling pathway was determined by comparing the effects of ET-1 on KV current under control conditions and after the cells were exposed for 5 min to the PLC inhibitor U-73122 (10 µM) or the PKC inhibitors staurosporine (1 nM) or GF 109203X (GFX; 30 nM). Staurosporine is thought to inhibit all isoforms of PKC, whereas GFX is a putative inhibitor of the Ca2+-dependent isoforms of PKC (31). The effect of PKC activation on the KV current was determined by exposure of PASMCs to either the biologically active diacylglycerol analog 1,2-dioctanoyl-sn-glycerol (1,2-DOG; 10 µM) or the phorbol ester phorbol 12-myristate 13-acetate (PMA; 500 nM). To further explore the Ca2+ dependence on the modulation of KV currents by ET-1, the currents were quantified before and after exposure to ET-1 when intracellular Ca2+ was buffered near 0 nM (no Ca2+ added to the pipette solution) and with external Ca2+ replaced with Mg2+.

Role of voltage-gated Ca2+ channels on ET-1-induced vasoconstriction. To determine whether membrane depolarization plays a functional role in the pressor response to ET-1, contraction in response to ET-1 (10-8 M) was determined in arterial segments in the absence and presence of the voltage-gated Ca2+-channel antagonist verapamil (10-5 M) and is expressed as a percentage of the contraction induced by 80 mM KCl.

Drugs and Chemicals

ET-1 and ChTX were obtained from American Peptides (Sunnyvale, CA). U-73122, GFX, PMA, and 1,2-DOG were obtained from CalBiochem (La Jolla, CA). Staurosporine, tetraethylammonium, 4-AP, and all other chemicals were obtained from Sigma (St. Louis, MO). Stock solutions of ET-1 (10-5 M) and ChTX (10-4 M) were made up in distilled water. Stock solutions of U-73122 (10-2 M), GFX (10-2 M), PMA (10-6 M), 1,2-DOG (10-2 M), and staurosporine (10-2 M) were made up in DMSO. Application of the vehicle used in the study alone at maximal concentration (1:1,000 dilution of DMSO in PSS) had no effect on KV currents. 4-AP was made up as a stock solution (10-1 M) in PSS, and the pH was adjusted to 7.4 with HCl. Stock solutions were divided into aliquots and kept frozen at -20°C. On the day of experiment, they were diluted as needed with PSS to the appropriate concentration.

Statistical Analysis

Statistical significance was determined with Student's t-test (paired and unpaired as applicable) and two-way ANOVA with repeated measures. A P value < 0.05 was accepted as statistically significant. In the text, data are expressed as means ± SE, and n refers to the number of cells tested. All experiments were conducted in cells from at least three different animals.

    RESULTS
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Abstract
Introduction
Methods
Results
Discussion
References

Effects of ET-1 and Antagonists of K+ Channels on Membrane Potential

Representative tracings of membrane potentials recorded in rat PASMCs during and after exposure to ET-1 and antagonists of K+ channels are shown in Fig. 1. Under control conditions, the resting membrane potential averaged -38.2 ± 2.4 mV, consistent with previously reported values (45, 52). ET-1 (10-8 M) caused a significant depolarization of the membrane potential to -19.0 ± 3.1 mV. This depolarization, which began 14 ± 6 s after application of ET-1, was partially reversed on removal of the drug. 4-AP, an inhibitor of KV currents, also caused a reversible depolarization (from -37.6 ± 2.6 to -15.2 ± 4.2 mV). In contrast, ChTX, an inhibitor of KCa currents, had no significant effect on the membrane potential (-32.9 ± 0.9 to -28.6 ± 1.8 mV). To eliminate possible involvement of KCa- and Ca2+-activated Cl- (ClCa)-channel activation in the ET-1-induced depolarization, the membrane potential was also measured in cells in which intracellular Ca2+ was buffered with BAPTA. Under these conditions, ET-1 still caused a significant membrane depolarization (from -31.3 ± 2.4 to -22.5 ± 3.0 mV; n = 5; P < 0.01).


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Fig. 1.   Representative tracings showing effect of endothelin (ET)-1 (A), 4-aminopyridine (4-AP; B), and charybdotoxin (ChTX; C) on pulmonary arterial smooth muscle cell membrane potential. D: mean depolarization induced by ET-1 (n = 6 cells from 4 animals), 4-AP (n = 4 cells from 4 animals), and ChTX (n = 9 cells from 5 animals). * Significant change in (Delta ) membrane potential, P < 0.01 by paired Student's t-test.

Characterization of Whole Cell K+ Currents

In the present study, the average cell capacitance of our freshly isolated rat PASMCs (n = 89) was 20.1 ± 1.6 pF, consistent with values previously reported in the rabbit (9) and rat (41, 45). Step depolarizations from a holding potential of -60 mV to test potentials between -30 and +60 mV elicited voltage-dependent outward currents (Fig. 2), which could be separated into at least two major components. A component of the K+ current that was inhibited by 100 nM ChTX, but not by 10 mM 4-AP, was observed at potentials positive to -10 mV. Isolation of this K+ current by subtracting the currents activated in the presence of ChTX from the control currents (Fig. 2C) showed that it was noninactivating and characterized by greater noise. These characteristics are consistent with those of the large-conductance KCa channels (15). Under our experimental conditions (intracellular ATP concentration = 5 mM and free [Ca2+]i ~ 75 nM), KCa current accounted for 43.5 ± 3.85 and 52.8 ± 2.6% (n = 24) of the total outward current at +40 mV when measured at peak current and at the end of the voltage-clamp pulse (steady-state current), respectively.


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Fig. 2.   Outward K+ currents from rat intrapulmonary arterial smooth muscle cells measured with whole cell voltage-clamp under control conditions (A) and in presence of ChTX (100 nM; B). C: subtracted difference between A and B, corresponding to ChTX-sensitive portion of current. D: current-voltage relationship before and after exposure to ChTX.

After inhibition of KCa currents with ChTX, a low-noise, voltage-gated K+ current with time-dependent activation and inactivation was observed (Fig. 2B). This current was activated at potentials positive to -30 mV. The magnitude of the current increased, whereas the time to peak decreased, with increasingly positive potentials (Fig. 3C). Time-dependent inactivation of this current was consistently observed at potentials positive to -10 mV, and the proportion of inactivating KV current increased with more positive potentials. 4-AP caused >95% inhibition of this K+ current (Fig. 3B), consistent with the KV current described in previous studies (1, 45).


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Fig. 3.   Characterization of voltage-dependent K+ (KV) current. A: inhibition of KV current by 10 mM 4-AP in presence of ChTX (n = 6 cells from 3 animals). B: mean current-voltage relationship before and after exposure to 4-AP. C: rate of KV-current activation as a function of test potential voltage (n = 6 cells).

The kinetics of KV current were further quantified by fitting the inactivating portions of the KV current with a biexponential equation (1, 51): I(t) = Ao + A1e(t/tau 1) + A2e(-t/tau 2), where I(t) is the current at time t, A o is the amplitude of the steady-state current, A1 and A2 are the amplitudes of the rapidly inactivating and slowly inactivating components, respectively, and tau 1 and tau 2 are the time constants of the rapidly inactivating and slowly inactivating components, respectively. This approach separated the KV current into the rapidly and slowly inactivating and noninactivating components (Table 1), with the average ratio of the components A o to A1 to A2 approximately equal to 2:1:1. The proportion of the three components varied among individual cells. In a few cells, the fast component was the major inactivating component, whereas in other cells, the slowly inactivating component was dominant. Nevertheless, all three components were sensitive to 4-AP (Fig. 3A), which reduced the peak current from 33.3 ± 6.4 to 2 ± 0.2 pA/pF (n = 6) and the steady-state current from 23.5 ± 6.4 to 1.3 ± 0.25 pA/pF.

                              
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Table 1.   Effect of ET-1 on KV current amplitudes and time constants of inactivation

Effects of ET-1 on KV Currents

After treatment with ChTX, application of ET-1 (10-9 M) to the PASMCs (n = 14) caused a significant reduction in the magnitude of the KV current (Fig. 4). The onset of the response was fast, within 5 s of ET-1 application, and required an average of 146.3 ± 15.7 s to reach a stable response of 25.4 ± 7.3% inhibition of peak KV current. Comparison of the time course of the effect of ET-1 on the membrane potential with that of the effect of ET-1 on the KV current suggests that inhibition of the KV current preceded depolarization. A partial reversal (70.6 ± 2.9%) of the inhibition of KV current was observed on washout of ET-1.


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Fig. 4.   Top: representative time course of change in peak KV current in response to ET-1. Measurements were made in presence of 100 nM ChTX. A: baseline. B: stable ET-1 response. C: recovery. Similar results were obtained in 7 other cells from 4 animals. Bottom: KV-current traces corresponding to baseline (A), stable ET-1 response (B), and recovery (C).

The effect of ET-1 was concentration dependent. As shown in Fig. 5, increasing concentrations of ET-1 (10-10 to 10-7 M) caused an increasing inhibition of KV current, as represented by the downward shifts in the I-V relationships. At 10-7 M, the highest concentration tested, ET-1 inhibited peak KV current by 50.9 ± 3.5% (from 57.1 ± 24.1 to 30.2 ± 14.4 pA/pF) at +40 mV. ET-1 did not alter time to peak at +40 mV, which averaged 32.4 ± 5.2 ms before ET-1 (10-9 M) and 30.0 ± 4.3 ms after ET-1.


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Fig. 5.   A: effect of ET-1 on whole cell KV current. Measurements were made in presence of 100 nM ChTX. B: mean current-voltage relationship at varying concentrations of ET-1. C: average percent inhibition of KV current induced by varying concentrations of ET-1 at +40 mV (n = 5 cells from 3 animals). * Significantly different from control value.

Analysis of the inactivation kinetics of KV currents indicated that Ao and both tau 1 and A1 decreased in the presence of 10-9 M ET-1 (Table 1). The effect of ET-1 on the overall inactivation of KV current is further illustrated by superimposing the peak normalized KV current in the presence and absence of ET-1 (Fig. 6), showing significant enhancement of inactivation and reduction in steady-state current in the presence of the peptide.


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Fig. 6.   Effect of ET-1 (10-9 M) on rate of KV-current inactivation. Measurements were made in presence of 100 nM ChTX. tau 1 and tau 2, time constants of rapidly inactivating and slowly inactivating components, respectively. Results are similar to those obtained in 14 other cells from 8 animals.

Effect of PLC and PKC Inhibitors on ET-1-Induced Inhibition of KV Currents

To test whether the inhibition of KV currents by ET-1 was mediated via the PLC and/or PKC pathway, we determined the effect of a specific PLC inhibitor, U-73122, on the inhibition of KV current induced by ET-1. Application of U-73122 (10 µM) had no significant effect on KV-current magnitude. In the presence of U-73122, ET-1 (10-9 M) failed to cause a significant effect on the time constants and amplitudes of inactivation, and ANOVA showed no difference in the I-V curves before and after exposure to ET-1 (Fig. 7).


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Fig. 7.   A: effect of phospholipase C inhibition with U-73122 on response of KV current to ET-1. Measurements were made in presence of ChTX. B: mean current-voltage relationship before and after exposure to ET-1 in presence of U-73122 (n = 7 cells from 5 animals).

To examine the possible involvement of PKC, PASMCs were exposed to staurosporine (1 nM), an isoform-nonselective PKC inhibitor, or GFX (30 nM), a putative inhibitor of the Ca2+-dependent isoforms of PKC (31). Staurosporine and GFX each caused some inhibition of KV currents (-11.9 ± 14.7 and -19.2 ± 7.5%, respectively, at +40 mV), although these effects did not achieve statistical significance. Subsequent exposure to ET-1 (10-9 M) had no significant effect on the I-V relationship in cells treated with staurosporine (Fig. 8A) or GFX (Fig. 8B). At potentials positive to +20 mV, both inhibitors markedly reduced the inhibitory effect of ET-1 on KV current (Fig. 9). Furthermore, there was no change in either the time constants or amplitudes of the fast or slow component of inactivation when ET-1 was applied in the presence of the PKC inhibitors.


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Fig. 8.   Effect of protein kinase C inhibitors staurosporine (Stauro; n = 9 cells from 5 animals; A) and GF 109203X (GFX; n = 6 cells from 5 animals; B) on response to ET-1. Measurements were made in presence of 100 nM ChTX.


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Fig. 9.   Effect of Stauro (n = 9 cells), GFX (n = 6 cells), and U-73122 (n = 6 cells) on response of KV current to ET-1 (n = 5 cells). * Significantly different from response to ET-1 under control conditions, P < 0.01 by Student's t-test. dagger  Significantly different from zero, P < 0.05 by paired Student's t-test.

To further test the possibility that inhibition of KV currents was mediated by PKC activation, we determined whether PKC activation by PMA and 1,2-DOG could mimic the ET-1-induced response. Similar to the results obtained with ET-1, application of either PMA or 1,2-DOG caused a significant decrease in peak KV current (at +40 mV) of 27 ± 11.4 and 28.5 ± 1.9%, respectively (Fig. 10). The effects of PMA and 1,2-DOG on the time constants of inactivation were also qualitatively similar to those of ET-1, in that PMA decreased tau 1 from 21.6 ± 4.3 to 15.9 ± 3.5 ms and tau 2 from 157.3 ± 37.9 to 86.8 ± 7.4 ms, whereas 1,2-DOG decreased tau 1 from 18.4 ± 3.2 to 16.7 ± 3.4 ms and tau 2 from 179.2 ± 59.9 to 118.9 ± 26.8 ms, although these reductions did not achieve statistical significance.


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Fig. 10.   Effect of protein kinase C activation with either phorbol 12-myristate 13-actetate (PMA; n = 8 cells from 7 animals; A) or 1,2-dioctanoyl-sn-glycerol (1,2-DOG; n = 7 cells from 6 animals; B) on KV current. Measurements were made in presence of 100 nM ChTX. C: percent inhibition of peak KV current at +40 mV by ET-1, PMA, and 1,2-DOG.

To determine whether the effects of ET-1 we observed in the rat PASMCs were Ca2+ dependent, KV currents were examined under Ca2+-free conditions. In these experiments, Ca2+ in the external solution was substituted with 2 mM Mg2+ plus 1 mM EGTA, and no Ca2+ was added to the pipette solution (0 mM Ca2+-10 mM BAPTA). Under these conditions, the inhibitory effect of ET-1 (10-7 M) on KV current was significantly reduced (Fig. 11). At a test potential of +40 mV, Ca2+-free conditions reduced the maximum inhibitory effect of ET-1 from -53.3 ± 5.2 to -27.1 ± 3.4%.


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Fig. 11.   Effect of intracellular Ca2+ concentration ([Ca2+]i) at physiological level (A) and after removal of extracellular Ca2+ and complete buffering of [Ca2+]i [10 mM 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid-0 mM Ca2+; n = 7 cells from 3 animals; B] on ET-1-induced inhibition of KV. Measurements were made in presence of 100 nM ChTX. C: percent inhibition of peak KV current at +40 mV under control and Ca2+-free conditions. * Significantly different from control value.

Effect of Ca2+-Channel Antagonist on ET-1-Induced Vasoconstriction

Depolarization resulting from ET-1-induced inhibition of KV channels could enhance vasoconstriction by promoting Ca2+ influx via voltage-gated Ca2+ channels. This possibility was tested by measuring the tension generated in response to ET-1 in rat intrapulmonary arterial segments in the presence and absence of verapamil, a Ca2+-channel antagonist. Verapamil (10-5 M) significantly reduced the peak contractile response to ET-1 (10-8 M) by 26.9 ± 8.8 and 38.0 ± 8.1% when measured at 15 and 25 min of exposure, respectively (Fig. 12), suggesting that enhanced Ca2+ influx through voltage-gated Ca2+ channels played a functional role in the pressor response to ET-1.


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Fig. 12.   A: effect of verapamil (n = 6 vessels from 6 animals) on ET-1-induced contraction in isolated rat intrapulmonary arteries. Contractile response to ET-1 is expressed as percent tension induced by 80 mM KCl (n = 6 vessels from 6 animals). B: percent inhibition of ET-1-induced contraction by verapamil 15 and 25 min after exposure to ET-1.

    DISCUSSION
Top
Abstract
Introduction
Methods
Results
Discussion
References

The major findings of this study in myocytes from rat intrapulmonary arteries of 300- to 800-µm diameter are that 1) ET-1 and 4-AP, but not ChTX, caused a marked membrane depolarization; 2) ET-1 caused a concentration-dependent inhibition of 4-AP-sensitive voltage-gated K+ currents, reducing both the rapidly inactivating and steady-state components of these currents and increasing the rate of current inactivation; and 3) these effects of ET-1 were attenuated by inhibitors of PLC and PKC, mimicked by PKC activation, and partially reduced in the absence of Ca2+. Additionally, in rat intrapulmonary arterial segments, Ca2+-channel antagonists attenuate the contractile response to ET-1. These results suggest that, in these cells, ET-1 causes membrane depolarization and inhibits KV current through activation of the PLC-PKC signal transduction pathway, possibly leading to activation of voltage-gated Ca2+ channels and contributing to ET-1-induced contraction.

In vascular smooth muscle, several subtypes of K+ channels have been identified, including KCa, KATP, inward rectifying K+, and KV channels. Consistent with previous reports (3, 40, 52), the predominant outward currents in our cells, which were dialyzed with a pipette solution containing ATP, were the large-conductance KCa and KV currents as identified by inhibition of the currents with ChTX and 4-AP, respectively. Although the KCa current comprised nearly one-half of the total outward current in our cells at positive potentials, our observation that 4-AP, but not ChTX, caused membrane depolarization confirmed the observation that KV, but not KCa, currents contributed significantly to regulation of the resting membrane potential in rat PASMCs (50). Although KV channels do not appear to be active at potentials negative to -30 mV, inhibition of these channels with 4-AP under baseline conditions caused substantial depolarization, suggesting that some of these channels were open. Given the high membrane input resistance (35), inhibiting only a few open channels could result in significant depolarization.

KV current in our rat intralobar pulmonary arterial myocytes consisted of at least three 4-AP-sensitive components: rapidly inactivating, slowly inactivating, and noninactivating. In our PASMCs, all components of KV current were abolished in the presence of 4-AP, indicating that the outward current was not contaminated by KCa or ClCa currents. The slowly inactivating and steady-state components closely resemble KV currents observed in other systemic (1, 21) and pulmonary (9, 52) arterial smooth muscle. In contrast, the rapidly inactivating component (15 ms < tau  < 40 ms), or A-type current, observed in our cells as well as in some other PASMCs (9, 22, 52), is not commonly observed in other vascular smooth muscle (44, 47), suggesting that this transient, rapidly inactivating outward current may be preferentially expressed in the pulmonary vasculature. The multiple components of KV current observed in this and other studies (1, 3, 9, 52) may suggest that multiple KV channel subtypes are present in PASMCs; however, the detailed distribution of KV channels in rat intrapulmonary arteries has yet to be determined.

In the present study, we evaluated the effect of ET-1 on 4-AP-sensitive KV currents as a whole as well as on the separate components. We found that ET-1 significantly inhibited KV current at concentrations between 10-10 and 10-7 M (EC50 ~ 10-9 M), with a maximum inhibition of ~50%. Both the rapidly inactivating and steady-state components were significantly attenuated, indicating that both the delayed rectifier and A-type currents were inhibited. The concentration dependence of ET-1 on the KV current was similar to that reported in contraction studies in isolated rat pulmonary arterial rings (6), and the effect of ET-1 was long lasting, requiring minutes for partial recovery. Our results are consistent with the observation of Salter and Kozlowski (42), who found that inhibition of KV current in myocytes from small arteries was due to activation of ETB receptors by ET-1 at concentrations between 10-11 and 10-8 M (EC50 ~ 0.5 nM), with a maximum inhibition of 25%. Although the concentration dependence and time course of the response were similar to our results, the greater inhibition by ET-1 in our PASMCs suggests that there may be differences in the reactivity to ET-1 in PASMCs from proximal and small arteries. This possibility is supported by findings that the contractile response to ET-1 varies with location in the vasculature (6, 24, 28). Thus, given possible heterogeneity in channel activity among species and vessels in the pulmonary vasculature (2, 3), further experiments will be necessary to determine whether these results are representative of pulmonary arteries in all species.

We also found that a moderate concentration of ET-1 (10-8 M) caused a marked depolarization in rat PASMCs, which is consistent with findings in pulmonary (43) and systemic (33) arterial smooth muscles. Given that the inhibition of KV current by ET-1 precedes the onset of depolarization and the inhibition of KV currents by 4-AP causes depolarization, it is possible that the inhibition of KV currents contributed to the depolarization observed in response to ET-1. However, in addition to KV and KCa channels, other channels may participate in ET-1-induced membrane depolarization (9, 43, 51). It has been proposed that inhibition of KATP channels by ET-1 or activation of ClCa channels secondary to ET-1-induced elevations in intracellular Ca2+ may contribute to membrane depolarization in coronary (33), aortic (46), and pulmonary (43) vascular smooth muscle cells. It is unlikely, however, that inhibition of KCa channels could account for the membrane depolarization observed in our PASMCs because inhibiting KCa channels with ChTX had little effect on membrane potential. Furthermore, the intracellular solution contained a high concentration of ATP, which should prevent KATP channels from participating in ET-1-induced depolarization. Finally, the effect of ET-1 on membrane potential persisted in cells in which intracellular Ca2+ was buffered with BAPTA, suggesting that activation of ClCa channels or inhibition of KV channels secondary to increased [Ca2+]i (12) was not the cause of the depolarization we observed. However, if lower concentrations of ATP were present and intracellular Ca2+ were not buffered, inhibition of KATP or activation of ClCa currents could act synergistically with the inhibition of KV current to potentiate ET-1-induced membrane depolarization.

Our results indicate that the inhibitory effect of ET-1 on KV current is mediated via the PLC-PKC signal transduction pathway. U-73122, staurosporine, and GFX, inhibitors of PLC and PKC, markedly reduced, whereas PMA and 1,2-DOG, activators of PKC, mimicked the effect of ET-1. The functional link between ET receptors and PLC activation in the systemic vasculature is well established (27, 30), and the involvement of the PLC-PKC pathway is consistent with previous observations (10) of contractile responses in other vascular preparations as well as with a report (5) in isolated perfused lungs that PKC inhibitors abolished the contractile effect of ET-1. Furthermore, our results are consistent with a recent study (1) in the rabbit portal vein that demonstrated that the PKC activators 4beta -phorbol 12,13-dibutyrate and 1,2-DOG caused an inhibition of KV currents that could be reversed by the PKC inhibitors chelerythrine and calphostin C. Although the presence of a small residual response to ET-1 in the presence of PKC inhibitors does not exclude the possibility that a PKC-independent pathway may play a minor role in the ET-1-induced inhibition of KV current, our results are in contrast to the observation that inhibition of KV current by ET-1 was related to a phosphorylation-independent transduction pathway between the ETB receptor and KV channels (42). This discrepancy may be related to the size of the vessels from which the cells were obtained. In our studies, cells were from proximal intrapulmonary arteries (300- to 800-µm OD), whereas cells for the other study were obtained from more distal arteries (200- to 400-µm OD). It has been suggested that smooth muscle cell phenotype (3) and ET receptor subtype distribution (28) may differ as a function of species or location in the pulmonary vascular tree, with predominantly ETA receptors in the main pulmonary arteries and ETB receptors in small pulmonary arteries. It will be important in future studies to determine the receptor subtype responsible for the effect of ET-1 in proximal pulmonary arteries.

Several PKC isoforms that can be classified by their dependence on Ca2+ have been identified in vascular smooth muscle. It is unclear which isoform was involved in the ET-1-induced inhibition of KV currents; however, removal of Ca2+ reduced, but did not eliminate, this inhibition. These results suggest that both Ca2+-dependent and -independent isoforms of PKC were involved, a finding that differs from the observations in rabbit portal arteries that suggested that Ca2+-independent isoforms of PKC mediated PKC-dependent inhibition of KV current (1). The mechanism by which PKC mediates inhibition of KV channels is unclear. It may involve direct phosphorylation of KV channels because multiple PKC phosphorylation sites are present in KV channels (38), or it may act indirectly through second messenger systems because several protein kinases can be phosphorylated by PKC and have been demonstrated to modulate KV-channel activity (17). In addition, recent evidence indicates that the interaction, or coexpression, of beta -subunits with the alpha -subunit of KV channels also enhances the rate of KV-channel inactivation (7, 34), a process that can be modulated by phosphorylation (25). More experiments are needed to elucidate the exact mechanisms responsible for this phenomenon.

Regardless of the exact mechanism by which ET-1 exerts its action on KV channels, reduction of KV current and membrane depolarization in response to ET-1 is likely to play an important role in vasoconstriction by activating voltage-gated Ca2+ channels, leading to enhanced Ca2+ influx and elevated intracellular Ca2+ levels. This notion is supported by our finding that inhibition of voltage-gated Ca2+ channels with verapamil significantly attenuated the pressor response to ET-1 in the rat intrapulmonary arteries from which our cells were isolated. Our results are consistent with those in isolated rabbit (29) and guinea pig (18) lung preparations that showed that Ca2+-channel blockade with verapamil and nifedipine, respectively, decreased the sustained vasopressor responses to ET-1 by 50% or more.

In summary, our results suggest that in rat PASMCs, ET-1 inhibits 4-AP-sensitive, voltage-activated K+ currents by a mechanism involving PKC and PLC. Both Ca2+-dependent and -independent pathways appear to be active because the effect of ET-1 on KV current was attenuated, but not abolished, in the absence of Ca2+. Although the mechanism by which ET-1 induces vasoconstriction is complex and likely to involve several pathways, these findings indicate a functional role for PLC and PKC in ET-1 signal transduction and suggest that ET-1 may modulate pulmonary vascular reactivity, in part, by inhibition of KV current and membrane depolarization.

    ACKNOWLEDGEMENTS

This work was supported by National Heart, Lung, and Blood Institute Grants HL-51912 and HL-09543.

    FOOTNOTES

Address for reprint requests: J. S. K. Sham, Division of Pulmonary and Critical Care Medicine, The Johns Hopkins Medical Institutions, 5501 Hopkins Bayview Circle, Baltimore, MD 21224.

Received 11 August 1997; accepted in final form 5 February 1998.

    REFERENCES
Top
Abstract
Introduction
Methods
Results
Discussion
References

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