Division of Pulmonary and Critical Care Medicine, Department of Medicine, The Johns Hopkins University, Baltimore, Maryland 21224
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ABSTRACT |
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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 (1010
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
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INTRODUCTION |
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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 1010 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-
(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.
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METHODS |
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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 byElectrophysiological 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 MIsolated 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 × 10Experimental 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
(108 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
(108 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 (10Statistical 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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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
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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
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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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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/1) + A2e(
t/
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
1 and
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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Effects of ET-1 on KV Currents
After treatment with ChTX, application of ET-1 (10
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The effect of ET-1 was concentration dependent. As shown in Fig.
5, increasing concentrations of ET-1
(1010 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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Analysis of the inactivation kinetics of
KV currents indicated that
Ao and both
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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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
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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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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 1 from 21.6 ± 4.3 to 15.9 ± 3.5 ms and
2 from 157.3 ± 37.9 to 86.8 ± 7.4 ms, whereas 1,2-DOG decreased
1 from 18.4 ± 3.2 to 16.7 ± 3.4 ms and
2 from 179.2 ± 59.9 to 118.9 ± 26.8 ms, although these reductions did not
achieve statistical significance.
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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
(107 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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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
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DISCUSSION |
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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 < < 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 1010 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
(108 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
4-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 -subunits with the
-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.
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ACKNOWLEDGEMENTS |
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This work was supported by National Heart, Lung, and Blood Institute Grants HL-51912 and HL-09543.
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FOOTNOTES |
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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.
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