Cellular mechanisms and role of endothelin-1-induced calcium
oscillations in pulmonary arterial myocytes
Jean-Marc
Hyvelin,
Christelle
Guibert,
Roger
Marthan, and
Jean-Pierre
Savineau
Laboratoire de Physiologie Cellulaire Respiratoire, Contrat de
Recherche Institut National de la Santé et de la Recherche
Médicale 9806, Université Bordeaux 2, 33076 Bordeaux,
France
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ABSTRACT |
The effect of endothelin (ET)-1 on both
cytosolic Ca2+ concentration
([Ca2+]i)
and membrane current in freshly isolated myocytes, as well as on the
contraction of arterial rings, was investigated in rat main pulmonary
artery (RMPA) and intrapulmonary arteries (RIPA). ET-1 (5-100 nM,
30 s) induced a first
[Ca2+]i
peak followed by 3-5 oscillations of decreasing amplitude. In
RMPA, the ET-1-induced
[Ca2+]i
response was fully abolished by BQ-123 (0.1 µM). In RIPA, the response was inhibited by BQ-123 in only 21% of the cells, whereas it
was abolished by BQ-788 (1 µM) in 70% of the cells. In both types of
arteries, the response was not modified in the presence of 100 µM
La3+ or in the absence of external
Ca2+ but disappeared after
pretreatment of the cells with thapsigargin (1 µM) or neomycin (0.1 µM). In RPMA myocytes clamped at
60 mV, ET-1 induced an
oscillatory inward current, the reversal potential of which was close
to the equilibrium potential for
Cl
. This current was
unaltered by the removal of external
Ca2+ but was abolished by niflumic
acid (50 µM). In arterial rings, the ET-1 (100 nM)-induced
contraction was decreased by 35% in the presence of either niflumic
acid (50 µM) or nifedipine (1 µM). These results demonstrate that
ET-1 via the ETA receptor only in
RMPA and both ETA and
ETB receptors in RIPA induce
[Ca2+]i
oscillations due to iterative Ca2+
release from an inositol trisphosphate-sensitive
Ca2+ store.
Ca2+ release secondarily activates
an oscillatory membrane Cl
current that can depolarize the cell membrane, leading to an influx of
Ca2+, this latter contributing to
the ET-1-induced vasoconstrictor effect.
cytosolic calcium concentration measurement; patch clamp; vascular
smooth muscle; niflumic acid; calcium-activated chloride currrent
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INTRODUCTION |
PULMONARY VASCULAR TONE is controlled by a variety of
circulating and locally released mediators, including the peptides
endothelin (ET)-1 and angiotensin II (ANG II), which are the most
potent vasoconstrictors (3). These two peptides are also implicated in
a variety of pulmonary pathophysiological processes such as cell
proliferation (7, 45) or pulmonary hypertension (11, 25). A better
knowledge of cellular and molecular mechanisms mediating the effect of
these peptides would be of interest to further understand pulmonary
vascular diseases.
At the vascular smooth muscle cell site, ET-1 and ANG II bind to
specific membrane receptors, inducing an increase in the cytosolic
Ca2+ concentration
([Ca2+]i)
that triggers the contractile response (28, 33). Unlike for ANG II
(15), the detailed mechanisms linking the binding of ET-1 to its
receptor to the activation process of the contractile apparatus in
pulmonary vascular smooth muscle are not fully elucidated. In all of
the different vascular beds, ET-1 produces slowly developing and
sustained vasoconstrictor responses. However, the sources of activator
Ca2+ and the cellular mechanisms
underlying these responses vary. ET-1 increases
[Ca2+]i
by either stimulating a Ca2+
influx through the plasmalemma (13) or releasing
Ca2+ from internal
Ca2+ stores (29, 41).
Ca2+ influx can result from the
opening of dihydropyridine-sensitive Ca2+ channels via a G protein (24)
or as a consequence of membrane depolarization due to the activation of
Cl
channels (26, 40),
nonselective cation channels (4), or inhibition of ATP-sensitive
K+ channels (31). More recently, a
receptor-mediated Ca2+-permeable
nonselective cation channel has been implicated in membrane
depolarization and Ca2+ entry
activated by ET-1 in aortic smooth muscle (32). Whatever the pathways
responsible for this
[Ca2+]i
increase, the pattern of ET-1-induced
[Ca2+]i
response generally corresponds to an initial peak followed by a
sustained plateau of a smaller amplitude (10). However, in myocytes
from small pulmonary arteries, oscillations in
[Ca2+]i
have been observed in response to ET-1 (2). This pattern of
[Ca2+]i
response mimics what we have previously described in the main pulmonary
artery upon ANG II stimulation (15). Whether ET-1-induced [Ca2+]i
oscillations are due to similar cellular mechanisms to those evoked by
ANG II is not known. Moreover, in the pulmonary circulation, ET-1-induced vasoconstriction results from the activation of both ETA and
ETB receptors, the ratio and the
efficacy of coupling of these receptors to the contractile apparatus
being species dependent (9) and varying throughout the pulmonary
arterial tree (18).
The present work was thus designed to investigate, in both rat main
pulmonary artery (RMPA) and intrapulmonary arteries (RIPA) 1) the subtype of ET-1 receptor
implicated in the
[Ca2+]i
response; 2) the sources of
Ca2+ and the cellular mechanisms
underlying this response; and 3) the
contribution of these mechanisms to the ET-1-induced contractile response. Indo 1 microspectrofluorimetry and the whole cell patch-clamp technique were used in freshly isolated myocytes to measure
[Ca2+]i
and membrane current, respectively. Isometric contraction was measured
in arterial rings from the same preparation.
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MATERIALS AND METHODS |
Tissue preparation. Wistar male rats
aged from 8 to 10 wk and weighing 280-340 g were anesthetized by
intraperitoneal injection of 40 mg of ethyl carbamate. Heart and lungs
were removed en bloc. The main and small intrapulmonary (277 ± 26.1 µm, n = 22) arteries were then
dissected under binocular control, and the adventitial and intimal
layers were removed. For contraction experiments, rings (3 mm in
length) were dissected from the main artery. For cell dissociation,
arteries were cut into several pieces (1 × 1 mm), incubated for
10 min in low-Ca2+ (200 µM)
physiological saline solution (PSS, composition given below) and then
incubated in low-Ca2+ PSS
containing 0.5 mg/ml collagenase, 0.4 mg/ml pronase, 0.06 mg/ml
elastase, and 3 mg/ml bovine serum albumin at 37°C for two successive periods of 20 min, using fresh enzymes each time. After this
sequence, the solution was removed and the arterial pieces were
incubated again in a fresh enzyme-free solution and triturated with a
fire-polished Pasteur pipette to release cells. Cells were stored on
glass coverslips at 4°C in PSS containing 0.8 mM
Ca2+ and used on the same day.
[Ca2+]i
measurements.
To assess the dynamic changes in
[Ca2+]i
of individual arterial myocytes, we used the
[Ca2+]i-
sensitive fluorophore indo 1. In most of the experiments, cells were
loaded with indo 1 by incubation in PSS containing 1 µM indo
1-pentaacetoxymethyl ester (indo 1-AM) for 25 min at room temperature
and then washed in PSS for 25 min. The coverslip with attached cells
was then mounted in a perfusion chamber and continuously perfused. In
combined experiments of
[Ca2+]i
and membrane current measurements, indo 1 (50 µM) was added to the
pipette solution and then entered the cells after establishment of the
whole cell recording mode (see below). The recording system included a
Nikon Diaphot inverted microscope fitted with epifluorescence (Nikon,
Tokyo, Japan). A single cell among those on the coverslip was tested
through a window slightly larger than the cell. As described previously
(15), the studied cell was illuminated at 360 nm and counted
simultaneously at 405 and 480 nm by two photomultipliers (P100; Nikon).
Voltage signals at each wavelength were stored in an IBM personal
computer for subsequent analysis. The fluorescence ratio
(405/480) was calculated on-line and displayed with the
two voltage signals on a monitor.
[Ca2+]i
was estimated from the 405-to-480 nm ratio (14) using a calibration for
indo 1 determined within cells (15).
Membrane current recordings. Membrane
currents were measured in the conventional whole cell current recording
mode (17) using a Biologic RK 400 patch-clamp amplifier. Whole cell
membrane currents were recorded with borosilicate patch pipettes of
3-7 M
resistance obtained with a vertical puller (Narishige,
Tokyo, Japan). Membrane currents were stored and analyzed using an IBM personal computer (pCLAMP System; Axon Instruments, Foster City, CA).
The currents were not corrected for leakage. Currents were recorded
under a constant voltage clamp at
60 mV and during a ramp pulse
(0.5 s in duration, 0.2 V/s) from
60 to +40 mV. The current-voltage
(I-V)
relation for ET-1-induced current was obtained by subtracting current
evoked by the same ramp pulse in the absence of ET-1. Generally, for
each experiment, five
I-V
curves were averaged, and the reversal potential
(Erev)
indicated is that of the mean
I-V
curve.
Solutions and application of ET-1. The
external PSS contained (in mM) 130 NaCl, 5.6 KCl, 1 MgCl2, 2 CaCl2, 11.1 D-glucose, and 10 HEPES, pH 7.4 with NaOH.
Ca2+-free PSS was prepared by
replacing CaCl2 with 0.4 mM EGTA.
The internal solution (the solution in the patch pipette and inside the
cell) contained (in mM) 120 CsCl, 10 NaCl, and 20 HEPES, pH 7.3 with
NaOH. ET-1 was applied to the recorded cell by pressure ejection from a
glass pipette located close to the cell for the period indicated on the
records. It was verified, in control experiments, that no change in
[Ca2+]i
was observed during test ejections of PSS. Generally, each record of
membrane current and
[Ca2+]i
response to ET-1 alone or in the presence of an additional substance
was obtained from a different cell. Each type of experiment was
repeated for the number of cells indicated in the text. Experiments were done at room temperature (20-22°C).
Isometric contraction measurements.
Isometric contraction was measured in rings from RMPA that were mounted
between two stainless steel clips in vertical 20-ml organ baths of a
computerized isolated organ bath system (IOS1; EMKA Technologies,
Paris, France). Baths were filled with Krebs-Henseleit (KH) solution
(composition in mM: 118.4 NaCl, 4.7 KCl, 2.5 CaCl2, 1.2 MgSO4, 1.2 KH2PO4,
25 NaHCO3, and 11.1 D-glucose, pH 7.4) maintained at 37°C and bubbled with
a 95% O2-5%
CO2 gas mixture. The upper
stainless clip was connected to an isometric force transducer (EMKA
Technologies). Tissues were set at optimal length by equilibration
against a passive load of 10 mN as determined in preliminary
experiments. At the beginning of each experiment,
K+-rich (80 mM) solution, obtained
by substituting an equimolar amount of KCl for NaCl from KH solution,
was repeatedly applied to obtain at least two contractions similar in
both amplitude and kinetics. This contraction served as a reference
response that was used to normalize subsequent contractile responses. A cumulative concentration-response curve (CCRC) to ET-1 (0.1-100 nM) was then constructed. A concentration increment was made once the
maximal contractile effect of the preceding concentration had been
recorded (generally 18-20 min). Fifteen minutes before the
beginning of the CCRC, the desired channel antagonist (nifedipine or
niflumic acid) was administered to one-half of the rings. The unexposed
rings served as temporal control.
Chemicals and drugs. Collagenase (type
CLS1) was from Worthington Biochemical (Freehold, NJ). Pronase (type
E), elastase (type 3), bovine serum albumin, ET-1, neomycin,
nifedipine, niflumic acid, phorbol 12,13-dibutyrate (PDBu), ruthenium
red, thapsigargin, and tetracaine were purchased from Sigma (Saint
Quentin Fallavier, France). BQ-123, BQ-788, and sarafotoxin (SRTX) S6c
were from RBI (Natick, MA). Caffeine was from Merck (Darmstadt,
Germany). Indo 1 was from Calbiochem (France Biochem, Meudon, France).
ET-1 was dissolved in distilled water to make aliquots of stock
solution (10
4 M) that were
kept frozen (
20°C) until the day of the experiment. Indo 1, niflumic acid, PDBu, and thapsigargin were dissolved in dimethyl
sulfoxide (DMSO). The maximal concentration of DMSO used in our
experiments was <0.1% and had no effect on the mechanical activity
of rings or on the resting value or the variation of the membrane
current and
[Ca2+]i
induced by agonists in cells.
Statistical analysis. Results are
expressed as means ± SE with n the
sample size. Significance was tested by means of Student's t-test at a
P value < 0.05. In contraction
experiments, EC50, the
concentration of ET-1 inducing 50% of the maximal response, was
graphically determined from the mean CCRC.
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RESULTS |
Characteristics of the ET-1-induced
[Ca2+]i
response.
Short application (30 s) of ET-1 (5-100 nM) induced cyclic
variations (oscillations) of
[Ca2+]i
in single myocytes from both RMPA and RIPA. The ET-1-induced [Ca2+]i
response was typically composed of four to six oscillations of constant
duration (6.48 ± 0.53 s, n = 25, and 6.73 ± 0.2 s, n = 40, in RMPA
and RIPA, respectively) but of decreasing amplitude (Fig.
1A; see
also Fig. 3A). The values of the
resting
[Ca2+]i,
the first peak in
[Ca2+]i,
the delay between the beginning of ET-1 ejection and this first peak,
and the frequency of oscillations are indicated in Table
1. Generally, the two last oscillations
occurred after the cessation of ET-1 microejection. The pattern of the
[Ca2+]i
response was relatively independent of the ejected ET-1 concentration (Fig. 1A). Nevertheless, the
percentage of cells that responded to ET-1 did depend on that
concentration (20, 84, and 100% of responding cells in RMPA and 32, 80, and 100% of responding cells in RIPA for 5, 50, and 100 nM ET-1,
respectively; Fig. 1B). Combination of the number of responding cells with the amplitude of the first [Ca2+]i
peak reveals a relationship between ET-1 concentration and the
[Ca2+]i
response (Fig. 1C). Although ET-1
induced a response for concentrations in the nanomolar range, in order
to assess the effect of ET-1 in conditions where all of the cells
respond, we thus used 100 nM ET-1 in subsequent experiments. This ET-1
concentration is also that inducing the maximal contractile response in
pulmonary arterial rings (see below).

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Fig. 1.
Effect of endothelin (ET)-1 on cytosolic
Ca2+ concentration
([Ca2+]i)
in pulmonary arterial myocytes. A:
short (30-s) ejection of ET-1 (0.01-0.1 µM) near the cell
induced oscillations in
[Ca2+]i
in the rat main pulmonary artery (RMPA).
B: relationship between ET-1
concentration ([ET-1]; abscissa) and the percentage of
cells generating oscillations in
[Ca2+]i
(responding cells, ordinate) in RMPA and rat intrapulmonary arteries
(RIPA). C: relationship
between [ET-1] (abscissa) and the amplitude of the first
[Ca2+]i
peak in RMPA and RIPA. Open bars represent data obtained solely from
responding cells; solid bars include data obtained from nonresponding
cells. For each ET-1 concentration, 25 cells were tested in
A and
B. Nos. in parentheses are sample
size.
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Table 1.
Effect of ET-1 receptor antagonists BQ-123 and BQ-788 on ET-1- and SRTX
S6c-induced [Ca2+]i response in
myocytes isolated from RMPA and RIPA
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Effect of ET-1 receptor modulators on the ET-1-induced
[Ca2+]i
response.
We investigated the effect of BQ-123 and BQ-788, two specific
inhibitors of type A and type B ET-1 receptors
(ETA,
ETB), respectively (19, 21), and
that of SRTX S6c, a specific agonist of
ETB receptors (42). BQ-123 and
BQ-788 alone did not modify the resting value of
[Ca2+]i
in myocytes from both RMPA and RIPA (Table 1).
In RMPA, BQ-123 (0.1-1 µM) fully abolished the ET-1-induced
[Ca2+]i
response (n = 15; Fig.
2A).
This effect was rapid since superfusion of cells for only 1 min with
the compound was enough to block the response. In contrast, superfusion
of the cells for 10 min with BQ-788 (1 µM) did not alter the pattern
of ET-1-induced
[Ca2+]i
response. The amplitude of the first rise as well as the duration and
the frequency of oscillations were not altered (Fig.
2B; Table 1). SRTX S6c (100 nM) did
not evoke a
[Ca2+]i
response (n = 15).

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Fig. 2.
Effect of ET-1 receptor antagonists on ET-1 (0.1 µM)-induced
[Ca2+]i
response in myocytes from RMPA. A:
superfusion of the cell with the
ETA receptor antagonist BQ-123
rapidly abolished the
[Ca2+]i
response. B: the
ETB receptor antagonist BQ-788 had
no effect on the ET-1-induced
[Ca2+]i
response. In A and
B, the first record
(a) is the control response obtained
in the absence of receptor antagonist. Times in parentheses indicate
duration of exposure to ET-1 receptor antagonist before ET-1 ejection.
Each trace was recorded from a different cell and is typical of 15 cells.
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In contrast, in RIPA, BQ-123 abolished the ET-1-induced
[Ca2+]i
response in only 21% of tested cells, and BQ-788 abolished the response in 70% of tested cells (Fig.
3B). In
the presence of the combination of the two antagonists, the
ET-1-induced response was inhibited in all cells (Fig. 3 and Table 1).
In responding cells, the presence of only one or the two antagonists
did not modify the ET-1-induced
[Ca2+]i
response when compared with the control response (Table 1). SRTX S6c
(100 nM) elicited in 71% of tested cells a
[Ca2+]i
response that was similar in amplitude and kinetics to the ET-1-induced
[Ca2+]i
response. The SRTX S6c-induced response was fully abolished by BQ-788
(1 µM; Fig. 4 and Table 1).

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Fig. 3.
Effect of ET-1 receptor antagonists on ET-1 (0.1 µM)-induced
[Ca2+]i
response in myocytes from RIPA. A:
a, control response obtained in the absence of receptor
antagonist; b, superfusion of the cell
with both the ETA receptor
antagonist BQ-123 and the ETB
receptor antagonist BQ-788 rapidly abolished the
[Ca2+]i
response in all tested cells. In the presence of only one antagonist,
ET-1 induced a
[Ca2+]i
response similar to the control response in the percentage of cells
indicated in B. Time in parentheses
indicates duration of exposure to ET-1 receptor antagonists before ET-1
ejection. Each trace was recorded from a different cell and is typical
of 15 cells. B : percentage of cells
responding to ET-1 (0.1 µM) with a
[Ca2+]i
signal similar to that illustrated in
Aa in the absence and in the presence
of ET-1 receptor antagonists.
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Fig. 4.
Effect of the ETB receptor agonist
sarafotoxin (SRTX) S6c on
[Ca2+]i
in myocytes from RIPA. A: in most of
the tested cells (71%), SRTX S6c (0.1 µM) induced a
[Ca2+]i
response similar to that evoked by ET-1
(a). SRTX S6c-induced response was
not altered in the presence of the
ETA receptor antagonist BQ-123
(b) but was inhibited by the
presence of the ETB receptor
antagonist BQ-788 (c). Time in
parentheses indicates duration of exposure to ET-1 receptor antagonist
before SRTX S6c ejection. Each trace was recorded from a different cell
and is typical of 20 and 10 cells, respectively.
B: percentage of tested cells
exhibiting a
[Ca2+]i
response in the presence of SRTX S6c alone and in the additional
presence of BQ-788.
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Source of
Ca2+ involved in
the ET-1-induced
[Ca2+]i
response.
In myocytes from both RMPA and RIPA, application of
La3+ (100 µM), a potent
inhibitor of Ca2+ entry pathways
(39), or removal of external Ca2+
(Ca2+-free PSS) did not
significantly modify the resting
[Ca2+]i
value (Table 2). Ten to twelve minutes
after the beginning of each of these pretreatments, ET-1 (100 nM)
induced an oscillatory [Ca2+]i
response, the pattern and the amplitude of which were not significantly different from the control response (Fig.
5, A and
B, and Table 2).
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Table 2.
Effect of La3+ or removal of external Ca2+
on the ET-1-induced [Ca2+]i response
in myocytes isolated from RMPA and RIPA
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Fig. 5.
Effect of La3+
(A),
Ca2+-free external solution (PSS;
B), and thapsigargin (TG;
C) on
[Ca2+]i
response induced by 0.1 µM ET-1 in myocytes from RMPA and RIPA.
Superfusion of arterial myocytes with 100 µM
La3+ or
Ca2+-free solution did not alter
[Ca2+]i
oscillations induced by ET-1. In contrast, superfusion of myocytes with
1 µM TG time dependently inhibited ET-1-induced
[Ca2+]i
response. In A-C, the first record
(a) is the control response obtained
in Ca2+-containing solution and in
the absence of La3+ and TG. Times
in parentheses indicate duration of exposure to modified solution
before ET-1 ejection. Each trace was recorded from a different cell and
is typical of 15-25 cells.
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In another set of experiments, we tested the effect of thapsigargin, a
specific inhibitor of the sarcoplasmic reticulum (SR) Ca2+ pump (37) that depletes the
intracellular Ca2+ store in the
pulmonary artery (12). Thapsigargin (0.1-1 µM) applied for
9-12 min led to the suppression of the ET-1-induced [Ca2+]i
response (n = 18; Fig.
5C).
Effect of modulators of signal transduction pathways on the
ET-1-induced
[Ca2+]i
response.
To investigate the cellular mechanisms of the ET-1-induced
[Ca2+]i
response, we used different compounds acting on signal transduction pathways in myocytes from RMPA. Neomycin (0.1-1 µM), an
inhibitor of phosphoinositide-phospholipase C (PI-PLC; see Ref. 6),
progressively abolished the ET-1-induced
[Ca2+]i
response (Fig.
6A and
Table 3). In
Ca2+-free PSS, PDBu (1 µM), a
potent protein kinase C (PKC) activator (35), time dependently
inhibited the ET-1-induced
[Ca2+]i
response (Fig. 6B and Table 3).

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Fig. 6.
Effect of neomycin (A) and phorbol
12,13-dibutyrate (PDBu; B) on
[Ca2+]i
response induced by 0.1 µM ET-1 in myocytes from RMPA. Superfusion of
arterial myocytes with 0.1 µM neomycin or 1 µM PDBu time
dependently inhibited
[Ca2+]i
oscillations induced by ET-1. In A and
B, the first record
(a) is the control response obtained
in the absence of the tested compound. Times in parentheses indicate
duration of exposure to the tested compound before ET-1 ejection. Each
trace was recorded from a different cell and is typical of 6-17
cells.
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Table 3.
Effect of various substances on the ET-1- and CAF-induced
[Ca2+]i responses in
myocytes isolated from RMPA
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In the subsequent series of experiments, we used tetracaine and
ruthenium red to assess the role of the
Ca2+-induced
Ca2+ release (CICR) mechanism in
the ET-1-induced
[Ca2+]i
response. We have previously demonstrated the ability of tetracaine to
block the caffeine-induced
[Ca2+]i
response in rat pulmonary artery myocytes (16). Pretreatment of the
cells with tetracaine (100-300 µM) for 10-12 min did not modify the ET-1-induced
[Ca2+]i
response (Table 3). Ruthenium red is also known to inhibit the
functioning of the ryanodine-sensitive
Ca2+ release channel in muscular
tissues (46), including smooth muscle (22). Pretreatment of RMPA
myocytes with 10-300 µM ruthenium red concentration dependently
inhibited the caffeine (5 mM)-induced [Ca2+]i
transient response but had no effect on the ET-1-induced response (Fig.
7, A and
B, and Table 3).

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Fig. 7.
Effect of ruthenium red and niflumic acid on
[Ca2+]i
response induced by 0.1 µM ET-1 in myocytes from RMPA.
A: control experiment showing that
superfusion of the cell with 200 µM ruthenium red inhibited the
transient
[Ca2+]i
response induced by 5 mM caffeine (CAF).
B: in contrast, 200 µM ruthenium red
did not alter the ET-1-induced
[Ca2+]i
response. C: a, control
response obtained in the absence of niflumic acid;
b and
c: superfusion of arterial myocytes
with 25 or 50 µM niflumic acid, respectively, did not alter
[Ca2+]i
oscillations induced by ET-1. Times in parentheses indicate duration of
exposure to niflumic acid before ET-1 ejection. Each trace was recorded
from a different cell and is typical of 5-20 cells.
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Finally, we used niflumic acid, a specific blocker of
Cl
channels (27).
Pretreatment of cells with 25 or 50 µM niflumic acid for 10-15
min had no significant effect on the ET-1-induced [Ca2+]i
response (Fig. 7C and Table
3).
Effect of ET-1 on the membrane
current. In myocytes from RMPA clamped at
60 mV,
a value close to that of the resting potential in these cells (5),
application (30 s) of ET-1 (100 nM) induced an oscillatory inward
current composed of a first large transient peak of 345 ± 44.2 pA
(n = 20) followed by four to six peaks
of decreasing amplitude (Fig.
8Aa).
Generally, the two last current peaks occurred after the cessation of
ET-1 microejection. The I-V
relation, obtained by applying a ramp pulse from
60 to +40 mV at
the peak of the first current oscillation, was mainly linear, and the
Erev was
2.3 mV, a value close to that of the theoretical equilibrium
potential for Cl
(ECl), which
was
2.1 mV (Fig. 8B). The
ET-1-induced current was not significantly altered in
Ca2+-free PSS
(n = 5; Fig.
8Ab) but vanished after
pretreatment of the cells with 1 µM thapsigargin
(n = 5, data not shown). Superfusion of the cell with niflumic acid (10-50 µM) concentration
dependently abolished the ET-1-induced membrane current (Fig.
8C). Combined recordings of membrane
current and
[Ca2+]i
showed that oscillatory inward current evoked by ET-1 appeared simultaneously with oscillations in
[Ca2+]i
(n = 4; Fig.
9A). The
first and large peak of the inward current (Fig.
9Aa) corresponded to the first and
large increase in
[Ca2+]i
(Fig. 9Ab), whereas subsequent peak
currents of decreasing amplitude were associated with subsequent
[Ca2+]i
oscillations also of decreasing amplitude. Combined recordings also
confirmed that niflumic acid (50 µM) fully inhibited ET-1-induced membrane current but had no effect on
[Ca2+]i
(n = 4; Fig.
9B), as mentioned above for
independent measurements.

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Fig. 8.
Characteristics of ET-1-induced membrane current in myocytes from RMPA.
A: ejection of 0.1 µM ET-1 evoked an
oscillatory inward current (I) in
both the presence (a) and the
absence (b) of external
Ca2+. Cells were voltage clamped
at 60 mV and were dialyzed with cesium solution.
B:
I-voltage
(V) relation obtained for a ramp
pulse (from 60 to +40 mV) delivered at the peak of the first
current oscillation induced by ET-1.
Erev is
2.3 mV. Curve is the mean of 5 experiments.
C: superfusion of cells with 25 (a) and 50 (b) µM niflumic acid inhibited the
ET-1-induced oscillatory current (compare with the control current
illustrated in Aa). Times in
parentheses indicate duration of exposure to
Ca2+-free solution or niflumic
acid before ET-1 ejection.
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Fig. 9.
Simultaneous recordings of membrane current and
[Ca2+]i
in myocytes from RMPA. A: 0.1 µM
ET-1 induced oscillations in both membrane current
(a) and
[Ca2+]i
(b).
B: superfusion of cells with
50 µM niflumic acid inhibited membrane current
(a) but not
[Ca2+]i
increases (b) induced by ET-1. Cells
were voltage clamped at 60 mV and were dialyzed with cesium
solution. Time in parentheses indicates duration of exposure to
niflumic acid before ET-1 ejection. Each combined trace was recorded
from a different cell and is typical of 4 cells.
|
|
Effect of ET-1 on the mechanical activity of pulmonary
artery. In rings isolated from RMPA, ET-1 (0.1-100
nM) concentration dependently induced slowly developing contractions.
The maximal ET-1-induced response was 144.7 ± 2.6%
(n = 6) of the 80 mM KCl-induced contraction, and the mean EC50 was
5.8 nM (Fig. 10). Pretreatment of the
arterial rings with niflumic acid (50 µM) for 15 min decreased the
amplitude of ET-1-induced contraction. The maximal force was reduced by
35.7 ± 8% (n = 6). A similar
inhibitory effect (34 ± 5%, n = 6) was observed in the presence of nifedipine (1 µM), a
voltage-activated Ca2+ channel
blocker (data not shown). When niflumic acid (50 µM) and nifedipine
(1 µM) were used in combination, the inhibitory effect on the
contraction was not significantly different (35 ± 6%,
n = 6) from that of each of the
channel antagonists (Fig. 10). In control experiments, we verified that
the 80 mM KCl-induced contraction was not altered by 50 µM niflumic
acid (n = 4), whereas it was fully
abolished by 1 µM nifedipine (n = 4;
data not shown).

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Fig. 10.
Effect of ET-1 on isometric contraction of rings isolated from RMPA.
Concentration-response curves for the action of ET-1 alone ( ) or in
the additional presence of niflumic acid ( ) or niflumic acid plus
nifedipine ( ). Ordinate, contraction expressed as a percentage of
the KCl (80 mM)-induced response obtained at the beginning of the
experiments. Data points are means ± SE;
n = 6 cells.
|
|
 |
DISCUSSION |
Characteristics of ET-1-induced
Ca2+ signaling
and ET-1 receptor subtypes involved.
The present work demonstrates that the vasoconstrictor effect of ET-1
in pulmonary arteries is underlined by a complex
Ca2+ signaling at the site of the
arterial myocyte. The induction of
[Ca2+]i
oscillations seems to be a property shared by a variety of pulmonary
vascular tone modulators (15, 16). As previously observed for ANG II in
RMPA myocytes, the pattern and the amplitude of ET-1-induced
[Ca2+]i
response are relatively independent of the ET-1 concentration, whereas
the percentage of responding cells increases with increasing ET-1
concentration. A similar lack of dependence between the amplitude of
agonist-induced Ca2+ release and
agonist concentration (the so called all-or-none response) has been
shown in other cell types. Nevertheless, combining the percentage of
responding cells with the all-or-none character of the ET-1 response
(Fig. 1C) reveals a
concentration-dependent relationship in the entire population of tested
cells and may account for the graduated physiological response in the
whole tissue. Unlike for ANG II- and ATP-induced
[Ca2+]i
oscillations in the same preparation (15, 16),
[Ca2+]i
did not return to its baseline value between each
[Ca2+]i
rise induced by ET-1. In this respect,
[Ca2+]i
oscillations observed in the present work exhibit an intermediary pattern between those generally observed in nonexcitable cells and
referred to as baseline spikes and sinusoidal oscillations (38).
Although the ET-1-induced
[Ca2+]i
response displays a similar pattern in RMPA and in RIPA, the type of
membrane receptor involved is different according to the site along the
pulmonary arterial tree. In RMPA, the
[Ca2+]i
response is entirely mediated by the A subtype receptor of ET-1
(ETA), since it is fully blocked
by the compound BQ-123 and, conversely, is not altered by the compound
BQ-788. This result is in accordance with contractile experiments
showing that the ET-1 mechanical response in RMPA is mediated by only
ETA receptors (18). This ET-1
receptor subtype distribution is similar to that observed in human
pulmonary arteries (9). In contrast, in RIPA, both
ETA and
ETB receptors are involved in the
ET-1-induced [Ca2+]i
response, with a major distribution of the B subtype. This is assessed
by the effect of SRTX S6c, which produces oscillations in 71% of the
cells, and that of BQ-123, the ETA
inhibitor and BQ-788, the ETB
inhibitor, which inhibit oscillations in 21 and 70% of the cells,
respectively. These findings suggest that ~70% of
myocytes mainly exhibit ETB
receptor and the others the ETA receptor. It is known that contraction in both the rabbit and the rat
small intrapulmonary arteries is mainly dependent on the activation of
ETB receptors (9, 18). Moreover,
in the rat small pulmonary arteries,
ETB-mediated electrophysiological
responses have also been described (34). The physiological meaning of this heterogeneity is unclear. However, a similar heterogeneity of
pulmonary myocytes for the expression of other membrane proteins along
the arterial tree has been previously reported in the case of potassium
and Ca2+ voltage-operated channels
(1, 8).
Mechanisms of ET-1-induced
[Ca2+]i
response.
Our results clearly show that the ET-1-induced
[Ca2+]i
response in myocytes from RMPA and RIPA involves the mobilization of an
intracellular Ca2+ source,
presumably the SR, since the response was not altered by
La3+ or in
Ca2+-free solution but vanished
after pretreatment of the cells with thapsigargin. The ET-1-induced
internal Ca2+ mobilization
operates via an inositol trisphosphate-sensitive signaling pathway,
since the ET-1-induced
[Ca2+]i
response was inhibited by neomycin, a selective inhibitor of PI-PLC
(6), and by the addition, in
Ca2+-free PSS, of PDBu, a potent
PKC activator in this preparation (35). In vascular smooth muscle, it
is known that phorbol ester-activated PKC negatively regulates the
activity of PI-PLC and inhibits agonist-induced inositol trisphosphate
production and the contractile response (30). Moreover, ET-1 increases
both
[Ca2+]i
and the production of inositol trisphosphate in a variety of vascular
preparations (23, 29). The oscillatory pattern of the ET-1-induced
[Ca2+]i
response could thus be in relation with a cyclic
Ca2+ release through the inositol
trisphosphate receptor Ca2+
release channel and explained by the well-known biphasic
Ca2+ regulation of the smooth
muscle inositol trisphosphate receptor activity (20). Evidence for such
a model of inositol trisphosphate-mediated Ca2+ oscillations has also been
provided recently in permeabilized epithelial cells (36). Although RMPA
myocytes contain another Ca2+
release channel in the SR membrane, i.e., the ryanodine receptor (16),
it is unlikely that such a channel is involved in the ET-1-induced
Ca2+ release, since tetracaine and
ruthenium red, two potent inhibitors of the CICR mechanism in smooth
muscle (16, 22), did not alter ET-1-induced
[Ca2+]i
oscillations. In this respect,
[Ca2+]i
oscillations in pulmonary vascular smooth muscle are different from
those observed in airway smooth muscle where it has been suggested that
the ryanodine receptor could contribute to acetylcholine-induced [Ca2+]i
oscillations (22).
Physiological implication of ET-1-induced
[Ca2+]i
oscillations.
Using electrophysiological and mechanical measurements, we tentatively
investigated the role of ET-1-induced
[Ca2+]i
oscillations in RMPA. In patch-clamped myocytes, ET-1 evoked an
oscillatory membrane current that was obviously carried by Cl
for the following
reasons. First, in our experimental conditions, i.e.,
ECl =
2.1
mV and holding potential =
60 mV, the current was inward as
expected from the large outward electrochemical gradient for
Cl
. Second, its
Erev was similar
to ECl. Third, it
was not altered in Ca2+-free
solution but was fully abolished by 50 µM niflumic acid, a selective
inhibitor of voltage- and agonist-activated
Cl
current in vascular
smooth muscle (27, 44). Simultaneous recordings of membrane current and
[Ca2+]i
showed that oscillations of the inward current coincided with oscillations in
[Ca2+]i.
Moreover, 1) both phenomena remained
unchanged in Ca2+-free solution
but disappeared after treatment of the cells with thapsigargin, and
2) niflumic acid, as mentioned
above, abolished only the oscillatory current but not
[Ca2+]i
oscillations. These results strongly suggest that oscillations in
membrane current are triggered by oscillations in
[Ca2+]i
and thus that ET-1-induced current in these conditions is a Ca2+-activated
Cl
current
[ICl(Ca)].
In vascular myocytes, it is generally proposed that the main
physiological role of agonist-induced
ICl(Ca) is to
depolarize the membrane to trigger the contraction by activation of a
voltage-dependent Ca2+ influx (27,
44). In the present work, niflumic acid reduced by 35% the amplitude
of ET-1-induced contraction. Interestingly, the same proportion of the
contractile response was inhibited by either nifedipine alone or by the
combination of both substances. These results confirm that
ET-1-activated
ICl(Ca)
contributes to depolarize the cell to the threshold activation of
voltage-dependent Ca2+ channels.
Nevertheless, although the resulting
Ca2+ influx plays a role in
ET-1-induced contraction, the main part of the contractile response
seems to be due to a Ca2+ release
from the internal store. The fact that the contribution of this Ca
influx is somewhat limited in the ET-1-induced response could explain,
at least in part, why it is difficult to demonstrate the presence of
such an influx in our experiments in isolated cells. Moreover,
1) in patch-clamp experiments,
myocytes were set at a membrane potential of
60 mV, a value much
more polarized than the threshold value for activating L-type
Ca2+ channels;
2) when myocytes were not clamped,
they may be slightly depolarized, the value of the membrane potential
then corresponding to inactivation of activating L-type
Ca2+ channels; and
3) finally, it is difficult to
separate a sustained small increase in
[Ca2+]i
related to Ca2+ influx when
oscillations are superimposed unless oscillations are blocked; however,
then ICl(Ca)
leading to Ca2+ influx is also
blocked. In the same preparation, we did perform such a separation in a
previous study in response to ATP (16); the
[Ca2+]i
response to ATP combines Ca2+
influx and Ca2+ oscillations, but
these are mediated by different membrane receptors, P2X and
P2U, respectively (16). In the
case of ET-1, the two mechanisms are linked and activated by the same
receptor. Finally, it must be kept in mind that additional cellular
mechanisms could participate in the ET-1-induced vasoconstrictor
response, such as an increase in the sensitivity to
Ca2+ of the contractile apparatus
as observed in rabbit mesenteric artery (43).
In conclusion, this study has demonstrated that ET-1, via the
activation of the receptor subtype A in RMPA and both receptor subtypes
A and B receptor subtypes in RIPA induce
[Ca2+]i
oscillations due to an inositol trisphosphate-mediated release from the
SR. Ca2+ release directly accounts
for the main part of the contractile response on the one hand and on
another hand activates an oscillatory ICl(Ca) that
depolarizes the cell to the threshold activation of voltage-dependent
Ca2+ channels. The resulting
Ca2+ influx is responsible for an
additional component of the ET-1-induced contraction.
 |
ACKNOWLEDGEMENTS |
This work was supported by grants from Contrat de Recherche
Institut National de la Santé et de la Recherche Médicale
9806, the Ministère de l'Environment [Agence De
l'Environement et de la Maîtrise de l'Energie (ADEME),
PRIMEQUAL 9593017], and Conseil Régional d' Aquitaine
(960301117). J.-M. Hyvelin is an ADEME studentship recipient.
 |
FOOTNOTES |
Address for reprint requests: J.-P. Savineau, Laboratoire de
Physiologie Cellulaire Respiratoire, Faculté de Médecine V. Pachon, Université Bordeaux 2, 146, rue Léo Saignat, 33076 Bordeaux Cédex, France.
Received 16 September 1997; accepted in final form 24 April 1998.
 |
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