| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
|
|
|
|||||||
|
|||||||||
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
 |
ABSTRACT |
|---|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
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
| |
INTRODUCTION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
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.
| |
MATERIALS AND METHODS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
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 (104 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.
| |
RESULTS |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
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).
|
|
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).
|
|
|
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).
|
|
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).
|
|
|
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.
|
|
|
| |
DISCUSSION |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
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)].
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.
| |
REFERENCES |
|---|
|
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
|
|---|
1.
Archer, S. L.,
J. M. C. Huang,
H. L. Reeve,
V. Hampl,
S. Tolarova,
Z. Michelakis,
and
E. K. Wier.
Differential distribution of electrophysiologically distinct myocytes in conduit and resistance arteries determines their response to nitric oxide and hypoxia.
Circ. Res.
78:
431-442,
1996
2.
Bakhramov, A.,
S. A. Hartley,
K. J. Salter,
and
R. Z. Kozlowski.
Contractile agonists preferentially activate Cl over K+ currents in arterial myocytes.
Biochem. Biophys. Res. Commun.
227:
168-175,
1996[Medline].
3.
Barnes, P. J.,
and
S. F. Liu.
Regulation of pulmonary vascular tone.
Pharmacol. Rev.
47:
87-131,
1995[Medline].
4.
Chen, C.,
and
P. K. Wagoner.
Endothelin induces a nonselective cation current in vascular smooth muscle cells.
Circ. Res.
69:
447-454,
1991
5.
Clapp, L. H.,
and
A. M. Gurney.
Outward currents in rabbit pulmonary artery cells dissociated with a new technique.
Exp. Physiol.
76:
677-693,
1991[Abstract].
6.
Cockcroft, S.,
and
B. D. Gomperts.
Role of guanine nucleotide binding protein in the activation of polyphosphoinositide phosphodiesterase.
Nature
314:
534-536,
1985[Medline].
7.
Dzau, V. J.
Cell biology and genetics of angiotensin in cardiovascular disease.
J. Hypertens. Suppl.
12:
S3-S10,
1994[Medline].
8.
Franco-Obregon, A.,
and
J. Lopez-Barneo.
Differential oxygen sensitivity of calcium channels in rabbit smooth muscle cells of conduit and resistance pulmonary arteries.
J. Physiol. (Lond.)
491:
511-518,
1996
9.
Fukuroda, T.,
M. Kobayashi,
S. Ozaki,
M. Yano,
T. Miyauchi,
M. Onizuka,
Y. Sugishita,
K. Goto,
and
M. Nishikibe.
Endothelin receptor subtypes in human versus rabbit pulmonary arteries.
J. Appl. Physiol.
76:
1976-1982,
1994
10.
Gardner, J. P.,
G. Tokudome,
H. Tomonari,
E. Maher,
D. Hollander,
and
A. Aviv.
Endothelin-induced calcium responses in human vascular smooth muscle cells.
Am. J. Physiol.
262 (Cell Physiol. 31):
C148-C155,
1992
11.
Giaid, A.,
M. Yanagisawa,
D. Langleben,
R. P. Michel,
R. Levy,
H. Shennib,
S. Kimura,
T. Masaki,
W. P. Duguid,
and
D. J. Stewart.
Expression of endothelin-1 in the lungs of patients with pulmonary hypertension.
N. Engl. J. Med.
328:
1732-1739,
1993
12.
Gonzalez De La Fuente, P.,
J. P. Savineau,
and
R. Marthan.
Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum Ca2+ pump blockers: thapsigargin and cyclopiazonic acid.
Pflügers Arch.
429:
617-624,
1995[Medline].
13.
Goto, K.,
Y. Kasuya,
N. Matsuki,
Y. Takuwa,
H. Kurihara,
T. Ishikawa,
S. Kimura,
M. Yanagisawa,
and
T. Masaki.
Endothelin activates the dihydropyridine-sensitive, voltage-dependent Ca2+ channel in vascular smooth muscle.
Proc. Natl. Acad. Sci. USA
86:
3915-3918,
1989
14.
Grynkiewicz, G.,
M. Poenie,
and
R. Y. Tsien.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J. Biol. Chem.
260:
3440-3450,
1985
15.
Guibert, C.,
R. Marthan,
and
J. P. Savineau.
Angiotensin II-induced Ca2+-oscillations in vascular myocytes from the rat pulmonary artery.
Am. J. Physiol.
270 (Lung Cell. Mol. Physiol. 14):
L637-L642,
1996
16.
Guibert, C.,
P. Pacaud,
G. Loirand,
R. Marthan,
and
J. P. Savineau.
Effect of extracellular ATP on cytosolic Ca2+ concentration in rat pulmonary artery myocytes.
Am. J. Physiol.
271 (Lung Cell. Mol. Physiol. 15):
L450-L458,
1996
17.
Hamill, O. P.,
A. Marty,
E. Neher,
B. Sakmann,
and
F. J. Sigworth.
Improved patch-clamp techniques for high-resolution current recording from cells and cell-free membrane patches.
Pflügers Arch.
391:
85-100,
1981[Medline].
18.
Higashi, T.,
T. Ishizaki,
K. Shigemori,
T. Nakai,
S. Miyabo,
T. Inui,
and
T. Yamamura.
Pharmacological heterogeneity of constrictions mediated by endothelin receptors in rat pulmonary arteries.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L287-L293,
1997
19.
Ihara, M.,
K. Noguchi,
T. Saeki,
T. Fukuroda,
S. Tsuchida,
S. Kimura,
T. Fukami,
K. Ishikawa,
M. Nishikibe,
and
M. Yano.
Biological profiles of highly potent novel endothelin antagonists selective for the ETA receptor.
Life Sci.
50:
247-255,
1992[Medline].
20.
Iino, M.
Biphasic Ca2+ dependence of inositol 1,4,5-trisphosphate-induced Ca release in smooth muscle cells of the guinea pig taenia caeci.
J. Gen. Physiol.
95:
1103-1122,
1990
21.
Ishikawa, K.,
M. Ihara,
K. Noguchi,
T. Mase,
N. Mino,
T. Saeki,
T. Fukuroda,
T. Fukami,
S. Ozaki,
T. Nagase,
M. Nishikibe,
and
M. Yano.
Biochemical and pharmacological profile of a potent and selective endothelin B-receptor antagonist, BQ-788.
Proc. Natl. Acad. Sci. USA
91:
4892-4896,
1994
22.
Kannan, M. S.,
Y. S. Prakash,
T. Brenner,
J. R. Mickelson,
and
G. C. Sieck.
Role of ryanodine receptor channels in Ca2+ oscillations of porcine tracheal smooth muscle.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L659-L664,
1997
23.
Kasuya, Y.,
Y. Takuwa,
M. Yanagisawa,
S. Kimura,
K. Goto,
and
T. Masaki.
Endothelin-1 induces vasoconstriction through two functionally distinct pathways in porcine coronary artery: contribution of phosphoinositide turnover.
Biochem. Biophys. Res. Commun.
161:
1049-1055,
1989[Medline].
24.
Kasuya, Y.,
Y. Takuwa,
M. Yanagisawa,
T. Masaki,
and
K. Goto.
A pertussis toxin-sensitive mechanism of endothelin action in porcine coronary artery smooth muscle.
Br. J. Pharmacol.
107:
456-462,
1992[Medline].
25.
Kiely, D. G.,
R. I. Cargill,
and
B. J. Lipworth.
Angiotensin II receptor blockade and effects on pulmonary hemodynamics and hypoxic pulmonary vasoconstriction in humans.
Chest
110:
698-703,
1996
26.
Klockner, U.,
and
G. Isenberg.
Endothelin depolarizes myocytes from porcine coronary and human mesenteric arteries through a Ca-activated chloride current.
Pflügers Arch.
418:
168-175,
1991[Medline].
27.
Large, W. A.,
and
Q. Wang.
Characteristics and physiological role of the Ca2+-activated Cl conductance in smooth muscle.
Am. J. Physiol.
271 (Cell Physiol. 40):
C435-C454,
1996
28.
Marrero, M. B.,
W. G. Paxton,
B. Schieffer,
B. N. Ling,
and
K. E. Bernstein.
Angiotensin II signalling events mediated by tyrosine phosphorylation.
Cell Signal.
8:
21-26,
1996[Medline].
29.
Marsden, P. A.,
N. R. Danthuluri,
B. M. Brenner,
B. J. Ballermann,
and
T. A. Brock.
Endothelin action on vascular smooth muscle involves inositol trisphosphate and calcium mobilization.
Biochem. Biophys. Res. Commun.
158:
86-93,
1989[Medline].
30.
McMillan, M.,
B. Chernow,
and
B. L. Roth.
Phorbol esters inhibit alpha 1-adrenergic receptor-stimulated phosphoinositide hydrolysis and contraction in rat aorta: evidence for a link between vascular contraction and phosphoinositide turnover.
Biochem. Biophys. Res. Commun.
134:
970-974,
1986[Medline].
31.
Miyoshi, Y.,
Y. Nakaya,
T. Wakatsuki,
S. Nakaya,
K. Fujino,
K. Saito,
and
I. Inoue.
Endothelin blocks ATP-sensitive K+ channels and depolarizes smooth muscle cells of porcine coronary artery.
Circ. Res.
70:
612-616,
1992
32.
Nakajima, T.,
H. Hazama,
E. Hamada,
S. N. Wu,
K. Igarashi,
T. Yamashita,
Y. Seyama,
M. Omata,
and
Y. Kurachi.
Endothelin-1 and vasopressin activate Ca(2+)-permeable non-selective cation channels in aortic smooth muscle cells: mechanism of receptor-mediated Ca2+ influx.
J. Mol. Cell. Cardiol.
28:
707-722,
1996[Medline].
33.
Pollock, D. M.,
T. L. Keith,
and
R. F. Highsmith.
Endothelin receptors and calcium signaling.
FASEB J.
9:
1196-1204,
1995[Abstract].
34.
Salter, K. J.,
and
R. Z. Kozlowski.
Endothelin receptor coupling to potassium and chloride channels in isolated rat pulmonary arterial myocytes.
J. Pharmacol. Exp. Ther.
279:
1053-1062,
1996
35.
Savineau, J. P.,
R. Marthan,
and
H. Crevel.
Contraction of vascular smooth muscle induced by phorbol 12,13 dibutyrate in human and rat pulmonary arteries.
Br. J. Pharmacol.
104:
639-644,
1991[Medline].
36.
Tanimura, A.,
and
R. J. Turner.
Inositol 1,4,5-trisphosphate-dependent oscillations of luminal [Ca2+] in permeabilized HSY cells.
J. Biol. Chem.
271:
30904-30908,
1996
37.
Thastrup, O.,
P. J. Cullen,
B. K. Drobak,
M. R. Hanley,
and
A. P. Dawson.
Thapsigargin, a tumor promoter, discharges intracellular Ca2+ stores by specific inhibition of the endoplasmic reticulum Ca2(+)-ATPase.
Proc. Natl. Acad. Sci. USA
87:
2466-2470,
1990
38.
Thomas, A. P.,
G. S. Bird,
G. Hajnoczky,
L. D. Robb-Gaspers,
and
J. W. Putney, Jr.
Spatial and temporal aspects of cellular calcium signaling.
FASEB J.
10:
1505-1517,
1996[Abstract].
39.
Van Breemen, C.,
B. R. Farinas,
R. Casteels,
P. Gerba,
F. Wuytack,
and
R. Deth.
Factors controlling cytoplasmic Ca2+ concentration.
Philos. Trans. R. Soc. Lond. B Biol. Sci.
265:
57-71,
1973[Medline].
40.
Van Renterghem, C.,
and
M. Lazdunski.
Endothelin and vasopressin activate low conductance chloride channels in aortic smooth muscle cells.
Pflügers Arch.
425:
156-163,
1993[Medline].
41.
Van Renterghem, C.,
P. Vigne,
J. Barhanin,
A. Schmid-Alliana,
C. Frelin,
and
M. Lazdunski.
Molecular mechanism of action of the vasoconstrictor peptide endothelin.
Biochem. Biophys. Res. Commun.
157:
977-985,
1988[Medline].
42.
Williams, D. L.,
K. L. Jones,
D. J. Pettibone,
E. V. Lis,
and
B. V. Clineschmidt.
Sarafotoxin S6c: an agonist which distinguishes between endothelin receptor subtypes.
Biochem. Biophys. Res. Commun.
175:
556-561,
1991[Medline].
43.
Yoshida, M.,
A. Suzuki,
and
T. Itoh.
Mechanisms of vasoconstriction induced by endothelin-1 in smooth muscle of rabbit mesenteric artery.
J. Physiol. (Lond.)
477:
253-265,
1994
44.
Yuan, X. J.
Role of calcium-activated chloride current in regulating pulmonary vasomotor tone.
Am. J. Physiol.
272 (Lung Cell. Mol. Physiol. 16):
L959-L968,
1997
45.
Zamora, M. A.,
E. C. Dempsey,
S. J. Walchak,
and
T. J. Stelzner.
BQ123, an ETA receptor antagonist, inhibits endothelin-1-mediated proliferation of human pulmonary artery smooth muscle cells.
Am. J. Respir. Cell Mol. Biol.
9:
429-433,
1993.
46.
Zucchi, R.,
and
S. Ronca-Testoni.
The sarcoplasmic reticulum Ca2+ channel/ryanodine receptor: modulation by endogenous effectors, drugs and disease states.
Pharmacol. Rev.
49:
1-51,
1997
This article has been cited by other articles:
|
|
 |
|
  J. F. Perez-Zoghbi and M. J. Sanderson Endothelin-induced contraction of bronchiole and pulmonary arteriole smooth muscle cells is regulated by intracellular Ca2+ oscillations and Ca2+ sensitization Am J Physiol Lung Cell Mol Physiol, October 1, 2007; 293(4): L1000 - L1011. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Ko, W. S. Park, J.-H. Ko, J. Han, N. Kim, and Y. E. Earm Endothelin-1 increases intracellular Ca2+ in rabbit pulmonary artery smooth muscle cells through phospholipase C Am J Physiol Heart Circ Physiol, October 1, 2005; 289(4): H1551 - H1559. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Kunichika, J. W. Landsberg, Y. Yu, H. Kunichika, P. A. Thistlethwaite, L. J. Rubin, and J. X.-J. Yuan Bosentan Inhibits Transient Receptor Potential Channel Expression in Pulmonary Vascular Myocytes Am. J. Respir. Crit. Care Med., November 15, 2004; 170(10): 1101 - 1107. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J.-M. Hyvelin, C. O'Connor, and P. McLoughlin Effect of changes in pH on wall tension in isolated rat pulmonary artery: role of the RhoA/Rho-kinase pathway Am J Physiol Lung Cell Mol Physiol, October 1, 2004; 287(4): L673 - L684. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Guibert, R. Marthan, and J.-P. Savineau 5-HT induces an arachidonic acid-sensitive calcium influx in rat small intrapulmonary artery Am J Physiol Lung Cell Mol Physiol, June 1, 2004; 286(6): L1228 - L1236. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 S. Kajioka, S. Nakayama, R. McCoy, G. McMurray, K. Abe, and A. F Brading Inward current oscillation underlying tonic contraction caused via ETA receptors in pig detrusor smooth muscle Am J Physiol Renal Physiol, January 1, 2004; 286(1): F77 - F85. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bonnet, J.-M. Hyvelin, P. Bonnet, R. Marthan, and J.-P. Savineau Chronic hypoxia-induced spontaneous and rhythmic contractions in the rat main pulmonary artery Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L183 - L192. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Bonnet, A. Belus, J.-M. Hyvelin, E. Roux, R. Marthan, and J.-P. Savineau Effect of chronic hypoxia on agonist-induced tone and calcium signaling in rat pulmonary artery Am J Physiol Lung Cell Mol Physiol, July 1, 2001; 281(1): L193 - L201. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. Janiak, S. M. Wilson, S. Montague, and J. R. Hume Heterogeneity of calcium stores and elementary release events in canine pulmonary arterial smooth muscle cells Am J Physiol Cell Physiol, January 1, 2001; 280(1): C22 - C33. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
S. Doi, D. S. Damron, K. Ogawa, S. Tanaka, M. Horibe, and P. A. Murray K+ channel inhibition, calcium signaling, and vasomotor tone in canine pulmonary artery smooth muscle Am J Physiol Lung Cell Mol Physiol, August 1, 2000; 279(2): L242 - L251. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 J.-P. Savineau and R. Marthan Cytosolic Calcium Oscillations in Smooth Muscle Cells Physiology, February 1, 2000; 15(1): 50 - 55. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
L. A. Shimoda, J. T. Sylvester, and J. S. K. Sham Mobilization of intracellular Ca2+ by endothelin-1 in rat intrapulmonary arterial smooth muscle cells Am J Physiol Lung Cell Mol Physiol, January 1, 2000; 278(1): L157 - L164. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
K.-X. Li, B. Fouty, I. F. McMurtry, and D. M. Rodman Enhanced ETA-receptor-mediated inhibition of Kv channels in hypoxic hypertensive rat pulmonary artery myocytes Am J Physiol Heart Circ Physiol, July 1, 1999; 277(1): H363 - H370. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Visit Other APS Journals Online |
) 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.