Laboratoire de Physiologie Cellulaire Respiratoire, Institut National de la Santé et de la Recherche Médicale (Equipe Mixte 9937), and Institut Fédératif de Recherche 4, Université Bordeaux 2, 33076 Bordeaux, France
![]() |
ABSTRACT |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The effect of chronic hypoxia (CH) for 14 days on Ca2+ signaling and contraction induced by agonists in the rat main pulmonary artery (MPA) was investigated. In MPA myocytes obtained from control (normoxic) rats, endothelin (ET)-1, angiotensin II (ANG II), and ATP induced oscillations in intracellular Ca2+ concentration ([Ca2+]i) in 85-90% of cells, whereas they disappeared in myocytes from chronically hypoxic rats together with a decrease in the percentage of responding cells. However, both the amount of mobilized Ca2+ and the sources of Ca2+ implicated in the agonist-induced response were not changed. Analysis of the transient caffeine-induced [Ca2+]i response revealed that recovery of the resting [Ca2+]i value was delayed in myocytes from chronically hypoxic rats. The maximal contraction induced by ET-1 or ANG II in MPA rings from chronically hypoxic rats was decreased by 30% compared with control values. Moreover, the D-600- and thapsigargin-resistant component of contraction was decreased by 40% in chronically hypoxic rats. These data indicate that CH alters pulmonary arterial reactivity as a consequence of an effect on both Ca2+ signaling and Ca2+ sensitivity of the contractile apparatus. A Ca2+ reuptake mechanism appears as a CH-sensitive phenomenon that may account for the main effect of CH on Ca2+ signaling.
angiotensin II; endothelin-1; calcium oscillations; vascular smooth muscle; pulmonary hypertension
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
IN MANY MAMMALIAN SPECIES including human, a prolonged decrease in alveolar oxygen tension induces a selective pulmonary vasoconstriction that is generally accompanied by an important vascular remodeling and a sustained pulmonary arterial hypertension (PAHT) (34, 35, 41). Chronic hypoxia (CH)-induced PAHT results from a combination of polycythemia and structural and functional changes in the pulmonary vascular bed (1, 17, 35). Structural changes involve cellular hypertrophy and hyperplasia and deposition of additional matrix that decreases the lumen of the vessels and reduces the elasticity of the arterial wall (17, 24, 46). These structural changes also contribute to the functional modification of pulmonary arterial reactivity. Despite their pathophysiological importance, the molecular and cellular mechanisms underlying these phenomena are not fully elucidated.
Under normoxic conditions, pulmonary arterial tone is controlled by
both membrane potential (8, 49) and a variety of circulating and locally released mediators such as endothelin (ET)-1,
angiotensin II (ANG II), serotonin, and ATP (4, 7, 25).
Several studies have investigated the effect of CH on membrane potential or ionic currents and reactivity to vasoconstrictors. In both
animals and humans, CH depolarizes pulmonary vascular smooth muscle
cells as a consequence of a dysfunction of voltage-gated K+
channels, and this depolarization secondarily increases the resting intracellular Ca2+ concentration
([Ca2+]i) (31, 32, 43, 45, 48).
It has also been demonstrated that CH in rats increases ET-1 gene
expression and plasma ET-1 levels as in PAHT in humans (1, 9, 10,
23). CH also increases ANG II-converting enzyme activity in rats
(29, 30, 50), suggesting that both mediators are
implicated in PAHT. Although ET-1 and ANG II inhibit voltage-gated
K+ channels under normoxic conditions, their effect is
differentially altered by CH (33, 37, 43). Regarding the
effect of CH on the reactivity to agonists, considerable variability is
evident in the literature depending on the animal species, the duration and degree of exposure to CH, and the considered portion of the pulmonary vascular bed (6, 22, 26-28). Moreover,
although an increase in [Ca2+]i is recognized
as the key step in the activation-contraction process and thus in the
vascular reactivity, to the best of our knowledge, little information
is available about the effect of CH on agonist-induced Ca2+
signaling in pulmonary vascular smooth muscle. Very recently, Shimoda
et al. (42) have shown an inhibitory effect of CH on the
ET-1-induced transient [Ca2+]i increase in
intrapulmonary arteries. Under normoxic conditions, our laboratory has
previously shown (13, 15, 20, 40) that ET-1 as
well as ANG II and ATP induces a complex
[Ca2+]i response in myocytes isolated from
the rat main pulmonary artery (MPA). This response is composed of a
series of [Ca2+]i peaks, the so-called
Ca2+ oscillations, which are mainly due to a cyclic release
of stored Ca2+ from the sarcoplasmic reticulum (SR) via an
inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-sensitive pathway
(13, 15, 20, 40). On one hand, these
Ca2+ oscillations directly account for the main part of the
contractile response, and on the other hand, they trigger an
oscillatory Ca2+-activated Cl current
(14, 20). This current depolarizes pulmonary arterial myocytes to the threshold activation of the voltage-dependent Ca2+ channel (3, 14, 20), thus inducing a
Ca2+ influx responsible for an additional component of the
agonist-induced contraction.
A better knowledge of the cellular effect of agonists under CH would be of interest to further understand the pathophysiology of the pulmonary circulation. The current study was thus designed to investigate, in the rat MPA, the effect of CH on both the agonist-induced contraction and the [Ca2+]i response. Tissues were obtained from rats maintained either in a hypobaric chamber (50.5 kPa) for 14 days (chronically hypoxic rats) or under normoxic conditions (control rats). Indo 1 microspectrofluorometry was used in freshly isolated myocytes to measure [Ca2+]i, and isometric contraction was measured in arterial rings. We have observed that the CH-induced decrease in the reactivity to ET-1 and ANG II was due to both an effect on Ca2+ signaling and a decrease in the Ca2+ sensitivity of the contractile apparatus. The main effect of CH on Ca2+ signaling, i.e., the loss of agonist-induced Ca2+ oscillations, appears to depend on an action at the site of Ca2+ reuptake mechanisms.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
CH. Adult male Wistar rats (aged 8-10 wk, weighing 220-240 g) were separated into two groups. One group (control or normoxic rats) was housed in room air at a normal atmospheric pressure (101 kPa). The other group (hypoxic rats) was maintained in a hypobaric chamber for 14 days. The pressure in the chamber was reduced to 0.5 atmosphere (50.5 kPa) with an electrically driven pump. The chamber was opened for 15-30 min twice a week. Pulmonary hypertension was assessed by measuring the ratio of right ventricle (RV) to left ventricle plus septum (LV+S) weight (5, 27, 43).
Tissue preparation. At completion of the exposure, the rats were anesthetized with an intraperitoneal injection of 40 mg of ethyl carbamate. The heart and lungs were removed en bloc. The MPA was then dissected under binocular control, and the adventitial and intimal layers were removed. For contraction experiments, rings (3 mm in length) were prepared. For cell dissociation, the MPA was cut into several pieces (1 × 1 mm), incubated for 10 min in low-Ca2+ (200 µM) physiological saline solution (PSS; composition given in Solutions and application of agonists), and then incubated in low-Ca2+ PSS containing 0.5 mg/ml of collagenase, 0.4 mg/ml of Pronase, 0.06 mg/ml of elastase, and 3 mg/ml of bovine serum albumin at 37°C for two successive periods of 20 min each, with 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 the cells. The cells were stored on glass coverslips at 4°C in PSS containing 0.8 mM Ca2+ and used on the same day.
Isometric contraction measurement. Isometric contraction was measured in rings from the MPA that were mounted between two stainless steel clips in vertical 20-ml organ baths of a computerized isolated organ bath system (IOX, EMKA Technologies, Paris, France). The baths were filled with Krebs-Henseleit 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). As determined in preliminary experiments, tissues were set at optimal length by equilibration against a passive load of 10 and 20 mN for rings obtained from normoxic and hypoxic rats, respectively. At the beginning of each experiment, a K+-rich (80 mM) solution obtained by substituting an equimolar amount of KCl for NaCl in the Krebs-Henseleit 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 to ANG II (0.1 nM to 1 µM) or 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. For ET-1, the maximal force was obtained after a 15- to 18-min application of ET-1, and the incremental concentration of ET-1 was added after such a duration in our experiments, whereas for ANG II, the incremental concentration was added at the top of the response to the preceding concentration obtained for a 4- to 5-min application because the ANG II-induced response is transient. Successful removal of the endothelium was confirmed by the inability of acetylcholine (1 µM) to induce >10% of relaxation in phenylephrine (1 µM)-contracted rings.
[Ca2+]i measurements. To assess the dynamic changes in [Ca2+]i of individual arterial myocytes, we used the [Ca2+]i-sensitive fluorophore indo 1. The cells were loaded with indo 1 by incubation in PSS containing 1 µM indo 1-AM for 25 min at room temperature and then washed in PSS for 25 min. The coverslip with the attached cells was then mounted in a perfusion chamber. 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. The studied cell was illuminated at 360 nm and counted simultaneously at 405 and 480 nm by two photomultipliers (P100, Nikon). The 405- to 480-nm fluorescence ratio was calculated on-line and displayed with the two voltage signals on a monitor. [Ca2+]i was estimated from the fluorescence ratio (12), with a calibration for indo 1 determined within the cells (14).
Solutions and application of agonists. 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. ET-1, ANG II, ATP, and caffeine were 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 [Ca2+]i response to the different agonists 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).
Chemicals and drugs. Collagenase (type CLS1) was from Worthington Biochemical (Freehold, NJ). Pronase (type E), elastase (type 3), bovine serum albumin, ANG II, ATP, cyclopiazonic acid (CPA), ET-1, methoxyverapamil (D-600), and thapsigargin were from Sigma (Saint Quentin Fallavier, France). Caffeine was from Merck (Darmstadt, Germany). Indo 1 was from Calbiochem (France Biochem, Meudon, France). CPA, D-600, indo 1, and thapsigargin were dissolved in DMSO. The maximal concentration of DMSO used in our experiments was <0.1% and had no effect on the mechanical activity of rings or the resting value of or the variation in [Ca2+]i induced by agonists in the cells.
Analysis of data.
[Ca2+]i responses to ANG II, ET-1, and ATP
were analyzed by comparing the resting
[Ca2+]i value, the relative amplitude of the
first peak of the response, the percentage of responding cells, the
percentage of cells generating [Ca2+]i
oscillations (oscillatory cells), and the number of Ca2+
oscillations in cells obtained from both control and CH rats. The
amount of cytosolic mobilized Ca2+ was estimated by
determining the area under the [Ca2+]i curve.
The falling part of the transient caffeine-induced
[Ca2+]i response was kinetically analyzed. It
was composed of two successive linear and exponential phases that were
described by the following equations as a function of time
(t): [Ca2+]i = A(t) + b and
[Ca2+]i = [Ca2+]i0 + Bet/
for phases 1 and
2, respectively, where A is the slope coefficient of phase 1, b is the peak
[Ca2+]i value in phase 1,
is
the time constant of phase 2,
[Ca2+]i0 is the
[Ca2+]i value at the end of phase
2, and B is the [Ca2+]i value
at the onset of phase 2. A and
were
compared between cells obtained in control and CH rats, respectively.
In contraction experiments, the concentration of ANG II or ET-1
inducing 50% of the maximal response (EC50) was
graphically determined from the mean cumulative concentration-response curve.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Effect of CH on RV weight. In rats exposed to hypobaric hypoxia for 14 days, RV-to-LV+S ratio values significantly increased compared with those in control rats (0.46 ± 0.2, n = 10, and 0.30 ± 0.01, n = 18, respectively; P < 0.05). This RV hypertrophy was the consequence of the development of PAHT.
Effect of CH on resting
[Ca2+]i value and ANG II-,
ET-1-, and ATP-induced
[Ca2+]i responses.
CH significantly increased the resting
[Ca2+]i value from 60-70 to 100-110
nM (Table 1). In MPA myocytes from
control rats, a short application (30 s) of ANG II (10 µM), ET-1 (0.1 µM), or ATP (100 µM) induced cyclic variations (oscillations) of
[Ca2+]i as previously described (13,
15, 20) (Fig. 1A). In
myocytes from CH rats, agonist-induced
[Ca2+]i responses were drastically altered.
The main changes were a decrease in the percentage of responding cells
and the disappearance of the oscillatory profile (Fig. 1B,
Table 1). However, in those cells that responded, the amount of
intracellular Ca2+ mobilized by the agonist was similar to
that in control cells as assessed by the measurement of the area under
the [Ca2+]i curve (Fig.
2).
|
|
|
Ca2+ sources implicated in ANG II-,
ET-1-, and ATP-induced
[Ca2+]i responses in
myocytes from CH rats.
In MPA myocytes from CH rats, removal of external Ca2+
(Ca2+-free PSS) decreased the resting
[Ca2+]i value from 95.8 ± 7.4 (n = 72) to 69 ± 26.8 nM (n = 72;
P < 0.05). However, ANG II-, ET-1-, or ATP-induced
[Ca2+]i responses were not significantly
altered in the absence of extracellular Ca2+ (Fig.
3, A-C). A
similar result was obtained in the presence of the
voltage-dependent Ca2+ channel blocker D-600 (10 µM;
n = 10 for each agonist; data not shown). In another
set of experiments, we tested the effect of thapsigargin, a specific
inhibitor of the SR Ca2+ pump in the pulmonary artery
(11). Thapsigargin (0.1-1 µM) applied for 10 min
suppressed the ATP-induced [Ca2+]i response
in myocytes from MPAs of CH rats (n = 10; Fig.
3D). Similar results were obtained with ET-1 and ANG II
(n = 10 in each case; data not shown).
|
Effect of CH on caffeine-induced
[Ca2+]i response.
As previously observed in MPA myocytes from control rats
(13), a short application (3-5 s) of caffeine (5 mM)
induced only one transient increase in
[Ca2+]i (Fig.
4A). CH altered neither the
relative amplitude of the caffeine-induced
[Ca2+]i response (673 ± 131 nM,
n = 10, and 584 ± 102 nM, n = 14)
nor the percentage of responding cells (90 and 85%) in MPAs from
control and hypoxic rats, respectively. However, the kinetics of the
falling part of the [Ca2+]i response was
modified (Fig. 4). The two phases were slowed down in cells from CH
rats. The slope coefficient (A) of phase 1 and
the time constant () of phase 2 were significantly
decreased and increased, respectively (Table
2).
|
|
Effect of CPA on the caffeine-induced
[Ca2+]i response.
In this set of experiments, the effect of CPA (5 and 10 µM), another
specific inhibitor of the SR Ca2+ pump in the pulmonary
artery (11), on the caffeine-induced [Ca2+]i response in MPA myocytes from control
rats was examined. CPA significantly increased the resting
[Ca2+]i value from 69 ± 11 (n = 86) to 118 ± 35 (n = 25) and
125 ± 43 (n = 15) nM in the presence of 5 and 10 µM CPA, respectively. As in myocytes from CH rats, the two phases of
the falling part of caffeine-induced [Ca2+]i
response were significantly delayed by CPA in myocytes from control
rats (Fig. 5, Table 2). Finally, CPA had
no effect on the falling part of the caffeine-induced
[Ca2+]i response in myocytes from CH rats.
|
Effect of CH on ANG II- and ET-1-induced contractile responses.
In MPA rings from control rats, ET-1 (0.1-100 nM) and ANG II
(0.1-1 µM) induced concentration-dependent contractions. The maximal ET-1- and ANG II-induced responses were 140.1 ± 12.6 (n = 6) and 72.63 ± 9.8% (n = 4), respectively, of the 80 mM KCl-induced contraction (Fig.
6). The mean EC50 values were
1.9 and 0.6 nM for ET-1 and ANG II, respectively. In MPA rings from CH
rats, the maximal forces induced by ET-1 and ANG II were reduced by 30 and 30.2%, respectively (Fig. 6, A and B,
respectively). CH also significantly increased the mean ANG II
EC50 to 6.6 nM (P < 0.05). We did verify
that the raw amplitude of the 80 mM KCl-induced contraction was not
different in MPA rings from control and CH rats (2,490.58 ± 403 mg, n = 13, and 2,335.53 ± 579.3 mg,
n = 16, respectively; P > 0.05).
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
The present study shows that in the rat MPA, CH alters both smooth muscle reactivity and Ca2+ signaling in response to a variety of agonists. In vascular tissues obtained from rats exposed for 14 days to CH, we observed a decrease in the reactivity to ET-1 and ANG II that can be ascribed to a complex combined effect on 1) the percentage of cells responding to agonists acting at plasmalemma membrane receptors, 2) Ca2+ signaling, and 3) Ca2+ sensitivity of the contractile apparatus. The Ca2+ reuptake mechanism appears as a CH-sensitive phenomenon that may account for the main effect of CH on Ca2+ signaling, i.e., the loss of agonist-induced Ca2+ oscillations.
A significant decrease in the percentage of responding cells was
observed regardless of the agonist used in the present study. Therefore, this effect was not restricted to a specific type of membrane receptor. Biochemical modulation of the agonist receptor binding step with either a decrease in the number of receptors expressed at the surface membrane or a decrease in the agonist binding
affinity may account for this effect. In the absence of binding
experiments, this hypothesis cannot be ruled out. However, again, it
should be kept in mind that such phenomena could occur in a similar way
for different receptors. Alternatively, we would favor the hypothesis
of a decrease in the
receptor-Ins(1,4,5)P3 formation
coupling as previously shown for serotonin receptors and
1-adrenoceptors in chronically hypoxic umbilical and
uterine vessels of sheep maintained in high altitude (18,
19).
The main effects of CH on Ca2+ signaling in the MPA were an increase in the resting [Ca2+]i value by ~60% (Table 1) as previously shown by others in intrapulmonary arteries (42, 47, 48) and, more specifically, a decrease in the percentage of cells generating Ca2+ oscillations. Indeed, in responding MPA myocytes from CH rats, the pattern of the [Ca2+]i response to the agonists acting on seven-transmembrane-domain G-coupled receptors was modified. Although in MPA myocytes from normoxic rats, agonists induce Ca2+ oscillations that are mainly dependent on a cyclic release of Ca2+ from an internal store, e.g., the SR (13, 15, 20), in MPA myocytes from CH rats, we observed that ET-1, ANG II, and ATP induced nonoscillating [Ca2+]i responses. This change was not due to a CH-induced change in the Ca2+ sources implicated in the [Ca2+]i responses. As in control conditions, agonist-induced [Ca2+]i responses in myocytes from CH rats were altered neither in a Ca2+-free solution nor in the presence of the voltage-dependent Ca2+ channel blocker D-600 but vanished after pretreatment of the cells with thapsigargin (Fig. 3), indicating that they also involved the mobilization of an intracellular Ca2+ source, presumably the SR. This change in the oscillating nature of the agonist-induced [Ca2+]i response could be due to either an alteration in the functioning of the Ins(1,4,5)P3 receptor and/or to a change in the subtype of Ins(1,4,5)P3 receptor involved in the response. In smooth muscle, the Ins(1,4,5)P3 receptor is biphasically regulated by the [Ca2+]i value (21), and this regulation accounts for the cyclic opening and closure of the associated Ca2+ channel (16) and, at least in part, for the so-called Ca2+ oscillations (40). It is unlikely that the amplitude of the CH-induced increase in the resting [Ca2+]i value, which averaged 60% but corresponded to an actual value far below 300 µM (21), could have modified the negative feed back of [Ca2+]i on the Ins(1,4,5)P3 receptor. In smooth muscle as in nonmuscle cells, the Ins(1,4,5)P3 receptor is encoded by three different genes, resulting in three isoforms, type 1, type 2, and type 3 Ins(1,4,5)P3 receptors. Recent studies performed on Ins(1,4,5)P3 receptors reconstituted in a lipid bilayer have revealed important functional differences between the three isoforms. Interestingly, only one type of Ins(1,4,5)P3 receptor, type 1, is the isoform that exhibits biphasic regulation by intracellular Ca2+ (16). It is thus tempting to speculate that CH could switch the Ins(1,4,5)P3 receptor from a biphasically to a nonbiphasically regulated subtype. This hypothesis requires further molecular biological investigations. Alternatively, this change in the oscillating nature of the agonist-induced [Ca2+]i response could be due to an alteration in the SR Ca2+ reuptake mechanisms that play an important role in Ca2+ oscillation generation (40). Such Ca2+ reuptake mechanisms can be examined by analyzing the falling part of the transient caffeine-induced [Ca2+]i response. First, CH did not modify the percentage of cells responding to caffeine, showing that it only interferes with the Ins(1,4,5)P3-sensitive Ca2+ release pathway and not with the caffeine- or ryanodine-sensitive Ca2+ release mechanism (Ca2+-induced Ca2+ release). Nevertheless, the recovery of the resting [Ca2+]i value after caffeine stimulation was clearly slowed down in MPA myocytes from CH rats. Mathematical fitting of the two phases of this recovery showed a significant decrease in the rate of restoration of the resting [Ca2+]i value, suggesting that CH alters the mechanisms of reuptake of Ca2+ into the SR, e.g., SR Ca2+ pump [sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)] or/and extrusion of Ca2+ [plasmalemma Ca2+ pump (plasma membrane Ca2+-ATPase) and Na+-Ca2+ exchange]. In the present study, the effect of CH on the kinetics of caffeine-induced [Ca2+]i response was mimicked by CPA (Fig. 5), a specific inhibitor of SERCA in this tissue (11). This finding suggests that CH acts at the site of the SR Ca2+ pump to delay the recovery of the resting [Ca2+]i value. Interestingly, it has been shown in cardiac muscle, including from humans, that SERCA expression changes during cardiac hypertrophy (2). We believe that this phenomenon accounts for the decrease in the number or disappearance of Ca2+ oscillations in responding cells to agonists acting at a membrane receptor. After the first Ca2+ increase, Ca2+ reuptake into the SR is slowed down, and hence the [Ca2+]i value remains elevated, thus modifying, in turn, the functioning of the Ins(1,4,5)P3 Ca2+ release channel.
CH-induced disappearance of Ca2+ oscillations in MPA myocytes may play a major role in CH-induced changes in MPA reactivity. Maximal ET-1- and ANG II-induced contraction was decreased as was the frequency of Ca2+ oscillations in response to the same agonists, whereas the amount of mobilized Ca2+ during the agonist-induced [Ca2+]i response remained unchanged in responding cells. Roux et al. (36) have previously demonstrated that rather than the amount of Ca2+, the frequency of Ca2+ oscillations plays a critical role in determining the amplitude of the maximal agonist-induced contraction in airway smooth muscle cells. Interestingly, Belouchi et al. (5) have recently shown that CH, which increases the sensitivity of the tracheal contractile response to cholinergic agonists, also increases the Ca2+ oscillation frequency in airway smooth muscle myocytes. In this latter smooth muscle, the alteration in contractility was also related to an alteration in Ca2+ signaling, although the overall effect was the opposite of that in pulmonary vascular smooth muscle. The reason for the differential effect of CH on the reactivity of the two smooth muscle types remains to be established.
The results of the present study indicate that CH decreases MPA reactivity via an additional mechanism, i.e., a decrease in the Ca2+ sensitivity of the contractile apparatus. This hypothesis is supported by the observations that 1) the D-600- and thapsigargin-resistant component of the contraction was decreased by ~40% in MPA rings from CH rats compared with that in control rats (Fig. 6); under these experimental conditions, agonists do not increase [Ca2+]i in MPA myocytes from either normoxic (13, 15, 20) or hypoxic rats (Fig. 3); and 2) CH had no effect on the contraction induced by an agonist that does not activate a membrane receptor (i.e., KCl). Therefore, the D-600- and thapsigargin-resistant component of the observed contraction is due to a sensitization of the contractile apparatus to Ca2+ (39, 40), and CH significantly decreases this sensitization. Interestingly, this latter present result observed in MPAs is opposite to that very recently suggested in intrapulmonary arteries (42). Both results do not indicate the specific pathway among the multiple ones implicated in the Ca2+ sensitivity of the smooth muscle contractile apparatus (39) targeted by CH. Again, as discussed above for airway smooth muscle (5), it appears that the effect of CH may vary depending not only on the type of smooth muscle but also on the site along the pulmonary vascular bed. It is also noteworthy that CH also diminishes the response to acute hypoxia in both the pulmonary artery (28) and cardiac muscle (44).
In conclusion, the present study indicates that CH alters pulmonary vascular smooth muscle reactivity as a consequence of an effect on both Ca2+ signaling and Ca2+ sensitivity of the contractile apparatus. A Ca2+ reuptake mechanism appears as a CH-sensitive phenomenon that may account for the main effect of CH on Ca2+ signaling, i.e., the loss of agonist-induced Ca2+ oscillations.
![]() |
ACKNOWLEDGEMENTS |
---|
We thank H. Crevel for technical assistance.
![]() |
FOOTNOTES |
---|
This work was supported by Conseil Régional d'Aquitaine Grant 20000301114.
Address for reprint requests and other correspondence: J. P. Savineau, Laboratoire de Physiologie Cellulaire Respiratoire, INSERM (EMI 9937), Université Bordeaux 2, 146 rue Léo-Saignat, 33076 Bordeaux, France (E-mail: jean-pierre.savineau{at}lpcr.u-bordeaux2.fr).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 28 November 2000; accepted in final form 22 February 2001.
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
1.
Aguirre, JI,
Morrell NW,
Long L,
Clift P,
Upton PD,
Polak JM,
and
Wilkins MR.
Vascular remodeling and ET-1 expression in rat strains with different responses to chronic hypoxia.
Am J Physiol Lung Cell Mol Physiol
278:
L981-L987,
2000
2.
Anger, M,
Lompre AM,
Vallot O,
Marotte F,
Rappaport L,
and
Samuel JL.
Cellular distribution of Ca2+ pumps and Ca2+ release channels in rat cardiac hypertrophy induced by aortic stenosis.
Circulation
98:
2477-2486,
1998
3.
Bakhramov, A,
Hartley SA,
Salter KJ,
and
Kozlowski RZ.
Contractile agonists preferentially activate Cl- over K+ currents in arterial myocytes.
Biochem Biophys Res Commun
227:
168-175,
1996[ISI][Medline].
4.
Barnes, PJ,
and
Liu SF.
Regulation of pulmonary vascular tone.
Pharmacol Rev
47:
87-131,
1995[ISI][Medline].
5.
Belouchi, NE,
Roux E,
Savineau JP,
and
Marthan R.
Effect of chronic hypoxia on calcium signalling in airway smooth muscle cells.
Eur Respir J
14:
74-79,
1999
6.
Bialecki, RA,
Fisher CS,
Murdoch WW,
Barthlow HG,
Stow RB,
Mallamaci M,
and
Rumsey W.
Hypoxic exposure time dependently modulates endothelin-induced contraction of pulmonary artery smooth muscle.
Am J Physiol Lung Cell Mol Physiol
274:
L552-L559,
1998
7.
Cassin, S,
Kristova V,
Davis T,
Kadowitz P,
and
Gause G.
Tone-dependent responses to endothelin in the isolated perfused fetal sheep pulmonary circulation in situ.
J Appl Physiol
70:
1228-1234,
1991
8.
Clapp, LH,
and
Gurney AM.
ATP-sensitive K+ channels regulate resting potential of pulmonary arterial smooth muscle cells.
Am J Physiol Heart Circ Physiol
262:
H916-H920,
1992
9.
Elton, TS,
Oparil S,
Taylor GR,
Hicks PH,
Yang RH,
Jin H,
and
Chen YF.
Normobaric hypoxia stimulates endothelin-1 gene expression in the rat.
Am J Physiol Regulatory Integrative Comp Physiol
263:
R1260-R1264,
1992
10.
Giaid, A,
Yanagisawa M,
Langleben D,
Michel RP,
Levy R,
Shennib H,
Kimura S,
Masaki T,
Duguid WP,
and
Stewart DJ.
Expression of endothelin-1 in the lungs of patients with pulmonary hypertension.
N Engl J Med
328:
1732-1739,
1993
11.
Gonzalez De La Fuente, P,
Savineau JP,
and
Marthan R.
Control of pulmonary vascular smooth muscle tone by sarcoplasmic reticulum Ca2+ pump blockers: thapsigargin and cyclopiazonic acid.
Pflügers Arch
429:
617-624,
1995[ISI][Medline].
12.
Grynkiewicz, G,
Poenie M,
and
Tsien RY.
A new generation of Ca2+ indicators with greatly improved fluorescence properties.
J Biol Chem
260:
3440-3450,
1985[Abstract].
13.
Guibert, C,
Marthan R,
and
Savineau JP.
Angiotensin II-induced Ca2+-oscillations in vascular myocytes from the rat pulmonary artery.
Am J Physiol Lung Cell Mol Physiol
270:
L637-L642,
1996
14.
Guibert, C,
Marthan R,
and
Savineau JP.
Oscillatory Cl- current induced by angiotensin II in rat pulmonary arterial myocytes: Ca2+ dependence and physiological implication.
Cell Calcium
21:
421-429,
1997[ISI][Medline].
15.
Guibert, C,
Pacaud P,
Loirand G,
Marthan R,
and
Savineau JP.
Effect of extracellular ATP on cytosolic Ca2+ concentration in rat pulmonary artery myocytes.
Am J Physiol Lung Cell Mol Physiol
271:
L450-L458,
1996
16.
Hagar, RE,
Burgstahler AD,
Nathanson MH,
and
Ehrlich BE.
Type III InsP3 receptor channel stays open in the presence of increased calcium.
Nature
396:
81-84,
1998[ISI][Medline].
17.
Haworth, SG.
Development of the normal and hypertensive pulmonary vasculature.
Exp Physiol
80:
843-853,
1995[Abstract].
18.
Hu, XQ,
Yang S,
Pearce WJ,
Longo LD,
and
Zhang L.
Effect of chronic hypoxia on alpha-1 adrenoceptor-mediated inositol 1,4,5-trisphosphate signaling in ovine uterine artery.
J Pharmacol Exp Ther
288:
977-983,
1999
19.
Hu, XQ,
and
Zhang L.
Chronic hypoxia suppresses pharmacomechanical coupling of the uterine artery in near-term pregnant sheep.
J Physiol (Lond)
499:
551-559,
1997[Abstract].
20.
Hyvelin, JM,
Guibert C,
Marthan R,
and
Savineau JP.
Cellular mechanisms and role of endothelin-1-induced calcium oscillations in pulmonary arterial myocytes.
Am J Physiol Lung Cell Mol Physiol
275:
L269-L282,
1998
21.
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[Abstract].
22.
Karamsetty, VS,
Kane KA,
and
Wadsworth RM.
The effects of chronic hypoxia on the pharmacological responsiveness of the pulmonary artery.
Pharmacol Ther
68:
233-246,
1995[ISI][Medline].
23.
Li, H,
Elton TS,
Chen YF,
and
Oparil S.
Increased endothelin receptor gene expression in hypoxic rat lung.
Am J Physiol Lung Cell Mol Physiol
266:
L553-L560,
1994
24.
Liu, SQ.
Alterations in structure of elastic laminae of rat pulmonary arteries in hypoxic hypertension.
J Appl Physiol
81:
2147-2155,
1996
25.
MacLean, MR,
Herve P,
Eddahibi S,
and
Adnot S.
5-Hydroxytryptamine and the pulmonary circulation: receptors, transporters and relevance to pulmonary arterial hypertension.
Br J Pharmacol
131:
161-168,
2000
26.
MacLean, MR,
and
McCulloch KM.
Influence of applied tension and nitric oxide on responses to endothelins in rat pulmonary resistance arteries: effect of chronic hypoxia.
Br J Pharmacol
123:
991-999,
1998[Abstract].
27.
McCulloch, KM,
Docherty C,
and
MacLean MR.
Endothelin receptors mediating contraction of rat and human pulmonary resistance arteries: effect of chronic hypoxia in the rat.
Br J Pharmacol
123:
1621-1630,
1998[Abstract].
28.
McMurtry, IF,
Petrun MD,
and
Reeves JT.
Lungs from chronically hypoxic rats have decreased pressor response to acute hypoxia.
Am J Physiol Heart Circ Physiol
235:
H104-H109,
1978
29.
Morrell, NW,
Morris KG,
and
Stenmark KR.
Role of angiotensin-converting enzyme and angiotensin II in development of hypoxic pulmonary hypertension.
Am J Physiol Heart Circ Physiol
269:
H1186-H1194,
1995
30.
Nong, Z,
Stassen JM,
Moons L,
Collen D,
and
Janssens S.
Inhibition of tissue angiotensin-converting enzyme with quinapril reduces hypoxic pulmonary hypertension and pulmonary vascular remodeling.
Circulation
94:
1941-1947,
1996
31.
Osipenko, ON,
Alexander D,
MacLean MR,
and
Gurney AM.
Influence of chronic hypoxia on the contributions of noninactivating and delayed rectifier K currents to the resting potential and tone of rat pulmonary artery smooth muscle.
Br J Pharmacol
124:
1335-1337,
1998[Abstract].
32.
Peng, W,
Hoidal JR,
Karwande SV,
and
Farrukh IS.
Effect of chronic hypoxia on K+ channels: regulation in human pulmonary vascular smooth muscle cells.
Am J Physiol Cell Physiol
272:
C1271-C1278,
1997
33.
Peng, W,
Michael JR,
Hoidal JR,
Karwande SV,
and
Farrukh IS.
ET-1 modulates KCa-channel activity and arterial tension in normoxic and hypoxic human pulmonary vasculature.
Am J Physiol Lung Cell Mol Physiol
275:
L729-L739,
1998
34.
Pierson, DJ.
Pathophysiology and clinical effects of chronic hypoxia.
Respir Care
45:
39-51,
2000[Medline].
35.
Rabinovitch, M,
Gamble W,
Nadas AS,
Miettinen OS,
and
Reid L.
Rat pulmonary circulation after chronic hypoxia: hemodynamic and structural features.
Am J Physiol Heart Circ Physiol
236:
H818-H827,
1979
36.
Roux, E,
Guibert C,
Savineau JP,
and
Marthan R.
[Ca2+]i oscillations induced by muscarinic stimulation in airway smooth muscle cells: receptor subtypes and correlation with the mechanical activity.
Br J Pharmacol
120:
1294-1301,
1997[Abstract].
37.
Salter, KJ,
and
Kozlowski RZ.
Endothelin receptor coupling to potassium and chloride channels in isolated rat pulmonary arterial myocytes.
J Pharmacol Exp Ther
279:
1053-1062,
1996[Abstract].
38.
Sauzeau, V,
Le Jeune H,
Cario-Toumaniantz C,
Smolenski A,
Lohmann SM,
Bertoglio J,
Chardin P,
Pacaud P,
and
Loirand G.
Cyclic GMP-dependent protein kinase signaling pathway inhibits RhoA-induced Ca2+ sensitization of contraction in vascular smooth muscle.
J Biol Chem
275:
21722-21729,
2000
39.
Savineau, JP,
and
Marthan R.
Modulation of the calcium sensitivity of the smooth muscle contractile apparatus: molecular mechanisms, pharmacological and pathophysiological implications.
Fundam Clin Pharmacol
11:
289-299,
1997[ISI][Medline].
40.
Savineau, JP,
and
Marthan R.
Cytosolic calcium oscillations in smooth muscle cells.
News Physiol Sci
15:
50-55,
2000
41.
Schulman, DS,
and
Matthay RA.
The right ventricle in pulmonary disease.
Cardiol Clin
10:
111-135,
1992[Medline].
42.
Shimoda, LA,
Sham JS,
Shimoda TH,
and
Sylvester JT.
L-type Ca2+ channels, resting [Ca2+]i, and ET-1-induced responses in chronically hypoxic pulmonary myocytes.
Am J Physiol Lung Cell Mol Physiol
279:
L884-L894,
2000
43.
Shimoda, LA,
Sylvester JT,
and
Sham JS.
Chronic hypoxia alters effects of endothelin and angiotensin on K+ currents in pulmonary arterial myocytes.
Am J Physiol Lung Cell Mol Physiol
277:
L431-L439,
1999
44.
Silverman, HS,
Wei SK,
Haigney MCP,
Ocampo CJ,
and
Stern MD.
Myocyte adaptation to chronic hypoxia and development of tolerance to subsequent acute severe hypoxia.
Circ Res
80:
699-707,
1997
45.
Smirnov, SV,
Robertson TP,
Ward JP,
and
Aaronson PI.
Chronic hypoxia is associated with reduced delayed rectifier K+ current in rat pulmonary artery muscle cells.
Am J Physiol Heart Circ Physiol
266:
H365-H370,
1994
46.
Stenmark, KR,
and
Mecham RP.
Cellular and molecular mechanisms of pulmonary vascular remodeling.
Annu Rev Physiol
59:
89-144,
1997[ISI][Medline].
47.
Vender, RL,
and
Saab EM.
Prolonged hypoxia increases pulmonary vascular smooth muscle cytosolic calcium.
In Vitro Cell Dev Biol Anim
30A:
485-487,
1994.
48.
Yuan, JX,
Aldinger AM,
Juhaszova M,
Wang J,
Conte JV, Jr,
Gaine SP,
Orens JB,
and
Rubin LJ.
Dysfunctional voltage-gated K+ channels in pulmonary artery smooth muscle cells of patients with primary pulmonary hypertension.
Circulation
98:
1400-1406,
1998
49.
Yuan, XJ.
Voltage-gated K+ currents regulate resting membrane potential and [Ca2+]i in pulmonary arterial myocytes.
Circ Res
77:
370-378,
1995
50.
Zhao, L,
Al-Tubuly R,
Sebkhi A,
Owji AA,
Nunez DJ,
and
Wilkins MR.
Angiotensin II receptor expression and inhibition in the chronically hypoxic rat lung.
Br J Pharmacol
119:
1217-1222,
1996[Abstract].