Sildenafil alters calcium signaling and vascular tone in pulmonary arteries from chronically hypoxic rats

Olivier Pauvert,1 Sébastien Bonnet,1 Eric Rousseau,2 Roger Marthan,1 and Jean-Pierre Savineau1

1Laboratoire de Physiologie Cellulaire Respiratoire, Institut National de la Santé et de la Recherche Médicale (E 356 and Institut Fédératif de Recherche 4), Université Bordeaux 2, 33076 Bordeaux, France; and 2Le Bilarium, Département de Physiologie et Biophysique, Université de Sherbrooke, Sherbrooke, Quebec J1H 5N4, Canada

Submitted 22 December 2003 ; accepted in final form 18 May 2004


    ABSTRACT
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Sildenafil, a potent type 5 nucleotide-dependent phosphodiesterase (PDE) inhibitor, has been recently proposed as a therapeutic tool to treat or prevent pulmonary artery hypertension (PAHT). We thus studied the effect of sildenafil on both the calcium signaling of isolated pulmonary artery smooth muscle cells (PASMCs) and the reactivity of pulmonary artery (PA) obtained from chronic hypoxia (CH)-induced pulmonary hypertensive rats compared with control (normoxic) rats. CH rats were maintained in an hypobaric chamber (50.5 kPa) for 3 wk leading to full development of PAHT. Intracellular calcium concentration ([Ca2+]i) was measured in PASMCs loaded with the calcium fluorophore indo 1. Unlike in control rats, sildenafil (10–100 nM) decreased the resting [Ca2+]i value in PASMCs obtained from CH rats. In PASMCs from both control and CH rats, sildenafil concentration dependently inhibited the [Ca2+]i response induced by G-coupled membrane receptor agonists such as angiotensin II and phenylephrine but had no effect on the amplitude of the [Ca2+]i response induced by caffeine. Sildenafil (0.1 nM–1 µM) concentration dependently reduced basal PA tone that is present in CH rats and relaxed PA rings precontracted with phenylephrine in both control and CH rats. These data show that sildenafil is a potent pulmonary artery relaxant in CH rats and that it normalizes CH-induced increases in resting [Ca2+]i and basal tone. Consequently, pharmacological inhibition of sildenafil-sensitive PDE5 downregulates the Ca2+ signaling pathway involved in this model of pulmonary hypertension.

pulmonary hypertension; vascular smooth muscle; phosphodiesterase 5; pulmonary vasodilator


INTRACELLULAR CYCLIC NUCLEOTIDE [cAMP and guanosine 3',5'-cyclic monophosphate (cGMP)] concentration is implicated in the control of the vascular smooth muscle tone including in the pulmonary artery (PA) (3, 9). Agents elevating cAMP or cGMP concentration relax precontracted PA (35, 36, 38). For instance, the endothelium-derived factor nitric oxide (NO) induces a dose-dependent increase in cGMP concentration and a step-wise relaxation of PA (30, 45). In smooth muscle cells, concentration of cGMP is mainly dependent on the balance between the production by guanylate cyclase and the degradation by phosphodiesterases (PDEs), which represent the unique degradation pathway for these intracellular compounds (34, 44). As a consequence, PDE activity is also implicated in the control of smooth muscle tone, and PDE inhibition relaxes smooth muscle (21, 41, 46). In PA from rats and humans, four types of PDE (PDE1, 3, 4, and 5) have been identified (23, 31, 34, 36). Hence, the pharmacological modulation of PDE activity (e.g., using selective PDE inhibitors) could be an effective means to control pulmonary vascular tone.

Pulmonary circulation develops a specific response to hypoxia, i.e., vasoconstriction, which can be maintained in patients suffering from chronic obstructive pulmonary diseases (COPD; e.g., chronic bronchitis) (24, 32), thus inducing, in turn, a sustained elevation in the pulmonary blood pressure. This PA hypertension (PAHT) leads to right ventricular hypertrophy (RVH), right heart failure, and ultimately death (12, 37). This pathophysiological adaptation of the pulmonary circulation to maintained hypoxia is a complex process. In these conditions, only long-lasting oxygen therapy slows down the progression of PAHT in COPD (2).

In pulmonary circulation, it has been shown that: 1) cGMP does play a major role on pulmonary vascular resistance (17); 2) PDE5, the enzyme that specifically hydrolyzes cGMP (44), is abundantly expressed in the whole lung and predominates in pulmonary artery smooth muscle cells (PASMCs) (18, 26, 31); 3) both the activity and the expression of PDE5 are increased in pulmonary arteries from rats with PAHT (23, 27); and 4) there is a direct correlation between the activity of PDE5 activity and pulmonary vascular resistance (18). More recently, sildenafil citrate, a potent and selective PDE5 inhibitor (1, 10) that is successfully used for the treatment of erectile dysfunction (8), has also been proposed to improve PAHT, and pioneer clinical trials with sildenafil have been performed both in animals and humans with PAHT (19, 25, 42, 47, 48). However, little information, at the cellular level, is available concerning the interactions of sildenafil and signaling pathway in the pulmonary vascular smooth muscle from chronically hypoxic (CH) animals.

The aims of the present study were thus to investigate the acute effect of sildenafil on both the calcium signaling pathway and the reactivity of the PA obtained from CH rats compared with that obtained from normoxic rats. Sildenafil effect was studied both in nonstimulated (resting state) and precontracted preparations with pulmonary vasoconstrictors such as phenylephrine (PE) and angiotensin II (ANG II). We used mirospectrofluorimetry (indo 1) to monitor variations of intracellular calcium concentration ([Ca2+]i) in PASMCs and measured isometric contraction in PA rings to assess the functional effect of sildenafil.


    MATERIALS AND METHODS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
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 21 days. The pressure in the chamber was reduced to 0.5 atm (50.5 kPa) by an electrically driven pump. The chamber was opened for 15–30 min twice a week. We assessed pulmonary hypertension by measuring the ratio of right ventricle (RV) to left ventricle plus septum (LV+S) weight.

Tissue preparation. At completion of the exposure, rats were anesthetized by intraperitoneal injection of 40 mg of ethylcarbamate. Heart and lungs were removed en bloc. The main part and branches of the PA were isolated and dissected under binocular control, and the adventitial and intimal layers were removed. For contraction measurements, PA rings (3 mm in length) were prepared. For measurement of [Ca2+]i, isolated PASMCs were obtained by an enzymatic dissociation method previously described (6). Cells were stored at 4°C and used between 2 and 8 h after isolation. Only elongated, smooth, and optically refractive cells were used for [Ca2+]i measurements.

[Ca2+]i measurements. The dynamic changes in [Ca2+]i of individual arterial myocytes were assessed with the [Ca2+]i-sensitive fluorophore indo 1. Cells were loaded with indo 1 by incubation in PSS containing 1 µM indo 1 pentaacetoxymethyl ester 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. 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 fluorescence ratio (405/480) was calculated on-line and displayed with the two voltage signals on a monitor. [Ca2+]i was estimated from the 405/480 ratio with the equation: [Ca2+]i = Kd{beta} (R – Rmin)/(Rmax – R) (15), where R is the experimental ratio value; Rmin and Rmax are the minimum and maximum fluorescence ratios, respectively; Kd is the dissociation constant for indo 1; and {beta} is the fluorescence ratio at 480 nm in the absence and in the presence of a saturating concentration of Ca2+. In control experiments, Rmin and Rmax were determined in permeabilized PASMCs in the presence of 5 mM EGTA and 5 mM Ca2+ added to PSS, respectively. PASMCs were permeabilized with 25 µM {beta}-escin for 90–120 s. In preliminary studies, we determined that such duration of exposure to the {beta}-escin solution was critical to ensure adequate subsequent retention of the dye indo 1. Rmin and Rmax were 0.07 ± 0.007 (n = 5) and 0.85 ± 0.05 (n = 7), respectively, values in good accordance with those previously reported in the literature for other vascular myocytes. Intermediary fluorescence ratio values were obtained from cells bathed in solutions of variable Ca2+ concentrations. The in vivo calibrating curve was then constructed, allowing the graphical determination of Kd{beta}. This method has the advantage of taking into account a possible difference in the Kd value between that in the cell in vivo and that determined in vitro (15).

Isometric contraction measurement. Isometric contraction was measured in rings from PA that were mounted between two stainless steel clips in vertical 5-ml organ baths of a computerized isolated organ bath system (IOX; 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 steel clip was connected to an isometric force transducer (EMKA Technologies). As determined in previous 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 (7). Some experiments were also conducted when both tissues were set at an identical preload value (i.e., 20 mN). At the beginning of each experiment, a 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. A cumulative concentration-response curve to sildenafil (0.1 nM–1 µM) was then constructed on PE-precontracted PA rings. Because reactivity to agonists is reduced in main PA rings from CH rats (4, 5, 40), 0.3 and 3 µM PE were used to obtain the same value of precontraction in rings from control and CH rats, respectively. Some experiments were also performed in endothelium-denuded rings. In this respect, the PA lumen was superfused with distilled water before being mounted in the bath. Successful removal of the endothelium was confirmed by the inability of acetylcholine (1 µM) to induce >10% of relaxation in PE (1 µM)-contracted rings.

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. PE, ANG II, 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. We assessed sildenafil's effect on agonist-induced [Ca2+]i response by preincubating PASMCs with sildenafil for 10 min. For experiments in permeabilized PASMCs, the free ion concentration of the physiological solution was calculated with a computer program adapted from that by Fabiato (13).

Chemicals and drugs. Acetylcholine and collagenase (type CLS1) were from Worthington Biochemical (Freehold, NJ). Pronase (type E), elastase (type 3), bovine serum albumin, ANG II, and PE were from Sigma (Saint Quentin Fallavier, France). Caffeine was from Merck (Darmstadt, Germany). Indo 1 was from Calbiochem (France Biochem, Meudon, France). Sildenafil was kindly provided by Pfizer (Sandwich, Kent, UK). Indo 1 was dissolved in dimethyl sulfoxide. All other materials were of reagent grade. All buffer solutions were prepared with deionized water from Millipore Milli Ro-Milli-Q-UF system (18 ± 0.2 M{Omega} x cm).

Analysis of data. [Ca2+]i response to PE and ANG II was analyzed compared with the resting [Ca2+]i value, the relative amplitude of the first peak of the response, the percentage of responding cells, and the number of calcium oscillations in PASMCs obtained from both control and CH rats. In the case of caffeine, the falling part of the transient [Ca2+]i response was kinetically analyzed, and the time in which [Ca2+]i decreases to 50% from the peak value (t50) was measured. Results are expressed as means ± SE with n as the sample size. Significance was tested by means of Student's t-test at a P value < 0.05. Regarding the number of experiments, n refers to the number of cells or rings (contraction experiments) and N to the number of animals.


    RESULTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
Effect of CH on RVH. In rats exposed to hypobaric hypoxia for 21 days, the ratio of RV/(LV+S) weight significantly increased compared with control rats: 0.58 ± 0.02 (N = 10) and 0.28 ± 0.01 (N = 8), respectively (P < 0.05). This RVH was the consequence of the development of a PAHT.

Effect of sildenafil on resting [Ca2+]i value of PASMCs from CH rats. Three weeks of CH significantly increased the resting [Ca2+]i value of PASMCs from 77 ± 6.2 nM (n = 20, N = 5) to 121 ± 5.8 nM (n = 20, N = 5, P < 0.05) (Fig. 1). Sildenafil concentration dependently decreased the resting [Ca2+]i value of PASMCs from CH rats (Fig. 1). Sildenafil at 100 nM normalized resting [Ca2+]i to 89 ± 6.3 nM (n = 15, N = 4), a value that was not significantly different (P > 0.05) from that of PASMCs from normoxic rats. In contrast, pretreatment of PASMCs from normoxic rats with sildenafil at 10 and 100 nM for 10 min had no effect on the resting [Ca2+]i value (Fig. 1).



View larger version (13K):
[in this window]
[in a new window]
 
Fig. 1. Effect of sildenafil on the resting intracellular calcium concentration ([Ca2+]i) value of pulmonary artery smooth muscle cells (PASMCs) obtained from chronically hypoxic (CH, filled bars) and normoxic (open bars) rats. PASMCs were incubated in the absence (control) or in the presence of sildenafil (10, 100 nM) for 10 min before the measurement of [Ca2+]i. Results are expressed as means ± SE (vertical bars); n = 20, N = 5–8. *P < 0.05 vs. control.

 
Effect of sildenafil on PE- and ANG II-induced [Ca2+]i response in PASMCs from CH rats. CH, per se, alters the pattern of PE- and ANG II-induced [Ca2+]i responses compared with responses obtained in PASMCs from normoxic rats (Figs. 2 and 3). The main changes were a decrease in the relative amplitude of the first [Ca2+]i peak, the disappearance of the oscillatory profile, and a decrease in the percentage of responding cells. In these cells, sildenafil (10 and 100 nM) concentration dependently decreased the amplitude of the [Ca2+]i response and the percentage of responding cells (Figs. 2Abc and 3Abc, Tables 1 and 2). Similar effects of sildenafil on [Ca2+]i response were observed when PASMCs from CH rats were stimulated by extracellular ATP (100 µM) (n = 12, N = 4; not shown). In PASMCs from control rats, sildenafil concentration dependently decreased the amplitude and the frequency of calcium oscillations, the relative amplitude of the first Ca2+ peak, and the percentage of responding cells (Figs. 2Bbc and 3Bbc, Tables 1 and 2). It could be noted that the relative amplitude of the first Ca2+ peak was less decreased by sildenafil in PASMCs from CH than from normoxic rats (Tables 1 and 2).



View larger version (14K):
[in this window]
[in a new window]
 
Fig. 2. Effect of sildenafil on the phenylephrine (PE)-induced [Ca2+]i response in PASMCs obtained from CH (A) and control (normoxic, B) rats. a: control traces in the absence of sildenafil. b and c: PASMCs were incubated with sildenafil for 10 min before application of PE. Each trace was recorded from a different cell and is typical of 15–20 cells.

 


View larger version (16K):
[in this window]
[in a new window]
 
Fig. 3. Effect of sildenafil on the ANG II-induced [Ca2+]i response in PASMCs obtained from CH (A) and control (normoxic, B) rats. a: control traces in the absence of sildenafil. b and c: PASMCs were incubated with sildenafil for 10 min before application of ANG II. Each trace was recorded from a different cell and is typical of 10–12 cells.

 

View this table:
[in this window]
[in a new window]
 
Table 1. Effect of sildenafil on phenylephrine-induced [Ca2+]i response in PA myocytes obtained from control and CH rats.

 

View this table:
[in this window]
[in a new window]
 
Table 2. Effect of sildenafil on angiotensin-II-induced [Ca2+]i response in PA myocytes obtained from control and CH rats

 
Effect of sildenafil on caffeine-induced [Ca2+]i response in PASMCs from CH rats. As previously observed in PASMCs from control rats (16), caffeine (5 mM), whatever its duration of application, induced only one transient increase in [Ca2+]i. CH had no effect on the relative amplitude of the caffeine-induced [Ca2+]i response or on the percentage of responding cells (Fig. 4 and Table 3). However, the kinetics of the falling part of the [Ca2+]i response was significantly slowed down (t50 was 8.1 ± 0.8 s and 5.9 ± 0.7 s, P < 0.05, in CH and normoxic rats, respectively; Fig. 4A and Table 3). Pretreatment of PASMCs from CH rats with sildenafil for 10 min restored the kinetics of the falling part of the caffeine-induced [Ca2+]i response. In PASMCs from control rats, sildenafil altered neither the relative amplitude of the caffeine-induced [Ca2+]i response nor the percentage of responding cells (Fig. 4B and Table 3).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 4. Effect of sildenafil on the caffeine (Caf)-induced [Ca2+]i response in PASMCs obtained from CH (A) and control (normoxic, B) rats. a: control traces in the absence of sildenafil. b: PASMCs were incubated with sildenafil (100 nM) for 10 min before application of caffeine for 5 s. Each trace was recorded from a different cell and is typical of 20–25 cells.

 

View this table:
[in this window]
[in a new window]
 
Table 3. Effect of sildenafil on caffeine-induced [Ca2+]i response in PA myocytes obtained from control and CH rats

 
Effect of sildenafil on PA basal tone. Unlike PA rings from normoxic rats, those from CH rats exhibited a spontaneous tone that was 15–18% of the amplitude of K+-rich (80 mM) solution-induced contraction. Sildenafil (1 nM–1 µM) concentration dependently decreased this tone, whereas it had no effect on the resting tension in PA rings from normoxic rats (Fig. 5A), whatever the applied resting load (10–20 mN, see MATERIALS AND METHODS).



View larger version (20K):
[in this window]
[in a new window]
 
Fig. 5. Relaxant effect of sildenafil in pulmonary artery (PA) rings obtained from CH and control (normoxic) rats. A: concentration-response curves for the effect of sildenafil on the resting tone in PA rings from CH ({square}) and normoxic ({bullet} for 10 mN preload, {circ} for 20 mN preload) rats. B: concentration-response curves for the effect of sildenafil on PE-precontracted PA rings from CH ({square}) and normoxic ({bullet} for 10 mN preload, {circ} for 20 mN preload and 3 µM PE) rats. Data points are means ± SE (n = 6, N = 6).

 
Relaxant effect of sildenafil on agonist-induced contraction in PA rings from normoxic and CH rats. Finally, we investigated the effect of sildenafil on PE-precontracted rings from CH rats. Sildenafil (0.1 nM–1 µM) concentration dependently relaxed PE-induced contraction. However, the concentration-response curve established in rings from CH rats was shifted to the right compared with that established in rings from control rats: 50% relaxation was obtained with 10 nM and 300 nM sildenafil, and the maximal value of relaxation was 85 and 75% in PA rings from normoxic and CH rats, respectively (Fig. 5B). Similar experiments were also conducted on endothelium-denuded PA rings. No significant difference with intact rings was observed in rings from both normoxic and CH rats (n = 6, N = 6 in each case, not shown). Also, a similar result was observed when tissues from normoxic animals were investigated under experimental conditions identical to those of CH animals (i.e., resting load 20 mN, PE 3 µM; Fig. 5B). Sildenafil (10–7 M) also decreased the amplitude of the contractile response induced by 3.10–8 M ANG II. The inhibition was 85 ± 5.8% and 54 ± 8.6% (n = 4, N = 3) in rings from normoxic and hypoxic rats, respectively. In contrast, sildenafil 10–7 M had no significant effect on the amplitude of the contractile response to caffeine (5 mM) in rings from both normoxic and hypoxic rats (n = 4, N = 4, not shown).


    DISCUSSION
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
The present work shows that sildenafil decreases resting tone and relaxes precontracted PA from CH rats. The relaxant effect of sildenafil appears mainly related to the inhibitory action of sildenafil on the calcium signaling pathway in PASMCs.

Sildenafil selectively decreases the resting [Ca2+]i value in PASMCs from CH rats. CH increases this resting value by ~60%, a typical feature of CH (7, 33, 43). In this connection, sildenafil also decreases resting tone in PA rings from CH rats. Whether these two phenomena are related remains to be established. However, it has been previously shown that both the elevated resting [Ca]i value and tone in tissues from CH rats are mainly due to an influx of extracellular calcium through the PASMC membrane (7, 43).

In PASMCs from CH rats, sildenafil inhibits PE- and ANG II-induced [Ca2+]i responses but had no effect on either the amplitude of caffeine-induced transient [Ca2+]i response or the percentage of responding cells to caffeine. Because we have previously demonstrated in PASMCs that [Ca2+]i responses induced by agonists acting via G protein-coupled receptors or by caffeine are due to the opening of the sarcoplasmic reticulum (SR) inositol trisphosphate (IP3)- and ryanodine-sensitive receptors, respectively (16, 39), the inhibitory effect of sildenafil on calcium signaling appears mainly related to the alteration of the IP3-mediated calcium release pathway. In PASMCs from CH rats, the kinetics of the agonist-induced [Ca2+]i response was altered: ANG II and PE induced nonoscillating [Ca2+]i responses in contrast to responses in PASMCs from control rats. This change in the oscillating nature of the agonist-induced [Ca2+]i responses is not due to a CH-induced change in the calcium sources implicated in the [Ca2+]i responses (5) but could result from an alteration in the functioning of the IP3 receptor (RIP3) and/or to a change in the subtype of RIP3 involved in the response (16, 39). In addition, in PASMCs from normoxic rats, agonist-induced calcium oscillations are due to the concerted functioning of RIP3, allowing a cyclic release of stored calcium from the SR and of the SR calcium pump [sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA)] (5, 16, 39, 43). Therefore, alternatively, the change in the oscillating nature of the agonist-induced [Ca2+]i response could be due to an alteration in the SR calcium reuptake mechanisms that play an important role in calcium oscillations (39). Such calcium reuptake mechanisms can be more easily examined in PASMCs by analyzing the kinetics of the falling part of the transient caffeine-induced [Ca2+]i response. In PASMCs from CH rats, sildenafil restored the kinetics of the falling phase of the caffeine-induced [Ca2+]i response, which had been slowed down by CH itself (Fig. 4). Interestingly, we have previously shown that cyclopiazonic acid, a selective SERCA inhibitor in PA (14), slows down the falling part of the caffeine-induced [Ca2+]i response in PASMCs from normoxic rats but has no further effect on this falling part in PASMCs from CH rats (5). Collectively, these findings suggest that, under CH, sildenafil acts at the site of the SERCA to activate the recovery of the resting [Ca2+]i value.

The various effects of sildenafil on the calcium signaling pathway appear very similar to that of 8-bromoguanosine 3',5'-cyclic monophosphate and of NO in the same preparation (30), suggesting that the effect of sildenafil on the calcium signaling pathway is cGMP mediated. This hypothesis is reinforced in PA by the fact that: 1) sildenafil is a potent inhibitor of purified PDE5 (IC50 = 3.4 nM) in PASMCs (29), 2) PDE5 isozyme is well expressed in rat PA (27, 29), and 3) sildenafil increased cGMP level in serum and decreases pulmonary vascular resistance in humans with PAHT (25). Sildenafil-induced alteration of calcium signaling in PASMCs could thus result from various cGMP-dependent mechanisms such as: phosphorylation of RIP3 (20), inhibition of PLC and thus decrease of IP3 formation (22), and activation of the SERCA (11). Although our data on the kinetics of caffeine-induced [Ca2+]i response are consistent with an effect of sildenafil on SERCA, further experiments are required to identify the true site of action of sildenafil along the IP3-dependent signaling pathway.

In PA from CH rats, sildenafil-induced inhibition of PE- and ANG II-induced [Ca2+]i response is correlated with inhibition of contractile response induced by these agonists, indicating that a decrease in calcium release from internal store is an important mechanism in sildenafil-induced pulmonary vasorelaxation. Moreover, it is noteworthy that the relaxant effect of sildenafil was effective in a range of concentrations similar to that inhibiting PE-induced [Ca2+]i response, i.e., in the nM range. However, additional mechanisms may be involved in the relaxant effect of sildenafil, e.g., cGMP-mediated activation of large-conductance calcium-sensitive K+ channels (26) and/or interaction between sildenafil and the calcium sensitivity of the contractile apparatus controlled by the Rho-Rho kinase signaling pathway. However, in the latter case, contrasting results have been reported by Sauzeau et al. (40), who show that CH decreased expression of small G protein RhoA that was prevented by a chronic treatment of animals by sildenafil, and by Nagaoka et al. (28), who suggest, in contrast, that a stimulation of this signaling pathway could be an important means for the increase in tone and reactivity to agonist during PAHT. The reasons for such a discrepancy from the same animal model are not evident.

In the present study, as for the effect of sildenafil on agonist-induced calcium responses, the relaxant effect of sildenafil in PA rings from CH rats was less important than that observed in normoxic ones. This latter finding did not depend on the experimental conditions. A similar effect was observed when tissues from normoxic animals were investigated under identical experimental conditions to those of CH animals (i.e., resting load 20 mN, PE 3 µM). The reduced amplitude of sildenafil's effect in tissues from CH rats may be ascribed to an increased activity and expression of PDE5 related to the effect of CH itself (23, 25).

In conclusion, the present work shows that sildenafil is a potent pulmonary vascular relaxant in PA obtained from CH rats. Its relaxant effect appears mainly related to action on calcium signaling via the IP3-dependent calcium release pathway and calcium reuptake mechanisms. The present work provides new information for the cellular basis of the mechanism of action of sildenafil and justifies clinical trials for better management of human PAHT.


    GRANTS
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 
This work was supported by Conseil Régional d'Aquitaine Grant 200220301301A and Ministère de l'Environnement ADEME-PRIMEQUAL 2 Grant 0262019.


    ACKNOWLEDGMENTS
 
E. Rousseau is a National Scholar and a member of the Health Respiratory Network of the Fond de la Recherche en Santé du Québec.


    FOOTNOTES
 

Address for reprint requests and other correspondence: J.-P. Savineau, Laboratoire de Physiologie Cellulaire Respiratoire, INSERM (E 356 and IFR 4), Université Bordeaux 2, 146 rue Léo Saignat, 33076 Bordeaux, France (E-mail: jean-pierre.savineau{at}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.


    REFERENCES
 TOP
 ABSTRACT
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 GRANTS
 REFERENCES
 

  1. Ballard SA, Gingell CJ, Tang K, Turner LA, Price ME, and Naylor AM. Effects of sildenafil on the relaxation of human corpus cavernosum tissue in vitro and on the activities of cyclic nucleotide phosphodiesterase isosymes. J Urol 159: 2164–2171, 1998.[ISI][Medline]
  2. Barberà JA, Peinado VI, and Santos S. Pulmonary hypertension in chronic obstructive pulmonary disease. Eur Respir J 21: 892–905, 2003.[Abstract/Free Full Text]
  3. Barnes PJ and Liu SF. Regulation of pulmonary vascular tone. Pharmacol Rev 47: 87–131, 1995.[ISI][Medline]
  4. 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.[Abstract/Free Full Text]
  5. Bonnet S, Belus A, Hyvelin JM, Roux E, Marthan R, and Savineau JP. Effect of chronic hypoxia on agonist-induced tone and calcium signaling in rat pulmonary artery. Am J Physiol Lung Cell Mol Physiol 281: L193–L201, 2001.[Abstract/Free Full Text]
  6. Bonnet S, Dubuis E, Vandier C, Marthan R, and Savineau JP,. Reversal of chronic hypoxia-induced alterations in pulmonary artery smooth muscle electromechanical coupling upon air breathing. Cardiovasc Res 53: 1019–1028, 2002.[CrossRef][ISI][Medline]
  7. Bonnet S, Hyvelin JM, Bonnet P, Marthan R, and Savineau JP. Chronic hypoxia-induced spontaneous and rhythmical contractions in the rat main pulmonary artery. Am J Physiol Lung Cell Mol Physiol 281: L183–L192, 2001.[Abstract/Free Full Text]
  8. Boolell M, Gepi-Attee S, Gingell JC, and Allen MJ. Sildenafil, a novel effective oral therapy for male erectile dysfunction. Br J Urol 78: 257–261, 1996.[ISI][Medline]
  9. Carvajal JA, Germain AM, Huidobro-Toro JP, and Weiner CP. Molecular mechanism of cGMP-mediated smooth muscle relaxation. J Cell Physiol 184: 409–420, 2000.[CrossRef][ISI][Medline]
  10. Corbin JD, Blount MA, Weeks JL, Beasley A, Kuhn KP, Ho YSJ, Daidi LF, Hurkey JH, Kotera J, and Francis SH. [3H] Sildenafil binding to phosphodiesterase-5 is specific, kinetically heterogeneous, and stimulated by cGMP. Mol Pharmacol 63: 1364–1372, 2003.[Abstract/Free Full Text]
  11. Cornvell TL, Pryzwansky KB, Wyatt TA, and Lincoln TM. Regulation of sarcoplasmic reticulum protein phosphorylation by localised cyclic GMP-dependent protein kinase in vascular smooth muscle cells. Mol Pharmacol 40: 923–931, 1991.[Abstract]
  12. Dawson CA. Role of pulmonary vasomotion in physiology of the lung. Physiol Rev 64: 544–616, 1984.[Free Full Text]
  13. Fabiato A. Computer programs for calculating total from specified free or free from specified total ionic concentrations in aqueous solutions containing multiple metals and ligands. Methods Enzymol 157: 378–417, 1988.[ISI][Medline]
  14. 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]
  15. 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]
  16. Guibert C, Marthan R, and Savineau JP. Angiotensin II-induced Ca(2+)-oscillations in vascular myocytes from the rat pulmonary artery. Am J Physiol Lung Cell Mol Physiol 270: L637–L642, 1996.[Abstract/Free Full Text]
  17. Hanson KA, Burns F, Rybalkin SD, Miller JW, Beavo J, and Clarke WR. Developmental changes in lung cGMP phosphodiesterase-5 activity, protein, and message. Am J Respir Crit Care Med 158: 279–288, 1998.[ISI][Medline]
  18. Hanson KA, Ziegler JW, Rybalkin SD, Miller JW, Abman SH, and Clarke WR. Chronic pulmonary hypertension increases fetal lung cGMP phosphodiesterase activity. Am J Physiol Lung Cell Mol Physiol 275: L931–L941, 1998.[Abstract/Free Full Text]
  19. Ichinose F, Erana-Garcia J, Hromi J, Raveh Y, Jones R, Krim L, Clark MW, Winkler JD, Bloch KD, and Zapol WM. Nebulized sildenafil is a selective pulmonary vasodilator in lambs with acute pulmonary hypertension. Crit Care Med 29: 1000–1005, 2001.[ISI][Medline]
  20. Komalavilas P and Lincoln TM. Phosphorylation of the inositol 1,4,5-trisphosphate receptor by cyclic GMP-dependent protein kinase. J Biol Chem 269: 8701–8707, 1994.[Abstract/Free Full Text]
  21. Komas N, Lugnier C, and Stoclet JC. Endothelium-dependent and independent relaxation of the rat aorta by cyclic nucleotide phosphodiesterase inhibitors. Br J Pharmacol 104: 495–503, 1991.[Abstract]
  22. Lincoln TM and Cornvell TL. Intracellular cyclic GMP receptor proteins. FASEB J 7: 328–338, 1993.[Abstract/Free Full Text]
  23. Maclean MR, Johnston ED, Mcculloch KM, Pooley L, Houslay MD, and Sweeney G. Phosphodiesterase isoforms in the pulmonary arterial circulation of the rat: changes in pulmonary hypertension. J Pharmacol Exp Ther 283: 619–624, 1997.[Abstract/Free Full Text]
  24. Marthan R, Castaing Y, Manier G, and Guenard H. Gas exchange alterations in patients with chronic obstructive lung disease. Chest 87: 470–475, 1985.[Abstract]
  25. Michelakis E, Tymchak W, Lien D, Webster L, Hashimoto K, and Archer S. Oral sildenafil is an effective and specific pulmonary vasodilator in patients with pulmonary arterial hypertension. Comparison with inhaled nitric oxide. Circulation 105: 2398–2403, 2002.[Abstract/Free Full Text]
  26. Michelakis E, Tymchak W, Noga M, Webster L, Wu XC, Lien D, Wang SH, Modry D, and Archer S. Long-term treatment with oral sildenafil is safe and improves functional capacity and hemodynamics in patients with pulmonary arterial hypertension. Circulation 108: 2066–2069, 2003.[Abstract/Free Full Text]
  27. Murray F, MacLean MR, and Pyne NJ. Increased expression on the cGMP-inhibited cAMP-specific (PDE3) and cGMP binding cGMP-specific (PDE5) phosphodiesterases in models of pulmonary hypertension. Br J Pharmacol 137: 1187–1194, 2002.[Abstract/Free Full Text]
  28. Nagaoka T, Morio Y, Casanova N, Bauer N, Gebb S, McMurtry I, and Oka M. Rho/Rho-kinase signaling mediates increased basal pulmonary vascular tone in chronically hypoxic rats. Am J Physiol Lung Cell Mol Physiol. In press.
  29. Pauvert O, Lugnier C, Keravis T, Marthan R, Rousseau E, and Savineau JP. Effect of sildenafil on cyclic nucleotide phosphodiesterase activity, vascular tone and calcium signaling in rat pulmonary artery. Br J Pharmacol 139: 513–522, 2003.[Abstract/Free Full Text]
  30. Pauvert O, Marthan R, and Savineau JP. NO-induced modulation of calcium-oscillations in pulmonary vascular smooth muscle. Cell Calcium 27: 329–338, 2000.[CrossRef][ISI][Medline]
  31. Pauvert O, Savail D, Rousseau E, Lugnier C, Marthan R, and Savineau JP. Characterization of cyclic nucleotide phosphodiesterase isoforms in the media layer of the main pulmonary artery. Biochem Pharmacol 63: 1763–1772, 2002.[CrossRef][ISI][Medline]
  32. Pierson DJ. Pathophysiology and clinical effects of chronic hypoxia. Respir Care 45: 39–51, 2000.[Medline]
  33. Platoshyn O, Yu Y, Golovina VA, McDaniel SS, Krick S, Li L, Wang JY, Rubin LJ, and Yuan JX. Chronic hypoxia decreases KV channel expression and function in pulmonary artery myocytes. Am J Physiol Lung Cell Mol Physiol 280: L801–L812, 2001.[Abstract/Free Full Text]
  34. Polson JB and Strada SJ. Cyclic nucleotide phosphodiesterases and vascular smooth muscle. Annu Rev Pharmacol Toxicol 36: 403–427, 1996.[CrossRef][ISI][Medline]
  35. Priest RM, Hucks D, and Ward JPT. Potentiation of cyclic AMP-mediated vasorelaxation by phenylephrine in pulmonary arteries of the rat. Br J Pharmacol 127: 291–299, 1997.
  36. Rabe KF, Tenor H, Dent G, Schudt C, Nakashima M, and Magnussen H. Identification of PDE isoenzymes in human pulmonary artery and effect of selective PDE inhibitors. Am J Physiol Lung Cell Mol Physiol 266: L536–L543, 1994.[Abstract/Free Full Text]
  37. 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.[Abstract/Free Full Text]
  38. Savineau JP, Gonzalez de la Fuente P, and Marthan R. Effect of vascular smooth muscle relaxants on the protein kinase C-mediated contraction in the rat pulmonary artery. Eur J Pharmacol 249: 191–198, 1993.[CrossRef][ISI][Medline]
  39. Savineau JP and Marthan R. Cytosolic calcium oscillations in smooth muscle cells. News Physiol Sci 15: 50–55, 2000.[ISI][Medline]
  40. Sauzeau V, Rolli-Dirkinderen M, Lehoux S, Loirand G, and Pacaud P. Sildenafil prevents change in RhoA expression induced by chronic hypoxia in rat pulmonary artery. Circ Res 93: 630–637, 2003.[Abstract/Free Full Text]
  41. Schoeffter P, Lugnier C, Demesy-Waeldele F, and Stoclet JC. Role of cyclic AMP- and cyclic GMP-phosphodiesterases in the control of cyclic nucleotide levels and smooth muscle tone in rat isolated aorta. A study with selective inhibitors. Biochem Pharmacol 36: 3965–72, 1987.[CrossRef][ISI][Medline]
  42. Sebkhi A, Strange JW, Phillips SC, Wharton J, and Wilkins MP. Phosphodiesterase type 5 as a target for the treatment of hypoxia-induced pulmonary hypertension. Circulation 107: 3230–3235, 2003.[Abstract/Free Full Text]
  43. Shimoda LA, Sham JS, Shimoda TH, and Sylvester JT. L-type Ca2+ channels, resting [Ca2+], and ET-1-induced responses in chronically hypoxic pulmonary myocytes. Am J Physiol Lung Cell Mol Physiol 279: L884–L894, 2000.[Abstract/Free Full Text]
  44. Stoclet JC, Keravis TH, Komas N, and Lugnier C. Cyclic nucleotide phosphodiesterases as therapeutic targets in cardiovascular diseases. Exp Opin Invest Drugs 4: 1081–100, 1995.
  45. Tamaoki J, Tagaya E, Nishimura K, Isono K, and Nagai A. Role of Na+-K+ ATPase in cyclic GMP-mediated relaxation of canine pulmonary artery smooth muscle cells. Br J Pharmacol 122: 112–116, 1997.[Abstract]
  46. Wagner RS, Smith CJ, Taylor A, and Roades RA. Phosphodiesterase inhibition improves agonist-induced relaxation of hypertensive pulmonary arteries. J Pharmacol Exp Ther 282: 1650–1657, 1997.[Abstract/Free Full Text]
  47. Wilkens H, Guth A, König J, Forestier N, Cremers B, Hennen B, Böhm M, and Sybrercht GW. Effect of inhaled iloprost plus oral sildenafil in patients with primary pulmonary hypertension. Circulation 104: 1218–1222, 2001.[Abstract/Free Full Text]
  48. Zhao L, Mason NA, Morrell NW, Kojonazarov B, Sadykov A, Maripov A, Mirrakhimov MM, Aldashev A, and Wilkins MR. Sildenafil inhibits hypoxia-induced pulmonary hypertension. Circulation 24: 424–428, 2001.




This Article
Abstract
Full Text (PDF)
All Versions of this Article:
287/3/L577    most recent
00449.2003v1
Alert me when this article is cited
Alert me if a correction is posted
Citation Map
Services
Email this article to a friend
Similar articles in this journal
Similar articles in ISI Web of Science
Similar articles in PubMed
Alert me to new issues of the journal
Download to citation manager
Search for citing articles in:
ISI Web of Science (3)
Google Scholar
Articles by Pauvert, O.
Articles by Savineau, J.-P.
Articles citing this Article
PubMed
PubMed Citation
Articles by Pauvert, O.
Articles by Savineau, J.-P.


HOME HELP FEEDBACK SUBSCRIPTIONS ARCHIVE SEARCH TABLE OF CONTENTS
Visit Other APS Journals Online
Copyright © 2004 by the American Physiological Society.