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Pauvert, Olivier, Sébastien Bonnet, Eric Rousseau, Roger Marthan, and Jean-Pierre Savineau. Sildenafil alters calcium signaling and vascular tone in pulmonary arteries from chronically hypoxic rats. Am J Physiol Lung Cell Mol Physiol 287: L577-L583, 2004. First published May 21, 2004 10.1152/ajplung.00449.2003.-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 ([Ca 2ϩ ]i) was measured in PASMCs loaded with the calcium fluorophore indo 1. Unlike in control rats, sildenafil (10 -100 nM) decreased the resting [Ca 2ϩ ]i value in PASMCs obtained from CH rats. In PASMCs from both control and CH rats, sildenafil concentration dependently inhibited the [Ca 2ϩ ]i response induced by G-coupled membrane receptor agonists such as angiotensin II and phenylephrine but had no effect on the amplitude of the [Ca 2ϩ ]i response induced by caffeine. Sildenafil (0.1 nM-1 M) 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 [Ca 2ϩ ]i and basal tone. Consequently, pharmacological inhibition of sildenafil-sensitive PDE5 downregulates the Ca 2ϩ signaling pathway involved in this model of pulmonary hypertension. pulmonary hypertension; vascular smooth muscle; phosphodiesterase 5; pulmonary vasodilator INTRACELLULAR CYCLIC NUCLEOTIDE [cAMP and guanosine 3Ј,5Ј-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 inhibitor...

1 Sildenafil (viagra) is a potent PDE5 inhibitor and thus a relaxant drug in corpus carvernosum smooth muscle. In the present work, we evidenced the presence of PDE5 isozyme and investigated the effect of sildenafil on the specific cyclic nucleotide phosphodiesterase (PDE) activity, smooth muscle tone and calcium signaling in the rat main pulmonary artery (MPA). 2 The PDE activity was measured in cytosolic and microsomal fractions. Total cAMP and cGMP-PDE activities were mainly present in the cytosolic fraction. Sildenafil (0.1 mM) reduced by 72% cGMP-PDE activity, whereas zaprinast (10 mM), a relatively selective PDE5 inhibitor, reduced this activity by 63%. Sildenafil (0.1 mM) also inhibited significantly (22%) the cAMP-PDE activity. 3 Western blot analysis revealed the expression of PDE5 mainly in the cytosolic fraction of MPA. Sildenafil concentration-dependently inhibited (IC 50 ¼ 3.4 nM) the activity of MPA PDE5 partially purified by HPLC. 4 Sildenafil (0.1 nM-50 mM) concentration-dependently relaxed MPA rings precontracted with phenylephrine (0.5 mM). The potency of sildenafil (IC 50 ¼ 11 nM) was similar to that of a nitric oxide donor, sodium nitroprusside, but higher than that of zaprinast (IC 50 ¼ 600 nM). The vasorelaxant effect of sildenafil was not altered by endothelium removal or in the presence of KT 5823 (1 mM) and H89 (1 mM), potent inhibitors of PKG and PKA, respectively. 5 In isolated MPA myocytes, which had been loaded with the calcium fluorophore indo-1, sildenafil (10 -100 nM) antagonized ATP-and endothelin-1-induced calcium oscillations but had no effect on the transient caffeine-induced [Ca 2+ ] i response. 6 This study demonstrates the presence of a functional and highly sildenafil-sensitive PDE5 isozyme in rat MPA. Inhibition of this isozyme mainly accounts for the potent pulmonary vasodilator action of sildenafil, which involves alteration in the inositol triphosphate-mediated calcium signaling pathway.

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