Effect of sildenafil on cyclic nucleotide phosphodiesterase activity, vascular tone and calcium signaling in rat pulmonary artery

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 muM) reduced by 72% cGMP-PDE activity, whereas zaprinast (10 muM), a relatively selective PDE5 inhibitor, reduced this activity by 63%. Sildenafil (0.1 muM) 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 (IC50 = 3.4 nM) the activity of MPA PDE5 partially purified by HPLC. 4 Sildenafil (0.1 nM - 50 muM) concentration-dependently relaxed MPA rings precontracted with phenylephrine (0.5 muM). The potency of sildenafil (IC50 = 11 nM) was similar to that of a nitric oxide donor, sodium nitroprusside, but higher than that of zaprinast (IC50 = 600 nM). The vasorelaxant effect of sildenafil was not altered by endothelium removal or in the presence of KT 5823 (1 muM) and H89 (1 muM), 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 muM) antagonized ATP- and endothelin-1-induced calcium oscillations but had no effect on the transient caffeine-induced [Ca2+](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.