Mechanisms underlying the vasorelaxant action of the pimarane ent-8(14),15-pimaradien-3β-ol in the isolated rat aorta

Pimarane-type diterpenes were described to exert antispasmodic and relaxant activities. Based on this observation we hypothesized that the diterpene ent-8(14),15-pimaradien-3β-ol (PA-3β-ol) induced vascular relaxation. With this purpose, the present work investigates the mechanisms involved in the vasorelaxant effect of the pimarane-type diterpene PA-3β-ol. Vascular reactivity experiments, using standard muscle bath procedures, were performed in isolated aortic rings from male Wistar rats. Cytosolic calcium concentration ([Ca2+]c) was measured by confocal microscopy using the fluorescent probe Fluo-3AM. PA-3β-ol (10, 50 and 100 µmol/l) inhibited phenylephrine and KCl-induced contraction in either endothelium-intact or denuded rat aortic rings. PA-3β-ol also reduced CaCl2-induced contraction in Ca2+-free solution containing KCl (30 mmol/l) or phenylephrine (0.1 µmol/l). PA-3β-ol (1–300 µmol/l) concentration dependently relaxed phenylephrine-pre-contracted rings with intact or denuded endothelium. The diterpene also relaxed KCl-pre-contracted rings with intact or denuded endothelium. Moreover, Ca2+ mobilization study showed that PA-3β-ol (100 µmol/l) and verapamil (1 µmol/l) inhibited the increase in Ca2+-concentration in smooth muscle and endothelial cells induced by phenylephrine (10 µmol/l) or KCl (60 mmol/l). Pre-incubation of intact or denuded aortic rings with NG-nitro-l-arginine methyl ester (L-NAME, 100 µmol/l) and 1H-[1,2,4]Oxadiazolo[4,3-a]quinoxalin-1-one (ODQ, 1 µmol/l) produced a rightward displacement of the PA-3β-ol concentration–response curves. On the other hand, 7-nitroindazole (100 µmol/l), 1400 W (1 µmol/l), indomethacin (10 µmol/l) and tetraethylammonium (1 mmol/l) did not affect PA-3β-ol-induced relaxation. Collectively, our results provide evidence that the effects elicited by PA-3β-ol involve extracellular Ca2+ influx blockade. Its effects are also partly mediated by the activation of NO-cGMP pathway.