Modulation of agonist‐induced calcium mobilisation in bovine aortic endothelial cells by phorbol myristate acetate and cyclic AMP but not cyclic GMP

Abstract

In bovine aortic endothelial cells (BAEC), thrombin (1 u ml−1), bradykinin (1–10 nm) and adenosine triphosphate (ATP) (0.3 μm–100 μm) each induced a biphasic elevation of cytosolic calcium ([Ca2+]i), consisting of an initial transient followed by a sustained plateau phase. Pretreatment of BAEC with 4β‐phorbol 12‐myristate 13‐acetate (PMA; 100 nm) reduced the magnitude of the initial transient elevation of [Ca2+]i, induced by thrombin (1 u ml−1), low concentrations of bradykinin (1 nm) or ATP (0.3 μm, 3 μm), but not by higher concentrations of the latter two agonists. Addition of PMA (100 nm) during the plateau phase of the increase in [Ca2+]i induced by thrombin (1 u ml−1), bradykinin (10 nm) or ATP (30 μm) resulted in a fall in [Ca2+]i. The inhibitory effects of PMA (100 nm) were inhibited by staurosporine (100 nm) but not mimicked by the inactive phorbol ester, 4α‐phorbol 12, 13‐didecanoate (4α‐PDD; 100 nm). Furthermore, staurosporine (100 nm) increased [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by thrombin or bradykinin. In contrast, staurosporine (100 nm) reduced [Ca2+]iWhen added during the plateau phase of the increase in [Ca2+]i induced by ATP (30 μm). Pretreatment with forskolin (10 μm) had no effect on the magnitude of the initial transient elevation of [Ca2+]i induced by thrombin (1 u ml−1), bradykinin (1 nm and 10 nm) or ATP (30 μm). In contrast, forskolin (10 μm) and isoprenaline (10 μm) each induced biphasic elevations of [Ca2+]i when added during the plateau phase of the increase in [Ca2+]i induced by the three agonists. Furthermore, in the presence of the inhibitor of calcium influx, nickel chloride (4 mm), these biphasic elevations were reduced to monophasic transient elevations. 8 Bromo cyclic GMP (30 μm), a membrane‐permeant analogue of guanosine 3′: 5′‐cyclic monophosphate (cyclic GMP), had no effect on the magnitude of the initial transient elevation of [Ca2+]i induced by thrombin (1 u ml−1), bradykinin (10 nm) or ATP (3 μm). Furthermore, 8 bromo cyclic GMP (30 μm) and sodium nitroprusside (1 μm), had no effect when added during the plateau phase of the increase in [Ca2+]i induced by the three agonists. NG‐nitro‐l‐arginine (50 μm), an inhibitor of nitric oxide synthase, had no effect on the magnitude of the initial transient elevation of [Ca2+]i induced by thrombin (1 u ml−1), bradykinin (1 nm) or ATP (3 μm), and had no effect on the plateau phase of the increase in [Ca2+]i induced by these agents. These findings suggest that while activation of protein kinase C inhibits and elevation of adenosine 3′:5′‐cyclic monophosphate (cyclic AMP) augments calcium mobilisation in bovine aortic endothelial cells, elevation of cyclic GMP appears to have no effect. 1991 British Pharmacological Society