Effect of forskolin, isoproterenol and IBMX on angiotensin converting enzyme and cyclic AMP production by cultured bovine endothelial cells

Abstract

The production of angiotensin converting enzyme (ACE) is known to be increased by glucocorticoids, thyroid hormones and converting enzyme inhibitors. We have recently reported that active cAMP analogues also stimulate production of the enzyme. The effect of stimulation of adenylate cyclase in cultured endothelial cells or of phosphodiesterase inhibition on ACE production was therefore evaluated. The phosphodiesterase inhibitor, isobutylmethylxanthine (IBMX) (10 −4 M), produced 10.5 ± 1.3 and 1.3 ± 0.1 ( P < 0.01 and P > 0.1) fold increases in extracellular and cellular cAMP levels and a 1.55 ± 0.10 ( P < 0.0001) fold increase in ACE accumulation. The adenylate cyclase stimulator, forskolin (0.01–10 μM), acutely stimulated cellular cAMP accumulation in a dose-dependent manner, reaching a 2.8 ± 0.1-fold increase at 10 μM. After 48 h exposure to 10 μM forskolin, significant increases in cellular (1.90 ± 0.38-fold increase, P < 0.0001) and extracellular cAMP (2.35 ± 0.26-fold increase, P < 0.0001) were also observed but ACE accumulation was unchanged (108 ± 10% of control, P > 0.5). The β-adrenoceptor agonist, isoproterenol (1–1000 nM), acutely stimulated cellular cAMP accumulation, with a threshold effect at 10 nM, an ED 50 of approximately 30 nM, and a plateau effect of 2.0 ± 0.13-fold increase by 100 nM. After 48 h exposure to isoproterenol (1 μM), extracellular cAMP levels were increased significantly (1.68 ± 0.33-fold increase, P < 0.01) but ACE production was slightly inhibited (83 ± 7% of control, P < 0.05). Thus chronic stimulation of adenylate cyclase by either isoproterenol or forskolin did not stimulate ACE production in these cells. These results therefore suggest that IBMX stimulates ACE production in these cells by a mechanism independent of cyclic AMP. Taken together with our previous findings, these data suggest that although cAMP analogues and IBMX stimulate ACE production in cultured endothelial cells, we are unable to demonstrate a role for endogenously generated cAMP in regulation of the enzyme.