Arachidonic acid metabolism in cultured aortic endothelial cells. Effect of cAMP and 3-isobutyl-1-methylxanthine.

To investigate the hypothesis that cyclic AMP (cAMP) regulates arachidonic acid metabolism in vascular tissue, we have studied the effects of forskolin (FSK), an activator of adenylate cyclase, and 3-isobutyl-1-methylxanthine (IBMX), a phosphodiesterase inhibitor, on hormone-stimulated prostacyclin (PGI2) synthesis in porcine aortic endothelial cells grown in culture. In these experiments, bradykinin (1 microgram/ml) and A23187 (0.2 microM) potently stimulated PGI2 biosynthesis (9- and 10-fold respectively). However, prostaglandin synthesis in response to either of these agents was not affected by FSK even though FSK elevated intracellular levels of cAMP 10-fold. IBMX failed to elevate basal cAMP levels when incubated with unstimulated cells. Stimulation of IBMX-treated (0.1 but not 1.0 or 4.0 mM) cells with bradykinin, however, did result in increased cAMP levels, presumably due to PGI2 formation and subsequent activation of adenylate cyclase. In addition to phosphodiesterase inhibition, IBMX inhibited PGI2 formation (72% at 1 mM) in a dose-dependent manner so that, at higher doses of IBMX, cAMP levels returned to baseline. Thus, prostacyclin synthesis inhibition by IBMX could not be attributed to elevated cAMP. In other experiments, IBMX (1 mM) was found to directly inhibit arachidonic acid release (32%) and arachidonic acid metabolism (65%) in endothelial cells and to inhibit arachidonic acid conversion to PGE2 by sheep seminal vesicle microsomes (65%). These data suggest that IBMX directly inhibits both phospholipase and cyclooxygenase activities. These experiments do not support the contention that cAMP regulates these enzymes in cultured aortic endothelial cells.