Distinct mechanisms of forskolin-stimulated cyclic AMP accumulation and forskolin-potentiated hormone responses in C6-2B cells

K Barovsky, C Pedone and G Brooker

Forskolin activates a variety of adenylate cyclase systems and acts synergistically with receptor-mediated agonists which stimulate cyclic AMP production. The mechanism(s) and site(s) of forskolin action remain unclear. In C6-2B rat astrocytoma cells, forskolin stimulated greater than a 100-fold increase in cellular cyclic AMP content with a half- maximally effective concentration (EC50) of greater than 50 microM. Incubation of C6-2B cells with forskolin plus (-)-isoproterenol resulted in an increase in (-)-isoproterenol efficacy and potency. The EC50 for the forskolin-induced increase in (-)-isoproterenol potency was 22 nM, greater than 3 orders of magnitude lower than the EC50 for direct forskolin-stimulated cyclic AMP accumulation. Forskolin had no effect on beta-receptor affinity for (-)-isoproterenol as measured by competition for (-)-[125I]iodopindolol binding sites. Forskolin also augmented the responses to prostaglandin E1 and cholera toxin. Inhibition of protein synthesis with cycloheximide markedly reduced forskolin-stimulated cyclic AMP accumulation with little or no effect on the responses to (-)-isoproterenol, prostaglandin E1, or cholera toxin. The ability of forskolin to act synergistically with these agents was unaffected by cycloheximide treatment. These observations are compatible with a two-site model of forskolin action in C6-2B cells: a low-affinity site which mediates the direct action of forskolin to increase cellular cyclic AMP accumulation and a high- affinity site which mediates the potentiative action of forskolin. The low-affinity forskolin site appears to reside on a protein which is closely associated with the catalytic adenylate cyclase moiety and has a relatively shorter half-life than other components of the cyclase system. The high-affinity site resides on a more stable component of the adenylate cyclase system. The synergistic action of forskolin may involve an enhancement of the interaction between the guanine nucleotide-binding regulatory component and the catalytic component of the adenylate cyclase complex.

Volume 25, Issue 2, pp. 256-260, 03/01/1984
Copyright © 1984 by American Society for Pharmacology and Experimental Therapeutics

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