Isolation of forskolin-resistant adrenal cells defective in the adenylate cyclase system.

Forskolin is an activator of adenylate cyclase in many cell types. In order to determine the mechanism of forskolin's action and to determine if this mechanism is shared by hormones and other agonists of the adenylate cyclase system, we isolated and partially characterized several spontaneous, forskolin-resistant mutants from the Y1 mouse adrenocortical tumor cell line. Forskolin increased adenylate cyclase activity in Y1 cell homogenates approximately 30-fold. By virtue of its effect on cAMP accumulation, forskolin (10 microM) also inhibited the growth of Y1 cells in monolayer culture. Using forskolin as a selective agent, spontaneous mutants capable of growth in the presence of 10 microM forskolin were isolated from the Y1 cell line at a frequency of 1-2/10(6) cells. In these mutants, resistance was stable, resulting from a defect in cAMP accumulation rather than cAMP action, and was associated with a reduced ability of forskolin to stimulate adenylate cyclase activity in cell homogenates. Whereas corticotropin stimulated adenylate cyclase activity over 35-fold in cell homogenates from the Y1 parent, ACTH had only marginal effects on the enzyme's activity in the mutant clones. Fluoride-stimulated adenylate cyclase activity seemed unimpaired. These results suggest that the resistance to forskolin resulted from a mutation in the adenylate cyclase system, not in the catalytic subunit, but at a locus related to the ACTH.