Stimulation of forskolin of intact S49 lymphoma cells involves the nucleotide regulatory protein of adenylate cyclase.

We have used wild type 549 lymphoma cells and S49 variants that have lesions in their ability to generate CAMP to explore the mechanism of activation of adenylate cyclase by the diterpene forskolin. In intact wild type cells, forskolin rapidly and reversibly stimulated CAMP accumulation several hundred-fold over basal levels. Simultaneous addition of forskolin and the padrenergic agonist isoproterenol gave greater than additive (Le. synergistic) responses. Forskolin lowered the Kact for isoproterenol-stimulated CAMP accumulation 2to %fold and increased maximal response to isoproterenol 8to 10-fold; isoproterenol decreased the K,,, for forskolin about 10-fold but had a much smaller effect (t2-fold) on maximal response. In competitive binding studies between (-)isoproterenol and [1261]iodocyanopindolol, forskolin more than doubled the fi-action of /%adrenergic receptors in a high affinity (-5-10 m) state, as assessed by computer modeling. Moreover, forskolin appeared to reverse desensitization of S49 cells to isoproterenol. UNC S49 cells, which have a defect in the ability of receptors to interact with the guanine nucleotide binding component (N) of adenylate cyclase, responded to forskolin alone as did wild type cells. By contrast, cycS49 cells, which appear to lack functional N, and H21a cells, which are defective in the coupling of N to the catalytic component of adenylate cyclase, responded to forskolin alone less than 15% as well as did wild type cells. HZla, but not UNC or cyc-, cells responded to isoproterenol when added together with forskolin. Thus, forskolin could partially restore response to the nonfunctional &adrenergic receptors of H21a cells. These results indicate that full expression of the twin actions of forskolin on intact 549 cells, stimulation of adenylate cyclase and potentiation of hormone-mediated response, require an intact N protein. The data further suggest that treatment of S49 cells with forskolin alters the interaction of N with both hormone receptors and the catalytic component of adenylate cyclase.