Influence of forskolin on the parathyroid hormone dependent adenylate cyclase system of canine kidney: evidence for noncatalytic effects of forskolin.

The present studies examined the effects of forskolin on the PTH responsive adenylate cyclase system of canine basolateral renal cortical membranes. This agent is a potent activator of adenylate cyclase in a wide variety of intact cells and broken cell preparations. Initially forskolin was believed to activate adenylate cyclase by direct stimulation of the catalytic unit of the enzyme. Several observations, however, have suggested that there may be additional noncatalytic sites of action. In the present studies, forskolin was found to increase PTH-stimulated adenylate cyclase activity by 4-fold associated with a 2-fold decrease in the Kact for PTH (dose of PTH required for half-maximal enzyme stimulation). Studies of the interaction of forskolin with magnesium revealed that forskolin resulted in a dose dependent increase in the affinity of adenylate cyclase for magnesium. A short lag was observed in the onset of enzyme activation by forskolin. The lag was decreased as Mg++ concentration was increased. The forskolin-induced increase in the affinity for Mg++ was similar to that produced by other activators of adenylate cyclase such as NaF and GTP, which interact with the nucleotide regulatory protein. Magnesium also markedly affected the affinity of the enzyme system for forskolin. Kact for forskolin was reduced from 10 microM to 0.1 microM as Mg++ concentration was raised from 0.5 mM to 40 mM. Since previous studies have shown that the allosteric effects of Mg++ are at, or closely related to, the nucleotide regulatory protein, these findings suggest that forskolin may also affect this site. In summary, in basolateral renal cortical membrane of canine kidney the effects of forskolin to increase the affinity of adenylate cyclase for PTH and to alter the allosteric effects of Mg++ on enzyme activity are indicative of noncatalytic effects of forskolin. The interpretation of action of forskolin on adenylate cyclase activity should not be restricted to direct stimulation of the catalytic unit.