MODULATION OF ADENYLYLCYCLASE BY PROTEIN-KINASE-C IN HUMAN NEUROTUMORSK-N-MC CELLS - EVIDENCE THAT THE ALPHA-ISOZYME MEDIATES BOTH POTENTIATION AND DESENSITIZATION

Exposure of human SK-N-MC neurotumor cells to 4 beta-phorbol 12-myrist ate 13-acetate (PMA) increased isoproterenol stimulation of cyclic AMP levels by severalfold. This potentiation was blocked by inhibitors of protein kinase C (PKC) and did not occur in cells in which PKC had be en down-regulated. PMA treatment also enhanced the stimulation by dopa mine, cholera toxin, and forskolin. Thus, the effect of PMA on the ade nylylcyclase system was postreceptor and involved either the guanine n ucleotide binding regulatory (G) proteins or the cyclase itself. As PM A treatment did not impair the inhibition of isoproterenol stimulation by neuropeptide Y, an involvement of the inhibitory G protein Gi was unlikely. Cholate extracts of membranes from control and PMA-treated c ells were equally effective in the reconstitution of adenylylcyclase a ctivity in S49 cyc(-) membranes, which lack the stimulatory G protein subunit G(s alpha); thus, G(s) did not appear to be the target of PMA action. Membranes from PMA-treated cells exhibited increased adenylylc yclase activity to all stimulators including Mn2+ and Mn2+ plus forsko lin. In addition, activity was increased when control membranes were i ncubated with ATP and purified PKC from rat brain. This is consistent with a direct effect of PKC on the adenylylcyclase catalyst in SK-N-MC cells. PMA treatment also resulted in a shift to less sensitivity in the K-act for isoproterenol but not for dopamine or CGP-12177 (a beta( 3)-adrenergic agonist) stimulation. Thus, the beta(1) but not the D1 o r beta(3) receptors were being desensitized by PKC activation. Analysi s of SK-N-MC cells by western blotting with antibodies against differe nt PKC isozymes revealed that both the alpha and zeta isozymes were pr esent in these cells. Whereas PKC-alpha was activated and translocated from cytosol to membrane by phorbol esters, the 5 isozyme was not. Th us, PKC-alpha, which has been implicated in desensitization in other c ell lines, also appears to potentiate adenylylcyclase activity.