THE ROLE OF PROTEIN KINASE-C-ALPHA IN THE ACTIVATION OF PARTICULATE GUANYLATE-CYCLASE BY 1-ALPHA,25-DIHYDROXYVITAMIN D-3 IN CACO-2 CELLS

Recent studies have implicated protein kinase-C (PKC) in the regulatio n of guanylate cyclase in several cell types. In view of prior experim ents by our laboratory which have demonstrated that l alpha a,25-dihyd roxyvitamin D-3 [l alpha,25-(OH)(2)D-3] can activate PKC in CaCo-2 cel ls, it was of interest to determine whether this secosteroid influence d particulate guanylate cyclase and, if so, to determine which isoform s of PKC were involved. To address these issues, CaCo-2 cells were tre ated with l alpha,25-(OH)(2)D-3 or other agents (see below), and crude membranes prepared from these cells were assayed for guanylate cyclas e activity. In several experiments, agents were added directly to isol ated membranes, and guanylate cyclase activity was then assayed. These studies demonstrated that 1) the addition of l alpha,25-(OH)(2)D-3 or 12-0-tetradecanoyl phorbol 13-acetate (TPA), a known activator of PKC , to intact CaCo-a cells stimulated particulate guanylate cyclase acti vity in a time-and concentration-dependent manner; 2) these agents ind uced the translocation of PKC alpha, but not PKC zeta from the cytosol ic to the membrane fraction of these cells; 3) preincubation of cells with staurosporine (50 nM), a PKC inhibitor, or U73122 (10 mu M), an i nhibitor of phospholipase-C-dependent processes, significantly reduced (P < 0.05) the stimulatory effect of l alpha,25-(OH)(2)D-3 (3 nM) on guanylate cyclase; 4) preincubation of isolated membranes with TPA, ca lcium, and Mg2+-ATP increased guanylate cyclase activity, an affect th at was augmented by purified rat brain PKC and inhibited by the PKC in hibitor peptide, PKC-(19-36); and 5) selective down-regulation of PKC alpha by treatment of cells with TPA (200 nM) for 24 h concomitantly a bolished the activation of guanylate cyclase by l alpha,25-(OH)(2)D-3. Taken together, these studies demonstrate that l alpha,25-(OH)(2)D-3 activates particulate guanylate cyclase at least in part via a PKC alp ha-dependent mechanism.