The role of protein kinase-Cα in the activation of particulate guanylate cyclase by 1α,25-dihydroxyvitamin D₃ in CaCo-2 cells

Recent studies have implicated protein kinase-C (PKC) in the regulation of guanylate cyclase in several cell types. In view of prior experiments by our laboratory which have demonstrated that 1α,25-dihydroxyvitamin D₃ [1α,25- (OH)₂D₃] can activate PKC in CaCo-2 cells, it was of interest to determine whether this secosteroid influenced particulate guanylate cyclase and, if so, to determine which isoforms of PKC were involved. To address these issues, CaCo-2 cells were treated with 1α,25-(OH)₂D₃ or other agents (see below), and crude membranes prepared from these cells were assayed for guanylate cyclase activity. In several experiments, agents were added directly to isolated membranes, and guanylate cyclase activity was then assayed. These studies demonstrated that 1) the addition of 1α,25-(OH)₂D₃ or 12-O- tetradecanoyl phorbol 13-acetate (TPA), a known activator of PKC, to intact CaCo-2 cells stimulated particulate guanylate cyclase activity in a time- and concentration-dependent manner; 2) these agents induced the translocation of PKCα, but not PKCζ, from the cytosolic to the membrane fraction of these cells; 3) preincubation of cells with staurosporine (50 nM), a PKC inhibitor, or U73122 (10 μM), an inhibitor of phospholipase-C-dependent processes, significantly reduced (P < 0.05) the stimulatory effect of 1α,25-(OH)₂D₃ (3 nM) on guanylate cyclase; 4) preincubation of isolated membranes with TPA, calcium, and Mg²⁺-ATP increased guanylate cyclase activity, an affect that was augmented by purified rat brain PKC and inhibited by the PKC inhibitor peptide, PKC-(19-36); and 5) selective down-regulation of PKCα by treatment of cells with TPA (200 nM) for 24 h concomitantly abolished the activation of guanylate clyclase by 1α,25-(OH)₂D₃. Taken together, these studies demonstrate that 1α,25-(OH)₂D₃ activates particulate guanylate cyclase at least in part via a PKCα-dependent mechanism.