Endocrinology, Vol 135, 277-283, Copyright © 1994 by Endocrine Society

ARTICLES

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

S Khare, XY Tien, D Wilson, RK Wali, BM Bissonnette, B Scaglione-Sewell, MD Sitrin and TA Brasitus
Department of Medicine, University of Chicago, Illinois 60637.

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 1 alpha,25- dihydroxyvitamin D3 [1 alpha,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 1 alpha,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 1 alpha,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 PKC alpha, but not PKC zeta, from the cytosolic to the membrane fraction of these cells; 3) preincubation of cells with staurosporine (50 nM), a PKC inhibitor, or U73122 (10 microM), an inhibitor of phospholipase-C-dependent processes, significantly reduced (P < 0.05) the stimulatory effect of 1 alpha,25- (OH)2D3 (3 nM) on guanylate cyclase; 4) preincubation of isolated membranes with TPA, calcium, and Mg(2+)-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 PKC alpha by treatment of cells with TPA (200 nM) for 24 h concomitantly abolished the activation of guanylate cyclase by 1 alpha,25-(OH)2D3. Taken together, these studies demonstrate that 1 alpha,25-(OH)2D3 activates particulate guanylate cyclase at least in part via a PKC alpha-dependent mechanism.

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