PROTEIN KINASE-C MEDIATES THE CALCIUM-INDUCED ACTIVATION OF RAT COLONIC PARTICULATE GUANYLATE-CYCLASE

Calcium can influence the cGMP/guanylate cyclase system in many tissue s, including rat colon. The mechanisms involved in this phenomenon, ho wever, are unclear. To further elucidate the mechanisms involved in th e Ca2+-induced activation of rat colonic particulate guanylate cyclase , isolated colonocytes were incubated with Ca2+ or other agents, and c rude membranes prepared and analyzed for particulate guanylate cyclase activity. Alternatively, the test agents were directly added to the g uanylate cyclase reaction mixture containing isolated membranes. The r esults of these studies demonstrated: (i) extracellular Ca2+ (1 and 2 mM) increased basal particulate guanylate cyclase activity; (ii) incre ases in intracellular Ca2+ induced by 10 mu M thapsigargin activated t his enzyme; (iii) preincubation of the cells with 50 nM staurosporine, a broad-spectrum inhibitor of protein kinases, including protein kina se C (PKC), or 5 mu M U73122, a specific inhibitor of phosphoinositide -phospholipase C-dependent processes, blocked the Ca2+-induced increas e in particulate guanylate cyclase activity; (iv) incubation of cells with 1 mu M 12-O-tetradecanoyl phorbol 13-acetate (TPA), an activator of PKC, stimulated guanylate cyclase; (v) no additivity in stimulation of this enzyme was observed when cells were concomitantly incubated w ith 1 mu M TPA and 2 mM extracellular Ca2+; (vi) incubation of membran es with 250 nM TPA, in the presence of 0.2 mM Ca2+, 6 mM Mg2+, and 1 m M ATP, activated guanylate cyclase; and (vii) incubation of membranes with purified rat brain PKC further augmented this stimulation. These results indicate that Ca2+ activates rat colonic particulate guanylate cyclase, at least in part, via a PKC-dependent mechanism. (C) 1994 Ac ademic Press, Inc.