| Abstrakt: |
Calcium can influence the cGMP/guanylate cyclase system in many tissues, including rat colon. The mechanisms involved in this phenomenon, however, are unclear. To further elucidate the mechanisms involved in the Ca2+-induced activation of rat colonic particulate guanylate cyclase, isolated colonocytes were incubated with Ca2+ or other agents, and crude membranes prepared and analyzed for particulate guanylate cyclase activity. Alternatively, the test agents were directly added to the guanylate cyclase reaction mixture containing isolated membranes. The results of these studies demonstrated: (i) extracellular Ca2+ (1 and 2 mM) increased basal particulate guanylate cyclase activity; (ii) increases in intracellular Ca2+ induced by 10 µM thapsigargin activated this enzyme; (iii) preincubation of the cells with 50 nM staurosporine, a broad-spectrum inhibitor of protein kinases, including protein kinase C (PKC), or 5 µM U73122, a specific inhibitor of phosphoinositide-phospholipase C-dependent processes, blocked the Ca2+-induced increase in particulate guanylate cyclase activity; (iv) incubation of cells with 1 µM 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 with 1 µM TPA and 2 mM extracellular Ca2+; (vi) incubation of membranes with 250 nM TPA, in the presence of 0.2 mM Ca2+, 6 mM Mg2+, and 1 mM 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.Copyright 1994, 1999 Academic Press, Inc. |
