Effects of trypsin on secretion stimulated by micromolar Ca2+ and phorbol ester in digitonin-permeabilized adrenal chromaffin cells

Publisher Website
Google Scholar
Abstract
Catecholamine secretion from digitonin-treated chromaffin cells is stimulated directly by micromolar Ca2+ in the medium. The permeabilized cells are leaky to proteins. In this study trypsin (30–50µg/ml) added to cells after digitonin treatment completely inhibited subsequent Ca2+-dependent catecholamine secretion. The same concentrations of trypsin did not inhibit secretion from permeabilized cells if trypsin was present only prior to cell permeabilization. The data indicate that trypsin entered digitonin-treated chromaffin cells which were capable of undergoing secretion and that an intracellular, trypsinsensitive protein is involved in secretion. Chymotrypsin was less potent but had effects similar to those of trypsin. The enhancement of Ca2+-dependent secretion from permeabilized chromaffin cells induced by the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) was inhibited by trypsin added simultaneously with Ca2+ to permeabilized cells at concentrations (3–10µg/ml) which had little or no effect on Ca2+-dependent secretion from cells untreated with TPA. Ca2+-dependent secretion in TPA-treated cells was reduced by trypsin only to the level that would have occurred in cells not treated with TPA. Trypsin reduced the large TPA-induced increment of membrane-bound protein kinase C. The data indicate that Ca2+-dependent secretion in the absence of TPA does not require aTPA-like effect of Ca2+ to activate protein kinase C. Protein kinase C activation by TPA probably enhances Ca2+-dependent secretion by modulating the normal Ca2+-dependent pathway or by activating another Ca2+-dependent pathway which functions in parallel to the normal pathway.