REGULATION OF MUCIN SECRETION IN T84 ADENOCARCINOMA CELLS BY FORSKOLIN - RELATIONSHIP TO CA2+ AND PKC

The relationship between the adenosine 3',5'-cyclic monophosphate-medi ated protein kinase A (PKA)-dependent stimulatory pathway for mucin se cretion and Ca2+-mediated and protein kinase C (PKC)-mediated secretio n was studied in T84 cells, using the postreceptor secretagogues forsk olin, A-23187, and phorbol 12-myristate 1S-acetate (PMA), the protein kinase inhibitors staurosporine and 1-(5-isoquinolinylsulfonyl)-2-meth ylpiperazine (H-7), high- and low-Ca2+ media, and the Ca2+ chelator ,2 -bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Stauros porine (10(-5) M) inhibited both PMA and forskolin at their maximally effective concentrations, whereas H-7 (5 x 10(-5) M) inhibited only PM A. Stimulation of mucin secretion by forskolin (5 x 10(-5) M) was not significantly affected by the reduction of medium Ca2+ to 47 and 129 n M, equivalent to published values for intracellular Ca2+ concentration ([Ca2+](i)). Stimulation by forskolin was reduced by preloading cells with BAPTA, but to a much smaller extent than Ca2+-dependent stimulat ion by A-23187. A-23187-mediated mucin secretion from BAPTA-loaded cel ls was augmented by high doses of forskolin. Similar concentrations of forskolin had no effect on A-23187-stimulated secretion in calcium-re plete cells. Our results indicate that forskolin does not stimulate mu cin secretion by increasing Ca2+ entry or releasing Ca2+ from intracel lular stores. Forskolin can stimulate mucin secretion in a Ca2+-indepe ndent manner but is apparently inhibited by high levels of intracellul ar Ca2+ induced by Ca2+ ionophores in 1.0 mM Ca2+ media. PKA and PKC p athways stimulate mucin secretion independently and supportively in th e presence and absence of Ca2+. The Ca2+-independent protein kinase-de pendent response to calcium ionophores is more susceptible to H-7 than secretion mediated by forskolin, making it unlikely that calcium iono phores stimulate secretion by activating PKA.