Ca2+-evoked serotonin secretion by parafollicular cells: Roles in signal transduction of phosphatidylinositol 3 '-kinase, and the gamma and zeta isoforms of protein kinase C

Parafollicular (PF) cells secrete 5-HT in response to stimulation of a G-pr otein-coupled Ca2+ receptor (CaR) by increased extracellular Ca2+ (up arrow [Ca2+](e)). We tested the hypothesis that protein kinase C (PKC) participat es in stimulus-secretion coupling. Immunoblots from membrane and cytosolic fractions of isolated PF cells revealed conventional (alpha, beta I, and ga mma), novel (delta and epsilon), and atypical (iota/lambda and zeta) PKCs. Only PKC gamma was found to have been translocated to the membrane fraction when secretion of 5-HT was evoked by up arrow[Ca2+](e) or phorbol esters. Although phorbol downregulation caused PKC gamma to disappear, secretion wa s only partially inhibited. A similar reduction of up arrow[Ca2+](e)-evoked secretion was produced by inhibitors of conventional and/or novel PKCs (Go 6976, calphostin C, and pseudoA), and these compounds did not inhibit secr etion at all when applied to phorbol-downregulated cells. In contrast, the phorbol downregulation-resistant component of secretion was abolished by ps eudoZ, which inhibits the atypical PKC zeta. Stimulation of PF cells with u p arrow[Ca2+](e) increased the activity of immunoprecipitated PKC zeta (but not PKC iota/lambda), and the activity of this PKC zeta was inhibited by p seudoZ. PF cells were found to express regulatory (p85) and catalytic (p110 alpha and p110 beta) subunits of phosphatidylinositol 3'-kinase (PI3'-kina se). up arrow[Ca2+](e) increased the activity of immunoprecipitated PI3'-ki nase; moreover, PI3'-kinase inhibitors (wortmannin and LY294002) antagonize d secretion. We suggest that PKC isoforms mediate secretion of 5-HT by PF c ells in response to stimulation of the CaR. PKC involvement can be accounte d for by PKC gamma and an isoform sensitive to inhibition by pseudoZ, proba bly PKC zeta, which is activated via PI3'-kinase.