Ca2+-Evoked Serotonin Secretion by Parafollicular Cells: Roles in Signal Transduction of Phosphatidylinositol 3′-kinase, and the γ and ζ Isoforms of Protein Kinase C

Parafollicular (PF) cells secrete 5-HT in response to stimulation of a G-protein-coupled Ca2+ receptor (CaR) by increased extracellular Ca2+(↑[Ca2+]e). We tested the hypothesis that protein kinase C (PKC) participates in stimulus–secretion coupling. Immunoblots from membrane and cytosolic fractions of isolated PF cells revealed conventional (α, βI, and γ), novel (δ and e), and atypical (ι/λ and ζ) PKCs. Only PKCγ was found to have been translocated to the membrane fraction when secretion of 5-HT was evoked by ↑[Ca2+]e or phorbol esters. Although phorbol downregulation caused PKCγ to disappear, secretion was only partially inhibited. A similar reduction of ↑[Ca2+]e-evoked secretion was produced by inhibitors of conventional and/or novel PKCs (Go6976, calphostin C, and pseudoA), and these compounds did not inhibit secretion at all when applied to phorbol-downregulated cells. In contrast, the phorbol downregulation-resistant component of secretion was abolished by pseudoZ, which inhibits the atypical PKCζ. Stimulation of PF cells with ↑[Ca2+]e increased the activity of immunoprecipitated PKCζ (but not PKCι/λ), and the activity of this PKCζ was inhibited by pseudoZ. PF cells were found to express regulatory (p85) and catalytic (p110α and p110β) subunits of phosphatidylinositol 3′-kinase (PI3′-kinase). ↑[Ca2+]e increased the activity of immunoprecipitated PI3′-kinase; moreover, PI3′-kinase inhibitors (wortmannin and LY294002) antagonized secretion. We suggest that PKC isoforms mediate secretion of 5-HT by PF cells in response to stimulation of the CaR. PKC involvement can be accounted for by PKCγ and an isoform sensitive to inhibition by pseudoZ, probably PKCζ, which is activated via PI3′-kinase.