The cortical F-actin cytoskeleton represents a negative control for secretion, and it must be locally disassembled to allow chromaffin vesicle exocytosis. Recombinant scinderin (a Ca(2+)-dependent F-actin-severing protein) potentiated Ca(2+)-evoked F-actin disassembly and exocytosis in permeabilized chromaffin cells, an effect blocked by peptides Sc-ABP1 and Sc-ABP2 (with sequences corresponding to two actin-binding sites of scinderin), exogenous gamma-actin, or phosphatidylinositol 4,5-bisphosphate (PIP2). PIP2 effect was blocked by peptide Sc-PIP2BP (with sequence corresponding to a PIP2-binding site of scinderin). Truncated scinderin254-715 (lacking actin-severing domains) did not potentiate exocytosis. Sc-ABP1, Sc-ABP2, and gamma-actin also inhibited exocytosis in the absence of recombinant scinderin, suggesting an inhibition of endogenous scinderin. Results suggest that scinderin-evoked cortical F-actin disassembly is required for secretion and that scinderin is an important component of the exocytotic machinery.

Histamine is a known chromaffin cell secretagogue that induces Ca" -dependent release of catecholamines . However, conflicting evidence exists as to the source of Ca t ' utilized in histamine-evoked secretion .Here we report that histamine-H, receptor activation induces redistribution of scinderin, a Ca t +-dependent Factin severing protein, cortical F-actin disassembly, and catecholamine release . Histamine evoked similar patterns of distribution of scinderin and filamentous actin . The rapid responses to histamine occurred in the absence of extracellular Ca t + and were triggered by release of Ca" from intracellular stores . The trigger for the release of Ca t ' was inositol 1,4,5-trisphosphate because U-73122, a phospholipase C inhibitor, but not its inactive isomer (U-73343), inhibited the increases in IP 3 and intracellular Ca" levels, scinderin redistribution, cortical F-actin disassembly, and catecholamine release in response to histamine . Thapsigargin, an agent known to mobilize intracellular Ca", blocked the rise in intracellular Ca t ' concentration, scinderin redistribution, F-actin disassembly, and catecholamine secretion in response to histamine . Calphostin C and chelerythrine, two inhibitors of protein kinase C, blocked all responses to histamine with the exception of the release of Ca" from intracellular stores . This suggests that protein kinase C is involved in histamine-induced responses . The results also show that in the absence of F-actin disassembly, rises in intracellular Ca t ' concentration are not by themselves capable of triggering catecholamine release .Abbreviations used : D,8H, dopamine ß-hydroxylase ; FITC, fluorescein isothioeyanate; IP3 , inositol 1,4,5-trisphosphate ; MARCKS, myristoylated alanine-rich C-kinase substrate ; NA, noradrenaline; PIP, phosphatidylinositol 4,5-bisphosphate ; PKC, protein kinase C; PLC, phospholipase C; PNMT, phenyl-N-methyltransferase ; U-73122, 1-16-l117[3-3-methoxyestra-1,3,5(10)-trien-17-yl]aminolhexyl ]-IH-pyrrole-2,5-dione .

In response to vessel injury or exposure to different substances, platelets undergo activation which consists of shape changes, formation of cellular pseudopodia, aggregation, and secretion. These dramatic changes are accompanied by cycles of actin depolymerization and polymerization. Previous work has shown the presence in platelets of gelsolin and scinderin, two Ca(2+)-dependent F-actin severing proteins. Recent published evidence suggests that scinderin is a component of the exocytotic machinery in chromaffin cells. The present work describes the preparation of recombinant scinderin and peptides Sc-ABP1 and Sc-ABP2 with sequences corresponding to two actin-binding sites of scinderin. Recombinant scinderin and peptides Sc-ABP1 and Sc-ABP2 were tested for their effects on Ca(2+)-induced serotonin release from digitonin permeabilized platelets. The results indicated that recombinant scinderin potentiates Ca(2+)-evoked serotonin release, an effect blocked in the presence of Sc-ABP1, Sc-ABP2, exogenous gamma-actin, or the addition of phosphatidylinositol 4,5-bisphosphate (PIP2). In the presence of a mismatched peptide (MMP) the potentiating effect of recombinant scinderin was not affected. Moreover, Sc-ABP1, Sc-ABP2, and gamma-actin inhibited Ca(2+)-induced release of serotonin in the absence of recombinant scinderin, suggesting an inhibition of platelet endogenous scinderin. MMP was ineffective under these conditions. The results suggest that F-actin disassembly, perhaps at a specific site, is required for platelet secretion and that scinderin might be an important component of the exocytotic machinery in platelets.