LOSS AND CA2+-DEPENDENT RETENTION OF SCINDERIN IN DIGITONIN-PERMEABILIZED CHROMAFFIN CELLS - CORRELATION WITH CA2+-EVOKED CATECHOLAMINE RELEASE

Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca2+-dependent actin-filament-severing protein), but not gelsolin, is among the proteins that leak out from digitonin-permeabilized cells. Chromaffin cells that were exposed to increasing concentrations (15-40 muM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 muM digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca2+ concentration in the permeabilizing medium produced a dose-dependent retention of scinderin. Results were confirmed by immunofluorescence microscopy of digitonin-permeabilized cells. Subcellular fractionation of permeabilized cells showed that scinderin leakage was mainly from the cytoplasm (80%); the remaining scinderin (20%) was from the microsomal fraction. Other Ca2+-binding proteins released by digitonin and also retained by Ca2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromaffin cell secretory response. Permeabilization in the presence of increasing free Ca2+ Concentrations produced a concomitant enhancement in the subsequent Ca2+-dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca2+, (2) permeabilization of chromaffin cells with digitonin in the presence of micromolar Ca2+ concentrations retained Ca2+-binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca2+-dependent catecholamine release observed in permeabilized chromaffin cells.