Premium
Loss and Ca 2+ ‐Dependent Retention of Scinderin in Digitonin‐Permeabilized Chromaffin Cells: Correlation with Ca 2+ ‐Evoked Catecholamine Release
Author(s) -
Vitale M. L.,
Castillo A. Rodríguez,
Trifaró J.M.
Publication year - 1992
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1992.tb11003.x
Subject(s) - digitonin , chromaffin cell , cytosol , biochemistry , intracellular , adrenal medulla , biophysics , chemistry , biology , depolarization , exocytosis , catecholamine , microbiology and biotechnology , enzyme , secretion , endocrinology
Exposure of chromaffin cells to digitonin causes the loss of many cytosolic proteins. Here we report that scinderin (a Ca 2+ ‐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 μM) of digitonin for 5 min released scinderin into the medium. One‐minute treatment with 20 μ digitonin was enough to detect scinderin in the medium, and scinderin leakage levelled off after 10 min of permeabilization. Elevation of free Ca 2+ 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 Ca 2+ ‐binding proteins released by digitonin and also retained by Ca 2+ were calmodulin, protein kinase C, and calcineurins A and B. Scinderin leakage was parallel to the loss of the chromamn cell secretory response. Permeabilization in the presence of increasing free Ca 2+ concentrations produced a concomitant enhancement in the subsequent Ca 2+ ‐dependent catecholamine release. The experiments suggest that: (1) scinderin is an intracellular target for Ca 2+ , (2) permeabilization of chromaffin cells with digitonin in the presence of micro‐molar Ca 2+ concentrations retained Ca 2+ ‐binding proteins including scinderin, and (3) the retention of these proteins may be related to the increase in the subsequent Ca 2+ ‐dependent catecholamine release observed in permeabilized chromaffin cells.