Loss and Ca(2+)-dependent retention of scinderin in digitonin-permeabilized chromaffin cells: correlation with Ca(2+)-evoked catecholamine release.
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 microM) of digitonin for 5 min released scinderin into the medium. One-minute treatment with 20 microM 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 Ca(2+)-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 Ca(2+)-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 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.
Vitale
M L
ML
Department of Pharmacology, Faculty of Medicine, University of Ottawa, Ontario, Canada.
Rodríguez Del Castillo
A
A
Trifaró
J M
JM
D000313:Adrenal Medulla / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D000818:Animals; D002118:Calcium / Q000502:physiology*; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002478:Cells, Cultured; D003600:Cytosol / Q000187:drug effects / Q000378:metabolism; D004072:Digitonin / Q000494:pharmacology*; D004305:Dose-Response Relationship, Drug; D018260:Gelsolin; D008840:Microfilament Proteins / Q000378:metabolism*; D013347:Subcellular Fractions / Q000378:metabolism
D002395:Catecholamines; D018260:Gelsolin; D008840:Microfilament Proteins; C065395:scinderin; D004072:Digitonin; D002118:Calcium