Peripheral actin filaments control calcium-mediated catecholamine release from streptolysin-O-permeabilized chromaffin cells.
Adrenal medullary chromaffin cells were permeabilized by treatment with a streptococcal cytotoxin streptolysin O (SLO) which generates pores of macromolecular dimensions in the plasma membrane. SLO did not provoke spontaneous release of catecholamines or chromogranin A, a protein marker of the secretory granule, showing the integrity of the secretory vesicle membrane. However, the addition of micromolar free calcium concentration induced the corelease of noradrenaline and chromogranin A, indicating that secretory products are liberated by exocytosis. Calcium-dependent exocytosis from SLO-permeabilized cells required Mg-ATP and could not occur in the presence of other nucleotides. The pores generated by the toxin were large enough to introduce proteins, e.g., immunoglobulins, but also caused efflux of the cytosolic marker lactate dehydrogenase. Despite this, the cells remained responsive to calcium for up to 30 min after permeabilization, indicating that they retained their secretory machinery. In the search for a functional role of cytoskeletal proteins in the secretory process, we used SLO-permeabilized cells to examine the localization of filamentous actin, using rhodamine-phalloidin, and that of the actin-severing protein, gelsolin, using specific antibodies. It was found that both F-actin and gelsolin were exclusively localized in the subplasmalemmal region of the cell. We examined the relationship between actin disassembly, the elevation of intracellular calcium and secretion in SLO-treated cells. F-Actin destabilizing agents such as cytochalasin D or DNase I were found to potentiate calcium-stimulated release. The maximal effect was observed at low calcium concentrations (1-4 microM) and at the later stages of the secretory response (after 10 min stimulation). In addition, using rhodamine-phalloidin, we observed that calcium provoked simultaneously both cortical actin disassembly and catecholamine release in SLO-permeabilized cells. These results demonstrate that a close relationship exists between the secretory response and actin disassembly and provide further evidence that intracellular calcium controls the subplasmalemmal cytoskeletal actin organization and thereby the access of secretory granules to exocytotic sites.
Sontag
J M
JM
Centre de Neurochimie du CNRS, Unité INSERM U44, Strasbourg/France.
Aunis
D
D
Bader
M F
MF
D000199:Actins / Q000378:metabolism / Q000502:physiology*; D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001426:Bacterial Proteins; D002118:Calcium / Q000494:pharmacology*; D002135:Calcium-Binding Proteins / Q000032:analysis; D002395:Catecholamines / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D002478:Cells, Cultured; D002838:Chromaffin System / Q000032:analysis / Q000166:cytology* / Q000187:drug effects / Q000648:ultrastructure; D015638:Cytochalasin D; D003572:Cytochalasins / Q000494:pharmacology; D003599:Cytoskeleton / Q000187:drug effects / Q000502:physiology; D003850:Deoxyribonuclease I / Q000494:pharmacology; D018260:Gelsolin; D008840:Microfilament Proteins / Q000032:analysis; D010590:Phalloidine / Q000494:pharmacology; D013301:Streptolysins / Q000494:pharmacology*
D000199:Actins; D001426:Bacterial Proteins; D002135:Calcium-Binding Proteins; D002395:Catecholamines; D003572:Cytochalasins; D018260:Gelsolin; D008840:Microfilament Proteins; D013301:Streptolysins; C025964:streptolysin O; D010590:Phalloidine; D015638:Cytochalasin D; D000255:Adenosine Triphosphate; D003850:Deoxyribonuclease I; D002118:Calcium