Morphology and secretory activity of digitonin- and alpha-toxin-permeabilized chromaffin cells.
An ultrastructural examination of cultured bovine chromaffin cells permeabilized with Staphylococcus aureus alpha-toxin or digitonin revealed differences in the preservation of cell morphology. The toxin-treated cells closely resembled control cultured cells whereas digitonin-treated cells showed gradations in cytoplasmic densities suggesting extraction, some swelling of the endoplasmic reticulum and, occasionally, discontinuities in the plasma membrane and free granules in the extracellular medium. In both cell models, there was a swelling of the mitochondria. Horseradish peroxidase labelling of permeabilized cells marked the cytoplasm of digitonin-treated cells but only the surface of toxin-treated cells, demonstrating that larger lesions were caused by digitonin. In stimulated cells, the decrease in volumetric density of chromaffin granules correlated well with catecholamine release. The sites of secretory activity could be demonstrated in toxin-treated cells using horseradish peroxidase as a surface marker. Although both cell systems secrete catecholamines in response to calcium stimulation, their calcium requirements and the kinetics of release were different. In alpha-toxin-treated cells, 100 microM free calcium induced maximal catecholamine release. In digitonin-treated cells, 20 microM evoked maximal release but secretion was blocked at 100 microM. Catecholamine release terminated in digitonin-treated cells within 10 min but continued in alpha-toxin-treated cells for at least 60 min. In addition, the maximal release observed in toxin-treated cells (50%) was always greater than that observed in digitonin-permeabilized cells (20%). The results suggest that both exocytosis and granule translocation are operational in alpha-toxin-treated cells, but that the translocation step or the docking of granules at the plasma membrane may be impaired in digitonin-treated cells.
Unité INSERM U44, Centre de Neurochimie du CNRS, Strasbourg, France.
N J
NJ
Grant
D
D
Aunis
M F
MF
Bader
D000313:Adrenal Medulla / Q000187:drug effects / Q000378:metabolism* / Q000648:ultrastructure; D000818:Animals; D001427:Bacterial Toxins / Q000494:pharmacology*; D002118:Calcium / Q000494:pharmacology; D002395:Catecholamines / Q000378:metabolism*; D002417:Cattle; D002463:Cell Membrane Permeability / Q000187:drug effects*; D002467:Cell Nucleus / Q000187:drug effects / Q000648:ultrastructure; D004072:Digitonin / Q000494:pharmacology*; D005089:Exocytosis / Q000187:drug effects; D006460:Hemolysin Proteins; D006735:Horseradish Peroxidase; D008855:Microscopy, Electron, Scanning
D001427:Bacterial Toxins; D002395:Catecholamines; D006460:Hemolysin Proteins; C021091:staphylococcal alpha-toxin; D006735:Horseradish Peroxidase; D004072:Digitonin; D002118:Calcium