Immunofluorescent evidence for exocytosis and internalization of secretory granule membrane in isolated chromaffin cells.
Cultured bovine adrenal medullary chromaffin cells were stimulated with the secretogogues Ba2+ or carbamyl choline plus Ca2+ in the presence of a monospecific rabbit IgG fraction directed against bovine dopamine beta-hydroxylase. The anti-dopamine beta-hydroxylase was labeled either with fluorescent protein A or with a fluorescent second antibody to rabbit IgG. Stimulation produced a patchy cell surface distribution of fluorescence. There was no noticeable internalization of the fluorescence for up to 2 h. In similar experiments using fluorescent monovalent fragments (Fab) of the same monospecificidopamine-beta-hydroxylase IgG, a more uniform distribution of the fluorescence was observed. A few min after a 5 min period of stimulation with Ba2+, the fluorescence appeared to be on or near the cell surface; however, after 20 min or more it was distributed throughout the cytoplasm except that the cell nuclei were not labeled. Thus, dopamine beta-hydroxylase which appeared on the cell surface as a consequence of exocytosis was internalized in the presence of monovalent antibody fragments, but not in the presence of the divalent (polyclonal) antibody, presumably because endocytosis of dopamine beta-hydroxylase was inhibited by crosslinking of the dopamine beta-hydroxylase molecules. The internalized anti-dopamine beta-hydroxylase Fab fragments were found to reappear on the cell surface during a second secretory response. It is concluded that the interior of the chromaffin granule membrane, for which dopamine beta-hydroxylase is a marker, becomes exposed on the surface of the cell during secretion and that the membrane is then retrieved back into the cell where it can be re-used in a further secretory cycle.
D000818:Animals; D000906:Antibodies / Q000276:immunology; D002417:Cattle; D002838:Chromaffin System / Q000166:cytology* / Q000502:physiology; D003594:Cytoplasmic Granules / Q000502:physiology*; D004299:Dopamine beta-Hydroxylase / Q000276:immunology; D000939:Epitopes; D005089:Exocytosis; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005455:Fluorescent Antibody Technique; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G / Q000276:immunology; D013861:Thiocyanates
D000906:Antibodies; D000939:Epitopes; D005452:Fluoresceins; D007140:Immunoglobulin Fab Fragments; D007074:Immunoglobulin G; D013861:Thiocyanates; D004299:Dopamine beta-Hydroxylase; D016650:Fluorescein-5-isothiocyanate