RECYCLING OF A SECRETORY GRANULE MEMBRANE-PROTEIN AFTER STIMULATED SECRETION

Recycling of a secretory granule membrane protein, dopamine-beta-hydro xylase, was examined in primary cultures of bovine adrenal chromaffin cells. Cells were stimulated to secrete in the presence of antibodies directed against the luminal domain of dopamine-beta-hydroxylase. The location of the antibodies after various times of reincubation and aft er a second secretory stimulus was assessed using immunofluorescence m icroscopy. Stimulation led to the exposure of dopamine-beta-hydroxylas e at the plasma membrane, which could be detected by a polyclonal anti body in living and fixed cells. The plasma membrane dopamine-beta-hydr oxylase, either alone or complexed with antibody, was rapidly internal ized after removal of the secretagogue. Internalized protein-antibody complex remained stable for at least 24 hours of reculture. Twenty fou r hours after stimulation the cells with internalized antibody could r espond to further stimulation and some of the antibody was re-exposed at the plasma membrane. These findings were confirmed using FACS analy sis. This suggests that the antibody-protein complex had returned to s ecretory granules that could respond to further secretagogue stimulati on.