Membrane recapture and early triggered secretion from the newly formed endocytotic compartment in bovine chromaffin cells

Abstract

1. Recycling of secretory vesicles in cultured bovine adrenal medullary cells was investigated. 2. Extracellular horseradish peroxidase (HRP), a fluid phase marker, was taken up into cultured adrenal medullary cells following carbamylcholine-induced secretion of catecholamine. 3. The endocytosed HRP remained compartmentalized within the cell, migrating to a low density band on a Percoll density gradient. The endocytotic compartment was distinct from the major pool of catecholamine-containing chromaffin granules, which were found at much higher densities on the Percoll gradient. 4. The chromaffin granule membrane marker dopamine beta-hydroxylase was associated with the endocytosed HRP compartment as well as with the heavier chromaffin granules. 5. A subsequent challenge of the cells with carbamylcholine triggered the release of up to forty per cent of the endocytosed HRP. 6. The time course for secretion of the fluid phase marker was similar to that for catecholamine secretion. 7. Triggered release of HRP was dependent on extracellular calcium. The dependence on the extracellular calcium concentration was similar to that of catecholamine release. 8. Release of HRP could be triggered from electropermeabilized cells by raising the intracellular Ca2+ into the micromolar range. The intracellular Ca2+ dependence of triggered HRP release was similar to that for catecholamine release. 9. HRP could be secreted as early as 5 min, and as late as 2 h after endocytosis. 10. These data provide evidence that endocytotic vesicles can rapidly re-enter the secretory cycle. Endocytosed vesicles may therefore not have to recycle via the trans-Golgi reticulum to form high-density chromaffin granules in order to re-enter the regulated secretory pathway.