Recapture after exocytosis causes differential retention of protein in granules of bovine chromaffin cells

Abstract

After exocytosis, chromaffin granules release essentially all their catecholamines in small fractions of a second, but it is unknown how fast they release stored peptides and proteins. Here we compare the exocytic release of fluorescently labelled neuropeptide Y (NPY) and tissue plasminogen activator from single granules. Exocytosis was tracked by measuring the membrane capacitance, and single granules in live cells were imaged by evanescent field microscopy. Neuropeptide Y left most granules in small fractions of a second, while tissue plasminogen activator remained in open granules for minutes. Taking advantage of the dependence on pH of the fluorescence of green fluorescent protein, we used rhythmic external acidification to determine whether and when granules re‐sealed. One‐third of them re‐sealed within 100 s and retained significant levels of tissue plasminogen activator. Re‐sealing accounts for only a fraction of the endocytosis monitored in capacitance measurements. When external [Ca 2+] was raised, even neuropeptide Y remained in open granules until they re‐sealed. It is concluded that a significant fraction of chromaffin granules re‐seal after exocytosis, and retain those proteins that leave granules slowly. We suggest that granules vary the stoichiometry of release by varying both granule re‐sealing and the association of proteins with the granule matrix.