Retention and stimulus-dependent recycling of dense core vesicle content in neuroendocrine cells

Abstract

We have used fluorescence imaging of individual exocytic events in combination with immunogold electron microscopy and FM1-43 photoconversion to study the stimulus-dependent recycling of dense core vesicle content in isolated rat pituitary lactotrophs. Secretory stimulation with high external [K+] resulted in 100 exocytic sites per cell that were labeled by extracellular antibodies against the peptide hormone prolactin. Morphological analysis demonstrated that the prolactin was retained and internalized in intact dense cores. Vesicles containing non-secreted, internalized prolactin did not colocalize with DiI-LDL that had been chased into lysosomes but did transiently colocalize with internalized transferrin. The recycling vesicles also trafficked through a syntaxin 6-positive compartment but not the TGN38-positive trans-Golgi. Recycling vesicles, which returned to the cell surface in a slow basal manner, could also be stimulated to undergo exocytosis with a high release probability during subsequent exocytic stimulation with external K+. These studies suggest a functional role for recycling vesicles that retain prolactin.

Retrieval and Reuse of Pituitary Secretory GranuleProteins

“…Together, our results indicate that after secretion the prolactin that remains associated with the cells is internalized and sorted to a secretory compartment, processes that imply its functional recycling. Although these findings are in agreement with the observations by others (34), it remains to be established whether functional recycling of unspent post-exocytotic hormone is peculiar to prolactin or a phenomenon common to other regulated secretory peptides.…”

“…It has been reported recently that, upon stimulation with high K ϩ at 22°C, prolactin cores remain associated with the plasma membrane, undergo internalization, and following restimulation, reappear at the cell surface (34). We found that in resting conditions 10% of the total prolactin was biotinylated in 30 min at 37°C, signaling its appearance at the cell surface.…”

“…In culture, the tonic dopaminergic inhibition of lactotropes (33) is removed, and basal release of PRL is high. In a recent report (34), upon depolarization with high K ϩ (in physiological saline and at 22°C), prolactin cores were detected at the plasma membrane, subsequently internalized, and reappeared at the cell surface after a second high potassium stimulation. These observations suggest that PRL packed in secretory granules can be reused if not spent during one round of exocytosis.…”

Dynamic Regulation of the Large Exocytotic Fusion Pore in Pancreatic Acinar Cells

“…Instead of granule collapse, fusion pore opening can be followed by closure. A final closure of the fusion pore is an essential step in the kiss-and-run model of exocytosis (Ceccarelli et al, 1972), where it is the transitional step between exocytosis and the endocytic recovery of the granule (Holroyd et al, 2002;Bauer et al, 2004;Tsuboi et al, 2004).…”

New insights into the control of secretion

“…1B). Significantly these recaptured vesicles can contain residual quantities of peptide hormones [13][14][15] suggesting that fusion pore dynamics is a limiting factor in the release of peptides. Some papers now suggest that fusion pore dynamics might specifically regulate the loss of low molecular weight (<200 Da) neurotransmitters.…”