Giovannucci, D. R. | Search bioRxiv

bioRxiv2024

A PACAP-activated network for secretion requires coordination of calcium influx and calcium mobilization

Chromaffin cells of the adrenal medulla transduce sympathetic nerve activity into stress hormone secretion. The two neurotransmitters principally responsible for coupling cell stimulation to secretion are acetylcholine and pituitary adenylate activating polypeptide (PACAP). In contrast to acetylcholine, PACAP evokes a persistent secretory response from chromaffin cells. However, the mechanisms by which PACAP acts are poorly understood. Here, it is shown that PACAP induces sustained increases in cytosolic Ca2+ which are disrupted when Ca2+ influx through L-type channels is blocked or internal Ca2+ stores are depleted. PACAP liberates stored Ca2+ via inositol trisphosphate receptors (IP3Rs) on the endoplasmic reticulum (ER), thereby functionally coupling Ca2+ mobilization to Ca2+ influx and supporting Ca2+-induced Ca2+-release. These Ca2+ influx and mobilization pathways are unified by an absolute dependence on phospholipase C epsilon (PLC{varepsilon}) activity. Thus, the persistent secretory response that is a defining feature of PACAP activity, in situ, is regulated by a signaling network that promotes sustained elevations in intracellular Ca2+ through multiple pathways.

cell biology↗

bioRxiv2019

Synaptotagmin-7 endows a population of chromaffin cell vesicles with enhanced calcium sensing and delayed content release properties

Synaptotagmin-7 (Syt-7) is one of two major calcium sensors for exocytosis in adrenal chromaffin cells, the other being synaptotagmin-1 (Syt-1). Despite its undoubted importance, questions remain as to the functional and physiological role of Syt-7 in secretion. We examined this issue using two distinct preparations - mouse chromaffin cells lacking endogenous Syt-7 (KO cells) and a reconstituted system employing cell-derived vesicles expressing either Syt-7 or Syt-1. First, we report using immunofluorescence that Syt-7 exhibits a punctate intracellular distribution consistent with its sorting to organelles, including dense core vesicles. We also find that the likelihood of vesicle fusion in KO cells is markedly lower than in WT cells. When fusion does occur, cargoes are discharged more rapidly when only Syt-1 is available to facilitate release. A distinctive characteristic of KO cells is that secretion runs down after prolonged cholinergic stimulation. In contrast, exocytosis persists in WT cells even with extended exposure to acetylcholine, suggesting a key role for Syt-7 in sustaining the secretory response. To determine the extent to which the aforementioned results are attributable purely to Syt-7, vesicles expressing only Syt-7 or Syt-1 were triggered to fuse on planar supported bilayers bearing plasma membrane SNARE proteins. Here, as in cells, Syt-7 confers substantially greater calcium sensitivity to vesicle fusion than Syt-1 and slows the rate at which cargos are released. Overall, this study demonstrates that by virtue of its high affinity for calcium, Syt-7 plays a central role in regulating secretory output from adrenal chromaffin cells.

biophysics↗