Delay between Fusion Pore Opening and Peptide Release from Large Dense-Core Vesicles in Neuroendocrine Cells

Abstract

Peptidergic neurotransmission is slow compared to that mediated by classical neurotransmitters. We have studied exocytotic membrane fusion and cargo release by simultaneous capacitance measurements and confocal imaging of single secretory vesicles in neuroendocrine cells. Depletion of the readily releasable pool (RRP) correlated with exocytosis of 10%-20% of the docked vesicles. Some remaining vesicles became releasable after recovery of RRP. Expansion of the fusion pore, seen as an increase in luminal pH, occurred after approximately 0.3 s, and peptide release was delayed by another 1-10 s. We conclude that (1) RRP refilling involves chemical modification of vesicles already in place, (2) the release of large neuropeptides via the fusion pore is negligible and only proceeds after complete fusion, and (3) sluggish peptidergic transmission reflects the time course of vesicle emptying.

Visualising insulin secretion. The Minkowski Lecture 2004

“…Note the two distinct phases of the response to glucose Ca 2+ concentration are usually monophasic or oscillatory [72]. As regards (ii) and (iii), electron microscopy showed that the morphologically docked pool comprised several hundred vesicles, a quantity significantly greater than the maximum number of vesicles in the readily releasable pool (~15) [71], although only a tiny fraction of the total beta cell vesicle complement (~13,000). Nevertheless, recent data in neurones [73] suggest that readily releasable vesicles are not localised close to the presynaptic membrane, but are instead dispersed through the vesicle cluster.…”

“…Three theories have been proposed: (i) time-dependent changes in the concentration of signalling molecules [69]; (ii) physical recruitment of vesicles from a reserve to a readily releasable pool [70]; and (iii) recruitment of vesicles to a readily releasable pool by other mechanisms (e.g. covalent modification of a vesicle protein) [71]. isoforms of each AMPK subunit exist [64].…”

Mechanisms of Exocytosis in Insulin‐Secreting B‐Cells and Glucagon‐Secreting A‐Cells

“…3). In B-cells, the size of the burst that can be released immediately upon Ca 2π -influx corresponds to less than 1% of the total number of granules (Barg et al 2001a(Barg et al , 2002a. Refilling of this functionally defined ''readily releasable pool'' is ATP-, Ca 2π -and temperature-dependent (Bittner & Holz 1992;Parsons et al 1995, Renström et al 1996a& b, Eliasson et al 1997Gromada et al 1999), and commonly referred to as ''priming'' (Hay & Martin 1992).…”

“…Refilling of this functionally defined ''readily releasable pool'' is ATP-, Ca 2π -and temperature-dependent (Bittner & Holz 1992;Parsons et al 1995, Renström et al 1996a& b, Eliasson et al 1997Gromada et al 1999), and commonly referred to as ''priming'' (Hay & Martin 1992). In the B-cells, the ''readily releasable pool'' can be depleted in less than a second (Barg et al 2001a(Barg et al & 2002a, whereas its refilling takes up to a minute Barg et al 2002a). Under conditions of prolonged stimulation, the priming of vesicles for exocytosis therefore becomes rate-limiting, which may account for the biphasic time course in glucose-stimulated insulin secretion.…”

“…Under conditions of prolonged stimulation, the priming of vesicles for exocytosis therefore becomes rate-limiting, which may account for the biphasic time course in glucose-stimulated insulin secretion. In this scenario, the early rapid component (1st phase secretion) corresponds to the Ca 2π -dependent release of granules already belonging to the ''readily releasable pool'', while the sustained component (2nd phase) reflects the relatively slow priming of granules and to some extent probably their translocation to the release site (docking) Barg et al 2002a docking of the granule at the release site and the ATP-dependence is therefore not due to the energy required for translocation. Several sequential steps are necessary to render the granule release-competent, and numerous ideas have been put forward to explain the ATP-dependence of priming.…”