Slow spontaneous secretion from single large dense‐core vesicles monitored in neuroendocrine cells

Abstract

Hormones are released from cells by passing through an exocytotic pore that forms after vesicle and plasma membrane fusion. In stimulated exocytosis vesicle content is discharged swiftly. Although rapid vesicle discharge has also been proposed to mediate basal secretion, this has not been studied directly. We investigated basal hormone release by preloading fluorescent peptides into single vesicles. The hormone discharge, monitored with confocal microscopy, was compared with the simultaneous loading of vesicle by FM styryl dye. In stimulated vesicles FM 4-64 (4 microM), loading and hormone discharge occurs within seconds. In contrast, in approximately 50% of spontaneously releasing vesicles, the vesicle content discharge and the FM 4-64 loading were slow (approximately 3 min). These results show that in peptide secreting neuroendocrine cells the elementary vesicle content discharge differs in basal and in stimulated exocytosis. It is proposed that the view dating back for some decades, which is that, at rest, the vesicle discharge of hormones and neurotransmitters is similar to that occurring after stimulation, needs to be extended. In addition to the classical paradigm that secretory capacity of a cell is determined by controlling the probability of occurrence of elementary exocytotic events, one will have to consider activity modulation of elementary exocytotic events as well.

A Fast Mode of Membrane Fusion Dependent on Tight SNARE Zippering

“…During full fusion, fusion pore dilation proceeds until completion of membrane merging, whereas it is restricted in KR/cavicapture (Harata et al, 2006). Evidence for both modes of fusion has been obtained in adrenal chromaffin cells and lactotrophs (Elhamdani et al, 2001(Elhamdani et al, , 2006Taraska et al, 2003;Stenovec et al, 2004;Fulop et al, 2005;Vardjan et al, 2007). KR dominates at low [Ca 2ϩ ] i , whereas full fusion increases with stimuli that generate high [Ca 2ϩ ] i (Fulop et al, 2005;Elhamdani et al, 2006;Vardjan et al, 2007).…”

Subnanometer Fusion Pores in Spontaneous Exocytosis of Peptidergic Vesicles

“…In neuroendocrine cells, stimulated discharge of vesicle content is some 10 -20 times faster than spontaneous discharge (Stenovec et al, 2004), indicating differences in the fusion pore properties under resting and stimulated conditions. In particular, electrophysiological measurements revealed regular repetitive transient fusion pore openings ("the pulsing pore") (Stenovec et al, 2004), suggesting that flickering activity of the fusion pore may be the constraint that causes slow release of vesicle content in resting neuroendocrine cells.…”

“…For decades, spontaneous exocytosis, although a lowprobability process, was thought to exhibit elementary properties similar, if not identical, to those of stimulated exocytosis (Katz, 1969). However, recent studies suggest that the exocytotic apparatus at rest differs from that under stimulation in many respects, including distinct protein requirements for vesicle trafficking (Wucherpfennig et al, 2003), fusion (Deitcher et al, 1998;Schoch et al, 2001;Sara et al, 2005), recycling (Sara et al, 2005), and the kinetics of vesicular content discharge (Stenovec et al, 2004).…”

“…In particular, electrophysiological measurements revealed regular repetitive transient fusion pore openings ("the pulsing pore") (Stenovec et al, 2004), suggesting that flickering activity of the fusion pore may be the constraint that causes slow release of vesicle content in resting neuroendocrine cells. However, the slower release might also reflect a narrow fusion pore under nonstimulated conditions.…”

Synaptic neuropeptide release induced by octopamine without Ca 2+ entry into the nerve terminal

“…However, because spontaneous release is functionally relevant only in specialized cases (1), neuromodulators are not believed to typically induce physiologically significant release in the absence of extracellular Ca 2+ . Studies of endocrine cells suggest that release of peptides packaged in large dense-core vesicles (LDCVs) also requires Ca 2+ entry because of the rarity of spontaneous LDCV fusion and the inefficient permeation of peptides through the LDCV-fusion pore (2,3). However, because it is difficult to measure peptide release at intact synapses, a much more limited dataset supports the conclusion that Ca 2+ influx into the nerve terminal is absolutely required for peptidergic transmission.…”