Neurotransmitter and hormone release requires the fusion of secretory vesicles with the plasma membrane of neurons and neuroendocrine cells (i.e. exocytosis). Exocytosis begins with the formation of a fusion pore, an aqueous channel between the vesicle and the plasma membrane through which cargo molecules diffuse out of the vesicle lumen to the cell exterior. After the fusion pore formation the pore either closes (transient fusion, "kiss-and-run" exocytosis), it fluctuates between an open and a closed state (fusion pore flickering, the pulsing fusion pore), or it fully opens leading to the merger of the vesicle with the plasma membrane (full fusion exocytosis).

Spontaneous hormone discharge from a single lactotroph vesicle of the anterior pituitary cell is 10-20 times slower than stimulated discharge because of the kinetic constraints of fusion pore flickering. To see whether the slow release at rest reflects also a relatively narrow fusion pore we analyzed the permeation of FM 4-64 dye (molecular diameter = 1 nm) and HEPES (molecular diameter = 0.5 nm) molecules through fusion pores in lactotroph vesicles expressing a pH-dependent fluorescent fusion marker synaptopHluorin. Confocal imaging showed that in 50% of the spontaneous exocytotic events fusion pore openings were associated with a change in synaptopHluorin fluorescence, indicating the efflux of protons, but the pore was impermeable to FM 4-64 and HEPES. These findings, confirmed with capacitance measurements, indicate a fusion pore diameter <0.5 nm, smaller than the neuropeptides stored in these vesicles (molecular diameter = 5.2 nm). In high potassium-stimulated cells, >70% of exocytotic events exhibited a larger pore permeable to FM 4-64 (>1 nm). Capacitance measurements showed that the majority of exocytotic events in spontaneous and stimulated conditions were transient. However, stimulation increased the frequency of transient events and their fusion pore dwell-time, but decreased the fraction of events with lowest measurable fusion pore.

Thus in lactotrophs transient mode of exocytosis is the dominant mode of peptidergic hormone release. Under stimulation, a pre-formed fusion pore may retain the transient nature, but with a prolonged dwell-time, increased frequency of re-openings and an increased fusion pore diameter. All of these changes facilitate the vesicle cargo release.