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NERVOUS SYSTEM CELL BIOLOGY

Hypotonicity and peptide discharge from a single vesicle

Jernej Jorgaevski,^1,* Matja Stenovec,^1,2,* Marko Kreft,^1,2 Aleksandar Baji,³ Botjan Rituper,¹ Nina Vardjan,^1,2 Stanko Stojilkovic,³ and Robert Zorec^1,2

¹Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia; ²Celica Biomedical Center, Ljubljana, Slovenia; and ³Section on Cellular Signaling, Developmental Neuroscience Program, National Institute of Child Health and Human Development, Bethesda, Maryland

Submitted 10 June 2008 ; accepted in final form 11 July 2008

Neuroendocrine secretory vesicles discharge their cargo in response to a stimulus, but the nature of this event is poorly understood. We studied the release of the pituitary hormone prolactin by hypotonicity, because this hormone also contributes to osmoregulation. In perfused rat lactotrophs, hypotonicity resulted in a transient increase followed by a sustained depression of prolactin release, as monitored by radioimmunoassay. In single cells imaged by confocal microscopy, hypotonicity elicited discharge of the fluorescently labeled atrial natriuretic peptide cargo from 2% of vesicles/cell. In contrast, KCl-induced depolarization resulted in a response of 10% of vesicles/cell, with different unloading/loading time course of the two fluorescent probes. In cell-attached studies, discrete changes in membrane capacitance were recorded in both unstimulated and stimulated conditions, reflecting single vesicle fusion/fissions with the plasma membrane. In stimulated cells, the probability of occurrence of full fusion events was low and unchanged, whereas over 95% of fusion events were transient, with the open fusion pore probability, the average pore dwell-time, the frequency of occurrence, and the fusion pore conductance increased. Hypotonicity only rarely elicited new fusion events in silent membrane patches. The results indicate that, in hypotonicity-stimulated lactotrophs, transient vesicle fusion mediates hormone release.

prolactin; hormone secretion; rat lactotrophs; fusion pore; postfusion release regulation