Hypotonicity and peptide discharge from a single vesicle.
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 approximately 2% of vesicles/cell. In contrast, KCl-induced depolarization resulted in a response of approximately 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.
Jorgacevski
Jernej
J
Laboratory of Neuroendocrinology-Molecular Cell Physiology, Faculty of Medicine, University of Ljubljana, Ljubljana, Slovenia.
Stenovec
Matjaz
M
Kreft
Marko
M
Bajić
Aleksandar
A
Rituper
Bostjan
B
Vardjan
Nina
N
Stojilkovic
Stanko
S
Zorec
Robert
R
D000818:Animals; D009320:Atrial Natriuretic Factor / Q000378:metabolism; D048429:Cell Size; D002478:Cells, Cultured; D025802:Electric Capacitance; D007038:Hypotonic Solutions; D052682:Lactotrophs / Q000187:drug effects / Q000378:metabolism*; D008297:Male; D008561:Membrane Fusion; D008564:Membrane Potentials; D018613:Microscopy, Confocal; D009997:Osmotic Pressure; D018408:Patch-Clamp Techniques; D010477:Perfusion; D011189:Potassium Chloride / Q000494:pharmacology; D011388:Prolactin / Q000378:metabolism*; D011863:Radioimmunoassay; D051381:Rats; D017208:Rats, Wistar; D022142:Secretory Vesicles / Q000187:drug effects / Q000378:metabolism*; D013997:Time Factors
D007038:Hypotonic Solutions; D011189:Potassium Chloride; D009320:Atrial Natriuretic Factor; D011388:Prolactin