Modulation of stimulus-secretion coupling in single rat gonadotrophs.
1. Exocytosis and intracellular [Ca2+] were determined simultaneously in single anterior pituitary gonadotrophs from ovariectomized female rats. Dispersed cells were cultured for 2-4 days with or without 0.2 nM oestradiol-17 beta (E2) before use. Cells were stimulated with either gonadotrophin releasing hormone (GnRH) or by membrane depolarization. Exocytosis was determined from the change in membrane capacitance (Cm) using the perforated-patch whole-cell recording technique. Intracellular [Ca2+] was measured using fura-2 fluorescence. 2. The exocytotic response to 1 nM GnRH was characterized by a wide spectrum of responses, ranging from exocytotic bursts to relatively slow, graded increases that were dependent on the evoked intracellular Ca2+ pattern. A kinetic model is presented that incorporates the observed steep dependence of exocytosis on measured intracellular [Ca2+]; simulated exocytosis reasonably approximated observed exocytotic responses, both kinetically and quantitatively. The model also suggests that the modulatory effects of E2 are brought about either by a change in the Ca2+ sensitivity of exocytosis or by a preferential clustering of docked-secretory granules close to sites of Ca2+ release. The results suggest that in gonadotrophs an oscillatory Ca2+ signal is sensed by the exocytotic apparatus in a modified form of digital encoding. 3. Exocytosis in E2-treated cells was 3-fold greater than in non-treated cells for GnRH-evoked secretion, and 38% greater for depolarization; however, there was no effect of E2 on the intracellular Ca2+ response to either stimulus. The results show that maximum expression of the effect of E2 on exocytosis requires activation of GnRH-dependent pathways.
Thomas
P
P
Department of Human Physiology, University of California, Davis 95616, USA.
Waring
D W
DW
D000818:Animals; D002118:Calcium / Q000502:physiology; D004558:Electric Stimulation; D004594:Electrophysiology; D004705:Endocytosis / Q000502:physiology; D004958:Estradiol / Q000494:pharmacology; D005089:Exocytosis / Q000502:physiology; D005260:Female; D007700:Kinetics; D008564:Membrane Potentials / Q000502:physiology; D008954:Models, Biological; D010052:Ovariectomy; D018408:Patch-Clamp Techniques; D010903:Pituitary Gland, Anterior / Q000166:cytology* / Q000187:drug effects; D051381:Rats; D017207:Rats, Sprague-Dawley
D004958:Estradiol; D002118:Calcium