Modulation of stimulus-secretion coupling in single rat gonadotrophs.

摘要

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.