Modulation of stimulus—secretion coupling in single rat gonadotrophs

1 Exocytosis and intracellular [Ca 2+ ] 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 n m oestradiol‐17 β (E 2 ) before use. Cells were stimulated with either gonadotrophin releasing hormone (GnRH) or by membrane depolarization. Exocytosis was determined from the change in membrane capacitance ( C m ) using the perforated‐patch whole‐cell recording technique. Intracellular [Ca 2+ ] was measured using fura‐2 fluorescence. 2 The exocytotic response to 1 n m 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 Ca 2+ pattern. A kinetic model is presented that incorporates the observed steep dependence of exocytosis on measured intracellular [Ca 2+ ]; simulated exocytosis reasonably approximated observed exocytotic responses, both kinetically and quantitatively. The model also suggests that the modulatory effects of E 2 are brought about either by a change in the Ca 2+ sensitivity of exocytosis or by a preferential clustering of docked‐secretory granules close to sites of Ca 2+ release. The results suggest that in gonadotrophs an oscillatory Ca 2+ signal is sensed by the exocytotic apparatus in a modified form of digital encoding. 3 Exocytosis in E 2 ‐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 E 2 on the intracellular Ca 2+ response to either stimulus. The results show that maximum expression of the effect of E 2 on exocytosis requires activation of GnRH‐dependent pathways.