Exocytosis and the cell-averaged cytosolic [Ca2+], [Ca2+](i), were tracked in single gonadotrophs. Cells released 100 granules/s at 1 μM [Ca2+](i) when gonadotropin-releasing hormone (GnRH) activated IP3- mediated Ca2+ release from internal stores, but only 1 granule/s when [Ca2+](i) was raised uniformly to 1 μM by other means. Strong exocytosis was then seen only at higher [Ca2+], (half-maximal at 16 μM). Parallel second messengers did not contribute to GaRH-induced exocytosis, because IP3 alone was as effective as GnRH, and because even GnRH failed to trigger rapid exocytosis when the [Ca2+](i) rise was blunted by EGTA. When [Ca2+](i) was released from stores, exocytosis depended on [Ca2+](i) rising rapidly, as if governed by Ca2+ flux into the cytosol. We suggest that IP3 releases Ca2+ selectively from subsurface cisternae, raising [Ca2+] near exocytic sites 5-fold above the cell average.
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