Association of intracellular calcium oscillations with release of gonadotropin-releasing hormone (GnRH) from GT1-1 neuronal cells

Abstract

GnRH is released from its nerve terminals via a calcium (Ca²⁺)-dependent mechanism. Oscillation in the intercellular Ca²⁺ [Ca²⁺][] concentration may encode signals that result in the release of GnRH. The objective of this study was to investigate the relationship of GnRH release with [Ca²⁺][] oscillations using the GT1-1 neuronal cell line. GnRH release was measured by radioimmunoassay of media incubated with GT1-1 neuronal cells for 1 hr with control or test substances to induce release of GnRH. [Ca[]][] oscillations were analyzed by confocal microscopy of fluorescence intensities, at 3 sec intervals, of GT1-1 neuronal cells loaded with fluo-3,AM dye. Depolarization (60 mMK+) induced a consistent 2-fold increase in GnRH release. The response of GT1-1 neuronal cells to treatment with NMDA varied between experiments. Studies with either PGE[] or PGE[] released GnRH in a dose-dependent manner, with PGE[] being more potent than PGE[]. 90% of GT1-1 neuronal cells examined exhibited intrinsic oscillations of [Ca²⁺][]. The frequency or pattern of [Ca²⁺][] oscillations was synchronized within an isolated aggregate of neuronal cells, but varied between unconnected aggregates of neuronal cells. Intrinsic [Ca²⁺][] oscillations disappeared when EGTA was added to the media. Depolarization evoked a transient spike in the [Ca²⁺][] at followed by a sustained elevated plateau of [Ca²⁺][]. NMDA did not significantly alter the frequency of [Ca²⁺][] oscillations. Both PGE[] and PGE[] induced an immediate increase in the frequency of [Ca²⁺][] oscillations. In summary, GT1-1 neuronal cells exhibit intrinsic [Ca²⁺][] oscillations whose frequency or pattern is synchronized between interconnected neuronal cells but vary between unconnected cells. Extracellular Ca²⁺ appears important for [Ca²⁺][] oscillations. Both PGE[] and PGE[], at doses that induce release of GnRH, induce a dramatic increase in the frequency of [Ca²⁺][] at oscillations. These results indicate that release of GnRH is associated with an increase frequency of [Ca²⁺][] oscillations and suggest that [Ca²⁺][] oscillations may be invovled in GnRH release.