Caffeine-stimulated GTH-II release involves Ca2+ stores with novel properties

Modulation of Ca2+ stores with 10 mM caffeine stimulates robust secretion of gonadotropin (GTH-II) from goldfish gonadotropes. Although both endogenous forms of gonadotropin-releasing hormone (GnRH) utilize a common intracellular Ca2+ store, sGnRH, but not cGnRH-II, uses an additional caffeine-sensitive mechanism. We examined caffeine signaling by using Ca2+ imaging, electrophysiology, and cell-column perifusion. Although caffeine inhibited K+ channels, this action appeared to be unrelated to caffeine-induced GTH-II release, because the latter was insensitive to tetraethylammonium. The effects of caffeine also were not mediated by the cAMP/protein kinase A pathway. Instead, caffeine-evoked GTH-II responses were Ca2+ signal dependent because they were abolished by 1,2-bis(2-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid loading. Caffeine generated localized Ca2+ signals that began near secretory granules. Surprisingly, caffeine-stimulated GTH-II release was insensitive to 100 muM ryanodine and, unlike GnRH action, was unaffected by inhibitors of voltage-gated Ca2+ channels or sarco(endo)plasmic reticulum Ca2+-ATPases. Collectively, these data indicate that caffeine-stimulated GTH-II release is not mediated by typical agonist-sensitive Ca2+ stores found in endoplasmic reticulum.