To determine the role of Ca2+ in LHRH release in vitro. RESULTS In a previous study we have reported that cultured LHRH neurons derived from the olfactory placode of monkey embryos release LHRH into media in a pulsatile manner at approximately 50-min intervals. As a first step in studying the mechanism of LHRH pulse generation, we examined the role of calcium in LHRH release using cultured LHRH neurons. The results are summarized as follows 1) Exposing the cells to a low Ca2+ (10 nM) buffer solution suppressed LHRH release; 2) LHRH release from cultured LHRH cells was stimulated by the L-type Ca2+ channel stimulant, Bay K 8644 (1 M), while it was suppressed by the L-type Ca2+ channel blocker, nifedipine (1 M), but not by the N-type channel blocker, w-conotoxin (1 M); 3) The intracellular Ca2+ ([Ca2+]i) transporting ATPase antagonist, thapsigargin (10 M), suppressed LHRH release, while the intracellular Ca2+ stimulant, ryanodine (1 M) stimulated LHRH release; 4) Carbonyl-cyanide-P-trifluoromethoxyphenyl hydrazone (FCCP, 1 mM), a mitochondrial Ca2+ mobilizer, tended to stimulate LHRH release. These results indicate that 1) release of LHRH is influenced by the presence of extracellular Ca2+ ([Ca2+]e), 2) Ca2+ enters the cell via L-type channels, but not N-type channels, and 3) mobilization of [Ca2+]i is important for LHRH release. It is concluded that the presence of [Ca2+]e and Ca2+ entry into the cell are essential for pulsatile LHRH release in cultured LHRH neurons. Moreover, mobilization of [Ca2+]i appears to contribute to LHRH release in cells. FUTURE DIRECTIONS We will examine if LHRH release is correlated to [Ca2+]i oscillations. KEY WORDS LHRH neurons, pulsatility, monkey embryos, LHRH release in vitro, intracellular Ca2+
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