To understand the mechanism of LHRH pulse generation, LHRH release pattern and the role of calcium in neurosecretion will be investigated in cultured LHRH neurons. Previously we have described primary LHRH cell cultures derived from the embryonic olfactory placode. The first study showed that cultured LHRH neurons release the decapeptide into media. It was found that LHRH cells release LHRH in a pulsatile manner at approximately 50-minute intervals. Further, LHRH release was stimulated by depolarization with high K+ and the Na+ channel opener, veratridine. However, while the Na+ channel blocker, tetrodotoxin (TTX) suppressed the effects of veratridine, TTX did not alter the effects of high K+. The second study examined the role of extracellular and intracellular Ca2+ in LHRH release. The results are summarized as follows 1) Exposing the cells to a low Ca2+ (20 nM) buffer solution suppressed LHRH release, while exposure to a normal Ca2+ solution (1.25 mM) maintained pulsatile LHRH release; 2) LHRH release from cultured LHRH cells was stimulated by the voltage sensitive L-type Ca2+ channel agonist, Bay K 8644 (10 5M), while it was suppressed by th e L-type Ca2+ channel blocker, nifedipine (1 5M), but not by the N-type channel blocker, (-conotoxin GVIA (1 5M); 3) The intracellular Ca2+ stimulant, ryanodine (1 5M) stimulated LHRH release, while the intracellular Ca2+ transporting ATPase antagonist, thapsigargin (1 and 10 5M), did not yield consistent results; 4) Carbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (FCCP, 1 (M), a mitochondrial Ca2+ mobilizer, stimulated LHRH release, while ruthenium red, a mitochondrial Ca2+ uptake inhibitor, did not induce consistent results. These results indicate that 1) the presence of extracellular Ca2+ is essential for LHRH neurosecretion, 2) Ca2+ enters the cell via L-type channels, but not N-type channels, and 3) mobilization of intracellular Ca2+ from inositol 1,4,5-triphosphate (IP3)-sensitive stores as well as mitochondrial stores, appears to contribute to LHRH release in these cells. FUTURE DIRECTIONS We will examine if LHRH release is correlated with intracellular calcium oscillations. KEY WORDS LHRH neurons, pulsatility, monkey embryos, LHRH release in vitro, intracellular Ca2+ FUNDING NIH HD15433 & RR00167 PUBLICATIONS Terasawa, E. 1998. Cellular mechanism of pulsatile LHRH release. Gen. Comp. Endocrinol. 112 283-295. [J]
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