Pulsatile release of luteinizing hormone-releasing hormone (LHRH) in cultured LHRH neurons derived from the embryonic olfactory placode of the rhesusmonkey

To study the mechanism of LH-releasing hormone (LHRH) pulse generation, the olfactory pit/placode and the migratory pathway of LHRH neurons from monke y embryos at embryonic age 35-37 were dissected out, under the microscope, and cultured on plastic coverslips coated with collagen in a defined medium for 2-5 weeks. First, we examined whether cultured neurons release the dec apeptide into media. It was found that LHRH cells release LHRH in a pulsati le manner at approximately 50-min intervals. Further, LHRH release was stim ulated by depolarization with high K+ and the Na+ channel opener, veratridi ne. However, whereas the Na+ channel blocker, tetrodotoxin suppressed the e ffects of veratridine, tetrodotoxin did not alter the effects of high K+. S ubsequently, the role of extracellular and intracellular Ca2+ in LHRH relea se was examined. The results are summarized as follows: 1) exposing the cel ls to a low Ca2+ (20 nM) buffer solution suppressed LHRH release, whereas e xposure to a normal Ca2+ solution (1.25 mM) maintained pulsatile LHRH relea se; 2) LHRH release from cultured LHRH cells was stimulated by the voltage- sensitive L-type Ca2+ channel agonist, Bay K 8644 (10 mu M), whereas it was suppressed by the L-type Ca2+ channel blocker, nifedipine (1 mu M), but no t by the N-type channel blocker, omega-conotoxin GVIA (1 mu M); 3) the intr acellular Ca2+ stimulant, ryanodine (1 mu M), stimulated LHRH release, wher eas the intracellular Ca2+ transporting adenosine triphosphatase antagonist , thapsigargin (1 and 10 mu M), did not yield consistent results; and 4) ca rbonyl cyanide p-trifluoromethoxyphenyl-hydrazone (1 mu M), a mitochondrial Ca2+ mobilizer, stimulated LHRH release, whereas ruthenium red, a mitochon drial Ca2+ uptake inhibitor, did not induce consistent results. These resul ts indicate that: 1) the presence of extracellular Ca2+ is essential for LH RH neurosecretion; 2) Ca2+ enters the cell via L-type channels but not N-ty pe channels; and 3) mobilization of intracellular Ca2+ from inositol 1,4,5- triphosphate-sensitive stores, as well as mitochondrial stores, seem to con tribute to LHRH release in these cells.