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Wisconsin Regional Primate Research Center (E.T., K.L.K., K.M., P.C.) and Department of Pediatrics (E.T.), University of Wisconsin, Madison, Wisconsin 53715
Address all correspondence and requests for reprints to: Ei Terasawa, Ph.D., Wisconsin Regional Primate Research Center, 1223 Capitol Court, Madison, Wisconsin 53715-1299. E-mail: terasawa{at}primate.wisc.edu
To study the mechanism of LH-releasing hormone (LHRH) pulse
generation, the olfactory pit/placode and the migratory pathway of LHRH
neurons from monkey 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 decapeptide into media. It was found that
LHRH cells release LHRH in a pulsatile manner at approximately 50-min
intervals. Further, LHRH release was stimulated by depolarization with
high K+ and the Na+ channel opener,
veratridine. However, whereas the Na+ channel blocker,
tetrodotoxin suppressed the effects of veratridine, tetrodotoxin did
not alter the effects of high K+. Subsequently, the role of
extracellular and intracellular Ca2+ in LHRH release was
examined. The results are summarized as follows: 1) exposing the cells
to a low Ca2+ (20 nM) buffer solution
suppressed LHRH release, whereas 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 µM), whereas it was suppressed by the L-type
Ca2+ channel blocker, nifedipine (1 µM), but
not by the N-type channel blocker, 
-conotoxin GVIA (1
µM); 3) the intracellular Ca2+ stimulant,
ryanodine (1 µM), stimulated LHRH release, whereas the
intracellular Ca2+ transporting adenosine triphosphatase
antagonist, thapsigargin (1 and 10 µM), did not yield
consistent results; and 4) carbonyl cyanide
p-trifluoromethoxyphenyl-hydrazone (1 µM), a
mitochondrial Ca2+ mobilizer, stimulated LHRH release,
whereas 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-sensitive stores, as
well as mitochondrial stores, seem to contribute to LHRH release in
these cells.
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J. E. Levine Editorial: Pulsatility in Primates--A Perspective from the Placode Endocrinology, March 1, 1999; 140(3): 1033 - 1035. [Full Text]
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E. A. Vitalis, J. L. Costantin, P.-S. Tsai, H. Sakakibara, S. Paruthiyil, T. Iiri, J.-F. Martini, M. Taga, A. L. H. Choi, A. C. Charles, et al. Role of the cAMP signaling pathway in the regulation of gonadotropin-releasing hormone secretion in GT1 cells PNAS, February 15, 2000; 97(4): 1861 - 1866. [Abstract] [Full Text] [PDF]
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