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Biology of Reproduction, Vol 21, 1073-1086, Copyright © 1979 by Society for the Study of Reproduction
1 Department of Physiology and Biophysics,
Colorado State University,
Fort Collins, Colorado 80523 The role of divalent cations, microtubules and microfilaments in mediating secretion of LH
from GnRH stimulated gonadotrophs was examined in ovine anterior pituitary cells cultured in
suspension. GnRH did not stimulate release of LH in suspensions of acutely dispersed cells. Release
of LH in response to GnRH was reestablished with time of culture; maximal responsiveness occurred after 18 h of culture. Conversely, release of cAMP in response to GnRH was increased 6-fold
in acutely dispersed cells. GnRH specific release of cAMP from cells immediately after dissociation
did not differ from GnRH specific release from cells cultured for 18 h. Culture of anterior pituitary
cells with actinomycin D or cycloheximide reduced total LH (media plus cells) following 18 h of
culture. GnRH specific release of LH, but not cAMP, was depressed relative to the 18 h control
from cells cultured for 18 h with inhibitors of transcription or translation. In a calcium-free environment, GnRH did not stimulate release of LH. Upon readdition of calcium, however, release of
LH from gonadotrophs stimulated by GnRH was elevated above basal levels with maximal release
occurring at concentrations of extracellular calcium of 1 mM and greater. Release of LH induced
by 73.5 mM K+ was also calcium dependent. High K+ stimulated release of LH above basal release
at 0.5 mM calcium. At no concentration of calcium did the release of LH induced by 73.5 mM K+
approach the release stimulated by 20 nM GnRH. Release of LH induced by 73.5 mM K+ and
20 nM GnRH was not additive. Increasing concentrations of magnesium (0-16 mM) had no
effect on specific release of LH stimulated by 200 nM GnRH, but increased the GnRH specific
release of cAMP 2.21-fold. Conversely, increasing concentrations of barium (0-4 mM) increased
GnRH specific release of LH 2.8-fold but depressed specific release of cAMP by 67%. GnRH specific release of LH from anterior pituitary cells was unaffected by coincubation with
colchicine (10 µM) or vinblastine (10 µM). Compared with controls, specific release of LH from
pituitary cells stimulated with 0.2 nM, 2.0 nM or 20 nM GnRH was reduced significantly during
coincubation with cytochalasin B (10 µg/ml). These results indicate that release of LH in response to GnRH is mediated by calcium and may
involve microfilaments but not microtubules. A model of stimulus-secretion coupling in the GnRH
activated gonadotroph is proposed and discussed.
Accepted on September 5, 1979
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