title: Altered rate of synthesis of gonadotropin-releasing hormone receptors: effects of homologous hormone appear independent of extracellular calcium.
abstract: Steady state levels of plasma membrane receptors (measured in radioligand assays) result from processes that contribute to the production of receptors (synthesis, recycling, and unmasking) as well as those that contribute to the loss of receptors (degradation, internalization, and inactivation). Accordingly, we have previously adapted the density shift technique to determine the contribution due to synthesis of GnRH receptors. In the present study we have evaluated the ability of homologous hormone to affect the rate of synthesis of these receptors. Additionally, because of its role in other actions of the releasing hormone, the requirement for extracellular Ca2+ to mediate the effects of GnRH was assessed. Cultures of pituitary cells, prepared from female weanling rats, were used for all studies. After treatment with GnRH, a GnRH antagonist, or the calcium ionophore A23187, with or without EGTA (a calcium chelator), cells were further cultured for up to 24 h in medium containing either dense or normal amino acids. After this treatment, receptors for GnRH were covalently linked to a radiolabeled photoaffinity probe [( 125I]Tyr5-[azido-benzoyl-D-Lys6-GnRH]) then solubilized in 1% sodium dodecyl sulfate. Receptors that had incorporated the dense amino acids (i.e. newly synthesized receptors) were separated from those that had been synthesized before the addition of dense amino acids by velocity sedimentation in sucrose gradients (0-20% sucrose, 1% sodium dodecyl sulfate, and 10 mM Tris-HCl, pH 7.0; centrifuged at 156,000 x g for 24 h). After centrifugation, gradients were fractionated, and the radioactivity in each fraction was quantified. GnRH treatment (10 or 0.1 nM) increased the rate at which dense amino acids were incorporated into GnRH receptors (t 1/2 = 13 +/- 2, 15 +/- 1, and 25 +/- 2 h for 0.1 nM GnRH, 10 nM GnRH, and control values, respectively). GnRH antagonist alone did not change the rate of GnRH receptor synthesis (t 1/2 = 22 +/- 3 h) compared to the control value (t 1/2 = 25 +/- 2 h) and was able to block the effects of GnRH. The effects of GnRH were not antagonized by inclusion of 3 mM EGTA during treatment (t 1/2 = 15 +/- 1 h vs. 13 +/- 2 h for 0.1 nM GnRH in the presence and absence of 3 mM EGTA, respectively).(ABSTRACT TRUNCATED AT 400 WORDS)
authors.affiliation: Department of Pharmacology, University of Iowa College of Medicine, Iowa City 52242-1109.
authors.forename: T D
authors.initials: TD
authors.lastname: Braden
authors.forename: P M
authors.initials: PM
authors.lastname: Conn
mesh_terms: D000345:Affinity Labels; D000818:Animals; D001386:Azides / Q000378:metabolism; D000001:Calcimycin / Q000494:pharmacology; D002118:Calcium / Q000494:pharmacology*; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D004533:Egtazic Acid / Q000494:pharmacology; D005260:Female; D007987:Gonadotropin-Releasing Hormone / Q000031:analogs & derivatives / Q000037:antagonists & inhibitors / Q000378:metabolism / Q000494:pharmacology*; D007700:Kinetics; D010902:Pituitary Gland / Q000187:drug effects / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D011966:Receptors, LHRH / Q000096:biosynthesis*
chemical_list: D000345:Affinity Labels; D001386:Azides; D011966:Receptors, LHRH; D007987:Gonadotropin-Releasing Hormone; D000001:Calcimycin; D004533:Egtazic Acid; C031737:LHRH, N-epsilon-azidobenzoyl-Lys(6)-; C042020:LHRH, acetyl-2-(2-naphthyl)-Ala(1)-4-F-Phe(2)-Trp(3)-Arg(6)-; D002118:Calcium