title: Homologous down-regulation of growth hormone-releasing hormone receptor messenger ribonucleic acid levels.
abstract: Repeated stimulation of pituitary cell cultures with GH-releasing hormone (GHRH) results in diminished responsiveness, a phenomenon referred to as homologous desensitization. One component of GHRH-induced desensitization is a reduction in GHRH-binding sites, which is reflected by the decreased ability of GHRH to stimulate a rise in intracellular cAMP. In the present study, we sought to determine if homologous down-regulation of GHRH receptor number is due to a decrease in GHRH receptor synthesis. To this end, we developed and validated a quantitative RT-PCR assay system that was capable of assessing differences in GHRH-R messenger RNA (mRNA) levels in total RNA samples obtained from rat pituitary cell cultures. Treatment of pituitary cells with GHRH, for as little as 4 h, resulted in a dose-dependent decrease in GHRH-R mRNA levels. The maximum effect was observed with 0.1 and 1 nM GHRH, which reduced GHRH-R mRNA levels to 49 +/- 4% (mean +/- SEM) and 54 +/- 11% of control values, respectively (n = three separate experiments; P < 0.05). Accompanying the decline in GHRH-R mRNA levels was a rise in GH release; reaching 320 +/- 31% of control values (P < 0.01). Because of the possibility that the rise in medium GH level is the primary regulator of GHRH-R mRNA, we pretreated pituitary cultures for 4 h with GH to achieve a concentration comparable with that induced by a maximal stimulation with GHRH (8 micrograms GH/ml medium). Following pretreatment, cultures were stimulated for 15 min with GHRH and intracellular cAMP accumulation was measured by RIA. GH pretreatment did not impair the ability of GHRH to induce a rise in cAMP concentrations. However, as anticipated, GHRH pretreatment (10 nM) significantly reduced subsequent GHRH-stimulated cAMP to 46% of untreated controls. These data suggest that GHRH, but not GH, directly reduces GHRH-R mRNA levels. To determine whether this effect was mediated through cAMP, cultures were treated with forskolin, a direct stimulator of adenylate cyclase. Forskolin (10 microM) significantly reduced GHRH-R mRNA concentrations (37 +/- 6% of control values) indicating that GHRH acts through the cAMP-second messenger system cascade to regulate GHRH-R mRNA. The somatostatin analogue, octreotide (10 nM), which has been previously reported to decrease adenylate cyclase activity, did not affect GHRH-R mRNA levels. Taken together, these results indicate that GHRH inhibits the production of its own receptor by a receptor-mediated, cAMP-dependent reduction of GHRH-R mRNA accumulation.
authors.affiliation: Department of Medicine, University of Illinois at Chicago 60612, USA.
authors.forename: G
authors.initials: G
authors.lastname: Aleppo
authors.forename: S F
authors.initials: SF
authors.lastname: Moskal
authors.forename: P A
authors.initials: PA
authors.lastname: De Grandis
authors.forename: R D
authors.initials: RD
authors.lastname: Kineman
authors.forename: L A
authors.initials: LA
authors.lastname: Frohman
mesh_terms: D000818:Animals; D002478:Cells, Cultured; D005576:Colforsin / Q000494:pharmacology; D016895:Culture Media, Serum-Free; D003907:Dexamethasone / Q000494:pharmacology; D015536:Down-Regulation; D005260:Female; D013007:Growth Hormone-Releasing Hormone / Q000494:pharmacology; D015282:Octreotide / Q000494:pharmacology; D010903:Pituitary Gland, Anterior / Q000166:cytology / Q000378:metabolism; D016133:Polymerase Chain Reaction; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D018013:Receptors, Neuropeptide / Q000235:genetics*; D018017:Receptors, Pituitary Hormone-Regulating Hormone / Q000235:genetics*; D014158:Transcription, Genetic
chemical_list: D016895:Culture Media, Serum-Free; D012333:RNA, Messenger; D018013:Receptors, Neuropeptide; D018017:Receptors, Pituitary Hormone-Regulating Hormone; D005576:Colforsin; D003907:Dexamethasone; D013007:Growth Hormone-Releasing Hormone; C048250:somatotropin releasing hormone receptor; D015282:Octreotide