HOMOLOGOUS DOWN-REGULATION OF GROWTH HORMONE-RELEASING HORMONE-RECEPTOR MESSENGER-RIBONUCLEIC-ACID LEVELS

Repeated stimulation of pituitary cell cultures with GH releasing horm one (GHRH) results in diminished responsiveness, a phenomenon referred to as homologous desensitization. One component of GHRH-induced desen sitization is a reduction in GHRH-binding sites, which is reflected by the decreased ability of GHRH to stimulate a rise in intracellular cA MP. In the present study, we sought to determine if homologous down-re gulation 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 m essenger RNA (mRNA) levels in total RNA samples obtained from rat pitu itary cell cultures. Treatment of pituitary cells with GHRH, for as li ttle as 4 h, resulted in a dose-dependent decrease in GHRH-R mRNA leve ls. The maximum effect was observed with 0.1 and 1 nM GHRH, which redu ced GHRH-R mRNA levels to 49 +/- 4% (mean +/- SEM) and 54 +/- 11% of c ontrol values, respectively (n = three separate experiments; P < 0.05) . Accompanying the decline in GHRH-R mRNA levels was a rise in GH rele ase, 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 a chieve a concentration comparable with that induced by a maximal stimu lation with GHRH (8 mu g GH/ml medium). Following pretreatment, cultur es were stimulated for 15 min with GHRH and intracellular cAMP accumul ation was measured by RIA. GH pretreatment did not impair the ability of GHRH to induce a rise in cAMP concentrations. However, as anticipat ed, GHRH pretreatment (10 mM) significantly reduced subsequent GHRH-st imulated cAMP to 46% of untreated controls. These data suggest that GH RH, but not GH, directly reduces GHRH-R mRNA levels. To determine whet her this effect was mediated through cAMP, cultures were treated with forskolin, a direct stimulator of adenylate cyclase. Forskolin (10 mu M) significantly reduced GHRH-IZ mRNA concentrations (37 +/- 6% of con trol values) indicating that GHRH acts through the cAMP-second messeng er system cascade to regulate GHRH-R mRNA. The somatostatin analogue, octreotide (10 nM), which has been previously reported to decrease ade nylate cyclase activity, did not affect GHRH-R mRNA levels. Taken toge ther, these results indicate that GHRH inhibits the production of its own receptor by a receptor-mediated, cAMP-dependent reduction of GHRH- R mRNA accumulation.