In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRH-Rs) on rat pituitary tumor (GH3) cells was preceded by a decrease in the activity of the mRNA for the TRH-R, as assayed in Xenopus oocytes. Here we report the effects of TRH, elevation of cytoplasmic free Ca2+ concentration, phorbol myristate acetate (PMA), and H-7 [1-(5-isoquinolinesulfonyl)2-methylpiperazine dihydrochloride], an inhibitor of protein kinases, on the levels of TRH-R mRNA, which were measured by Northern analysis and in nuclease protection assays using probes made from mouse pituitary TRH-R cDNA, in GH3 cells. These agents were studied to gain insight into the mechanism of the TRH effect, because signal transduction by TRH involves generation of inositol 1,4,5-trisphosphate and elevation of cytoplasmic free Ca2+ concentration, which leads to activation of Ca2+/calmodulin-dependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 microM TRH, a maximally effective dose) caused a marked transient decrease in TRH-R mRNA that attained a nadir of 20-45% of control by 3-6 h, increased after 9 h, but was still below control levels after 24 h. Elevation of the cytoplasmic free Ca2+ concentration had no effect on TRH-R mRNA. A maximally effective dose of PMA (1 microM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 microM TRH. H-7 (20 microM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents.(ABSTRACT TRUNCATED AT 250 WORDS)