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Thyrotropin-Releasing Hormone (TRH) and Phorbol Myristate Acetate Decrease TRH Receptor Messenger RNA in Rat Pituitary GH₃ Cells: Evidence That Protein Kinase-C Mediates the TRH Effect

Division of Endocrinology and Metabolism, Departments of Medicine and Cell Biology and Anatomy, New York Hospital, Cornell University Medical College New York, New York 10021

Address requests for reprints to: Dr. Marvin C. Gershengorn, Room A328, 1300 York Avenue, New York, New York 10021.

Abstract

In a previous report we showed that TRH-induced down-regulation of the density of its receptors (TRHRs) 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⁺/calmodulindependent protein kinase, and of 1,2-diacylglycerol, which leads to activation of protein kinase-C. TRH (1 µM 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 µM) caused decreases in TRH-R mRNA that were similar in magnitude and time course to those induced by 1 µM TRH. H-7 (20 µM) blocked the effects of TRH and PMA to lower TRH-R mRNA to similar extents. These data show that TRH and PMA decrease the levels of TRH-R mRNA in GH₃ cells and are consistent with the idea that the effect of TRH is via a mechanism that is mediated by protein kinase-C.

FOOTNOTES

This work was supported by NIH Grant DK-43036.

^* Present address: Molecular Genetics Laboratory, Columbia University College of Physicians and Surgeons, Box 58, 722 West 168th Street, New York, New York 10032.

Received for publication June 6, 1991. Revision received July 31, 1991. Accepted for publication July 31, 1991.

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