ACTIVATION OF THYROTROPIN-RELEASING-HORMONE GENE-EXPRESSION IN CULTURED FETAL DIENCEPHALIC NEURONS BY DIFFERENTIATING AGENTS

The present studies were undertaken to investigate the effect of 5-bro mo-2'-deoxyuridine (BrdU; 50 mu M) or forskolin/3-isobutyl-1-methylxan thine (F/I; 10/500 mu M) on TRH gene expression in cultured fetal dien cephalic cells. BrdU as well as drugs such as F/I that raise intracell ular cAMP levels had been previously termed differentiating agents bec ause they cause morphological and functional differentiation of IMR-32 neuroblastoma cells. We postulated that neurons of fetal diencephalon s may remain relatively undifferentiated in vitro and that this might be the reason for low or undetectable TRH production. We hypothesized that treatment with differentiating agents might increase neuropeptide expression. Both BrdU and F/I dramatically (P < 0.01) raised intracel lular TRH and pro-TRH messenger RNA concentrations in cultured diencep halic neurons. Although a short BrdU exposure during the first 4 days of culture was sufficient to irreversibly change TRH neurons and to ca use maintenance of high TRH levels after withdrawal of the drug, F/I h ad to be present continuously throughout the observation period of 16 days to significantly elevate TRH expression. This suggests that BrdU and F/I act at different intracellular sites to activate TRH expressio n in cultured diencephalic neurons. The reduction of glial cells that occurs concurrent with the BrdU treatment was not observed after F/I e xposure, and therefore, this effect does not appear to be a key factor for the induction of TRH expression. As the intracellular accumulatio n of somatostatin and arginine vasopressin, which were determined in p arallel, was similarly enhanced after treatment with BrdU or F/I, our culture system might provide a valuable tool for the study of these an d possibly other neuropeptides in vitro.