REGULATION OF TYROSINE-HYDROXYLASE GENE-EXPRESSION BY THE ML MUSCARINIC ACETYLCHOLINE-RECEPTOR IN RAT PHEOCHROMOCYTOMA CELLS

Tyrosine hydroxylase (TH) gene transcription rate is increased in rat adrenal medulla after administration of muscarinic agonists. In order to study this muscarinic regulation of TH gene expression in more deta il, we have generated a rat pheochromocytoma PC18 cell line that stabl y expresses the mouse mi muscarinic acetylcholine receptor. Treatment of this cell line, designated PC18/ml-13, with carbachol leads to rapi d increases in phosphatidylinositol turnover and intracellular [Ca2+]( i); these increases are totally blocked by the muscarinic antagonist a tropine. Carbachol produces no changes in cAMP levels or protein kinas e A activity in PC18/ml-13 cells. TH mRNA levels in PC18/ml-13 cells i ncrease approximately 3-fold after 6 h of treatment with carbachol. Th is induction of TH mRNA is also completely inhibited by simultaneous t reatment with atropine. Transient transfection assays using a TH gene promoter-chloramphenicol acetyltransferase (TH-CAT) construct demonstr ate that sequences within the most proximal 272 bp of the TH gene 5'-f lanking region are responsive to carbachol in PC18/ml-13 cells. Studie s using TH-CAT constructs with site-directed mutations within the TH g ene promoter indicate that the responsiveness of the promoter to carba chol is mediated primarily by the cAMP response element; however, the API site also participates to a lesser extent in this response. The ca rbachol-mediated stimulation of TH gene promoter activity is partially inhibited by down-regulation of protein kinase C (PKC) or by treatmen t with the Ca2+/calmodulin-dependent protein kinase inhibitor, KN62. T hese results are consistent with the hypothesis that agonist occupatio n of mi muscarinic receptors stimulates the TH gene via signal transdu ction pathways that are initiated by activation of PKC and Ca2+/calmod ulin-dependent protein kinase, leading to activation of transcription factors that interact with the TH CRE and API sites.