Abstract
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 detail, we have generated a rat pheochromocytoma PC18 cell line that stably expresses the mouse m1 muscarinic acetylcholine receptor. Treatment of this cell line, designated PC18/m1-13, with carbachol leads to rapid increases in phosphatidylinositol turnover and intracellular [Ca 2+ ] i ; these increase are totally blocked by the muscarinic antagonist atropine. Carbachol produces no changes in cAMP levels or protein kinase A activity in PC18/m1-13 cells. TH mRNA levels in PC18/m1-13 cells increase approximately 3-fold after 6 h of treatment with carbachol. This induction of TH mRNA is also completely inhibited by simultaneous treatment with atropine. Transient transfection assays using a TH gene promoter-chloramphenicol acetyltransferase (TH-CAT) construct demonstrate that sequences within the most proximal 272 bp of the TH gene 5′-flanking region are responsive to carbachol in PC18/m1-13 cells. Studies using TH-CAT constructs with site-directed mutations within the TH gene promoter indicate that the responsiveness of the promoter to carbachol is mediated primarily by the cAMP response element; however, the AP1 site also participates to a lesser extent in this response. The carbachol-mediated stimulation of TH gene promoter activity is partially inhibited by down-regulation of protein kinase C (PKC) or by treatment with the Ca 2+ /calmodulin-dependent protein kinase inhibitor, KN62. These results are consistent with the hypothesis that agonist occupation of m1 muscarinic receptors stimulates the TH gene via signal transduction pathways that are initiated by activation of PKC and Ca 2+ /calmodulin-dependent protein kinase, leading to activation of transcription factors that interact with the TH CRE and AP1 sites.
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