Neural regulation of phenylethanolamine N-methyltransferase (PNMT) gene expression in bovine chromaffin cells differs from other catecholamine enzyme genes.
Expression of the gene encoding the epinephrine-synthesizing enzyme phenylethanolamine N-methyltransferase (PNMT) is regulated by hormonal and neural stimuli. Because the 5'-upstream regions of the PNMT do not contain sequences analogous to those demonstrated to convey neural regulation to the tyrosine hydroxylase (TH) and dopamine beta-hydroxylase (DBH) catecholamine-synthesizing enzyme genes, functional and biochemical analyses have been utilized to characterize PNMT promoter responses to cholinergic and depolarizing agents. In primary cultures of bovine adrenal medullary chromaffin cells, reporter gene expression from transiently transfected 3- and 0.9-kb-containing PNMT promoter constructs is stimulated approximately twofold by nicotine and muscarine. Depolarizing concentrations of K+ produce fourfold increases in expression. These responses are not detected with constructs containing the proximal 0.3-kb promoter, indicating that the regions between -273 and -877 bp convey neural responsiveness for the PNMT gene in bovine chromaffin cells. Electrophoretic mobility shift assays (EMSAs) with oligonucleotides encoding these regions of the PNMT promoter revealed distinctions in migration of nuclear protein complexes formed following treatment of chromaffin cells with nicotine, muscarine, or 50 mM K+. Thus, the PNMT promoter between 0.3 and 0.9 kb contains sequences capable of responding to cholinergic and depolarization stimuli. Moreover, these treatments influence the interactions of specific nuclear proteins with this region of the PNMT promoter.
Lee
Y S
YS
Department of Pediatrics, SUNY at Stony Brook, NY 11794-8111, USA.
Raia
G
G
Tönshoff
C
C
Evinger
M J
MJ
D000313:Adrenal Medulla / Q000201:enzymology*; D000818:Animals; D001483:Base Sequence; D001665:Binding Sites; D002417:Cattle; D002467:Cell Nucleus / Q000502:physiology; D002478:Cells, Cultured; D019439:Chromaffin Cells / Q000201:enzymology*; D004299:Dopamine beta-Hydroxylase / Q000235:genetics; D015971:Gene Expression Regulation, Enzymologic / Q000187:drug effects; D017930:Genes, Reporter; D008564:Membrane Potentials / Q000187:drug effects; D008969:Molecular Sequence Data; D009116:Muscarine / Q000494:pharmacology*; D009538:Nicotine / Q000494:pharmacology*; D009687:Nuclear Proteins / Q000378:metabolism; D010625:Phenylethanolamine N-Methyltransferase / Q000235:genetics*; D011188:Potassium / Q000494:pharmacology; D011401:Promoter Regions, Genetic / Q000187:drug effects; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic / Q000187:drug effects; D014162:Transfection; D014446:Tyrosine 3-Monooxygenase / Q000235:genetics
D009687:Nuclear Proteins; D014157:Transcription Factors; D009538:Nicotine; D009116:Muscarine; D014446:Tyrosine 3-Monooxygenase; D004299:Dopamine beta-Hydroxylase; D010625:Phenylethanolamine N-Methyltransferase; D011188:Potassium