Inhibition of biopterin synthesis and DOPA production in PC-12 pheochromocytoma cells induced by 6-aminonicotinamide.
Pheochromocytoma cells (clone PC-12) were treated with 6-aminonicotinamide. Tetrahydrobiopterin content and DOPA production of the cells were determined by reverse-phase HPLC and subsequent electrochemical detection. The same chromatographic system was used to determine total biopterin (tetrahydrobiopterin, dihydrobiopterin and quinoide dihydrobiopterin) by fluorescence detection. Tetrahydrobiopterin plays a decisive role as cofactor of tyrosine hydroxylase for the biosynthesis of DOPA and dopamine. Addition of 6-aminonicotinamide to the culture medium resulted in the accumulation of 6-phosphogluconate, suggesting that PC-12 cells synthesize 6-aminonicotinamide-adenine-dinucleotide-phosphate (6-ANADP) by a glycohydrolase localized in the endoplasmic reticulum. This substance is known to be a strong inhibitor of 6-phosphogluconate dehydrogenase and leads to a blockade of the pentose phosphate pathway. In our experiments, the synthesis of biopterins was depressed after application of 6-aminonicotinamide. The decrease of intracellular tetrahydrobiopterin and total biopterin by 6-aminonicotinamide at different concentrations was strongly correlated with a reduced cellular DOPA production. The decreased content of biopterin cofactor was compensated by addition of the precursor sepiapterin, indicating that the NADPH2-dependent reductases in biopterin synthesis are not inhibited by the antimetabolite. However, DOPA production remained suppressed at the same time. After application of NADH2, we observed an increased DOPA production though the decreased biopterin levels remained almost unchanged. The results imply that the first step in the synthesis of biopterin from GTP as well as the recycling pathways of the oxidized cofactor might be the site of action of the antimetabolite.(ABSTRACT TRUNCATED AT 250 WORDS)
Jung
W
W
Institut für Pharmakologie, Freie Universität, Berlin.
Herken
H
H
D015120:6-Aminonicotinamide / Q000494:pharmacology*; D000429:Alcohol Oxidoreductases / Q000378:metabolism; D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000032:analysis / Q000096:biosynthesis*; D002851:Chromatography, High Pressure Liquid; D004093:Dihydropteridine Reductase / Q000378:metabolism; D004295:Dihydroxyphenylalanine / Q000096:biosynthesis*; D005942:Gluconates / Q000378:metabolism; D009536:Niacinamide / Q000031:analogs & derivatives*; D010673:Pheochromocytoma / Q000378:metabolism*; D051381:Rats; D013762:Tetrahydrofolate Dehydrogenase / Q000378:metabolism; D014407:Tumor Cells, Cultured / Q000187:drug effects / Q000378:metabolism
D005942:Gluconates; D001708:Biopterins; D009536:Niacinamide; D015120:6-Aminonicotinamide; D004295:Dihydroxyphenylalanine; D000429:Alcohol Oxidoreductases; C022630:sepiapterin reductase; D013762:Tetrahydrofolate Dehydrogenase; D004093:Dihydropteridine Reductase; C003402:sapropterin; C008884:6-phosphogluconic acid