Evaluation of neurotropic drug actions on tyrosine hydroxylase activity and dopamine metabolism in clonal cell lines.
Two clonal cell lines (the pheochromocytoma clone PC-12 and the neuroblastoma clone N1E-115) were used to compare direct and indirect drug effects on tyrosine hydroxylase and dopamine turnover. Both clones contain the cofactor of tyrosine hydroxylase, tetrahydrobiopterin, in sufficient concentrations. 2,4-Diamino-6-hydroxy-pyrimidine (DAO-Pyr), an inhibitor of GTP cyclohydrolase, which is the rate-limiting enzyme in tetrahydrobiopterin biosynthesis, lowers DOPA production indicating that cofactor supply is a limiting factor for catecholamine synthesis. DOPA synthesis in the PC-12 cells can be stimulated by incubation with the natural cofactor tetrahydrobiopterin, but also by its possible precursors sepiapterin and dihydrobiopterin or the analogs methyl-tetrahydropterin and dihydropterin. The regulating enzyme for DOPA synthesis, tyrosine hydroxylase, can be inhibited by certain drugs either directly or indirectly by increasing dopamine concentrations in the cytoplasm after release from its vesicular stores. Using the neuroblastoma clone N1E-115 which lacks DOPA decarboxylase and thus contains only low levels of dopamine the site of action of certain drugs could be determined. Drugs affecting the tyrosine hydroxylase directly (alpha-methyl-para-tyrosine, apomorphine) decreased DOPA production in both clones, while drugs acting via interference with the vesicular stores (reserpine, amphetamine, nigericin) were effective only in the PC-12 cells. After total depletion of dopamine by nigericin at high concentrations or long-term incubation with 3-hydroxybenzyl-hydrazine (NSD 1015), DOPA production increased in the PC-12 cells indicating a usually occurring regulation of tyrosine hydroxylase by cytoplasmic dopamine. Dopamine concentration in the cytoplasm was calculated to be in the range of 1 X 10(-6) mol/l.(ABSTRACT TRUNCATED AT 250 WORDS)
Bräutigam
M
M
Kittner
B
B
Herken
H
H
D000818:Animals; D001708:Biopterins / Q000031:analogs & derivatives / Q000378:metabolism; D002396:Catechols / Q000378:metabolism; D002460:Cell Line; D002999:Clone Cells; D004295:Dihydroxyphenylalanine / Q000096:biosynthesis; D004298:Dopamine / Q000378:metabolism*; D005910:Glioma; D051379:Mice; D009447:Neuroblastoma; D009474:Neurons / Q000187:drug effects* / Q000201:enzymology / Q000378:metabolism; D014446:Tyrosine 3-Monooxygenase / Q000037:antagonists & inhibitors*
D002396:Catechols; D001708:Biopterins; D004295:Dihydroxyphenylalanine; D014446:Tyrosine 3-Monooxygenase; C003402:sapropterin; D004298:Dopamine