Competitive irreversible inhibition of dopamine uptake by 6-hydroxydopamine.
We examined the effects of 6-hydroxydopamine (6-OHDA) treatment on the human neuroblastoma cell line SK-N-SH-SY5Y (SY5Y) and the rat pheochromocytoma cell line, PC12. Structural and metabolic integrity was tested by measuring the ability of cells to transport the non-metabolizable amino acid analogue [3H]-alpha-aminoisobutyric acid (AIB). We determined that treatment with 6-OHDA at concentrations of 49 microM and 62 microM inhibited 50% of the AIB uptake in SY5Y and PC12 cells, respectively. Inhibition of AIB uptake was prevented by the addition of catalase, but was not influenced by the addition of 1 mM dopamine. This indicated that cell damage resulted from the generation of H2O2 and was independent of the catecholamine uptake system. Effects directly on the catecholamine uptake system were observed by measuring the uptake of 3H-dopamine. In contrast to the effects on amino acid uptake, dopamine uptake was significantly inhibited by 6-OHDA treatment, and this inhibition was not prevented by the addition of catalase. The results indicate a Ki of 430 microM for inhibition of dopamine uptake by 6-OHDA treatment of PC12 cells. The results are consistent with a competitive irreversible inhibition of the dopamine uptake sites by 6-OHDA or one of its metabolites. Thus, the lack of a catecholamine uptake-dependent cellular toxicity appears to result from the direct inactivation of catecholamine uptake sites. Similarly, the inhibition of dopamine uptake in vivo by 6-OHDA may be explained, at least in part, by direct inactivation of dopamine uptake sites rather than exclusively by intracellular transport and action of 6-OHDA.
Decker
D E
DE
Upjohn Company, Kalamazoo, MI 49001.
Althaus
J S
JS
Buxser
S E
SE
VonVoigtlander
P F
PF
Ruppel
P L
PL
D000310:Adrenal Gland Neoplasms / Q000378:metabolism; D000621:Aminoisobutyric Acids / Q000037:antagonists & inhibitors / Q000378:metabolism; D000818:Animals; D001667:Binding, Competitive; D001692:Biological Transport; D002374:Catalase / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism*; D018492:Dopamine Antagonists; D006801:Humans; D009447:Neuroblastoma / Q000378:metabolism; D016627:Oxidopamine / Q000494:pharmacology*; D010673:Pheochromocytoma / Q000378:metabolism; D051381:Rats; D014407:Tumor Cells, Cultured
D000621:Aminoisobutyric Acids; D018492:Dopamine Antagonists; D016627:Oxidopamine; D002374:Catalase; D004298:Dopamine