Hydrogen peroxide induces loss of dopamine transporter activity: a calcium-dependent oxidative mechanism.
H2O2 dose dependently inhibited dopamine uptake in PC12 cells and in striatal synaptosomes. Treatment with H2O2 resulted in a reversible reduction in Vmax, with no effect on its Km value. This suppressive effect of H2O2 could be relieved by reducing agents (dithiothreitol and cysteine). Furthermore, an oxidizer (dithiodipyridine) also markedly suppressed the dopamine transporter (DAT). Oxidative stress therefore might contribute to the action of H2O2. H2O2 appeared to modify DAT at both extracellular and intracellular sites because cumene-H2O2 (a radical generator mostly restricted to plasma membranes) at high concentrations also slightly suppressed DAT activity and the intracellular overexpression of catalase ameliorated the inhibitory effect of H2O2. Internalization was unlikely to be involved because concanavalin A, which blocked endocytosis, did not prevent the H2O2-evoked inhibition of DAT activity. Interestingly, H2O2 treatment evoked a Ca2+ influx in PC12 cells. Moreover, removal of external calcium by EGTA or reduction in the intracellular calcium level using BAPTA-AM reversed the inhibitory effect of H2O2. Conversely, depletion of intracellular calcium stores using thapsigargin did not affect the reduction in DAT activity by H2O2. Collectively, our results indicate that the DAT, one of the most important proteins controlling the dopaminergic system, is also a redox sensor. In addition, H2O2 might suppress the DAT by a Ca2+-dependent oxidative pathway.
Huang
Chuen-Lin
CL
Institute of Biomedical Sciences, Academia Sinica, Taipei 11529, Taiwan, Republic of China.
Huang
Nai-Kuei
NK
Shyue
Song-Kun
SK
Chern
Yijuang
Y
D000818:Animals; D002118:Calcium / Q000378:metabolism*; D002478:Cells, Cultured; D003042:Cocaine / Q000494:pharmacology; D003342:Corpus Striatum / Q000737:chemistry; D004298:Dopamine / Q000493:pharmacokinetics; D050483:Dopamine Plasma Membrane Transport Proteins; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006801:Humans; D006861:Hydrogen Peroxide / Q000494:pharmacology*; D017665:Hydroxyl Radical / Q000378:metabolism; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins / Q000187:drug effects* / Q000235:genetics / Q000378:metabolism*; D009419:Nerve Tissue Proteins; D016877:Oxidants / Q000494:pharmacology*; D010084:Oxidation-Reduction / Q000187:drug effects; D018384:Oxidative Stress / Q000187:drug effects; D016716:PC12 Cells; D051381:Rats; D017207:Rats, Sprague-Dawley; D013574:Synaptosomes / Q000737:chemistry / Q000187:drug effects / Q000378:metabolism; D014162:Transfection
D050483:Dopamine Plasma Membrane Transport Proteins; D004791:Enzyme Inhibitors; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D016877:Oxidants; C493214:SLC6A3 protein, human; C493216:Slc6a3 protein, rat; D017665:Hydroxyl Radical; D006861:Hydrogen Peroxide; D003042:Cocaine; D002118:Calcium; D004298:Dopamine