Dopamine-dependent iron toxicity in cells derived from rat hypothalamus.
We report a new and specific mechanism for iron-mediated neurotoxicity using RCHT cells, which were derived from rat hypothalamus. RCHT cells exhibit immunofluorescent-positive markers for dopamine beta-hydroxylase and the norepinephrine transporter, NET. In the present study, we observed that iron-induced neurotoxicity in RCHT cells was dependent on (i) formation of an Fe-dopamine complex (100 microM FeCl3:100 microM dopamine); (ii) specific uptake of the Fe-dopamine complex into RCHT cells via NET (79+/-2 pmol 59Fe/mg/min; P<0.05), since the uptake of the 59Fe-dopamine complex by the cells was inhibited by 30 microM reboxetine, a specific NET inhibitor (78% inhibition, P<0.001); and (iii) intracellular oxidation of dopamine present in the Fe-dopamine complex to aminochrome; (iv) inhibition of DT-diaphorase, since incubation of RCHT cells with 100 microM Fe-dopamine complex in the presence of 100 microM dicoumarol, an inhibitor of DT-diaphorase, induced significant cell death (51+/-5%; P<0.001). However, this cell death was reduced by 75% when the cells were incubated in the presence of 30 microM reboxetine (P<0.01). No significant cell death was observed when the cells were incubated with 100 microM dopamine, 100 microM Fe-Dopamine complex, 100 microM dicoumarol, or 100 microM FeCl3 (8.3+/-2, 9+/-4, 8.5+/-3, or 9.7+/-2% of control, respectively). ESR studies using the spin trapping agent DMPO showed no formation of hydroxyl radicals when the cells were incubated with 100 microM FeCl3 alone. However, using the same ESR technique, the formation of hydroxyl radicals and a carbon-centered radical was detected when the cells were incubated with 100 microM Fe-dopamine complex in the presence of 100 microM dicoumarol. These studies suggest that iron can induce cell toxicity by a mechanism that requires the formation and NET-mediated uptake of an Fe-dopamine complex, ultimately resulting in the intracellular formation of reactive species.
Paris
Irmgard
I
Program of Molecular and Clinical Pharmacology, ICBM, Faculty of Medicine, and Department of Biophysics, Faculty of Chemistry and Pharmacy, University of Chile, Casilla 70000, Santiago-7, Chile.
Martinez-Alvarado
Pedro
P
Cárdenas
Sergio
S
Perez-Pastene
Carolina
C
Graumann
Rebecca
R
Fuentes
Patricio
P
Olea-Azar
Claudio
C
Caviedes
Pablo
P
Segura-Aguilar
Juan
J
D018759:Adrenergic Uptake Inhibitors / Q000494:pharmacology; D000818:Animals; D016923:Cell Death / Q000187:drug effects; D002460:Cell Line; D002712:Chlorides; D001728:Dicumarol / Q000494:pharmacology; D004298:Dopamine / Q000378:metabolism* / Q000494:pharmacology; D004299:Dopamine beta-Hydroxylase / Q000378:metabolism; D005290:Ferric Compounds / Q000378:metabolism*; D005455:Fluorescent Antibody Technique; D007031:Hypothalamus / Q000187:drug effects* / Q000201:enzymology / Q000473:pathology; D045563:Indolequinones / Q000378:metabolism; D007501:Iron / Q000378:metabolism / Q000633:toxicity*; D018613:Microscopy, Confocal; D009025:Morpholines / Q000494:pharmacology; D016660:NAD(P)H Dehydrogenase (Quinone) / Q000037:antagonists & inhibitors; D050484:Norepinephrine Plasma Membrane Transport Proteins; D051381:Rats; D011916:Rats, Inbred F344; D000077593:Reboxetine; D027981:Symporters / Q000378:metabolism
D018759:Adrenergic Uptake Inhibitors; D002712:Chlorides; D005290:Ferric Compounds; D045563:Indolequinones; D009025:Morpholines; D050484:Norepinephrine Plasma Membrane Transport Proteins; C493226:Slc6a2 protein, rat; D027981:Symporters; C091584:aminochrome 1; D001728:Dicumarol; D000077593:Reboxetine; D007501:Iron; D004299:Dopamine beta-Hydroxylase; D016660:NAD(P)H Dehydrogenase (Quinone); C024555:ferric chloride; D004298:Dopamine