Free radical production induced by methamphetamine in rat striatal synaptosomes.
The pro-oxidative effect of methamphetamine (METH) in dopamine terminals was studied in rat striatal synaptosomes. Flow cytometry analysis showed increased production of reactive oxygen species (ROS) in METH-treated synaptosomes, without reduction in the density of dopamine transporters. In synaptosomes from dopamine (DA)-depleted animals, METH did not induce ROS production. Reserpine, in vitro, completely inhibited METH-induced ROS production. These results point to endogenous DA as the main source of ROS induced by METH. Antioxidants and inhibitors of neuronal nitric oxide synthase and protein kinase C (PKC) prevented the METH-induced oxidative effect. EGTA and the specific antagonist methyllycaconitine (MLA, 50 microM) prevented METH-induced ROS production, thus implicating calcium and alpha7 nicotinic receptors in such effect. Higher concentrations of MLA (>100 microM) showed nonspecific antioxidant effect. Preincubation of synaptosomes with METH (1 microM) for 30 min reduced [(3)H]DA uptake by 0%. The METH effect was attenuated by MLA and EGTA and potentiated by nicotine, indicating that activation of alpha(7) nicotinic receptors and Ca(2+) entry are necessary and take place before DAT inhibition. From these findings, it can be postulated that, in our model, METH induces DA release from synaptic vesicles to the cytosol. Simultaneously, METH activates alpha(7) nicotinic receptors, probably inducing depolarization and an increase in intrasynaptosomal Ca(2+). This would lead to DAT inhibition and NOS and PKC activation, initiating oxidation of cytosolic DA.
Pubill
David
D
Unitat de Farmacologia i Farmacognòsia, Facultat de Farmàcia, Nucli Universitari de Pedralbes, Universitat de Barcelona, 08028 Barcelona, Spain. d.pubill@ub.edu
Chipana
Carlos
C
Camins
Antonio
A
Pallàs
Mercè
M
Camarasa
Jordi
J
Escubedo
Elena
E
D000818:Animals; D000697:Central Nervous System Stimulants / Q000633:toxicity*; D004298:Dopamine / Q000378:metabolism; D050483:Dopamine Plasma Membrane Transport Proteins; D005609:Free Radicals / Q000378:metabolism*; D066298:In Vitro Techniques; D008297:Male; D008562:Membrane Glycoproteins / Q000378:metabolism; D026901:Membrane Transport Proteins / Q000378:metabolism; D008694:Methamphetamine / Q000633:toxicity*; D017072:Neostriatum / Q000187:drug effects* / Q000378:metabolism; D009419:Nerve Tissue Proteins / Q000378:metabolism; D051381:Rats; D017207:Rats, Sprague-Dawley; D017382:Reactive Oxygen Species / Q000378:metabolism*; D011978:Receptors, Nicotinic / Q000187:drug effects / Q000378:metabolism; D013574:Synaptosomes / Q000187:drug effects* / Q000378:metabolism; D018675:Toxicity Tests; D064569:alpha7 Nicotinic Acetylcholine Receptor
D000697:Central Nervous System Stimulants; C579165:Chrna7 protein, rat; D050483:Dopamine Plasma Membrane Transport Proteins; D005609:Free Radicals; D008562:Membrane Glycoproteins; D026901:Membrane Transport Proteins; D009419:Nerve Tissue Proteins; D017382:Reactive Oxygen Species; D011978:Receptors, Nicotinic; C493216:Slc6a3 protein, rat; D064569:alpha7 Nicotinic Acetylcholine Receptor; D008694:Methamphetamine; D004298:Dopamine