Iron-induced lipid peroxidation and inhibition of dopamine synthesis in striatum synaptosomes

Crude striatum synaptosomes (P2 fraction) from Fischer 344 female rats were incubated in the presence of ADP-chelated Fe3 (0.5-50 microM) and ascorbate (250 microM). Intrasynaptosomal conversion of tyrosine to dopamine (DA) was measured by 14CO2 evolution from L-[1-14C]tyrosine in the absence of added cofactors and DOPA decarboxylase. Malondialdehyde (MDA) was measured as an index of lipid peroxidation. A concentration-dependent inhibition of DA synthesis by ADP-Fe3./ascorbate was found with 50% inhibition occurring at 2.5 microM Fe3 concentration. This was accompanied by marked accumulation of MDA. Ascorbate or ADP alone did not affect DA synthesis and ADP-Fe3 in the absence of exogenous ascorbate was effective only above 25 microM. Exogenously added MDA did not inhibit DA synthesis. Purified synaptosomes were isolated from peroxidized and control P2 actions using sucrose gradients. Membrane microviscosity of the purified synaptosomes was assessed by nitroxyl spin labels of stearic acid using electron paramagnetic resonance techniques. There was a significant increase in membrane microviscosity as a result of ADP-Fe3./ascorbate induced peroxidation. Maleimide nitroxide spin-label binding to protein sulfhydryls was significantly modified by peroxidation of striatum synaptosomes. The weakly immobilized component of the sulfhydryl spin-label (w) was drastically decreased whereas the strongly immobilized component (s) was modified less, thus leading to a marked reduction of w/s ratio. The exposure of striatum synaptosomes to the peroxidizing system resulted in a significant increase in total iron and in a 25% decrease in protein sulfhydryl content. It is concluded that iron-induced damage to the DA synthetic system is mediated by alterations of the structural properties of nerve ending membranes.