Figure 2.

Modification of parkin by 4-HNE in striatal synaptosomes in METH-treated rats. Rats were given saline (1 mL/kg) or METH (4 × 10 mg/kg. i.p., every 2 hrs) and killed at 1 hr after the last injection. Parkin was immunoprecipitated from striatal and cerebellar synaptosomes (A) In the striatum, METH decreased the levels of parkin (blots on the left, top panel) and increased the levels of 4-HNE-parkin (bottom panel), but not S-nitrosocysteine-parkin (middle panel) at 1 hr after the last dose of the drug as compared to saline controls. Omission of parkin antibody or addition of parkin peptide corresponding to the epitope of the parkin antibody during immunoprecipitation did not produce bands at 52 kDa neither with antibody against parkin nor with antibody against 4-HNE-protein conjugates (blots on the right). (B) Illustrated are ratios of 4-HNE to parkin immunoreactivity normalized to saline controls. Data are expressed as mean ± SEM. 4-HNE/parkin ratio was augmented by METH in striatal (+40%, p<0.05, Student’s t-test, n=3–6), but not cerebellar, synaptosomes. (C) Incubation of striatal synaptosomes from saline-treated animals with H₂O₂/Fe²⁺ (200/10 µM) for 1 hr at 37°C decreased parkin immunoreactivity (blot on the left) and increased 4-HNE immunoreactivity on immunoprecipitated parkin (blot on the right). In samples on the right, the levels of parkin were equalized to show the difference in 4-HNE levels. * Significantly different from saline controls. Abbreviations: SAL, saline; METH, methamphetamine; IP, immunoprecipitation; WB, western blotting; 4-HNE, 4-hydroxy-2-nonenal; STR, striatum; CER, cerebellum; ctrl, control.