6‐Hydroxydopamine increases the hydroxylation and nitration of phenylalanine <i>in vivo</i>: implication of peroxynitrite formation

In the present study, we investigated the effect of the dopaminergic neurotoxin 6‐hydroxydopamine (6‐OHDA) on hydroxyl free radical and peroxynitrite formation in vivo using d ‐phenylalanine as a novel mechanistic probe. In vivo microdialysis was carried out in the striatum of freely moving male Wistar rats. The microdialysis probes were perfused with artificial cerebrospinal fluid containing 5 m m d ‐phenylalanine (flow rate 2 µL/min). After obtaining a stable baseline 6‐OHDA was delivered into the striatum via reverse microdialysis for 60 min. HPLC measurements of the effluent were performed using photodiode array detection for determination of phenylalanine derived o ‐tyrosine and m ‐tyrosine (as hydroxylation markers) as well as of nitrotyrosine and nitrophenylalanine (as nitration markers). The basal levels of the hydroxylation derived products of phenylalanine were approximately 100‐fold higher than those of the nitration derived products. 6‐OHDA (0.1, 1, 10 m m ) significantly increased o ‐ and m ‐tyrosine up to nine‐ and 13‐fold, respectively, whereas levels of 3‐nitrotyrosine and 4‐nitrophenylalanine were significantly increased up to 422‐ and 358‐fold, respectively. The results demonstrate that phenylalanine is a sensitive in vivo marker for 6‐OHDA‐induced hydroxylation and nitration reactions which are clearly concentration dependent. We conclude that peroxynitrite formation is involved in 6‐OHDA‐induced neurochemical effects.