6-Hydroxydopamine increases the hydroxylation and nitration of phenylalanine in vivo: implication of peroxynitrite formation

In the present study, we investigated the effect of the dopaminergic neurot oxin 6-hydroxydopamine (6-OHDA) on hydroxyl free radical and peroxynitrite formation in vivo using D-phenylalanine as a novel mechanistic probe. In vi vo microdialysis was carried out in the striatum of freely moving male Wist ar rats. The microdialysis probes were perfused with artificial cerebrospin al fluid containing 5 MM D-phenylalanine (flow rate 2 muL/min). After obtai ning a stable baseline 6-OHDA was delivered into the striatum via reverse m icrodialysis for 60 min. HPLC measurements of the effluent were performed u sing photodiode array detection for determination of phenylalanine derived o-tyrosine and m-tyrosine (as hydroxylation markers) as well as of nitrotyr osine and nitrophenylalanine (as nitration markers). The basal levels of th e hydroxylation derived products of phenylalanine were approximately 100-fo ld higher than those of the nitration derived products. 6-OHDA (0.1, 1, 10 mm) significantly increased o- and m-tyrosine up to nine- and 13-fold, resp ectively, whereas levels of 3-nitrotyrosine and 4-nitrophenylalanine were s ignificantly increased up to 422- and 358-fold, respectively. The results d emonstrate that phenylalanine is a sensitive in vivo marker for 6-OHDA-indu ced hydroxylation and nitration reactions which are clearly concentration d ependent. We conclude that peroxynitrite formation is involved in 6-OHDA-in duced neurochemical effects.