L-NAME reverses quinolinic acid-induced toxicity in rat corticostriatal slices: Involvement of src family kinases.

Quinolinic acid (QA) is an endogenous excitotoxin acting on N-methyl-d-aspartate receptors (NMDARs) that leads to the pathologic and neurochemical features similar to those observed in Huntington's disease (HD). The mechanism of QA toxicity also involves free radicals formation and oxidative stress. NMDARs are particularly vulnerable to the action of reactive oxygen species (ROS) and reactive nitrogen species (RNS) that can act as modulators of the activity of protein tyrosine kinases (PTKs) and phosphotyrosine phosphatases (PTPs). Because QA is able to activate neuronal nitric oxide synthase (nNOS) as well as to stimulate the NMDARs, we evaluated the effect of Nomega-Nitro-l-arginine-methyl ester (l-NAME), a selective nNOS inhibitor, on QA-induced neurotoxicity in rat corticostriatal slices. In electrophysiologic experiments we observed that slice perfusion with QA induced a strong reduction of field potential (FP) amplitude, followed by a partial recovery at the end of the QA washout. In the presence of l-NAME the recovery of FP amplitude was significantly increased with respect to QA alone. In synaptosomes, prepared from corticostriatal slices after the electrophysiologic recordings, we observed that l-NAME pre-incubation reversed the QA-mediated inhibitory effects on protein tyrosine phosphorylation pattern, c-src, lyn, and fyn kinase activities and tyrosine phosphorylation of NMDAR subunit NR2B, whereas the PTP activity was not recovered in the presence of l-NAME. These findings suggest that NO plays a key role in the molecular mechanisms of QA-mediated excitotoxicity in experimental model of HD.

Quinolinic acid modulates the activity of src family kinases in rat striatum: in vivo and in vitro studies.

Quinolinic acid (QA) has been shown to evoke neurotoxic events via NMDA receptor (NMDAR) overactivation and oxidative stress. NMDARs are particularly vulnerable to free radicals, which can modulate protein tyrosine kinase (PTK) and phosphotyrosine phosphatase (PTP) activities. The src family of tyrosine kinases are associated with the NMDAR complex and regulate NMDA channel function. Because QA is an NMDAR agonist as well as a pro-oxidant agent, we investigated whether it may affect the activity of PTKs and PTPs in vivo and in vitro. In synaptosomes prepared from striata dissected 15 min, 30 min or 15 days after bilateral injection of QA we observed modulation of the phosphotyrosine pattern; a significant decrease in PTP activity; and a sustained increase in c-src and lyn activity at 15 and 30 min after treatment with QA, followed by a decrease 2 weeks later. Striatal synaptosomes treated in vitro with QA showed time- and dose-dependent modulation of c-src and lyn kinase activities. Moreover, the nitric oxide synthase inhibitor NG-nitro-L-arginine-methyl ester, the NMDAR antagonist d-2-amino-5-phosphonovaleric acid and pyruvate suppressed the QA-induced modulation of c-src activity. These findings suggest a novel feature of QA in regulating src kinase activity through the formation of reactive radical species and/or NMDAR overactivation.

Protein phosphatase 1alpha is tyrosine-phosphorylated and inactivated by peroxynitrite in erythrocytes through the src family kinase fgr.

Protein serine/threonine phosphorylation is a significant component of the intracellular signal that together with tyrosine phosphorylation regulates several processes, including cell-cycle progression, muscle contraction, transcription, and neuronal signaling. Cross-talk between phosphoserine/threonine- and phosphotyrosine-mediated pathways is not yet well understood. In this study we found that peroxynitrite, a physiological oxidant formed by the fast radical-radical reaction between the nitric oxide and the superoxide anion, induced tyrosine phosphorylation of the serine/threonine protein phosphatase 1alpha (PP1alpha) in human erythrocytes through activation of src family kinases. We have previously shown in mouse red cells that upregulation of the src kinase fgr phosphorylates PP1alpha, acting as an upstream negative regulator of PP1alpha, and downregulates K-Cl cotransport. Here we found that PP1alpha is a selective substrate of peroxynitrite-activated fgr and that tyrosine phosphorylation of PP1alpha corresponds to an inhibition of its enzymatic activity. Despite fgr activation and PP1alpha downregulation, peroxynitrite stimulated in a dose-dependent fashion the function of the K-Cl cotransporter. In an attempt to understand the mechanism of K-Cl cotransport activation, we found that the effect of peroxynitrite is completely reversed by dithriothreitol, suggesting that peroxynitrite acts as an oxidizing agent by an SH-dependent and PP1alpha-independent mechanism. These findings highlight a novel function of peroxynitrite in regulating the intracellular signal transduction pathways involving serine/threonine phosphorylation and the functional role of proteins that are targets of these phosphatases.

Peroxynitrite activates kinases of the src family and upregulates tyrosine phosphorylation signaling.

The hypothesis that peroxynitrite may act as a signaling molecule able to upregulate protein tyrosine phosphorylation is discussed. This article focuses on the mechanisms for activating kinases of the src family, an important class of nonreceptor tyrosine kinases implicated in the regulation of cell communication, proliferation, migration, differentiation, and survival. Recent in vitro findings show that in erythrocytes, synaptosomes, and cerebellar primary culture cells peroxynitrite is able to inhibit phosphatases and to activate different members of the src kinase family through different mechanisms involving cysteine-dependent and -independent processes. The ability of nitrotyrosine-containing peptides with SH2 binding affinity to activate src kinases is also discussed.

Peroxynitrite affects exocytosis and SNARE complex formation and induces tyrosine nitration of synaptic proteins.

The reactive species peroxynitrite, formed via the near diffusion-limited reaction of nitric oxide and superoxide anion, is a potent oxidant that contributes to tissue damage in neurodegenerative disorders. Peroxynitrite readily nitrates tyrosine residues in proteins, producing a permanent modification that can be immunologically detected. We have previously demonstrated that in the nerve terminal, nitrotyrosine immunoreactivity is primarily associated with synaptophysin. Here we identify two other presynaptic proteins nitrated by peroxynitrite, Munc-18 and SNAP25, both of which are involved in sequential steps leading to vesicle exocytosis. To investigate whether peroxynitrite affects vesicle exocytosis, we used the fluorescent dye FM1-43 to label a recycling population of secretory vesicles within the synaptosomes. Bolus addition of peroxynitrite stimulated exocytosis and glutamate release. Notably, these effects were strongly reduced in the presence of NaHCO(3), indicating that peroxynitrite acts mainly intracellularly. Furthermore, peroxynitrite enhanced the formation of the sodium dodecyl sulfate-resistant SNARE complex in a dose-dependent manner (100-1000 microm) and induced the formation of 3-nitrotyrosine in proteins of SNARE complex. These data suggest that modification(s) of synaptic vesicle proteins induced by peroxynitrite may affect protein-protein interactions in the docking/fusion steps, thus promoting exocytosis, and that, under excessive production of superoxide and nitric oxide, neurons may up-regulate neuronal signaling.