As a measure of ROS formation we measured

As a measure of ROS formation we measured the extent by which Cu(I) affected the production of hydrogen peroxide and, at the same time, to ensure that there would not be shortage of NADH substrate. Our results show that H2O2 production is increased over time by LPS stimulation but it is not when microglia are not activated. As expected, the higher dose of LPS (although non-cytotoxic) elicits a slightly higher (albeit nonsignificant) release of H2O2, an increase that is most likely due to increased cellular respiration. In addition, results pertaining to the effects of Cu(I) on H2O2 production mirror those of nitrite release in that, while a high dose of 100 μM Cu(I) drastically reduces H2O2 production, a low dose of 1 μM Cu(I) does not. We next sought to determine the extent to which Cu(I) exposure would affect SNO formation in activated microglia. SNOs are generated by the covalent, but reversible, addition of an NO moiety to reactive sulfhydryl residues of peptides and proteins. The fine equilibrium between NO production and SNO reduction is catalyzed by de-nitrosylating enzymes including the NADH-dependent GSNOR (Foster et al., 2009) which, ultimately, reduces nitrosative stress-induced toxicity (Liu et al., 2001) by metabolizing GSNO to glutathione disulfide and a glutathione N-hydroxysulfenamide intermediate, before being reduced to ammonia. Therefore, GSNOR regulates cellular concentrations of GSNO thus resulting in the regulation of protein S-nitrosylation-directed signaling. The S-nitrosylation of proteins of LPS-activated microglia by treatment with a high dose of 100 μM Cu(I) could be attributable to the direct cleavage of SNO bonds. Although it is not clear to us why the 100 μM Cu(I) treatment would abrogate expression of GSNOR protein irrespective of LPS stimulation while control microglia express measurable GSNOR activity and protein, we would exclude that complete abrogation of GSNOR protein be attributed to either an inhibited gaba agonist or to cell death, as indicated by the intact GAPDH expression in those conditions. Furthermore, although GSNOR expression is reduced by the high dose of Cu(I), the reduction in GSNOR activity is only observed in microglia stimulated with 100 ng/mL LPS but not by 1000 ng/mL LPS nor by 100 μM Cu(I) alone, both of which, although not reaching significance, follow the same decreasing trend as for the 100 ng/mL LPS. We speculate that expression of GSNOR protein ceases to be detectable by immunoblotting while NADH consumption is still measurable and quantifying mRNA levels of GSNOR over time would provide some insight as to this discrepancy. Except for the lower expression of iNOS levels in the 100 μM Cu(I) alone, Cu(I) did not affect expression in LPS-stimulated microglia, suggesting that NO is being released at an equivalent rate, at least, within the cell. Our results also show that Cu(II) has no effect on either GSNOR activity or protein, confirming that the reduction in GSNOR is dependent on the redox state of Cu. One-thousand ng/mL LPS elicited a statistically significant drop in GSNOR activity without affecting either GSNOR protein expression or iNOS expression suggesting that Cu(I) affects GSNOR directly. As expected, 1000 ng/mL LPS resulted in increased SNO protein expression, which was inhibited by 1400 W and by 100 μM Cu(I) but not by 1 μM Cu(I). In contrast to the lack of effect on the S-nitrosylation of proteins by a high dose of Cu(I), a low dose of 1 μM Cu(I) elicited a robust expression which might be due to a low and sustained production of intracellular NO (as suggested by the weak iNOS expression in Fig. 5) which, in turn, would only partially perturb GSNOR. Alternatively, the addition of Cu(I) could greatly enhance S-nitrosothiols (RSNO) reduction and biotin labeling (Wang et al., 2008), thereby affecting the S-nitrosothiols decomposition by Cu(I) ions; this one electron reduction of the RSNO group would result in free thiol, NO and Cu(II), which, subsequently, would be reduced to Cu(I).