Differential regulation of brain protein phosphatase activities by nitric oxide and nitroxyl anion

Abstract

While nitric oxide (NO) is a physiological messenger molecule, increasing evidence suggests that its one‐electron reduction product, nitroxyl anion (NO–), may be particularly cytotoxic. We have examined the differential regulation of redox‐sensitive protein phosphatase activities in a rat brain soluble fraction by the nitric oxide donor dethylamine NONOate and the nitroxyl anion donor Angeli's salt using p‐nitrophenylphosphate (p‐NPP) as a nonspecific substrate. We report that micromolar NONOate but not Angeli's salt produced a 2–3‐fold increase in a calcineurin (CaN) phosphatase activity, defined as Ni2± dependent, calmidazolium‐sensitive, p‐NPPase activity. Activation of CaN by NONOate was (i) enhanced by carboxy‐PTIO, which promotes formation of the S‐nitrosylating agent N2O3 (ii) mimicked by S‐nitrosoglutathione (iii) partially reversed by the sulfhydryl reductant DTT, and (iv) accompanied by protein S‐nitrosylation. NONOate also produced a modest, largely DTT‐reversible, inhibition of the vanadate‐sensitive fraction of p‐NPPase that was attributed to protein tyrosine phosphatase (PTP) activity. We propose that regulation of protein phosphatase activities by reversible sulfhydryl modulation may contribute to the physiological actions of nitric oxide. In contrast to NONOate, Angeli's salt produced a marked and irreversible inhibition of PTP activity consistent with a possible higher order oxidation of an active‐site cysteine residue. Notably, Angeli's salt was at least a 10‐fold more potent inhibitor of PTP activity than was hydrogen peroxide. We propose that PTP inactivation by nitroxyl anion may promote excessive and neurotoxic protein tyrosine phosphorylation.