S‐Nitrosoglutathione in Rat Cerebellum: Identification and Quantification by Liquid Chromatography‐Mass Spectrometry

Abstract

Given the extreme lability and the facile inactivation of the messenger nitric oxide (NO) by many reactive biochemical species, it has been suggested that some intermediate compounds, for example, S‐nitrosothiols, may act to stabilize NO and at the same time to preserve its biological activity. To test this hypothesis, we investigated if the S‐nitrosothiol of glutathione, which is the predominant low molecular weight thiol in CNS, is present in the rat brain. The HPLC analysis of cerebellar extract from [35S]cysteine‐prelabeled slices suggested that S‐nitrosoglutathione (GSNO) was indeed present in rat brain. To detect endogenous GSNO, a methodology based on liquid chromatography‐mass spectrometry was developed. Besides an unequivocal identification of the endogenous GSNO, this method also permitted its precise quantification using 15N‐labeled GSNO ([15N]‐GSNO) as internal standard. GSNO level in adult cerebellum amounts to 15.4 ± 1.4 pmol/mg of protein. This is the first direct demonstration of the presence of endogenous GSNO in CNS. The packaging of NO in the form of GSNO might serve to facilitate its transport, prolong its life, and target its delivery to specific effectors.

Nitric oxide S -nitrosylates serine racemase, mediating feedback inhibition of d -serine formation

“…Basal S-nitrosylation of SR is observed in the wild-type but not in the nNOS knockout brain. decompose into NO but is in the range of GSNO levels reported in brain tissue (20). As such, the effective NO levels are lower and the actual IC 50 is also less than what we observed.…”

SNOSID, a proteomic method for identification of cysteine S-nitrosylation sites in complex protein mixtures

“…As an initial test of the potential for SNOSID to specify SNO-Cys residues, experiments were performed with cerebellum lysates in which SNO-protein levels were modestly enriched by treatment with relatively low levels of GSNO. Notably, GSNO has been detected in rat cerebellum at an estimated 6 -8 M concentration (16) and is implicated as an effector of mammalian NO͞cGMP signaling (17). A physiological role of GSNO in regulating the extent of protein S-nitrosylation in vivo is provided by the observed up-regulation of SNO-protein levels in mice that are nullozygous for an enzyme that selectively denitrosylates GSNO (GSNOR) (18).…”

S-Nitrosothiol measurements in biological systems

“…Data derived from experimental conditions or using standard curves, in which SNO concentrations are 10-1,000 fold higher than those measured in biology, are both irrelevant and misleading [10,11,12]. It is mandatory to generate a standard curve using the specific SNO of interest in the biological range (usually 10 nM -5μM) [1,2,5,[11][12][13][14][15][16].…”

“…Certain high-quality standards -such as Snitrosoglutathione (GSNO) -are commercially available. Others, including S-NO modified L-and D-isomers of cysteine, 15 NO + modified species and S-NO proteins [25], will need to be synthesized. Protocols for SNO synthesis and characterization have been extensively detailed elsewhere [25][26][27] and are not the subject of this paper.…”