Nitric oxide (NO) physiologically stimulates the sarco/endoplasmic reticulum calcium (Ca(2+)) ATPase (SERCA) to decrease intracellular Ca(2+) concentration and relax cardiac, skeletal and vascular smooth muscle. Here, we show that NO-derived peroxynitrite (ONOO(-)) directly increases SERCA activity by S-glutathiolation and that this modification of SERCA is blocked by irreversible oxidation of the relevant cysteine thiols during atherosclerosis. Purified SERCA was S-glutathiolated by ONOO(-) and the increase in Ca(2+)-uptake activity of SERCA reconstituted in phospholipid vesicles required the presence of glutathione. Mutation of the SERCA-reactive Cys674 to serine abolished these effects. Because superoxide scavengers decreased S-glutathiolation of SERCA and arterial relaxation by NO, ONOO(-) is implicated as the intracellular mediator. NO-dependent relaxation as well as S-glutathiolation and activation of SERCA were decreased by atherosclerosis and Cys674 was found to be oxidized to sulfonic acid. Thus, irreversible oxidation of key thiol(s) in disease impairs NO-induced relaxation by preventing reversible S-glutathiolation and activation of SERCA by NO/ONOO(-).

Cysteine thiol modifications are increasingly recognized to occur under both physiological and pathophysiological conditions, making their accurate detection, identification, and quantification of growing importance. Amongst free cysteines, the bulk of modifications occur on a subset of cysteines that are more reactive. These exist as thiolate anions at physiological pH because of their surrounding electrostatic environment. Reagents with iodoacetamide active groups can be used to selectively label these reactive thiols with a high degree of selectivity. Thiol adducts can be detected by the failure to label with iodoacetamide or other reagents; restoration of labeling by specific reducing agents (eg. ascorbate or glutaredoxin), can be used to detect reversible S-nitroso and S-glutathione adducts. These adducts also may be detected with radiolabels and antibodies. S-glutathiolation in response to physiological stimuli may be detected in cells and tissues with glutathione ester labeled with biotin. Mass spectrometry can identify thiol modifications with precision, and with isotope-coded affinity tags, used to quantify modification of specific thiols. Combinations of these methods increase sensitivity and specificity, and enable quantification and precise identification of thiol modifications that occur under physiological and pathological conditions.

Nitration of protein tyrosine residues (nY) is a marker of oxidative stress and may alter the biological activity of the modified proteins. The aim of this study was to develop antibodies toward site-specific nY-modified proteins and to use histochemistry and immunoblotting to demonstrate protein nitration in tissues. Affinity-purified polyclonal antibodies toward peptides with known nY sites in MnSOD nY-34 and of two adjacent nY in the sarcoplasmic endoplasmic reticulum calcium ATPase (SERCA2 di-nY-294,295) were developed. Kidneys from rats infused with ANG II with known MnSOD nY and aorta from atherosclerotic rabbits and aging rat skeletal and cardiac sarcoplasmic reticulum with known SERCA di-nY were used for positive controls. Staining for MnSOD nY-34 was most intense in distal renal tubules and collecting ducts. Staining of atherosclerotic aorta for SERCA2 di-nY was most intense in atherosclerotic plaques. Aging rat skeletal muscle and atherosclerotic aorta and cardiac atrium from human diabetic patients also stained positively. Staining was decreased by sodium dithionite, which chemically reduces nitrotyrosine to aminotyrosine, and the antigenic nY-peptide blocked staining for each respective nY site but not for the other. As previously demonstrated, immunoblotting failed to detect these modified proteins in whole tissue lysates but did when the proteins were concentrated. Immunohistochemical staining for specific nY-modified tyrosine residues offers the ability to assess the effects of oxidant stress associated with pathological conditions on individual proteins whose function may be affected in specific tissue sites.