ARTICLES 1200 VOLUME 10 | NUMBER 11 | NOVEMBER 2004 NATURE MEDICINE Sequestration of Ca 2+ by SERCA mediates smooth, cardiac and skele- tal muscle relaxation, and regulates many cellular processes, including apoptosis and proliferation 1 . We and others have reported that NO decreases intracellular Ca 2+ by accelerating sarcoplasmic reticulum Ca 2+ uptake in smooth muscle 2,3 , platelets 4 and cardiac myocytes 5,6 , but the molecular mechanism is unknown. Although cyclic GMP and protein kinase G mediate many of the actions of NO, the activation of SERCA by NO in smooth muscle and platelets occurs independently of this second messenger system 2–4 . NO itself is a weak thiol oxidant, but physiological regulation of ion channels and transporters by S- nitrosation (SNO-) or S-glutathiolation (GSS-) of reactive thiols may occur through the secondary generation of reactive nitrogen species (RNS) including • NO 2 ,N 2 O 3 , NO – or ONOO – (refs. 7–11). In the absence of glutathione (GSH), RNS cause the oxidative modifications of SERCA1 in skeletal muscle in vitro. Diethylamine NONOate (0.1 mM) or ONOO – (100 μM) modified or S-nitrosated multiple cys- teine residues, but Ca 2+ -uptake activity was not increased 9,12–14 . Notably, ONOO – (450 μM) not only oxidized cysteines (Cys636, Cys670 and Cys674) located in the key hinge region of the molecule, but in the presence of GSH formed GSS-Cys residues at Cys344, Cys349, Cys364, Cys498, Cys525 and Cys614 (refs. 13,14). Because ONOO – has been suggested as an endogenous vasodilator 15–17 and positive inotropic agent 5 , we hypothesized that ONOO – might regu- late SERCA activity by these thiol modifications, possibly at low con- centrations that could be generated physiologically from endothelium-derived NO. Atherosclerosis is associated with chronic augmentation of reactive oxygen and nitrogen species (ROS/RNS) 18 , as evidenced by increased 3-nitrotyrosine content of proteins in general, and of SERCA in par- ticular, in rabbit and human atherosclerotic aorta 19,20 . The impair- ment of SERCA activity associated with ROS/RNS generation in atherosclerosis is also a feature of other pathological states, including heart failure 21,22 and aging of skeletal muscle 23 . Because high concen- trations of ROS/RNS have been found to oxidize cysteines of SERCA in vitro, as discussed above 13,14,22 , we have analyzed the thiol-redox state of SERCA to understand the mechanisms regulating the physio- logical and pathological smooth muscle response to NO. We aimed to elucidate the molecular mechanism by which NO/ONOO – regulates SERCA. Our results show a dual role for ROS/RNS in regulating protein function. Physiologically, NO and superoxide anion, through the formation of ONOO – , activate SERCA by reversible S-glutathiolation, but chronically elevated levels of ROS/RNS in atherosclerosis oxidize the responsible thiols and block NO-induced S-glutathiolation. RESULTS ONOO – increases SERCA activity by S-glutathiolation We obtained highly purified SERCA2 from a rabbit heart membrane fraction on a reactive-red column that has affinity for ATPase(s) 24 (Fig. 1a). We labeled free reactive thiols on SERCA with biotinylated iodoacetamide (IAM) at pH 7.0 (refs. 25,26). ONOO – (10–50 μM) alone decreased the amount of IAM-labeled SERCA thi- 1 Vascular and Myocardial Biology Units, Whitaker Cardiovascular Institute, Boston University Medical Center, X707, 650 Albany Street, Boston, Massachusetts 02118-2393, USA. 2 Department of Pharmaceutical Chemistry, University of Kansas School of Pharmacy, 2095 Constant Avenue, Lawrence, Kansas, 66047, USA. Correspondence should be addressed to T.A.(Tadachi@sc.itc.keio.ac.jp) or R.A.C. (racohen@bumc.bu.edu). Published online 17 October 2004; doi:10.1038/nm1119 S-Glutathiolation by peroxynitrite activates SERCA during arterial relaxation by nitric oxide Takeshi Adachi 1 , Robert M Weisbrod 1 , David R Pimentel 1 , Jia Ying 1 , Victor S Sharov 2 , Christian Schöneich 2 & Richard A Cohen 1 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 – . © 2004 Nature Publishing Group http://www.nature.com/naturemedicine