371 Nitric Oxide Prevents Myoglobin/tert-Butyl Hydroperoxide–Induced Inhibition of Ca 2+ Transport in Skeletal and Cardiac Sarcoplasmic Reticulum ELIZABETH V. MENSHIKOVA, a VLADIMIR B. RITOV, b NIKOLAJ V. GORBUNOV, b GUY SALAMA, a H. GREGG CLAYCAMP, b AND VALERIAN E. KAGAN b-d Departments of a Cell Biology and Physiology, b Environmental and Occupational Health, and c Pharmacology, University of Pittsburgh, Pittsburgh, Pennsylvania 15238, USA ABSTRACT: Interaction of hydrogen peroxide or organic hydroperoxides with hemoproteins is known to produce oxoferryl hemoprotein species that act as very potent oxidants. Since skeletal and cardiac muscle cells contain high concentrations of myoglobin this reaction may be an important mechanism of initiation or enhancement of oxidative stress, which may impair their Ca 2+ transport systems. Using skeletal and cardiac sarcoplasmic reticulum (SR) ves- icles, we demonstrated by EPR the formation of alkoxyl radicals and protein- centered peroxyl radicals in the presence of myoglobin (Mb) and tert-butyl hy- droperoxide (t-BuOOH). The low temperature EPR signal of the radicals was characterized by a major feature at g = 2.016 and a shoulder at g = 2.036. In the presence of SR vesicles, the magnitude of the protein-centered peroxyl rad- ical signal decreased, suggesting that the radicals were involved in oxidative modification of SR membranes. This was accompanied by SR membrane oxi- dative damage, as evidenced by accumulation of 2-thiobarbituric acid–reactive substances (TBARS) and the inhibition of Ca 2+ transport. We have shown that nitric oxide (NO), reacting with redox-active heme iron, can prevent peroxyl radical formation activated by Mb/t-BuOOH. Incubation of SR membranes with an NO donor, PAPA/NO (a non-thiol compound that releases NO) at 200- 500 mM completely prevented the t-BuOOH–dependent production of peroxyl radicals and formation of TBARS, and thus protected against oxidative inhibi- tion of Ca 2+ transport. INTRODUCTION Metabolic activation of molecular oxygen by hemoproteins (e.g., cytochrome P 450 , cytochrome oxidase, peroxidases, hemoglobin, and myoglobin) produces ex- tremely potent oxidants—oxoferryl species. 1,2 Oxoferryl hemoproteins can be de- tected as catalytic intermediates of enzymatic reactions 3 or found in normal human blood (oxoferryl species formed via autoxidation of hemoglobin). 4 The formation and reactivity of oxoferryl hemoglobin and myoglobin, which occur as a result of in- d Address correspondence to: Dr. Valerian E. Kagan, Department of Environmental and Occu- pational Health, University of Pittsburgh, 260 Kappa Drive, RIDC Park, Pittsburgh, PA 15238; Telephone: (412) 967-6516; Fax: (412) 624-1020.