| The sarcoplasmic endoplasmic reticulum Ca2+ ATPase (SERCA) lowers intracellular free Ca2+ by increasing Ca2+ uptake and inhibiting extracellular Ca2+ influx. Nitric oxide (NO) stimulates SERCA activity via both cyclic GMP-dependent and -independent mechanisms, leading to NO-induced vasodilatation. Our previous studies showed that NO causes cyclic GMP-independent activation of SERCA activity by inducing reversible S-glutathiolation at cysteine-674 (C674). The current studies aimed to assess the significance of cysteine-674 in NO-mediated regulation of SERCA activity in physiological and pathological situations.; To test the role of C674 in physiological situations, overexpression of either wild type (WT) or mutant SERCA in which cysteine-674 was mutated to serine (C674S) was accomplished by stable transfection of HEK 293 or adenoviral expression in rat aortic smooth muscle cells (VSMC). In the cell models expressing C674S SERCA, biotinylated-iodoacetamide (BIAM) and biotinylated-glutathione labeling of SERCA was markedly decreased, and NO failed to increase SERCA activity or decrease Ca2+ influx. The NO donor, S-nitrosopenicillamine (SNAP) inhibited migration of cells with WT SERCA, but had no effect on the migration of either HEK cells or VSMC with C674S SERCA. The same result was obtained in VSMC in which endogenous NO was produced by iNOS induced by IL-1beta. Blocking cyclic GMP did not prevent the inhibition of migration by NO. Thus, C674 of SERCA is required for cyclic GMP-independent, NO mediated inhibition of cell Ca2+ and migration.; Using mass spectrometry and a novel SERCA C674SO3H antibody, we detected sulfonic acid on cysteine-674 of SERCA in diabetic pig hypercholesterolemic pig aorta and in arteries exposed to high glucose. This irreversible oxidation of SERCA cysteine-674 is related to the decreased SERCA activity and the impaired vasodilatation in diseased arteries. Furthermore, finding the oxidized cysteine-674 in a lower molecular weight SERCA fragment suggests the association between the irreversible oxidation of SERCA C674 and the accelerated degradation of SERCA.; In conclusion, C674 is required for NO-induced redox regulation of SERCA activity, which is important for NO-mediated cellular physiological functions. In pathological situations, SERCA C674 is irreversible oxidized, which blocks NO-mediated regulation of SERCA and may lead to accelerated degradation of SERCA. |
