Nitric oxide synthase generates superoxide and nitric oxide in arginine-depleted cells and this causes cell injury

In addition to synthesizing nitric oxide (NO)₁ purified nitric oxide synthase (NOS) can produce Superoxide (O₂) at lower arginine concentration. But it is not known if NOS-mediated O₂ generation occurs in intact cells and the significance of this O₂ has not been established. Therefore, we characterized the formation of NO and O₂ in neural NOS-transfected 293 cells by direct measurement techniques using EPR spin-trapping and studied the role of these radicals in cell injury. In control cells, Ca²⁺ ionophore A23187 (1 uM) triggered NO generation but no O₂ signals were seen. When cytosolic arginine (arg) was depleted by incubating cells in arg free media, O₂ formation was observed and increased as the cytosolic arg levels (measured by HPLC) decreased. This O₂ formation was inhibited by the NOS blocker, L-NAME (1 mM). Conversely, NO formation in these arg-depleted cells decreased along with the cytosolic arg depletion. Marked nitrotyrosine formation, a peroxynitrite oxidization product, was found on arg-depleted cells but not on control cells. Moreover, activation of NOS with A23187 triggered cytotoxic effects on arg-depleted cells with marked LDH release (5-fold increases i/s control, P<0.01). Thus, with arg depletion NOS switches from NO to O₂ production in intact cells and this process was controlled by cytosolic arg levels. NOS-mediated NO and O₂ interact to form peroxynitrite, which in turn causes cell injury. This mechanism could happen in ischémie tissues or wound injury where cellular arg depletion may occur.