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.
|Original language||English (US)|
|State||Published - Dec 1 1996|
ASJC Scopus subject areas
- Molecular Biology