Change of membrane fluidity and protein conformation involved in [Ca2+](i)overload in neuronal cells induced by ONOO-

Considerable evidence indicates that the formation of peroxynitrite (ONOO-) with superoxide anion (O-2(-.)) may be involved in the neuronal toxicity o f NO. Here, the effects of ONOO- on intracellular free calcium concentratio n ([Ca2+](i)) in single MN9D cells was studied by the Fura-2 microfluoromet ric technique. The results showed that [Ca2+](i) was increased dose-depende ntly with the addition of ONOO- (0-40 mu mol/l) after 5 s and then decrease d rapidly back to the basal level after ONOO- was removed. [Ca2+](i) respon se to ONOO- can be blocked by removing Ca2+ from the bath or adding L-type calcium channel antagonist nifedipine (10 mu mol/l) to the bath. [Ca2+](i) transients by ONOO- were substantially inhibited by dithiothreitol (DTT), w hich indicated ONOO- may alter the L-type calcium channel on neuronal cell by thiol oxidation. To elucidate the mechanism of ONOO on [Ca2+](i), the el ectron spin resonance spin-labeling technique was used to study the effects of ONOO- on the membrane fluidity and the membrane protein conformation on freshly dissociated neurons. The results indicate that ONOO- decreases mem brane fluidity both near the surface and deep in the membrane and affects p rotein conformation. The fact that DTT effectively inhibits the deteriorati on supports the conclusion that the change of membrane fluidity and protein conformation is involved in [Ca2+](i) overload in neuronal cells induced b y ONOO.