Change of membrane fluidity and protein conformation involved in [Ca²⁺]_i overload in neuronal cells induced by ONOO⁻

Abstract

Considerable evidence indicates that the formation of peroxynitrite (ONOO⁻) with superoxide anion (O ^−⊙₂ ot) may be involved in the neuronal toxicity of NO. Here, the effects of ONOO⁻ on intracellular free calcium concentration ([Ca²⁺]_i) in single MN9D cells was studied by the Fura-2 microfluorometric technique. The results showed that [Ca²⁺]_i was increased dose-dependently with the addition of ONOO⁻ (0–40 μmol/l) after 5 s and then decreased rapidly back to the basal level after ONOO⁻ was removed. [Ca²⁺]_i response to ONOO⁻ can be blocked by removing Ca²⁺ from the bath or adding L-type calcium channel antagonist nifedipine (10 μmol/l) to the bath. [Ca²⁺]_i transients by ONOO⁻ were substantially inhibited by dithiothreitol (DTT), which 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 electron 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 membrane fluidity both near the surface and deep in the membrane and affects protein conformation. The fact that DTT effectively inhibits the deterioration supports the conclusion that the change of membrane fluidity and protein conformation is involved in [Ca²⁺]_i overload in neuronal cells induced by ONOO⁻.