Release of intracellular Zn super(2+) in cultured neurons after brief exposure to low concentrations of exogenous nitric oxide

Several studies have shown intracellular Zn super(2+) release and concomitant cell death after prolonged exposure to exogenous NO. In the present study, we investigated whether cortical neurons briefly exposured to exogenous NO would demonstrate similar levels of intracellular Zn super(2+) release and subsequent cell death. Cortical neurons were loaded with the Zn super(2+) selective fluorophore FluoZin-3 and treated with various concentrations of the NO generator, spermine NONOate. Fluorescence microscopy was used to detect and quantify intracellular Zn super(2+) levels. Concomitant EDTA perfusion was used to eliminate potential effects of extracellular Zn super(2+). Neurons were perfused with the heavy metal chelator TPEN to selectively eliminate Zn super(2+) induced fluorescence changes. A significant increase of intracellular fluorescence was detected during a 5 min perfusion with spermine NONOate. The increase in intracellular Zn super(2+) release appeared to peak at 1 mu M spermine NONOate (123.8 plus or minus 28.5%, increase above control n = 20, P < 0.001). Further increases in spermine NONOate levels as high as 1 mM failed to further increase detectable intracellular Zn super(2+) levels. The NO scavenger hemoglobin blocked the effects of spermine NONOate and the inactive analog of the spermine NONOate, spermine, was without effect. No evidence of cell death induced by any of the brief treatments with exogenous NO was observed; only prolonged incubation with much larger amounts of exogenous NO resulted in significant cell death. These data suggest that in vivo release of NO may cause elevations of intracellular Zn super(2+) in cortical neurons. The possibility that release of intracellular Zn super(2+) in response to NO could play a role in intracellular signaling is discussed.