Release of intracellular Zn(2+) in cultured neurons after brief exposure to low concentrations of exogenous nitric oxide.
Several studies have shown intracellular Zn(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(2+) release and subsequent cell death. Cortical neurons were loaded with the Zn(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(2+) levels. Concomitant EDTA perfusion was used to eliminate potential effects of extracellular Zn(2+). Neurons were perfused with the heavy metal chelator TPEN to selectively eliminate Zn(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(2+) release appeared to peak at 1 microM spermine NONOate (123.8 +/- 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(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(2+) in cortical neurons. The possibility that release of intracellular Zn(2+) in response to NO could play a role in intracellular signaling is discussed.
Lin
Wei
W
Department of Biological Sciences, Ohio University, Athens, OH 45701, USA.
Mohandas
Bhavana
B
Fontaine
Charles P
CP
Colvin
Robert A
RA
D000818:Animals; D002478:Cells, Cultured; D006454:Hemoglobins / Q000737:chemistry; D008668:Metallothionein / Q000737:chemistry*; D008856:Microscopy, Fluorescence / Q000379:methods; D009474:Neurons / Q000378:metabolism*; D009569:Nitric Oxide / Q000378:metabolism*; D051381:Rats; D017207:Rats, Sprague-Dawley; D015398:Signal Transduction; D013096:Spermine / Q000737:chemistry; D013778:Tetrazolium Salts / Q000494:pharmacology; D013844:Thiazoles / Q000494:pharmacology; D013997:Time Factors; D015032:Zinc / Q000737:chemistry*
D006454:Hemoglobins; D013778:Tetrazolium Salts; D013844:Thiazoles; D013096:Spermine; D009569:Nitric Oxide; D008668:Metallothionein; C022616:thiazolyl blue; D015032:Zinc