DISRUPTED [CA2-OXIDE IN CULTURED HIPPOCAMPAL-NEURONS(](I) HOMEOSTASISCONTRIBUTES TO THE TOXICITY OF NITRIC)

Nitric oxide (NO) has been shown to be an important mediator in severa l forms of neurotoxicity. We previously reported that NO alters intrac ellular Ca2+ concentration ([Ca2+](i)) homeostasis in cultured hippoca mpal neurons during 20-min exposures. In this study, we examine the re lationship between late alterations of [Ca2+](i) homeostasis and the d elayed toxicity produced by NO. The NO-releasing agent S-nitrosocystei ne (SNOC; 300 mu M) reduced survival by about one half 1 day after 20- min exposures, as did other NO-releasing agents. SNOC also was found t o produce prolonged elevations of [Ca2+](i), persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca2+](i) elevati on and the delayed toxicity of SNOC. Removal of extracellular Ca2+ dur ing the 20-min SNOC treatment failed to prevent the late [Ca-2+](i) el evations and did not prevent the delayed toxicity, but removal of extr acellular Ca2+ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an incre ased proteolytic breakdown of the structural protein spectrin, generat ing a fragment with immunoreactivity suggesting activity of the Ca2+-a ctivated protease calpain. The spectrin breakdown and the toxicity of SNOC were inhibited by treatment with calpain antagonists. We conclude that exposures to toxic levels of NO cause prolonged disruption of [C a2+](i) homeostatic mechanisms, and that the resulting persistent [Ca2 +](i) elevations contribute to the delayed neurotoxicity of NO.