Disrupted [Ca²⁺]_i Homeostasis Contributes to the Toxicity of Nitric Oxide in Cultured Hippocampal Neurons

Abstract

Nitric oxide (NO) has been shown to be an important mediator in several forms of neurotoxicity. We previously reported that NO alters intracellular Ca2+ concentration ([Ca2+]i) homeostasis in cultured hippocampal neurons during 20‐min exposures. In this study, we examine the relationship between late alterations of [Ca2+]i homeostasis and the delayed toxicity produced by NO. The NO‐releasing agent S‐nitrosocysteine (SNOC; 300 µM) reduced survival by about one half 1 day after 20‐min exposures, as did other NO‐releasing agents. SNOC also was found to produce prolonged elevations of [Ca2+]i, persisting at 2 and 6 h. Hemoglobin, a scavenger of NO, blocked both the late [Ca2+]i elevation and the delayed toxicity of SNOC. Removal of extracellular Ca2+ during the 20‐min SNOC treatment failed to prevent the late [Ca2+]i elevations and did not prevent the delayed toxicity, but removal of extracellular Ca2+ for the 6 h after exposure as well blocked most of the toxicity. Western blots showed that SNOC exposure resulted in an increased proteolytic breakdown of the structural protein spectrin, generating a fragment with immunoreactivity suggesting activity of the Ca2+‐activated 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 [Ca2+]i homeostatic mechanisms, and that the resulting persistent [Ca2+]i elevations contribute to the delayed neurotoxicity of NO.

Modulation of mitochondrial Ca2+ by nitric oxide in cultured bovine vascular endothelial cells

“…NO acts at the level of cytochrome oxidase, reducing the affinity of the enzyme for O 2 (12,16). Disruption of mitochondrial respiration by NO has been proposed to be partially responsible for the energy depletion in neurons (10) that led to the disruption of Ca 2ϩ homeostasis (11). We have demonstrated previously that in vascular endothelial cells, NO inhibits capacitative Ca 2ϩ entry and enhances endoplasmic reticulum Ca 2ϩ uptake; however, the effect of NO on mitochondrial Ca 2ϩ homeostasis has not been evaluated in details (19).…”

Nitric oxide, cell bioenergetics and neurodegeneration

“…In neurons, NO administered exogenously (Brorson and Zhang 1997; Brorson et al . 1997) or generated endogenously after NMDA receptor stimulation (Marks et al .…”

Synergism of Nitric Oxide and Iron in Killing the Transformed Murine Oligodendrocyte Cell Line N20.1

“…Application of SNAP or SNP did not disrupt Ca 2ϩ homeostasis in N20.1 cells within our time of observation (up to 15 min). NO induces a rise in intracellular Ca 2ϩ concentration in several cell types, including hepatocytes, the murine macrophage cell line PU5-1.8, pancreatic islet cells, hippocampal neurons, and oligodendrocytes (Hartmann et al, 1994;Kong et al, 1994;Richter et al, 1994;Brorson and Zhang, 1997;Nedelkoska et al, 1997). These studies showed that the origin of the increased intracellular Ca 2ϩ level may be extracellular, intracellular, or sometimes both.…”