Excitotoxic mitochondrial depolarisation requires both calcium and nitric oxide in rat hippocampal neurons.
1. Glutamate neurotoxicity has been attributed to cellular Ca2+ overload. As mitochondrial depolarisation may represent a pivotal step in the progression to cell death, we have used digital imaging techniques to examine the relationship between cytosolic Ca2+ concentration ([Ca2+]c) and mitochondrial potential (DeltaPsim) during glutamate toxicity, and to define the mechanisms underlying mitochondrial dysfunction. 2. In cells of > 11 days in vitro (DIV), exposure to 50 mM potassium or 100 microM glutamate had different consequences for DeltaPsim. KCl caused a small transient loss of DeltaPsim but in response to glutamate there was a profound loss of DeltaPsim. In cells of 7-10 DIV, glutamate caused only a modest and reversible drop in DeltaPsim. 3. Using fura-2 to measure [Ca2+]c, responses to KCl and glutamate did not appear significantly different. However, use of the low affinity indicator fura-2FF revealed a difference in the [Ca2+]c responses to KCl and glutamate, which clearly correlated with the loss of DeltaPsim. Neurons exhibiting a profound mitochondrial depolarisation also showed a large secondary increase in the fura-2FF ratio. 4. The glutamate-induced loss of DeltaPsim was dependent on Ca2+ influx. However, inhibition of nitric oxide synthase (NOS) by L-NAME significantly attenuated the loss of DeltaPsim. Furthermore, photolysis of caged NO at levels that had no effect alone promoted a profound mitochondrial depolarisation when combined with high [Ca2+]c, either in response to KCl or to glutamate in cultures at 7-10 DIV. 5. In cells that showed only modest mitochondrial responses to glutamate, induction of a mitochondrial depolarisation by the addition of NO was followed by a secondary rise in [Ca2+]c. These data suggest that [Ca2+]c and nitric oxide act synergistically to cause mitochondrial dysfunction and impaired [Ca2+]c homeostasis during glutamate toxicity.
Keelan
J
J
Department of Physiology, University College London, Gower Street, London WC1E 6BT, UK. j.keelan@ucl.ac.uk
Vergun
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O
Duchen
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MR
D000818:Animals; D002118:Calcium / Q000378:metabolism / Q000502:physiology*; D046508:Culture Techniques; D003600:Cytosol / Q000378:metabolism; D004594:Electrophysiology; D005456:Fluorescent Dyes; D016257:Fura-2; D018698:Glutamic Acid / Q000506:poisoning*; D006624:Hippocampus / Q000166:cytology / Q000502:physiology*; D008928:Mitochondria / Q000187:drug effects / Q000502:physiology*; D009474:Neurons / Q000502:physiology*; D009569:Nitric Oxide / Q000502:physiology*; D009994:Osmolar Concentration; D051381:Rats; D020112:Rhodamine 123
D005456:Fluorescent Dyes; D020112:Rhodamine 123; D009569:Nitric Oxide; D018698:Glutamic Acid; D002118:Calcium; D016257:Fura-2