Neuronal intracellular pH directly mediates nitric oxide-induced programmed cell death

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Abstract
Neuronal injury is intricately linked to the activation of three distinct neuronal endonucleases. Since these endonucleases are exquisitely pH dependent, we investigated in primary rat hippocampal neurons the role of intracellular pH (pHi) regulation during nitric oxide (NO)-induced toxicity. Neuronal injury was assessed by both a 0.4% Trypan blue dye exclusion survival assay and programmed cell death (PCD) with terminal deoxynucleotidyl transferase nick-end labeling (TUNEL) 24 h following treatment with the NO generators sodium nitroprusside (300 μM), 3-morpholinosydnonimine (300 μM), or 6-(2-hyrdroxy-1-methyl-2-nitrosohydrazino)-N-methyl-1-hexanamine (300 μM). The pHi was measured using the fluorescent probe BCECF. NO exposure yielded a rapid intracellular acidification during the initial 30 min from pHi 7.36 ± 0.01 to approximately 7.00 (p < .0001). Within 45 min, a biphasic alkaline response was evident, with pHi reaching 7.40 ± 0.02, that was persistent for a 6-h period. To mimic the effect of NO-induced acidification, neurons were acid-loaded with ammonium ions to yield a pHi of 7.09 ± 0.02 for 30 min. Similar to NO toxicity, neuronal survival decreased to 45 ± 2% (24 h) and DNA fragmentation increased to 58 ± 8% (24 h) (p < .0001). Although neuronal caspases did not play a dominant role, neuronal injury and the induction of PCD during intracellular acidification were dependent upon enhanced endonuclease activity. Furthermore, maintenance of an alkaline pHi of 7.60 ± 0.02 during the initial 30 min of NO exposure prevented neuronal injury, suggesting the necessity for the rapid but transient induction of intracellular acidification during NO toxicity. Through the identification of the critical role of both NO-induced intracellular acidification and the induction of the neuronal endonuclease activity, our work suggests a potential regulatory trigger for the prevention of neuronal degeneration. © 1999 John Wiley & Sons, Inc. J Neurobiol 40: 171–184, 1999

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