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

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

Role of poly(ADP-ribose) polymerase in rapid intracellular acidification induced by alkylating DNA damage

“…Therefore, extensive PARP activation can consume a large amount of NAD and release a substantial amount of protons in a very short period; this may have the potential to cause cellular acidification after DNA damage. Indeed, oxidative damage that also activates PARP has earlier been shown to cause rapid acidification of certain cells that are pathophysiologically exposed to oxidants, such as cardiac myocytes or myoblasts (14), aortic endothelial cells (15,16), renal epithelial cells (17), and hippocampal neurons (18). Neuronal acidification has also been reported in other models that indirectly implicate oxidant damage, such as cerebral ischemia (19) or treatment with N-methyl-D-glucamine (20).…”

“…Because acidosis or holding cells in acidic medium can induce cell death by apoptosis or necrosis (18,(32)(33)(34), impact of MNNG-induced acidification on the mode of cell death was examined by using the pH clamp method to either suppress or introduce acidification. Molt 3 cells were clamped at pH 7.4 and exposed to 100 M MNNG to prevent the early acidification response.…”

Alterations of intracellular pH homeostasis in apoptosis: origins and roles

“…NO: nitric oxide; H 2 O 2 : hydrogen peroxide; PARP: poly(ADP-ribose) polymerase. Inspired from Matsuyama et al, 21 Hirpara et al, 25 Marches et al, 26 Vincent et al 29 and Koike et al 30 Intracellular pH in apoptosis D Lagadic-Gossmann et al cyclosporin A, used in that study as a specific inhibitor of calcineurin phosphatase, inhibited this decrease, the authors then postulated that NHE1 dephosphorylation was dependent on the action of this phosphatase. However, downregulation of NHE-controlling kinases might also be involved.…”

“…25 It should nevertheless be emphasized that the way pH i varies following mitochondrial production of H 2 O 2 might depend upon intracellular H 2 O 2 concentration; indeed, as observed by Clé ment et al, 27 an acidification was induced by apoptotic doses of exogenous H 2 O 2 (0.25-0.5 mM) whereas necrotic concentrations (41 mM) promoted intracellular alkalinization. Besides H 2 O 2 , other factors, such as nitric oxide, which has been shown to be proapoptotic under certain circumstances 28 and to induce cytosolic acidification, for example, in neurons, 29 or more simply a collapse of the sodium gradient, 30 might also be involved in the downregulation of NHE activity ( Figure 4); this remains as yet to be tested. In addition to NHE, it is important to stress at this point that other membrane transporters or channels as well as lactate production by glycolysis also deserve attention in the future, since they might also participate, in some situations, in the development of cytosolic acidification.…”

Intracellular Acidification Is Associated with Changes in Free Cytosolic Calcium and Inhibition of Action Potentials in Rat Trigeminal Ganglion

“…Many biological processes, such as enzyme activity, ionic conductance, and activity of membrane transporters, are pH-sensitive (3,4). Thus, the regulation of pH i in neurons is of critical importance; failure to maintain pH i may lead to numerous pathophysiological conditions (5,6). Neurons may become acidified in response to neurotransmitters and chemical compounds (7)(8)(9), and this intracellular acidification has been linked to the activity of the plasma membrane Ca 2ϩ /ATPase (PMCA) (10,11).…”