Original Contribution INTRACELLULAR pH-DEPENDENT PEROXYNITRITE-EVOKED SYNERGISTIC DEATH OF GLUCOSE-DEPRIVED ASTROCYTES CHUNG JU, * ,y YOUNG J. OH, z BYUNG H. HAN, § HEE-SUN KIM, * , y HYOUNG-CHUN KIM, b and WON-KI KIM * , y *Department of Pharmacology, College of Medicine and y Laboratory of Neurodegenerative Diseases, Ewha Institute of Neuroscience, Ewha WomenTs University; z Department of Biology, Yonsei University College of Science; § College of Pharmacy and Natural Products Research Institute, Seoul National University, Seoul; and b Department of Pharmacy, College of Pharmacy, Kwangwon National University, Chunchon, Kwangwon-do, Republic of Korea (Received 19 August 2003; Revised 22 June 2004; Accepted 8 July 2004) Available online 4 August 2004 Abstract—Previously, we reported that glucose-deprived astrocytes were highly vulnerable to peroxynitrite (ONOO  ). Here we demonstrate that the increased vulnerability caused by glucose deprivation and ONOO  depends on intracellular pH. The ONOO  releasing reagent 3-morpholinosydnonimine (SIN-1) markedly induced the release of lactate dehydrogenase (LDH, the marker of cytotoxicity) in glucose-deprived astrocytes. Morphological studies and caspase activity assay showed that astrocytes treated together with glucose deprivation and ONOO  died mostly in a necrotic mode. Alkalinization of pH from 7.4 to 7.8 increased LDH release, whereas acidification from pH 7.4 to 7.0 decreased it. However, intracellular pH (pH i ), not extracellular pH (pH e ), appeared to play a critical role in the synergistic death. Thus, without a change in pH e (7.4) cytosolic acidification by a weak acid salt, sodium acetate, and a Na + /H + antiporter inhibitor, amiloride, reduced LDH release. In contrast, a weak base, NH 4 Cl, and a Na + /H + antiporter stimulator, monensin, increased pH i and greatly enhanced LDH release. The augmented death was found to be due, in part, to the preceding decrease in the level of reduced glutathione, the ONOO  scavenger, and collapse of the mitochondrial transmembrane potential at alkaline pH. D 2004 Elsevier Inc. All rights reserved. Keywords—Astrocytes, Peroxynitrite (ONOO  ), pH, Mitochondrial permeability transition, Mitochondrial transmembrane permeability (Dc m ), Free radical INTRODUCTION Small changes in intracellular pH (pH i ) can greatly affect brain function via alterations of cell membrane excit- ability and metabolism [1, 2]. Thus, pH i in normal brain is tightly regulated at ~7.04, whereas interstitial pH is ~7.3 [3]. In cerebral ischemic insults, a large amount of lactic acid is produced by anaerobic glycolysis, resulting in an acidosis. However, reperfusion after ischemia causes a sudden pH shift leading to a transient intra- cellular alkaline pH [4]. Areas of chronic infarction have been shown to be alkaline relative to normal brain [5]. Both acidosis and alkalosis have been implicated in ischemic cerebral infarction. Acidosis attenuated neuro- nal injury or death caused by glutamate or oxygen– glucose deprivation in murine cortical neuronal cultures [6]. Moderate increase in extracellular pH (pH e 7.7–8.0) exacerbated injury and augmented excitotoxic calcium accumulation in cultured cortical neurons treated with oxygen–glucose deprivation [7]. In dogs metabolic alkalemia also impaired recovery from global ischemia through a NMDA receptor-mediated mechanism [8]. In contrast to neuronal cells, astrocytes were shown to be very susceptible to acidosis, rather than alkalemia. Thus, cultured astrocytes were reported to die after brief exposures to pH e below 5.3 [9]. However, such extremes of tissue pH may not be encountered during in vivo ischemia [10,11]. Some experiments have even been carried out at a pH e below 5.0. In ischemic areas pH was only moderately lowered for many hours; pH e fell during global ischemia from 7.3 to 6.8 in normoglycemic animals and to as low as 6.2 in hyperglycemic animals [10,12]. Furthermore, although the preischemic glucose 1160 Address correspondence to: Won-Ki Kim, Ph.D., Laboratory of Neurodegenerative Diseases, Ewha Women’s University, 70 Chongro- 6-ga, Chongro-ku, Seoul 110-783, Republic of Korea; Fax: (82) 2 760 5524; E-mail: wonki@mm.ewha.ac.kr. doi:10.1016/j.freeradbiomed.2004.07.011 Free Radical Biology & Medicine, Vol. 37, No. 8, pp. 1160–1169, 2004 Copyright D 2004 Elsevier Inc. Printed in the USA. All rights reserved 0891-5849/$-see front matter