Following ischaemic reperfusion, large amounts of superoxide anion (.O2−), hydroxyl radical (.OH) and H2O2 are produced, resulting in brain oedema and changes in cerebral vascular permeability. We have found that H2O2 (100 μm) induces a significant intracellular acidosis in both cultured rat cerebellar astrocytes (0.37 ± 0.04 pH units) and C6 glioma cells (0.33 ± 0.07 pH units). Two membrane‐crossing ferrous iron chelators, phenanthroline and deferoxamine, almost completely inhibited H2O2‐induced intracellular acidosis, while the non‐membrane‐crossing iron chelator apo‐transferrin had no effect. Furthermore, the acidosis was completely inhibited by two potent membrane‐crossing .OH scavengers, N‐(2‐mercaptopropionyl)‐grycine (N‐MPG) and dimethyl thiourea (DMTU). Since .OH can be produced during iron‐catalysed H2O2 breakdown (Fenton reaction), we have shown that a large reduction in pH1 in glial cells can result from the production of intracellular .OH via H2O2 oxidation. We have ruled out the possible involvement of: (i) an increase in intracellular Ca2+ levels; and (ii) inhibition of oxidative phosphorylation. Our results suggest that .OH inhibits glycolysis, leading to ATP hydrolysis and intracellular acidosis. This conclusion is based on the following observations: (i) in glucose‐free medium, or in the presence of iodoacetate or 2‐deoxy‐D‐glucose, H2O2‐induced acidosis is completely suppressed; (ii) H2O2 and iodoacetate both produce an increase in levels of intracellular free Mg2+, an indicator of ATP breakdown; and (iii) direct measurement of intracellular ATP levels and lactate production show 50 and 55% reductions in ATP content and lactate production, respectively, following treatment with 100 μm H2O2. Inhibition of the pH1 regulators (i.e. the Na+–H+ exchange and possibly the Na+–HCO3−–dependent pH1 transporters) resulting from H2O2‐induced intracellular ATP reduction may also be involved in the H2O2‐evoked intracellular acidosis in glial cells.

Tumor-associated macrophages play an important role in tumor progression, but whether they exert a tumor-progressive effect remains controversial. Here, we demonstrated that activated macrophage-conditioned medium (AMCM) obtained from RAW macrophages (RAW/AMCM) induced epithelial-mesenchymal transition (EMT) and stimulated the migratory and invasive activities of HepG2 cells, whereas control conditioned media had no effect. Epithelial-cadherin (E-cadherin) and b-catenin staining patterns were altered at the adherens junctions by RAW/AMCM treatment, with an approximately 50% decrease in E-cadherin and b-catenin in the cell membrane. Importantly, levels of b-cateninassociated E-cadherin were also decreased. Following RAW/ AMCM treatment, enhanced activation of c-Src was seen prior to increased tyrosine phosphorylation of b-catenin, and this led to the destabilization of adherens junctions. Pretreatment of HepG2 cells with the Src kinase inhibitor, PP2, completely abolished the effects of RAW/AMCM on the EMT, migration, invasion, and expression and association of E-cadherin and bcatenin. AMCMs obtained from human THP-1 monocytes and mouse peritoneal macrophages also caused disassembly of the adherens junctions and migration of HepG2 cells. Furthermore, inhibition of the epidermal growth factor receptor (EGFR) with gefitinib partially prevented the downregulation of E-cadherin and b-catenin at the adherens junctions and migration behavior induced by RAW/AMCM. Our results suggest that activated macrophages have a tumor-progressive effect on HepG2 cells which involves the c-Src-and EGFR-dependent signaling cascades.

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After a transient ischemic attack of the cardiac vascular system, reactive oxygen-derived free radicals, including the superoxide (O2-.) and hydroxyl (.OH) radicals can be easily produced during reperfusion. These free radicals have been suggested to be responsible for reperfusion-induced cardiac stunning and reperfusion-induced arrhythmia. Hydrogen peroxide (H2O2) is often used as an experimental source of oxygen-derived free radicals. Using freshly dissociated single rat cardiac myocytes and the rat cardiac myoblast cell line, H9c2, we have shown, for the first time, that an intriguing pHiota acidification (approximately 0.24 pH unit) is induced by the addition of 100 micromol/L H2O2 and that this dose is without effect on the intracellular free Ca2+ levels or viability of the cells. Using H9c2 as a model cardiac cell, we have shown that it is the intracellular production of .OH, and not O2-. or H2O2, that results in this acidification. We have excluded any involvement of (1) the three known cardiac pHi regulators (the Na+-H+ exchanger, the Cl--HCO3 exchanger, and the Na+-HCO3 co-transporter), (2) a rise in intracellular Ca2+ levels, and (3) inhibition of oxidative phosphorylation. However, we have found that H2O2-induced acidosis is due to inhibition of the glycolytic pathway, with hydrolysis of intracellular ATP and the resultant intracellular acidification. In cardiac muscle and in skinned cardiac muscle fiber, it has been shown that a small intracellular acidification may severely inhibit contractility. Therefore, the sustained pHi decrease caused by hydroxyl radicals may contribute, in some part, to the well-documented impairment of cardiac mechanical function (ie, reperfusion cardiac stunning) seen during reperfusion ischemia.