Mechanism of Hydrogen Peroxide and Hydroxyl Free Radical–Induced Intracellular Acidification in Cultured Rat Cardiac Myoblasts

Abstract

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.

Mechanism of oxidative stress‐induced intracellular acidosis in rat cerebellar astrocytes and C6 glioma cells

“…In summary, we have shown that in glial cells, HµOµ induces a marked intracellular acidosis, probably via inhibition of glycolysis by ·OH. Since this acidosis is similar to that seen in cardiac myoblasts (Wu, Tsai, Wang, Wu, Wang & Lee, 1996) it is possible that this phenomenon exists in many other cells. This intracellular acidosis may influence the ability of glial cells to regulate the pHï of the microenviroment and may probably contribute, in part, to the neuronal activity and cytotoxic cell oedema seen during brain ischaemic reperfusion.…”

A cytosolic source of calcium unveiled by hydrogen peroxide with relevance for epithelial cell death

“…These compartments store high mM levels of calcium, and a functional exchange of Ca 2 þ for H þ occurs at both endosomes 31 and lysosomes. 32 Due to its induction of fast ATP hydrolysis, hydrogen peroxide rapidly decreases cytosolic pH, 25 thus setting the right conditions for accelerated proton/calcium exchange at these organelles. However, abrogation of peroxide-induced cytosolic acidification in HeLa cells using a pH clamp did not significantly affect the calcium rise, showing that proton/ calcium exchange is not an important mechanism of oxidative calcium release.…”

“…Lysosomal calcium, which has been recently measured at about 500 mM, is much less sensitive to extracellular calcium depletion. 32 Similar to endosomal calcium, lysosomal calcium varies reciprocally with organelle proton concentration; so the large cytosolic acidification resulting from peroxide exposure 25 may in principle release the divalent in exchange for protons. An important source of protons in this case is the hydrolysis of ATP due to glycolytic inhibition by peroxide.…”

“…An important source of protons in this case is the hydrolysis of ATP due to glycolytic inhibition by peroxide. 25 In HeLa cells coloaded with 2 0 7 0 -bis(2-carboxyethyl)-5(6)-carboxyfluorescein (BCECF) and Rhod-2 ( Figure 5a), exposure to H 2 O 2 caused a significant decrease in pH, which started simultaneously with the calcium increase ( Figure 5c). In order to investigate a possible role for protons as calcium agonists, we inhibited the acidification induced by peroxide by applying a þ /K þ exchanger that clamps proton concentration to the reciprocal of the ratio between intracellular and extracellular potassium concentration.…”

Natural (ghrelin) and synthetic (hexarelin) GH secretagogues stimulate H9c2 cardiomyocyte cell proliferation

“…It has been used in several studies to investigate different cellular mechanisms in cardiomyocytes (Sipido & Marban 1991, Wu et al 1996, Chen et al 2000 and has been shown to be responsive to IGF-I (Chen et al 1995, Bahr et al 1997.…”