![]() The 490/440 emission ratio from the intracellular BCECF was calculated and converted to a linear pH scale (see below) by in situ calibration at pH 4.5 and 9.5 performed at the end of the experiment using the nigericin technique, described elsewhere. The excitation light was transmitted to the cell under study using a 510-nm dichroic mirror under the microscope nosepiece, and the resulting fluorescence was collected by a ×40 oil-immersion lens. The cells were then washed with the same solution and excited alternately by 490- and 440-nm wavelength light, using a filter wheel (Cairn Research) rotating at 32 Hz. 26 In brief, single cardiac myocytes or H9c2 cells, grown on a cover glass, were loaded with 5 μmol/L BCECF-AM (Molecular Probes) for 5 to 10 minutes at room temperature in HEPES-buffered solution. Measurement of pH i has been described in detail elsewhere. OH radicals in the presence of ferrous ions 22 23 :.16 21 It can readily cross the cell membrane 22 and be converted, via the Fenton reaction, to the more toxic H 2O 2 is an important tool for studying the effects of oxygen-derived free radicals on the reperfused ischemic myocardium. 10 11 16 17 During ischemia or reperfusion, intracellular measurements show that the free Mg 2+ level in cardiac tissues increases as a result of the depletion of intracellular ATP. OH, are implicated as the major factors responsible for reperfusion-induced cardiac arrhythmia, 13 14 cardiac ultrastructural abnormalities, 15 reduction of the Ca 2+ transient and contractility, 16 increased diastolic Ca 2+ levels, 16 inhibition of glycolysis and oxidative phosphorylation and intracellular ATP depletion. ![]() 1 10 11 12 Moreover, both the oxygen-derived free radicals, O 2 − OH that is suggested as having the most damaging effects, including cytotoxicity and cardiac stunning, during ischemic reperfusion.has been implicated as a mediator of ischemia/reperfusion-induced leukocyte adhesion in postcapillary venules 8 and of increased microvascular permeability, 9 but it is.3 These ROS are derived from a variety of sources, such as activated neutrophils, 4 5 leakage of electrons from mitochondria, 6 the xanthine oxidase system, 1 and the cyclooxygenase pathway of arachidonic acid metabolism. 1 2 3 The initial burst of ROS production peaks 2 minutes after reflow, and production continues for up to 3 hours. OH) radicals, and hydrogen peroxide (H 2O 2) are formed during reperfusion or reoxygenation of ischemic or hypoxic myocardium.It is well documented that ROS, such as the superoxide (O 2 − Therefore, the sustained pH i 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. In cardiac muscle and in skinned cardiac muscle fiber, it has been shown that a small intracellular acidification may severely inhibit contractility. However, we have found that H 2O 2-induced acidosis is due to inhibition of the glycolytic pathway, with hydrolysis of intracellular ATP and the resultant intracellular acidification. We have excluded any involvement of (1) the three known cardiac pH i regulators (the Na +-H + exchanger, the Cl −-HCO 3 exchanger, and the Na +-HCO 3 cotransporter), (2) a rise in intracellular Ca 2+ levels, and (3) inhibition of oxidative phosphorylation. or H 2O 2, that results in this acidification. ![]() Using H9c2 as a model cardiac cell, we have shown that it is the intracellular production of 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 pH i acidification (≈0.24 pH unit) is induced by the addition of 100 μmol/L H 2O 2 and that this dose is without effect on the intracellular free Ca 2+ levels or viability of the cells. Hydrogen peroxide (H 2O 2) is often used as an experimental source of oxygen-derived free radicals. These free radicals have been suggested to be responsible for reperfusion-induced cardiac stunning and reperfusion-induced arrhythmia.
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