OXIDATIVE STRESS-INDUCED PERTURBATIONS OF CALCIUM HOMEOSTASIS AND CELL-DEATH IN CULTURED MYOCYTES - ROLE OF EXTRACELLULAR CALCIUM

The role of extracellular calcium in the process of oxidative stress-i nduced calcium overload and cell death was investigated in cultured ne onatal rat myocytes. Oxidative stress was induced by addition of cumen e hydroperoxide (CHPO), a toxic organic hydroperoxide, in combination with varying extracellular calcium concentrations (1. normal calcium b uffer: 2.5 mM Ca2+, 2. low calcium buffer: 5 mu M Ca2+, 3, zero calciu m buffer: 2.5 mM EGTA, no CaCl2). Intracellular free calcium ion conce ntration ([Ca2+](i)) was measured with fura-2 using a spectrofluoromet er. To study the toxicant-induced changes in [Ca2+](i) in more detail, single cell imaging was performed using digital imaging fluorescence microscopy (DIFM). In control experiments (in the absence of CHPO, but under different extracellular Ca2+ conditions) the [Ca2+](i) remained at the basal level and cell viability was preserved. Administration o f CHPO (50 mu M) to the myocyte cultures generated a sustained increas e in [Ca2+](i) followed by loss of cell viability. A low extracellular calcium concentration (5 mu M), in the absence or presence of diltiaz em (10 mu M), induced a delay in the rise in [Ca2+](i) but was not abl e to prevent the CHPO-induced calcium overload and cell necrosis. Addi tion of EGTA (2.5 mM) to the low calcium buffer resulted also in CHPO- induced cell death, although no increase in [Ca2+](i) was observed. In normal and low calcium buffers, DIFM revealed that CHPO produced a te mporally and spatially heterogeneous distribution of [Ca2+](i) in a gr oup of myocytes. So, in the presence of normal or low extracellular Ca 2+, CHPO intoxication of cultured myocytes leads to an increase of [Ca 2+](i) prior to the onset of cell death. If extracellular Ca2+ is chel ated by EGTA, CHPO also induces cell death which is not preceded by ce llular calcium overload. Apparently a disturbance in the calcium homeo stasis is not causally related with oxidative stress-induced myocardia l cell death.