Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells

The effect of exposure of cultured P388D1 cells to H2O2 on intracellular free calcium ([Ca++]/sub i/) was investigated utilizing the intracellular fluorescent calcium chelator Quin 2. [Ca++]/sub i/ rose from approximately 150 nM to >2 μM over a time course that was strongly dependent on the concentration of H2O2 used. After exposure of P388D1 cells to 5 x 10-3 M H2O2, Quin 2 was fully saturated between 15 and 30 min exposure. During this time, no apparent change in the rate of equilibration of 45Ca++ from the extracellular medium could be detected, whereas in cells preloaded with 45Ca, net 45Ca was lost from the cells at a greater rate than controls. Measurements of total cellular calcium by atomic absorption spectroscopy confirmed that there was a net loss of calcium from the cells during the first 30 min. During the rise of intracellular [Ca++]/sub i/, cell morphology was observed by both light and scanning electron microscopy and revealed that surface blebs appeared during this phase of injury. From these results the authors conclude the following, 1) H2O2 exposure induces a dose-dependent disturbance of intracellular calcium homeostatis; 2) the rise in [Ca++]/sub i/ is mediated by exposure to H2O2 in the early phase of the injury, and is not dependent on the continuing presence of the oxidant; 3) the rate of rise of [Ca++]/sub i/ is largely independent of the quantity of calcium mobilized to the Quin 2 pool; 4) during the early phase (<30 min) of rise of [Ca++]/sub i/, only intracellular calcium is involved in the response; 5) these events occur concomitantly with gross morphological changes to the plasma membrane; and finally, 6) these events precede loss of integrity of the plasma membrane as a permeability barrier