Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D 1 cells

The effects of exposure of cultured P388D 1 cells to H 2 O 2 on intracellular free calcium ([Ca ++ ] i ) was investigated utilizing the intracellular fluorescent calcium chelator “Quin 2.” [Ca ++ ] i rose from approximately 150 nM to >2 μM over a time course that was strongly dependent on the concentration of H 2 O 2 used (5 × 10 −5 to 5 × 10 −3 M). After exposure of P388D 1 cells to 5 × 10 −3 M H 2 O 2 , Quin 2 was fully saturated between 15 and 30 min exposure. During this time, no apparent change in the rate of equilibration of 45 Ca ++ from the extracellular medium could be detected, whereas in cells preloaded with 45 Ca, net 45 Ca was lost from the cells at a greater rate than controls. Measurements of total cellular calcium by atomic absorption spetcroscopy confirmed that there was a net loss of calcium from the cells during the first 30 min. At time points >45 min after exposure to H 2 O 2 the influx of extracellular 45Ca and net intracellular Ca ++ , Na + and K + rapidly increased. Half times for H 2 O 2 catabolism by the cells varied from about 8 min at 5.0 × 10 −4 M H 2 O 2 to 14.0 min at 5.0 × 10 −3 M. When the total [Ca ++ ] i ‐buffering capacity of the Quin 2 pool was varied by increasing the loading of intracellular Quin 2 by 68‐fold (1.1 × 10 2 ‐ 7.6 × 10 3 amol per cell), the rate of rise of [Ca ++ ] i was depressed by only 1.6‐fold following exposure to 5 mM H 2 O 2 . During the rise of intracellular [Ca ++ +] i , cell morphology was observed by both light and scanning electron microscopy and revealed that “surface blebs” appeared during this phase of injury. Both the rise in [Ca ++ +] i and “blebbing” were observable before any loss in cell viability was detected by either loss of Trypan blue exclusion or loss of preloaded 51 Cr from the cells. From these results we conclude the following, (1) H 2 O 2 exposure induces a dose‐dependent disturbance of intracellular calcium homeostatis; (2) the rise in[Ca ++ +] i is mediated by exposure to H 2 O 2 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 ++ +] 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 ++ +] 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.