Intracellular calcium homeostasis during hydrogen peroxide injury to cultured P388D1 cells

Abstract

The effects of exposure of cultured P388D₁ cells to H₂O₂ 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 H₂O₂ used (5 × 10⁻⁵ to 5 × 10⁻³M). After exposure of P388D₁ cells to 5 × 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 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 H₂O₂ the influx of extracellular 45Ca and net intracellular Ca⁺⁺, Na⁺ and K⁺ rapidly increased. Half times for H₂O₂ catabolism by the cells varied from about 8 min at 5.0 × 10⁻⁴ M H₂O₂ 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 × 10² - 7.6 × 10³ amol per cell), the rate of rise of [Ca⁺⁺]_i was depressed by only 1.6-fold following exposure to 5 mM H₂O₂. 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 ⁵¹Cr from the cells. From these results we conclude the following, (1) H₂O₂ exposure induces a dose-dependent disturbance of intracellular calcium homeostatis; (2) the rise in[Ca⁺⁺+]_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⁺⁺+]_i is largely independent of the quantity of calcium mobilized to the Quin 2 pool; (4) during the early phase (+++]_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.