There is evidence to suggest that cell injury induced in alveolar macrophages (AM) following phagocytic activation by silica particles may be mediated through changes in intracellular free calcium [Ca2+]i. However, the mechanism of silica-induced cytotoxicity relative to [Ca2+]i overloading is not yet clear. To provide a better insight into this mechanism, isolated rat AMs were exposed to varying concentrations of crystalline silica (particle size < 5 mum in diameter) and the fluctuation in their [Ca2+]i and cell integrity were quantitatively monitored with the fluorescent calcium probe, Fura-2 AM, and the membrane integrity indicator, propidium iodide (PI). Results from this study indicate that silica can rapidly increase [Ca2+]i in a dose-dependent manner with a characteristic transient calcium rise at low doses (<0.1 mg/ml) and an elevated and sustained rise at high doses (>0.1 mg/ml). Depletion of extracellular calcium [Ca2+]o markedly inhibited the [Ca2+]i rise (almost-equal-to 90%), suggesting that Ca2+ influx from extracellular source is a major mechanism for silica-induced [Ca2+]i rise. When used at low doses but sufficient to cause a transient [Ca2+]i rise, silica did not cause significant increase in cellular PI uptake during the time of study, suggesting the presevation of membrane integrity of AMs under these conditions. At high doses of silica, however, a marked increase in PI nuclear fluorescence was observed. Depletion of [Ca2+]o greatly inhibited cellular PI uptake, induced by 0.1 mg/ml or higher doses of silica. This suggests that Ca2+ influx, as a result of silica activation, is associated with cell injury. Indeed, our results further demonstrated that the low dose effect of silica on Ca2+ influx is inhibited by the Ca2+ channel blocker nifedipine. At high doses of silica (>0.1 mg/ml), cell injury was not prevented by nifedipine or extracellular Ca2+ depletion, suggesting that other cytotoxic mechanisms, i.e., nonspecific membrane damage due to lipid peroxidation, are also responsible for the silica-induced cell injury. Silica had no significant effect on cellular ATP content during the time course of the study, indicating that the observed silica-induced [Ca2+]i rise was not due to the impairment of Ca2+-ATPase pumps, which restricts Ca2+ efflux. Pretreatment of the cells with cytochalasin B to block phagocytosis failed to prevent the effect of silica on [Ca2+]i rise. Taken together, these results suggest that the elevation of [Ca2+]i caused by silica is due mainly to Ca2+ influx through plasma membrane Ca2+ channels and nonspecific membrane damage (at high doses). Neither ATP depletion nor Ca2+ leakage during phagocytosis was attributed to the silica-induced [Ca2+]i rise.
