Rojanasakul Y, Wang L, Malanga C J, Ma J Y, Banks D E, Ma J K
School of Pharmacy, West Virginia University, Morgantown 26506.
J Cell Physiol. 1993 Feb;154(2):310-6. doi: 10.1002/jcp.1041540214.
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 microns 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 (approximately 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 preservation 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 Ca(2+)-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).(ABSTRACT TRUNCATED AT 400 WORDS)
有证据表明，二氧化硅颗粒吞噬激活后在肺泡巨噬细胞（AM）中诱导的细胞损伤可能通过细胞内游离钙[Ca2+]i的变化介导。然而，相对于[Ca2+]i过载，二氧化硅诱导细胞毒性的机制尚不清楚。为了更好地了解这一机制，将分离的大鼠AMs暴露于不同浓度的结晶二氧化硅（粒径<5微米），并用荧光钙探针Fura-2 AM和膜完整性指示剂碘化丙啶（PI）定量监测其[Ca2+]i波动和细胞完整性。本研究结果表明，二氧化硅能以剂量依赖的方式迅速增加[Ca2+]i，低剂量（<0.1 mg/ml）时钙有特征性的短暂升高，高剂量（>0.1 mg/ml）时则有升高且持续的升高。细胞外钙[Ca2+]o的耗尽显著抑制了[Ca2+]i的升高（约90%），表明细胞外来源的Ca2+内流是二氧化硅诱导[Ca2+]i升高的主要机制。低剂量使用但足以引起[Ca2+]i短暂升高时，在研究期间二氧化硅未导致细胞PI摄取显著增加，表明在这些条件下AMs的膜完整性得以保留。然而，高剂量的二氧化硅会导致PI核荧光显著增加。[Ca2+]o的耗尽极大地抑制了由浓度为0.1 mg/ml或更高剂量的二氧化硅诱导的细胞PI摄取。这表明，二氧化硅激活导致的Ca2+内流与细胞损伤有关。实际上，我们的结果进一步证明，Ca2+通道阻滞剂硝苯地平可抑制二氧化硅对Ca2+内流的低剂量效应。高剂量的二氧化硅（>0.1 mg/ml）时，硝苯地平或细胞外Ca2+耗尽并不能预防细胞损伤，这表明其他细胞毒性机制，即脂质过氧化导致的非特异性膜损伤，也与二氧化硅诱导的细胞损伤有关。在研究过程中，二氧化硅对细胞ATP含量没有显著影响，这表明观察到的二氧化硅诱导的[Ca2+]i升高不是由于限制Ca2+外流的Ca(2+)-ATP酶泵受损所致。用细胞松弛素B预处理细胞以阻断吞噬作用并不能预防二氧化硅对[Ca2+]i升高的影响。综上所述，这些结果表明，二氧化硅引起的[Ca2+]i升高主要是由于Ca2+通过质膜Ca2+通道内流以及非特异性膜损伤（高剂量时）。（摘要截短至400字）
