pH-dependent regulation of lysosomal calcium in macrophages.
pH-dependent regulation of lysosomal calcium in macrophages.
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发表时间：
2002-02
影响因子：
4
通讯作者：
K. Christensen;Jesse T. Myers;J. Swanson
中科院分区：
文献类型：
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作者：
K. Christensen;Jesse T. Myers;J. Swanson
在活细胞的酸性液泡区室中的钙测量很少，主要是因为钙的荧光探针的校准需要了解pH和探针钙结合亲和力的pH依赖性。在这里，我们报告的pH值校正测量游离钙浓度在小鼠巨噬细胞的溶酶体，使用比例和时间分辨荧光显微镜的pH值和钙的探针。巨噬细胞溶酶体中的平均游离钙浓度为4- 6x 10（-4）M，不到细胞外钙浓度的一半，但远高于胞浆钙水平。在不同的细胞外钙浓度下孵育细胞不会改变溶酶体pH，并且对溶酶体钙浓度仅具有适度的影响，表明细胞外液的内吞作用对溶酶体钙浓度提供了小但可测量的贡献。相比之下，由巴弗洛霉素A（1）或氯化铵介导的溶酶体pH值升高，使溶酶体钙浓度降低了几个数量级。溶酶体的再酸化允许溶酶体钙浓度快速恢复至更高浓度。溶酶体钙浓度的pH依赖性降低似乎是由于钙从溶酶体移动到细胞质中，因为在溶酶体碱化后可以检测到细胞溶质钙水平的增加。这些研究表明，溶酶体钙浓度很高，并且部分地由穿过溶酶体膜的质子梯度维持。此外，溶酶体可以提供细胞内钙离子水平的生理增加的来源。
Calcium measurements in acidic vacuolar compartments of living cells are few, primarily because calibration of fluorescent probes for calcium requires knowledge of pH and the pH-dependence of the probe calcium-binding affinities. Here we report pH-corrected measurements of free calcium concentrations in lysosomes of mouse macrophages, using both ratiometric and time-resolved fluorescence microscopy of probes for pH and calcium. Average free calcium concentration in macrophage lysosomes was 4-6x10(-4) M, less than half of the extracellular calcium concentration, but much higher than cytosolic calcium levels. Incubating cells in varying extracellular calcium concentrations did not alter lysosomal pH, and had only a modest effect on lysosomal calcium concentrations, indicating that endocytosis of extracellular fluid provided a small but measurable contribution to lysosomal calcium concentrations. By contrast, increases in lysosomal pH, mediated by either bafilomycin A(1) or ammonium chloride, decreased lysosomal calcium concentrations by several orders of magnitude. Re-acidification of the lysosomes allowed rapid recovery of lysosomal calcium concentrations to higher concentrations. pH-dependent reductions of lysosomal calcium concentrations appeared to result from calcium movement out of lysosomes into cytoplasm, since increases in cytosolic calcium levels could be detected upon lysosome alkalinization. These studies indicate that lysosomal calcium concentration is high and is maintained in part by the proton gradient across lysosomal membranes. Moreover, lysosomes could provide an intracellular source for physiological increases in cytosolic calcium levels.