Ultrastructural demonstration of calcium loss from local regions of the plasma membrane of surface-stimulated human granulocytes.
Ultrastructural demonstration of calcium loss from local regions of the plasma membrane of surface-stimulated human granulocytes.
复制标题
表面刺激的人粒细胞质膜局部区域钙损失的超微结构演示。
作者：
S. Hoffstein
大多数细胞相关钙以结合钙而不是游离钙的形式存在。由于钙和其他阳离子是多形核白细胞(PMN)功能的重要调节因子，我们用超微结构细胞化学方法研究了静息PMN中结合钙的定位及其在各种免疫刺激下的再分布。在一些实验中，使用焦锑酸钾原位沉淀阳离子；在另一些实验中，细胞在固定过程中负载外源性钙以定位结合部位。这两种方法都导致沿着静息细胞质膜出现离散的电子致密沉积。在这两种情况下，沉淀物都可以用3 mM的EGTA和EDTA去除。额外的焦锑酸盐主要在细胞核的异染色质区域和嗜天青颗粒中沉淀，但不在细胞液中。PMN与颗粒刺激物预先孵育，如调理酵母多糖、免疫复合物沉淀物或ConA共价结合到琼脂糖珠上，导致质膜上与结合颗粒接近的区域的焦锑可沉淀阳离子迅速(不到10秒)的节段性丢失，而不是来自较远的膜区域。在细胞的其余部分，包括质膜上不与颗粒接触的区域，沉淀物的分布没有改变。将整个细胞表面暴露在可溶性刺激下，如ConA、趋化肽F-Met-leu-Phe或钙离子载体A23187，会导致整个质膜上可沉淀的焦锑酸盐钙沿圆周方向丢失，但不会从细胞内的任何位置损失。这些数据表明，在足够高的浓度下，阳离子与静息PMN的质膜结合，以沉淀焦锑，这些与膜相关的阳离子是从受体-配体相互作用的部位释放出来的。
Most cell-associated calcium exists as bound rather than free ionized calcium. Since calcium and other cations are important regulators of polymorphonuclear leukocyte (PMN) function, the localization of bound calcium in resting PMN and its redistribution after exposure to a variety of immune stimuli was investigated by means of ultrastructural cytochemistry. Potassium pyroantimonate was used to precipitate cations in situ in some experiments; in others, cells were loaded with exogenous calcium during fixation to localize binding sites. Both methods resulted in the appearance of discrete electrondense deposits along the plasmalemma of resting cells. The deposits could in both cases be removed with 3 mM EGTA as well as EDTA. Additional pyroantimonate was precipitated primarily in the heterochromatin regions of the nucleus, and in azurophile granules, but not in the cell sap. Preincubation of PMN with particulate stimuli, such as opsonized zymosan, immune complex precipitates, or Con A covalently bound to Sepharose beads, resulted in the rapid (less than 10 sec) segmental loss of pyroantimonate precipitable cations from regions of plasma membrane that were in close apposition to the bound particle but not from more distant areas of membrane. The distribution of precipitates in the remainder of the cell, including regions of plasmalemma not in contact with particles, was unaltered. Exposure of the entire cell surface to soluble stimuli such as Con A, the chemotactic peptide F-met-leu-phe, or the calcium ionophore A23187, resulted in the circumferential loss of pyroantimonate precipitable calcium from the entire plasma membrane but not from any intracellular location. The data suggest that cations are associated with the plasma membrane of resting PMN in concentrations sufficiently high to precipitate pyroantimonate and that these membrane-associated cations are released from sites of receptor-ligand interaction.