Ultrastructural Demonstration of Calcium Loss from Local Regions of the Plasma Membrane of Surface-Stimulated Human Granulocytes

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Abstract
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.