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Abstract
Neutrophils loaded with the calcium indicator quin-2 and challenged with the ionophore ionomycin or the chemotactic peptide fMet-Leu-Phe were examined in the light of a theory that relates time-dependent changes in the fluorescence of the indicator to cytosolic calcium fluxes and levels. The cytosolic binding capacity was estimated from the theory to be 1.5 +/- 0.6 X 10(8) sites/cell (0.76 mM based on a cell volume of 330 micron 3, irrespective of water content and the distribution of sites), each site having an apparent average single class dissociation constant of 0.55 +/- 0.2 microM. Some 20% of the total available cytosolic calcium sites of the normal resting cell appear to be occupied when no quin-2 is present. In a calcium-free medium, the amount of calcium released by fMet-Leu-Phe from storage pool locations that are distinct from the cytosolic sites is sufficient to further raise the cytosolic site occupancy level to 50%, at which point the calcium buffering capacity of the cytosol is maximal. In a calcium-containing medium, however, simultaneous influx from the outside appears to supply enough additional calcium to saturate most of the remaining sites. The combined initial rate of storage pool calcium release plus influx through the plasma membrane was roughly twice the initial rate at which calcium was released from storage locations alone, suggesting that stimulus-induced influx from the outside may be comparable in importance to storage pool mobilization in determining physiological calcium levels in stimulated cells.
Highlights
Neutrophils loaded with the calcium indicator quin- capacity for buffering calcium, a value that has been previ
The relation between the fluorescence intensity of intracellular quin-2 and cytosolic calcium as givenby Equations 4 and 5 indicates that fluorescence changes must be interpreted in terms of the amount of quin-2 present
Changes in thetotal cytosolic calcium concentrationunderthese conditions are proportional to theobserved change in quin-2 fluorescence, and the calcium fluxes can be monitored directly by quin-2 fluorescence
Summary
From the Theodor Kocher Institute, University of Bern, CH-3000 Bern 9, Switzerlund. Neutrophils loaded with the calcium indicator quin- capacity for buffering calcium, a value that has been previ-. One of the earliest effects observed following neutrophil more than 95% neutrophils, which were kept in the medium at room stimulation is a rise in cytosolic calcium concentration [1,2,3,4] This was studied by measuring the distribution of radioactive calcium (l),and by using the fluorescent calcium indicator quin-2 [2,3,4] which is presumed to be confined exclusively to temperature until use. R. Squibb & Sons, Princeton, NJ; 500 pmol/million neutrophils) was added to loaded cells in the presence of high external calcium levels to bring about complete saturation of intracellular quin-2, and thefluorescence was taken asFmm.All quin fluorescence was quenched by addition ofMnC12, and the background fluorescence (cell autofluorescence) was taken as Fo. In agreement with earlier work [8, 16], the fluorescence of the calciumquin-2 complex was found to be six times that of free quin-2, so that. Whereas Equation contains the information about the amounts of calcium in the cytosol, Equation 5 provides the theoretical basis for understanding the time dependence of the calcium fluxes
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