Lymphocyte calcium extrusion: kinetic and thermodynamic measurements using ratiometric dual-emission...

A method is described for monitoring intracellular ionized calcium (Ca2+) and determining kinetic and thermodynamic parameters of Ca2(+)-extrusion from intact lymphocytes. The method uses ratiometric spectrofluorometry and the fluorescent Ca2+ dye indo-1. Lymphocytes were loaded with calcium and placed in a low calcium medium. A novel formula for calculation of intracellular Ca2+ that corrects for background fluorescence and fluorescence quenching was used. Calcium extrusion resulted in exponential decrease in cytoplasmic Ca2+ with a rate constant of 0.031 +/- 0.003 sec-1, maximal rate of 23 +/- 7 nM/sec, dissociation constant of 366 +/- 63 nM, Hill coefficient of 2.3 +/- 0.4, Q10 of 2.58 +/- 0.28, and activation energy of 18.3 Kcal/mol. This method should allow for characterization of the Ca2(+)-extrusion system of lymphocytes and may be applicable to other blood cell types.