Measurement of specific [3H]-ouabain binding to different types of human leucocytes

We have studied the specific binding of [3H]-ouabain to intact mononuclear leucocytes (82% lymphocytes) and polymorphonuclear leucocytes. In both types of cells [3H]-ouabain binding was saturable, confined to a single site of high affinity, slow to reach equilibrium, slow to reverse, temperature-dependent, competitively antagonized by potassium, and facilitated by the presence of divalent cations. The equilibrium dissociation constants were 2.4 +/- 0.7 nmol/l (polymorphs) and 2.4 +/- 0.4 nmol/l (mononuclear cells) (NS). The values of maximal specific ouabain binding, measured by Scatchard analysis of concentration vs binding curves (Bmax), were 33.9 +/- 6.0 fmol/10(6) cells (polymorphs) and 59.3 +/- 11.6 fmol/10(6) cells (mononuclear cells) (P less than 0.02). The corresponding numbers of sites per cell were 20415 +/- 3616 and 35712 +/- 6986 respectively (P less than 0.02). When the numbers of binding sites were expressed per square micron of cell surface area the difference between the two cell types was proportionately greater (83 and 186 sites per micron 2 respectively). We conclude that the [3H]-ouabain binding sites on mononuclear and polymorphonuclear leucocytes are similar in nature, but different in both number and density on the cell surface. Measurements of Bmax in mixed cell populations should therefore take account of cell type as well as cell size and number.