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

Abstract

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.

Discrepancy between the short and long term effects of ouabain on the sodium pumps of human cells grown in culture

“…As the apparent sensitivity of the HeLa Na', K+-ATPase in short term experiments is similar to that of other types of human cells (heart cell membranes; Erdmann, 1981: leucocytes;Boon et al, 1984); it may well be that the true sensitivity of these other cells is also some 4 to 10 times the apparent value. This sensitivity is such that if this isoform is present in human heart the normal plasma concentrations of cardiac glycosides of a few nm should give a substantial block of the sodium pumps.…”

Ouabain inhibition of the sodium‐potassium pump: estimation of ED50 in different types of human leucocytes in vitro.

“…This could be explained by different potassium permeabilities of the plasma membranes of MNL and PMNL. Uncoupled sodium-efflux in MNL may also be considered as a possible mechanism for the decreased uptake of K+ (86Rb+) by MNL (Glynn & Karlish, 1975 , 1984;Clausen, 1986;Ford et al, 1979). Qualitative differences in the pump units from different tissues may also occur.…”

“…The observed rates of pump inhibition reflect a low rate of association of ouabain with pump. Ligand-binding studies with tritiated ouabain have previously demonstrated low rates of association of ouabain with the Na+-K+ pump of human leucocytes at ouabain concentrations of 108mol 1-1 or less (Boon et al, 1984;Pedersen & Klitgaard, 1983). Except for the extreme concentrations of ouabain studied, which caused either no inhibition (10'0°mol 1-l) or an immediately obtained maximal inhibition (105mol L-), a steady-state level of pump inhibition was observed after about 2.5 h incubation of both MNL and PMNL with ouabain.…”

“…Suspensions of intact human mononuclear and polymorphonuclear leucocytes were prepared from whole blood by centrifugation on a discontinuous gradient of Percoll following an initial sedimentation step with dextran, described by Boon et al (1984). Whole blood was obtained from healthy volunteers by venepuncture using 1% (w/v) EDTA for anticoagulation when the cells were suspended in Ringer solution, and heparin (125 i.u.…”

“…Ouabain binding to the pump has been used to estimate the number of pump units, because of the 1:1 molar stoichiometry (Glynn & Karlish, 1975), and the binding characteristics of ouabain to various types of leucocytes have been described (Boon et al, 1984;Pedersen & Klitgaard, 1983). However, binding characteristics have not been compared with measurements of the effect of ouabain on pump activity.…”