The characteristics of [3H]‐ouabain binding to human lymphocytes.

Abstract

sodium/potassium pump sites were 43154 + 8037 molecules/cell (n = 25) in lymphocytes and 75474 ± 6764 (n = 9) molecules/cell in mixtures of mononucleated cells.5 Direct determination of the [3H]-ouabain binding capacity of lymphocytes can be performed with acceptable accuracy and precision using 30 ml whole blood. Provided high cell purity, this method may be useful, when studying sodium/potassium pump function in clinical settings.

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

“…In lymphocytes glycoside binding has been shown to be saturable, confined to a single site of high affinity, temperature-sensitive, slow to reach equilibrium, slow to reverse, and competitively inhibited by potassium (Quastel & Kaplan, 1975;Pedersen & Klitgaard, 1983). Our work has confirmed these qualitative observations in intact mononuclear leucocytes (the majority of which were lymphocytes), and has demonstrated the same properties in intact polymorphonuclear leucocytes.…”

“…The equilibrium dissociation constants calculated from our data are similar to those found by Erdmann & Hasse (1975) in human erythrocytes (2.8 x 10-9 mol/l) and cardiac membranes (2.8 x 10-9 mol/l). Although other workers have reported values ranging from 2.9 to 7.8 x 10-9 mol/l for human erythrocytes we believe that these figures are based on experiments in which binding was not at equilibrium, and that a value of about 1 to 2 x 10-9 mol/l is a more accurate assessment of the true KD of glycoside binding to the membranes of intact erythrocytes (see Ford et al, 1979 (Baker & Willis, 1972 (Wiley et al, 1979;Pedersen & Klitgaard, 1983 ouabain binding characteristics in these, and other, cells is the very slow rate of both association and dissociation. The potential problems of studying a slowly equilibrating ligand-receptor interaction have been discussed by Aranyi (1979Aranyi ( , 1980 (Aranyi, 1979).…”

“…However, these discrepancies may reflect the fact that other workers have tended to use shorter incubation times and may not have reached true equilibrium; they may also be due to differences in the definition of specific binding and differences in the cell population under study. For example, Arnott et al (1982), Wiley et al (1979) and Pedersen & Klitgaard (1983) (Oh et al, 1983), and this may at least partly explain their high value for Bmax.…”

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

“…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.…”

“…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.…”