Modes of association of indometacin with human polymorphonuclear leucocytes

The basic transport properties of indometacin (INDO) were investigated in human blood polymorphonuclear leucocytes (PMNs) of healthy volunteers. The silicone oil cushion centrifugation method was used as ligand-binding assay for both the measurements of the cellular association of INDO and the intracellular volume. This assay enabled us to calculate the intracellular INDO concentrations. A considerable amount of INDO accumulates in the PMNs to cause cell/medium ratios between 20 and 90. At low INDO levels (1-10 nmol/l) the cell/medium ratio appeared to be higher than at high INDO levels (greater than 2 mumol/l). This finding suggests that the cell accumulation of INDO comprises saturable binding and/or transport components. Scatchard analysis of the association isotherm of INDO after incubation for 60 min at 37 degrees C reveals apparent KD values of 3.7 mumol/l (low affinity) and 1.2 nmol/l (high affinity). The number of high-affinity association sites (60 fmol/3 X 10(6) PMNs) was 1,000-2,000 times lower than the number of low-affinity association sites (103 pmol/3 X 10(6) PMNs). INDO cell association is suggested to be a net result of diffusive and mediated influx and efflux mechanisms, and of binding to (plasma) membranes. The capacity of the PMN to accumulate INDO is enhanced upon (1) establishment of an inward gradient of [H+], and (2) activation of the Na(+)-H+ antiport by chemotactic peptide. The possible mechanisms of incorporation of INDO in the PMN are discussed in the scope of the proposed PMN-related anti-inflammatory action of INDO.