Interaction of [3H]dilazep at nucleoside transporter-associated binding sites on S49 mouse lymphoma cells

Dilazep, a tertiary amine that is greater than 96% protonated at pH 7.4, is a potent inhibitor of facilitated diffusion (equilibrative) nucleoside transport (NT) in animal cells. In this study, saturable reversible binding of [3H]dilazep was demonstrated at sites on S49 mouse lymphoma cells but not in AE1 cells, an NT-deficient mutant of S49 cells. Mass law analysis of dilazep binding under equilibrium conditions revealed two saturable components, representing binding sites that differed about 50-fold in affinity for dilazep (Kd values of 0.21 and 10 nM). At pH 7.4, the low affinity sites were more abundant (Bmax, 3.5 X 10(5) sites/cell) than the high affinity site (Bmax, 3.0 X 10(4) sites/cell). Binding of dilazep was pH dependent; at pH 9.0, binding at the high affinity sites predominated, whereas, at pH 5.0, the low affinity component predominated, suggesting that these components represented binding of nonprotonated and protonated dilazep molecules, respectively. Nitrobenzylthioinosine (NBMPR) and physostigmine selectively blocked binding of nonprotonated and protonated species of dilazep, respectively, at pH 7.4, yielding Scatchard plots that were similar to control plots obtained at pH 5.0 and 9.0. First-order plots of the dissociation of [3H]dilazep-binding site complexes in the presence of excess nonradioactive dilazep at pH 7.4 were nonlinear and were resolved into rapid (rate constant, 3.4-4.7 min-1) and slow (rate constant, 0.13-0.15 min-1) components. In the presence of site-saturating concentrations of NBMPR or high concentrations of nucleoside permeants, dissociation of site-bound [3H]dilazep was incomplete and only the slow component of dissociation was apparent (rate constant, 0.11-0.19 min-1). The combined presence of nonradioactive dilazep and NBMPR yielded time courses of [3H]dilazep-site dissociation equivalent to those obtained in the presence of nonradioactive dilazep alone. These results are consistent with a model in which protonated and nonprotonated species of dilazep bind at separate sites on S49 cells. The absence of both high and low affinity sites on AE1 cells suggests that, in S49 cells, both populations of sites are associated with NT polypeptides. The high affinity sites that bind nonprotonated species of dilazep appear to overlap with NBMPR binding sites on these cells.