Characteristics of the binding of [3H]‐mepyramine to intact human U373 MG astrocytoma cells: evidence for histamine‐induced H 1 ‐receptor internalisation

1 The kinetics of the binding of 5 n m [ 3 H]‐mepyramine to sites on intact human U373 MG astrocytoma cells, sensitive to inhibition by 2 μ m pirdonium, were temperature‐dependent. At 37°C the half‐time for association was 0.9 ± 0.4 min and at 4°C 19 ± 3 min. Dissociation of bound [ 3 H]‐mepyramine was fast at 37°C, t 0.5 1.5 ± 0.3 min, but at 6°C dissociation initiated by dilution or addition of unlabelled mepyramine was negligible over 120 min. The very slow dissociation at 6°C made it possible to reduce the level of pirdonium‐insensitive binding from 56 ± 5% to 39 ± 5% by washing the cells in ice‐cold medium before filtration. 2 The binding of [ 3 H]‐mepyramine sensitive to 2 μ m temelastine, measured after 10 min equilibration at 37°C, failed to saturate and was resolved into an hyperbola and an apparently linear component, whereas the fit to the binding of [ 3 H]‐mepyramine sensitive to 2 μm pirdonium was not significantly improved over that to an hyperbola. The mean K d for the binding of [ 3 H]‐mepyramine to the saturable component, 2.5 ± 0.4 n m , was in close agreement with the value of 3.5 n m for mepyramine derived from inhibition of histamine H 1 receptor‐mediated inositol phosphate formation in U373 MG cells. 3 Curves for the inhibition of the binding of 5 n m [ 3 H]‐mepyramine to U373 MG cells by histamine H 1 ‐receptor antagonists were biphasic and were fitted to a two site‐model. Affinities calculated from the best‐fit IC 50 values for the nigh‐affinity site correlated well with those expected for binding to H 1 receptors. 4 The percentages of the high‐affinity site in curves of the inhibition of [ 3 H]‐mepyramine binding to intact U373 MG cells by two tertiary amine antagonists, nor pirdonium and 4‐methyldiphenhydramine, 68 ± 3 and 63 ± 4, were significantly greater than the percentages of the high‐affinity site in the inhibition curves of their quaternary derivatives, 50 ± 1 and 45 ± 3, respectively. Similarly, the percentage of the high‐affinity site for unlabelled mepyramine, 65 ± 7, was greater than for the non‐cell penetrant H 1 ‐antagonist temelastine, 42 ± 5%. 5 Incubation of U373 MG cells with 100 μm histamine at 37°C, followed by washing twice in ice‐cold medium and then incubation with 1 −15 n m [ 3 H]‐mepyramine for 120 min at 4°C, resulted in a decrease in the binding of [ 3 H]‐mepyramine sensitive to 2 μ m pirdonium, compared to control cells not exposed to histamine. The binding of [ 3 H]‐mepyramine in the absence of pirdonium was not altered by histamine pretreatment, whereas the level of the pirdonium‐insensitive binding was significantly increased, except after 1 min exposure to histamine. The decreases in the pirdonium‐sensitive binding after 5, 10 and 60 min incubation with 100 μ m histamine were 41 ± 6, 56 ± 6 and 67 ± 8, respectively, but the decrease after 1 min incubation with histamine, 16 ± 8, was not statistically significant. 6 The results are consistent with the binding of [ 3 H]‐mepyramine to intact U373 MG cells being to both plasma membrane and intracellular histamine H 1 receptors. The high‐affinity binding sensitive to the non‐cell penetrant quaternary compounds and to temelastine is thus to plasma membrane H 1 receptors. On exposure to 100 μ m histamine receptors are translocated to the intracellular pool, since the change in the high‐affinity binding of [ 3 H]‐mepyramine is primarily in the level of the pirdonium‐insensitive binding, rather than in the total binding.