Characteristics of the binding of [3H]‐mepyramine to intact human U373 MG astrocytoma cells: evidence for histamine‐induced H₁‐receptor internalisation

Abstract

1 The kinetics of the binding of 5 nM [3Hl-mepyramine to sites on intact human U373 MG astrocytoma cells, sensitive to inhibition by 2 gM pirdonium, were temperature-dependent. At 370C the half-time for association was 0.9 ±0.4 min and at 40C 19±3 min. Dissociation of bound [3H]-mepyramine was fast at 370C, tO.5 1.5 +0.3 min, but at 60C dissociation initiated by dilution or addition of unlabelled mepyramine was negligible over 120 min. The very slow dissociation at 60C 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. 4 The percentages of the high-affinity site in curves of the inhibition of [3H]-mepyramine binding to intact U373 MG cells by two tertiary amine antagonists, norpirdonium 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 Hi-antagonist temelastine, 42 + 5%.5 Incubation of U373 MG cells with 100 gM histamine at 37°C, followed by washing twice in ice-cold medium and then incubation with 1-15 nM [3H]-mepyramine for 120 min at 4°C, resulted in a decrease in the binding of [3H]-mepyramine sensitive to 2 gM pirdonium, compared to control cells not exposed to histamine. The binding of [3H]-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 gM 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 [3H]-mepyramine to intact U373 MG cells being to both plasma membrane and intracellular histamine HI-receptors. The high-affinity binding sensitive to the non-cell penetrant quaternary compounds and to temelastine is thus to plasma membrane H,-receptors. On exposure to 100 gM histamine receptors are translocated to the intracellular pool, since the change in the high-affinity binding of [3H]-mepyramine is primarily in the level of the pirdoniuminsensitive binding, rather than in the total binding.

Intact Cell Binding for In Vitro Prediction of Sedative and Non-sedative Histamine H1–Receptor Antagonists Based on Receptor Internalization

“…High and low affinity sites for olopatadine, epinastine, or fexofenadine showed exactly the same difference observed between tertiary amine antagonists and their quaternary derivatives, including pirdonium and norpirdonium (17), yet the percentage of the binding of [ 3 H]mepyramine insensitive to inhibition is approximately the same, irrespective of whether the antagonists are tertiary or quaternary amines (17) or sedative or non-sedative (Table 1). This suggests that the secondary site is intracellular, but there is only sufficient intracellular compound for inhibition to be observed during the experiment at higher extracellular concentrations of antagonist.…”

“…Human U373 MG astrocytoma cells (National Culture Collection, Porton Down, UK) were cultured in 150-cm 2 culture flasks as described previously (17). Dissociated cells were suspended in normal HEPES buffer (120 mM NaCl, 5.4 mM KCl, 1.6 mM MgCl 2 , 1.8 mM CaCl 2 , 11 mM D-glucose, and 25 mM HEPES, pH 7.4 at 37°C) and kept at 37°C for 30 min for equilibration.…”

“…We have established an intact cell binding method to evaluate the number of G q proteincoupled histamine H 1 receptors on the cell surface in human U373 MG astrocytoma cells by using pirdonium, an H 1 -receptor antagonist with a permanently charged quaternary ammonium group, to define the binding of a cell-penetrant radioligand, [ 3 H]mepyramine, to cell surface H 1 receptors (17,18). In these measurements, internalization of H 1 receptors was monitored as a decrease in pirdonium-sensitive […”

Histamine hyperpolarizes human glioblastoma cells by activating the intermediate-conductance Ca2+-activated K+ channel

“…H 1 and H 2 receptors have been reported in both native astrocytes and in astrocytoma cell lines (12)(13)(14)29). Using the selective antagonists of the different histamine receptors, we showed that the H 1 receptor is involved.…”

“…Histamine binds to three types of membrane receptors: H 1 receptors, usually coupled to phospholipase C activation and internal Ca 2ϩ mobilization; H 2 receptors, connected with adenylate cyclase; and H 3 receptors, controlling histamine turnover and release (11,21). Astrocytes as well as astrocytoma cell lines have been demonstrated to express both H 1 and H 2 histamine receptors (12,13,14,29). Stimulation of H 1 receptors results in an increased intracellular Ca 2ϩ concentration ([Ca 2ϩ ] i ) in glioma cells, caused by an initial transient phase of Ca 2ϩ mobilization from intracellular stores and a second sustained phase mediated by Ca 2ϩ influx (1,10,24,25,31,41,43).…”