We investigated the regulatory pathways responsible for agonist-induced internalization and down-regulation of G q protein-coupled histamine H 1 -receptors in Chinese hamster ovary cells. Histamine-induced internalization and down-regulation of H 1 -receptors were detected as the loss of [ 3 H]mepyramine binding sites on intact cells accessible to hydrophilic and hydrophobic H 1 -receptor antagonists, pirdonium and mepyramine, respectively. Pretreatment of cells with 0.1 mM histamine for 30 min at 37°C induced internalization as well as down-regulation of H 1 -receptors, both of which were inhibited either in the presence of an inhibitor against G protein-coupled receptor kinases (ZnCl 2 ) or under hypertonic conditions where clathrin-dependent endocytosis is known to be inhibited, but were not affected by inhibitors against caveolae/raft-dependent endocytosis (filipin and nystatin). Down-regulation of H 1 -receptors, but not their internalization, was inhibited by protein kinase C inhibitors (chelerythrin or GF109203X), a ubiquitin E1 inhibitor (UBEI-41) and proteasome inhibitors (lactacystin and MG-132). Neither a Ca 2 + /calmodulin-dependent protein kinase II inhibitor (KN-62) nor lysosomal protease inhibitors (E-64, leupeptin, chloroquine and NH 4 Cl) affected the internalization and down-regulation of H 1 -receptors. These results suggest that H 1 -receptors internalize upon agonist stimulation via G protein-coupled receptor kinase/clathrin-dependent but caveolae/raft-independent mechanisms and are delivered to proteasomes, preferentially to lysosomes, for their prompt down-regulation.

Abstract. We evaluated changes in the binding properties of sedative and non-sedative histamine H 1 -receptor antagonists induced by internalization of H 1 receptors in intact human U373 MG astrocytoma cells. Internalization of H 1 receptors was induced without their degradation by treatment with 0.1 mM histamine for 30 min at 37°C, and then the intact cell binding assay was performed at 4°C. The binding properties of [3 H]mepyramine, a cell-penetrating radioligand for H 1 receptors, were not changed by histamine pretreatment. Displacement curves for sedative H 1 -receptor antagonists (diphenhydramine, chlorpheniramine, promethazine, ketotifen, azelastine and oxatomide) against [ 3 H]mepyramine binding were not changed by histamine pretreatment. In contrast, the displacement curves for non-sedative H 1 -receptor antagonists (mequitazine, bepotastine, olopatadine, epinastine, carebastine, desloratadine and fexofenadine) were changed by histamine pretreatment: two types of changes, i.e. a rightward shift in the monophasic curve or an increase in the proportion of the low affinity component of the biphasic curve, were prevented under hypertonic conditions, in which clathrin-mediated receptor internalization is known to be inhibited. Thus, internalization-mediated changes in the binding properties of H 1 -receptor antagonists were well correlated with their sedative and nonsedative behaviors, which might confirm their permeability through the biomembrane and possibly the blood brain barrier.

G q/11 protein-coupled human histamine H 1 receptors in Chinese hamster ovary cells stimulated with histamine undergo clathrin-dependent endocytosis followed by proteasome/lysosome-mediated down-regulation. In this study, we evaluated the effects of a sustained increase in intracellular Ca