Ca 2+ ‐dependent down‐regulation of human histamine H 1 receptors in Chinese hamster ovary cells

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 2+ concentrations induced by a receptor‐bypassed stimulation with ionomycin, a Ca 2+ ionophore, on the endocytosis and down‐regulation of H 1 receptors in Chinese hamster ovary cells. All cellular and cell‐surface H 1 receptors were detected by the binding of [ 3 H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H 1 receptor ligands, mepyramine and pirdonium, respectively. The pretreatment of cells with ionomycin markedly reduced the mepyramine‐ and pirdonium‐sensitive binding sites of [ 3 H]mepyramine, which were completely abrogated by the deprivation of extracellular Ca 2+ and partially by a ubiquitin‐activating enzyme inhibitor ( UBEI ‐41), but were not affected by inhibitors of calmodulin (W‐7 or calmidazolium) and protein kinase C (chelerythrine or GF 109203X). These ionomycin‐induced changes were also not affected by inhibitors of receptor endocytosis via clathrin (hypertonic sucrose) and caveolae/lipid rafts (filipin or nystatin) or by inhibitors of lysosomes (E‐64, leupeptin, chloroquine, or NH 4 Cl), proteasomes (lactacystin or MG ‐132), and a Ca 2+ ‐dependent non‐lysosomal cysteine protease (calpain) ( MDL 28170). Since H 1 receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H 1 receptors, even after the ionomycin treatment, H 1 receptors appeared to exist in a form to which [ 3 H]mepyramine was unable to bind. These results suggest that H 1 receptors are apparently down‐regulated by a sustained increase in intracellular Ca 2+ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.