Ca2+-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 Ca2+ concentrations induced by a receptor-bypassed stimulation with ionomycin, a Ca2+ 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 [H-3]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 [H-3]mepyramine, which were completely abrogated by the deprivation of extracellular Ca2+ 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 GF109203X). 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 NH4Cl), proteasomes (lactacystin or MG-132), and a Ca2+-dependent non-lysosomal cysteine protease (calpain) (MDL28170). 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 [H-3]mepyramine was unable to bind. These results suggest that H-1 receptors are apparently down-regulated by a sustained increase in intracellular Ca2+ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.