Ca2+ -dependent down-regulation of human histamine H1 receptors in Chinese hamster ovary cells

G_q/11 protein-coupled human histamine H₁ 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 H₁ receptors in Chinese hamster ovary cells. All cellular and cell-surface H₁ receptors were detected by the binding of [³ H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H₁ 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 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 NH₄ Cl), proteasomes (lactacystin or MG-132), and a CA²⁺ -dependent non-lysosomal cysteine protease (calpain) (MDL28170). Since H₁ receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H₁ receptors, even after the ionomycin treatment, H₁ receptors appeared to exist in a form to which [³ H]mepyramine was unable to bind. These results suggest that H₁ receptors are apparently down-regulated by a sustained increase in intracellular CA²⁺ concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.

Ca2+; down-regulation; endocytosis; histamine H1 receptor; internalization; ionomycin.