Hishinuma Shigeru, Komazaki Hiroshi, Tsukamoto Hayato, Hatahara Hirokazu, Fukui Hiroyuki, Shoji Masaru
Department of Pharmacodynamics, Meiji Pharmaceutical University, Tokyo, Japan.
Department of Molecular Studies for Incurable Diseases, Institute of Biomedical Sciences, Tokushima University Graduate School, Tokushima, Japan.
J Neurochem. 2018 Jan;144(1):68-80. doi: 10.1111/jnc.14245. Epub 2017 Nov 21.
G protein-coupled human histamine H 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 concentrations induced by a receptor-bypassed stimulation with ionomycin, a Ca ionophore, on the endocytosis and down-regulation of H receptors in Chinese hamster ovary cells. All cellular and cell-surface H receptors were detected by the binding of [ H]mepyramine to intact cells sensitive to the hydrophobic and hydrophilic H receptor ligands, mepyramine and pirdonium, respectively. The pretreatment of cells with ionomycin markedly reduced the mepyramine- and pirdonium-sensitive binding sites of [ H]mepyramine, which were completely abrogated by the deprivation of extracellular Ca 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 NH Cl), proteasomes (lactacystin or MG-132), and a Ca -dependent non-lysosomal cysteine protease (calpain) (MDL28170). Since H receptors were normally detected by confocal immunofluorescence microscopy with an antibody against H receptors, even after the ionomycin treatment, H receptors appeared to exist in a form to which [ H]mepyramine was unable to bind. These results suggest that H receptors are apparently down-regulated by a sustained increase in intracellular Ca concentrations with no process of endocytosis and lysosomal/proteasomal degradation of receptors.
在中国仓鼠卵巢细胞中，用组胺刺激的G蛋白偶联人组胺H受体经历网格蛋白依赖性内吞作用，随后由蛋白酶体/溶酶体介导下调。在本研究中，我们评估了用离子霉素（一种钙离子载体）进行受体旁路刺激诱导的细胞内钙离子浓度持续升高对中国仓鼠卵巢细胞中H受体的内吞作用和下调的影响。通过[³H]美吡拉敏分别与对疏水性和亲水性H受体配体美吡拉敏和匹多宁敏感的完整细胞结合来检测所有细胞和细胞表面的H受体。用离子霉素预处理细胞显著降低了[³H]美吡拉敏对美吡拉敏和匹多宁敏感的结合位点，细胞外钙离子缺失可使其完全消除，泛素激活酶抑制剂（UBEI-41）可使其部分消除，但不受钙调蛋白抑制剂（W-7或氯米帕明）和蛋白激酶C抑制剂（白屈菜红碱或GF109203X）的影响。这些离子霉素诱导的变化也不受网格蛋白介导的受体内吞作用抑制剂（高渗蔗糖）、小窝/脂筏抑制剂（制霉菌素或制假丝菌素）、溶酶体抑制剂（E-64、亮抑酶肽、氯喹或氯化铵）、蛋白酶体抑制剂（乳胞素或MG-132）以及钙离子依赖性非溶酶体半胱氨酸蛋白酶（钙蛋白酶）抑制剂（MDL28170）的影响。由于即使在离子霉素处理后，通过共聚焦免疫荧光显微镜用抗H受体抗体仍能正常检测到H受体，H受体似乎以[³H]美吡拉敏无法结合 的形式存在。这些结果表明，H受体明显因细胞内钙离子浓度持续升高而下调，且不存在受体内吞作用以及受体的溶酶体/蛋白酶体降解过程。
