Kameyama Yoshio, Yamashita Keiko, Kobayashi Kaoru, Hosokawa Masakiyo, Chiba Kan
Laboratory of Pharmacology and Toxicology, Graduate School of Pharmaceutical Sciences, Chiba University, Chiba, Japan.
Pharmacogenet Genomics. 2005 Jul;15(7):513-22. doi: 10.1097/01.fpc.0000170913.73780.5f.
SLCO1B15 and SLCO1B115 have been reported to reduce the clearance of pravastatin in healthy volunteers. However, there remains controversy in the effects of SLCO1B15 on the activity of OATP1B1 in vitro. In addition, the effect of SLCO1B115 on the function of OATP1B1 has not been studied using cDNA-expression systems. Object of the present study was to study the influence of SLCO1B1*5, *15 and *15+C1007G, a novel haplotype found in a patient with pravastatin-induced myopathy, on the functional properties of OATP1B1 by transient expression systems of HEK293 and HeLa cells using endogenous conjugates and statins as substrates.
Transporting assays for endogenous substrates were performed using tritium labeled estradiol-17beta-D-glucuronide and estrone-3-sulfate. Quantitation of pravastatin, atorvastatin, cerivastatin and simvastatin were carried out using HPLC tandem mass spectrometry.
The transporting activities of cells expressing SLCO1B1*5, *15 and 15+C1007G decreased significantly but those of SLCO1B11b, *1a+C1007G and 1b+C1007G were not altered for all of the substrates tested except for simvastatin. Kinetic analysis of pravastatin and atorvastatin showed that Km values were not altered but Vmax values decreased significantly in cells expressing SLCO1B15, *15 and 15+C1007G. Immunocytochemical study showed that SLCO1B15, *15 and *15+C1007G proteins are localized not only at the plasma membrane but also in the intracellular space.
These findings suggest that 521T>C, existing commonly in SLCO1B1*5, *15 and 15+C1007G, is the key single nucleotide polymorphism (SNP) that determines the functional properties of SLCO1B15, *15 and *15+C1007G allelic proteins and that decreased activities of these variant proteins are mainly caused by a sorting error produced by this SNP.
据报道，SLCO1B15和SLCO1B115可降低健康志愿者体内普伐他汀的清除率。然而，SLCO1B15对体外OATP1B1活性的影响仍存在争议。此外，尚未使用cDNA表达系统研究SLCO1B115对OATP1B1功能的影响。本研究的目的是通过HEK293和HeLa细胞的瞬时表达系统，以内源性结合物和他汀类药物为底物，研究SLCO1B1*5、15以及在普伐他汀诱导的肌病患者中发现的新型单倍型15 + C1007G对OATP1B1功能特性的影响。
使用氚标记的雌二醇-17β-D-葡萄糖醛酸和硫酸雌酮进行内源性底物的转运试验。使用HPLC串联质谱法定量普伐他汀、阿托伐他汀、西立伐他汀和辛伐他汀。
除辛伐他汀外，表达SLCO1B15、15和15 + C1007G的细胞对所有测试底物的转运活性均显著降低，但表达SLCO1B11b、1a + C1007G和1b + C1007G的细胞的转运活性未改变。普伐他汀和阿托伐他汀的动力学分析表明，表达SLCO1B15、15和15 + C1007G的细胞中，Km值未改变，但Vmax值显著降低。免疫细胞化学研究表明，SLCO1B15、15和15 + C1007G蛋白不仅定位于质膜，也定位于细胞内空间。
这些发现表明，常见于SLCO1B15、15和15 + C1007G中的521T>C是决定SLCO1B15、15和15 + C1007G等位基因蛋白功能特性的关键单核苷酸多态性（SNP），这些变异蛋白活性降低主要是由该SNP产生的分选错误所致。
