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Pharmacogentics of Na~+/taurocholate Cotransporting Polypeptide (NTCP) and Apical Sodium-dependent Bile Acid Transporter (ASBT)

Author: PanZuo
Tutor: ZhouHongZuo;Jae-Gook Shin
School: Central South University
Course: Pharmacology
Keywords: Na~+/taurocholate cotransporting polypeptide apical sodium-dependent bile salt transporter genetic polymorphism uptake herb extract
CLC: R96
Type: PhD thesis
Year: 2007
Downloads: 120
Quote: 0
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Abstract


BACKGROUND: Bile formation in the liver is essential for normalintestinal lipid digestion and absorption. After secreted from the liver, the bilesalts undergo enterohepatic circulation several times per day. This process isdependent on several transport systems, among which the twosodium-dependent bile salt cotransporters of the SLC10 family, theNa~+/taurocholate cotransporting polypeptide (NTCP; SLC10A1) and the apicalsodium-dependent bile salt transporter (ASBT; SLC10A2) play a crucial role.One potential source of altered protein function may be genetic heterogeneityin this transporter related to the presence of single nucleotide polymorphisms,or SNPs. However, up to date, we know little about the genetic polymorphismthese two genes.ASBT promotes reabsorption of bile salts from the intestinal tract.Inhibiton of ASBT will induced the more lose of bile salts in the intestine,which will be compensated by the increased metabolism of cholesterol intobile acid. Thus, the inhibitors of ASBT are potentially a new class of plasmacholesterol lowering drugs. We scereened 400 herb extract to search the ASBTinhibitors.METHODS: (1) Genetic variants in exons and upstream domain ofSLC10A1 and SLC10A2 gene were detected by direct sequencing in 50 Koreansubjects. The novel nonsynonymous mutation was further screened bypyrosequencing in Chinese and Vietnamese population. (2) NTCP G190A(novel) and C800T (*2) were transfected into MDCK cells. The uptake studywas conducted by using [~3H] taurocholic acid and [~3H] estron sulfate as probesubstrates. The transport activity of NTCP wild type and two mutants werealso checked in X. Laevis oocytes injected with cRNA. (3) For thehigh-throughput screening of inhibitory effect of herb extracts to ASBT transporter, ASBT stably-transfected MDCK cells were grown in 96 wellplates and the uptake of [~3H] taurocholic acid was determined in the presenceand absence of various herb extracts.RESULTS: (1) Sequence analysis revealed two upstream SNPs and 4code region SNPs in SLC10A1 gene, including a novel nonsynonymous SNPG190A (Ala64Thr) with 1%allele frequency in Korean (n=150). However,this SNP was not found in 366 Chinese and 152 Vietnamese subjects. Thealleles frequency of reported C800T (Ser267Phe) SNP were found 3.1%, 7.4%and 9.2%in Korean, Chinese and Vietnamese subjects respectively.(2) In accordance with the data of others, C800T (Ser267Phe) exhibiteda near complete loss of function for bile acid uptake yet fully normal transportfunction for the nonbile acid substrate estrone sulfate. In contrast,G190A(Ala64Thr) had decreased uptake activity of both [~3H] taurocholic acidand [~3H] estrone sulfate substrates when compared with wild type though theyhad same expression level. However, both two mutants (Ala64Thr,Ser267Phe) and wild type were all inhibited by the known NTCP inhibitor(taurocholic acid, bromsulphalein and cyclosporin A) no matter using [~3H]taurocholic acid and [~3H] estron sulfate as substrate.(3) Some of 400 herb ethanol extracts showed inhibition on ASBT.Concentration dependency of the inhibitory effect of 24 herb extracts withmost remarkable inhibition were further determined. IC50 values were rangedfrom 5-60μg/ml. The main ingredients of some of these extracts, for examplehoelen, glycyrrhizae radix, salviae radix, cirsii radix share the similarconstructs with bile acids, and moreover glycyrrhizae radix, salviae radix andbupleuri radix are already known to have cholesterol lowering effect. TheMTT assay showed that the choosen 4 herb extracts had no celluar toxicity.CONCLUSION: (1) We found a novel nonsynonymous SNP ofSLC10A1 gene, G190A (Ala64Thr), which showed a Korean-specific distribution. This novel variant showed impaired uptake activity to thesubstrates, taurocholic acid and estrone sulfate. (2) Several extracts showedASBT inhibitory effect in vitro, some of which may have potential for thedevelopment of new cholesterol lowering drugs.

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