胆汁盐的荧光衍生物。III. 通过胆酰牛磺酸转运系统将7β-NBD-NCT摄取到分离的肝细胞中。

Fluorescent derivatives of bile salts. III. Uptake of 7 beta-NBD-NCT into isolated hepatocytes by the transport systems for cholyltaurine.

作者信息

Schramm U, Fricker G, Buscher H P, Gerok W, Kutz G

机构信息

Institut für Organische Chemie und Biochemie, Universität Freiburg, Germany.

出版信息

J Lipid Res. 1993 May;34(5):741-57.

PMID:8509713

Abstract

Uptake of 7 beta-NBD-NCT ([N-[7-(4-nitrobenzo-2-oxa-1,3-diazol)]-7 beta-amino-3 alpha,12 alpha-dihydroxy-5 beta-cholan-24-oyl)-2'-aminoethanesulfonate) in isolated rat liver hepatocytes occurs by saturable transport without being superimposed by simple diffusion. The dependency of flux rate of uptake on the concentration of 7 beta-NBD-NCT in the presence of Na+ (143 mM) and with Na+ depletion (1 mM) is best described by the assumption of two simple transport systems. Maximal flux rates of uptake Jn and half-saturation constants KT for 7 beta-NBD-NCT are in presence of Na+ for transport system 1 J1(Na+ 143) = 0.15 +/- 0.03 nmol/(min.mg protein) and KT1(Na+ 143) = 3.5 +/- 0.5 microM and for transport system 2 J2(Na+ 143) = 1.0 +/- 0.1 nmol/(min.mg protein) and KT2(Na+ 143) = 190 +/- 25 microM, and in case of Na+ depletion J1(Na+ 1) = 0.1 +/- 0.03 nmol/(min.mg protein), KT1(Na+ 1) = 3.0 +/- 0.5 microM, and J2(Na+ 1) = 0.85 +/- 0.9 nmol/(min.mg protein) and KT2(Na+ 1) = 195 +/- 27 microM. Uptake of 7 beta-NBD-NCT by both transport systems is competitively inhibited by cholyltaurine in the presence of Na+ and with Na+ depletion. Two transport systems are likewise involved in the uptake of cholyltaurine in the presence of Na+ as well as in case of Na+ depletion. Their kinetic parameters are in presence of Na+ J'1(Na+ 143) = 1.55 +/- 0.14 nmol/(min.mg protein) and K'T1(Na+ 143) = 16.1 +/- 3.0 microM, and J'2(Na+ 143) = 0.51 +/- 0.05 nmol/(min.mg protein) and K'T2(Na+ 143) = 38.0 +/- 4.1 microM, and in case of Na+ depletion J'1(Na+ 1) = 0.10 +/- 0.02 nmol/(min.mg protein), K'T1(Na+ 1) = 7.7 +/- 1.2 microM, and J'2(Na+ 1) = 0.40 +/- 0.03 nmol/(min.mg protein) and K'T2(Na+ 1) = 41.0 +/- 4.2 microM. Uptake of cholyltaurine by both transport systems is competitively inhibited by 7 beta-NBD-NCT in the presence of Na+ as well as in case of Na+ depletion. In both cases the inhibition constants are practically identical with the KT values for uptake of 7 beta-NBD-NCT. Photoaffinity labeling of isolated hepatocytes using 7,7-ACT (400 microM) resulted in the irreversible inhibition of uptake of both bile salts to similar extents, confirming the kinetic data that 7 beta-NBD-NCT is a true analogue of cholyltaurine.

摘要

7β-NBD-NCT（[N-[7-(4-硝基苯并-2-恶唑-1,3-二氮杂环)]-7β-氨基-3α,12α-二羟基-5β-胆烷-24-酰基]-2'-氨基乙磺酸盐）在离体大鼠肝脏肝细胞中的摄取通过可饱和转运发生，不存在简单扩散的叠加。在存在Na⁺（143 mM）和Na⁺缺失（1 mM）的情况下，摄取通量率对7β-NBD-NCT浓度的依赖性最好通过两个简单转运系统的假设来描述。7β-NBD-NCT在Na⁺存在下的最大摄取通量率Jn和半饱和常数KT，对于转运系统1，J1(Na⁺ 143) = 0.15 ± 0.03 nmol/(min·mg蛋白)，KT1(Na⁺ 143) = 3.5 ± 0.5 μM；对于转运系统2，J2(Na⁺ 143) = 1.0 ± 0.1 nmol/(min·mg蛋白)，KT2(Na⁺ 143) = 190 ± 25 μM。在Na⁺缺失的情况下，J1(Na⁺ 1) = 0.1 ± 0.03 nmol/(min·mg蛋白)，KT1(Na⁺ 1) = 3.0 ± 0.5 μM，J2(Na⁺ 1) = 0.85 ± 0.9 nmol/(min·mg蛋白)，KT2(Na⁺ 1) = 195 ± 27 μM。在Na⁺存在和Na⁺缺失的情况下，两个转运系统对7β-NBD-NCT的摄取均受到胆酰牛磺酸的竞争性抑制。在Na⁺存在以及Na⁺缺失的情况下，两个转运系统同样参与胆酰牛磺酸的摄取。它们的动力学参数在Na⁺存在时，J'1(Na⁺ 143) = 1.55 ± 0.14 nmol/(min·mg蛋白)，K'T1(Na⁺ 143) = 16.1 ± 3.0 μM，J'2(Na⁺ 143) = 0.51 ± 0.05 nmol/(min·mg蛋白)，K'T2(Na⁺ 143) = 38.0 ± 4.1 μM；在Na⁺缺失的情况下，J'1(Na⁺ 1) = 0.10 ± 0.02 nmol/(min·mg蛋白)，K'T1(Na⁺ 1) = 7.7 ± 1.2 μM，J'2(Na⁺ 1) = 0.40 ± 0.03 nmol/(min·mg蛋白)，K'T2(Na⁺ 1) = 41.0 ± 4.2 μM。在Na⁺存在以及Na⁺缺失的情况下，两个转运系统对胆酰牛磺酸的摄取均受到7β-NBD-NCT的竞争性抑制。在两种情况下，抑制常数实际上与7β-NBD-NCT摄取的KT值相同。使用7,7-ACT（400 μM）对离体肝细胞进行光亲和标记导致两种胆汁盐摄取的不可逆抑制程度相似，证实了动力学数据，即7β-NBD-NCT是胆酰牛磺酸的真正类似物。