Simmons T W, Hinchman C A, Ballatori N
Department of Biophysics, University of Rochester School of Medicine, NY 14642.
Biochem Pharmacol. 1991 Nov 6;42(11):2221-8. doi: 10.1016/0006-2952(91)90359-d.
Mechanisms of hepatic glutathione and glutathione S-conjugate efflux were investigated in isolated hepatocytes and perfused liver of the little skate (Raja erinacea). Glutathione was released by isolated skate hepatocytes at a rate of 0.12 +/- 0.03 nmol.hr-1.(mg protein)-1. In the perfused liver, glutathione concentrations in bile were high (approximately 0.7 mM) compared to hepatic tissue levels (0.61 +/- 0.11 mumol.g-1). During the first hour of perfusion, the biliary glutathione excretion rate was 3 nmol.hr-1.(g liver)-1, whereas glutathione accumulated in the recirculating perfusate at a rate of only 1.5 nmol.hr-1.(g liver)-1. Release of glutathione by isolated hepatocytes and perfused liver was not affected by the addition of acivicin, an inhibitor of gamma-glutamyltransferase (EC 2.3.2.2), to cell suspension medium or liver perfusate. 1-Chloro-2,4-dinitrobenzene (CDNB) was taken up by isolated hepatocytes, conjugated to glutathione, and released as S-(2,4-dinitrophenyl) (DNP)-glutathione. After infusion of 0.5 mumol CDNB in perfused liver, S-DNP-glutathione was concentrated in bile (0.5 mM) and was associated with choleresis. S-DNP-Conjugates of cysteinylglycine, cysteine and N-acetylcysteine, were also found in bile, suggesting intrahepatic breakdown of S-DNP-glutathione and subsequent acetylation of the resulting cysteine conjugate to form the mercapturic acid, S-DNP-N-acetylcysteine. This mercapturic acid accounted for 31% of the total S-DNP-conjugates collected in bile. In contrast, neither S-DNP-glutathione nor other S-DNP-conjugates were detected in the perfusate (less than 0.5 microM). These findings demonstrate that biliary excretion is the predominant route for efflux of glutathione and a glutathione S-conjugate from skate liver. The results also identify an intrahepatic pathway for mercapturic acid biosynthesis facilitated by biliary glutathione S-conjugate excretion.
在小斑鳐（Raja erinacea）的分离肝细胞和灌注肝脏中研究了肝脏谷胱甘肽和谷胱甘肽S-共轭物的外排机制。分离的鳐肝细胞释放谷胱甘肽的速率为0.12±0.03 nmol·hr⁻¹·(mg蛋白质)⁻¹。在灌注肝脏中，胆汁中的谷胱甘肽浓度（约0.7 mM）高于肝组织水平（0.61±0.11 μmol·g⁻¹）。在灌注的第一个小时内，胆汁中谷胱甘肽的排泄速率为3 nmol·hr⁻¹·(g肝脏)⁻¹，而谷胱甘肽在循环灌注液中的积累速率仅为1.5 nmol·hr⁻¹·(g肝脏)⁻¹。向细胞悬浮培养基或肝脏灌注液中添加γ-谷氨酰转移酶（EC 2.3.2.2）抑制剂阿西维辛，对分离的肝细胞和灌注肝脏释放谷胱甘肽没有影响。1-氯-2,4-二硝基苯（CDNB）被分离的肝细胞摄取，与谷胱甘肽结合，并以S-(2,4-二硝基苯基)（DNP）-谷胱甘肽的形式释放。在灌注肝脏中注入0.5 μmol CDNB后，S-DNP-谷胱甘肽在胆汁中浓缩（0.5 mM），并与胆汁分泌增加有关。胆汁中还发现了半胱氨酰甘氨酸、半胱氨酸和N-乙酰半胱氨酸的S-DNP-共轭物，这表明S-DNP-谷胱甘肽在肝内分解，随后生成的半胱氨酸共轭物乙酰化形成巯基尿酸S-DNP-N-乙酰半胱氨酸。这种巯基尿酸占胆汁中收集的总S-DNP-共轭物的31%。相比之下，在灌注液中未检测到S-DNP-谷胱甘肽或其他S-DNP-共轭物（低于0.5 μM）。这些发现表明，胆汁排泄是鳐肝脏中谷胱甘肽和谷胱甘肽S-共轭物外排的主要途径。研究结果还确定了一条由胆汁谷胱甘肽S-共轭物排泄促进的肝内巯基尿酸生物合成途径。
