Mrp2 is involved in benzylpenicillin-induced choleresis

Benzylpenicillin (PCG; 180 μmol/kg), a classic β-lactam antibiotic, was intravenously given to Sprague-Dawley (SD) rats and multidrug resistance-associated protein 2 (Mrp2)-deficient Eisai hyperbilirubinemic rats (EHBR). A percentage of the [3H]PCG was excreted into the bile of the rats within 60 min (SD rats: 31.7% and EHBR: 4.3%). Remarkably, a transient increase in the bile flow (∼2-fold) and a slight increase in the total biliary bilirubin excretion were observed in SD rats but not in the EHBR after PCG administration. This suggests that the biliary excretion of PCG and its choleretic effect are Mrp2-dependent. Positive correlations were observed between the biliary excretion rate of PCG and bile flow ( r2 = 0.768) and more remarkably between the biliary excretion rate of GSH and bile flow ( r2 = 0.968). No ATP-dependent uptake of [3H]PCG was observed in Mrp2-expressing Sf9 membrane vesicles, whereas other forms of Mrp2-substrate transport were stimulated in the presence of PCG. GSH efflux mediated by human MRP2 expressed in Madin-Darby canine kidney II cells was enhanced in the presence of PCG in a concentration-dependent manner. In conclusion, the choleretic effect of PCG is caused by the stimulation of biliary GSH efflux as well as the concentrative biliary excretion of PCG itself, both of which were Mrp2 dependent.

Endotoxin-induced reduction in biliary indocyanine green excretion rate in a chronically catheterized rat model

Using a nonstressed chronically catheterized rat model in which the common bile duct was cannulated, we studied endotoxin-induced alterations in hepatic function by measuring changes in the maximal steady-state biliary excretion rate of the anionic dye indocyanine green (ICG). Biliary excretion of ICG was calculated from direct measurements of biliary ICG concentrations and the bile flow rate during a continuous vascular infusion of ICG. Despite significant elevations in mean peak serum tumor necrosis factor-α (TNF-α) concentrations (90.9 ± 16.2 ng/ml), there was no effect on mean rates of bile flow or biliary ICG clearance after administration of 100 μg/kg endotoxin at 6 or 24 h. Significant differences from mean baseline rates of bile flow and biliary ICG excretion did occur after administration of 1,000 μg/kg endotoxin (mean peak TNF-α 129.6 ± 24.4 ng/ml). Furthermore, when rats were treated with up to 16 μg/kg of recombinant TNF-α, there was no change in mean rates of bile flow or ICG biliary clearance compared with baseline values. These data suggest that the complex regulation of biliary excretion is not mediated solely by TNF-α.

Effect of Hypothermia on the Hepatic Uptake and Biliary Excretion of Vecuronium in the Isolated Perfused Rat Liver

Background Hypothermia prolongs the time course of action of nondepolarizing muscle relaxants. It is not known whether this prolongation is caused by a reduced rate of extrahepatic distribution or elimination, liver uptake, metabolic clearance, or biliary excretion. Therefore, the authors studied the effects of hypothermia on the net hepatic uptake, metabolism, and biliary excretion of vecuronium in isolated perfused rat liver. Methods Livers of Wistar rats were perfused with Krebs Ringer solution (1% albumin, 3.3% carbon dioxide in oxygen, pH 7.36-7.42, 38 degrees C). Each perfusion experiment (recirculatory perfusion system) was divided into three phases. In phase 1, a bolus dose of vecuronium (950 microg) was followed by a continuous infusion of vecuronium (63 microg/min) throughout the perfusion experiment. In phase 2, the temperature was reduced to 28 degrees C. In phase 3, temperature was restored. In controls, the temperature was kept constant throughout the perfusion. Concentrations of vecuronium and its metabolites were measured in perfusion medium, bile, and liver homogenate. Parameters of a multicompartmental liver model were fitted to the concentration patterns in perfusion medium and in bile. Results Hypothermia increased vecuronium concentrations in the perfusion medium from 4.0 microg/ml (range, 2.5-6.6) to 15.6 microg/ml (11.5-18.4 microg/ml; P = 0.018). Hypothermia reduced the biliary excretion rate of 3-desacetyl vecuronium from 18% (range, 6-37%) to 16% (range, 4-19%) of that of vecuronium (P = 0.018). Pharmacokinetic analysis confirmed that hypothermia reduced the rate constants of hepatic uptake and metabolism from 0.219 to 0.053 and from 0.059 to 0.030, respectively. Conclusions Hypothermia significantly and reversibly reduced the net hepatic uptake of vecuronium. Hypothermia reduced the metabolism of vecuronium and the biliary excretion rate of 3-desacetyl vecuronium.

Thermally Modulated Biliary Excretion of [14C]Taurocholate in Rainbow Trout (Salmo gairdneri) and the Na+,K+-ATPase

Biliary excretion rate of [14C]taurocholate was 53–63% greater in rainbow trout (Salmo gairdneri) acclimated to and tested at 18 than 14 or 10 °C. Acute 4° temperature shifts up or down increased or decreased respectively biliary excretion rate by 40–53%. Furthermore, 10 °C acclimated fish shifted to 14 °C had 125% greater biliary excretion rate than 18 °C acclimated fish shifted to 14 °C. Apparent hepatic blood flow was greater in fish acclimated to and measured at 18 than 14 or 10 °C but was not different when 10 or 18 °C acclimated fish were measured at 14 °C. Acclimation to 10 versus 18 °C did not affect the Vmax or the temperature/activity relationship for the liver plasma membrane (LPM) Na+,K+-ATPase or the LPM Mg2+ -ATPase but did cause a major shift in the Km of the Na+,K+-ATPase (0.14 and 1.11 mM, respectively). This Km shift was observed at an 18 °C but not a 37 °C assay temperature. Thus, acclimation-induced changes in this enzyme were only observable at physiologically relevant temperature and substrate conditions. Depending on the thermal treatment, both hepatic blood flow and LMP Na+,K+-ATPase may module biliary excretion.

Cholestasis induced by Sulphated Glycolithocholic Acid in the Rat: Protection by Endogenous Bile Acids

1. Sulphated glycolithocholic acid (SGLC) causes cholestasis in experimental animals, despite its sulphated form. In the present study, the cholestatic potency and the pharmacokinetics of SGLC were investigated in rats under two conditions: (a) in the presence of an intact circulating bile acid pool and (b) after exhaustion of the bile acid pool by 24 h of bile diversion. 2. Intravenous administration of SGLC (8 μmol/ 100 g body weight) to rats with an intact bile acid pool did not cause cholestasis. However, biliary phospholipid and cholesterol concentrations were reduced by 40% and 29% respectively during the first hour after administration. When the same dose of the bile acid was injected in rats with a 24 h biliary drainage, a complete cessation of bile production was observed within 1 h. Twelve hours after the onset of cholestasis, bile production gradually increased again, showed a marked overshoot, and reached control levels after 3 days. In the recovery phase, biliary phospholipid and cholesterol concentrations were greatly reduced. 3. The absence of endogenous bile acids did not change the hepatic clearance rate of a tracer dose of radiolabelled SGLC, but markedly decreased its biliary excretion rate. 4. It was concluded that the hepatotoxic effect of SGLC is much more pronounced in rats with an exhausted bile acid pool, possibly due to a slower biliary excretion of the toxic compound. This phenomenon may have clinical implications for patients with a contracted bile acid pool.

Importance of perfusate hematocrit for insulin- and glucagon-induced choleresis in the perfused rat liver

Insulin and glucagon stimulate bile production in the intact rat without affecting the biliary excretion rate of bile acids. This effect was not demonstrable in rat liver perfused with human erythrocytes suspended in a Krebs-Henseleit-bicarbonate buffer at a hematocrit of about 18%. The present experiments demonstrate that the choleretic effect of insulin and glucagon observed in the intact rat is reproducible in perfused rat liver if the hematocrit of the perfusate is raised to about 35%. An increase in perfusate hematocrit is also accompanied by a 65% rise in hepatic oxygen consumption and a 27% rise in the basic production of bile, due to an increase in bile acid-independent bile formation. The mechanism by which these changes in perfusate hematocrit influence the function of the perfused liver is obscure. The difference in oxygen content of the perfusates appears to be without importance. Judged from lactate-pyruvate and beta-hydroxybutyrate-acetoacetate ratios of perfusate as well as electron microscopy, maldistribution of perfusate flow and local hypoxia were not evident in perfusions with low hematocrit. A part of the increase in hepatic oxygen consumption seen with a rise in perfusate hematocrit can, however, be explained by an increase in lactate supply from erythrocyte glycolysis. The results stress the importance of perfusate hematocrit for optimal bile production of the perfused rat liver.

Influence of bile salts on hepatic transport of dibromosulphthalein.

The influence of bile salts on hepatic transport of the organic anion dibromosulphthalein (DBSP) was investigated in rats. Bile salts influence the hepatic uptake, intracellular binding, and biliary excretion of DBSP. The overall effect depends on the administered dose of bile salts and DBSP. High doses of bile salts inhibited hepatic uptake of DBSP, whereas low doses of bile salts stimulated bile flow and simultaneously increased maximal biliary excretion of DBSP. The uncharged nonbile salt choleretic ouabain also stimulated biliary DBSP excretion. In contrast, the anionic nonbile salt choleretics, ethacrynic acid and theophylline, did not stimulate biliary excretion of DBSP. Because DBSP inhibited biliary excretion of ethacrynic acid and its metabolites, the lack of a stimulatory effect of ethacrynic acid choleresis might be explained by concomitant inhibition of biliary excretion of DBSP, masking the stimulatory effect of ethacrynic acid. Biliary transport maximum of DBSP was highly correlated with bile flow. The biliary clearance (Vmax/Km) was only moderately changed by bile salt administration, whereas the increase in the maximal biliary excretion rate was more pronounced, implying that the apparent Km for biliary excretion of DBSP was also increased by the bile salts. It is inferred that the stimulation of net biliary excretion of DBSP by bile salts may be due to a diminished transport from bile into the hepatocytes as the consequence of the decreased biliary concentration caused by the choleresis.