[34] Preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes

doi:10.1016/0076-6879(90)92092-R

Methods in Enzymology

Volume 192, 1990, Pages 534-545

https://doi.org/10.1016/0076-6879(90)92092-R Get rights and content

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Hepatocytes represent highly polarized secretory cells that exhibit eificient transport of a wide variety of endogenous and exogenous compounds from blood into bile. While sinusoidal and lateral surfaces (i.e., the "basolateral" pole of hepatocytes) provide for etficient exchange of various ions, organic solutes, and proteins with blood plasma, the bile canalicular or apical pole of the cell is separated from the plasma space by tight junctions and is highly specialized for the primary secretion of bile. To be able to separately study basolateral and canalicular membrane transport processes without interference of intracellular metabolic events, it is necessary to selectively isolate basolateral (blLPM) and/or canalicular (cLPM) liver plasma membrane vesicles. Hence, for in vitro transport studies isolation procedures are still required that result in both high yields and high purification of basolateral and canalicular membrane subfractions within reasonable working hours. The procedure described here represents an extensive modification of the Evan's procedure using a combination of rate zonal flotation and high speed discontinuous sucrose gradient centrifugation techniques. The method permits the simultaneous isolation of bILPM and cLPM vesicles from the same homogenate in a yield sufficient for a large number of individual transport studies to be carried out in both plasma membrane subfractions.

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Hepatic HAX-1 inactivation prevents metabolic diseases by enhancing mitochondrial activity and bile salt export
2020, Journal of Biological Chemistry
Citation Excerpt :
Livers were excised and homogenized in ice-cold radioimmune precipitation assay buffer containing protease and phosphatase inhibitor mixture. For assessment of BSEP expression, canalicular plasma membrane was isolated as described previously (59). Samples were diluted with a 4× volume of SDS sample buffer (40% (v/v) glycerol, 240 mm Tris-HCl (pH 6.8), 8% SDS, 0.04% bromphenol blue, 5% β-mercaptoethanol) and heated at 95 °C for 5 min.
Increasing hepatic mitochondrial activity through pyruvate dehydrogenase and elevating enterohepatic bile acid recirculation are promising new approaches for metabolic disease therapy, but neither approach alone can completely ameliorate disease phenotype in high-fat diet–fed mice. This study showed that diet-induced hepatosteatosis, hyperlipidemia, and insulin resistance can be completely prevented in mice with liver-specific HCLS1-associated protein X-1 (HAX-1) inactivation. Mechanistically, we showed that HAX-1 interacts with inositol 1,4,5-trisphosphate receptor-1 (InsP3R1) in the liver, and its absence reduces InsP3R1 levels, thereby improving endoplasmic reticulum–mitochondria calcium homeostasis to prevent excess calcium overload and mitochondrial dysfunction. As a result, HAX-1 ablation activates pyruvate dehydrogenase and increases mitochondria utilization of glucose and fatty acids to prevent hepatosteatosis, hyperlipidemia, and insulin resistance. In contrast to the reduction of InsP3R1 levels, hepatic HAX-1 deficiency increases bile salt exporter protein levels, thereby promoting enterohepatic bile acid recirculation, leading to activation of bile acid–responsive genes in the intestinal ileum to augment insulin sensitivity and of cholesterol transport genes in the liver to suppress hyperlipidemia. The dual mechanisms of increased mitochondrial respiration and enterohepatic bile acid recirculation due to improvement of endoplasmic reticulum–mitochondria calcium homeostasis with hepatic HAX-1 inactivation suggest that this may be a potential therapeutic target for metabolic disease intervention.
Biliary secretion and excretion in health and disease: Current concepts
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All of these tools have led to an enormous understanding of the transport proteins of the hepatocyte. Another conceptual advance was the recognition that hepatocytes are polarized, and this led in time, to methods15 permitting separation of basolateral domains (the membrane of the hepatocyte facing the sinusoid) and of apical domains (canalicular membranes). The ability to transfect cells has led to the creation of what may be considered artificial hepatocytes in which either a basolateral (sinusoidal) or apical (canalicular) transporter or both are transfected.
Biliary secretion in health and disease is reviewed. The powerful techniques of molecular biology have enabled cloning of the transporters involved in biliary secretion and the enterohepatic circulation of bile acids. This, in turn has permitted elucidation of their function as well as their regulation by nuclear receptors. Bile acid secretion is required for efficient lipid absorption, and bile acids also possess powerful direct and indirect antimicrobial functions in the small intestine. The enterohepatic circulation results from efficient ileal absorption, and is highly regulated at two sites. In the hepatocyte, biosynthesis of bile acids is regulated in negative feedback manner by the nuclear receptor FXR as well as by cytokines and by a peptide (FGF-19) liberated by bile acids from the ileal enterocyte. In the ileal enterocyte, bile acid reclamation is regulated in negative feedback manner by FXR and other nuclear receptors. The bile salt export pump (BSEP) mediates uphill canalicular bile acid secretion. Inborn defects in its function cause intrahepatic cholestasis in infants; inhibition of its function by drugs causes hepatotoxicity. Bile acid therapy is based on correction of bile acid deficiency by supplemental bile acids or displacement in which a noncytotoxic bile acid (ursodeoxycholic acid, ursodiol, UDCA) is administered and dilutes out the endogenous cytotoxic bile acids. Administration of primary bile acids may be lifesaving in inborn defects of bile acid biosynthesis. A synthetic bile acid, norUDCA is absorbed by the biliary ductules after secretion and cures the peribiliary fibrosis occurring in the MDR2^-/- mouse which lacks biliary phospholipid.
An integrated approach to model hepatic drug clearance
2006, European Journal of Pharmaceutical Sciences
It has been well accepted that hepatic drug extraction depends on the blood flow, vascular binding, transmembrane barriers, transporters, enzymes and cosubstrate and their zonal heterogeneity. Models of hepatic drug clearances have been appraised with respect to their utility in predicting drug removal by the liver. Among these models, the “well-stirred” model is the simplest since it assumes venous equilibration, with drug emerging from the outflow being in equilibrium with drug within the liver, and the concentration is the same throughout. The “parallel tube” and dispersion models, and distributed model of Goresky and co-workers have been used to account for the observed sinusoidal concentration gradient from the inlet and outlet. Departure from these models exists to include heterogeneity in flow, enzymes, and transporters. This article utilized the physiologically based pharmacokinetic (PBPK) liver model and its extension that include heterogeneity in enzymes and transporters to illustrate how in vitro uptake and metabolic data from zonal hepatocytes on transport and enzymes may be used to predict the kinetics of removal in the intact liver; binding data were also necessary. In doing so, an integrative platform was provided to examine determinants of hepatic drug clearance. We used enalapril and digoxin as examples, and described a simple liver PBPK model that included transmembrane transport and metabolism occurring behind the membrane, and a zonal model in which the PBPK model was expanded three sets of sub-compartments that are arranged sequentially to represent zones 1, 2, and 3 along the flow path. The latter model readily accommodated the heterogeneous distribution of hepatic enzymes and transporters. Transport and metabolic data, piecewise information that served as initial estimates, allowed for the unknown efflux and other intrinsic clearances to be estimated. The simple or zonal PBPK model provides predictive views on the hepatic removal of drugs and metabolites.
Use of canalicular membrane vesicles (CMVs) from rats, dogs, monkeys and humans to assess drug transport across the canalicular membrane
2006, Journal of Pharmacological and Toxicological Methods
A novel application of a Ultrafree filter cartridge/centrifugation method was evaluated to determine uptake in canalicular membrane vesicles (CMVs) from SD rats, beagle dogs, cynomolgus monkeys (common safety species in the pharmaceutical industry) and humans to assess biliary transport.
CMVs prepared from fresh livers of rats, dogs, monkeys and humans (four donors) were characterized for enrichment, basolateral and Golgi contamination and orientation. The presence of MRP2 and p-glycoprotein (P-gp) were confirmed by Western blots. Uptake of [³H]-leukotriene C₄ (LTC₄) and [³H]-estradiol-17β-d-glucuronide (E₂-Gluc) was determined at a low substrate concentration and/or by kinetic measurements (K_m and V_max). Correlation of in vitro data with in vivo findings was achieved by determining the biliary clearance of E₂-Gluc in rats after a single i.v. dose and with literature in vivo data for LTC₄.
CMVs were highly enriched and minimally contaminated based on marker enzyme activities. Uptake clearance among different species varied by approximately ten-fold (rat > dog = human > monkey) for LTC₄ and less than two-fold for E₂-Gluc. The lower uptake of LTC₄ by human than rat CMVs may be attributed to a higher K_m value for human than rat CMVs. Uptake of LTC₄ or E₂-Gluc by human CMVs showed little inter-subject variability (2-5-fold). Differences in in vitro uptake clearance (10-fold) between LTC₄ and E₂-Gluc in rat CMVs seemed to correlate with differences in their biliary clearance (4-fold) in rats, consistent with LTC₄ and E₂-Gluc being a high and a low clearance substrate, respectively.
A novel application of a Ultrafree filter cartridge/centrifugation method was developed to determine uptake in CMVs from different preclinical animal safety species and humans, and may represent a useful approach to study the mechanism of biliary excretion during drug discovery and development.
Modulation of organic anion transporting polypeptide 1 and multidrug resistance protein 3 expression in the liver and kidney of Gunn rats
2004, Hepatology Research
Background: Gunn rat is an animal model of Crigler–Najjar syndrome (CNS) type I that develops jaundice due to defect of bilirubin conjugation. Bilirubin UDP-glucuronosyltransferase (UGT1A1), which plays a critical role in bilirubin glucuronidation, has been reported to be deficient in CNS type 1. On the other hand, little is known about the expression of organic anion transporters in Gunn rats. In the present study, we evaluated expressions of organic anion transporting polypeptide (oatp) 1 and 2, multidrug resistance-associated protein (mrp) 2 and mrp3 in the liver and kidney of Gunn rats. Methods: Serum samples, liver and kidney tissues were obtained from Gunn rats and normal SD rats (n=6, in each group). Semi-quantitative mRNA expression of oatp1, oatp2, mrp2, and mrp3 mRNA was evaluated by constructed reverse transcription-polymerase chain reaction (RT-PCR). Protein expressions were determined by Western blotting and by immunohistochemistry. Results: Marked elevation of serum unconjugated bilirubin concentration (129.4±34.8 μmol/l) was observed in Gunn rats. Hepatic expression of oatp1 and oatp2 mRNA was 44% (P<0.01) and 35% (P<0.01) lower in Gunn rats than in SD rats, respectively. Hepatic oatp1 protein expression was 37% (P<0.05) lower in Gunn rats than in SD rats. In contrast to oatp1, hepatic expression of mrp3 mRNA and protein was 76% (P<0.01) and 557% (P<0.01) higher in Gunn rats than in SD rats, respectively. Hepatic expression of oatp2 and mrp2 protein was not significantly different between Gunn rats and SD rats. Like the Western blot analysis, immunohistochemical staining disclosed decrease of oatp1 and increase of mrp3 protein expressions in the liver of Gunn rats. Decrease of oatp1 and increase of mrp3 expressions were also observed in the kidney of Gunn rats. Conclusion: Decreased expression of oatp1 and increased expression of mrp3 were observed in the liver and kidney of Gunn rats. Deficient UGT1A1 activity-associated retention of unconjugated bilirubin in the hepatocytes may modulate the expressions of these transporters in Gunn rats.
Regulation of rat organic anion transporters in bile salt-induced cholestatic hepatitis: Effect of ursodeoxycholate
2003, Hepatology
Hepatic uptake of organic anions, including bile salts, is mediated by members of the organic anion-transporting polypeptide (Oatp) family. In rat liver, Oatp1 (Slc21a1), Oatp2 (Slc21a5), and Oatp4 (Slca10) are expressed at the basolateral membrane of hepatocytes and may be differentially regulated under pathophysiologic conditions such as cholestasis. The aim of this study was to determine the effects of cholic acid (CA) and ursodeoxycholic acid (UDCA) on the expression of Oatp4 compared with Ntcp, Oatp1, and Oatp2. Wistar rats were fed with CA (0.5%) or both CA (0.5%) and UDCA (0.25%) for 3 weeks. Oatp expression was studied by Northern and Western blot analysis as well as immunofluorescence analysis. Transport function was compared measuring biliary secretion of ¹⁴C-CA and ¹⁴C-taurocholic acid (TCA). In CA-fed animals, biliary secretion of ¹⁴C-CA and ¹⁴C-TCA was markedly delayed over 40 minutes compared with controls. Accordingly, Oatp4 protein was significantly down-regulated in CA-fed animals together with Oatp1 and Ntcp. Cofeeding of CA plus UDCA prevented the impairment of ¹⁴C-CA and ¹⁴C-TCA secretion and the down-regulation of Oatp4. Oatp4 messenger RNA (mRNA) levels did not differ significantly between bile salt-fed groups, suggesting a posttranscriptional effect of CA on Oatp4 expression. In contrast to Oatp1 and Oatp4, Oatp2 protein expression was increased by CA feeding, indicating a differential regulation of Oatp transporters. In conclusion, we show that CA feeding may cause cholestasis associated with a posttranscriptional down-regulation of Oatp4. UDCA may prevent impairment of hepatic function by restoring hepatic transporter expression. (Hepatology 2003;38:187-195.)

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[34] Preparation of basolateral (sinusoidal) and canalicular plasma membrane vesicles for the study of hepatic transport processes

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Biochim. Biophys. Acta

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