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St-Pierre, M. V., M. A. Serrano, R. I. R. Macias, U. Dubs, M. Hoechli, U. Lauper, P. J. Meier, and J. J. G. Marin. Expression of members of the multidrug resistance protein family in human term placenta. Am J Physiol Regulatory Integrative Comp Physiol 279: R1495-R1503, 2000.--The placenta serves, in part, as a barrier to exclude noxious substances from the fetus. In humans, a single-layered syncytium of polarized trophoblast cells and the fetal capillary endothelium separate the maternal and fetal circulations. P-glycoprotein is present in the syncytiotrophoblast throughout gestation, consistent with a protective role that limits exposure of the fetus to hydrophobic and cationic xenobiotics. We have examined whether members of the multidrug resistance protein (MRP) family are expressed in term placenta. After screening a placenta cDNA library, partial clones of MRP1, MRP2, and MRP3 were identified. Immunofluorescence and immunoblotting studies demonstrated that MRP2 was localized to the apical syncytiotrophoblast membrane. MRP1 and MRP3 were predominantly expressed in blood vessel endothelia with some evidence for expression in the apical syncytiotrophoblast. ATP-dependent transport of the anionic substrates dinitrophenyl-glutathione and estradiol-17-[Beta]-glucuronide was also demonstrated in apical syncytiotrophoblast membranes. Given the cellular distribution of these transporters, we hypothesize that MRP isoforms serve to protect fetal blood from entry of organic anions and to promote the excretion of glutathione/glucuronide metabolites in the maternal circulation. placenta transport; human multidrug resistance protein 1; human multidrug resistance protein 2; human multidrug resistance protein 3

Physiological cholestasis linked to immature hepatobiliary transport systems for organic anions occurs in rat and human neonates. In utero, the placenta facilitates vectorial transfer of certain fetal-derived solutes to the maternal circulation for elimination. We compared the ontogenesis of organic anion transporters in the placenta and the fetal liver of the rat to assess their relative abundance throughout gestation and to determine whether the placenta compensates for the late maturation of transporters in the developing liver. The mRNA of members of the organic anion transporting polypeptide (Oatp) superfamily, the multidrug resistance protein (Mrp) family, one organic anion transporter (OAT), and the bile acid carriers Na(+)-taurocholate cotransporting polypeptide (Ntcp) and bile salt export pump (Bsep) was quantified by real-time PCR. The most abundant placental transporters were Oatp4a1, whose mRNA increased 10-fold during gestation, and Mrp1. Mrp1 immunolocalized predominantly to epithelial cells of the endoplacental yolk sac, suggesting an excretory role that sequesters fetal-derived solutes in the yolk sac cavity, and faintly to the basal syncytiotrophoblast surface. The mRNA levels of Oatp2b1, Mrp3, and Bsep in the placenta exceeded those in the fetal liver until day 20 of gestation, suggesting that the fetus relies on placental clearance of substrates when expression in the developing liver is low. Mrp3 immunolocalized to the epithelium of the endoplacental yolk sac and less abundantly in the labyrinth zone and endothelium of the maternal arteries. The placental expression of Oatp1a1, Oatp1a4, Oatp1a5, Oatp1b2, Oat, Ntcp, Mrp2, and Mrp6 was low.

More and more patients are trying out herbal medicine. It is estimated that half of the population have used alternative products at least once in their live. Gastrointestinal diseases often require long-lasting treatments involving many side-effects that can impair the patient's motivation. The majority of persons with symptoms of the irritable bowel syndrome or chronic liver disease resort to non-conventional therapies. However, potential hepatotoxicity of herbal products should not be underestimated. In this article, we discuss herbal preparations in specific gastrointestinal and hepatological indications, concentrating on products that have been tested in randomized, controlled clinical trials. Effective symptomatic treatment of obstipation, irritable bowel and inflammatory bowel disease has been demonstrated with plant-derived preparations. On the other hand phytotherapeutic preparations can not be recommended at present for the treatment of cirrhosis or chronic viral hepatitis based on the available data.

Bile acids are steroidal amphipathic molecules derived from the catabolism of cholesterol. They modulate bile flow and lipid secretion, are essential for the absorption of dietary fats and vitamins, and have been implicated in the regulation of all the key enzymes involved in cholesterol homeostasis. Bile acids recirculate through the liver, bile ducts, small intestine and portal vein to form an enterohepatic circuit. They exist as anions at physiological pH and, consequently, require a carrier for transport across the membranes of the enterohepatic tissues. Individual bile acid carriers have now been cloned from several species. Na+-dependent transporters that mediate uptake into hepatocytes and reabsorption from the intestine and biliary epithelium and an ATP-dependent transporter that pumps bile acids into bile comprise the classes of transporter that are specific for bile acids. In addition, at least four human and five rat genes that code for Na+-independent organic anion carriers with broad multi-substrate specificities that include bile acids have been discovered. Studies concerning the regulation of these carriers have permitted identification of molecular signals that dictate eventual changes in the uptake or excretion of bile acids, which in turn have profound physiological implications. This overview summarizes and compares all known bile acid transporters and highlights findings that have identified diseases linked to molecular defects in these carriers. Recent advances that have fostered a more complete appreciation for the elaborate disposition of bile acids in humans are emphasized.

The bile salt export pump (Bsep), a member of the ATP-binding cassette superfamily of transporters, mediates the ATP-dependent canalicular secretion of bile salts. We have cloned and expressed the mouse Bsep (mBsep) protein in Sf9 insect cells, and characterized its transport and ATPase properties. Because its deduced amino acid sequence predicts multiple phosphorylation sites for protein kinase A, protein kinase C (PKC) and Ca(2+)-calmodulin dependent kinase II, we have also tested whether mBsep undergoes phosphorylation. MBsep transports both glycine and taurine conjugated bile salts. Sf9 cell membranes that express mBsep exhibit higher basal ATPase activity than control membranes, and this is further stimulated by bile salts and inhibited by vanadate. Taurochenodeoxycholate is transported with the highest affinity and is the most potent inducer of ATPase activity. Cyclosporin A, glibenclamide and rifamycin SV, all competitive inhibitors of Bsep transport, also reduced the bile salt-stimulated ATPase activity. MBsep exists as a phospho-protein when expressed in Sf9 cells and the immunoprecipitated mBsep complex is a substrate for the catalytic subunit of PKC. When mBsep and the alpha-isoform of mouse PKC are co-expressed in Sf9 cells, a ninefold stimulation of phosphorylation occurs. This is further increased to 18-fold after activation by phorbol ester. Given that bile salts activate selected PKC isoforms in hepatocytes, including the alpha isoform, the phosphorylation of mBsep by PKCalpha may represent a point of regulation for this transporter that is mediated by its own substrate.

Cholestasis of pregnancy induces alterations in bile acid transport by human trophoblast plasma membrane (TPM) vesicles. We investigated whether maternal cholestasis affects the overall ability of the rat placenta to carry out vectorial bile acid transfer from the fetus to the mother. Complete obstructive cholestasis (OCP) was maintained during the last week of pregnancy and released at term (day 21), before experiments were performed. In situ single-pass perfusion of one placenta per rat with 250 nmol [(14)C]glycocholic acid (GC) revealed an impaired uptake in OCP rats (2.28 vs. 5.53 nmol in control rats). Approximately 100% of GC taken up by control placentas was secreted in maternal bile over 120 minutes (5.38 nmol), whereas this was only 61% (1.40 nmol) of the GC taken up by OCP placentas. When 5 nmol GC was administered through the jugular vein no significant difference between both groups in total GC bile output was found. The efficiency (V(max)/K(M)) of adenosine triphosphate (ATP)-dependent GC transport by vesicles from the maternal side of TPM was decreased (-41%) in OCP. Moreover, histological examination of the placentas suggested a reduction in the amount of functional trophoblast in the OCP group. This was consistent with a lower antipyrine diffusion across the placenta in these animals. In sum, our results indicate that maternal cholestasis affects the ability of the placenta to efficiently carry out bile acid transfer from fetal to maternal blood. Changes in both the structure and the functionality of the chorionic tissue may account for this impairment.

Background & Aims: Guanosine 3',5'-cyclic monophosphate (cGMP), whose production is stimulated by the interaction of nitric oxide, natriuretic peptides, and guanylin with their respective guanylate cyclases, activates secretion through ion channels in several epithelia. Cl- channels have been identified in the apical membrane of biliary epithelial cells. The aim of this study was to investigate the production of cGMP and its effects on Cl- permeability in biliary epithelial cells., Methods: Halide efflux measurement, whole-cell patch clamp recording, radioimmunoassay, and reverse-transcription polymerase chain reaction using two human biliary cell lines (H69 and Mz-ChA-1) were performed., Results: In cells equilibrated with 125I, bromo-cGMP stimulated halide efflux by 22%. In whole-cell patch clamp recordings, the addition of cGMP intracellularly, or of atrial natriuretic peptide extracellularly, stimulated inward currents at negative membrane potentials, consistent with Cl- efflux through open channels. In H69 cells, atrial and C-type natriuretic peptides stimulated production of cGMP. Mz-ChA-1 responded only to atrial natriuretic peptide. Both cell lines expressed messenger RNA for the guanylate cyclase type A receptor and the guanylate cyclase free-clearance receptor., Conclusions: These data suggest that natriuretic peptide stimulates cGMP production in human biliary epithelial cells, which in turn may regulate ductular bile formation through the opening of Cl- channels.

Previous mouse liver studies with diazepam (DZ), N-desmethyldiazepam (NZ), and temazepam (TZ) confirmed that under first-order conditions, DZ formed NZ and TZ in parallel. Oxazepam (OZ) was generated via NZ and not TZ despite that preformed NZ and TZ were both capable of forming OZ. In the present studies, the concentration-dependent sequential metabolism of DZ was studied in perfused mouse livers and microsomes, with the aim of distinguishing the relative importance of NZ and TZ as precursors of OZ. In microsomal studies, the Kms and Vmaxs, corrected for binding to microsomal proteins, were 34 microM and 3.6 nmole/min per mg and 239 microM and 18 nmole/min per mg, respectively, for N-demethylation and C3-hydroxylation of DZ. The Kms and Vmaxs for N-demethylation and C3-hydroxylation of TZ and NZ, respectively, to form OZ, were 58 microM and 2.5 nmole/min per mg and 311 microM and 2 nmole/min per mg, respectively. The constants suggest that at low DZ concentrations, NZ formation predominates and is a major source of OZ, whereas at higher DZ concentrations, TZ is the important source of OZ. In livers perfused with DZ at input concentrations of 13 to 35 microM, the extraction ratio of DZ (E[DZ]) decreased from 0.83 to 0.60. NZ was the major metabolite formed although its appearance was less than proportionate with increasing DZ input concentration. By contrast, the formation of TZ increased disproportionately with increasing DZ concentration, whereas that for OZ decreased and paralleled the behavior of NZ. Computer simulations based on a tubular flow model and the in vitro enzymatic parameters provided a poor in vitro-organ correlation. The E[DZ], appearance rates of the metabolites, and the extraction ratio of formed NZ (E[NZ, DZ]) were poorly predicted; TZ was incorrectly identified as the major precursor of OZ. Simulations with optimized parameters improved the correlations and identified NZ as the major contributor of OZ. Saturation of DZ N-demethylation at higher DZ concentrations increased the role of TZ in the formation of OZ. The poor aqueous solubility (limiting the concentration range of substrates used in vitro), avid tissue binding and the coupling of enzymatic reactions in liver, favoring sequential metabolism, are possible explanations for the poor in vitro-organ correlation. This work emphasizes the complexity of the hepatic intracellular milieu for drug metabolism and the need for additional modeling efforts to adequately describe metabolite kinetics.

Liver clearance models serve to improve our understanding of the relationships between the physiological determinants and hepatic clearance and predict changes in the disposition of substrates when homeostasis of the organ is perturbed. Their ability to describe metabolism was presently extended to the sequential formation and elimination of primary (M1), secondary (M2), and tertiary (M3) metabolites during a single passage of drug (P) across the liver, under steady state and first-order conditions. The well-stirred model is distinct from other models in that metabolite formation and elimination is independent of enzymic distributions, the number of steps involved in metabolite formation, and the intrinsic clearances of the precursors. This model predicts that the extraction ratio of a formed primary metabolite derived from drug (E[M1, P]) is identical to that for the preformed primary metabolite (E[M1]), and that the extraction ratios of a secondary metabolite derived from drug (E[M2, P]) and primary metabolite (E[M2, M1]) or preformed secondary metabolite (E[M2]) are identical. For the more physiologically acceptable, parallel-tube and dispersion models, metabolite sequential elimination is highly influenced by the intrinsic clearances of the precursors and the enzymic distributions that mediate removal of precursor species and the metabolites. Furthermore, the extent of sequential metabolism recedes as the number of steps involved for metabolite formation increases. These models predict that E[M1, P] less than E[M1], and E[M2, P] less than E[M2, M1] less than E[M2], with the magnitude of the changes being less for the dispersion model than for the parallel-tube model. Competing pathways that divert substrate from entering the sequential pathway were found to exert only minimal influence on the sequential pathway.

The disposition of tracer doses of 3H-oxazepam was studied in the recirculating perfused mouse liver preparation. 3H-Oxazepam was biotransformed primarily to the diastereomeric 3H-oxazepam glucuronides, which either effluxed into the circulation or underwent biliary excretion. Three additional, unknown metabolites constituted a small fraction (5-10%) of the total radioactivity recovered in bile (7% of dose); no other metabolite was detected in perfusate. A physiologically based model, comprising the reservoir, liver blood and tissue, and bile, was fitted to reservoir concentrations of 3H-oxazepam and 3H-oxazepam glucuronides, and the cumulative amount excreted into bile. The model allowed for consideration of elimination pathways other than glucuronidation and the presence of a transport barrier for the oxazepam glucuronides across the hepatocyte membrane. The fitted results suggest a slight barrier existing for the transport of metabolites across the sinusoidal membrane, inasmuch as the transmembrane clearance was comparable to liver blood flow rate. Upon further comparison of estimates of formation, biliary, and transmembrane clearances for the oxazepam glucuronides, the rate-limiting step in the overall (biliary) clearance appears to be a poor capacity for biliary excretion. The influence of the cumulative volume loss that a recirculating perfused organ system incurs upon repeated sampling was discussed, and a compartmental method of correcting the observed concentrations of drug and generated metabolite was presented.

The temporal aspects of theophylline disposition are of interest, as there are predictable time-dependent fluctuations in the pulmonary function of patients with asthma and theophylline serum concentrations may vary throughout a 24-hour period. We studied the extent to which there are significant temporal changes in theophylline kinetics and the relative contribution of distribution, metabolism, and excretion to this phenomenon. Eight healthy men received an intravenous dose (6 mg/kg) of theophylline at 8 AM and 8 PM at 1-week intervals. Serum and urine were analyzed for theophylline and its three major metabolites by HPLC. Distribution volumes and total body and nonrenal clearances showed no differences between morning and evening dosing. The elimination rate was 12% greater after morning dosing. Renal clearance was 24% greater after morning dosing and was accompanied by an increased excretion fraction of unchanged theophylline. Based on total urinary metabolite excretion and the metabolite serum AUCs, there was no evidence of time-dependent variation in theophylline biotransformation. Although theophylline renal clearance is greater after morning dosing, it is only a small fraction of the overall drug elimination and does not change the total body clearance after morning or evening dosing.

Because of the propensity of nascently formed metabolites towards sequential metabolism within formation organs, theoretical and experimental treatments that achieve mass conservation must recognize the various sources contributing to primary, secondary, and tertiary metabolite formation. A simple one-compartment open model, with first-order conditions and the liver as the only organ of drug disappearance and metabolite formation, was used to illustrate the metabolism of a drug to its primary, secondary, and tertiary metabolites, encompassing the cascading effects of sequential metabolism. The concentration-time profiles of the drug and metabolites were examined for two routes of drug administration, oral and intravenous. Formation of the primary metabolite from drug in the gut lumen, with or without further absorption, and metabolite formation arising from first-pass metabolism of the drug and the primary metabolite during oral absorption were considered. Mass balance equations, incorporating modifications of the various absorption and conversion rate constants, were integrated to provide the explicit solutions. Simulations, with and without consideration of the sources of metabolite formation other than from its immediate precursor, were used to illustrate the expected differences in circulating metabolite concentrations. However, a simple relationship between the area under the curve of any metabolite, M, or [AUC (m)], its clearance [CL(m)], and route of drug administration was found. The drug dose, route, fraction absorbed into the portal circulation, Fabs, fraction available of drug from the liver, F, availabilities of the metabolites F(m) from formation organs, and CL(m) are determinants of the AUC(m)'s. After iv drug dosing, the area of any intermediary metabolites is determined by the iv drug dose divided by the (CL(m)/F(m] of that metabolite. When a terminal metabolite is not metabolized, its area under the curve becomes the iv dose of drug divided by the clearance of the terminal metabolite since the available fraction for this metabolite is unity. Similarly, after oral drug administration, when loss of drug in the gut lumen does not contribute to the appearance of metabolites systematically, the general solution for AUC(m) is the product of Fabs and oral drug dose divided by [CL(m)/F(m)]. A comparison of the area ratios of any metabolite after po and iv drug dosing, therefore, furnishes Fabs. When this fraction is divided into the overall systemic availability or Fsys, the drug availability from the first-pass organs, F, may be found.(ABSTRACT TRUNCATED AT 400 WORDS)

A sensitive, simple high-performance liquid chromatographic assay, capable of simultaneously measuring diazepam, its active metabolites oxazepam, temazepam and N-desmethyldiazepam and two phenyl hydroxylated metabolites, 4'-hydroxy-N-desmethyldiazepam and 4'-hydroxydiazepam, is described. The assay is easily modified to include separation of additional metabolite(s), e.g. oxazepam glucuronide(s). A thin-layer chromatographic assay, which resolves diazepam, the active metabolites and the two phenyl hydroxylated derivatives in one solvent system, is also reported. Application of these procedures to the quantitation of diazepam and its metabolites was shown, after delivery of diazepam (5 micrograms/ml or 16 microM) at a constant flow-rate (10 ml/min per liver) through the single-pass perfused rat liver preparation. Blood perfusion medium and bile were analysed for parent drug and metabolites before and after enzyme hydrolysis. These assay methods are found to be particularly pertinent and useful in providing a more comprehensive metabolic profile of diazepam metabolism, especially when aromatic hydroxylation pathways predominate.

The technique of normal and retrograde rat liver perfusion has been widely used to probe zonal differences in drug-metabolizing activities. The validity of this approach mandates the same tissue spaces being accessed by substrates during both normal and retrograde perfusions. Using the multiple-indicator dilution technique, we presently examine the extent to which retrograde perfusion alters the spaces accessible to noneliminated references. A bolus dose of 51Cr-labeled red blood cells, 125I-albumin, 14C-sucrose and 3H2O was injected into the portal (normal) or hepatic (retrograde) vein of rat livers perfused at 10 ml per min per liver. The outflow perfusate was serially collected over 220 sec to characterize the transit times and the distribution spaces of the labels. During retrograde perfusion, red blood cells, albumin and sucrose profiles peaked later and lower than during normal perfusion, whereas the water curves were similar. The transit times of red blood cells, albumin and sucrose were longer (p less than 0.005), whereas those for water did not change. Consequently, retrograde flow resulted in significantly larger sinusoidal blood volumes (45%), albumin Disse space (42%) and sucrose Disse space (25%) than during normal flow, whereas the distribution spaces for total and intracellular water remained unaltered. The distension of the vascular tree was confirmed by electron microscopy, by which occasional isolated foci of widened intercellular recesses and spaces of Disse were observed. Cellular ultrastructure was otherwise unchanged, and there was no difference found between normal and retrograde perfusion for bile flow rates, AST release, perfusion pressure, oxygen consumption and metabolic removal of ethanol, a substrate with flow-limited distribution, which equilibrates rapidly with cell water (hepatic extraction ratios were virtually identical: normal vs. retrograde, 0.50 vs. 0.48 at 6 to 7.4 mM input concentration). These findings suggest that the functional and metabolic capacities of the liver remain unperturbed during retrograde perfusion, rendering the technique suitable for the investigation of zonal differences in drug-metabolizing enzymes.