AAVrh.10-mediated genetic delivery of bevacizumab to the pleura to provide local anti-VEGF to suppress growth of metastatic lung tumors.

Vascular endothelial growth factor (VEGF) produced by tumor cells has a central role in stimulating angiogenesis required for tumor growth. Humanized monoclonal anti-VEGF antibody (bevacizumab, Avastin), approved as a treatment for non-squamous, non-small cell lung cancer, requires administration every 3 weeks. We hypothesized that an intrapleural administration of an adeno-associated virus (AAV) vector expressing an anti-VEGF-A antibody equivalent of bevacizumab would result in sustained anti-VEGF-A localized expression within the lung and suppress metastatic tumor growth. The AAV vector AAVrh.10alphaVEGF encodes the light chain and heavy chain complementary DNAs of monoclonal antibody A.4.6.1, a murine antibody that specifically recognizes human VEGF-A with the same antigen-binding site as bevacizumab. A metastatic lung tumor model was established in severe combined immunodeficient mice by intravenous administration of human DU145 prostate carcinoma cells. Intrapleural administration of AAVrh.10alphaVEGF directed long-term expression of the anti-human VEGF-A antibody in lung, as shown by sustained, high-level anti-human VEGF titers in lung epithelial lining fluid for 40 weeks, which was the duration of the study. In the AAVrh.10alphaVEGF-treated animals, tumor growth was significantly suppressed (P<0.05), the numbers of blood vessels and mitotic nuclei in the tumor was decreased (P<0.05) and there was increased survival (P<0.05). Thus, intrapleural administration of an AAVrh.10 vector, encoding the murine monoclonal antibody equivalent of bevacizumab, effectively suppresses the growth of metastatic lung tumors, suggesting AAV-mediated gene transfer to the pleura to deliver bevacizumab locally to the lung as a novel alternative platform to conventional monoclonal antibody therapy.

Persistent expression of biologically active anti-HER2 antibody by AAVrh.10-mediated gene transfer.

Trastuzumab (Herceptin) is a recombinant humanized monoclonal antibody (mAb) directed against an extracellular region of the human epidermal growth-factor receptor type 2 (HER2) protein. We hypothesized that a single adeno-associated virus (AAV)-mediated genetic delivery of an anti-HER2 antibody should be effective in mediating long-term production of anti-HER2 and in suppressing the growth of human tumors in a xenograft model in nude mice. The adeno-associated virus gene transfer vector AAVrh.10alphaHER2 was constructed based on a non-human primate AAV serotype rh.10 to express the complementary DNAs for the heavy and light chains of mAb 4D5, the murine precursor to trastuzumab. The data show that genetically transferred anti-HER2 selectively bound human HER2 protein and suppressed the proliferation of HER2(+) tumor cell lines. A single administration of AAVrh.10alphaHER2 provided long-term therapeutic levels of anti-HER2 antibody expression without inducing an anti-idiotype response, suppressed the growth of HER2(+) tumors and increased the survival of tumor bearing mice. In the context that trastuzumab therapy requires frequent and repeated administration, this strategy might be developed as an alternate platform for delivery of anti-HER2 therapy.

Affinity maturation of an anti-V antigen IgG expressed in situ through adenovirus gene delivery confers enhanced protection against Yersinia pestis challenge.

Genetic transfer of neutralizing antibodies (Abs) has been shown to confer strong and persistent protection against bacterial and viral infectious agents. Although it is well established that for many exogenous neutralizing Abs increased antigen affinity correlates with protection, the effect of antigen affinity on Abs produced in situ after adenoviral gene transfer has not been examined. The mouse IgG2b monoclonal Ab, 2C12.4, recognizes the Yersinia pestis type III secretion apparatus protein, LcrV (V antigen), and confers protection in mice when administered as an IgG intraperitoneally or after genetic immunization with engineered, replication-defective serotype 5 human adenovirus (Ad). The 2C12.4 Ab was expressed as a single-chain variable fragment (scFv) in Escherichia coli and was shown to display an equilibrium dissociation constant (K(D))=3.5 nM by surface plasmon resonance analysis. The 2C12.4 scFv was subjected to random mutagenesis, and variants with increased affinity were isolated by flow cytometry using the anchored periplasmic expression bacterial display system. After a single round of mutagenesis, variants displaying up to 35-fold lower K(D) values (H8, K(D)=100 pM) were isolated. The variable domains of the H8 scFv were used to replace those of the parental 2C12.4 IgG encoded in the Ad vector, AdalphaV, giving rise to AdalphaV.H8. The two adenoviral vectors resulted in similar titers of anti-V antigen Abs 3 days after immunization, with 10(9), 10(10) or 10(11) particle units (pu). After intranasal challenge with 363 LD(50) (lethal dose, 50%) of Y. pestis CO92, 54% of the mice immunized with 10(10) pu of AdalphaV.H8 survived through the 14 day end point compared with only 15% survivors for the group immunized with AdalphaV expressing the lower-affinity 2C12.4 (P<0.04; AdalphaV versus AdalphaV.H8). These results indicate that affinity maturation of a neutralizing Ab delivered by genetic transfer may confer increased protection not only for Y. pestis challenge but also possibly for other pathogens.

Clinical trial: modulation of human placental multidrug resistance proteins in cholestasis of pregnancy by ursodeoxycholic acid.

BACKGROUND: The effects of ursodeoxycholic acid on human placental bile acids and bilirubin transporters in intrahepatic cholestasis of pregnancy are still undefined. AIM: To evaluate whether ursodeoxycholic acid affects MRP2, MRP3 and MRP4 expression in the placenta. MATERIALS AND METHODS: Forty-three pregnant women were enrolled; fourteen subjects had physiological pregnancies. Intrahepatic cholestasis of pregnancy patients were divided into two groups: (i) 13 received ursodeoxycholic acid (20 mg/kg/day) and (ii) 16 untreated. Total bile acid and bilirubin in serum and cord blood were determined in each subject. Multidrug resistance proteins expression (immunoblot, quantitative real-time PCR) was evaluated in placentas collected at delivery. anova test was used for statistical analysis of data. RESULTS: Ursodeoxycholic acid administration significantly improved maternal serum bile acid and cord blood bilirubin and bile acid levels. MRP2 protein and RNA expression was significantly increased in placentas from treated patients compared to controls (P < 0.001 and P < 0.01, respectively). MRP3 protein expression was not significantly different between the groups while RNA expression was significantly decreased in treated patients (P < 0.01). MRP4 did not show significant differences between the groups. CONCLUSIONS: Ursodeoxycholic acid administration induces placental MRP2 expression, and reduces bilirubin and bile acid levels in cord blood.

The organic anion transport polypeptide 1d1 (Oatp1d1) mediates hepatocellular uptake of phalloidin and microcystin into skate liver.

Organic anion transporting polypeptides (rodent Oatp; human OATP) mediate cellular uptake of numerous organic compounds including xenobiotic toxins into mammalian hepatocytes. In the little skate Leucoraja erinacea a liver-specific Oatp (Oatp1d1, also called sOatp) has been identified and suggested to represent an evolutionarily ancient precursor of the mammalian liver OATP1B1 (human), Oatp1b2 (rat), and OATP1B3 (human). The present study tested whether Oatp1d1 shares functional transport activity of the xenobiotic oligopeptide toxins phalloidin and microcystin with the mammalian liver Oatps/OATPs. The phalloidin analogue [(3)H]-demethylphalloin was taken up into skate hepatocytes with high affinity (Km approximately 0.4 microM), and uptake could be inhibited by phalloidin and a variety of typical Oatp/OATP substrates such as bromosulfophthalein, bile salts, estrone-3-sulfate, cyclosporine A and high concentrations of microcystin-LR (Ki approximately 150 microM). When expressed in Xenopus laevis oocytes Oatp1d1 increased uptake of demethylphalloin (Km approximately 2.2 microM) and microcystin-LR (Km approximately 27 microM) 2- to 3-fold over water-injected oocytes, whereas the alternative skate liver organic anion transporter, the dimeric Ostalpha/beta, exhibited no phalloidin and only minor microcystin-LR transport. Also, the closest mammalian Oatp1d1 orthologue, the human brain and testis OATP1C1, did not show any phalloidin transport activity. These results demonstrate that the evolutionarily ancient Oatp1d1 is able to mediate uptake of cyclic oligopeptide toxins into skate liver. The findings support the notion that Oatp1d1 is a precursor of the liver-specific mammalian Oatps/OATPs and that its transport properties are closely associated with certain forms of toxic liver injury such as for example protein phosphatase inhibition by the water-borne toxin microcystin.

The Comparative Toxicogenomics Database (CTD): a resource for comparative toxicological studies.

The etiology of most chronic diseases involves interactions between environmental factors and genes that modulate important biological processes (Olden and Wilson, 2000. Nat Rev Genet 1(2):149-153). We are developing the publicly available Comparative Toxicogenomics Database (CTD) to promote understanding about the effects of environmental chemicals on human health. CTD identifies interactions between chemicals and genes and facilitates cross-species comparative studies of these genes. The use of diverse animal models and cross-species comparative sequence studies has been critical for understanding basic physiological mechanisms and gene and protein functions. Similarly, these approaches will be valuable for exploring the molecular mechanisms of action of environmental chemicals and the genetic basis of differential susceptibility.

(N)-methanocarba-2MeSADP (MRS2365) is a subtype-specific agonist that induces rapid desensitization of the P2Y1 receptor of human platelets.

Adenosine diphosphate (ADP) initiates and maintains sustained aggregation of platelets through simultaneous activation of both the Gq-coupled P2Y1 receptor and the Gi-coupled P2Y12 receptor. We recently described the synthesis and P2Y1 receptor-specific agonist activity of (N)-methanocarba-2MeSADP (MRS2365). Consequences of selective activation of the P2Y1 receptor by MRS2365 have been further examined in human platelets. Whereas MRS2365 alone only induced shape change, addition of MRS2365 following epinephrine treatment, which activates the Gi/z-linked, alpha2A-adrenergic receptor, resulted in sustained aggregation that was indistinguishable from that observed with ADP. Conversely, the platelet shape change promoted by ADP in the presence of the GPIIb/IIIa antagonist eptifibatide was similar to that promoted by MRS2365. Preaddition of the high affinity P2Y1 receptor antagonist MRS2500 inhibited the effect of MRS2365, whereas addition of MRS2500 subsequent to MRS2365 reversed the MRS2365-induced shape change. Preactivation of the P2Y1 receptor with MRS2365 for 2 min resulted in marked loss of capacity of ADP to induce aggregation as evidenced by a greater than 20-fold rightward shift in the concentration effect curve of ADP. This inhibitory effect of P2Y1 receptor activation was dependent on the concentration of MRS2365 (EC50 = 34 nm). The inhibitory effect of preincubation with MRS2365 was circumvented by activation of the Gq-coupled 5-HT2A receptor suggesting that MRS2365 induces loss of the ADP response as a consequence of desensitization of the Gq-coupled P2Y1 receptor. The time course of MRS2365-induced loss of aggregation response to epinephrine was similar to that observed with ADP. These results further demonstrate the P2Y1 receptor selectivity of MRS2365 and illustrate the occurrence of agonist-induced desensitization of the P2Y1 receptor of human platelets studied in the absence of P2Y12 receptor activation .

Rat hepatocyte engraftment in severe combined immunodeficient x beige mice using mouse-specific anti-fas antibody.

BACKGROUND: Hepatocyte transplantation holds promise as a treatment for acute and chronic liver failure; however, robust model systems needed to study xenogeneic hepatocyte transfer are lacking. Severe combined immunodeficient x beige (SCID/bg) hybrid mice readily accept foreign tissue. Repopulation of C.B-17 SCID/bg mouse liver with rat hepatocytes was studied following induction of mouse hepatocyte apoptosis using an anti-mouse agonistic fas monoclonal antibody (Jo2 mAb) that does not engage xenogeneic fas. METHODS: SCID/bg mice were transplanted with 1 x 10(6) fresh adult rat hepatocytes intrasplenically and treated with various doses, routes and frequencies of Jo2 mAb. Rat cell repopulation was characterized by quantitative immunofluorescent antibody (q-IFA) staining specific for rat dipeptidyl peptidase type IV (DPP-IV) and leucine amino peptidase, amplification of rat genomic DNA using polymerase chain reaction and histopathological and serum biochemistry analyses. RESULTS: Analysis of liver sections from mice treated twice weekly for 12 weeks with 0.4 mg/kg Jo2 mAb intraperitoneally consistently demonstrated >50% rat hepatocytes in the parenchymal mass by q-IFA. Rat hepatocyte engraftment protected mice from Jo2 mAb-mediated liver hemorrhage and hepatocyte apoptosis. Serum liver enzyme levels did not increase in Jo2 mAb-treated mice that were highly engrafted with rat hepatocytes, in contrast to matched non-engrafted mice. At 12 weeks post-engraftment, minimal fibrosis and inflammation were apparent and liver architecture had returned to near normal. Jo2 mAb did not induce histopathological abnormalities in other tissues known to express fas antigen (i.e. heart, lung). CONCLUSIONS: This novel model represents a simple and robust system of xenogeneic hepatocyte transplantation that could be applied to studies of liver biology, regeneration and hepatocyte transplantation.

Adaptive regulation of bile salt transporters in kidney and liver in obstructive cholestasis in the rat.

BACKGROUND & AIMS: Cholestasis results in adaptive regulation of bile salt transport proteins in hepatocytes that may limit liver injury. However, it is not known if changes also occur in the expression of bile salt transporters that reside in extrahepatic tissues, particularly the kidney, which might facilitate bile salt excretion during obstructive cholestasis. METHODS: RNA and protein were isolated from liver and kidney 14 days after common bile duct ligation in rats and assessed by RNA protection assays, Western analysis, and tissue immunofluorescence. Sodium-dependent bile salt transport was also measured in brush border membrane vesicles from the kidney. RESULTS: After common bile duct ligation, serum bile salts initially rose and then declined to lower levels after 3 days. In contrast, urinary bile salt excretion rose progressively over the 2-week period. By that time, the ileal sodium-dependent bile salt transporter messenger RNA and protein expression in total liver had increased to 300% and 200% of controls, respectively, while falling to 46% and 37% of controls, respectively, in the kidney. Sodium-dependent uptake of (3)H-taurocholate in renal brush border membrane vesicles was decreased. In contrast, the multidrug resistance-associated protein 2 expression in the kidney was increased 2-fold, even 1 day after ligation. Immunofluorescent studies confirmed the changes in the expression of these transporters in liver and kidney. CONCLUSIONS: These studies show that the molecular expression of bile salt transporters in the kidney and cholangiocytes undergo adaptive regulation after common bile duct obstruction in the rat. These responses may facilitate extrahepatic pathways for bile salt excretion during cholestasis.

Acyclic and cyclopropyl analogues of adenosine bisphosphate antagonists of the P2Y1 receptor: structure-activity relationships and receptor docking.

The activation of P2Y1 receptors in platelets contributes to platelet aggregation, and selective antagonists are sought as potential antithrombotic agents. We reported (Kim et al. J. Med. Chem. 2000, 43, 746-755) that acyclic analogues of adenine nucleotides, containing two phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of adenine, are moderately potent P2Y1 receptor antagonists. In this study we have varied the chain structure, to include asymmetric substitution, olefinic, and cyclopropyl groups. These antagonists inhibited the stimulation of phospholipase C in turkey erythrocyte membranes induced by 30 nM 2-MeS-ADP in the micromolar range. In the series of symmetrically branched aliphatic groups substituted with two phosphate groups, the optimal antagonist potency occurred with the 2-methylpropyl group. A 2-chloro-N(6)-methyladenine derivative, 2-[2-(2-chloro-6-methylaminopurin-9-yl)methyl]propane-1,3-bisoxy(diammoniumphosphate) (7), was a full antagonist at the P2Y1 receptor with an IC(50) value of 0.48 microM. Esterification of one of the phosphate groups or substitution with O-acetyl greatly reduced the antagonist potency at the P2Y1 receptor. Removal of a methylene group of 7 or inclusion of an olefinic or cyclopropyl group also reduced potency. A pair of enantiomeric glycerol derivatives demonstrated a 5-fold stereoselectivity for the S-isomer. Stereoisomerically defined analogues of 7 containing a cyclopropyl group in place of the branched carbon were less potent than 7 as antagonists, with IC(50) values of 2-3 microM. No agonist activity was observed for these analogues. A new rhodopsin-based molecular model of the P2Y1 receptor indicated that the optimal docked orientation of the two monophosphate moieties relative to the adenine N(6) (compared to a rigid, bicyclic analogue) was consistent with the dependence of antagonist potency on chain length. The 3'-phosphate was predicted to occupy a restricted space, deeper in the binding cleft than the 5'-phosphate location. In summary, modification of the flexible spacer chain linking bisphosphate groups to the adenine moiety provided many moderately potent antagonists.

Bile salt export pump is highly conserved during vertebrate evolution and its expression is inhibited by PFIC type II mutations.

Bile secretion is a fundamental function of the liver of all vertebrates and is generated by ATP-dependent transport proteins at the canalicular membrane of hepatocytes, particularly by the bile salt export pump BSEP. To determine the evolutionary origin and structure-function relationship of this transport mechanism, a liver cDNA library from the marine skate Raja erinacea, a 200 million-year-old vertebrate, was screened for BSEP orthologues. A full-length clone was isolated that encodes for 1,348 amino acids and shares 68.5% identity to human BSEP. Northern blot analysis revealed a 5-kb transcript only in skate liver. Expression of skate Bsep in Sf9 cells demonstrated a sixfold stimulation of ATP-dependent taurocholate transport compared with controls, with a Michaelis-Menten constant of 15 microM, which is comparable to rat Bsep. Sequences at the site of published mutations in human BSEP are also conserved in skate Bsep. When two of these mutations were introduced into the skate Bsep cDNA, this resulted in defective expression of the mutant proteins in Sf9 cells. These studies demonstrate that Bsep is a liver-specific ATP-dependent export pump that is highly conserved throughout evolution and provide insights into critical determinants for the function of this transporter in higher vertebrates.

Cl(-)-dependent secretory mechanisms in isolated rat bile duct epithelial units.

Cholangiocytes absorb and secrete fluid, modifying primary canalicular bile. In several Cl(-)-secreting epithelia, Na(+)-K(+)-2Cl(-) cotransport is a basolateral Cl(-) uptake pathway facilitating apical Cl(-) secretion. To determine if cholangiocytes possess similar mechanisms independent of CO2/HCO, we assessed Cl(-)-dependent secretion in rat liver isolated polarized bile duct units (IBDUs) by using videomicroscopy. Without CO2/HCO, forskolin (FSK) stimulated secretion entirely dependent on Na(+) and Cl(-) and inhibited by Na(+)-K(+)-2Cl(-) inhibitor bumetanide. Carbonic anhydrase inhibitor ethoxyzolamide had no effect on FSK-stimulated secretion, indicating negligible endogenous CO2/HCO transport. In contrast, FSK-stimulated secretion was inhibited approximately 85% by K(+) channel inhibitor Ba(2+) and blocked completely by bumetanide plus Ba(2+). IBDU Na(+)-K(+)-2Cl(-) cotransport activity was assessed by recording intracellular pH during NH4Cl exposure. Bumetanide inhibited initial acidification rates due to NH entry in the presence and absence of CO2/HCO. In contrast, when stimulated by FSK, a 35% increase in Na(+)-K(+)-2Cl(-) cotransport activity occurred without CO2/HCO. These data suggest a cellular model of HCO-independent secretion in which Na(+)-K(+)-2Cl(-) cotransport maintains high intracellular Cl(-) concentration. Intracellular cAMP concentration increases activate basolateral K(+) conductance, raises apical Cl(-) permeability, and causes transcellular Cl(-) movement into the lumen. Polarized IBDU cholangiocytes are capable of vectorial Cl(-)-dependent fluid secretion independent of HCO. Bumetanide-sensitive Na(+)-K(+)-2Cl(-) cotransport, Cl(-)/HCO exchange, and Ba(2+)-sensitive K(+) channels are important components of stimulated fluid secretion in intrahepatic bile duct epithelium.

Expression cloning of two genes that together mediate organic solute and steroid transport in the liver of a marine vertebrate.

Uptake of organic solutes and xenobiotics by mammalian cells is mediated by ATP-independent transporters, and four families of transporters have now been identified. To search for novel organic solute transporters, a liver cDNA library from an evolutionarily primitive marine vertebrate, the little skate Raja erinacea, was screened for taurocholate transport activity by using Xenopus laevis oocytes. In contrast to the organic anion transporters identified to date, a transport activity was identified in this library that required the coexpression of two distinct gene products, termed organic solute transporter alpha and beta (Ostalpha, Ostbeta). Ostalpha cDNA encodes for a protein of 352 aa and seven putative transmembrane (TM) domains. Ostbeta contains 182 aa and has at least one and perhaps two TM domains. There is no significant sequence identity between Ostalpha and Ostbeta, and only low identity with sequences in the databases; however, Ostalpha bears a resemblance to some G protein-coupled receptors, and Ostbeta exhibits 22% amino acid identity with the C-terminal TM and intracellular domains of protocadherin-gamma, a cell surface glycoprotein. Xenopus oocytes injected with the cRNA for both Ostalpha and Ostbeta, but not each separately, were able to take up taurocholate, estrone sulfate, digoxin, and prostaglandin E(2), but not p-aminohippurate or S-dinitrophenyl glutathione. Transport was sodium-independent, saturable, and inhibited by organic anions and steroids, including the major skate bile salt, scymnol sulfate. These results identify an organic anion transporter composed of a putative seven-helix TM protein and an ancillary membrane polypeptide.

Structure-activity relationships of pyridoxal phosphate derivatives as potent and selective antagonists of P2X1 receptors.

Novel analogues of the P2 receptor antagonist pyridoxal-5'-phosphate 6-azophenyl-2',5'-disulfonate (2) were synthesized and studied as antagonists in functional assays at recombinant rat P2X1, P2X2, and P2X3 receptors expressed in Xenopus oocytes (ion flux stimulation) and at turkey erythrocyte P2Y1 receptors (phospholipase C activation). Selected compounds were also evaluated as antagonists of ion flux and the opening of a large pore at the recombinant human P2X7 receptor. Modifications were made in the 4-aldehyde and 5'-phosphate groups of the pyridoxal moiety: i.e. a CH2OH group at the 4-position in pyridoxine was either condensed as a cyclic phosphate or phosphorylated separately to form a bisphosphate, which reduced potency at P2 receptors. 5-Methylphosphonate substitution, anticipated to increase stability to hydrolysis, preserved P2 receptor potency. At the 6-position, halo, carboxylate, sulfonate, and phosphonate variations made on the phenylazo ring modulated potency at P2 receptors. The p-carboxyphenylazo analogue, 4, of phosphate 2 displayed an IC50 value of 9 nM at recombinant P2X1 receptors and was 1300-, 16-, and > 10,000-fold selective for P2X1 versus P2X2, P2X3, and P2Y1 subtypes, respectively. The corresponding 5-methylphosphonate was equipotent at P2X1 receptors. The 5-methylphosphonate analogue containing a 6-[3,5-bis(methylphosphonate)]phenylazo moiety, 9, had IC50 values of 11 and 25 nM at recombinant P2X1 and P2X3 receptors, respectively. The analogue containing a phenylazo 4-phosphonate group, 11, was also very potent at both P2X1 and P2X3 receptors. However, the corresponding 2,5-disulfonate analogue, 10, was 28-fold selective for P2X1 versus P2X3 receptors. None of the analogues were more potent at P2X7 and P2Y1 receptors than 2, which acted in the micromolar range at these two subtypes.

Structurally related nucleotides as selective agonists and antagonists at P2Y1 receptors.

The P2Y1 receptor responds to adenine nucleotides and is present in platelets, heart, smooth muscles prostate, ovary, and brain. A selective antagonist may be useful as an antithrombotic agent. We have analyzed the binding site of this G protein-coupled receptor using ligand design, site-directed mutagenesis, and homology modeling based on rhodopsin. We have designed and synthesized a series of deoxyadenosine 3',5'-bisphosphate derivatives that act as antagonists, or, in some cases with small structural changes, as agonists or partial agonists. The 2-position accommodates Cl or thioethers, whereas the N6-position is limited to Me or Et. 2'-Substitution with OH or OMe increases agonist efficacy over 2'-H. Using molecular modeling of the binding site, the oxygen atoms of the ribose moiety were predicted to be non-essential, i.e. no specific H-bonds with the receptor protein appear in the model. We have, therefore, substituted this moiety with carbocylics, smaller and larger rings, conformationally constrained rings, and acyclics, with retention of affinity for the receptor. With simplified pharmacophores we are exploring the steric and electronic requirements of the receptor binding site, and the structural basis of receptor activation.

Cellular localization and up-regulation of multidrug resistance-associated protein 3 in hepatocytes and cholangiocytes during obstructive cholestasis in rat liver.

The hepatic expression of the ATP-dependent conjugate export pump multidrug resistance-associated protein 2 (Mrp2) is diminished in experimentally induced models of cholestasis. In this study we have examined the localization and expression of Mrp3, another member of the multidrug resistance-associated protein family, in normal liver and after obstructive cholestasis in the rat. Indirect immunofluorescence and confocal microscopy were used to determine the tissue localization and Western blot analysis was performed to quantitate the expression. In normal rat liver Mrp3 was found on the basolateral membrane of cholangiocytes and a single layer of hepatocytes surrounding the central vein. Three and 7 days after bile duct ligation Mrp3 expression was significantly increased, predominantly in hepatocytes in the pericentral region. By 14 days all hepatocytes showed basolateral membrane labeling for Mrp3 at a time when apical Mrp2 staining was significantly diminished. Proliferating bile ducts continued to stain positive, although the intensity of staining did not seem to vary. After 14 days Western blot quantitation showed that Mrp3 had increased approximately 30-fold in total liver membranes. Quantitation of Mrp3 in membranes from isolated hepatocytes of livers of sham and common bile duct-ligated (CBDL) animals showed a significant up-regulation beginning at 1 day and continuing to increase through 14 days postligation. This was in contrast to the progressive decrease in Mrp2 protein. Because Mrp3 is capable of transporting toxic bile acids, up-regulation of Mrp3 may compensate for the down-regulation of Mrp2 in obstructive cholestasis.

Isolation of functional polarized bile duct units from mouse liver.

The development of genetically altered murine animals has generated a need for in vitro systems in the mouse. We have now characterized a novel isolated bile duct unit (IBDU) preparation from the mouse to facilitate such studies. The mouse IBDU is isolated by portal perfusion of collagenase, blunt dissection, further enzymatic digestions, filtering through sized mesh, and culturing on Matrigel for 16-72 h. This mouse IBDU forms a central, enclosed lumen lined by polarized cytokeratin-19-positive cholangiocytes with numerous microvilli on the apical membrane. The IBDU responds to secretory stimuli, including secretin, vasoactive intestinal peptide, IBMX, and forskolin, resulting in expansion of the central lumen from secretion as quantified by videomicroscopy. The secretory response to secretin is dependent on Cl- and HCO3-in the perfusate. These findings indicate that mouse IBDUs are intact, polarized, functional bile duct secretory units that permit quantitative measurements of fluid secretion from mouse bile duct epithelium for the first time. This method should facilitate studies of cholangiocyte secretion in genetically altered murine animal models.

Role of sodium/hydrogen exchanger isoform NHE3 in fluid secretion and absorption in mouse and rat cholangiocytes.

Na+/H+ exchanger (NHE) isoforms play important roles in intracellular pH regulation and in fluid absorption. The isoform NHE3 has been localized to apical surfaces of epithelia and in some tissues may facilitate the absorption of NaCl. To determine whether the apical isoform NHE3 is present in cholangiocytes and to examine whether it has a functional role in cholangiocyte fluid secretion and absorption, immunocytochemical studies were performed in rat liver with NHE3 antibodies and functional studies were obtained in isolated bile duct units from wild-type and NHE3-/- mice after stimulation with forskolin, using videomicroscopic techniques. Our results indicate that NHE3 protein is present on the apical membranes of rat cholangiocytes and on the canalicular membrane of hepatocytes. Western blots also detect NHE3 protein in rat cholangiocytes and isolated canalicular membranes. After stimulation with forskolin, duct units from NHE3-/- mice fail to absorb the secreted fluid from the cholangiocyte lumen compared with control animals. Similar findings were observed in isolated bile duct units from wild-type mice and rats in the presence of the Na+/H+ exchanger inhibitor 5-(N-ethyl-N-isopropyl)-amiloride. In contrast, we could not demonstrate absorption of fluid from the canalicular lumen of mouse or rat hepatocyte couplets after stimulation of secretion with forskolin. These findings indicate that NHE3 is located on the apical membrane of rat cholangiocytes and that this NHE isoform can function to absorb fluid from the lumens of isolated rat and mouse cholangiocyte preparations.