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.

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.

Structural integrity of hepatocyte tight junctions.

The significance of discontinuities frequently found in freeze-fracture replicas of the tight junction was evaluated using complementary replicas of hepatocyte junctions from control and bile duct-ligated rats. An extensive analysis of complementary replicas using rotary platinum shadowing indicates that discontinuities in the protoplasmic (P) fracture face do not represent structural breaks in the tight-junctional network. In no case did P-face discontinuities correspond with interruptions in the groove network on the complementary extracellular (E) face. Quantitative analysis of replicas shows that P-face discontinuities result in part from "transfer" of material to the complementary E face (approximately 7% of the junctional length). However, many P-face discontinuities (7-30% of the junctional length) are matched only by a groove on the complementary E face. This finding demonstrates that a significant amount of material can be lost during freeze-fracture. An analysis of junctions from bile duct-ligated rats, which are known to have an increased paracellular permeability, shows comparable transfer and loss of material. However, the number of junctional elements and the tight-junction network density was significantly reduced by bile duct ligation. These observations indicate that discontinuities in tight-junctional elements result during the preparation of freeze-fracture replicas and are not physiologically important features of the junctional barrier. Variation in the number of elements provides the best explanation for observed differences in tight-junction permeability.

Structural and functional polarity of canalicular and basolateral plasma membrane vesicles isolated in high yield from rat liver.

A method has been developed for routine high yield separation of canalicular (cLPM) from basolateral (blLPM) liver plasma membrane vesicles of rat liver. Using a combination of rate zonal floatation (TZ-28 zonal rotor, Sorvall) and high speed centrifugation through discontinuous sucrose gradients, 9-16 mg of cLPM and 15-28 mg of blLPM protein can be isolated in 1 d. cLPM are free of the basolateral markers Na+/K+-ATPase and glucagon-stimulatable adenylate cyclase activities, but are highly enriched with respect to homogenate in the "canalicular marker" enzyme activities leucylnaphthylamidase (48-fold), gamma-glutamyl-transpeptidase (60-fold), 5'-nucleotidase (64-fold), alkaline phosphatase (71-fold), Mg++-ATPase (83-fold), and alkaline phosphodiesterase I (116-fold). In contrast, blLPM are 34-fold enriched in Na+/K+-ATPase activity, exhibit considerable glucagon-stimulatable adenylate cyclase activity, and demonstrate a 4- to 15-fold increase over homogenate in the various "canalicular markers." cLPM have a twofold higher content of sialic acids, cholesterol; and sphingomyelin compared with blLPM. At least three canalicular-(130,000, 100,000, and 58,000 mol wt) and several basolateral-specific protein bands have been detected after SDS PAGE of the two LPM subfractions. Specifically, the immunoglobin A-binding secretory component is restricted to blLPM as demonstrated by immunochemical techniques. These data indicate virtually complete separation of basolateral from canalicular LPM and demonstrate multiple functional and compositional polarity between the two surface domains of hepatocytes.

Localization of Na+,K+-ATPase alpha-subunit to the sinusoidal and lateral but not canalicular membranes of rat hepatocytes.

Controversy has recently developed over the surface distribution of Na+,K+-ATPase in hepatic parenchymal cells. We have reexamined this issue using several independent techniques. A monoclonal antibody specific for the endodomain of alpha-subunit was used to examine Na+,K+-ATPase distribution at the light and electron microscope levels. When cryostat sections of rat liver were incubated with the monoclonal antibody, followed by either rhodamine or horseradish peroxidase-conjugated goat anti-mouse secondary, fluorescent staining or horseradish peroxidase reaction product was observed at the basolateral surfaces of hepatocytes from the space of Disse to the tight junctions bordering bile canaliculi. No labeling of the canalicular plasma membrane was detected. In contrast, when hepatocytes were dissociated by collagenase digestion, Na+,K+-ATPase alpha-subunit was localized to the entire plasma membrane. Na+,K+-ATPase was quantitated in isolated rat liver plasma membrane fractions by Western blots using a polyclonal antibody against Na+,K+-ATPase alpha-subunit. Plasma membranes from the basolateral domain of hepatocytes possessed essentially all of the cell's estimated Na+,K+-ATPase catalytic activity and contained a 96-kD alpha-subunit band. Canalicular plasma membrane fractions, defined by their enrichment in alkaline phosphatase, 5' nucleotidase, gamma-glutamyl transferase, and leucine aminopeptidase had no detectable Na+,K+-ATPase activity and no alpha-subunit band could be detected in Western blots of these fractions. We conclude that Na+,K+-ATPase is limited to the sinusoidal and lateral domains of hepatocyte plasma membrane in intact liver. This basolateral distribution is consistent with its topology in other ion-transporting epithelia.

Chlorpromazine and its metabolites alter polymerization and gelation of actin.

Hepatic hydroxylated metabolites of chlorpromazine (10(-5)M to 10(-4)M), a frequently used phenothiazine tranquilizer, produce solid gel formation with filamentous actin, but the less toxic chlorpromazine sulfoxide metabolite does not. At higher concentrations (5 x 10(-4)M) chlorpromazine inhibits actin polymerization. These dose-response relationships parallel the drug's hepatic toxicity in vivo and suggest that interactions between chloropromazine or chlorpromazine metabolites and actin could be an underlying mechanism of cell injury.

The effect of thyroid hormone on bile salt-independent bile flow and Na+, K+ -ATPase activity in liver plasma membranes enriched in bile canaliculi.

The relationship between bile salt-independent canalicular flow and ATPase activity in liver plasma membranes (LPM) enriched in bile canaliculi, was studied in control, hyperthyroid, and hypothyroid rats. Canalicular bile production was significantly increased in hyperthyroid rats (3.19 +/- 0.23 mul/min per g liver) compared to controls (2.27 +/- 0.24 mul/min per g liver), while it diminished in hypothyroid animals (1.58 +/- 0.17 mul/min per g liver). Although bile salt excretion was also increased in hyperthyroid animals (62.4 +/- 13.3 vs. 41.2 +/- 8.4 nmol/min per g liver), the stimulation in canalicular secretion was primarily related to enhancement of the bile salt-independent fraction of flow (2.47 mul/min per g liver in hyperthyroid rats vs. 1.67 mul/min per g liver in controls). LPM Na+, K+-ATPase activity doubled in hyperthyroid animals (21.5 +/- 5.8 vs. 10.7 +/- 3.1 mumol Pi/mg protein per h) while Mg++-ATPase activity remained unchanged and 5'-nucleotidase activity increased to a small but significant extent. In hypothyroid rats, bile salt excretion remained unchanged from control values so that the reduced secretion was entirely secondary to an inhibition of bile salt-independent secretion (1.19 mul/min per g liver). Na+, K+-ATPase activity in the LPMs from hypothyroid animals decreased by nearly 50% (5.4 +/- 1.6 mumol Pi/mg protein per h), although comparable reductions in the specific activity of Mg++-ATPase and 5'-nucleotidase were also observed. Administration of L-thyroxine to hypothyroid animals restored both bile salt-independent canalicular secretion and membrane enzymes to control values within 2 and 4 days, respectively. Sodium dodecyl sulfate gel electrophoresis demonstrated no significant changes in LPM protein fractions from any of the treatment groups. These studies indicate that thyroid hormone has a parallel effect on bile salt-independent canalicular secretion and LPM Na+, K+-ATPase activity, supporting the hypothesis that Na+ transport and Na+, K+-ATPase may be determinants of bile salt-independent canalicular flow.

Glucose reabsorption from bile. Evidence for a biliohepatic circulation.

Glucose is absent from human bile and present in low concentrations in bile from the rat. To study the mechanisms of this blood-bile glucose concentration difference, infusions of glucose were administered i.v. to 300-400 g male Sprague-Dawley rats with ligated renal pedicles and to two postcholecystectomy patients with indwelling t-tubes. Glucose was assayed in plasma, bile, and rat liver by a hexokinase method specific for D-glucose. In man, glucose was detected in bile when plasma glucose increased above 350 mg/100 ml. In animals studies, low concentrations of bile glucose were observed at plasma levels between 100 and 300 mg/100 ml. However, when plasma concentrations increased between 400 and 900 mg/100 ml, glucose appeared more rapidly in bile, defining by extrapolation an apparent plasma glucose threshold of 280 mg/100 ml. Intraportal phlorizin, a competitive inhibitor of glucose transport, significantly increased bile glucose concentrations. Plasma-bile concentration differences were also observed in rats after i.v. [3-14C]O-methyl glucose (3-O-MG) but not after [3H]mannitol. Hepatic glucose levels were never lower than plasma levels and liver-plasma 3-O-MG ratios were 0.92 +/- 0.22 indicating that entry of glucose and 3-O-MG into hepatocyte water was not limiting. Furthermore, when sodium dehydrocholate augmented canalicular secretion, biliary glucose excretion increased proportionally suggesting that glucose entry into bile was not impeded. When estimates of hepatic glucose secretion were compared with biliary glucose excretion, the latter increased progressively when estimated secretion rates exceeded 50 micrograms/min or when phlorizin was given. Finally, during bile stop-flow experiments, [3-14C]O-MG and [14C]glucose were selectively removed from bile compared with [3H]mannitol. The findings suggest that glucose and 3-O-MG are reabsorbed from bile after entry at the hepatocyte, accounting for their low bile-plasma ratio. The biliary glucose transport process may be described by Michaelis-Menten kinetics and is analogous to recently defined kinetics for renal tubular reabsorption of glucose. These studies provide evidence that certain products of bile secretion may undergo a "biliohepatic" circulation.

Canalicular bile secretion in man. Studies utilizing the biliary clearance of (14C)mannitol.

[(14)C]Mannitol was administered i.v. as a bolus injection to five postcholecystectomy patients with indwelling T-tubes and re-established enterohepatic circulations to evaluate the biliary clearance of [(14)C]mannitol as a means of estimating canalicular bile flow in man. [(14)C]Mannitol appeared in collections of bile 9-22.5 min after intravenous injection, rose to a peak, and thereafter paralleled the plasma [(14)C]mannitol disappearance curve. Bile-plasma [(14)C]mannitol ratios and [(14)C]mannitol clearances were determined during control and choleretic periods after correction of the bile [(14)C]mannitol points for the transit time of a given sample. After i.v. injection of sodium dehydrocholate in five studies, bile flow and mannitol clearance increased proportionately. However, when ductular secretion was stimulated with an i.v. bolus of secretin in three other studies, [(14)C]mannitol clearance remained essentially unchanged, indicating that [(14)C]mannitol entered bile at the level of the hepatocyte and could be utilized as a marker of canalicular flow in man. During control studies, when bile drained spontaneously from biliary fistulae in fasting patients, bileplasma [(14)C]mannitol ratios averaged 0.62+/-0.18 and canalicular flow, as estimated by [(14)C]mannitol clearance. (0.27+/-0.16 ml/min) accounted for 44-95% of total bile production (0.43+/-0.12 ml/min). When the rate of bile flow was plotted as a function of bile salt excretion after correction for the effects of biliary dead space, linear regression analysis revealed that approximately 7 mul of bile were secreted with each mumol of bile salt. Estimates of bile salt-independent canalicular flow accounted for at least one-third of the estimated 24-h bile production (604 ml) in these patients, indicating that this fraction of canalicular flow is a significant source of bile secretion in man.

Cytochemical localization of Na+, K+-ATPase in the rat hepatocyte.

The enzyme Na+,5+-ATPase was cytochemically localized in the rat hepatocyte by a modification of the Ernst potassium-dependent nitrophenyl phosphatase technique. Measurement of nitrophenol release from 50-micrometer liver slices confirmed the presence of ouabain-inhibitable nitrophenyl phosphatase activity that increased over the 30-min incubation period. Electron micrographs demonstrated that sinusoidal and lateral membrane reaction product deposition was K+-dependent, Mg++-dependent, inhibited by ouabain but not by alkaline phosphatase inhibitors, and was localized to the cytoplasmic side of the membrane. In contrast, canalicular reaction product was K+-independent, Mg++-dependent, inhibited by alkaline phosphatase inhibitors but not by ouabain, and was localized to the luminal side of the membrane. These findings indicate that Na+,K+-ATPase is localized to the sinusoidal and lateral portions of the rat hepatocyte plasma membrane and is not detectable on the bile canaliculus where alkaline phosphatase is confined. This basolateral localization of Na+,K+-ATPase is similar to that found in epithelia where secretion is also directed across the apical membrane.

Intracellular pH regulation in isolated rat bile duct epithelial cells.

To evaluate ion transport mechanisms in bile duct epithelium (BDE), BDE cells were isolated from bile duct-ligated rats. After short-term culture pHi was measured with a single cell microfluorimetric set-up using the fluorescent pHi indicator BCECF, and calibrated with nigericin in high K+ concentration buffer. Major contaminants were identified using vital markers. In HCO3(-)-free media, baseline pHi (7.03 +/- 0.12) decreased by 0.45 +/- 0.18 pH units after Na+ removal and by 0.12 +/- .04 after amiloride administration (1 mM). After acid loading (20 mM NH4Cl) pHi recovery was inhibited by both Na+ removal and amiloride (JH+ = 0.74 +/- 1.1, and JH+ = 2.28 +/- 0.8, respectively, vs. 5.47 +/- 1.97 and 5.97 +/- 1.76 mM/min, in controls, respectively). In HCO3- containing media baseline pHi was higher (7.16 +/- 0.1, n = 36, P less than 0.05) and was decreased by Na+ substitution but not by amiloride. Na+ removal inhibited pHi recovery after an intracellular acid load (0.27 +/- 0.26, vs. 7.7 +/- 4.1 mM/min, in controls), whereas amiloride reduced JH+ only by 27%. pH recovery was inhibited by DIDS (0.5-1 mM), but not by Cl- depletion. Finally, acute Cl- removal increased pHi by 0.18 pH units in the absence but not presence of DIDS. These data indicate that BDE cells possess mechanisms for Na+/H+ exchange, Na+:HCO3- symport and Cl-/HCO3 exchange. Therefore BDE may be capable of transepithelial H+/HCO3- transport.

Bicarbonate-dependent and -independent intracellular pH regulatory mechanisms in rat hepatocytes. Evidence for Na+-HCO3- cotransport.

Using the pH-sensitive dye 2,7-bis(carboxyethyl)-5(6)-carboxy-fluorescein and a continuously perfused subconfluent hepatocyte monolayer cell culture system, we studied rat hepatocyte intracellular pH (pHi) regulation in the presence (+HCO3-) and absence (-HCO3-) of bicarbonate. Baseline pHi was higher (7.28 +/- 09) in +HCO3- than in -HCO3- (7.16 +/- 0.14). Blocking Na+/H+ exchange with amiloride had no effect on pHi in +HCO3- but caused reversible 0.1-0.2-U acidification in -HCO3- or in +HCO3- after preincubation in the anion transport inhibitor 4,4'-diisothiocyano-2,2'-disulfonic acid stilbene (DIDS). Acute Na+ replacement in +HCO3- alos caused acidification which was amiloride independent but DIDS inhibitible. The recovery of pHi from an intracellular acid load (maximum H+ efflux rate) was 50% higher in +HCO3- than in -HCO3-. Amiloride inhibited H+ effluxmax by 75% in -HCO3- but by only 27% in +HCO3-. The amiloride-independent pHi recovery in +HCO3- was inhibited 50-63% by DIDS and 79% by Na+ replacement but was unaffected by depletion of intracellular Cl-, suggesting that Cl-/HCO3- exchange is not involved. Depolarization of hepatocytes (raising external K+ from 5 to 25 mM) caused reversible 0.05-0.1-U alkalinization, which, however, was neither Na+ nor HCO3- dependent, nor DIDS inhibitible, findings consistent with electroneutral HCO3- transport. We conclude that Na+-HCO3- cotransport, in addition to Na+/H+ exchange, is an important regulator of pHi in rat hepatocytes.

Direct determination of the driving forces for taurocholate uptake into rat liver plasma membrane vesicles.

To determine directly the driving forces for bile acid entry into the hepatocyte, the uptake of [3H]taurocholic acid into rat liver plasma membrane vesicles was studied. The membrane preparation contained predominantly right-side-out vesicles, and was highly enriched in plasma membrane marker enzymes. The uptake of taurocholate at equilibrium was inversely related to medium osmolarity, indicating transport into an osmotically sensitive space. In the presence of an inwardly directed sodium gradient (NaCl or sodium gluconate), the initial rate of uptake was rapid and taurocholate was transiently accumulated at a concentration twice that at equilibrium (overshoot). Other inwardly directed cation gradients (K+, Li+, choline+) or the presence of sodium in the absence of a gradient (Na+ equilibrated) resulted in a slower initial uptake rate and did not sustain an overshoot. Bile acids inhibited sodium-dependent taurocholate uptake, whereas bromsulphthalein inhibited both sodium-dependent and sodium-independent uptake and D-glucose had no effect on uptake. Uptake was temperature dependent, with maximal overshoots occurring at 25 degrees C. Imposition of a proton gradient across the vesicle (pHo less than pHi) in the absence of a sodium gradient failed to enhance taurocholate uptake, indicating that double ion exchange (Na+-H+, OH- -anion) is unlikely. Creation of a negative intravesicular potential by altering accompanying anions or by valinomycin-induced K+-diffusion potentials did not enhance taurocholate uptake, suggesting an electroneutral transport mechanism. The kinetics of taurocholate uptake demonstrated saturability with a Michaelis constant at 52 microM and maximum velocity of 4.5 nmol X mg-1 X protein X min-1. These studies provide definitive evidence for a sodium gradient-dependent, carrier-mediated, electrically neutral transport mechanism for hepatic taurocholate uptake. These findings are consistent with a model for bile secretion in which the basolateral enzyme Na+,K+-ATPase provides the driving force for "uphill" bile acid transport by establishing a trans-membrane sodium gradient.

Bile formation in the rat: the role of the paracellular shunt pathway.

Transepithelial movement of water and solute occurs both through the cell membrane as well as across the intercellular junctional complex (paracellular shunt pathways). Permeability of paracellular shunt pathways is increase by transmucosal osmotic gradients, and in certain epithelia these changes are associated with bullous-like deformations (blisters) of the zonula occludens and localization of lanthanum within junctional complexes. Although bile acids increase biliary secretion by osmotic forces, the source of this water movement into bile is not known. In the present studies we examined whether a choleretic infusion of sodium dehydrocholic acid (DHC) or its taurine conjugate, taurodehydrocholate, altered the solute permeability characteristics and morphologic appearance of the junctional complexes of rat hepatocytes. Animals were continuously infused for 1 hr with 1% albumin--0.9% NaCl alone or 120 mumol of DHC and bile flow and biliary clearance of [14C]sucrose, an indirect marker of biliary permeability were measured. The number of intercellular blisters adjacent to the bile canaliculus were counted in an unbiased manner from photographs obtained with scanning electron microscopy. Bile flow and the biliary sucrose clearance remained unchanged in control animals whereas DHC infusions resulted in a progressive increase in the biliary clearance of [14C]sucrose during the 60 min of infusion even though the choleretic response to DHC was stable during the final 30 min of infusion. DHC infusions produced surface invaginations, or blisters, (0.1--0.7 micrometer in diameter) which were located immediately adjacent to the hemi-bile canaliculus and occurred with a frequency of 1.62 +/- 0.08 per hepatocyte surface, which was fivefold greater than observed in controls. In separate groups of animals 5 mM ionic lanthanum chloride was perfused intraportally after taurodehydrocholate infusions, and the number of junctional complexes that contained the electron dense marker were quantitated by transmission electron microscopy. Localization of lanthanum in the junctional complexes of fasted control animals was not observed, whereas approximately equal to 50% of the zonula occludens in DHC-infused animals contained lanthanum which was also occasionally identified within the lumen of the bile canaliculus. These results indicate that infusions of DHC cause blisters adjacent to the junctional complex of rat hepatocytes in association with changes in solute conductivity of the zonula occludens to cations such as ionic lanthanum chloride, and presumably to larger solutes such as sucrose. Qualitatively similar morphologic findings were also observed during the infusion of sodium taurocholate at physiologic rate (40 mumol/h). These studies suggest that the paracellular shunt pathway in the liver is an important site for bile acid-induced water and solute movement into bile.

The effect of sodium taurocholate on the hepatic metabolism of sulfobromophthalein sodium (BSP). The role of bile flow.

The influence of bile salts on the hepatic metabolism of sulfobromophthalein sodium (BSP) was studied in the perfused rat liver. During sodium taurocholate infusions, hepatic uptake of BSP from plasma was increased and appeared to be related to an enhanced transit of BSP from liver into bile. BSP-glutathione conjugation was not affected by the bile salt infusions, although bile salts inhibited the enzyme system in vitro. The major effect of bile salts was to increase the BSP transport maximum (Tm). When sodium taurocholate was infused in saline at a rate of 30 mumoles/hr, both bile flow and the BSP Tm increased, and remained at peak levels of 1.5 +/-0.12 mul/min per g liver and 21 +/-3.0 mug/min per g liver, respectively. In contrast, during saline infusion alone both levels were significantly lower (1.06 +/-0.17 mul/min per g liver and 15.8 +/-4.16 mug/min per g liver, respectively), and both fell progressively after the 2nd hr of perfusion. This decline in bile flow and BSP Tm was associated with a decrease in biliary bile salt excretion and was reversed by adding bile salts to the perfusate. Since the biliary concentration of BSP remained within a narrow range in all experiments, the BSP Tm was primarily determined by the rate of bile flow. Dependence of BSP Tm on the rate of bile production was further confirmed by changing the temperature of the perfusate during a constant infusion of taurocholate. BSP Tm paralleled temperature-induced changes in bile flow irrespective of changes in the level of bile salt excretion. Since the biliary concentration of BSP remained within a narrow range in all experiments, the concentrating capacity for BSP in bile may be the major limiting factor in BSP transport. Thus two independent factors appear to determine the BSP Tm: the bile BSP concentration, and the rate of bile production. Because taurocholate enhanced BSP transport only when it increased bile production, its effect on the BSP Tm appears to be attributable to its choleretic properties.

Tauroursodeoxycholic acid stimulates hepatocellular exocytosis and mobilizes extracellular Ca++ mechanisms defective in cholestasis.

To assess the effects of tauroursodeoxycholic acid (TUDCA) on bile excretory function, we examined whether TUDCA modulates vesicular exocytosis in the isolated perfused liver of normal rats in the presence of high (1.9 mM) or low (0.19 mM) extracellular Ca++ and in cholestatic rats 24 h after bile duct ligation. In addition, the effects of TUDCA on Ca++ homeostasis were compared in normal and in cholestatic hepatocytes. In the isolated perfused rat liver, TUDCA (25 microM) stimulated a sustained increase in the biliary excretion of horseradish peroxidase, a marker of the vesicular pathway, in the presence of high, but not low extracellular Ca++ or in the cholestatic liver. In contrast, TUDCA stimulated bile flow to the same extent regardless of the concentration of extracellular Ca++ or the presence of cholestasis. In indo-1-loaded hepatocytes, basal cytosolic free Ca++ ([Ca++]i) levels were not different between normal and cholestatic cells. However, in cholestatic cells [Ca++]i increases induced by TUDCA (10 microM) and its 7 alpha-OH epimer taurochenodeoxycholic acid (50 microM) were reduced to 22% and 26%, respectively, compared to normal cells. The impairment of TUDCA-induced [Ca++]i increase in cholestatic cells could be mimicked by exposing normal cells to low extracellular Ca++ (21%) or to the Ca++ channel blocker NiCl2 (23%). These data indicate that (a) dihydroxy bile acid-induced Ca++ entry may be of functional importance in the regulation of hepatocellular vesicular exocytosis, and (b) this Ca++ entry mechanism across the plasma membrane is impaired in cholestatic hepatocytes. We speculate that the beneficial effect of ursodeoxycholic acid in cholestatic liver diseases may be related to the Ca+(+)-dependent stimulation of vesicular exocytosis by its conjugate.

Irreversible binding of conjugated bilirubin to albumin in cholestatic rats.

A diazo-positive fraction of serum bilirubin that is irreversibly bound to albumin has been shown to accumulate in serum of patients with cholestasis. In the present study, a cholestatic animal model was used to determine the chemical nature of the bilirubin species involved in its formation. The data indicate that conjugated bilirubin is the precursor of "albumin-bound bilirubin" and that the presence or absence of light does not affect its formation. An albumin-bound bilirubin-complex indistinguishable from the complex detected in cholestatic sera from patients or in bile duct-ligated Sprague-Dawley rats can be formed in vitro in sera enriched in conjugated bilirubin at 37 degrees C, pH 7.4.

Quantitative assessment of canalicular bile formation in isolated hepatocyte couplets using microscopic optical planimetry.

Isolated rat hepatocyte couplets (IRHC) are primary units of bile secretion that accumulate fluid in an enclosed canalicular space with time in culture. We have quantitated the rate of canalicular secretion in IRHC cultured for 4-8 h by measuring the change in canalicular space volume by video-microscopic optical planimetry using high resolution Nomarski optics. Electron microscopic morphometric studies revealed significant increases in canalicular membrane area after 4-6 h in culture. Canalicular secretion in basal L-15 medium (3.8 +/- 1.3 fl/min) increased significantly with the choleretic bile salts (10 microM), taurocholate, and ursodeoxycholate (14 +/- 7 fl/min each). Secretion rates after exposure to bile acids correlated directly with the canalicular surface area before stimulation. In contrast, expansion times after stimulation varied inversely with initial canalicular volumes. Ursodeoxycholic acid failed to produce a hypercholeresis at 10-, 100-, or 200-microM concentrations compared with taurocholate, either in normal or taurine-depleted IRHC. The present findings establish that rates of canalicular bile secretion can be quantitated in IRHC by serial optical planimetry, both in the basal state and after stimulation with bile acids. Furthermore, ursodeoxycholate does not acutely induce hypercholeresis at the canalicular level in this model. Rather, both taurocholic and ursodeoxycholic acids induced secretion in proportion to the surface area of the canalicular membrane. The IRHC are a useful model to identify canalicular choleretics and for studies of canalicular bile formation.

Effect of secretion on intracellular pH regulation in isolated rat bile duct epithelial cells.

The effects of secretin on ion transport mechanisms involved in regulation of intracellular pH (pHi) and HCO3- excretion were characterized in bile duct epithelial (BDE) cells isolated from normal rat liver. pHi was measured with 2,7-bis(carboxy-ethyl)-5(6)-carboxy-fluorescein-acetomethylester (BCECF-AM) using a microfluorimetric method. Basal pHi of BDE was 7.04 +/- 0.06 in Hepes and 7.16 +/- 0.10 in KRB and was unaffected by secretin (50-200 nM). Recovery rates from an acid load in Hepes or in KRB media (with and without amiloride) were also not altered by secretin, indicating that Na+/H+ exchange and Na+/HCO3- cotransport were not affected by this hormone. After acute Cl- removal, pHi rose 0.24 +/- 0.08 pHU at a maximal rate of 0.125 +/- 0.06 pHU/min (H+ flux rates = 6.02 +/- 3.27 mM/min) and recovered after Cl- readmission (0.188 +/- 0.08 pHU/min; H+ flux rates = 11.82 +/- 5.34 mM/min). Pretreatment with 1 mM DIDS inhibited the effects of Cl- removal, while valinomycin, which induces cell depolarization, enhanced these effects, probably by stimulating electrogenic HCO3- influx. Secretin significantly increased both the maximal rate of alkalinization after Cl- removal (P < 0.012) and of pHi recovery after Cl- readmission (P < 0.025), indicating stimulation of Cl-/HCO3- exchange activity. These findings were reproduced with N6,2'-O-Dibutyryladenosine-3',5'-cyclic monophosphate (DBcAMP). The Cl- channel blocker 5-nitro-2'-(3-phenylpropylamino)-benzoate (NPPB, 10 microM) significantly decreased the effects of secretin and DBcAMP on the pHi changes promoted by acute Cl- removal/readmission. These findings establish that secretin stimulates the activity of the Cl-/HCO3- exchanger in BDE cells, probably by activating Cl- channels via the intracellular messenger cAMP. This in turn depolarizes the cell, stimulating electrogenic Na+/HCO3- symport. The cell depolarization induced by Cl- channel activation should enhance HCO3- entrance through electrogenic Na+/HCO3- symport, which in turn stimulates the Cl-/HCO3- exchange. These mechanisms could account for secretin stimulated bicarbonate secretion in bile.

Ursodeoxycholate stimulates Na+-H+ exchange in rat liver basolateral plasma membrane vesicles.

Na+:H+ and Cl-:HCO3- exchange are localized, respectively, to basolateral (blLPM) and canalicular (cLPM) rat liver plasma membranes. To determine whether these exchangers play a role in bile formation, we examined the effect of a choleretic agent, ursodeoxycholate (UDCA), on these exchange mechanisms. 22Na (1 mM) and 36Cl (5 mM) uptake was determined using outwardly directed H+ and HCO3- gradients, respectively. Preincubation of blLPM vesicles with UDCA (0-500 microM) resulted in a concentration-dependent increase in initial rates of amiloride-sensitive pH-driven Na+ uptake, with a maximal effect at 200 microM. UDCA (200 microM) increased Vmax from 23 +/- 2 (control) to 37 +/- 7 nmol/min per mg protein; apparent Km for Na+ was unchanged. Preincubation with tauroursodeoxycholate (200 microM), taurocholate (10-200 microM) or cholate, chenodeoxycholate, or deoxycholate (200 microM) had no effect on pH-driven Na+ uptake. UDCA (200 microM) had no effect on either membrane lipid fluidity, assessed by steady-state fluorescence polarization using the probes 1,6-diphenyl-1,3,5-hexatriene, 12-(9-anthroyloxy) stearic acid, and 2-(9-anthroyloxy) stearic acid (2-AS), or Na+,K+-ATPase activity in blLPM vesicles. In cLPM vesicles, UDCA (0-500 microM) had no stimulatory effect on initial rates of HCO3(-)-driven Cl- uptake. Enhanced basolateral Na+:H+ exchange activity, leading to intracellular HCO3- concentrations above equilibrium, may account for the bicarbonate-rich choleresis after UDCA infusion.