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.

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.

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.

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.

Molecular identification and functional characterization of Mdr1a in rat cholangiocytes.

BACKGROUND & AIMS: The multidrug resistance P-glycoprotein 170 gene products (mdr1a and 1b) are glycosylated plasma membrane proteins that function as adenosine triphosphate-dependent transmembrane export pumps for lipophilic xenobiotics of widely different structure. We assessed whether these P-glycoproteins are functionally expressed in cholangiocytes. METHODS: A reverse-transcription polymerase chain reaction was performed on RNA from a normal rat cholangiocyte cell line using mdr1-specific primers. Northern and Western blot analyses were performed on cholangiocytes immunoisolated from 2-week bile duct-ligated rats and cholangiocytes and isolated cholangiocyte membrane subfractions, respectively. Functional assays were performed in isolated bile duct units from bile duct-ligated rats and incubated with rhodamine 123, a P-glycoprotein substrate, with or without the P-glycoprotein inhibitors verapamil or GF120918. RESULTS: A 400-base pair fragment with 99% homology to the cytosolic domain of rat intestinal mdr1a (5' 1953-2350 3') was identified that hybridized to a 5.2-kilobase RNA transcript in a normal rat cholangiocyte cell line, isolated rat cholangiocytes, and ileum. Western analysis localized mdr1 to the apical membrane of cholangiocytes. Confocal microscopy showed active secretion of rhodamine 123 into the lumen of isolated bile duct units that was abolished by vanadate and P-glycoprotein competitive antagonists, verapamil and GF120918, in a dose-dependent manner. CONCLUSIONS: These findings provide the first molecular and functional evidence for the expression of mdr1a on the luminal membrane of cholangiocytes, where it may have a protective role.

Communication via gap junctions modulates bile secretion in the isolated perfused rat liver.

BACKGROUND & AIMS: Bile secretion is regulated in part by adenosine 3',5'-cyclic monophosphate (cAMP) and cytosolic Ca2+ (Ca2+i). Hormone receptors that link to these second messengers are not uniformly distributed across the hepatic lobule, but both cAMP and Ca2+i cross gap junctions, so we tested whether gap junctional communication plays a role in changes in bile flow induced by the activation of these receptors. METHODS: cAMP levels in isolated perfused rat livers were increased by using glucagon, because glucagon receptors are predominantly on pericentral hepatocytes, or by using dibutyryl cAMP, which acts on hepatocytes throughout the hepatic lobule. Ca2+i concentration was increased by using vasopressin, because V1a receptors are most heavily expressed on pericentral hepatocytes, or by using 2,5-di(tert-butyl)-1, 4-benzo-hydroquinone (t-BuBHQ), which increases the Ca2+i concentration in hepatocytes throughout the hepatic lobule. We used 18alpha-glycyrrhetinic acid (alphaGA) to block gap junction conductance, which was assessed by fluorescence recovery after photobleaching. RESULTS: alphaGA blocked fluorescence recovery after photobleaching without altering the basal rate of bile flow. Glucagon and dibutyryl cAMP increased bile flow; alphaGA blocked the glucagon-induced increase but not that induced by dibutyryl cAMP. Vasopressin and t-BuBHQ decreased bile flow; alphaGA exacerbated the decrease induced by vasopressin but not by t-BuBHQ. CONCLUSIONS: Glucagon and vasopressin modulate bile flow in a manner that depends in part on gap junctional communication, even though the two hormones activate second messengers with opposing effects on bile flow. The organization of second messenger signals across the hepatic lobule may be an important component of hormonal regulation of bile secretion.

Nitric oxide and guanosine 3',5'-cyclic monophosphate stimulate bile secretion in isolated rat hepatocyte couplets, but not in isolated bile duct units.

Nitric oxide (NO) and guanosine 3',5'-cyclic monophosphate (cGMP) have recently been shown to stimulate bile acid-independent bile flow in the isolated perfused rat liver (IPRL). However, the cellular origin and mechanisms of this choleresis have not yet been determined. To address these questions, we examined the effects of NO and cGMP on bile secretion in isolated rat hepatocyte couplets (IRHC) and in isolated bile duct units (IBDU), both of which are isolated cell systems in which cell polarity is maintained and secretion can be measured directly. Changes in the area of the canalicular and ductular lumens were determined in IRHC and IBDU, respectively, as indicators of the rate of fluid secretion using video microscopy. In addition, Cl-/HCO3- exchanger activity in IBDU was evaluated by measuring changes in intracellular pH (pHi) after Cl- removal/readmission by microfluorometric methods. In the presence of HCO3-, both the NO donor, S-nitroso-acetyl-penicillamine (SNAP), and the cell-permeant cGMP analogue, dibutyryl cGMP (DBcGMP), stimulated canalicular bile secretion (P <.05), as did the cell-permeant cAMP analogue, dibutyryl cAMP (DBcAMP) (P <.05). Removal of HCO3- from the buffer completely abolished the choleretic effects of DBcGMP, but had no effect on NO-induced choleresis. In contrast, secretion in IBDU was not stimulated following incubations with SNAP or DBcGMP over 30 minutes, whereas DBcAMP and secretin, a cholangiocyte secretagogue and cAMP agonist, both had a marked effect on ductular secretion over this same time interval (P <.05). SNAP also had no effect on Cl-/HCO3- exchanger activity in IBDU, and inhibition of endogenous NO synthesis by NG-monomethyl-L-arginine (L-NMMA) did not alter secretin-induced stimulation of ductular bile secretion and Cl-/HCO3- exchanger activity. In summary, NO and cGMP stimulate bile secretion exclusively at the the level of hepatocytes, whereas cAMP mediates choleresis at both hepatocyte and bile duct levels. These findings may have important implications for the regulation of ductular bile secretion by hormones and neuropeptides, as well as under pathological conditions with increased hepatic NO synthesis.

Intracellular pH regulation in bombesin-stimulated secretion in isolated bile duct units from rat liver.

Bombesin, a neuropeptide, stimulates fluid and HCO-3 secretion from cholangiocytes, but the underlying mechanisms are poorly understood. In this study, we aimed to examine the effects of bombesin on ion transport processes involved in the regulation of intracellular pH (pHi) and HCO-3 secretion in polarized cholangiocytes. Isolated bile duct units from normal rat liver were used to measure pHi by 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein 495 nm-to-440 nm dual ratio methods. Bombesin increased Cl--HCO-3 exchange activity but did not affect basal pHi or the activities of Na+/H+ exchange or Na+-HCO-3 symport. Depolarization of cholangiocytes increased basal pHi and the activity of Cl-/HCO-3 exchange, suggesting that an electrogenic Na+-HCO-3 symport might function as a counterregulatory pHi mechanism. Na+-independent acid-extruding mechanisms were not observed. We conclude that bombesin stimulates biliary secretion from cholangiocytes by activating luminal Cl-/HCO-3 exchange, which may be coupled to basolateral electrogenic Na+-HCO-3 symport.

Role of glutathione in hepatic bile formation during reperfusion after cold ischemia of the rat liver.

BACKGROUND/AIMS: Liver reperfusion following cold ischemia is frequently associated with diminished bile flow in patients undergoing liver transplantation. Glutathione is a major determinant of bile-acid independent bile flow, and the effects of cold ischemia on biliary glutathione excretion are unknown. METHODS: We examined the effects of cold ischemia (University of Wisconsin solution (4 degrees C), 24 h) with subsequent reperfusion (100 min) on biliary glutathione excretion in a recirculating system. Since glutathione might represent an important antioxidant within the biliary tract and oxidative stress in the biliary tract during reperfusion could contribute to the pathogenesis of bile duct injury after liver transplantation, we also assessed bile duct morphology in reperfused livers of mutant TR- -rats, in whom biliary excretion of glutathione is already impaired. RESULTS: Hepatic bile formation was diminished in reperfused Wistar rat livers after cold ischemia. Biliary glutathione concentrations and output were significantly decreased and correlated with postischemic changes in bile secretion. An increased biliary oxidized glutathione/glutathione ratio, indicating oxidative stress, was detected only immediately after the onset of reperfusion. Basal bile flow rates in TR- -rat livers which were already markedly reduced in control-perfused livers, decreased further during the early but not the later reperfusion period. Reperfusion of both Wistar and TR- -rat livers was not associated with electron microscopic evidence of bile duct damage. CONCLUSIONS: We conclude that impaired biliary excretion of glutathione contributes to decreased bile flow after cold ischemia. The absence of biliary glutathione does not appear to promote ultrastructural evidence of bile duct injury during reperfusion in the isolated perfused rat liver.

Stimulation of bile duct epithelial secretion by glybenclamide in normal and cholestatic rat liver.

Cholestasis is a cardinal complication of liver disease, but most treatments are merely supportive. Here we report that the sulfonylurea glybenclamide potently stimulates bile flow and bicarbonate excretion in the isolated perfused rat liver. Video-microscopic studies of isolated hepatocyte couplets and isolated bile duct segments show that this stimulatory effect occurs at the level of the bile duct epithelium, rather than through hepatocytes. Measurements of cAMP, cytosolic pH, and Ca2+ in isolated bile duct cells suggest that glybenclamide directly activates Na+-K+-2Cl- cotransport, rather than other transporters or conventional second-messenger systems that link to secretory pathways in these cells. Finally, studies in livers from rats with endotoxin- or estrogen-induced cholestasis show that glybenclamide retains its stimulatory effects on bile flow and bicarbonate excretion even under these conditions. These findings suggest that bile duct epithelia may represent an important new therapeutic target for treatment of cholestatic disorders.

Role of kinases and phosphatases in the regulation of fluid secretion and Cl-/HCO3- exchange in cholangiocytes.

The role of protein kinase A (PKA), protein kinase C (PKC), and protein phosphatases in the process of secretin stimulation of fluid and bicarbonate secretion from biliary epithelium was examined using a novel isolated bile duct unit (IBDU) model from rat liver. Sp-adenosine 3',5'-cyclic monophosphothiolate (Sp-cAMPS), 100 microM, a PKA-specific agonist, significantly increased secretion during a 30-min perfusion (+61%, P < 0.01). In contrast, preincubation and perfusion of Rp-cAMPS, 100 microM, a specific PKA inhibitor, reduced the ability of secretin to stimulate both fluid secretion (111 vs. 25%; P < 0.01) and Cl-/HCO3- exchanger activity (80 vs. 28%). Neither the PKC agonist phorbol 12-myristate 13-acetate, 10 microM, nor the PKC antagonist staurosporine showed any effect on either basal or secretin-stimulated fluid secretion or Cl-/HCO3- exchange activity in IBDU. Okadaic acid, a specific inhibitor of protein phosphatases 1 and 2A, also had no effect on basal fluid secretion or on the basal activity of the Cl-/HCO3- exchanger. However, okadaic acid resulted in persistence of secretion after removal of secretin, in contrast to the reduction in secretion observed in controls. These findings indicate that PKA but not PKC is involved in the signal transduction of secretin-stimulated fluid secretion and Cl-/HCO3- exchange activity in rat bile duct epithelium, a process inactivated by dephosphorylation by protein phosphatases 1 and/or 2A.

Bombesin stimulates bicarbonate secretion from rat cholangiocytes: implications for neural regulation of bile secretion.

BACKGROUND & AIMS: Bombesin is a neuropeptide with many biological functions and is known to stimulate bile secretion. The aim of this study was to determine the role of bombesin in bile secretion and its site of action. METHODS: The effects of bombesin on bile secretion were examined using isolated perfused rat livers, hepatocyte couplets, and isolated bile duct units (IBDU) from rat liver. RESULTS: Bombesin (100 nmol/L) increased bile pH, bicarbonate concentration, and output in isolated perfused rat livers from both normal and 2-week bile duct-ligated rats, although bile flow increased only in the latter model. Bombesin (10-100 nmol/L) also had no effect on canalicular bile secretion in isolated hepatocyte couplets. However, bombesin produced a dose-dependent increase in secretion in IBDU, which was inhibited almost completely by a specific bombesin receptor inhibitor, [Tyr4, D-Phe12]-bombesin (1 micromol/L). This bombesin (10 nmol/L)-stimulated secretion in IBDU was accompanied by an increase in luminal pH and was dependent on bicarbonate and chloride in the medium. Somatostatin but not substance P inhibited the bombesin response. CONCLUSIONS: Neuropeptides such as bombesin can directly stimulate fluid and bicarbonate secretion at the level of cholangiocytes, suggesting that neuropeptides play an important regulatory role in biliary transport and secretion.

Nitric oxide donors stimulate bile flow and glutathione disulfide excretion independent of guanosine 3',5'-cyclic [corrected] monophosphate in the isolated perfused rat liver.

Nitric oxide (NO) modulates several metabolic functions in hepatocytes, but the role of NO in bile secretion has not been clearly defined. In the present study, we examined the effects of NO on bile flow and biliary HC03- and glutathione excretion in the isolated perfused rat liver and assessed the role of guanosine 3',5'-cyclic monophosphate (cGMP) in mediating these effects. The NO donors sodium nitroprusside (SNP) and S-nitroso-acetyl-penicillamine stimulated bile flow and increased both HCO3- and glutathione excretion. Increases in bile flow were linearly related to increases in biliary glutathione concentration and output (P < .0001), which were almost entirely caused by glutathione disulfide, whereas the excretion of reduced glutathione remained unchanged. NO donors increased cGMP concentrations in bile and perfusate, and the membrane-permeant cGMP analogue dibutyryl cGMP was also found to stimulate bile flow and HCO3- excretion. However, in contrast to the NO donors, dibutyryl cGMP did not increase glutathione excretion. Furthermore, the NO donors failed to stimulate bile flow in mutant TR- rats in which the canalicular transport of glutathione and glutathione conjugates is deficient, although dibutyryl cGMP increased bile flow and HCO3- excretion in the mutant rats as in normals. These findings indicate that exogenous sources of NO increase bile acid-independent bile flow by stimulating glutathione disulfide excretion, effects that are independent of cGMP.

Liver sinusoidal endothelial cells are responsible for nitric oxide modulation of resistance in the hepatic sinusoids.

The mechanisms that regulate vascular resistance in the liver are an area of active investigation. Previously, we have shown that nitric oxide (NO) modulates hepatic vascular tone in the normal rat liver. In this study, the production of NO is examined in further detail by isolating sinusoidal endothelial cells (SEC) from the rat liver. Endothelial NO synthase (eNOS) was present in SEC based on Western blotting and confocal immunofluorescence microscopy. Exposure of SEC to flow increased the release of NO. To investigate the relevance of these in vitro findings to the intact liver, we modified an in situ perfusion system to allow for direct measurement of NO release from the hepatic vasculature. NO was released from the hepatic vasculature in a time-dependent manner, and administration of N-monomethyl-L-arginine reduced NO release and increased portal pressure. Immunostaining of intact liver demonstrated eNOS localization to endothelial cells lining the hepatic sinusoids. These findings demonstrate that SEC in vitro and in vivo express eNOS and produce NO basally, and increase their production in response to flow. Additionally, an increase in portal pressure concomitant with the blockade of NO release directly demonstrates that endogenous endothelial-derived NO modulates portal pressure.

Characterization of cytosolic Ca2+ signaling in rat bile duct epithelia.

Bile duct epithelia play an important role in the formation and conditioning of bile. However, hormonal responses in this epithelial tissue are incompletely understood. Secretin increases ductular secretion through the intracellular messenger adenosine 3',5'-cyclic monophosphate (cAMP), but whether hormones increase cytosolic Ca2+ (Ca2+(i)) in these cells and whether Ca2+(i) regulates duct secretion is unknown. To address these questions, we examined Ca2+(i) signaling in isolated rat bile duct units using ratio microspectrofluorometry and confocal microscopy. We also used videomicroscopy to examine secretion and cell volume in isolated bile duct cells and duct units. Acetylcholine (ACh) and ATP both increased Ca2+(i) in bile duct units and elicited patterns of Ca2+(i) increases and oscillations that were distinct and dose dependent. In contrast, Ca2+(i) was not increased by the hepatocyte Ca2+(i) agonists vasopressin, angiotensin, and phenylephrine or by the exocrine pancreas agonists cholecystokinin (CCK) and bombesin. In addition, secretin did not increase Ca2+(i) in the isolated bile duct units, whereas ACh did not increase Ca2+(i) in isolated hepatocytes. Mobilization of internal, thapsigargin-sensitive Ca2+ stores contributed more than influx of extracellular Ca2+ to the Ca2+(i) increases induced in the duct units, and ATP-induced increases in Ca2+(i) could be blocked by microinjection of heparin but not de-N-sulfated heparin. ACh transiently decreased bile flow in the isolated perfused rat liver, although neither ACh nor ATP altered secretion in isolated ducts or changed the volume of single isolated bile duct cells. These findings demonstrate that bile duct epithelial cells possess both muscarinic and purinergic receptors that activate Ca2+(i) signaling pathways similar to those seen in other types of epithelia, but that the two types of receptors elicit distinct patterns of Ca2+(i) signals. Increases in Ca2+(i) have minimal direct effects on bile duct secretion, although it remains to be determined whether such signals selectively modulate other aspects of bile duct epithelial cell function.

Ca2+ waves are organized among hepatocytes in the intact organ.

Hormone-induced increases in cytosolic Ca2+ (Cai2+) begin as Cai2+ waves in cells isolated from most types of tissue (1, 11), but whether such waves actually occur in vivo is unknown. To investigate this, we examined vasopressin-induced Cai2+ signals in hepatocytes within the perfused rat liver. Using confocal fluorescence video microscopy, we found that increases in Cai2+ began as waves that usually originated in hepatocytes near central venules, then spread opposite to the direction of blood flow, to hepatocytes near portal venules. We used immunochemistry to determine that the liver vasopressin V1a receptor is most concentrated among hepatocytes in the pericentral region, providing the mechanism by which Cai2+ waves originate there. Pericentral-to-periportal Cai2+ waves may direct peristaltic flow of bile, since Cai2+ induces contraction of the apical pole of hepatocytes and since peristaltic contractions in liver also occur in a pericentral-to-periportal direction. The organization of Cai2+ waves among cells in intact tissue may be a means by which an integrative, organ-level response is provided in response to hormonal stimuli.

Isolation of small polarized bile duct units.

Fragments of small interlobular bile ducts averaging 20 microns in diameter can be isolated from rat liver. These isolated bile duct units form luminal spaces that are impermeant to dextran-40 and expand in size when cultured in 10 microM forskolin for 24-48 hr. Secretion is Cl- and HCO3- dependent and is stimulated by forskolin > dibutyryl cAMP > secretion but not by dideoxyforskolin, as assessed by video imaging techniques. Secretin stimulates Cl-/HCO3- exchange activity, and intraluminal pH increases after forskolin administration. These studies establish that small polarized physiologically intact interlobular bile ducts can be isolated from rat liver. These isolated bile duct units should be useful preparations for assessing the transport properties of small bile duct segments, which are the primary site of injury in cholestatic liver disorders, known as "vanishing bile duct syndromes."

Effect of cerulein hyperstimulation on the paracellular barrier of rat exocrine pancreas.

BACKGROUND/AIMS: Cerulein-induced pancreatitis causes a rapid increase in pancreatic enzyme levels in serum and decreases in pancreatic duct secretion and interstitial edema. One mechanism to explain these early events is disruption of the actin tight junction paracellular seal of acinar and intralobular pancreatic duct cells. METHODS: To examine the paracellular barrier of the proximal exocrine pancreas, rats were hyperstimulated with 5.0 micrograms.kg-1.h-1 of cerulein. Actin was visualized with rhodamine phalloidin and by electron microscopy and tight junctions were visualized with antibodies to the tight-junction protein ZO-1. Paracellular permeability was measured by movement of horseradish peroxidase from interstitium into duct or acinar lumens. RESULTS: In controls, linear actin and ZO-1 staining occurred along the apical membrane of intralobular duct cells and extended to the apical pole of acinar cells. Hyperstimulation caused progressive disruption of the linear staining of f-actin and ZO-1. Actin disruption in duct cells was confirmed by electron microscopy. Horseradish peroxidase entered intralobular ducts and acinar lumens of hyperstimulated animals more frequently than those of controls. CONCLUSIONS: The structure and function of the paracellular barrier of acinar and intralobular pancreatic duct cells are disrupted early during cerulein pancreatitis and may contribute to early clinical features.

Altered expression and function of hepatocyte gap junctions after common bile duct ligation in the rat.

Gap junction channels allow intercellular exchange of ions and small molecules between adjacent cells. Such communication coordinates cellular and organ function in tissues, although it is unclear if altered gap junction expression and communication contribute to organ dysfunction in pathological states. We examined the immunofluorescent (IF) localization and mRNA and protein levels of the two hepatocyte gap junction proteins connexin 32 and connexin 26, after hepatic injury induced by common bile duct ligation (CBDL) in the rat. Intercellular communication was measured by comparing gap junction-mediated coordination of hormone-induced Ca2+ signals in isolated rat hepatocyte couplets from control and CBDL animals. Connexin 32 plasma membrane IF, protein, and mRNA levels decreased markedly early after CBDL and remained low at 14 days. Connexin 26 plasma membrane IF and protein levels also decreased markedly after CBDL, but mRNA levels rose, and a partial return in membrane IF and protein levels was noted at 9 and 14 days. Coordination of vasopressin-induced Ca2+ signals between cells in isolated rat hepatocyte couplets 1 day after CBDL was significantly impaired compared with controls. These results demonstrate that hepatocyte gap junction communication is impaired early after CBDL because of decreased connexin protein levels. Disruption of gap junctions after CBDL may contribute to loss of hepatic functions that depend on gap junction communication.