Proinflammatory cytokines inhibit secretion in rat bile duct epithelium.

BACKGROUND AND AIMS: Cholestatic disorders often are associated with portal inflammation, but whether or how inflammation contributes to cholestasis is unknown. Thus we studied the effects of proinflammatory cytokines on bile duct epithelia secretory mechanisms. METHODS: Isolated bile duct units (IBDUs) were cultured with interleukin (IL)-6, interferon gamma, tumor necrosis factor (TNF)-alpha, and IL-1 alone or in combination. Ductular secretion was measured using video-optical planimetry. Bicarbonate and Cl(-) transport were assessed microfluorimetric measuring pH(i) (BCECF) and [Cl(-)](i) transients (MEQ). Expression of Cl(-)/HCO(3)(-) exchanger (AE-2), cystic fibrosis transmembrane conductance regulator (CFTR), and the secretin receptor (SR) were assessed by ribonuclease protection assay. Cellular cyclic adenosine monophosphate (cAMP) levels were studied by enzymatic immunoassay. Paracellular permeability was assessed using fluorescein-labeled dextrans (FD) in cholangiocyte monolayers (NRC-1). RESULTS: Although not effective when given alone, each combination of IL-6, interferon gamma, IL-1, and TNF-alpha inhibited secretion in IBDU. Cytokines inhibited cAMP formation, AE-2 activity, and cyclic AMP-dependent Cl(-) efflux, but not that induced by purinergic agonists. AE-2 gene expression was unaffected by proinflammatory cytokines, whereas CFTR and SR expression was increased. In addition, paracellular transit of FD across NRC-1 monolayers was increased. CONCLUSIONS: Inflammatory cytokines inhibit cAMP-dependent fluid secretion in cholangiocytes and impair the barrier functions of biliary epithelia. These changes may represent the molecular mechanisms by which inflammation leads to ductular cholestasis in vivo.

Pathophysiology of the intrahepatic biliary epithelium.

The intrahepatic bile duct epithelium modulates the fluidity and alkalinity of the primary hepatocellular bile from which it reabsorbs fluids, amino acids, glucose and bile acids, while secreting water, electrolytes and immunoglobulin A. The transport function of the intrahepatic biliary epithelium is finely regulated by a number of gastrointestinal hormones, neuropeptides and neurotransmitters that promote either secretion or absorption. The intrahepatic biliary epithelium appears to be a primary target in a broad group of chronic cholestatic disorders that represent an important cause of morbidity and mortality. The spectrum of cholangiopathies ranges from conditions in which a normal epithelium is damaged by disordered autoimmunity, infectious agents, toxic compounds or ischaemia, to genetically determined disorders arising from an abnormal bile duct biology, such as cystic fibrosis or biliary atresia. Probably as a result of the known heterogeneity in cholangiocyte function, different portions of the biliary tree appear to be preferentially affected in specific cholangiopathies. From a pathophysiological point of view, cholangiopathies are characterized by the coexistence of cholangiocyte loss (by apoptotic or lytic cell death) with cholangiocyte proliferation and various degrees of portal inflammation, fibrosis and cholestasis. These basic disease mechanisms are discussed in detail. Better understanding of cholangiocyte pathophysiology, in particular the immune regulation of cholangiocyte function, will help in designing newer genetic or pharmacological approaches to treat cholangiopathies.

Functional polarity of Na+/H+ and Cl-/HCO3- exchangers in a rat cholangiocyte cell line.

Intrahepatic bile duct cells (cholangiocytes) play an important role in the secretion and alkalinization of bile. Both Na+/H+ exchange (NHE) and Cl-/HCO-3 exchange (AE) contribute to these functions, but their functional distribution between the apical and basolateral membrane domains remains speculative. We have addressed this issue in a normal rat cholangiocyte cell line (NRC-1), which maintains a polarized distribution of membrane markers. Gene expression of AE and NHE isoforms was studied by RT-PCR. For functional studies, cells were placed in a chamber that allowed separate perfusion of the apical and basolateral aspect of the epithelial sheet; intracellular pH (pHi) was measured by 2', 7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein microfluorometry. In HCO-3-CO2free medium and in the presence of apical amiloride, pHi recovery from an acid load was Na+ dependent and was inhibited by basolateral amiloride and by HOE-642 (10 microM), consistent with basolateral localization of the NHE1 isoform, which had clearly expressed mRNA. Apical Na+ readmission induced a slow pHi recovery that was inhibited by apical administration of 1 mM HOE-642 or amiloride. Among the apical NHE isoforms, NHE2 but not NHE3 gene expression was detected. The AE1 gene was not expressed, but two different variants of AE2 mRNAs (AE2a and AE2b) were detected; pHi experiments disclosed AE activities at both sides of the membrane, but only apical AE was activated by cAMP. In conclusion, these studies provide the first functional description of acid-base transporters in a polarized cholangiocyte cell line. NHE1, NHE2, AE2a, and AE2b isoforms are expressed and show different membrane polarity, functional properties, and sensitivity to inhibitors. These observations add a considerable level of complexity to current models of electrolyte transport in cholangiocytes.

Purinergic regulation of acid/base transport in human and rat biliary epithelial cell lines.

Biliary epithelial cells (cholangiocytes) are responsible for rapid regulation of bile volume and alkalinity. Secretin and other hormones raising intracellular cyclic adenosine monophosphate (cAMP) concentrations promote biliary HCO3 secretion by stimulating apical Cl- channels and Cl-/HCO3- exchange (AE2). Cholangiocyte ion transport may also be stimulated by locally acting mediators; for example, adenosine 5'-triphosphate (ATP), a secretagogue that can be released into the bile by hepatocytes and cholangiocytes, activates Cl- conductances and Na+/H+ exchange (NHE) in cholangiocyte cell lines. To further explore the role of extracellular ATP in the paracrine regulation of carrier mechanisms regulating cholangiocyte H+/HCO3- secretion, we investigated the effects of nucleotides on intracellular pH regulation (measured by microfluorimetry with 2'7'-bis(2-carboxyethyl)-5,6,carboxyfluorescein [BCECF]) in human (MZ-ChA-1) and rat (NRC-1) cholangiocyte cell lines. In MZ-ChA-1 cells, 10 mol/L ATP, uridine 5'-triphosphate (UTP), and ATPgammas significantly increased NHE activity. The pharmacological profile of agonists was consistent with that anticipated for receptors of the P2Y2 class. ATP did not increase AE2 activity, but, when given to cells pretreated with agents raising intracellular cAMP, had a synergistic stimulatory effect that was inhibited by amiloride. To assess the polarity of purinergic receptors, monolayers of NRC-1 cells were exposed to apical or basolateral nucleotides. Apical administration of purinergic agonists, but not adenosine, increased basolateral NHE activity (ATPgammaS > UTP > ATP). Basolateral administration of purinergic agonists induced a weaker activation of NHE, which was instead strongly stimulated by adenosine and by adenosine receptor agonists (NECA = R-PIA = S-PIA). In conclusion, this study demonstrates that, consistent with the proposed role for biliary ATP in paracrine and autocrine control of cholangiocyte ion secretion, extracellular ATP stimulates cholangiocyte basolateral NHE activity through P2Y2 receptors that are predominantly expressed at the apical cell membrane.

Na(+)-dependent and -independent Cl-/HCO-3 exchange mediate cellular HCO3- transport in cultured human intrahepatic bile duct cells.

Biliary epithelial cells (cholangiocytes) modulate bile fluidity and alkalinity absorbing and/or secreting fluid and electrolytes, particularly HCO3- and Cl-. Mechanisms responsible for transepithelial H+/HCO3- secretion in human cholangiocytes are largely unknown. Human cholangiocytes isolated by enzymatic digestion and immunomagnetic purification from normal liver tissue obtained from reduced grafts used for pediatric liver transplantation were cultured in the presence of human hepatocyte growth factor. Maintenance of cholangiocyte phenotypic features was assessed using markers such as cytokeratin 19, gamma-glutamyltranspeptidase, vimentin, factor VIII-related antigen, desmin, epithelial membrane antigen (EMA), and human epithelial antigen (HEA) 125. Intracellular pH (pHi) transients were measured microfluorimetrically 2'7'-Bis(2-carboxyethyl)-5,6, carboxyfluorescein-acetossimethylester (BCECF). In the absence of HCO3-, pHi recovery from an intracellular acid load (ammonia pre-pulse technique) was Na(+)-dependent and amiloride-inhibitable. No Na(+)-independent recovery was recorded even after stimulation with agents raising intracellular cyclic adenosine monophosphate (cAMP) concentrations. In the presence of HCO3-, recovery from an intracellular acid load required Na+, but was only partly inhibited by amiloride. In these conditions H+ extrusion was inhibited by 4,4-diisothiocyan atostilben-2,2-disulfonic acid (DIDS) and by intracellular Cl- depletion. Acute removal of extracellular Cl induced a pHi alkalinization that was inhibited by DIDS. pHi recovery from an intracellular alkaline load (isohydric CO2 changes) was Cl(-)-dependent and DIDS-inhibitable. Administration of agents raising intracellular cAMP concentrations increased both Na(+)-dependent and Na(+)-independent Cl-/HCO-3 exchange activity. Stimulation of Cl-/HCO3- exchange activity was not prevented by the Cl- channel inhibitor 5'-nitro-2(2)-phenylpropyl-amino-benzoate(NPPB). In conclusion, human cholangiocytes possess two acid extruders (Na+/H+exchanger and Na(+)-dependent Cl-/HCO3- exchange) and an acid loader (Cl-/HCO3- exchange), whereas no evidence was found for cAMP activated H(+)-ATPase. Bicarbonate influx is thus mainly mediated by Na-dependent Cl-/HCO3- exchange, whereas Na+:HCO-3 cotransport is not active in the physiological range of pHi. Stimulation of Na(+)-independent Cl-/HCO3- exchanger by cAMP does not require activation of Cl- conductances. These mechanisms may underlay hormone-regulated biliary HCO3- secretion in the human biliary tree.

Intracellular pH regulation in Hep G2 cells: effects of epidermal growth factor, transforming growth factor-alpha, and insulinlike growth factor-II on Na+/H+ exchange activity.

Intracellular pH (pHi) plays an important role in the metabolic activation of quiescent cells after a proliferative stimulus, and Na+/H+ exchange activity is required for growth in some extrahepatic tumors. To investigate intracellular acid/base homeostasis in hepatoma cells and the effects of putative liver growth factors on Na+/h+ exchange activity, we have studied intracellular pH (pHi) regulation in Hep G2 cells, a well-differentiated hepatoma cell line, both in resting conditions and after administration of epidermal growth factor (EGF), transforming growth factor-alpha (TGF alpha), and insulinlike growth factor-II (IGF-II). The effects of fetal calf serum, TGF alpha, and amiloride on 3H-Thymidine incorporation were also studied. Amiloride (1 mmol/L) and external Na+ removal decreased baseline pHi in both HEPES and KRB. In HEPES, cells recovered from an acid load (20 mmol/L NH4Cl) by an amiloride inhibitable Na+/H+ exchange. In KRB, an additional, DIDS-inhibitable, Na(+)- and HCO3- dependent, but Cl(-)-independent acid extruder (Na:HCO3 cotransport) was activated. No evidence was found for a Cl/HCO3 exchange acting as acid loader. Administration of EGF and TGF alpha, but not of IGF-II, induced a dose-dependent, amiloride-inhibitable increase in baseline pHi, together with an increase in Na+/H+ exchange activity, shifting to the right the JH/pHi curve. Finally, 3H-thymidine incorporation in Hep G2 cells, in the presence of FCS or TGF alpha, was strongly inhibited by amiloride. In conclusion, in Hep G2 cells, pHi is mainly regulated by Na+/H+ exchange, which activity can be stimulated by EGF and TGF alpha, but not by IGF-II. Administration of TGF alpha stimulates DNA synthesis, an effect that is blocked by amiloride, an inhibitor of Na+/H+ exchanger. These data suggest that Na+/H+ exchange activation may play a critical role in the growth of some hepatic tumors.

Effect of ursodeoxycholic acid on intracellular pH in a bile duct epithelium-like cell line.

Recent studies in perfused livers and isolated hepatocytes indicate that ursodeoxycholic acid-induced HCO3-rich hypercholeresis originates at the ductule/duct level. The bile duct epithelium may be involved in bile alkalinization by passively reabsorbing the protonated unconjugated ursodeoxycholic acid, by directly secreting in response to an ursodeoxycholic acid-induced increase in acid/base transporter activity or by taking up UDCA- in exchange for a base equivalent. To investigate these processes in more detail, we studied the effects of ursodeoxycholic acid on intracellular pH in SK-ChA-1, a well-differentiated human cholangiocarcinoma cell line similar to bile duct epithelium in terms of intracellular pH regulatory mechanisms and morphological markers. Intracellular pH changes were monitored with a microfluorimetric setup using the fluorescent indicator 2'-7'-bis(2-carboxyethyl)-5,6,carboxy fluorescein. Administration of 50 to 1,000 mumol/L UDCA in the absence of HCO3 caused dose-dependent intracellular acidification (intracellular pH = -0.13 +/- 0.03 pH/U after 500 mumol/L ursodeoxycholic acid). Acidification was not prevented by preincubation of cells with 0.5 mmol/L 4,4-diisothiocyanatostilbene-2,2,-disulfonic acid (DIDS) for 30 min or by furosemide administration (1 mmol/L), thus ruling out the stimulation of Cl/HCO3 exchange or the presence of an ursodeoxycholic acid/base exchange. Ursodeoxycholic acid also acidified human fibroblasts, a cell type with no transport capability for ursodeoxycholic acid. In addition, direct measurement of the activities of the three major acid/base transporters in Sk-ChA-1 cells (Na+/H+ exchange, sodium-dependent and sodium-independent Cl/HCO3 exchange) failed to show significative differences between cells treated with 500 mumol/L UDCA and controls.(ABSTRACT TRUNCATED AT 250 WORDS)