Oxidative Stress Activates Membrane Ion Channels in Human Biliary Epithelial Cancer Cells (Mz-Cha-1).

Oxidative stress is known to contribute to cell damage. In several cell types, one of the earliest effects of oxidative stress is a rapid and substantial decrease in cell volume, which in turn regulates a broad range of cell functions, including development of apoptosis. Since volume regulation is closely coupled to membrane ion permeability, activation of ion channels may play an important role in oxidative stress-related cell injury. Oxidative stress plays a major role in a variety of liver diseases and bile duct epithelia cells (BDE) represent an important site of injury. We, therefore, investigated the functional interactions of oxidative stress, cell volume and ion permeability in a BDE model. Whole-cell patch clamp studies were performed in the human Mz-Cha 1 cell line. Oxidative stress was produced by addition of H2O2 to the bath solution. Changes of intracellular Ca(2+) concentration and of crosssectional area (for calculating cell volume) were monitored by laser scanning microscopy. Exposure of Mz-Cha 1 cells to H2O2 resulted in cell shrinkage and increase of the intracellular Ca(2+) concentration. Patch-clamp studies revealed that exposure to H2O2 also resulted in the activation of ion currents with a threshold of 10(-6) M H2O2. Ion substitution studies and blocker experiments identified the currents as representing an increase in membrane K(+) and Cl-permeability. Interestingly both ion channel activation and cell shrinkage had a close relationship to the applied H2O2 concentration and were significantly inhibited by intracellular Ca(2+) chelation. These data imply that in a BDE model, oxidative stress leads to cell shrinkage through activation of Ca(2+)-dependent K(+) and Cl(-) currents. Since cell shrinkage has been associated with increased cell damage, the opening of these ion channels might contribute to the high susceptibility of biliary epithelial cancer cells to oxidative stress.

Extracellular ATP induces cytoplasmic and nuclear Ca2+ transients via P2Y2 receptor in human biliary epithelial cancer cells (Mz-Cha-1).

Extracellular nucleotides such as adenosine triphosphate (ATP) play a role in biliary epithelial cell function. Since nucleotide receptors are potential targets for various diseases related to epithelial cell dysfunction and cancer, the purpose of this study was to investigate the expression and to functionally characterize the nucleotide receptor subtypes in biliary epithelial cancer cells (Mz-Cha-1). Extracellular ATP dose-dependently resulted in an intracellular Ca(2+) increase (mean effective concentration (EC(50)) 40 µM). Uridine triphosphate (UTP) produced a similar Ca(2+) response and cross-desensitation was observed. The rank order of tested agonists was ATP=UTP>> adenosine>ADP=AMP>α,ß-methylene-ATP. This confirms the functional expression of purinoceptor P2Y2 and P2Y4 in biliary epithelial cancer cell membranes. mRNAs for P2Y1, P2Y2, P2Y4 and P2Y6 purinergic receptor subtypes were found, whereas western blot analysis suggested only the expression of P2Y2 receptors. Confocal imaging and nuclear staining was used to compartmentalize ATP-induced cytosolic and nuclear Ca(2+)-transients, indicating a role for secretory ATP in regulating nuclear function, by increasing nuclear Ca(2+) concentrations. These data define the expression profile of P2Y receptors on human biliary epithelial cancer cells and indicate P2Y2 receptors as being potential targets in new treatment strategies for biliary cancer.

Oxidative stress reduces Na+/H+ exchange (NHE) activity in a biliary epithelial cancer cell line (Mz-Cha-1).

In cholangiocarcinogenesis, chronic inflammation and oxidative stress play a key role. The Na(+)/H(+) exchanger (NHE) forms a potential link between control of intra- and pericellular pH and tumor development. Therefore, the effects of oxidant stress were determined by the use of tert-butyl hydroperoxide (t-BOOH) on Na(+)/H(+) exchange in a biliary epithelial cancer cell line (Mz-Cha-1). The cells were exposed to the hydroperoxide and the rate of recovery from acidosis was determined by the use of the pH-sensitive fluorochrome 2',7'-bis(carboxyethyl)-5(6)-carboxyfluorescein acetoxymethyl ester (BCECF/AM). t-BOOH reduced Na(+)/H(+) exchange activity in a dose-dependent manner. At 4 mM t-BOOH, Na(+)/H(+) exchange activity was virtually absent. This was accompanied by an increase in cytotoxicity (MTT assay). Glutathione repletion and intracellular Ca(++) chelation partially restored the Na(+)/H(+) exchange activity. Hydroperoxide seemed neither to alter the intracellular signal transduction pathways (cAMP and Ca(++) oscillations) nor the membrane distribution of the exchanger (immunostaining). Decrease in Na(+)/H(+) exchange activity in this model of oxidant stress may represent an early perturbation of membrane function, and the functional integrity of Na(+)/H(+) exchange could therefore be dependent on the glutathione redox system.

Antibiotic prophylaxis after variceal hemorrhage reduces incidence of early rebleeding.

BACKGROUND/AIMS: This study aims to evaluate the role of new onset infection in the initiation of early rebleeding after variceal hemorrhage in patients with liver cirrhosis and the effect of prophylactic antibiotic treatment. METHODOLOGY: Two hundred and twenty-one consecutive admissions for variceal bleeding with no signs of infection at the time of admission were evaluated retrospectively. RESULTS: Systemic antibiotic prophylaxis was administered in 126 cases and significantly reduced the overall incidence of new onset infections (19.8% vs. 34.71%; p<0.01) and of early rebleeding (17.5% vs. 32.6%; p<0.01). Multivariate analysis showed strong correlation of rebleeding with new onset infection (p<0.001) and lack of prophylactic antibiotic treatment (p<0.05). Child-Pugh C cirrhosis, ventilatory assistance, and balloon tamponade were independent predictors of new onset infection (p<0.001, respectively). In the subgroup of patients with at least one predictor prophylactic treatment nearly halved the incidence of infections and rebleedings while in patients without predictors it had no significant effect. CONCLUSIONS: For the first time our data indicate a role for new onset infections in initiating early rebleedings. Immediate prophylactic antibiotic treatment for patients at high risk of infection might be effective in lowering both, the risk of acquiring infections and early rebleeding.

Guanylin regulates chloride secretion in the human gallbladder via the bile fluid.

BACKGROUND & AIMS: The biliary epithelium of bile ducts and gallbladder modifies the composition of primary hepatic bile by absorption and secretion of an electrolyte-rich fluid. The underlying transport mechanisms, however, are still incompletely understood. We investigated the expression, the cellular localization, and the functional role of guanylin, a bioactive intestinal peptide involved in the cystic fibrosis transmembrane conductance regulator (CFTR)-regulated electrolyte/water secretion, in the human gallbladder. METHODS: Peptide-specific antibodies were raised to localize guanylin and its affiliated signaling proteins, i.e., the guanylin receptor, guanylate cyclase C (GC-C), cGMP-dependent protein kinase type II (cGKII), and CFTR in the human gallbladder and cholangiocarcinoma cells (Mz-Cha-1) by RT-PCR, Western blot, and immunocytochemistry. A sensitive ELISA was used to assess the range of guanylin concentration in human bile fluid. The functional role of guanylin was investigated in subconfluent Mz-Cha-1 cell monolayers by isotope efflux experiments. RESULTS: Guanylin and its affiliated signaling proteins are highly expressed in the human gallbladder. Guanylin is localized to secretory epithelial cells of the gallbladder and is present in the bile, whereas GC-C, cGKII, and CFTR are confined exclusively to the apical membrane of the same epithelial cells. Functional studies in Mz-Cha-1 cells identify guanylin as a specific regulator of biliary Cl(-) secretion that very likely is mediated by an intracellular increase of cGMP-concentration. CONCLUSIONS: Based on the present findings and on the functional role of guanylin in other epithelia, it is likely that gallbladder epithelial cells synthesize and release guanylin into the bile to regulate electrolyte secretion by a paracrine/luminocrine signaling pathway.

Regulation of transferrin-induced endocytosis by wild-type and C282Y-mutant HFE in transfected HeLa cells.

The hereditary hemochromatosis protein HFE is known to complex with the transferrin receptor; however, its function regarding endocytosis of transferrin is unclear. We performed patch-clamp capacitance measurements in transfected HeLa cells carrying wild-type or C282Y-mutant HFE cDNA under the control of a tetracycline-sensitive promoter. Whole cell experiments in cells with suppressed expression of wild-type HFE revealed a decrease in membrane capacitance, reflecting predominance of endocytosis in the presence of transferrin. Cells overexpressing C282Y-mutant HFE displayed less intense capacitance decreases, whereas no significant decrease was observed in cells overexpressing wild-type HFE. The formation of single endocytic vesicles in cells with suppressed expression of wild-type HFE was greatly increased in the presence of transferrin as revealed by cell-attached recordings. According to their calculated diameters, many of these vesicles corresponded to clathrin-coated vesicles. These results suggest that wild-type HFE negatively modulates the endocytic uptake of transferrin. This inhibitory effect is attenuated in cells expressing C282Y-mutant HFE. Time-resolved measurements of cell membrane capacitance provide a powerful tool to study transferrin-induced endocytosis in single cells.

Molecular characterization of volume-sensitive SK(Ca) channels in human liver cell lines.

In human liver, Ca(2+)-dependent changes in membrane K(+) permeability play a central role in coordinating functional interactions between membrane transport, metabolism, and cell volume. On the basis of the observation that K(+) conductance is partially sensitive to the bee venom toxin apamin, we aimed to assess whether small-conductance Ca(2+)-sensitive K(+) (SK(Ca)) channels are expressed endogenously and contribute to volume-sensitive K(+) efflux and cell volume regulation. We isolated a full-length 2,140-bp cDNA (hSK2) highly homologous to rat brain rSK2 cDNA, including the putative apamin-sensitive pore domain, from a human liver cDNA library. Identical cDNAs were isolated from primary human hepatocytes, human HuH-7 hepatoma cells, and human Mz-ChA-1 cholangiocarcinoma cells. Transduction of Chinese hamster ovary cells with a recombinant adenovirus encoding the hSK2-green fluorescent protein fusion construct resulted in expression of functional apamin-sensitive K(+) channels. In Mz-ChA-1 cells, hypotonic (15% less sodium glutamate) exposure increased K(+) current density (1.9 +/- 0.2 to 37.5 +/- 7.1 pA/pF; P < 0.001). Apamin (10-100 nM) inhibited K(+) current activation and cell volume recovery from swelling. Apamin-sensitive SK(Ca) channels are functionally expressed in liver and biliary epithelia and likely contribute to volume-sensitive changes in membrane K(+) permeability. Accordingly, the hSK2 protein is a potential target for pharmacological modulation of liver transport and metabolism through effects on membrane K(+) permeability.