ABSTRACT
Derived from bile duct epithelia (BDE), secretion by liver cyst-lining epithelia is positioned to drive cyst expansion but the responsible ion flux pathways have not been characterized. Cyst-lining epithelia were isolated and cultured into high resistance monolayers to assess the ion secretory pathways. Electrophysiologic studies showed a marked rate of constitutive transepithelial ion transport, including Cl(-) secretion and Na(+) absorption. Na(+) absorption was amiloride-sensitive, suggesting the activation of epithelial sodium channels (ENaC). Further, both cAMP(i) and extracellular ATP induced robust secretory responses. Western blotting and immunohistologic analysis of liver cyst epithelia demonstrated expression of P2X4, a potent purinergic receptor in normal BDE. Luminometry and bioassaying measured physiologically relevant levels of ATP in a subset of liver cyst fluid samples. Liver cyst epithelia also displayed a significant capacity to degrade extracellular ATP. In conclusion, regulated ion transport pathways are present in liver cyst epithelia and are positioned to direct fluid secretion into the lumen of liver cysts and promote increases in liver cyst expansion and growth.
Subject(s)
Bile Ducts/metabolism , Cysts/metabolism , Epithelial Cells/metabolism , Epithelial Sodium Channels/metabolism , Liver Neoplasms/metabolism , Adenosine Triphosphate/analysis , Adenosine Triphosphate/metabolism , Adenosine Triphosphate/pharmacology , Amiloride/pharmacology , Animals , Bile Ducts/pathology , Chlorine/metabolism , Cyclic AMP/pharmacology , Cysts/chemistry , Epithelial Sodium Channels/drug effects , Ion Transport , Liver Neoplasms/chemistry , Mice , Mice, Inbred Strains , Receptors, Purinergic P2/metabolism , Receptors, Purinergic P2X4 , Sodium/metabolismABSTRACT
The P2X family of ligand-gated cation channels is comprised of seven distinct isoforms activated by binding of extracellular purines. Although originally identified in neurons, there is increasing evidence for expression of P2X receptors in epithelia as well. Because ATP is released by both hepatocytes and cholangiocytes, these studies were performed to evaluate whether P2X receptors are present in cholangiocytes and contribute to local regulation of biliary secretion and bile formation. RT-PCR of cDNA from cultured normal rat cholangiocytes detected transcripts for P2X receptors 2, 3, 4, and 6; products from P2X3 and P2X4 were robust and always detectable. In cholangiocyte lysates, P2X4 protein was readily detected, and immunohistochemical staining of intact rat liver revealed P2X4 protein concentrated in intrahepatic bile ducts. To assess the functional significance of P2X4, isolated Mz-ChA-1 cells were exposed to the P2X4-preferring agonist 2',3'-O-(4-benzoyl-benzoyl)-ATP (BzATP), which activated inward currents of -18.2 + 3.0 pA/pF. In cholangiocyte monolayers, BzATP but not P2X3 agonists elicited robust Cl(-) secretory responses (short-circuit current) when applied to either the apical (DeltaI(sc) 22.1 +/- 3.3 microA) or basolateral (18.5 +/- 1.6 microA) chamber, with half-maximal stimulation at approximately 10 microM and approximately 1 microM, respectively. The response to BzATP was unaffected by suramin (not significant) and was inhibited by Cu(2+) (P < 0.01). These studies provide molecular and biochemical evidence for the presence of P2X receptors in cholangiocytes. Functional studies indicate that P2X4 is likely the primary isoform involved, representing a novel and functionally important component of the purinergic signaling complex modulating biliary secretion.
Subject(s)
Adenosine Triphosphate/analogs & derivatives , Bile Ducts, Intrahepatic/metabolism , Receptors, Purinergic P2/metabolism , Adenosine Triphosphate/pharmacology , Animals , Bile/metabolism , Bile Ducts, Intrahepatic/cytology , Bile Ducts, Intrahepatic/drug effects , Bile Ducts, Intrahepatic/pathology , Cell Line, Tumor , Cells, Cultured , Chlorides/metabolism , Electrophysiology , Humans , Immunohistochemistry/methods , Protein Isoforms/genetics , RNA, Messenger/metabolism , Rats , Receptors, Purinergic P2/genetics , Receptors, Purinergic P2X , Receptors, Purinergic P2X4 , Staining and LabelingABSTRACT
BACKGROUND & AIMS: Increases in intracellular Ca 2+ are thought to complement cAMP in stimulating Cl - secretion in cholangiocytes, although the site(s) of action and channels involved are unknown. We have identified a Ca 2+ -activated K + channel (SK2) in biliary epithelium that is inhibited by apamin. The purpose of the present studies was to define the role of SK channels in Ca 2+ -dependent cholangiocyte secretion. METHODS: Studies were performed in human Mz-Cha-1 cells and normal rat cholangiocytes (NRC). Currents were measured by whole-cell patch clamp technique and transepithelial secretion by Ussing chamber. RESULTS: Ca 2+ -dependent stimuli, including purinergic receptor stimulation, ionomycin, and increases in cell volume, each activated K + -selective currents with a linear IV relation and time-dependent inactivation. Currents were Ca 2+ dependent and were inhibited by apamin and by Ba 2+. In intact liver, immunoflourescence with an antibody to SK2 showed a prominent signal in cholangiocyte plasma membrane. To evaluate the functional significance, NRC monolayers were mounted in a Ussing chamber, and the short-circuit current ( I sc ) was measured. Exposure to ionomycin caused an increase in I sc 2-fold greater than that induced by cAMP. Both the basal and ionomycin-induced I sc were inhibited by basolateral Ba 2+, and approximately 58% of the basolateral K + current was apamin sensitive. CONCLUSIONS: These studies demonstrate that cholangiocytes exhibit robust Ca 2+ -stimulated secretion significantly greater in magnitude than that stimulated by cAMP. SK2 plays an important role in mediating the increase in transepithelial secretion due to increases in intracellular Ca 2+. SK2 channels, therefore, may represent a target for pharmacologic modulation of bile flow.
