Cyclic AMP Regulates Bicarbonate Secretion in Cholangiocytes Through Release of ATP Into Bile
2007; Elsevier BV; Volume: 133; Issue: 5 Linguagem: Inglês
10.1053/j.gastro.2007.08.020
ISSN1528-0012
AutoresNoritaka Minagawa, Jun Nagata, Kazunori Shibao, Anatoliy I. Masyuk, Dawidson Assis Gomes, Michele Ângela Rodrigues, Gene LeSage, Yasutada Akiba, Jonathan D. Kaunitz, Barbara E. Ehrlich, Nicholas F. LaRusso, Michael H. Nathanson,
Tópico(s)Cystic Fibrosis Research Advances
ResumoBackground & Aims: Bicarbonate secretion is a primary function of cholangiocytes. Either adenosine 3′,5′-cyclic monophosphate (cAMP) or cytosolic Ca2+ can mediate bicarbonate secretion, but these are thought to act through separate pathways. We examined the role of the inositol 1,4,5-trisphosphate receptor (InsP3R) in mediating bicarbonate secretion because this is the only intracellular Ca2+ release channel in cholangiocytes. Methods: Intrahepatic bile duct units (IBDUs) were microdissected from rat liver then luminal pH was examined by confocal microscopy during IBDU microperfusion. Cyclic AMP was increased using forskolin or secretin, and Ca2+ was increased using acetylcholine (ACh) or adenosine triphosphate (ATP). Apyrase was used to hydrolyze extracellular ATP, and suramin was used to block apical P2Y ATP receptors. In selected experiments, IBDUs were pretreated with short interfering RNA (siRNA) to silence expression of specific InsP3R isoforms. Results: Both cAMP and Ca2+ agonists increased luminal pH. The effect of ACh on luminal pH was reduced by siRNA for basolateral (types I and II) but not apical (type III) InsP3R isoforms. The effect of forskolin on luminal pH was reduced by a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and by siRNA for the type III InsP3R. Luminal apyrase or suramin blocked the effects of forskolin but not ACh on luminal pH. Conclusions: Cyclic AMP-induced ductular bicarbonate secretion depends on an autocrine signaling pathway that involves CFTR, apical release of ATP, stimulation of apical nucleotide receptors, and then activation of apical, type III InsP3Rs. The primary role of CFTR in bile duct secretion may be to regulate secretion of ATP rather than to secrete chloride and/or bicarbonate. Background & Aims: Bicarbonate secretion is a primary function of cholangiocytes. Either adenosine 3′,5′-cyclic monophosphate (cAMP) or cytosolic Ca2+ can mediate bicarbonate secretion, but these are thought to act through separate pathways. We examined the role of the inositol 1,4,5-trisphosphate receptor (InsP3R) in mediating bicarbonate secretion because this is the only intracellular Ca2+ release channel in cholangiocytes. Methods: Intrahepatic bile duct units (IBDUs) were microdissected from rat liver then luminal pH was examined by confocal microscopy during IBDU microperfusion. Cyclic AMP was increased using forskolin or secretin, and Ca2+ was increased using acetylcholine (ACh) or adenosine triphosphate (ATP). Apyrase was used to hydrolyze extracellular ATP, and suramin was used to block apical P2Y ATP receptors. In selected experiments, IBDUs were pretreated with short interfering RNA (siRNA) to silence expression of specific InsP3R isoforms. Results: Both cAMP and Ca2+ agonists increased luminal pH. The effect of ACh on luminal pH was reduced by siRNA for basolateral (types I and II) but not apical (type III) InsP3R isoforms. The effect of forskolin on luminal pH was reduced by a cystic fibrosis transmembrane conductance regulator (CFTR) inhibitor and by siRNA for the type III InsP3R. Luminal apyrase or suramin blocked the effects of forskolin but not ACh on luminal pH. Conclusions: Cyclic AMP-induced ductular bicarbonate secretion depends on an autocrine signaling pathway that involves CFTR, apical release of ATP, stimulation of apical nucleotide receptors, and then activation of apical, type III InsP3Rs. The primary role of CFTR in bile duct secretion may be to regulate secretion of ATP rather than to secrete chloride and/or bicarbonate. See editorial on page 1726. See editorial on page 1726. The biliary tree plays an important role in conditioning the canalicular bile that is secreted by hepatocytes. Although diseases of the biliary tree can be due to a range of etiologies, their common end point is cholestasis. Chronic cholestatic conditions in turn are a frequent indication for liver transplantation. According to the organ procurement transplantation network (http://www.optn.org), nearly 1 in 7 liver transplantations performed in the United States in 2005 were on patients whose primary diagnosis was a cholestatic disorder such as primary biliary cirrhosis or primary sclerosing cholangitis. Cholangiocytes are the polarized, secretory epithelia that line the biliary tree and are the targets of disease in most chronic cholestatic disorders. Cholangiocytes are capable of increasing the rate of bile flow by as much as 50%,1Nathanson M.H. Burgstahler A.D. Mennone A. et al.Stimulation of bile duct epithelial secretion by glybenclamide in normal and cholestatic rat liver.J Clin Invest. 1998; 101: 2665-2676Crossref PubMed Scopus (23) Google Scholar but, under normal conditions, the primary role of cholangiocytes is to modify the composition rather than the volume of bile flow.2Hirata K. Nathanson M.H. Bile duct epithelia regulate biliary bicarbonate excretion in normal rat liver.Gastroenterology. 2001; 121: 396-406Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar In particular, ductular secretion is responsible for regulating the concentration of biliary bicarbonate.2Hirata K. Nathanson M.H. Bile duct epithelia regulate biliary bicarbonate excretion in normal rat liver.Gastroenterology. 2001; 121: 396-406Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar Biliary bicarbonate secretion occurs through 2 separate pathways. The first pathway is stimulated by hormones such as secretin and is mediated by adenosine 3′,5′-cyclic monophosphate (cAMP). The downstream effect of cAMP is to activate the cystic fibrosis transmembrane conductance regulator (CFTR), the apical chloride channel that is defective in cystic fibrosis. Chloride released into the ductular lumen then is thought to be exchanged for bicarbonate via a chloride-bicarbonate exchanger.3Boyer J.L. Nathanson M.H. Bile formation.in: Schiff E.R. Sorrell M.F. Maddrey W.C. Diseases of the liver. Lippincott-Raven, New York, New York1999: 119-146Google Scholar, 4Lazaridis K.N. Strazzabosco M. Larusso N.F. The cholangiopathies: disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 5Kanno N. LeSage G. Glaser S. et al.Regulation of cholangiocyte bicarbonate secretion.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G612-G625PubMed Google Scholar In some systems, bicarbonate may be released directly via CFTR, and limited evidence suggests this may occur in cholangiocytes as well.2Hirata K. Nathanson M.H. Bile duct epithelia regulate biliary bicarbonate excretion in normal rat liver.Gastroenterology. 2001; 121: 396-406Abstract Full Text Full Text PDF PubMed Scopus (62) Google Scholar The cAMP/CFTR pathway is thought to be of primary importance in maintaining ductular bile flow, and, indeed, cystic fibrosis can result in chronic cholestasis and secondary biliary cirrhosis.6Colombo C. Battezzati P.M. Crosignani A. et al.Liver disease in cystic fibrosis: a prospective study on incidence, risk factors, and outcome.Hepatology. 2002; 36: 1374-1382Crossref PubMed Scopus (301) Google Scholar The second secretory pathway in cholangiocytes is stimulated by neurotransmitters such as acetylcholine (ACh) and autocrine agents such as adenosine triphosphate (ATP) and is mediated by cytosolic Ca2+.7Nathanson M.H. Burgstahler A.D. Mennone A. et al.Characterization of cytosolic Ca2+ signaling in rat bile duct epithelia.Am J Physiol. 1996; 271: G86-G96PubMed Google Scholar The downstream effect of Ca2+ is to activate Ca2+-dependent chloride channels on the apical plasma membrane, which in turn release chloride that is exchanged for bicarbonate via a chloride-bicarbonate exchanger.3Boyer J.L. Nathanson M.H. Bile formation.in: Schiff E.R. Sorrell M.F. Maddrey W.C. Diseases of the liver. Lippincott-Raven, New York, New York1999: 119-146Google Scholar, 4Lazaridis K.N. Strazzabosco M. Larusso N.F. The cholangiopathies: disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 5Kanno N. LeSage G. Glaser S. et al.Regulation of cholangiocyte bicarbonate secretion.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G612-G625PubMed Google Scholar The relative importance of cAMP-dependent and Ca2+-dependent ductular secretion is not understood. However, loss of expression of the inositol 1,4,5-trisphosphate (InsP3) receptor (InsP3R) appears to be a general feature of cholestatic conditions.8Shibao K. Hirata K. Robert M.E. et al.Loss of inositol 1,4,5-trisphosphate receptors from bile duct epithelia is a common event in cholestasis.Gastroenterology. 2003; 125: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar Because the InsP3R is the only intracellular Ca2+ release channel in cholangiocytes,9Hirata K. Dufour J.F. Shibao K. et al.Regulation of Ca(2+) signaling in rat bile duct epithelia by inositol 1,4,5-trisphosphate receptor isoforms.Hepatology. 2002; 36: 284-296Crossref PubMed Scopus (70) Google Scholar this suggests that secretion mediated by cAMP and CFTR must depend in part on InsP3R expression. The purpose of this study was to determine whether and how each of the 3 isoforms of the InsP3R affects cAMP- and CFTR-mediated bicarbonate secretion in cholangiocytes. Male Sprague-Dawley rats (200–250 g, Charles River Laboratories, Boston, MA) were used for all studies. Animals were maintained on a standard diet and housed under a 12-hour light-dark cycle. ACh, ATP, apyrase, suramin, and the adenylyl cyclase activator forskolin were purchased from Sigma Chemical Co (St. Louis, MO). The membrane-impermeant, pH-sensitive dye 2′,7′-bis(2-carboxyethyl-5-(and-6)-carboxyfluorescein (BCECF) dextran (70,000 mol wt), fluo-4/acetoxymethyl ester (AM), 2-bis(2-aminophenoxy)-ethane-N,N,N′N′-tetraacetic acid (BAPTA)/AM, the lipophilic membrane dye DiD, and the nuclear stain TO-PRO-3 were obtained from Invitrogen (Eugene, OR). The selective, small-molecule CFTR inhibitor CFTRinh-172 was chemically synthesized and purified by high-performance liquid chromatography (HPLC) as described previously10Akiba Y. Jung M. Ouk S. et al.A novel small molecule CFTR inhibitor attenuates.Am J Physiol Gastrointest Liver Physiol. 2005; 289: G753-G759PubMed Google Scholar, 11Ma T. Thiagarajah J.R. Yang H. et al.Thiazolidinone CFTR inhibitor identified by high-throughput screening blocks cholera toxin-induced intestinal fluid secretion.J Clin Invest. 2002; 110: 1651-1658Crossref PubMed Scopus (591) Google Scholar and was microperfused into the lumen of bile ducts 30 minutes before the start of experiments. Type I InsP3R antibodies from affinity-purified specific rabbit polyclonal antiserum directed against the 19 C-terminal residues of the mouse type I InsP3R were commercially obtained from Affinity Bioreagents (Golden, CO). Type II InsP3R antibodies from affinity-purified specific rabbit polyclonal antiserum directed against the 18 C-terminal residues of the rat type II insP3R were kindly provided by Richard Wojcikiewicz (State University of New York, Syracuse). Monoclonal antibodies directed against the N-terminus of the type III InsP3R were commercially obtained (Transduction Laboratories, Lexington, KY). All other chemicals were of the highest quality commercially available. Intrahepatic bile duct units (IBDUs) were microdissected from normal rat liver as described previously.12Masyuk A.I. Gong A.Y. Kip S. et al.Perfused rat intrahepatic bile ducts secrete and absorb water, solute, and ions.Gastroenterology. 2000; 119: 1672-1680Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Briefly, rats were anesthetized with pentobarbital sodium (50 mg/kg intraperitoneally [IP]) then the portal vein was exposed and cannulated, and the liver was perfused with ice-cold saline. Subsequently, 2–3 mL liquid trypan blue agar was injected into the portal vein to facilitate identification of portal tracts. The liver was surgically removed and immersed in ice-cold, preoxygenated HEPES-buffered saline (HBS, pH 7.4). After mechanical removal of the hepatic capsule and surface hepatocytes, intrahepatic bile ducts were exposed and microdissected using a Zeiss Stemi SV11 dissection microscope (Zeiss, Inc, Thornwood, NY). The IBDUs were cut into 1.0- to 1.5-mm segments and transferred to a culture chamber. Viability was assessed by trypan blue exclusion; only IBDUs without evidence of trypan blue uptake were used. A modified approach to this was taken to isolate bile duct segments for Ca2+ measurements.8Shibao K. Hirata K. Robert M.E. et al.Loss of inositol 1,4,5-trisphosphate receptors from bile duct epithelia is a common event in cholestasis.Gastroenterology. 2003; 125: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Hirata K. Dufour J.F. Shibao K. et al.Regulation of Ca(2+) signaling in rat bile duct epithelia by inositol 1,4,5-trisphosphate receptor isoforms.Hepatology. 2002; 36: 284-296Crossref PubMed Scopus (70) Google Scholar Briefly, rat livers were isolated and then perfused with buffer containing collagenase (Boehringer Manheim Biochemicals, Indianapolis, IN). The portal tissue residue was separated mechanically and then cut into strips. DiD and fluo-4/AM were first coinjected into the common bile duct to facilitate identification of bile duct epithelia within portal strips. Potential target sites within the rat InsP3R genes were selected and then searched with BlastN (NCBI, Bethesda, MD) to confirm specificity for each InsP3R isoform as described previously.13Mendes C.C. Gomes D.A. Thompson M. et al.The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria.J Biol Chem. 2005; 280: 40892-40900Crossref PubMed Scopus (227) Google Scholar The short interfering RNA (siRNA) for the types I, II, and III InsP3R were prepared by a transcription-based method using the Silencer kit according to the manufacturer’s instruction (Ambion, Austin, TX). The sense and antisense oligonucleotides of siRNA were, respectively, as follows: type I, 5′-AAAGTTGTAGCTGCTGGTGCTCCTGTCCTC-3′ and 5′-AAAGCACCAGCAG CTACAACTCCTGTCCTC-3′; type II, 5′-AACAGCCTAATCAAGATCTCCCCTG TCTC-3′ and 5′-AAGGAGA TCTTGATTAGGCTGCCTGTCTC-3′; type III, 5′-ATGGTGCTGGCAAACTTGTTTCCTGTCCTC-3′ and 5′-AAACAAGTTTGCCA GCACCATCCTGTCCTC-3′; scrambled (siRNA negative control), 5′-AACACCTATAACAACGGTAGTC CTGTCTC-3′ and 5′-AAACTACCGTTGTTATAGGTGCCTGTCTC-3′. siRNA constructs were tested for efficacy in CHO cells as described previously13Mendes C.C. Gomes D.A. Thompson M. et al.The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria.J Biol Chem. 2005; 280: 40892-40900Crossref PubMed Scopus (227) Google Scholar and then used in IBDUs.14Splinter P.L. Masyuk A.I. LaRusso N.F. Specific inhibition of AQP1 water channels in isolated rat intrahepatic bile duct units by small interfering RNAs.J Biol Chem. 2003; 278: 6268-6274Crossref PubMed Scopus (58) Google Scholar IBDUs were maintained in culture with siRNA or the corresponding scrambled siRNA for 24 hours at 37°C prior to use in microperfusion studies. siRNA were delivered to both CHO cells and cholangiocytes using the TransMessenger Transfection Reagent lipid carrier (Qiagen, Valencia, CA). The luminal pH of perfused IBDUs was measured using the cell-impermeant pH-sensitive dye BCECF dextran as an index of ductular bicarbonate secretion.12Masyuk A.I. Gong A.Y. Kip S. et al.Perfused rat intrahepatic bile ducts secrete and absorb water, solute, and ions.Gastroenterology. 2000; 119: 1672-1680Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar, 15Dranoff J.A. Masyuk A.I. Kruglov E.A. et al.Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G1059-G1067PubMed Google Scholar, 16Nathanson M.H. Burgstahler A.D. Masyuk A. et al.Stimulation of ATP secretion in the liver by therapeutic bile acids.Biochem J. 2001; 358: 1-5Crossref PubMed Scopus (69) Google Scholar Luminal pH is an indirect measure of bicarbonate secretion, and other transport processes in cholangiocytes can increase luminal pH as well, but available evidence suggests that bicarbonate secretion is the primary process to increase luminal pH in cholangiocytes stimulated with the agonists used in this study.3Boyer J.L. Nathanson M.H. Bile formation.in: Schiff E.R. Sorrell M.F. Maddrey W.C. Diseases of the liver. Lippincott-Raven, New York, New York1999: 119-146Google Scholar, 4Lazaridis K.N. Strazzabosco M. Larusso N.F. The cholangiopathies: disorders of biliary epithelia.Gastroenterology. 2004; 127: 1565-1577Abstract Full Text Full Text PDF PubMed Scopus (310) Google Scholar, 5Kanno N. LeSage G. Glaser S. et al.Regulation of cholangiocyte bicarbonate secretion.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G612-G625PubMed Google Scholar Individual isolated IBDUs were placed in a specially designed, temperature-controlled chamber mounted on the stage of a Zeiss LSM 510 Laser Scanning Confocal Microscope equipped with a krypton/argon mixed gas laser (Zeiss, Inc.). Concentric glass pipettes made from soft fine glass tubes (Drummond Scientific Co., Broomall, PA) were attached to a microperfusion apparatus built to specification and used to position and perfuse the IBDUs. One end of an individual IBDU was drawn into the tip of a glass holding pipette by gentle suction. The lumen was then cannulated with a concentric perfusion pipette that contained the perfusion solution. Solutions were delivered near the tip of the perfusion pipette at a rate of 1 μL/min via a fluid-exchange pipette system connected with a variable-speed syringe pump (model 55-2222 Harvard microliter syringe pump; Harvard Apparatus, Inc., Holliston, MA). The opposite end of an IBDU was stabilized using a holding pipette. The external surface of the microperfused IBDU was simultaneously bathed in a buffer that was continuously oxygenated and exchanged. The temperature of the fluid was maintained at 37°C. Luminal BCECF dye was excited at a wavelength of 488 nm, and fluorescence emission was monitored at 505–550 nm. Confocal images were obtained at a rate of 5–20 images/min. In situ calibration curves were generated at the end of each experiment by perfusion with 10 μmol/L nigericin plus monensin in serial solutions of pH 6.8, 7.4, and 8.0, as described.12Masyuk A.I. Gong A.Y. Kip S. et al.Perfused rat intrahepatic bile ducts secrete and absorb water, solute, and ions.Gastroenterology. 2000; 119: 1672-1680Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar Nonratio images of BCECF-dextran were collected to determine luminal pH, and potential errors from such measurements include photobleaching and changes in the volume of distribution of the dye. Luminal fluorescence decreased by only 0.2% after 5 minutes of continuous imaging (P = .3; n = 6 IBDU) so that photobleaching was negligible in this experimental system. Similarly, lumen width did not increase after 25 minutes of stimulation with either forskolin or ACh (86 ± 11 μm both before and after stimulation; P > .95; n = 6). Therefore, quantitative, calibrated measurements of luminal pH were made using this approach. Western blots were performed as previously described13Mendes C.C. Gomes D.A. Thompson M. et al.The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria.J Biol Chem. 2005; 280: 40892-40900Crossref PubMed Scopus (227) Google Scholar using protein from CHO cells to test the efficacy of isoform-specific siRNA for InsP3Rs. Protein was extracted by lysing CHO cells with M-PER mammalian protein extraction reagent (Pierce, Rockford, IL). The samples were subjected to sodium-dodecyl-sulphate polyacrylamide gel electrophoresis (SDS-PAGE) in 10% Tris-HCl gels. Gels were transferred to a nitrocellulose membrane (Bio-Rad, Hercules, CA), and then membranes were blocked with 5% nonfat dry milk in Tris-buffered saline (TBS-T; 25 mmol/L Tris-HCl, pH 7.5, 150 mmol/L NaCl) plus 0.1% Tween 20 for 1 hour. Blots were incubated with InsP3R isoform-specific antibody (antitype I InsP3R [1:1000], antitype II InsP3R [1:100], or antitype III InsP3R [1:1000]) in 5% nonfat dry milk in TBS-T at 4°C overnight, followed by incubation for 1 hour with peroxidase-conjugated immunoglobulin G secondary antibody (Bio-Rad; anti-rabbit [1:4000] or anti-mouse [1:5000]) in TBS-T. Blots were visualized by enhanced chemiluminescence using the ECL plus kit (Amersham Biosciences, Arlington Heights, IL). A Bio-Rad GS-700 imaging densitometer was used for quantitative analysis of the blots. Confocal immunofluorescence was performed as described previously.8Shibao K. Hirata K. Robert M.E. et al.Loss of inositol 1,4,5-trisphosphate receptors from bile duct epithelia is a common event in cholestasis.Gastroenterology. 2003; 125: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar IBDUs were incubated in normal rat cholangiocyte medium with or without InsP3R-siRNA (5 μmol/L) for 24 hours at 37°C. IBDUs were then fixed with cold acetone for 10 minutes and then permeabilized in 0.1% Triton X-100 at room temperature. After blocking steps, IBDUs were labeled with primary antibodies (antitype I InsP3R rabbit polyclonal [1:100], antitype II InsP3R rabbit polyclonal [1:5], or antitype III mouse monoclonal [1:100]). Following the primary antibody incubation, IBDUs were washed with phosphate-buffered saline (PBS) and incubated with secondary antibody. Secondary antibodies were Alexa 488 anti-mouse (1:500) or Alexa 555 anti-rabbit (1:500). For negative control studies, IBDUs were incubated with secondary antibodies, but anti-InsP3R (primary) antibodies were omitted. IBDUs also were labeled with the nuclear stain TO-PRO-3 (Invitrogen; 1:200). Some specimens were triple labeled to detect types I and III InsP3R and the nucleus or types II and III InsP3R and the nucleus. Triple-labeled specimens were serially excited at 488 nm and observed at 505–550 nm to detect Alexa 488, excited at 543 nm and observed at >585 nm to detect Alexa 555, and excited at 633 nm and observed at >650 nm to detect TO-PRO-3. Double labeling was performed to detect type III InsP3R and the nucleus. Double-labeled specimens were excited at 488 nm and observed at 505–550 nm to detect Alexa 488 then excited at 633 nm and observed at >650 nm to detect TO-PRO-3. All images were collected using a Zeiss LSM 510 Meta Laser Scanning Confocal Microscope (Zeiss, Inc.). DiD (25 μmol/L) and fluo-4/AM (18 μmol/L) were coinjected into the common bile duct, and then bile duct segments were isolated as described above, transferred to a perfusion chamber, and observed using a Zeiss LSM 510 confocal imaging system. Tissue was excited at 647 nm and observed at greater than 680 nm to identify individual DiD-labeled bile duct cells, then cytosolic Ca2+ was monitored in these cells by exciting the specimen at 488 nm and detecting fluo-4 emission signals above 515 nm. Increases in Ca2+ were expressed as percentage increases in fluorescence intensity of fluo-4.8Shibao K. Hirata K. Robert M.E. et al.Loss of inositol 1,4,5-trisphosphate receptors from bile duct epithelia is a common event in cholestasis.Gastroenterology. 2003; 125: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Hirata K. Dufour J.F. Shibao K. et al.Regulation of Ca(2+) signaling in rat bile duct epithelia by inositol 1,4,5-trisphosphate receptor isoforms.Hepatology. 2002; 36: 284-296Crossref PubMed Scopus (70) Google Scholar In separate studies, cytosolic Ca2+ was measured in confluent monolayers of polarized rat cholangiocytes in culture.17Alpini G. Phinizy J.L. Glaser S. et al.Development and characterization of secretin-stimulated secretion of cultured rat cholangiocytes.Am J Physiol Gastrointest Liver Physiol. 2003; 284: G1066-G1073Crossref PubMed Scopus (28) Google Scholar The cells were plated on a 96-well microtiter plate at 5 to 10 × 103 cells/well and grown overnight. Cells were loaded with the Ca2+-sensitive ratio dye fura-2/AM, and fluorescence was monitored using a Pathway Imaging System (BD Biosciences, Rockville, MD). The cells were alternately exposed to excitation wavelengths of 340 and 380 nm, and the fluorescence emission was collected at 510 nm. Measurements were obtained from 30–80 individual regions of interest (including both single cells and groups of cells) spanning 6–8 individual wells. Results are expressed as mean values ± SEM, except where otherwise noted. SigmaPlot version 9.01 (Systat Software, San Jose, CA) and Prism version 3.02 (GraphPad Software, San Diego, CA) were used for data analysis. Statistical significance was tested using Student t test or 1-way ANOVA, and a P value <.05 was taken to indicate a significant difference. Intrahepatic segments of bile ducts were microdissected from rat liver as described previously12Masyuk A.I. Gong A.Y. Kip S. et al.Perfused rat intrahepatic bile ducts secrete and absorb water, solute, and ions.Gastroenterology. 2000; 119: 1672-1680Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar then perfused with the cell-impermeant pH dye BCECF-dextran while examined by confocal microscopy to monitor bicarbonate secretion into the lumen (Figure 1A). In selected experiments, IBDU were maintained in culture overnight prior to microperfusion, which enabled IBDU to be treated with siRNA constructs prior to study. Responses to the cAMP agonist forskolin (100 μmol/L) were similar among freshly isolated IBDU and IBDU that had been in culture for 24 hours (Figure 1B). Cyclic AMP-mediated secretion was induced by stimulation with forskolin (100 μmol/L), which activates adenylyl cyclase directly. Forskolin induced a sustained increase in luminal pH (n = 3; Figure 1B), similar to what has been reported previously.12Masyuk A.I. Gong A.Y. Kip S. et al.Perfused rat intrahepatic bile ducts secrete and absorb water, solute, and ions.Gastroenterology. 2000; 119: 1672-1680Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar This response was similar to the pattern observed during stimulation with secretin (100 nmol/L; data not shown), which activates Gs and then adenylyl cyclase via the secretin receptor.3Boyer J.L. Nathanson M.H. Bile formation.in: Schiff E.R. Sorrell M.F. Maddrey W.C. Diseases of the liver. Lippincott-Raven, New York, New York1999: 119-146Google Scholar Treatment of IBDU with the CFTR inhibitor CFTRinh-172 blocked forskolin-induced increases in luminal pH (P < .05, n = 3; Figure 1B). Ca2+-mediated secretion was induced either by stimulation with ACh (100 μmol/L; n = 3), which activates M3 muscarinic receptors, or with ATP (100 μmol/L; n = 3), which activates P2Y nucleotide receptors. Cholangiocytes express both of these receptors, each of which links to Gq, phospholipase C activation, and then InsP3 formation to increase cytosolic Ca2+.15Dranoff J.A. Masyuk A.I. Kruglov E.A. et al.Polarized expression and function of P2Y ATP receptors in rat bile duct epithelia.Am J Physiol Gastrointest Liver Physiol. 2001; 281: G1059-G1067PubMed Google Scholar, 18Alvaro D. Alpini G. Jezequel A.M. et al.Role and mechanisms of action of acetylcholine in the regulation of rat cholangiocyte secretory functions.J Clin Invest. 1997; 100: 1349-1362Crossref PubMed Scopus (121) Google Scholar Stimulation with either Ca2+ agonist induced a sustained increase in luminal pH (Figure 2A and B). Treatment of IBDU with the CFTR inhibitor CFTRinh-172 did not block ACh-induced increases in luminal pH (n = 3; Figure 2A). These findings show that confocal imaging of luminal pH in microperfused IBDU can be used to monitor agonist-induced ductular bicarbonate secretion and demonstrate that cAMP-mediated secretion requires CFTR. Ca2+ signaling in cholangiocytes is mediated entirely by the InsP3R because this is the only type of intracellular Ca2+ release channel expressed in these cells.9Hirata K. Dufour J.F. Shibao K. et al.Regulation of Ca(2+) signaling in rat bile duct epithelia by inositol 1,4,5-trisphosphate receptor isoforms.Hepatology. 2002; 36: 284-296Crossref PubMed Scopus (70) Google Scholar Cholangiocytes express all 3 isoforms of the InsP3R; the types I and II InsP3R are found throughout the cell, whereas the type III isoform is concentrated in the apical region.9Hirata K. Dufour J.F. Shibao K. et al.Regulation of Ca(2+) signaling in rat bile duct epithelia by inositol 1,4,5-trisphosphate receptor isoforms.Hepatology. 2002; 36: 284-296Crossref PubMed Scopus (70) Google Scholar To understand the relative role of each isoform in the regulation of bicarbonate secretion, we used isoform-specific siRNA13Mendes C.C. Gomes D.A. Thompson M. et al.The type III inositol 1,4,5-trisphosphate receptor preferentially transmits apoptotic Ca2+ signals into mitochondria.J Biol Chem. 2005; 280: 40892-40900Crossref PubMed Scopus (227) Google Scholar to eliminate them from IBDU. The combination of siRNA for types I and II InsP3R was effective in simultaneously reducing expression of both isoforms in CHO cells (Figure 3A), whereas siRNA for type III InsP3R selectively reduced expression of that isoform (Figure 3B). Confocal immunofluorescence of IBDU in overnight culture demonstrated that the types I and II InsP3R are in the basolateral region of cholangiocytes (Figure 4A and B), whereas the type III InsP3R is concentrated in the apical region (Figure 4C), similar to what has been observed in cholangiocytes in the intact liver.8Shibao K. Hirata K. Robert M.E. et al.Loss of inositol 1,4,5-trisphosphate receptors from bile duct epithelia is a common event in cholestasis.Gastroenterology. 2003; 125: 1175-1187Abstract Full Text Full Text PDF PubMed Scopus (103) Google Scholar, 9Hirata K. Dufour J.
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