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Early prediction of response to 40 kDa peginterferon alfa-2A (PEGASYS®) plus ribavirin (RBV) in patients with chronic hepatitis C (CHC)
HEPATOLOGY Vol. 34, NO. 4, Pt. 2, 2001
AASLD ABSTRACTS
351A
715
716
IMMUNOMODULATORY ACTIVITIES OF VIRAMIDINE, A LIVER-TARG E T I N G RIBAVIRIN PRODRUG, I N V I T R O AND I N VIVO. Robert Tam, Charmaine Lira, Josie Bard, Parmala Bagha, Johnson Y Lau, Zhi Hong, ICN Pharmaceuticals, Costa Mesa, CA
EARLY PREDICTION OF RESPONSE TO 40 KDA PEGINTERFERON
Background: Ribavirin is a purine nucleoside analog approved for the treatment of hepatitis C in combination with interferon-a. It is believed that ribavirin achieves its clinical benefits by inducing a Type i cytokine bias that favors antiviral cellular immunity. To improve the toxicity profile of ribavirin and alleviate the adverse effects of ribavirin therapies such as hemolytic anemia, several nucleoside analogs were synthesized and their immunomodulatory activities were evaluated. Aim: To determine whether viramidine, a liver-targeting prodrug analog of ribavirin, has similar i m m u n o m o d u l a t o r y activity to ribavirin in vitro and in vivo. Methods: The in vitro effect of viramidine on Type 1 cytokine production (IL-2, IFN% TNFc~) in activated h u m a n T cells was determined by ELISA. The in vfvo effect in a murine model was assessed by Type 1 cytokine-mediated contact hypersensitivity (CHS) responses to dinitrofluorobenzene (DNFB). Results: Similar to ribavirin, viramidine enhanced SEB-stimulated Type 1 cytokine expression (IL-2, IFN 7 and T N F a ) by h u m a n T ceils in a dose-dependent m a n n e r (0,67-I0/zM). The viramidine concentration that gave the peak cytokine responses showed donor-to-donor variability (similar to ribavirin) with the highest frequency at 5 to 10/zM in 15 donors. The magnitude of the induction of Type 1 cytokines by viramidine was similar to that observed for ribavirin in T cells from the same donors. Viramidine (admimstered ip at 0.28, 0.56 and 1.12 mg/kg) enhanced Type 1 cytokinemediated CHS responses to DNFB (as measured by the inflammatory ear response to contact allergen) in BALB/c mice in a dose dependent manner. The magnitude and peak dose (0.5-1 mg/kg) of the effect of viramidine on CHS responses was similar to ribavirin. Using a MTT assay for T cell proliferation, high doses ( > 20/.LM) of viramidine showed similar anti-proliferative effects to ribavirin at doses which showed no effect on h u m a n T cell viability (assessed by propidium iodide exclusion). Conclusion: Viramidine showed similar immunomodulatory properties to ribavirin w h e n administered to in vitro stimulated h u m a n T cells and in vivo mouse model for a Type 1 cytokine-mediated i m m u n e response.
ALFA-2A (PEGASYS ®) PLUS RIBAVIRIN (RBV) IN PATIENTS WITH CHRONIC HEPATITIS C (CHC). Peter Ferenci, University of Vienna, Wien Austria; Mitchell L Shiffman, Medical College of Virginia, Richmond, VA; Michael W Fried, University of North Carolina, Chapel Hill, NC; Mark S Sulkowski, Johns Hopkins Center for Viral Hepatitis, Baltimore, MD; Dieter Haeussinger, University of Duesseldorf, Duesseldorf Germany; Jean-Pierre Zarski, CHU de Grenoble, Grenoble France; Fernando GoncalesJr., University of Campinas, Campinas Spain; Donald M Jensen, Rush-Presbyterian St Luke's Hospital, Chicago, IL; Ferrucio Bonino, Azienda Ospedaliera Univ Pisana, Pisa Italy; Daniel Dhumeaux, H6pital Henri Mondor, Creteil France; Steven Blother, Joseph Hoffman, Joseph Oliveto, Hoffmann-La Roche, Nutley, NJ Background: The administrationof 40 kDa peginterferonalfa-2a (PEGASYS®) is associatedwith early viral decline in the majorityo[ CYICpatients.We have demonstratedthat failureto achieve an early virologicalresponse (EVR), defined as ->2 logto decline in HCV RNA from baselineby week 12 of treatment,is highlypredictivefor a lack of sustained virologicalresponse (SVR)among patients treated with 40 kDa peginterferon alfa-2a alone (Lee SS et al. Hepatology. 2000; 32 [supplJ:370A), or treatedwith the combinationof 40 kDa peginterferonalfa-2a + ribavirin(Fried MW et al. DDW. 2001). Objective:The aim of the present studywas to determinethe effectof HCV genotypeand adherenceto study regimenon the positiveand negativepredictivevalues(PPVand NPV) for SVRusing EVRin patients treatedwith combinationof 40 kDa peginterferon alfa-2a 180 /zg qw + RBV1000-1200rag qd. Methods:A retrospectiveanalysiswas performedon data froma large randomized trial comparing the combination of 40 kDa peginterferon alfa-2a + RBV (n=453) vs IFN a-2b + RBVvs 40 kDa peginterferonalfa-2a + placeboin CHC.EVRwas defined as HCV RNAbelow levelof detection (<600 IU/mL,COBASAMPLICORHCVMONITOR~"Test, v 2.0) or -->2Iogtodecline in HCV RNA from baselineby week 12. PPV and NPV for SVRwere calculated for all patients in the 40 kDa peginterferonalfa-2a + RBVarm (n=453) and according to genotype.PatientsachievingEVRwere further evaluatedfor the effectof adherence (A->80=at least 80% of assignedtreatmentreceived)on SVR.Results: Of all patients treated,86% had an EVR and 56%achievedSVR.The EVR-PPVwas 65%and the EVR-NPVw-as97%.For patients with EVR who were adherent to the study regimen(A>80%),SVRwas 75% comparedto only 48% in EVR patients who were not adherent (A<80%). EVRwas achievedin 81% of genotype 1 patients and 97% of genotype2 or 3 patients. The EVR-PPVfor SVRwas 67% and 88% for adherent patients with genotype 1 and genotypes2 or 3, respectively(Table). Conclusions: Treatmentwith 40 kDa peginterferonalfa-2a + ribavirininduces an EVRin 86% of patients by week 12. EVRis positively predictivefor the achievementof SVR.Failure to achieveEVRstronglypredicts a poor virological outcome and alternativemanagementapproachesshould be considered, especiallyin genotype 1 patients. Adherenceto therapy plays a major role in achievingSVR. The likelihood of achieving SVR is enhanced in EVR patients who follow the treatment regimen, Continued adherence to treatment should be encouragedin those patients who demonstratean early response to therapy.
n All Patients Genotype I Geno~pes2, 3
453 298 140
SVR (%) 53/R(%) EVR-PPV(%) EVR-NPV(%) 56 46 76
86 81 97
65 57 77
97 98 75
SVR (%) (EV~,A~80L 75 67 88
717
718
CYCLIC NUCLEOTIDE-GATED CATION CHANNELS CONTRIBUTE T O
EXPRESSION, SUBCELLULAR LOCALIZATION AND FUNCTION OF AQUAPORINS IN RAT HEPATOCYI'ES. Robert C Huebert, Patrick L Splinter, Mayo Medical Sch, Clinic and Fdn, Rochester, MN; Raul A Marinelli, Fabiana A Garcia, Facuhad de Ciencias Bioquimicas; Suipacha 570, Rosario, Santa Fe Argentina; Nicholas F LaRusso, Mayo Medical Sch, Clinic and Fdn, Rochester, MN
VOLUME-SENSITIVE NA + INFLUX IN LIVER CELLS. Jeffrey C Dunkelberg, J G Fitz, University of Colorado H e a h h Sciences Center, Denver, CO Regulated changesin membraneNa+ permeabilityplay-a key role in the modulation of livercell volume. Changesin cell volumerepresent a signal that modulatesa broad range of liver cell and organ functions. Failure to regulatecell volumehas been implicatedin liver cell injury associated with alcohol, ischemia-reperfusion,and organ preservation.Hepatocellularnon-selectivecation (NSC) currents mediateNa+ influx and cellvolume increasein response to cell shrinkagecaused by oxidative or osmolar stress. However,the molecular basis of this conductance has not been defined. We demonstratethat cyclicnucleotide-gated(CNG)cationchannelsprovidea molecular basis for regulatedNa+ influx and cell volumehomeostasisin hepatocytes.METHODS.Cell lines: isolated rat hepatocytes,HTC cells from rat hepatoma,and HEK293cells. RT-PCR,Northern blot analysis, CNG channel cDNA cloning, and cell transfections were performed using standard protocols. Membranecation currents were measuredand analyzedusing whole-cellpatch clamp techniques. RESULTS:(1) Cyclicnucleotides increase HTC cell membranecation permeability. Intracellulardialysiswith 500 tzM cAMPincreasedHTC cell cation permeability~20-fold over 5-10 min. Dialysiswith cGMPincreasedNa+ currents in a dose-dependentfashionwith activation of an NSC conductance permeableto Na+~K+>Tris+. (2) Intracellulardialysiswith a selective CNG channel agonistactivatesan analogouscation conductance. PEG-2000-(cGMP)2,a synthetic cGMPdimer constructed as a potent and selectiveagonist for CNG channels, activatedthe endogenous cation-selectivecurrent of HTC cells. (3) Molecular identification of a cDNA encoding primary rat hepatocyteand HTC cell CNG channels. CNG-1channel gene expressionwas identifiedin rat hepatocytesby RT-PCRand Northern blot analysis.CNG-1cDNAwas clonedfromHTC cellsby RT-PCRand 5' RACE.HTC CNG-1 expressionvectorswere created.(4) cAMP-dependent opening of heterologouslyexpressed HTC CNG-1 channels. The HTC CNG-1 cDNA was expressed in HEK293cells, which have no endogenousCNG channel activity.Na+ influx could be induced in CNG-t-expressingcells by inclusion of 500 g~McAMPin the patch pipette solution while no control HEK293cells exhibited such currents. (5) Votume-dependemopening of haterologously-expressed HTC CNG-1 channels. In liver cells, NSC conductance is activated in response to decreasesin cell volume. To evaluatewhether CNG channels behave similarly, CNGexpressing HEK293 cells were evaluated by patch clamp analysis. In CNG-expressingcells, exposure to hypertoinc buffer to decreasecell size was followedby activationot currents in 4/4 cells. Currents were abrogatedwhen extracellularNa+ was replaced by Tris+. No Na+ currents were induced in control HEK293 cells or HEK293 cells expressing green fluorescent protein treated analogously.These findings demonstratethat HTC CNG-1 channels open in response to decreases in cell volume and, therefore,may have a role in mediatingthe hepatocyteregulatory volumeincrease(RVI)response. SUMMARY.(1) HTCcell NSC conductanceis activatedby cAMP and cGMP. (2) A selectiveCNG channelagonist activatesan analogouscation influx in HTCcells. (3) CNG-1 channel mRNAis expressed in primary rat hepatocytesand HTC cells. (4) Heterologously-expressedHTC CNG-1channel mediatescAMP-inducedNa+ influx. (5) Heterologouslyexpressed HTC CNG-1 channel mediates Na+ influx in response to cell shrinkage caused by hyperosmotic stress. CONCLUSION:These findings indicate that CNG-t-related channels are expressed in HTC cells and mediate the influx of Na+ ions. Moreover, the similar biophysical properties of CNG channel conductance to NSC conductancesuggests that CNG channels may contribute to regulation of cell volume in response to hormones (e.g. vasopressin,insulin) and other stimuli (oxidativestress) known to regulateliver function through effectson cell volume.
Formation of hepatic bile requires that large volumes of water be rapidly transported across liver epithelia, including hepatocytes. While the transepithelial pathways (i.e., paracellular or transcellular) and the molecular mechanisms (e.g., diffusion, channel-mediated, etc.) by which water is secreted into bile are obscure, it is being increasingly recognized that channel-mediated, transcellular water transport is an important mechanism in water transporting epithelia. The aquaporins (AQPs) are a family of ten channel-forming, integral membrane proteins of - 2 8 k D a numbered 0 - 9 that allow water to rapidly traverse epithelial barriers in several organs including kidney, eye, and brain. The aim of this study was to identify which AQPs are expressed in hepatocytes, quantitate their message levels, determine the subcellular localization of AQP proteins, and demonstrate that AQPs are involved in canalicular bile secretion. Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative ribonuclease protection assays (Q-RPAs) confirmed and quantified the levels of three AQP mRNAs in hepatocytes isolated from normal rat liver (AQP8 > AQP9 > AQP0). Immunoblots of hepatocyte membrane fractions showed enrichment of AQP0 and AQP8 in intracellular vesicles and on the canalicular plasma membrane (CPM); in contrast, AQP9 was enriched on the basolateral plasma membrane (BPM). Immunohistochemistry of whole liver and immunofluorescence of isolated hepatocyte couplets confirmed the intracellular and canalicular localization of AQP0 and AQP8 and the basolateral localization of AQP9. Changes in canalicular volume were measured in isolated hepatocyte couplets after exposure to hypoosmotic stress. Secretion into the canalicular space was markedly increased by pre-incubation of the couplets with dibutyryl cyclic AMP, an effect which was blocked by incubation with the AQP inhibitors, dimethylsulfoxide and phloretin. The data show that hepatocytes selectively express message and protein of three of the AQP water channels and that these AQPs are differentially localized in distinct subcellular compartments. In addition, canalicular secretion is shown to be increased upon addition of a choleretic stimulus and this effect is abolished in the presence of AQP inhibitots. These results provide morphologic, biochemical, and functional evidence for the presence of AQPs in hepatocytes and suggest a channel-mediated component of water transport across the hepatocyte epithelial barrier.
AASLD ABSTRACTS
351A
715
716
IMMUNOMODULATORY ACTIVITIES OF VIRAMIDINE, A LIVER-TARG E T I N G RIBAVIRIN PRODRUG, I N V I T R O AND I N VIVO. Robert Tam, Charmaine Lira, Josie Bard, Parmala Bagha, Johnson Y Lau, Zhi Hong, ICN Pharmaceuticals, Costa Mesa, CA
EARLY PREDICTION OF RESPONSE TO 40 KDA PEGINTERFERON
Background: Ribavirin is a purine nucleoside analog approved for the treatment of hepatitis C in combination with interferon-a. It is believed that ribavirin achieves its clinical benefits by inducing a Type i cytokine bias that favors antiviral cellular immunity. To improve the toxicity profile of ribavirin and alleviate the adverse effects of ribavirin therapies such as hemolytic anemia, several nucleoside analogs were synthesized and their immunomodulatory activities were evaluated. Aim: To determine whether viramidine, a liver-targeting prodrug analog of ribavirin, has similar i m m u n o m o d u l a t o r y activity to ribavirin in vitro and in vivo. Methods: The in vitro effect of viramidine on Type 1 cytokine production (IL-2, IFN% TNFc~) in activated h u m a n T cells was determined by ELISA. The in vfvo effect in a murine model was assessed by Type 1 cytokine-mediated contact hypersensitivity (CHS) responses to dinitrofluorobenzene (DNFB). Results: Similar to ribavirin, viramidine enhanced SEB-stimulated Type 1 cytokine expression (IL-2, IFN 7 and T N F a ) by h u m a n T ceils in a dose-dependent m a n n e r (0,67-I0/zM). The viramidine concentration that gave the peak cytokine responses showed donor-to-donor variability (similar to ribavirin) with the highest frequency at 5 to 10/zM in 15 donors. The magnitude of the induction of Type 1 cytokines by viramidine was similar to that observed for ribavirin in T cells from the same donors. Viramidine (admimstered ip at 0.28, 0.56 and 1.12 mg/kg) enhanced Type 1 cytokinemediated CHS responses to DNFB (as measured by the inflammatory ear response to contact allergen) in BALB/c mice in a dose dependent manner. The magnitude and peak dose (0.5-1 mg/kg) of the effect of viramidine on CHS responses was similar to ribavirin. Using a MTT assay for T cell proliferation, high doses ( > 20/.LM) of viramidine showed similar anti-proliferative effects to ribavirin at doses which showed no effect on h u m a n T cell viability (assessed by propidium iodide exclusion). Conclusion: Viramidine showed similar immunomodulatory properties to ribavirin w h e n administered to in vitro stimulated h u m a n T cells and in vivo mouse model for a Type 1 cytokine-mediated i m m u n e response.
ALFA-2A (PEGASYS ®) PLUS RIBAVIRIN (RBV) IN PATIENTS WITH CHRONIC HEPATITIS C (CHC). Peter Ferenci, University of Vienna, Wien Austria; Mitchell L Shiffman, Medical College of Virginia, Richmond, VA; Michael W Fried, University of North Carolina, Chapel Hill, NC; Mark S Sulkowski, Johns Hopkins Center for Viral Hepatitis, Baltimore, MD; Dieter Haeussinger, University of Duesseldorf, Duesseldorf Germany; Jean-Pierre Zarski, CHU de Grenoble, Grenoble France; Fernando GoncalesJr., University of Campinas, Campinas Spain; Donald M Jensen, Rush-Presbyterian St Luke's Hospital, Chicago, IL; Ferrucio Bonino, Azienda Ospedaliera Univ Pisana, Pisa Italy; Daniel Dhumeaux, H6pital Henri Mondor, Creteil France; Steven Blother, Joseph Hoffman, Joseph Oliveto, Hoffmann-La Roche, Nutley, NJ Background: The administrationof 40 kDa peginterferonalfa-2a (PEGASYS®) is associatedwith early viral decline in the majorityo[ CYICpatients.We have demonstratedthat failureto achieve an early virologicalresponse (EVR), defined as ->2 logto decline in HCV RNA from baselineby week 12 of treatment,is highlypredictivefor a lack of sustained virologicalresponse (SVR)among patients treated with 40 kDa peginterferon alfa-2a alone (Lee SS et al. Hepatology. 2000; 32 [supplJ:370A), or treatedwith the combinationof 40 kDa peginterferonalfa-2a + ribavirin(Fried MW et al. DDW. 2001). Objective:The aim of the present studywas to determinethe effectof HCV genotypeand adherenceto study regimenon the positiveand negativepredictivevalues(PPVand NPV) for SVRusing EVRin patients treatedwith combinationof 40 kDa peginterferon alfa-2a 180 /zg qw + RBV1000-1200rag qd. Methods:A retrospectiveanalysiswas performedon data froma large randomized trial comparing the combination of 40 kDa peginterferon alfa-2a + RBV (n=453) vs IFN a-2b + RBVvs 40 kDa peginterferonalfa-2a + placeboin CHC.EVRwas defined as HCV RNAbelow levelof detection (<600 IU/mL,COBASAMPLICORHCVMONITOR~"Test, v 2.0) or -->2Iogtodecline in HCV RNA from baselineby week 12. PPV and NPV for SVRwere calculated for all patients in the 40 kDa peginterferonalfa-2a + RBVarm (n=453) and according to genotype.PatientsachievingEVRwere further evaluatedfor the effectof adherence (A->80=at least 80% of assignedtreatmentreceived)on SVR.Results: Of all patients treated,86% had an EVR and 56%achievedSVR.The EVR-PPVwas 65%and the EVR-NPVw-as97%.For patients with EVR who were adherent to the study regimen(A>80%),SVRwas 75% comparedto only 48% in EVR patients who were not adherent (A<80%). EVRwas achievedin 81% of genotype 1 patients and 97% of genotype2 or 3 patients. The EVR-PPVfor SVRwas 67% and 88% for adherent patients with genotype 1 and genotypes2 or 3, respectively(Table). Conclusions: Treatmentwith 40 kDa peginterferonalfa-2a + ribavirininduces an EVRin 86% of patients by week 12. EVRis positively predictivefor the achievementof SVR.Failure to achieveEVRstronglypredicts a poor virological outcome and alternativemanagementapproachesshould be considered, especiallyin genotype 1 patients. Adherenceto therapy plays a major role in achievingSVR. The likelihood of achieving SVR is enhanced in EVR patients who follow the treatment regimen, Continued adherence to treatment should be encouragedin those patients who demonstratean early response to therapy.
n All Patients Genotype I Geno~pes2, 3
453 298 140
SVR (%) 53/R(%) EVR-PPV(%) EVR-NPV(%) 56 46 76
86 81 97
65 57 77
97 98 75
SVR (%) (EV~,A~80L 75 67 88
717
718
CYCLIC NUCLEOTIDE-GATED CATION CHANNELS CONTRIBUTE T O
EXPRESSION, SUBCELLULAR LOCALIZATION AND FUNCTION OF AQUAPORINS IN RAT HEPATOCYI'ES. Robert C Huebert, Patrick L Splinter, Mayo Medical Sch, Clinic and Fdn, Rochester, MN; Raul A Marinelli, Fabiana A Garcia, Facuhad de Ciencias Bioquimicas; Suipacha 570, Rosario, Santa Fe Argentina; Nicholas F LaRusso, Mayo Medical Sch, Clinic and Fdn, Rochester, MN
VOLUME-SENSITIVE NA + INFLUX IN LIVER CELLS. Jeffrey C Dunkelberg, J G Fitz, University of Colorado H e a h h Sciences Center, Denver, CO Regulated changesin membraneNa+ permeabilityplay-a key role in the modulation of livercell volume. Changesin cell volumerepresent a signal that modulatesa broad range of liver cell and organ functions. Failure to regulatecell volumehas been implicatedin liver cell injury associated with alcohol, ischemia-reperfusion,and organ preservation.Hepatocellularnon-selectivecation (NSC) currents mediateNa+ influx and cellvolume increasein response to cell shrinkagecaused by oxidative or osmolar stress. However,the molecular basis of this conductance has not been defined. We demonstratethat cyclicnucleotide-gated(CNG)cationchannelsprovidea molecular basis for regulatedNa+ influx and cell volumehomeostasisin hepatocytes.METHODS.Cell lines: isolated rat hepatocytes,HTC cells from rat hepatoma,and HEK293cells. RT-PCR,Northern blot analysis, CNG channel cDNA cloning, and cell transfections were performed using standard protocols. Membranecation currents were measuredand analyzedusing whole-cellpatch clamp techniques. RESULTS:(1) Cyclicnucleotides increase HTC cell membranecation permeability. Intracellulardialysiswith 500 tzM cAMPincreasedHTC cell cation permeability~20-fold over 5-10 min. Dialysiswith cGMPincreasedNa+ currents in a dose-dependentfashionwith activation of an NSC conductance permeableto Na+~K+>Tris+. (2) Intracellulardialysiswith a selective CNG channel agonistactivatesan analogouscation conductance. PEG-2000-(cGMP)2,a synthetic cGMPdimer constructed as a potent and selectiveagonist for CNG channels, activatedthe endogenous cation-selectivecurrent of HTC cells. (3) Molecular identification of a cDNA encoding primary rat hepatocyteand HTC cell CNG channels. CNG-1channel gene expressionwas identifiedin rat hepatocytesby RT-PCRand Northern blot analysis.CNG-1cDNAwas clonedfromHTC cellsby RT-PCRand 5' RACE.HTC CNG-1 expressionvectorswere created.(4) cAMP-dependent opening of heterologouslyexpressed HTC CNG-1 channels. The HTC CNG-1 cDNA was expressed in HEK293cells, which have no endogenousCNG channel activity.Na+ influx could be induced in CNG-t-expressingcells by inclusion of 500 g~McAMPin the patch pipette solution while no control HEK293cells exhibited such currents. (5) Votume-dependemopening of haterologously-expressed HTC CNG-1 channels. In liver cells, NSC conductance is activated in response to decreasesin cell volume. To evaluatewhether CNG channels behave similarly, CNGexpressing HEK293 cells were evaluated by patch clamp analysis. In CNG-expressingcells, exposure to hypertoinc buffer to decreasecell size was followedby activationot currents in 4/4 cells. Currents were abrogatedwhen extracellularNa+ was replaced by Tris+. No Na+ currents were induced in control HEK293 cells or HEK293 cells expressing green fluorescent protein treated analogously.These findings demonstratethat HTC CNG-1 channels open in response to decreases in cell volume and, therefore,may have a role in mediatingthe hepatocyteregulatory volumeincrease(RVI)response. SUMMARY.(1) HTCcell NSC conductanceis activatedby cAMP and cGMP. (2) A selectiveCNG channelagonist activatesan analogouscation influx in HTCcells. (3) CNG-1 channel mRNAis expressed in primary rat hepatocytesand HTC cells. (4) Heterologously-expressedHTC CNG-1channel mediatescAMP-inducedNa+ influx. (5) Heterologouslyexpressed HTC CNG-1 channel mediates Na+ influx in response to cell shrinkage caused by hyperosmotic stress. CONCLUSION:These findings indicate that CNG-t-related channels are expressed in HTC cells and mediate the influx of Na+ ions. Moreover, the similar biophysical properties of CNG channel conductance to NSC conductancesuggests that CNG channels may contribute to regulation of cell volume in response to hormones (e.g. vasopressin,insulin) and other stimuli (oxidativestress) known to regulateliver function through effectson cell volume.
Formation of hepatic bile requires that large volumes of water be rapidly transported across liver epithelia, including hepatocytes. While the transepithelial pathways (i.e., paracellular or transcellular) and the molecular mechanisms (e.g., diffusion, channel-mediated, etc.) by which water is secreted into bile are obscure, it is being increasingly recognized that channel-mediated, transcellular water transport is an important mechanism in water transporting epithelia. The aquaporins (AQPs) are a family of ten channel-forming, integral membrane proteins of - 2 8 k D a numbered 0 - 9 that allow water to rapidly traverse epithelial barriers in several organs including kidney, eye, and brain. The aim of this study was to identify which AQPs are expressed in hepatocytes, quantitate their message levels, determine the subcellular localization of AQP proteins, and demonstrate that AQPs are involved in canalicular bile secretion. Reverse transcription-polymerase chain reaction (RT-PCR) and quantitative ribonuclease protection assays (Q-RPAs) confirmed and quantified the levels of three AQP mRNAs in hepatocytes isolated from normal rat liver (AQP8 > AQP9 > AQP0). Immunoblots of hepatocyte membrane fractions showed enrichment of AQP0 and AQP8 in intracellular vesicles and on the canalicular plasma membrane (CPM); in contrast, AQP9 was enriched on the basolateral plasma membrane (BPM). Immunohistochemistry of whole liver and immunofluorescence of isolated hepatocyte couplets confirmed the intracellular and canalicular localization of AQP0 and AQP8 and the basolateral localization of AQP9. Changes in canalicular volume were measured in isolated hepatocyte couplets after exposure to hypoosmotic stress. Secretion into the canalicular space was markedly increased by pre-incubation of the couplets with dibutyryl cyclic AMP, an effect which was blocked by incubation with the AQP inhibitors, dimethylsulfoxide and phloretin. The data show that hepatocytes selectively express message and protein of three of the AQP water channels and that these AQPs are differentially localized in distinct subcellular compartments. In addition, canalicular secretion is shown to be increased upon addition of a choleretic stimulus and this effect is abolished in the presence of AQP inhibitots. These results provide morphologic, biochemical, and functional evidence for the presence of AQPs in hepatocytes and suggest a channel-mediated component of water transport across the hepatocyte epithelial barrier.