Mechanosensitive Cl secretion in biliary epithelium mediated through TMEM16A

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1 m J Physiol Gstrointest Liver Physiol 34: G87 G98, 213. First pulished Octoer 25, 212; doi:1.1152/jpgi Mechnosensitive l secretion in iliry epithelium medited through TMEM16 ml K. Dutt, 1 Kngmee Woo, 1 l-krim Khimji, 2 hrles Kresge, 1 nd ndrew P. Fernchk 1 1 Deprtment of Peditrics, University of Texs Southwestern Medicl enter, Dlls, Texs; nd 2 Deprtment of Internl Medicine, University of Texs Southwestern Medicl enter, Dlls, Texs Sumitted 19 pril 212; ccepted in finl form 19 Octoer 212 Dutt K, Woo K, Khimji, Kresge, Fernchk P. Mechnosensitive l secretion in iliry epithelium medited through TMEM16. m J Physiol Gstrointest Liver Physiol 34: G87 G98, 213. First pulished Octoer 25, 212; doi:1.1152/jpgi ile formtion y the liver is initited y cnliculr trnsport t the heptocyte memrne, leding to n increse in ductulr ile. Thus, ile duct epithelil cells (cholngiocytes), which contriute to the volume nd dilution of ile through regulted l trnsport, re exposed to chnges in nd sher force t the picl memrne. The im of the present study ws to determine if fluid, or sher stress, is signl regulting cholngiocyte trnsport. The results demonstrte tht, in humn nd mouse iliry cells, fluid, or sher, increses l currents nd identify TMEM16, 2 -ctivted l chnnel, s the opertive chnnel. Furthermore, ctivtion of TMEM16 y is dependent on PK through process involving extrcellulr TP, inding purinergic P2 receptors, nd increses in intrcellulr 2 concentrtion. These studies represent the initil chrcteriztion of mechnosensitive l currents medited y TMEM16. Identifiction of this novel mechnosensitive secretory pthwy provides new insight into ile formtion nd suggests new therpeutic trgets to enhnce ile formtion in the tretment of cholesttic liver disorders. liver; cholngiocyte; purinergic signling; TP relese; chloride chnnel; noctmin-1 HOLESTTI LIVER DISESES, ssocited with poor ile, comprise significnt proportion of liver disorders in children nd dults. While diverse in etiology, these disorders result in significnt decrese in ductulr ile. It hs een proposed tht decrese in ile my directly ffect ile production through mechnicl effects on the ile duct epithelium, including norml cili function nd norml 2 signling (23). However, potentil direct effects of fluid, or sher, on memrne trnsport nd secretion re unknown. Intrheptic ile duct epithelil cells, or cholngiocytes, represent n importnt component of the ile secretory unit. While ile formtion is initited t the heptocyte cnliculr memrne, cholngiocytes susequently modify the volume nd composition of ile through regulted ion nd wter secretion. l chnnels in the picl memrne of cholngiocytes provide the driving force for ductulr secretion (17, 18), nd two complementry l chnnels hve een identified on moleculr sis: FTR, cmp-ctivted l chnnel (16, 19), nd TMEM16, 2 -ctivted l chnnel (9). FTR is found on the picl memrne of cholngiocytes forming the medium- nd lrge-sized, ut not smll-sized, ile ducts nd is regulted vi inding of the hormone secretin to solterl ddress for reprint requests nd other correspondence:. K. Dutt, UT Southwestern Medicl enter, 5323 Hrry Hines ld., Dlls, TX (e-mil: ml.dutt@utsouthwestern.edu). receptors, increses in intrcellulr cmp concentrtion, nd PK-dependent chnnel phosphoryltion (18). In contrst, TMEM16 is found on the smll-, medium-, nd lrge-sized ile ducts nd is ctivted y increses in intrcellulr 2 concentrtion ([ 2 ] i ) (9), lthough the physiologicl events mediting chnnel regultion hve not een defined. TMEM16, 114-kD memrne protein with eight puttive trnsmemrne domins, hs een estlished s 2 - ctivted l chnnel in other epitheli, lthough its regultion is fr from understood. dditionlly, TMEM16 hs een shown to regulte trchel crtilge development (39), ppers to ply role in cncer progression (12), nd my contriute to the regultion of lood (4, 7). The diverse clinicl significnce of this protein is just eginning to e pprecited. While TMEM16 is found in iliry epithelium, its regultion nd overll contriution to iliry secretion nd ile formtion re unknown. Mechnicl fluid (sher) ctivtes memrne trnsport in epithelil nd endothelil cells (2, 31, 33, 48). In vsculr endothelil cells, sher stress hs een shown to ctivte simultneously n inwrd-rectifying K chnnel nd n outwrd-rectifying l chnnel (22). Similrly, in kidney, stimultes K secretion in mmmlin corticl collecting ducts (32). iliry epithelil cells re lso exposed to plsm memrne-directed forces, including /sher forces t the picl memrne due to chnges in ile. The hormone secretin, for exmple, increses ile from.67 to 1.54 ml/min in humns (3), representing potentil increse in -induced force t the picl cholngiocyte memrne. In isolted rt ile duct segments nd confluent iliry epithelil monolyers, the increse in results in significnt increses in [ 2 ] i (34, 48). The moleculr identifiction of the protein(s) contriuting to mechnosensitive cholngiocyte trnsport hs not een defined. Our present studies, in humn nd mouse iliry epithelil models, demonstrte for the first time tht the force of t the surfce of the plsm memrne is significnt nd physiologicl stimulus for TMEM16-medited l trnsport through process dependent on TP relese, inding of purinergic P2 receptors, nd increses in [ 2 ] i. We therefore propose tht the mechnicl force generted y my directly regulte cholngiocyte secretory events nd ile formtion. METHODS ell models. Studies were performed in humn nd mouse models, including humn intrheptic iliry epithelil H69 cells, humn Mz-h-1 cells, nd mouse smll nd lrge cholngiocytes (MS nd ML, respectively). The H69 cholngiocyte cell line is SV-4- trnsformed humn intrheptic ile duct epithelil cell line originlly Downloded from y on Decemer 1, /13 opyright 213 the mericn Physiologicl Society G87

2 G88 derived from norml liver hrvested for liver trnsplnttion (25). These cells retin phenotypic mrkers of primry humn cholngiocytes, including functionl memrne chnnels nd trnsporters (24, 38). Humn Mz-h-1 cells lso exhiit phenotypic fetures of differentited iliry epithelium (3, 29) nd hve een utilized s models for iliry purinergic signling nd 2 -stimulted secretion (14, 15, 41, 42, 47). Immortlized MS nd ML demonstrte properties identicl to those of freshly isolted MS nd ML (21). Importntly, MS do not express secretin receptors, FTR, or the HO 3 /l exchnger nd do not exhiit secretory response to secretin, while ML do (21). ells were mintined in culture, s descried elsewhere (21, 46). Perfusion system. Sher ws pplied to cells in prllel-plte chmer ( mm; model R-25F, Wrner Instruments, Hmden, T) for 2 imging nd to cells in n open-top chmer for ptch-clmp recording. In ech cse, ws pplied y dul-syringe pump (model 33, Hrvrd pprtus, Holliston, M). The eqution relting sher stress to volumetric rte through the chmers is s follows: w 6 Q / 2, where is viscosity of the solution (poise), Q is rte (ml/s), is chmer height (cm), nd is chmer width (cm). 2 imging. ells were cultured for 48 h on 15-mm glss coverslips nd then loded with 2.5 g/ml fur 2-M (TEF Ls, ustin, TX) in isotonic extrcellulr uffer contining (in mm) 14 Nl, 4 Kl, 2 l 2, 1 Mgl 2,1KH 2PO 4, 5 glucose, nd 1 HEPES (ph 7.4) supplemented with.1% Pluronic F-127 for 3 min t 22. In selected studies, EGT (2 mm) ws used to remove 2 from the th nd perfusing solutions. The coverslip ws plced in the perfusion chmer on the stge of n inverted fluorescence microscope (Nikon TE2), nd the in nd out ports were ttched to Mz-h1 7-7 H s.3 n 2 ms 6 s.1 n 1 ms Flow D Mz-h urrent density (-/pf) % of control 12 s cftr-inh s 1 5 n. cid Downloded from y on Decemer 1, 217 Fig. 1. hrcteriztion of -stimulted currents in humn iliry epithelil cells. Whole cell currents were mesured during sl conditions nd during exposure to of isotonic extrcellulr uffer. nd : representtive whole cell recordings of n Mz-h-1 cell nd n H69 cell. urrents were mesured t 8 (Œ), representing l current, nd t ( ), representing K current. Flow exposure (sher. 24 dyn/cm 2 ) is indicted y horizontl r. urrents were ctivted within 2 min of the onset of nd were prtilly reversile when ws stopped. voltge-step protocol (inset in ), from holding potentil of 4 with 1-ms steps from 1 to 1 in 2- increments, ws otined t nd, representing sl nd mximl inwrd currents, respectively. urrents demonstrted time-dependent ctivtion t memrne potentils 6. Zero-current level is indicted y dotted lines. urrent-voltge (I-V) plot ws generted from these protocols during sl ( ) nd -stimulted (Œ) conditions. : representtive whole cell current recordings of Mz-h-1 cells in the presence or sence of the 2 -ctivted l chnnel inhiitor niflumic cid (n. cid, 1 M, top) nd in the presence of the FTR inhiitor FTR(inh)-172 (5 M, ottom). Flow is indicted y horizontl r. D: cumultive dt demonstrting percentge of mximum -stimulted currents in the presence or sence of the l chnnel inhiitors 5-nitro-2-(3-phenylpropylmino)enzoic cid (NPP, 1 M), niflumic cid (1 M), DIDS (1 M), FTR(inh)-172 (5 M), nd mlonic hydrzide (MlH, 5 M). Vlues represent percentge of current density mesured t 8 (n 4 5 ech). Significntly different from control (P.5). JP-Gstrointest Liver Physiol doi:1.1152/jpgi

3 the syringe pump. hnges in [ 2 ] i were mesured t excittion wvelength of 34 nm for 2 -ound fur 2-M nd 38 nm for 2 -free fur 2-M t emission wvelength of 51 nm. fter sutrction of ckground fluorescence, [ 2 ] i ws clculted ccording to the Grynkiewicz eqution (44): [ 2 ] i (nm) K d [(R R min)/(r mx R)] S f, where K d is the dissocition constnt (145 nm t 22 ) nd S f is the rtio of seline fluorescence (38 nm) under the 2 -free condition to fluorescence under the 2 - ound condition. Experiments were performed t room temperture nd t 37. Mesurement of -stimulted currents. Memrne currents were mesured using whole cell ptch-clmp techniques. ells on coverslip were mounted in the chmer, nd whole cell currents were mesured during sl nd perfused conditions with stndrd extrcellulr solution contining (in mm) 14 Nl, 4 Kl, 1 l 2,2 Mgl 2,1KH 2PO 4, 1 glucose, nd 1 HEPES/NOH (ph 7.4). The stndrd intrcellulr (pipette) solution for whole cell recordings contined (in mm) 13 Kl, 1 Nl, 2 Mgl 2, 1 HEPES/KOH,.5 l 2, nd 1 EGT (ph 7.3), corresponding to free 2 concentrtion of 1 nm (6, 13). Ptch pipettes, which were pulled from orning 752 glss, hd resistnce of 3 1 M. Recordings were mde with n xoptch ID mplifier (xon Instruments, Foster ity, ), digitized (2 khz), nd nlyzed using plmp version 1 (xon Instruments, urlingme, ), s previously descried (13, 2). Two voltge protocols were utilized: 1) holding potentil of 4 with 2-ms steps to nd 8 t 1-s intervls nd 2) holding potentil of 4 with 4-ms steps from 1 to 1 in 2- increments. urrent-voltge reltions were generted from the step protocol. Pipette voltges re referred to the th. Results re compred with control studies mesured on the sme dy to minimize effects of dy-to-dy vriility. While men cell cpcitnce ws pf (n 11), results re reported s current density (/pf) to normlize for differences in cell size (13). TMEM16 nd FTR silencing. TMEM16 ws suppressed y specific nti-tmem16 smll interfering RN (sirn; TMEM16- HSS12394), s descried in our previous studies (9). riefly, 25- nucleotide sirns were designed nd synthesized y Invitrogen [G UU GUG GG UG GU GGG (ntisense) nd GGU U G U U U U U (sense)] nd trnsfected using FuGENE (5 g/1 l). Noncoding Stelth RNi (medium gunine-cytosine duplex, Invitrogen) ws utilized in control (mock) trnsfections. Similrly, FTR ws suppressed y specific nti-ftr sirn (ctlog no , Life Technologies). LOK-iT Fluorescent Oligo (ctlog no. 213, Invitrogen) ws used to optimize trnsfection conditions nd to select trnsfected cells for whole cell ptch-clmp recording. Whole cell ptch-clmp experiments were performed h fter trnsfection. Trnsfection efficiency nd the degree of TMEM16 nd FTR silencing were mesured t the messge level y rel-time PR nd t the protein level y Western lot nlysis (9). Regents. The FTR inhiitors FTR(inh)-172 nd mlic hydrzide (MlH) were kind gifts from Drs. Nitin Sonwne nd ln Verkmn (University of liforni, Sn Frncisco, ). nti-ftr (clone M37) monoclonl ntiody (ctlog no ) ws purchsed from Millipore. ll other regents were otined from Sigm- ldrich (St. Louis, MO). Sttistics. Vlues re mens SE, with n representing the numer of culture pltes or repetitions for ech ssy. Sttisticl nlysis included Fisher s pired nd unpired t-test nd NOV for multiple comprisons to ssess sttisticl significnce. P.5 ws considered to e sttisticlly significnt. RESULTS Flow (sher) ctivtes memrne l currents. To chrcterize the iophysicl nd phrmcologicl properties of memrne l currents in response to sher, whole cell ptchclmp studies were performed in single Mz-h-1 nd H69 cells nd MS nd ML in the presence or sence of defined sher. Representtive trces of Mz-h-1 cell nd H669 cell re shown in Fig. 1. Under sl conditions with stndrd intr- nd extrcellulr uffers, l current ws smll ( /pf). Exposure to (sher.24 dyn/cm 2 ) resulted in ctivtion of currents within s, incresing current density to /pf t 8 (P.1, n 13 for Mz-h-1 cells; P.5, n 4 for H69 cells). The currents were sustined for the durtion of exposure nd were fully reversile within 5 min of cesstion. Interestingly, currents demonstrted two distinct ptterns. In the mjority ( 85%) of studies, the currents exhiited reversl ner [l reversl (equilirium) potentil], outwrd rectifiction, nd time-dependent ctivtion t depolrizing potentils ove 6 (Fig. 1), chrcteristics ssocited with 2 - ctivted l currents previously descried in these cells (9, 16). However, in minority ( 15%) of studies, currents demonstrted time-dependent inctivtion t positive depolrizing potentils ove 6 (Fig. 2). In some studies, currents with oth types of iophysicl properties were oserved in the sme cell (5 of 35). Inclusion of MgTP 2 in the ptch pipette incresed (from 15% to 38%) the reltive percentge of the currents displying time-dependent inctivtion. Therefore, to minimize the currents demonstrting time-dependent inctivtion, the mjority of studies were performed without dditionl MgTP 2 in the pipette. MS nd ML lso demonstrted -stimulted l currents with iophysicl properties similr to those of the humn iliry cells (Fig. 3). In numer of studies the -stimulted l currents were preceded y smll K currents, which s.5 n 2 ms Fig. 2. Flow-stimulted l currents demonstrte second nd distinct iophysicl profile, s shown in representtive whole cell recording of Mz-h-1 cell. urrents were mesured t 8 (Œ), representing l current, nd t ( ), representing K current. Flow exposure (sher. 24 dyn/cm 2 ) is indicted y horizontl r. voltge-step protocol s descried in Fig. 1 legend (test potentils etween 1 nd 1 in 2- increments) ws otined t (sl) nd (mximl current response). urrents demonstrte time-dependent inctivtion t memrne potentils 6. Zero-current levels re indicted y dotted lines. I-V plot ws generted from these protocols during sl ( ) nd -stimulted (Œ) conditions. G89 Downloded from y on Decemer 1, 217 JP-Gstrointest Liver Physiol doi:1.1152/jpgi

4 G9 MS 15 ML urrent density (-/pf) s 12 s Flow FTR-inh 172 (5 µm) ML Flow MS Fig. 3. hrcteriztion of -stimulted currents in mouse lrge nd smll cholngiocytes (ML nd MS, respectively). : representtive whole cell current recordings from MS nd ML. urrents in ML were mesured in the presence of FTR chnnel inhiitor FTR(inh)-172 (5 M) t 8 (Œ) nd( ). Flow exposure (sher. 18 dyn/cm 2 ) is indicted y horizontl r. urrents were ctivted within 2 min of the onset of. voltge-step protocol (test potentils etween 1 nd 1 in 2- increments) ws otined t (sl) nd (mximl current response). urrents demonstrte time-dependent ctivtion t memrne potentils 6. Zero-current levels re indicted y dotted lines. I-V plot ws generted from these protocols during sl ( ) nd -stimulted (Œ) conditions. : cumultive dt demonstrting mximum current density in response to ddition of the FTR-ctivting cocktil (chlorophenylthio-cmp, forskolin, nd IMX) or exposure to (sher.18 dyn/cm 2 ) in ML nd MS. Flow-stimulted currents were recorded in the presence or sence of niflumic cid (1 M) or FTR(inh)-172 (5 M). Vlues represent mximum current density mesured t 8 (n 4 11 ech). FTR-ctivting cocktil significntly incresed currents from sl in ML (P.5). Flowstimulted currents were significntly inhiited y niflumic cid in ML (P.5). ns, Not significnt. n.s. occurred erly nd were trnsient. The iophysicl properties of these K currents were consistent with the smll- or intermedite-conductnce 2 -ctivted K chnnels previously descried in iliry cells (1, 14). Flow-stimulted l currents re independent of FTR. To determine if FTR contriutes to the -stimulted l current, three complementry strtegies were utilized: 1) phrmcologicl inhiition, 2) differentil models of FTR expression, nd 3) moleculr silencing. 1) Flow-stimulted currents MW (kd) sirn FTR urrent density (-/pf) 6 s n 1 ms sirn FTR FTR β ctin control sirn FTR control Fig. 4. Flow-stimulted currents re independent of FTR chnnel. : representtive Western lot demonstrting FTR protein levels in Mz-h-1 cells trnsfected with nontrgeting smll interfering RN (sirn, mock) nd nti-ftr sirn. ctin ws used s loding control. : representtive whole cell current recordings from Mz-h-1 cells trnsfected with FTR sirn under sl nd -stimulted conditions. Flow exposure (sher.18 dyn/cm 2 ) is indicted y horizontl r. urrents were mesured t 8 (Œ) nd( ). Voltge-step protocols were otined t (sl) nd (mximl current response). urrents demonstrte time-dependent ctivtion t memrne potentils 6. I-V plots were generted from the step protocols during sl ( ) nd -stimulted (Œ) conditions. : cumultive dt demonstrting mximl current density mesured t 8 in response to in cells trnsfected with nontrgeting sirn (mock) or cells trnsfected with nti-ftr sirn. Vlues re mens SE; n 4 5. P not significnt. Downloded from y on Decemer 1, 217 JP-Gstrointest Liver Physiol doi:1.1152/jpgi

5 G91 were inhiited y the nonspecific l chnnel locker 5-nitro- 2-(3-phenylpropylmino)enzoic cid nd y the 2 -ctivted l chnnel lockers niflumic cid nd DIDS (Fig. 1, nd D) ut were unffected y phrmcologicl inhiitors of FTR, including FTR(inh)-172 nd MlH (Fig. 1, nd D). 2) omprison studies were performed in mouse cholngiocytes with nd without FTR expression. MS ( 8 m), isolted from the epithelium forming the smll intrheptic ile ducts, do not express FTR (46), while ML ( 14 m), isolted from the epithelium forming the lrge ile ducts, do. In f34 / f38 rtio f 34 / f 38 rtio [ 2+ ] i (nm) ML MS time (seconds) MS 16 ML time (seconds) ionomycin EGT ionomycin EGT / D urrent density (-/pf) Extrcellulr 2+ free 12 s Intrcellulr 2+ free 12 s oth side 2+ free 12 s 2 1 # Downloded from y on Decemer 1, 217 sher:.2 dyne/cm 2.9 dyne/cm 2 Fig. 5. Flow-stimulted currents re dependent on intrcellulr 2 concentrtion ([ 2 ] i). : representtive studies demonstrting the increse in [ 2 ] i, ssocited with in ML nd MS. Mximl nd miniml [ 2 ] i ws otined y exposure to ionomycin (2 M) nd EGT (1 mm), respectively. F 34/F 38, rtio of fluorescence t 34 nm to fluorescence t 38 nm. : cumultive dt demonstrting -stimulted increse in [ 2 ] i in ML nd MS (n 1 ech) Significnt increse in [ 2 ] i t sher.9 dyn/cm 2 compred with sher.2 dyn/cm 2 (P.5). No difference in [ 2 ] i etween MS nd ML t either sher (P not significnt). : representtive whole cell current recordings from Mz-h-1 cells with removl of extrcellulr 2 (1 mm EGT in th, top), removl of intrcellulr 2 (2 mm EGT in pipette, middle), nd removl of extr- nd intrcellulr 2 (ottom). urrents were mesured t 8 ( ) nd(œ). Flow exposure (sher.24 dyn/cm 2 ) is indicted y horizontl r. D: cumultive dt demonstrting mximum current density with removl of extrcellulr [(E) 2 free], intrcellulr [(I) 2 free], or oth ( 2 free) compred with control. Vlues represent mximum current density mesured t 8 (n 4 8 ech). Flow-stimulted currents were significntly inhiited: P.1, #P.1. JP-Gstrointest Liver Physiol doi:1.1152/jpgi

6 G92 MS, exposure to FTR-ctivting cocktil (1 M forskolin, 1 M IMX, nd 5 M chlorophenylthio-cmp) did not increse l currents from the sl level, consistent with the sence of FTR chnnel protein (Fig. 3). However, exposure to (sher.12 dyn/cm 2 ) significntly incresed current density (Fig. 3). In comprison, ddition of the FTR-ctivting cocktil to ML incresed l current density (from to /pf), consistent with functionl FTR expression. Interestingly, susequent exposure to (sher.12 dyn/cm 2 ) in the sme cell further incresed the mgnitude of l currents ( /pf). The FTR inhiitor FTR(inh)-172 completely inhiited the currents in response to the FTR cocktil ut hd no effect on the -stimulted currents (Fig. 3, nd ). The iophysicl properties of the -stimulted currents in oth cell types were identicl to those of the 2 -ctivted nd TP-stimulted l currents previously reported in these cells (9), with time-dependent ctivtion, outwrd rectifiction, nd reversl t. While FTR(inh)-172 hd no effect, the 2 -ctivted l chnnel locker niflumic cid significntly inhiited -stimulted l currents in the ML (Fig. 3). Thus, exposure resulted in ctivtion of l currents in mouse cholngiocytes lcking FTR expression (MS) nd in the presence of phrmcologicl inhiitors of FTR in FTRexpressing cholngiocytes (ML). 3) Studies were performed in humn Mz-h-1 cells with nd without FTR silencing. Trnsfection of cells with FTR sirn significntly decresed expression of FTR protein y 54 6%; however, no significnt differences in the mgnitude of l currents in response to (sher.18 dyn/cm 2 ) were oserved compred with cells trnsfected with nontrgeting sirn (Fig. 4). Together, these findings provide evidence tht FTR contriutes negligily to -stimulted l currents nd suggest the existence of lternte (non-ftr) chnnels, which contriute to stimulted l trnsport. Flow-stimulted currents re dependent on [ 2 ] i. It hs previously een shown tht fluid (sher) increses [ 2 ] i in rt ile duct epithelil cells (34, 48), isolted duct segments (34), nd single Mz-h-1 cells (48). To confirm tht sher increses [ 2 ] i in mouse cholngiocytes, studies were performed in MS nd ML. In oth cell types, exposure to resulted in n increse in [ 2 ] i (Fig. 5). The increse in [ 2 ] i ws sher-dependent, s [ 2 ] i ws incresed to significntly greter extent y exposure to sher of.9 thn.2 dyn/cm 2 in oth cell types. However, there ws no significnt difference in the mgnitude of the -stimulted [ 2 ] i etween MS nd ML t the sme rtes (Fig. 5). In prllel whole cell ptch-clmp studies, -stimulted l currents were dependent on 2, s removl of 2 from the ptch pipette (2 mm EGT) nd th completely inhiited l currents. To determine the reltive contriutions of intr- vs. extrcellulr 2 to sher-stimulted l currents, 2 ws individully removed from the pipette or the th nd l currents were mesured in response to sher. While removl of extrcellulr 2 hd little effect on the mgnitude of l currents, removl of intrcellulr 2 significntly decresed sher-stimulted l currents (Fig. 5, nd D), suggesting tht the opertive chnnels re primrily dependent on intrcellulr 2 stores for ctivtion. Functionl expression of TMEM16 in humn intrheptic cholngiocytes. We previously demonstrted tht iliry epithelil cells (including humn, rt, nd mouse) express the 2 -ctivted l chnnel TMEM16 (9). We performed dditionl studies to determine if H69 humn intrheptic cholngiocytes lso express functionl TMEM16 chnnel proteins. 1) Expression of TMEM16 ws confirmed y Western lotting (Fig. 6). 2) Whole cell currents were mesured in response to incresing [ 2 ] i. s shown in Fig. 6, ddition of 1 M free 2 in the ptch pipette incresed l currents from.9.3 to /pf (P.1, n 5). 2 -ctivted currents exhiited time-dependent ctivtion t memrne potentils ove 6, outwrd rectifiction, 2-2 urrent density (-/pf) Mzh1 6 s.5 n ms H69 TMEM16 β ctin Fig. 6. Functionl expression of TMEM16 chnnels in humn intrheptic iliry epithelil (H69) cells. : representtive Western lot demonstrting TMEM16 protein in H69 nd Mz-h-1 cells. : representtive whole cell current recordings. urrents were mesured in stndrd extrcellulr (th) nd intrcellulr (pipette) solution uffered with 1 M free intrcellulr 2 in the pipette. urrents were mesured t 8 (Œ), representing l current, nd( ), representing K current. Voltge-step protocols were otined t (initil) nd (mximl current response). urrents demonstrte time-dependent ctivtion t memrne potentils 6. I-V plots were generted from step protocols during sl ( ) nd 2 -stimulted (Œ) conditions. : cumultive dt demonstrting mximl current density in H69 cells mesured t 8 in response to [ 2 ] i (1 M) or (sher.18 dyn/cm 2 ). Vlues re mens SE; n 4 5. Downloded from y on Decemer 1, 217 JP-Gstrointest Liver Physiol doi:1.1152/jpgi

7 nd reversl (equilirium) potentil (E l ) ner, iophysicl properties consistent with TMEM16. Thus, humn intrheptic H69 cells express TMEM16 nd functionl 2 -ctivted l currents similr to other iliry models. To our knowledge, these findings represent the first iophysicl chrcteriztion of 2 -ctivted l chnnels in humn intrheptic iliry epithelil cells. Flow-stimulted l currents re medited y TMEM16. To determine if TMEM16 represents the -stimulted l chnnel, studies were performed in Mz-h-1 cells with nd without TMEM16 silencing. Trnsfection of cells with nti- TMEM16 sirn significntly decresed expression of TMEM16 mrn y 66 13% nd protein y 64 8% nd significntly inhiited l currents in response to (sher.12 dyn/cm 2 ) compred with cells trnsfected with nontrgeting sirn (Fig. 7). Together, these studies demonstrte tht TMEM16 contriutes to -stimulted l currents in humn iliry cells. While TMEM16 sirn significntly inhiited -stimulted currents with iophysicl properties control-mock 225 urrent density (-/pf) n 6 s 2 ms control-mock sl TMEM16-siRN sl consistent with the 2 -ctivted l chnnel, in 14% of cells, currents with time-dependent inctivtion (s shown in Fig. 2) were still oserved (dt not shown). The trnsient -ctivted K currents oserved in some trils were not significntly ffected y TMEM16 silencing (dt not shown). Together, these studies demonstrte tht TMEM16 contriutes to -stimulted l currents in humn iliry cells. Flow-stimulted l currents re dependent on sher rte, ut not viscosity. s shown in Fig. 8, exposure to very low sher (.1 dyn/cm 2 ) did not ctivte currents in most of the whole cell current recordings. urrents were not oserved until sher of.2 dyn/cm 2 ws pplied (Fig. 8). Whole cell l currents (mesured t 8 ) reched mximl vlues t sher rte of.12 dyn/cm 2. Higher rtes did not result in further increse in the mgnitude of currents. The clculted sher rte t hlf-mximl current (k ½mx ) ws.5 dyn/cm 2 (Fig. 8). s sher is determined not only y rte, ut lso y viscosity, the effects of incresing viscosity on the mgni- TMEM16-siRN D TMEM16 (reltive density).2 n 6 s 2 ms control mock sirn TMEM16 β ctin control mock sirn Fig. 7. TMEM16 contriutes to -stimulted currents. Representtive whole cell current recordings from Mz-h-1 cells trnsfected with nontrgeting sirn [control-mock ()] or TMEM16 sirn () under sl nd -stimulted conditions. Flow exposure (sher.12 dyn/cm 2 ) is indicted y horizontl r. urrents were mesured t 8 (Œ)ndt( ). Voltge-step protocols were otined t (sl) nd (mximl current response). urrents demonstrte time-dependent ctivtion t memrne potentils 6. I-V plots were generted from step protocols during sl ( ) nd -stimulted (Œ) conditions. : cumultive dt demonstrting mximl current density mesured t 8 in response to in cells trnsfected with nontrgeting sirn (mock) or TMEM16 sirn. Vlues re mens SE; n 5. P.5 vs. control. D: representtive Western lot nd cumultive dt demonstrting TMEM16 protein levels in control cells, cells trnsfected with nontrgeting sirn (mock), nd cells trnsfected with TMEM16 sirn. P.5 vs. control or mock. -ctin ws used s loding control. G93 Downloded from y on Decemer 1, 217 JP-Gstrointest Liver Physiol doi:1.1152/jpgi

8 G urrent density (-/pf) Normlized current s Sher:.3,.6 dyne/cm 2 K 1/2 =.45 dyne/cm dyne/cm 2 n. s..24 n. s. -8 Pre ml/min % Dextrn Fig. 8. Flow-stimulted currents re dependent on rte. : representtive whole cell recording. urrents were mesured t 8 (Œ) nd( ). Flow exposure (sher. 3 or.6 dyn/cm 2 ) is indicted y horizontl r. : (sher) rte-response curve for l currents. Dt re plotted from mximum current density mesured t 8 in response to different rtes. Vlues re mens SE (n 4 15) fit to dt for sher ctivtion of whole cell currents s follows: y V mx x n /(K n x n ), where y is the current density, x is sher, V mx is mximum current density t 8, K is hlf-mximum sher, nd n is the Hill coefficient. : cumultive dt demonstrting normlized l currents reltive to sl vlues in response to low (.5 ml/min) nd high (2 ml/min) with nd without 5% dextrn (n 5 1). ddition of dextrn did not increse mgnitude of -stimulted currents (P not significnt). tude of currents were determined. These studies were designed to determine if the ctivtion of l chnnels y ws due to direct mechnicl effect on the chnnel or n indirect effect of on the delivery of solule fctor to the picl memrne. Incresing viscosity y inclusion of 5% dextrn to the uffer solution (incresing viscosity from.95 to 2.8 cp) did not increse the mgnitude of currents t low (.5 ml/min) or high (2 ml/min) rte (Fig. 8). Thus the currents re dependent on the rte, ut not viscosity. This is consistent with the delivery of solule sustnce in the perfuste to the site of chnnel ctivtion. Flow-stimulted l currents re medited y extrcellulr TP. We previously showed tht -stimulted increses in [ 2 ] i re medited in prt y relese of TP, inding of TP to P2Y receptors, nd relese of 2 from intrcellulr stores (48). To determine if -stimulted TMEM16 currents re dependent on ctivtion of memrne P2Y receptors, studies were performed in the presence or sence of P2Y receptor ntgonists. In the presence of the nonspecific P2 receptor inhiitor surmin or the structurlly unrelted P2Y inhiitor rective lue 2, -stimulted increses in [ 2 ] i nd l currents were inhiited (Fig. 9). Similrly, in the presence of pyrse in the th nd perfuste to hydrolyze TP, stimulted increses in [ 2 ] i nd l currents were inhiited. Together, these studies demonstrte tht the TMEM16medited l currents stimulted y re dependent on extrcellulr TP nd P2Y receptor stimultion. Flow-stimulted l currents re regulted y PK. PK hs een shown to e involved in signl trnsduction in response to mechnicl stimuli in epithelil nd endothelil cells (9, 27, 4, 48). Swelling in Mz-h-1 cells results in rpid trnsloction of PK to the plsm memrne, nd inhiition of PK locks volume-stimulted TP relese nd volume-stimulted l currents (4). In contrst, -stimulted TP relese is regulted y PK (45). Thus, distinct PK isoforms pper to e involved in trnsducing different mechnosensitive stimuli to intrcellulr signls involved in purinergic signling. To evlute potentil role of these PK isoforms in -stimulted l chnnel regultion, studies were performed in the presence or sence of specific ntgonists. 1) Whole cell ptch-clmp studies were performed utilizing intrcellulr dilysis to deliver specific inhiitor of PK, PK pseudosustrte (45), directly to the cell interior. Intrcellulr dilysis with the PK pseudosustrte or scrmled PK peptide s control did not ffect the stimulted l currents (Fig. 1). 2) Flow-stimulted currents were mesured in the presence or sence of the PK inhiitor Gö 6976 (1 M). In contrst to PK inhiition, inhiition of PK completely nd reversily inhiited stimulted l currents (Fig. 1). Thus, while -stimulted TP relese is, in prt, dependent on PK (48), -stimulted l chnnels re regulted y the conventionl isoform PK. DISUSSION The present studies provide evidence tht fluid is stimulus for TMEM16 ctivtion nd tht TMEM16 represents the downstrem effector pthwy mediting purinergic (P2) receptor-medited secretion. Evidence for this includes the following. 1) Fluid ctivtes l currents with outwrd rectifiction, time-dependent ctivtion t depolrizing potentils ove 6, nd reversl potentil ner, iophysicl properties consistent with TMEM16 (5, 43, 49). 2) Flow-stimulted currents re inhiited y the 2 -ctivted l chnnel lockers niflumic cid nd DIDS, ut not y the FTR inhiitors FTR(inh)-172 nd MlH. 3) MS, which express TMEM16, ut not FTR, exhiit roust -stimulted l currents. 4) Fluid increses [ 2 ] i, nd inhiition of the -stimulted increse in [ 2 ] i prevents current ctivtion. 5) Flow-stimulted currents exhiit iophysicl properties similr to those ctivted y extrcellulr TP (9) nd removl of TP, or lockde of P2 receptors olishes -stimulted currents. 6) FTR sirn hs no effect on the -stimulted currents, while TMEM16 Downloded from y on Decemer 1, 217 JP-Gstrointest Liver Physiol doi:1.1152/jpgi

9 G urrent density (-/pf) 12 s pyrse 12 s R2 12 s 14 7 sirn significntly inhiits the -stimulted currents. Together, the findings re consistent with TMEM16 s the opertive l chnnel responsile for the -stimulted memrne currents through pthwy involving TP relese, inding P2 receptors, nd increses in [ 2 ] i. Thus, TMEM16 plys criticl role in mechnosensitive signling nd is, therey, n importnt regultor of iliry epithelil secretion nd ile formtion. In light of recent studies demonstrting tht the mechnicl effects of fluid, or sher stress, t the picl memrne of iliry epithelil cells re roust stimulus for TP relese (48), model emerges in which mechnosensitive TP relese nd l secretion represent dominnt pthwy regulting iliry secretion. While FTR hs recently een shown to e mechnosensitive in other model systems (5), there is no evidence in the present studies tht FTR contriutes to the -stimulted response. 1) The iophysicl properties of the -stimulted l currents re distinct from FTR, which generlly exhiits liner current-voltge reltion nd no time dependence (28). 2) Phrmcologicl inhiition, or moleculr silencing, of FTR ffected neither the ctivtion nor the mgnitude of D F34/f38 rtio F34/f38 rtio [ 2+ ] i nm Time (s) surmin Time (s) Fig. 9. Flow-stimulted l currents re dependent on extrcellulr TP-medited P2 receptor stimultion. : representtive whole cell ptch-clmp recordings in response to (sher.18 dyn/cm 2, indicted y horizontl r) in control conditions (top), in the presence of the TP-hydrolyzing enzyme pyrse (5 U/ml, middle), nd in the presence of the P2 receptor ntgonist rective lue 2 (R2, 25 M, ottom). : cumultive dt representing mximum current density mesured t 8. Vlues re mens SE (n 4 11 ech). pyrse, surmin, nd R2 significntly inhiited -stimulted currents (P.5). : -stimulted increses in fur 2 fluorescence in control conditions (top) nd in the presence of the P2Y receptor ntgonist surmin (1 M, ottom). D: cumultive dt representing mximum increse in [ 2 ] i. Vlues re mens SE (n 6). Significntly different from control [ 2 ] i (P.5). -stimulted currents. 3) MS, which do not express FTR, still exhiited -stimulted currents. While previous studies hve highlighted the importnce of FTR in driving ductulr secretion, recent evidence suggests tht l secretion medited y extrcellulr TP is functionlly of greter significnce. In response to TP, the unitry currents from single cells nd the short-circuit current response from intct iliry epithelil monolyers re severlfold greter thn those medited y increses in cmp (11). dditionlly, even cmp-medited secretion requires extrcellulr TP (37). Our present findings provide further evidence tht l secretion medited y non- FTR pthwys is functionlly importnt nd, in fct, my e the predominnt pthwy mediting ductulr ile formtion. The mgnitude of -stimulted currents incresed with incresing sher rte, with clculted k ½mx of.5 dyn/cm 2 nd steep ctivtion curve. We did not oserve n increse in the mgnitude of -stimulted currents when sher force ws incresed y incresing the viscosity of the perfuste y the ddition of dextrn. Thus the currents re dependent on sher rte, ut not viscosity. This is consistent with the delivery of solule sustnce (TP) in the perfuste to the site of Downloded from y on Decemer 1, 217 JP-Gstrointest Liver Physiol doi:1.1152/jpgi

10 G96 6 Gö 6976 Gö s Fig. 1. Flow-stimulted l currents re regulted y PK. : representtive whole cell ptch-clmp recordings of -stimulted currents (sher.12 dyn/cm 2, indicted y horizontl r) in the presence or sence Gö 6976 (1 M) in th solution (top) or with PK-specific peptides in the ptch pipette: PK pseudosustrte (2 M, middle) or PK scrmle (2 M, ottom). urrents were mesured t 8 (Œ) nd( ). : cumultive dt demonstrting mximum current density in the presence or sence of Gö 6976 (1 M, left)orpk pseudosustrte (2 M) nd control PK scrmled peptide (2 M; right). Vlues (mens SE; n 4 5 ech) represent mximum current density mesured t 8. Flow-stimulted currents were significntly inhiited y Gö 6976 (P.5) urrent density (-/pf) 3 15 urrent density (-/pf) 8 PKζ pseudosustrte ontrol peptide 4 Downloded from y on Decemer 1, 217 chnnel ctivtion (memrne P2 receptor). Thus the currents pper to e dependent on the rte-driven delivery of TP to the memrne (sher rte), nd not on direct mechnicl stimultion (sher stress) on the chnnel itself. In the sence of TP (pyrse in the perfuste), -stimulted currents were significntly reduced. Furthermore, there is no evidence tht TMEM16 is itself mechnosensitive chnnel, nd while TMEM16 is ctivted y memrne distension due to cell swelling in other models (1), this my occur vi swellingstimulted TP relese nd utocrine stimultion of memrne P2 receptors (41). If these studies, performed in humn nd mouse iliry epithelil cells, trnslte to in vivo conditions, severl points, s well s uncertinties, deserve to e highlighted. First, the ctul rtes nd sher force long the intrheptic ile ducts re unknown. The rtes used in this study were sed on oservtions, s well s clcultions, derived from previous studies of cell nd niml models. Flow-stimulted TP relese from humn nd rt iliry cells occurs t sher rte of.16 dyn/cm 2 (27, 48), similr to the sher rtes used in these studies. Msyuk et l. (36) utilized three-dimensionl imging of the rt intrheptic iliry tree to evlute ile duct size nd then used mthemticl clcultions to estimte the rtes in the ducts. On the sis of these clcultions, the estimted rte ws 11.1 nl/min in smll (5 m) ile ducts nd 1,64 nl/min in lrger (225 m) ducts, corresponding to sher force of.14 dyn/cm 2. These clcultions utilized constnt vlue for viscosity nd, hence, presumed wll sher stress tht is constnt throughout the system. While our present studies utilized sher forces within this clculted rnge, direct evlution of rtes nd sher force in the smll intrheptic ile ducts requires technicl dvnces. JP-Gstrointest Liver Physiol doi:1.1152/jpgi

11 Second, while the mjority of -stimulted l currents demonstrted outwrd rectifiction nd time-dependent ctivtion t depolrizing potentils ove 6, in 15% of cells the -stimulted l currents demonstrted slightly different iophysicl properties (time-dependent inctivtion) tht were unffected y TMEM16 silencing. Thus distinct l chnnel, of unknown moleculr identity, ppers lso to contriute to the -stimulted currents. These iophysicl properties re consistent with the swelling-ctivted l currents previously descried in iliry epithelil cells (42). In other cell types, currents with these iophysicl properties hve een ttriuted to l chnnel protein 3 (l-3) (26); however, l-3 hs not een definitively identified in iliry epithelium. The possiility tht other TMEM16 isoforms (f, j, nd k), previously identified in iliry epithelium (9), form novel l -permele heteromultimers with distinct iophysicl properties cnnot e excluded. Third, in the mjority of studies, -ctivted l currents were preceded y ctivtion of K currents. When they were oserved, the K currents ctivted rpidly in response to nd were trnsient. In secretory epithelium, it is elieved tht ctivtion of K chnnels leds to memrne hyperpolriztion to mintin the electricl driving force for continued l efflux (8). While we previously identified the 2 -ctivted K chnnels SK2 nd IK1 in iliry epithelium (1, 14), the moleculr identifiction of the -stimulted K chnnels is unknown. Fourth, the mechnism y which iliry epithelium senses nd initites the mechnosensory signling cscde is unknown. holngiocytes express primry cilium, mechnosensory orgnelle tht trnsltes to increses in [ 2 ] i nd my ply role in mechnosensitive TP relese (48). However, Mz-h-1 cells do not express primry cilium, despite exhiiting roust -stimulted TP relese, [ 2 ] i increses, nd l secretion, suggesting the existence of other mechnosensors in these cells. Furthermore, removl of the primry cilium from rt cholngiocytes (chlorl hydrte) decreses, ut does not eliminte, mechnosensitive TP relese (48). In other epitheli, mechnicl stimultion my e trnsduced through microvilli, the cytoskeleton, or other memrne proteins. The identifiction of the mechnosensors in cholngiocytes will e n importnt re for future investigtion of mechnosensitive signling in the liver. Lstly, normlities in mechnosensitive signling long the ile duct my hve importnt implictions during cholesttic liver disese. decrese in ile ssocited with cholestsis would inhiit mechnosensitive TP relese, 2 signling, nd l secretion, thus decresing ductulr secretion nd ffecting the volume nd composition of ile. onversely, trgeting the elements of the mechnosensitive signling pthwy, including TP relese, P2 receptors, nd/or TMEM16, my provide new nd innovtive strtegies to increse ductulr ile formtion for the tretment of cholesttic liver diseses. KNOWLEDGMENTS MS nd ML were kindly provided y Dr. Ginfrnco lpini (Texs & M Helth Science enter ollege of Medicine, Temple, TX). GRNTS This study ws supported y mericn Liver Foundtion Liver Scholr wrd (. K. Dutt), the hildren s Medicl enter Foundtion (. K. Dutt), G97 ystic Firosis Foundtion Grnt FERN8G (. P. Fernchk), nd Ntionl Institute of Dietes, Digestive, nd Kidney Diseses Grnt DK (. P. Fernchk). DISLOSURES No conflicts of interest, finncil or otherwise, re declred y the uthors. UTHOR ONTRIUTIONS.K.D. nd.p.f. re responsile for conception nd design of the reserch;.k.d., K.W.,.-K.K., nd.k. performed the experiments;.k.d., K.W.,.-K.K.,.K., nd.p.f. nlyzed the dt;.k.d., K.W.,.-K.K.,.K., nd.p.f. interpreted the results of the experiments;.k.d. nd.p.f. prepred the figures;.k.d. nd.p.f. drfted the mnuscript;.k.d. nd.p.f. edited nd revised the mnuscript;.k.d. nd.p.f. pproved the finl version of the mnuscript. 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