Evidence of hydrogen ion secretion from the human gall bladder in vitro

Transcription

1 554 Department of Mediine, Royal Infirmary of dinburgh, and Department of Pathology, University of dinburgh, Sotland J N Plevris P Hayes I A D Bouhier Department of Pathology D J Harrison orrespondene to: Dr J N Plevris, Department of Mediine, Royal Infirmary of dinburgh, Lauriston Plae, dinburgh H8 9YW. Aepted for publiation 11 July 1991 Gut, 1992,33, videne of hydrogen ion seretion from the human gall bladder in vitro J N Plevris, P Hayes, D J Harrison, I A D Bouhier Abstrat Gail bladder bile is more aid that hepati bile and this has been attributed to biarbonate absorption by the gall bladder epithelium. he aim of this study was to investigate in vitro the aid base hanges that our aross the human gall bladder muosa. Fresh gall bladder tissue was obtained at holeystetomy and plaed in an Ussing hamber and perfused with Ringer- Krebs gluose biarbonate solution. he viability of the gall bladder was assessed by measuring the potential differenes aross the epithelium and by the morphology of the epithelial ells at the end of the experiments. Aliquots from the solutions were taken at two, 45 and 7 minutes and po2, hydrogen ion and biarbonate onentrations were measured. In the muosal side of the hamber a onsistent and signifiant derease was observed from two minutes to 7 minutes in biarbonate onentration while po2 and hydrogen ion onentrations signifiantly inreased. he degree of inflammation orrelated weil with the ability for aidifiation, the more inflamed the tissue the less its ability to aidify. When the gail bladder was exposed to amiloride or sodium free solution aidifiation was abolished in the muosal side. When tissue metabolism was irreversibly inhibited by exposure to formaldehyde, hydrogen ion onentration and po2 were signifiantly dereased in the muosal side of the hamber ompared with the viable gail bladder. he human gail bladder is apable of sereting aid and this may be an important mehanism for preventing alium preipitation and gail stone formation. Gall stone disease is a ommon ause of morbidity and mortality and holoystetomy represents a signifiant perentage of major upper abdominal operations in western soiety. Although onsiderable researh has been onentrated on the pathogenesis of gall stone formation, this has often foused on the biohemial hanges that our in the bile. It is only within the last 1 years that the importane of the events in the gall bladder and in partiular the possible ontributions of the gall bladder muosa to lithogenesis has been reognised. he gall bladder muosa has one of the highest rates of water absorption in the body and an 8 to 9% derease in the initial volume of bile is ahieved within the gall bladder. his is ahieved by oupling of ative sodium transport and passive water absorption resulting in isotoni fluid absorption. ' Fluid transport is subjet to physiologial regulation and is higher in the daytime.23 letrolyte transport has been extensively investigated and hloride is atively absorbed in exhange for biarbonate.4 Potassium moves passively from the muosa to the serosa aording to eletrohemial gradients,5 and alium is also absorbed and its distribution aross the gall bladder epithelium is onsistent with passive transport.6 Its ontribution to gall stone formation is inreasingly reognised. Most of the organi omponents of bile - namely, bile aids, leithin, bilirubin, and holesterol are absorbed to a limited degree by the human gall bladder muosa.57 8 here is more reent evidene that the human gall bladder has not only absorptive but also seretory properties. Fluid absorption an be reversed to net seretion with feeding in hroni inflammation or with the use of pharmaologial agents (prostaglandins, prostaylin, gut hormones, et).9 An inreasing number of biliary proteins are known to be sereted from the gall bladder muosal ells suh as muin, immunoglobulins, et.-'>2 In man as well as in other speies there is deline in the ph of the gall bladder bile ompared with hepati bile. Initially this was thought to be the result of biarbonate reabsorption by the gall bladder muosa.' 5 Studies in rabbit, guinea pig, and neturus gall bladders have produed evidene for muosal hydrogen seretion during sodium reabsorption, probably representing a sodium/ hydrogen exhange.'3-'7 In a more reent study it has been suggested that the anine gall bladder has the ability to serete hydrogen ions in vivo. " here are, however, no studies on the human gall bladder muosa dealing with the mehanisms of bile aidifiation and it is not lear whether this is beause of hydrogen ion seretion or biarbonate reabsorption. Aid seretion by the gall bladder has important impliations for gall stone formation beause the majority of gall stones ontain alium arbonate and hanges in the ph of the bile are of ritial importane in influening the alium solubility in bile. '1 he present study was undertaken to investigate the role of the gall bladder epithelium in the aidifiation proess of bile in vitro using fresh viable human gall bladder muosa obtained at holeystetomy. Methods Part of this work has been presented at the British Soiety of Gastroenterologv Studies were done using fresh human gall Autumn Meeting, Sheffield 1988, and has been published as an Abstrat in Gut (Otober 1988). bladder muosa obtained at the time of eletive Gut: first published as /gut on 1 April Downloaded from on 18 August 218 by guest. Proteted by opyright.

2 videne ofhydrogen ion seretionfrom the human gall bladder in vitro U,._ (U a) n holeystetomy usually for gall stone disease. Patient's sex, age, and type of gall stones, if any, were reorded. he gall bladder tissue was retrieved within 15 minutes of the ligation of the ysti artery. A irular piee of the gall bladder wall (d=12 mm) was removed using a tissue punh and plaed in Ringer-Krebs gluose biarbonate solution ( 9% Nal (-154 M), 1-15% K1 (-154 M), 1-22% a12 (-11 M), 2-11% KH2PO4 (154 M), 3-8% MgSO4. 7H2 (-154 M), 1-3% NaHO3, 5 4% gluose ( 3 M)) at 4 and preoxygenated with 95% 2. 5% O2. o minimise tissue hypoxia, this was done as soon as the gall bladder was removed. he gall bladder tissue was transferred to the laboratory and within five minutes the muosa was stripped by blunt dissetion from the musular part of the wall, rinsed with Ringer- Krebs biarbonate gluose (RKGB) solution to remove debris and bile and mounted in an Ussing hamber. he Ussing hamber onsisted of two 2 ml volume ompartments eah of whih ommuniated with a glass tube above through two plasti tubes (inlet and outlet) (Fig 1). he gall bladder tissue separated the two ompartments reating a muosal ompartment at the muosal site and a serosal ompartment on the opposite side. he term 'serosa' is used throughout to indiate the non-luminal surfae of the muous membrane. Both ompartments were filled with 1 ml RKGB through the glass tubes (2 ml) in eah ompartment of the hamber and 8 ml at eah glass tube). he muosal ompartment was sealed while 95% 2/5% O2 (21/min) was bubbled through to the serosal ompartment only. Preliminary experiments had shown that PO2 between muosal and serosal ompartments did not differ signifiantly if 95% 2/5% O2 was bubbled into the serosal ompartment only and this was preferred in order to avoid disruption of the 'unstirred water layer' in the muosal side.s-' he whole system was overed by a thermostati water jaket to maintain a onstant temperature of 37 throughout ime (minutes) Figure 1: Potential differene aross the human gall bladder muosa; (A) represents a viable gall bladder with an initial potential differene of 7 mv. Oxygen deprivation or immersion in formaldehyde resulted in a drop in the potential differene aross the epithelium but reovered when oxygen was reintrodued. (B) represents a gall bladder with an initial potential differene ofless than 2 mv. Although potential differene reovered after 15 minutes, oxygen deprivation resulted in a non-reversible fall in the potential differene. Gall bladders with initial potential differene <2 mv were exluded from further study. 555 the experiment. A pair of silver/silver hloride matrix 1 mm eletrodes (lark letromedial Instruments, Reading, Berks, UK) monitored the potential differene aross the two sites of the tissue and used as an index of viability of the tissue. ah study lasted for 7 minutes. One millilitre of the solution was removed through a miropipette from eah ompartment of the Ussing hamber at two minutes, 45 minutes and 7 minutes and was immediately analysed in an 132 ph/blood gas analyser (Instrumentation Laboratory System, Lexington, MA, USA) for PO2, po2, hydrogen ion ([H+]), and biarbonate ([HO3-I) onentrations. Gall bladders whih were marosopially grossly distorted or damaged were exluded from further study. XPRIMNS Forty gall bladders were studied. he experiments were divided into three groups. First group Five gall bladders were studied. he effet of oxygen deprivation and immersion in formaldehyde 4% on the resting potential differene was observed. 95% 2/5% O2 was bubbled at a steady flow rate of 2 1/min from time to 2 minutes, then stopped for 1 minutes from time 2 to 3 minutes and subsequently ontinued at the same flow rate until the end of the experiment. hereafter the gall bladder muosa was exposed to 4% formaldehyde for two minutes, rinsed with RKGB solution and remounted to the Ussing hamber and studied for further 1 minutes. he potential differenes were monitored ontinuously throughout the experiment. Seond group wenty five gall bladders were studied. wenty one were proessed aording to the standard protool to maintain viability and four were immersed in formaldehyde 4% for two minutes before being mounted on the Ussing hamber. he hydrogen ion onentration, biarbonate onentration po2 and P2 were measured at the beginning (two minutes) at 45 minutes and the end of the experiments (7 minutes). he resting transepithelial potential differene was monitored throughout. he aim was to study the aidifiation apaity of the viable gall bladder ompared with non-viable tissue. hird group en gall bladders were studied. In the first set of experiments involving six gall bladders the muosal bathing solution was replaed by sodium free Krebs-Ringer solution (Nal and NaHO3 removed) and the analytes mentioned above were measured. In a seond set of experiments involving four gall bladders 2 mm amiloride (a speifi Na+/H+ inhibitor) was introdued into the muosal ompartment and its effet on aidifiation was observed after 45 minutes. Gut: first published as /gut on 1 April Downloaded from on 18 August 218 by guest. Proteted by opyright.

3 556 Plevris, Hayes, Harrison, Bouhier +- I 16 'a -I A 'O[ m U -5._ ~~~~~~a) (. m. B (.) 554-l -1 B -74 L 1-33 Muosal Serosal 'a I -5 F N,. 5r S5F n,. _Z -gi I -} - l- }Z 17 Viable Non-viable Viable Non-viable Viable Non-viable gall bladder gall bladder gall bladder gall bladder gall bladder gall bladder Figure 2: Differenes in hydrogen ion onentration (A), biarbonate onentration (B), andpo2 () between 7 and two minutes in the muosal and serosal side ofthe viable and non-viable gall bladders (p< 5, +p<-3, + +p<-2, p<.1). Figure 3: Differenes in hydrogen ion seretion with histology (grade I (mild) to grade 3 (severe) holeystitis) p<.1 4-.)- o'.- ~ I PAHOLOGY A small sample of the tissue under study was fixed in 1% buffered formalin before eah Ussing hamber experiment. he rest of the tissue was also fixed in 1% buffered formalin at the end of the experiment. An experiened histopathologist examined eah gall bladder blindly on two oasions and reported on the degree of holeystitis and the viability of the tissue aording to ell morphology at the beginning and end of the experiments. he degree of holeystitis was graded from 1 (mild) to 3 (severe) aording to the appearane of the muosa, musle layer thikness, presene of Rokitansky-Ashoff sinuses and degree of inflammatory proess.22 he morphology hanges were reorded as (healthy looking ells), 1 (mild morphologial hanges suh as ell oedema, presene of granules, vauolation), 2 (moderate but without evidene of ell nerosis). he gall bladders used in the above experiments were seleted aording to the following riteria: (a) satisfatory marosopi appearane with no obvious uleration or fibrosis at the beginning of the study and no obvious damage during the study; (b) expression of a resting potential differene more than 2 mv of serosa positive; () data from muosa with severe hanges in ell morphology were exluded from further analysis. SAISIAL ANALYSIS Data are expressed as mean standard error of mean (SM). he data were not normally distri- 2 r- uraue Z (n 6) = Degree of holeystitis Grade 3 (n = 6) buted and were analysed using non-parametri tests; Mann-Whitney test was used for unpaired data and Wiloxon's test for paired data. A p value of -5 was taken as signifiant. Results In the first set of experiments the gall bladders (five) doumented a mean potential differene of 75 ± 3 5 mv serosa positive after five minutes in the hamber. his potential differene remained quite stable throughout the ensuing 15 minutes. here was a signifiant drop in the potential differene when 95% oxygen 5% O2 was disontinued, but when reapplied the potential differene reovered to levels similar to those before oxygen deprivation indiating that hypoxia had a diret effet on reduing the resting potential differene. When those gall bladders were immersed in formaldehyde 4% and remounted on the Ussing hamber, however, there was a dramati and irreversible drop in the potential differene indiating nonviability of the tissue (Fig 1). he P2 was measured in both ompartments at two minutes, 45 minutes, and 7 minutes. Although 95% 2/ 5% O2 was bubbled in the serosal ompartment only, there was no differene in the P2 between the muosal and serosal ompartment (25-3 (2-1) pka v 23-5 (2-2) pka respetively) whih shows that the diffusion of oxygen through the gall bladder muosa was adequate. It was thought therefore unneessary to oxygenate the muosal ompartment diretly, as this would disrupt the 'unstirred water layer'. All gall bladders showed evidene of hroni holeystitis (three grade 1, two grade 2) without any signifiant hanges in ell morphology at the beginning and end of the experiments. In the seond set of experiments 25 gall bladders were studied (18 women, seven men; 13 ontained holesterol stones, nine pigment stones, three biliary sludge). Four of those were immersed in formaldehyde and used as ontrols and from the remaining 21, six showed evidene of grade 3 hroni holeystitis and were not inluded in the analysis outlined below. here was a signifiant inrease in the hydrogen ion onentration from two minutes to 45 minutes.6 Gut: first published as /gut on 1 April Downloaded from on 18 August 218 by guest. Proteted by opyright.

4 videne ofhydrogen ion seretion from the human gall bladder in vitro Figure 4: Differenes in hydrogen ion seretion with ell morphology hanges (grade : healthy ells, grade 1: mild morphologial hanges suggesting 'sik ells', grade 2: moderate hanges without evidene of nerosis) p<1. a) L-, F and to 7 minutes observed in the muosal ompartment (p<1) while in the serosal ompartment there was a signifiant derease in hydrogen onentration (p<5). By ontrast in the non-viable gall bladder (those immersed in formaldehyde) there was a signifiant drop in hydrogen onentration on the muosal site without any signifiant hange of hydrogen onentration in the serosal side (Fig 2a). In the viable gall bladder biarbonate onentration signifiantly dereased in the muosal ompartment from two minutes to 45 and 7 minutes (p<2) while on the serosal side there was a signifiant inrease of biarbonate onentration from two minutes to 7 minutes. In the nonviable gall bladder an inrease of biarbonate onentration was seen in both ompartments (Fig 2b). In the viable gall bladder there was a signifiant inrease in po2 between two and 7 minutes on the muosal site (p<3) while in the serosal ompartment a signifiant derease in PO2 was observed (p<5). In the non-viable gall bladder there was a drop in po2 in both ompartments being signifiant in the muosal side (p<1) (Fig 2). In the viable gall bladder the mean potential differene did not signifiantly hange throughout the experiments. here was, however, a signifiant differene in the potential differene between the viable and the non-viable gall bladders studied (6&3 (2 6) to 1L3 (-9), p<.1) at five minutes. he potential differene of the non-viable gall bladders rapidly delined to. When the aidifiation ability of the 21 gall bladders was plotted against the degree of hroni holeystitis there was a progressive and statistially signifiant derease of hydrogen ion seretion from grade 1 (mild) to grade 3 (severe) holeystitis (Fig 3). he same was true when hydrogen ion seretion was plotted against the degree of ell morphology hanges graded from to 2 indiating that gall bladder epithelia with impaired viability had a tendeny to serete less hydrogen ions (Fig 4). here were no differenes in the aidifiation apaity of the gall bladders with sex or type of gall stones, although epithelia from gall bladders with pigment stones had a higher ability to absorb biarbonate (A[HO3]=-1 68 ( 49) mmol/l (pigment) v 3-8 ( 74) mmol/l (holesterol), p< 1). In the third set of experiments (1) RKGB solution was replaed with sodium free isotoni solution in the muosal side. No signifiant Grade (n = 5) Grade 1 (n = 7) Grade 2 (n = 3) Degree of ell morphology hanges 2r I) -1 _--7 A [H+] A [Ho3- A lpo2i (nmol/l) (mmol/l) (mm Hg) Amiloride 2 (mmol/l) (muosal side) (n = 4) Figure 5: ffet ofmuosal appliation ofamiloride (2 mm) on hydrogen ion seretion. 557 differenes in the [H+], [HO3 ] and po2 were observed in any site of the Ussing hamber, between two and 7 minutes of the experiment. Similarly, the use of amiloride (2 mm) in the muosal side abolished aidifiation (Fig 5). Disussion he Ussing hamber method has been adapted and used by many investigators sine its introdution by Ussing and Zerhan in It is a valid method of keeping a biologial preparation viable during the period of investigation and has been used to study eletrophysiologial, seretory, and absorptive properties of tissues in vitro. issues previously used inlude frog skin, intestine, bladder, and animal and human gall bladder. For years it has been known that gall bladder bile is more aid ompared with hepati and the differene in the ph has been attributed to biarbonate absorption.' 5 In this study we have shown that fresh viable human gall bladder muosa is apable of aidifying physiologial solutions in vitro. his aidifiation is a funtion of viable tissue; it was redued in more inflammed gall bladders or when the epithelial ells beame 'sik' during the study and was lost when the muosa was non-viable. Aidifiation was abolished when the gall bladder epithelium was exposed to. sodium free solution or in the presene of high onentration of amiloride in the muosal ompartment. In our study where human gall bladders were used to investigate aid seretion in vitro, the gall bladder epithelium appeared apable of inreasing the hydrogen onentration in muosal side with simultaneous derease of hydrogen onentration in the serosal side whih suggests that hydrogen ions were transferred from the serosal to muosal side of the tissue. he onomminant derease of biarbonate onentration with inreased po2 in the muosal side indiates that this aidifiation is not simply the result of biarbonate reabsorption. It appears that hydrogen ions produed by the muosal ells reat with biarbonate to form o2 and water with a derease in biarbonate ions. he observed differenes in po2 between the viable and non-viable gall bladders ould be explained on the basis that a non-viable gall bladder muosa loses its ability to serete aid therefore less hydrogen ions are available to reat with biarbonate and also that the prodution of O2 from the muosal ell metabolism is depressed. Studies on gall bladders of other animal 1-9 Gut: first published as /gut on 1 April Downloaded from on 18 August 218 by guest. Proteted by opyright.

5 558 Plevris, Hayes, Ham'son, Bouhier speies suh as rabbit, guinea pig, neturus, and dog have shown that these tissues are apable of aidifying the muosal solutions in vitro and there is evidene that a sodium/hydrogen antiport is present at the apial site of the epithelial ell. In our experiments the use of sodium free solutions and amiloride, whih is a speifi sodium/hydrogen inhibitor, abolished aidifiation whih suggests that hydrogen seretion in the human gall bladder depends upon a sodium/ hydrogen antiport. A potential problem, with Ussing hamber studies is related to the viability of the tissue reeived at holeystetomy. he ligation of ysti artery whih is part of the operative proedure usually takes plae between five to 2 minutes before the removal of the gall bladder. he effet of hypoxia during this period has been studied24 and about 7% of the gall bladders examined immediately after the operation showed evidene of mitohondrial hange whih was attributed to anoxia and mehanial damage. he human as well as animal gall bladder, however, is a durable organ whih an rapidly reover from hypoxia and an be preserved viable for in vitro experiments for up to four hours. ' he gall bladder has been used for many years as a model to study ion transport aross the epithelia. In this study all the gall bladders showed a transmural potential differene ofmore than 2-3 mv (range mv) serosa positive. his potential differene remained stable throughout the period of experiment and we, as well as other workers,25 27 were able to show that anoxia resulted in a drop at the transmural potential diffferene refleting a reduing viability of the tissue whih was reversible if the anoxi period was less than 1 minutes. Similar results were produed after immersion in formaldehyde with a permanent drop of the potential differene indiating loss of viability. he ontinuous transpotential differene monitoring therefore has been used as a reliable method of monitoring the viability of the tissue and allow further study of the muosal funtion. he transmural potential differene of the gall bladders under study was in aordane with the literature. Ross and others25 arried out studies on the eletrial properties of the human gall bladder and on 46 gall bladders the mean transpotential differene was 7-6 mv while other investigators26 later showed that inflamed gall bladders have a lower resting transpotential differene whih is dependent upon the degree of inflammation, the variation in time between the ligation of ysti artery, and the transfer to the laboratory and the methodology used for studying the eletrial properties of the tissue. For instane, tissues whih are lamped at the edges as in the Ussing hamber tehnique suffer from edge damage whih redues the transpotential differene.27 Another potential problem with physiologial studies using tissues from routine holoystetomy is that most are not histologially normal. Although it is not appropriate to extrapolate to normal gall bladders in respet of hydrogen ion seretion, the fat that we ould show hanges in the aidifiation apaity with histology, the more inflamed gall bladders being less apable to serete aid, implies that hydrogen ion seretion is a funtion of the normal human gall bladder muosa. he aidifiation apaity was impaired when the ell morphology was abnormal and was abolished in the non-viable gall bladder; these suggest that hydrogen ion seretion is a funtion of a viable tissue. here is reent supportive evidene from animal and human studies that the normal gall bladder seretes aid. Moore et al showed that the anine gall bladder is apable of sereting hydrogen ions. I Preliminary in vivo studies from the same group have shown that the diseased human gall bladder is assoiated with dereased aid output.2829 heir onlusions, however, were not based on studies in human gall bladder muosa diretly, but were inferred from biohemial analysis of gall bladder biles from laparotomy. Our data would support the hypothesis by Moore and olleagues that redued gall bladder hydrogen ion seretion is assoiated with gall stone formation. It is postulated that the observed ph hanges in our experiments with diseased gall bladders represent only a fration of the apaity whih the normal human gall bladder epithelium might possess to serete aid. More studies on fresh normal tissue are required to test this suggestion. It is now aepted that the sequene of events in the proess of gall stone formation is supersaturation of bile, nuleation, preipitation, and subsequent growth to stone sized aggregates.3 Supersaturation of bile with holesterol is present in the vast majority of patients with gall stone disease3"; but 4 to 8% of normal individuals may have supersaturated bile without ever forming gall stones.3233 herefore the proess of nuleation is important and this depends upon the presene of ertain nuleating agents or the absene of the naturally ouring inhibitors of rystal formation. alium bilirubinate or muous gluoproteins ould serve as nuleating fators34 while a bile protein whih is a holesterol rystal formation inhibitor has been proposed by Holzbah as a gall stone formation protetive protein.35 alium has long been impliated in the pathogenesis of holesterol and pigment gall stones. Pigmented gall stones are predominantly omposed of the alium salts of arbonate, bilirubinate, phosphate, and long hain fatty aids and to a lesser extent arbonate.3638 alium arbonate also preipitates on to the surfae of the holesterol gall stones and is present in most holesterol gall stones.8 he regulation of alium onentration in the gall bladder bile is therefore of ritial importane. It is postulated that aid seretion may be biologially important beause a redution in the ph of the gall bladder bile effetively lowers the biarbonate and may redue the risk of insoluble arbonate formation. As a result, the onentration of ionised alium is inreased in bile. he gall bladder epithelium, however, has the ability to absorb alium and an redue its onentration in bile by more than 5%8; bile aids also buffer the remaining ionised alium. onsequently less alium is available to form insoluble salts.39 he mehanism of aid seretion also merits Gut: first published as /gut on 1 April Downloaded from on 18 August 218 by guest. Proteted by opyright.

6 videne ofhydrogen ion seretion from the human gall bladder in vitro 559 further investigation. In this study we have shown that it is sodium dependant, therefore it is losely linked with the onentrating ability of the gall bladder beause it is known that water is passively absorbed onsequent upon the absorption of sodium from the epithelial ell. It is, however, neessary to identify other fators whih may influene aid seretion and further studies are under way to investigate this. In onlusion we have shown in this study that the viable human gall bladder is apable of sereting aid. his aid seretion probably ours through an apial Na+/H+ exhange at the muosal site of the gall bladder epithelial ell. his may represent a protetive mehanism against alium preipitation. Further studies are needed to investigate more fully the proess and identify those fators whih regulate aid seretion. We would like to thank Dr P W Flatman, Dept Physiology for advising on the Ussing hamber method, Mr B White for his tehnial assistane and the surgeons and staff of 9/1 and 7/8 operating theatres, RI for providing the gall bladders. We would also like to thank Mrs V ampbell for typing this manusript. Dr J N Plevris is a reipient of William Gibson Fellowship, Faulty of Mediine, University of dinburgh. his study was partly supported by a grant from the Lothian Health Board. 1 Diamond JM. ook F, ed. ransport mehanisms in the gallbladder. Handbook of physiology: alimentary anal. Washington D: Amerian Physiologial Soiety 1968: Diamond J. he mehanism of isotoni water transport. ] Gen Physiol 1964; 48: Diamond J. ransport of salt and water in rabbit and guinea pig gall bladder. J Gen Physiol 1964; 48: Heintze K, Petersen KU, Olles P, Saverymuttu SH, Wood JR. ffets of biarbonate on fluid and eletrolyte transport by the guinea pig gallbladder: a biarbonate-hloride exhange. J Memor Biol 1979; 45: Rose R. Absorptive funtions of the gallbladder. In: Johnson LR, ed. Physiology of the gastrointestinal trat. Vol 2. New York: Raven Press, 1981; Rege RV, Nahrwold DL, Moore W. Absorption of biliary alium from the anine gallbladder; protetion against the formation of alium-ontaining gallstones. ] Lab lin Med 1987; 11: Jayna MR, Ross P, Bakar MA, Hopwood D, Bouhier IAD. harateristis of holesterol absorption by human gallbladder: relevane to holesterolosis. J lin Pathol 1987; 4: onter RL, Roslyn JL, Porter-Fink V, DenBesten L. Gallbladder absorption inreases during early holesterol gallstone formation. AmJ Surg 1986; 151: Wood JR, Svanvik J. Gallbladder water and eletrolyte transport and its regulation. Gut 1983; 24: Wahlin, hornell, Jivegard L, Svanvik J. ffets of intraluminal prostaglandin 2 in vivo on seretory behaviour and ultrastrutural hanges in mouse gallbladder epithelium. Gastroenterology 1988; 95: Lee SP, LaMont J, arey M. 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