canalicular retention as anin vitroassay of tight junctional permeability in isolated hepatocyte...

FUNDAMENTAL AND APPLIED TOXICOLOGY 37, 71–81 (1997)ARTICLE NO. FA972309

Canalicular Retention as an in Vitro Assay of Tight JunctionalPermeability in Isolated Hepatocyte Couplets: Effectsof Protein Kinase Modulation and Cholestatic Agents

Marcelo G. Roma, Dominic J. Orsler, and Roger Coleman

School of Biochemistry, The University of Birmingham, Edgbaston, Birmingham B15 2TT, United Kingdom

Received August 29, 1996; accepted March 21, 1997

compounds (Watanabe et al., 1987; Kitamura et al., 1990;Canalicular Retention as an in Vitro Assay of Tight Junctional Fentem et al., 1990; Boyer, 1993; Wilton et al., 1994; Mag-

Permeability in Isolated Hepatocyte Couplets: Effects of Protein lova et al., 1995), lipid secretion (Crawford and Gollan,Kinase Modulation and Cholestatic Agents. Roma, M. G., Orsler,

1988), cytoskeleton function (Oshio and Phillips, 1981; Wa-D. J., and Coleman, R. (1997). Fundam. Appl. Toxicol. 37, 71–81.tanabe and Phillips, 1984), gap junction function (Reverdinand Weingart, 1988; Saez, et al., 1989), and electrophysio-A simple, fast method to evaluate acute changes of tight junc-

tional permeability in isolated hepatocyte couplets is proposed. logical investigations on membrane potential and electrolyteThe method consists of the recording of the number of canalicular transport (Graf et al., 1984, 1987; Boyer, 1993). In addition,vacuoles able to retain the previously accumulated fluorescent bile this preparation has proved to be a very useful tool for theacid analogue cholyl-lysyl-fluorescein (CLF), as visualized by in- study of the disruption of canalicular function induced byverted fluorescent microscopy, following acute exposure to the hepatotoxic (Fentem et al., 1990; Stone et al., 1994; Cole-compounds under study. The method was validated by (i) making

man et al., 1995) and cholestatic agents (Thibault et al.,a systematic documentation of the effect on CLF retention of a

1992; Roman and Coleman, 1994).variety of hormonal modulators (vasopressin and phorbol esters),Hepatocyte couplets retain the structural and functionalas well as several cholestatic (taurolithocholic acid, cyclosporin A,

polarity of intact hepatocytes. Electron microscopy studiesand estradiol 17b-glucuronide) and hepatotoxic agents (menadi-on plasma membrane re-assembling (Boyer et al., 1985;one, A23187, and t-butyl hydroperoxide), all known to affect bili-Gautam et al., 1987) and immunolocalization of the tightary permeability in intact liver, and (ii) carrying out a comparative

analysis of the results obtained with those recorded using rapid junction-associated protein ZO-1 (Boyer, 1993) revealed thatcanalicular access of horseradish peroxidase (HRP) as an alterna- the canalicular membrane reorganizes within 4–5 hr of cul-tive procedure. The compounds tested all decreased canalicular ture, reestablishing the biliary pole between the two adjacentvacuolar retention of CLF in a dose-dependent manner. Vasopres- cells as a vacuole surrounded by a belt of tight junctionalsin- and phorbol ester-induced decline in CLF retention were pre- cell membrane contact. This structure seals the canalicularvented by pretreatment with the protein kinase C inhibitors

space from the extracellular medium, limiting the access ofH-7 and staurosporine, thus confirming a role for this enzyme in

extracellular markers into the canalicular space. For exam-canalicular permeability regulation. A significant direct correlationple, ruthenium red, an electrondense, ionic dye, is excluded(rÅ 0.934, põ 0.001) was obtained when the decrease in canalicu-from the canalicular space of 90% of couplets (Gautam etlar retention of CLF was compared with the increment in theal., 1987; Boyer, 1993), and the same holds true for thecanalicular access of HRP. Image analysis revealed that cellularprotein, horseradish peroxidase (HRP)1 (Nathanson et al.,fluorescence was not increased following exposure to these com-

pounds, suggesting a paracellular rather than transcellular route 1992; Boyer, 1993). Resealing of the canalicular tight junc-for CLF egress. These results all support canalicular vacuolar re- tional apparatus is indispensable for the secretory unit totention of CLF as a suitable method to readily evaluate acute retain previously secreted materials in its newly formed can-changes in tight junctional permeability in isolated hepatocyte alicular vacuole. Indeed, following recovery of cell polarity,couplets induced by physiological modulators or hepatotoxicagents. q 1997 Society of Toxicology.

1 Abbreviations used: CLF, cholyl-lysyl-fluorescein; cVR, canalicularvacuolar retention; CyA, cyclosporin A; DAB, 3,3*-diaminobenzidine tetra-hydrochloride; DMSO, dimethyl sulfoxide; 17b-EG, estradiol 17b-glucuro-nide; H-7, 1-(5-isoquinolinylsulfonyl)-2-methyl piperazine; HRP, horserad-Isolated hepatocyte couplets provide an accessible, func-ish peroxidase; L-15, Leibovitz-15; PDB, phorbol 12,13-dibutyrate; PKC,

tional, primary unit of canalicular bile formation. This model protein kinase C; PMA, phorbol 12-myristate 13-acetate; t-BuHP, t-butylhas been employed for the study of a variety of hepatocyte hydroperoxide; TDHC, taurodehydrocholate; TLC, taurolithocholate; VP,

vasopressin.physiological functions comprising transport of cholephilic

71 0272-0590/97 $25.00Copyright q 1997 by the Society of Toxicology.All rights of reproduction in any form reserved.

AID FAAT 2309 / 6k1c$$$101 05-16-97 03:55:37 ftoxa

72 ROMA, ORSLER, AND COLEMAN

of Mills et al. (1991); the synthetic procedures gave a high yield of CLF,hepatocyte couplets are able to retain, in the canalicularwhich appeared as a single spot after HPTLC. Collagenase type A fromvacuole, organic anions such as fluorescein or fluoresceinClostridium histolyticum was purchased from Gibco (Paisley, UK). Tauroli-

derivatives of bile acids, either after ionophoresis through thocholic acid (sodium salt) (TLC) and taurodehydrocholic acid (sodiummicroelectrodes or following uptake and secretion of these salt) (TDHC) were from Calbiochem-Novabiochem Ltd. (Nottingham, UK).

CyA was from Sandoz A.G. (Basel, Switzerland). Leibovitz-15 (L-15) tissuecompounds (Boyer, 1993; Wilton et al., 1993b). In turn,culture medium, bovine serum albumin (fraction V), 3,3*-diaminobenzidineresealing of the canalicular space represents the first step fortetrahydrochloride (DAB), HRP, phorbol 12,13-dibutyrate (PDB), phorbolthe overall rearrangement of the canalicular and basolateral12-myristate 13-acetate (PMA), vasopressin (VP), staurosporine, 1-(5-iso-

membranes following loss of polarity induced by cell isola- quinolinylsulfonyl)-2-methyl piperazine (H-7), 2-methyl-1,4-naphtoqui-tion, including expression of the respective membrane trans- none (menadione), A23187, t-butyl hydroperoxide (t-BuHP) and estradiol

17b-glucuronide (17b-EG) were obtained from Sigma Chemical Co. (Poole,port systems in these surface domains (Gautam et al., 1987;Dorset, UK). All other chemicals were of reagent grade.Boyer, 1993; Roelofsen et al., 1995).

Animals. Male Wistar rats bred at in the University of BirminghamSimilarities between the permeability properties of the(220–240 g) were used throughout. Before the experiments, the animalshepatocyte couplet paracellular barrier and those of the intactwere maintained on a standard laboratory diet (41B maintenance diet, Pils-

liver make the former a good potential tool for the study of bury, Birmingham, UK) and tap water ad libitum. Rats were anaesthetisedbiogenesis, regulation, and integrity of hepatocellular tight using Ketalar (ketamine hydrochloride, 6 mg/100g body wt) with Domitor

(medetomidine, 25 mg/100g body wt). Surgery was started between 8:00junctional structures, without the interference of other com-AM and 9:00 AM to minimize circadian variations.plicating factors such as extrahepatic influences or hepatic

Isolation, enrichment, and culture of hepatocyte couplets. Hepatocytehemodynamic changes. Despite these advantages, only a fewcouplets were obtained from rat liver according to the two-step collagenasemethodological approaches have been described to evaluateperfusion procedure described by Wilton et al. (1993b), adapted from Gau-

tight junctional permeability in couplets, and no systematic tam et al. (1989). Following a perfusion (20 ml/min) with a Ca2/- andanalysis of the employed methods was performed. Penetra- Mg2/-free Hanks’ balanced salt solution into the portal vein for 12.5 min,

approximately 0.02% (depending on batch) of collagenase in Hanks’ bal-tion of the extracellular marker, ruthenium red, into the cana-anced salt solution was infused for 4.5 min. The liver was then removed,licular vacuole, as assessed by electron microscopy, has beenchopped, and agitated in ice-cold Krebs–Henseleit solution for 5 min. Theregarded as indicative of the tightness of tight junctionalfinal suspension was filtered through 150-mm nylon gauze and the remaining

structures (Gautam et al., 1987), but this procedure only tissue was reincubated in the collagenase solution for approx 7 min (de-permits qualitative rather than quantitative evaluation of this pendant upon collagenase batch activity) at 377C to liberate a second cell

preparation with a high overall viability (ú90%), as assessed by trypanproperty. An alternative quantitative approach was intro-blue exclusion using an improved Neubauer hemocytometer. This initialduced by Nathanson et al. (1992), who recorded changes inpreparation contained 24 { 4% (n Å 20) of couplets with high viabilityparacellular permeability induced by hormonal messengers,(ú93%). If either cell of a couplet stained positively with trypan blue, then

by assessing the percentage of couplets which were pene- the whole unit was considered nonviable. Couplets were further enrichedtrated by exogenously administered HRP. In preliminary using centrifugal elutriation as described by Wilton et al. (1991). The re-

sulting preparation, containing 69 { 5% of couplets (n Å 20), was platedstudies from our laboratory (Stone et al., 1994; Roman andin L-15 containing 50 U/ml penicillin and 50 mg/ml streptomycin onto 35-Coleman, 1994) we observed that canalicular retention of themm plastic culture dishes (2 ml/dish) at a density of 0.5 1 105 units/ml,preaccumulated, fluorescent, conjugated bile acid analogueand incubated at 377C for 5 hr. This time was described to be appropiate

cholyl-lysyl-fluorescein (CLF) was impaired by 2-methyl- for couplets to reach maximal capability to transport and accumulate CLF1,4-naphthoquinone (menadione) and cyclosporin A (CyA), into their canalicular vacuoles (Wilton et al., 1993b). Since this capability

then remains almost constant for at least 4 hr (Wilton et al., 1993b), experi-two compounds known to affect biliary permeability in intactments were performed within this time period.liver (Kan and Coleman, 1990; Lora et al., 1991). Since

Assessment of tight junctional permeability. Paracellular permeabilitycanalicular retention of cholephilic compounds requires tightin hepatocyte couplets was assessed by two methods, namely, (i) canalicular

junctions to be sealed (Wilton et al., 1993b), these observa- vacuole retention (cVR) of CLF and (ii) rapid appearance of HRP withintions raise the interesting possibility that changes in canalicu- the canalicular space. A schematic representation describing both methods

is shown in Fig. 1.lar retention of CLF may actually reflect modifications ofcVR of CLF was assessed by determining the percentage of coupletsparacellular permeability in couplets. To investigate this pos-

able to retain this fluorescent bile acid analogue (Roman and Coleman,sibility further, the present study provides a systematic docu-1994; Stone et al., 1994). For this purpose, CLF (2 mM, final concentration

mentation of the effect on CLF retention of a variety of in the dishes) was added to each plate and incubated at 377C for 15 minhormonal modulators and hepatotoxic compounds known before washing twice at 377C with 2 ml of L-15. This time was chosen

because canalicular accumulation of CLF was shown to reach an apparentto affect biliary permeability in intact liver. A comparativesteady state within this time (Wilton et al., 1993b). Where ‘‘hormones’’ oranalysis of the results with those recorded using rapid cana-cholestatic or hepatotoxic compounds were studied (see later section onlicular access of HRP is also provided.treatments), they were added at this point into the dishes containing 2 mlof L-15 medium after removing CLF by washing twice with 2 ml of L-15

METHODS at 377C.cVR of CLF was monitored by using an inverted fluorescent microscope

(Olympus IMT2-RFL; Olympus Optical Ltd., London, UK) equipped withMaterials. CLF, kindly provided by Dr. Charles O. Mills (Birmingham,UK), was synthesized and its purity confirmed according to the methods a 100 W mercury light source and an incubator to maintain the cells at

AID FAAT 2309 / 6k1c$$$101 05-16-97 03:55:37 ftoxa

73CANALICULAR RETENTION AND PERMEABILITY IN COUPLETS

FIG. 1. Schematic representation of the different assays employed to evaluate paracellular permeability in isolated hepatocyte couplets. Under normalconditions, tight junctional apparatus reseals after a short-term culture. This resealing is indispensable for the couplets to retain previously secretedmaterials in, and to exclude extracellular material from, their newly formed canalicular vacuole. Opening of tight junctions would cause accumulatedmaterials to escape from the canalicular vacuole and extracellular material to penetrate into this space. Opening of tight junctions can therefore bequantified by counting the number of couplets able to retain previously secreted cholyl-lysyl-fluorescein (CLF) (retention assay) or the number of coupletswhich were penetrated by exogenously administered horseradish peroxidase (HRP) (penetration assay).

377C during observation. Preaccumulated CLF is easily visualized as a couplets and results were expressed as the percentage of the total coupletsanalysed.brightly fluorescent vacuole between adjacent hepatocytes. Due to progres-

sive bleaching of the fluorophore with continuous exposure to ultraviolet Treatments. Changes in cVR of CLF or HRP penetration in responselight, such fluorescent microscopy can only be used as a series of ‘‘snap- to a number of hormones and second messengers acting through proteinshots’’ rather than continuous recording. Therefore, the estimation of cVR kinase C (PKC) were studied. The compounds tested comprised (final con-of CLF was determined by randomly choosing a field of vision and all the centrations in plate, volume of vehicle used to deliver): VP (1007–10011

couplets visible in the field (ú30) were then counted within 30–45 sec.M, 1 ml in acetic acid 0.05%), PDB (1005–1009 M, 2 ml in dimethyl sulfoxide,

Results were expressed as the percentage of total couplets counted dis- DMSO), and PMA (1005–1009 M, 2 ml in DMSO), with or without theplaying sufficient CLF to be visible in the canalicular vacuole. PKC inhibitors H-7 (1004 M, 10 ml in saline) or staurosporine (1006 M, 10

In some experiments, canalicular and cellular couplet fluorescence were ml in saline). Stocks of these compounds were prepared in the solventsquantified by performing image analysis. For this purpose, fluorescent mi- indicated above and stored in frozen aliquots until use. Cells incubated withcrophotographs from randomly chosen fields of vision were digitalized to VP, PDB, or PMA were exposed to these agents for 30 sec and coupletsobtain computer images on which cellular and canalicular CLF fluorescence pretreated with H-7 or staurosporine received the inhibitor for 1 min priorin the couplets could be quantified by densitometry with the aid of an image to stimulation. These doses and times of exposure were similar to thoseanalysis program. The intensity of cellular fluorescence (as a measure of employed Nathanson et al. (1992) to assess the effect of hormonal messen-CLF content in the cell body) was derived by dividing the amount of gers on canalicular permeability in isolated hepatocyte couplets.fluorescence inside the cells by total couplet area. Canalicular fluorescence A variety of hepatotoxic and cholestatic agents were also tested for their(as a quantitative estimation of CLF retention in the canalicular vacuole) ability to affect cVR of CLF. They included (final concentrations in plate)was expressed as a percentage of total (canalicular plus cellular) fluores- CyA (25–1000 nM), 17b-EG (50–400 mM), TLC (0.5–5 mM), A23187cence. All the couplets in a field (ú30) were examined, irrespective of

(0.125–5 mM), menadione (5–100 mM), and t-BuHP (25–350 mM). Thewhether they displayed canalicular fluorescence.

compounds were all dissolved in DMSO and added in a volume of 10 mlAssessment of rapid canalicular HRP penetration was based on the

of stock solutions, which were stored in frozen aliquots until use. The timemethod described by Nathanson et al. (1992). The cells were exposed to a

of exposure of the compounds was 15 min for CyA and menadione and 304 mg/ml solution of HRP in L-15 for 1 min. The cells were then washed once

min for 17b-EG, TLC, A23187, and t-BuHP. These times of exposure werewith L-15 and fixed by incubating for 30 min with 4% paraformaldehyde in

chosen on the basis of preliminary experiments showing that a maximal0.1 M phosphate buffer, pH 7.2, at room temperature. The cells were then

effect had been reached at these respective time periods. For every experi-washed twice with 0.1 M phosphate buffer (pH 7.4) and left overnight at

ment, a control dish receiving vehicle alone was treated simultaneously,47C in 0.1 M phosphate/2.5% sucrose buffer (pH 7.4). HRP was developed

thus taking into account any change in cVR of CLF with time.via the DAB reaction. For this purpose, the cells were washed once with

Viability was systematically assessed before and after each treatment0.1 M Tris–HCl buffer (pH 7.4) and left for 5 min at room temperature.(trypan blue exclusion assay) and showed no significant change.The buffer solution was then removed and the cells exposed consecutively

When rapid access of HRP into the canaliculus was determined, only theto a 0.2% DAB solution in 0.1 M Tris–HCl buffer (pH 7.4) for 30 min andmaximal dose of each compound was tested.to the same solution containing 0.01% H202 for 10 min. Finally, the cells

Statistical analysis. Results are expressed as means { SE and werewere washed twice with 0.1 M Tris–HCl buffer (pH 7.4) and observedconsidered significant when the p value was õ 0.05 using paired Student’susing light microscopy (Olympus IMT2-RFL, Olympus Optical Ltd.). At test. Regression line analysis was carried out using the least squaresdarkened canaliculus was considered indicative of the presence of HRP/

DAB reaction product. Canalicular HRP was determined in 200 consecutive method and significance of the difference against zero of the correlation

AID FAAT 2309 / 6k1c$$$101 05-16-97 03:55:37 ftoxa


induced decrease of cVR of CLF. Complete prevention ofthis effect was seen when couplets were pretreated witheither inhibitor at these concentrations. H-7 and stauro-sporine alone had no independent effect on cVR of CLF(data not shown).

Effect of Hepatotoxic and Cholestatic Agentson cVR of CLF

Figure 5 depicts the dose–response effect on cVR of CLFof different compounds known to affect tight junctional per-meability in intact liver. These agents comprise the chole-static compounds CyA, 17b-EG, and TLC, the calcium iono-phore A23187, and the oxidative stress inducers menadioneand t-BuHP. None of these compounds affected cell viabilityover the entire range of doses tested. Instead, these com-pounds all reduced the percentage of canaliculi retainingCLF in a dose-dependent manner. Although different pat-FIG. 2. Time course of the decline in canalicular vacuolar retention ofterns of dose–response effect were observed, the rate ofpreviously accumulated cholyl-lysyl-fluorescein (CLF) following removal

of the fluorescent bile acid from the incubation medium in hepatocyte decrease of this parameter was generally higher at lowercouplets. Couplets were allowed to accumulate CLF (2 mM, final concentra- doses and then decreased slower as the dose increases totion in the dishes) for 15 min and then the fluorescent bile acid was removed approach an apparent minimal value. This asymptotic valuewith two washes of L-15 medium (time, 0 min). Canalicular CLF retention

appeared to be different depending on the considered com-was expressed as the percentage of total couplets retaining CLF referred topound. In fact, according to the patterns obtained, two groupsthe initial value. All values are means { SE of four experiments. *Signifi-

cantly different from the initial value (p õ 0.05). could be differentiated. CyA, 17b-EG, and menadione seemto reach a relatively high plateau (cVR of CLF at the highestdose: 42.6, 35.7, and 40.5%, respectively), whereas thisasymptotic value appears to be lower for TLC, A23187, andcoefficient r was calculated using appropriate tables (Snedecor and Cochran,t-BuHP (cVR of CLF at the highest dose: 23.5, 16.5, and1969).12.7%, respectively).

RESULTSComparison of cVR of CLF and HRP Penetration

to Evaluate Tight Junctional PermeabilityStability of cVR of CLFin Hepatocyte Couplets

Time-dependent changes in the capability of hepatocyteComparative data of the number of canaliculi retainingcouplets to retain previously accumulated CLF are shown

CLF and the values recorded using HRP canalicular penetra-in Fig. 2. The number of couplets retaining CLF remainedtion as a reference method following exposure to the differ-around 75% following 15 min of CLF exposure (Table 1).ent compounds tested in this study are summarized in TableFollowing CLF removal from the incubation medium, this1. As shown in Fig. 6, a clear linear correlation was obtainedvalue (expressed as percentage of original value) remainedbetween the values recorded using both methods, as sug-roughly stable for 30 min, but then declined gradually sogested by the high correlation coefficient obtained (r Åthat after 2 hr, canaliculi retaining CLF were approximately0.934, p õ 0.001).half of the initial value (Fig. 2).

Distribution of CLF Fluorescence in Isolated HepatocyteEffect of Vasopressin (VP) and Phorbol EstersCoupletson cVR of CLF

As can be seen in Fig. 3, both VP and the phorbol esters Fluorescence microphotographs exhibiting retention ofCLF in the canalicular lumen of control couplets and in cellsPDB and PMA induced a dose–response decrease of the

number of canaliculi retaining CLF after 15 min. This de- treated with VP (1008M), 17b-EG (400 mM), and A23187

(5 mM) are shown in Fig. 7. CLF accumulation into thecline was apparent at 10010 and 1007M for VP and phorbol

esters, respectively, although it reached statistical signifi- canalicular space can be easily visualised as a brightly fluo-rescent vacuole between adjacent cells. A clear diminutioncance at doses of one order of magnitude higher than these.

Figure 4 shows the effect of the PKC inhibitors H-7 (1004 in the number of canaliculi containing CLF can be observedfollowing exposure to all the three tested compounds.M) and staurosporine (1006

M) on the VP- or phorbol ester-

AID FAAT 2309 / 6k1c$$$101 05-16-97 03:55:37 ftoxa


TABLE 1Comparison between CLF Retention and Rapid HRP Access into the Canalicular Vacuole to Evaluate Changes in Tight Junctional

Permeability in Hepatocyte Couplets as Modified by Different Treatments

Canaliculi retaining CLF Canaliculi accumulating HRP

% of total % of change % of total % of change

Control (DMSO, 10 ml, 30 sec) 78.2 { 1.2 — 12.4 { 1.9 —VP (1008 M, 30 sec) 50.2 { 3.7* 036 { 2 22.3 { 3.0* /80 { 10PDB (1 mM, 30 sec) 54.0 { 1.9* 031 { 2 19.2 { 2.3* /55 { 7PMA (1 mM, 30 sec) 53.2 { 5.0* 032 { 3 21.5 { 3.2* /73 { 10

Control (DMSO, 10 ml, 15 min) 76.3 { 4.5 — 13.7 { 1.5 —CyA (1 mM, 15 min) 35.3 { 3.7* 054 { 2 33.6 { 2.9* /145 { 12Menadione (100 mM, 15 min) 26.5 { 2.2* 065 { 2 30.8 { 3.7* /125 { 13

Control (DMSO, 10 ml, 30 min) 74.7 { 6.0 — 14.4 { 1.7 —17b-EG (400 mM, 30 min) 26.3 { 2.8* 065 { 2 32.3 { 3.2* /124 { 15TLC (5 mM, 30 min) 20.7 { 2.5* 073 { 1 40.2 { 3.4* /179 { 16A23187 (5 mM, 30 min) 12.5 { 0.9* 083 { 3 50.8 { 2.7* /257 { 14t-BuHP (350 mM, 30 min) 12.3 { 1.7* 085 { 2 46.9 { 1.9* /230 { 10

Note. Hepatocyte couplets were exposed to the compounds for 30 sec (VP, PDB, and PMA), 15 min (CyA and menadione), or 30 min (17b-EG, TLC,A23187, and t-BuHP). Percentage of change for each compound was calculated on the base of its respective control. Data are expressed as means { SE(n Å 4–5). CLF, cholyl-lysyl-fluorescein; CyA, cyclosporin A; DMSO, dimethyl sulfoxide; 17b-EG, estradiol 17b-glucuronide; HRP, horseradishperoxidase; PDB, phorbol, 12,13-dibutyrate; PMA, phorbol 12-myristate 13-acetate; t-BuHP, t-butyl hydroperoxide; TLC, taurolithocholate; VP, vaso-pressin.

* Significantly different from the respective control (p õ 0.05).

The effect of five representative compounds comprising essential. The investigation of functional modulation of tightjunctions and their impairment following toxicological insulttwo ‘‘hormonal’’ modulators (VP and PDB) and three hepa-

totoxicants (17b-EG, TLC, and A23187) on CLF distribu- is one such application.tion was quantitatively examined in hepatocyte couplets by The present work proposes a simple, fast method to assessimage analysis. Results are summarized in Table 2. Image tight junctional permeability in hepatocyte couplets. Thisanalysis revealed that both canalicular fluorescence com- procedure takes advantage of the modified technique of cou-pared to total couplet fluorescence (as a quantitative estima- plet preparation and further purification by centrifugal elutri-tion of CLF retention in the canalicular space) and cellular ation described by our laboratory (Wilton et al., 1991), whichfluorescence per unit of total couplet area (as a measure of enables an examination of the behaviour of a large, represen-CLF content in the cellular body) were decreased by all five tative number of canalicular vacuoles rather than of prese-compounds. Furthermore, the decrease in cellular fluores- lected, individual units. The method involves the recordingcence appeared to correlate directly with the decrease in of the number of canalicular vacuoles present in a field ofcanalicular fluorescence (r Å 0.992, p õ 0.001). In turn, vision that are able to retain the previously accumulatedwhen cVR of CLF was compared with the decrease in cellu- fluorescent bile acid analogue CLF, which is used here aslar fluorescence, a good correlation was also obtained (r Å an indicator of canalicular accumulation. Our approach is0.997, p õ 0.001). based on the widely held view that cholephilic compounds

require integrity of the structures that seal the canaliculus,to be retained in this space. Therefore, any perturbation in-DISCUSSIONvolving the opening of tight junctions would cause preaccu-mulated materials to escape from the canalicular lumen. Fur-The need to study hepatocyte function in a rapid andthermore, taking into account that CLF is negatively chargedreproducible manner, avoiding the use of a large number ofand recognizing the reported permselectivity of the bile cana-animals, has encouraged the development of alternatives inliculus (Bradley and Herz, 1978), this method would alsovitro that mimic hepatocyte function in vivo. Among these,offer an opportunity to examine changes in permselectivitythe hepatocyte couplet model has a preferential place sincenot associated with changes in permeability. Any such per-this preparation retains the structural and functional polarityturbations would be ultimately visualized as a reduction inof the hepatic epithelium, making this system particularly

useful for studies in which preservation of these features is the number of canalicular vacuoles retaining the fluorescent

AID FAAT 2309 / 6k1c$$$101 05-16-97 03:55:37 ftoxa


The validity of the method to evaluate tight junctionalpermeability was tested (i) by making a systematic documen-tation of the effect on CLF retention of a variety of hormonalmessengers and hepatotoxic agents known to affect biliarypermeability in intact liver; and (ii) by carrying out a com-

FIG. 3. Dose–response curves of the effect of VP, PDB, or PMA oncanalicular vacuolar retention of previously accumulated CLF in isolatedrat hepatocyte couplets. The cells were exposed to the hormonal modulators(or vehicle alone in controls) for 30 sec. Canalicular CLF retention wasexpressed as the percentage of total couplets retaining CLF referred to thecontrol values. All values are means { SE (n Å 4–5). *Significantly differ-ent from control values (p õ 0.05). CLF, cholyl-lysyl-fluorescein; PDB,phorbol 12,13-dibutyrate; PMA, phorbol 12-myristate 13-acetate; VP, vaso-pressin.

bile acid analogue, compared with an initial high proportion(ú70%) of couplets being able to accumulate the marker in

FIG. 4. Effect of the PKC inhibitors H-7 (1004 M) and staurosporinecontrol conditions.(1006 M) on VP (1009 M), PDB (1006 M), or PMA (1006 M) induced decrease

This parameter appears to provide similar information to of canalicular vacuolar retention of previously accumulated CLF in isolatedother, more quantitative, but also more laborious, alterna- rat hepatocyte couplets. Cells incubated with VP, PDB, or PMA were

exposed to these agents (or vehicle alone in controls) for 30 sec and cellstives using image analysis. As indicated by the good correla-pretreated with H-7 or staurosporine received the inhibitor for 1 min priortion seen between the number of canalicular vacuoles re-to stimulation. Canalicular CLF retention was expressed as the percentagetaining CLF and the percentage of canalicular fluorescenceof total couplets retaining CLF referred to the control values. All values

expressed as a proportion of total couplet fluorescence, no are means { SE (n Å 4–5). *Significantly different from control valuefurther effort to quantify intensity of canalicular fluorescence (p õ 0.05). CLF, cholyl-lysyl-fluorescein; PDB, phorbol 12,13-dibutyrate;

PMA, phorbol 12-myristate 13-acetate; VP, vasopressin.seems necessary to improve our methodology.

AID FAAT 2309 / 6k1c$$$101 05-16-97 03:55:37 ftoxa


FIG. 5. Dose–response curves of the effect of different cholestatic or hepatotoxic agents on canalicular vacuolar retention of previously accumulatedCLF in isolated rat hepatocyte couplets. Canalicular CLF retention was assessed following 15 min (CyA, menadione) or 30 min (17b-EG, TLC, A23187,t-BuHP) of exposure to the toxicants (or vehicle in controls) and expressed as the percentage of total couplets retaining CLF referred to control value.All values are means { SE (n Å 4–6). *Significantly different from control value (p õ 0.05). CLF, cholyl-lysyl-fluorescein; CyA, cyclosporin A; 17b-EG, estradiol 17b-glucuronide; TLC, taurolithocholate; t-BuHP, t-butyl hydroperoxide.

parative analysis of the results obtained with those recorded firmed by experiments in isolated perfused rat liver (Loweet al., 1988) and in hepatocyte couplets (Nathanson et al.,using rapid canalicular access of HRP as an alternative ap-

proach. 1992). These studies pointed to the involvement of PKC inthis pathway, as indicated by the actions of the PKC activa-Hormonal regulation of hepatic tight junctional permeabil-

ity is a well-documented phenomenon which has been con- tors VP and PDB. We took advantage of this observation to

AID FAAT 2309 / 6k1c$$$101 05-16-97 03:55:37 ftoxa


(Reichen and Le, 1983), induced no effect at concentrationsshowing impairment of cVR of CLF when TLC was the bilesalt administered (results not shown). These results empha-size the suitability of the method to selectively detectchanges of paracellular permeability following hepatotoxicinsult. It should be pointed out, however, that this methodol-ogy requires hepatocellular function to be unaffected in orderto permit normal accumulation of CLF. Therefore, this ap-proach cannot be used to evaluate the effect of in vivo pre-treatments resulting in altered hepatocyte transport function.In this case, alternative methodologies assessing entrance ofpoorly permeable solutes like HRP need to be used. Ourmethodology, instead, is particularly useful in complement-ing studies of canalicular function in which acute effects oftoxicants on canalicular vacuole accumulation of CLF areevaluated (see Coleman et al., 1995). In fact, impairment of

FIG. 6. Correlation between the percentage of decrease of cholyl-lysyl-the couplet’s ability to accumulate CLF may involve alter-fluorescein (CLF) canalicular retention and the percentage of increase ofation of a number of processes, namely, uptake, intracellularrapid horseradish peroxidase (HRP) canalicular access in the experimental

conditions detailed in Table 1. Correlation coefficient (r Å 0.934) was transport, canalicular excretion, and intracanalicular reten-significantly different from zero (p õ 0.001). tion. This last possibility can be investigated by analyzing

changes in cVR of CLF. It is important to note that, in thiscase, control experiments in which couplets are exposed to

analyse the suitability of cVR of CLF to visualize thethe vehicle for a similar period should be simultaneously

changes in canalicular permeability induced by these com-run, since time-dependent changes in this parameter occur

pounds, adding PMA as a third PKC activator. This experi-spontaneously (see Fig. 2).mental test is somewhat of a ‘‘tour de force’’ for the system,

The pattern of cVR reduction induced by these compoundssince the effect induced by these hormonal modulators isshowed an exponential profile, the rate of decrease beingextremely fast (õ 30 sec), and this time period could belower as the dose increases, finally approaching an apparentregarded as too short to permit the diffusion of canalicularasymptotic value (see Fig. 5). This kind of nonlinear patternmaterials from the canalicular vacuole. However, a signifi-suggests heterogeneity in the susceptibility of the cell popu-cant, dose-dependent, reduction of cVR of CLF was ob-lation to the hepatotoxicants, some couplets being fully resis-served following this short-term exposure (see Fig. 3), andtant to the effect of some hepatotoxicants, even at high doses.such an effect was completely prevented by previous expo-Couplet preparations are mixed populations of periportal,sure of the cells to the PKC inhibitors H-7 and staurosporinemid-zone, and perivenous hepatocytes, which do not neces-(see Fig. 4). These results confirm and extend previous ob-sarily have similar sensitivities to modulatory or toxicologi-servations pointing to a role of PKC on hepatic tight junc-cal perturbations. In line with this view, menadione, which,tional permeability and, more importantly for our purpose,together with CyA and 17b-EG, appears to be able to affectvalidate cVR of CLF as a suitable method to detect rapid,only a limited proportion of couplets (see Fig. 5), has beenmodulatory changes of this function.shown to affect differentially periportal from perivenousChanges in biliary permeability are a frequent feature incouplets, as judged by their ability to impair canalicular CLFhepatotoxicity and cholestasis. We analyzed the effect onaccumulation (Wilton et al., 1993a). Extension of this studycVR of CLF of a variety of toxicants which were shown toto the analysis of cVR of CLF for this and the other toxicantsaffect biliary permeability in intact liver by different mecha-in subpopulations of periportal and perivenous couplets ob-nisms. They comprise the cholestatic agents CyA (Lora ettained by centrifugal elutriation (Wilton et al., 1993a) shouldal., 1991), 17b-EG (Kan et al., 1989), and TLC (Boyer etcontribute to a validation of this observation.al., 1976; Vu et al., 1992), as well as the calcium ionophore

A previous study by Nathanson et al. (1992) has evaluatedA23187 (Kan and Coleman, 1988) and the oxidative stressparacellular permeability in couplets by determining theinducers menadione (Kan and Coleman, 1990) and t-BuHPrapid access (approx 1 min) into the canalicular space of the(Ballatori and Truong, 1989). These compounds all de-exogenously added protein HRP. This method relies on thecreased cVR of CLF in a dose-dependent manner and atfact that in intact liver, HRP was shown to pass mostlyconcentrations at or below those that had been instrumentalthrough the paracellular route following a short-term (õ5in impairing biliary permeability in the studies quoted above.min) exposure (Lowe et al., 1985). HRP is a protein of 40Instead, TDHC, a nontoxic bile salt which had been shown

not to affect biliary permeability in isolated perfused rat liver kDa and, therefore, severe restrictions to its passage are

AID FAAT 2309 / 6k1c$$$101 05-16-97 03:55:37 ftoxa


FIG. 7. Fluorescent microphotographs showing control isolated rat hepatocyte couplets which were allow to accumulate CLF (2 mM, final concentrationin the dishes) for 15 min (A) and couplets exposed to VP (0.01 mM, 30 sec) (B), 17b-EG (400 mM, 30 min) (C), or A23187 (5 mM, 30 min) (D).Preaccumulated CLF is visualised as a brightly fluorescent vacuole between adjacent cells. Note the reduction in the number of canaliculi retaining CLFfollowing exposure to the above mentioned agents. CLF, cholyl-lysyl-fluorescein; 17b-EG, estradiol 17b-glucuronido; VP, vasopressin.

expected to occur during its movement through the tight Even when all the evidence points to a preferential egressof CLF from the bile canaliculus through the paracellularjunctional region. This explains the relatively low (õ15%)

percentage of canalicular vacuoles exhibiting HRP reaction pathway following tight junction disruption, the possibilityalso exists that the accumulated bile acid analogue leavesproduct under control conditions (see Nathanson et al., 1992,

and Table 1). We took advantage of this alternative approach the canalicular lumen through a transcellular route, by reen-tering one or both of the hepatocytes. However, this is un-to validate the results obtained using cVR of CLF. The good

correlation between the results obtained using both methods likely to contribute significantly to the observed effect be-cause, if this happens, it would have been expected to cause(see Fig. 6) further supports the credibility of our method.

AID FAAT 2309 / 6k1c$$$101 05-16-97 03:55:37 ftoxa


TABLE 2Quantitative Assessment of CLF Distribution in Hepatocyte Couplets as Modified by Different Treatments

Canaliculi retaining Canalicular fluorescence Cellular fluorescenceCLF

% of total Cellular fluorescence per% of % of couplet % of unit of cellular area % oftotal control fluoresce control (arbitrary units) control

Control (DMSO, 10 ml, 30 sec) 77 100 41.1 { 3.0 100 11.6 { 0.7 100VP (1008 M, 30 sec) 47 61 27.4 { 3.8* 67 10.6 { 0.5 91PDB (1 mM, 30 sec) 55 71 32.9 { 3.1* 80 10.9 { 0.8 94

Control (DMSO, 10 ml, 30 min) 75 100 40.4 { 2.7 100 11.3 { 0.6 10017b-EG (400 mM, 30 min) 28 37 17.7 { 4.1* 45 9.5 { 0.8* 84TLC (5 mM, 30 min) 20 27 13.3 { 2.1* 33 9.1 { 1.1* 81A23187 (5 mM, 30 min) 13 17 9.3 { 3.7* 23 8.6 { 1.0* 76

Note. Hepatocyte couplets were exposed to the compounds for 30 sec (VP, PDB), or 30 min (17b-EG, TLC, A23187). Then, microphotographs ofrandomly selected fields were taken and the number of canaliculi retaining CLF counted. Every fluorescent photomicrograph was then digitalized toobtain a computer image on which canalicular fluorescence (as a percentage of total couplet fluorescence) and cellular fluorescence per unit of coupletarea could be quantified with the aid of an image analysis program as indicated under Methods. Number of couplets analyzed was ú30. Data areexpressed as means { SE. CLF, cholyl-lysyl-fluorescein; DMSO, dimethyl sulfoxide; 17b-EG, estradiol 17b-glucuronide; PDB, phorbol 12,13-dibutyrate;TLC, taurolithocholate; VP, vasopressin.

* Significantly different from the respective control (p õ 0.05).

Boyer, J. L., Layden, T. J., and Hruban, Z. (1976). Mechanism of cholesta-an increase (or no change, if canalicular ingress is simultane-sis: Taurolithocholate alters canalicular membrane composition, structureously counterbalanced by basolateral egress) rather than aand permeability. In Membrane Alterations as Basis of Liver Injury (H.

decrease in cellular fluorescence following exposure to the Popper, L. Bianchi, and W. Reutter, Eds.), pp. 353–369. MTP Press,tested substances. The reported decrease in cellular fluores- Lancaster.cence is compatible rather with increased secretion of CLF Boyer, J. L., Ng, O.-C., and Gautam, A. (1985). Formation of canalicularinto the canalicular space driven by the continuous loss of spaces in isolated rat hepatocyte couplets. Trans. Assoc. Am. Phys. 98,

21–29.CLF from the space via the paracellular pathway.In conclusion, assessment of cVR of CLF provides a Boyer, J. L. (1993). Isolated rat hepatocyte couplets: A model for the study

of bile secretory function. In Hepatic Transport and Bile secretion (N.means to evaluate, rapidly, acute changes in tight junctionalTavoloni and P. D. Berk, Eds.), pp. 597–606. Raven Press, New York.permeability induced by hepatotoxic agents or hormonal

Bradley, S. E., and Herz, R. (1978). Permselectivity of biliary canalicularmodulators in a reproducible and sensitive manner, withoutmembrane in rats: Clearance probe analysis. Am. J. Physiol. 254, E570–requiring laborious microscopic techniques involving fixa-E576.

tion and staining. Its application to the analysis of tightColeman, R., Wilton, J. C., Stone, V., and Chipman, J. K. (1995). Hepatobil-junction performance may aid in our understanding of the

iary function and toxicity in vitro using isolated hepatocyte couplets.role of this structure in hepatocellular function, its modula- Gen. Pharmacol. 26, 1445–1453.tion by hormonal agents, and its impairment following toxi- Crawford, J. M., Vinter, D. W., and Gollan, J. L. (1988). Taurocholate in-cological insult. duces pericanalicular localization of C6-NBD-ceramide in isolated hepa-

tocyte couplets. Am. J. Physiol. 260, G119–G132.

ACKNOWLEDGMENTS Fentem, J. H., Mills, C. O., Coleman, R., and Chipman, J. K. (1990). Biliaryexcretion of fluorescent cholephiles in hepatocyte couplets: An in vitro

M. G. Roma, from the Institute of Physiology, University of Rosario- model for hepatobiliary and hepatotoxicity studies. Toxicol. in Vitro 4,CONICET, Argentina, was a recipient of a postdoctoral fellowship of The 452–457.Royal Society-CONICET Interchange Program. D. J. Orsler is in receipt of Gautam, A., Ng, O.-C., and Boyer, J. L. (1987). Isolated hepatocyte coupletsan MRC collaborative Ph.D. studentship with Astra Charnwood. Laboratory in short culture: Structural characteristics and plasma membrane reorgani-expenses were supported by The Wellcome Trust, The Royal Society, and zation. Hepatology 7, 216–223.Astra Charnwood. The authors thank Mr. Jose Manuel Pellegrino for his

Gautam, A., Ng, O.-C., Strazzabosco, M., and Boyer, J. L. (1989). Quantita-assistance in image analysis and graphic design.tive assessment of canalicular bile formation in isolated hepatocyte cou-plets using microscopic optical planimetry. J. Clin. Invest. 83, 565–573.

REFERENCESGraf, J., Gautam, A., and Boyer, J. L. (1984). Isolated rat hepatocyte cou-

plets: A primary unit for electrophysiological studies of bile secretoryBallatori, N., and Truong, A. T. (1989). Altered hepatic junctional perme-function. Proc. Natl. Acad. Sci. USA 81, 6516–6520.ability, bile acid excretion and glutathione efflux during oxidative chal-

lenge. J. Pharmacol. Exp. Ther. 251, 1069–1075. Graf, J., Henderson, R. M., Krumpholz, B., and Boyer, J. L. (1987). Cell

AID FAAT 2309 / 6k1c$$$101 05-16-97 03:55:37 ftoxa


membrane and transepithelial voltages and resistances in isolated rat Roman, I. D., and Coleman, R. (1994). Disruption of canalicular functionin isolated rat hepatocyte couplets caused by cyclosporin A. Biochem.hepatocyte couplets. J. Membr. Biol. 95, 241–254.Pharmacol. 48, 2181–2188.Kan, K. S., and Coleman, R. (1988). The calcium ionophore A23187 in-

Roman, I. D., Johnson, G. D., and Coleman, R. (1996). S-Adenosyl-L-me-creases the tight-junctional permeability in rat liver. Biochem. J. 256,thionine prevents disruption of canalicular function and pericanalicular1039–1041.cytoskeleton integrity caused by cyclosporin A in isolated rat hepatocyteKan, K. S., Monte, M., Parslow, R. A., and Coleman, R. (1989). Oestradiolcouplets. Hepatology 24, 134–140.17b-glucuronide increases tight junctional permeability in rat liver. Bio-

Saez, J. C., Gregory, W. A., Watanabe, T., Dermietzel, R., Hertzberg, E.,chem. J. 261, 297–300.Bennett, M. V., and Spray, D. C. (1989). cAMP delays disappearance ofKan, K. S., and Coleman, R. (1990). Menadione increases hepatic tightgap junctions between pairs of rat hepatocytes in primary culture. Am.junctional permeability. Its effect can be decreased by butylated hydroxy-J. Physiol. 236, C1–C11.toluene and verapamil. Biochem. J. 270, 241–243.

Snedecor, G. W., and Cochran, W. G. (1969). Statistical Methods. The IowaKitamura, T., Gatmaitan, Z., and Arias, I. M. (1990). Serial quantitativeState Univ. Press, Ames, IA.analysis and confocal microscopy of hepatic uptake, intracellular distribu-

Stone, V., Johnson, G. D., Wilton, J. C., Coleman, R., and Chipman, J. K.tion and biliary secretion of a fluorescent bile acid analog in rat hepatocyte(1994). Effect of oxidative stress and disruption of Ca2/ homeostasis ondoublets. Hepatology 12, 1358–1364.hepatocyte canalicular function in vitro. Biochem. Pharmacol. 47, 625–

Lora, L., Martines, D., Plebani, M., Floreani, A. R., Salugnini, M., and632.

Naccarato, R. (1991). Cyclosporin A increases tight junctional permeabil-Thibault, N., Claude, J. R., and Ballet, F (1992). Actin filament alterationity in rat liver. J. Hepatol. 13, S45. [Abstract]

as a potential marker for cholestasis: a study in isolated rat hepatocyteLowe, P. J., Kan, K. S., Barnwell, S. G., Sharma, R. K., and Coleman, R.

couplets. Toxicology 73, 269–279.(1985). Transcytosis and paracellular movements of horseradish peroxi-

Vu, D. D., Tuchweber, B., Raymond, P., and Yousef, I. M. (1992). Tightdase across liver parenchymal tissue from blood to bile. Effect of a-junction permeability and plasma membrane fluidity in lithocholate-in-naphthylisothiocyanate and colchicine. Biochem. J. 229, 529–537.duced cholestasis. Exp. Mol. Pathol. 57, 47–61.

Lowe, P. J., Miyai, K., Steinbach, J. H., and Hardison, W. G. M. (1988).Watanabe, S., and Phillips, M. J. (1984). Calcium causes active contractionHormonal regulation of hepatocyte tight junctional permeability. Am. J.

of bile canaliculi: Direct evidence from microinjection studies. Proc.Physiol. 255, G454–G461.Natl. Acad. Sci. USA 81, 6164–6168.

Maglova, L. M., Jackson, A. M., Meng, X.-J., Carruth, M. W., Schteingart,Watanabe, S., Tomono, M., Hirose, M., Takeuchi, M., Kitamura, T., and

C. D., Ton-Nu, H.-T., Hoffmann, A. F., and Weinman, S. A. (1995).Nishima, T. (1987). Microscopic fluorometric analysis of Na-fluorescein

Transport characteristics of three fluorescent conjugated bile acid analogstransport in the smallest secretory unit (couplet hepatocytes) and the

in isolated rat hepatocyte couplets. Hepatology 22, 637–647.effect of the cytochalasin D. Lab. Invest. 56, 146–150.

Nathanson, M. H., Gautam, A., Ng, O. C., Bruck, R., and Boyer, J. L. Wilton, J. C., Williams, D. E., Strain, A. J., Parslow, R. A., Chipman, J. K.,(1992). Hormonal regulation of paracellular permeability in isolated rat and Coleman, R. (1991). Purification of hepatocyte couplets by centrifu-hepatocyte couplets. Am. J. Physiol. 262, G1079–G1086. gal elutriation. Hepatology 14, 180–183.

Oshio, C., and Phillips, M. J. (1981). Contractility of bile canaliculi: impli- Wilton, J. C., Chipman, J. K., Lawson, C. J., Strain, A. J., and Coleman, R.cations for liver function. Science 212, 1041–1042. (1993a). Periportal- and perivenous-enriched hepatocyte couplets: Differ-

Reichen, J., and Le, M. (1983). Taurocholate, but not taurodehydrocholate, ences in canalicular activity and in response to oxidative stress. Biochem.increases biliary permeability to sucrose. Am. J. Physiol. 245, G651– J. 292, 773–779.G655. Wilton, J. C., Coleman, R., Lankester, D. J., and Chipman, J. K. (1993b).

Reverdin, E. C., and Weingart, R. (1988). Electrical properties of the gap Stability and optimization of canalicular function in hepatocyte couplets.junctional membrane studied in rat liver cell pair. Am. J. Physiol. 254, Cell Biochem. Funct. 11, 179–185.C226–C234. Wilton, J. C., Matthews, G. M., Burgoyne, R. D., Mills, C. O., Chipman,

J. K., and Coleman, R. (1994). Fluorescent choleretic and cholestatic bileRoelofsen, H., Bakker, C. T. M., Schoemaker, B., Heijn, M., Jansen,P. L. M., and Oude Elferink, R. P. J. (1995). Redistribution of canalicular salts take different paths across the hepatocyte: Transcytosis of glycoli-

thocholate leads to an extensive redistribution of annexin II. J. Cell Biol.organic anion transport activity in isolated and cultured rat hepatocytes.Hepatology 21, 1649–1657. 127, 401–410.

AID FAAT 2309 / 6k1c$$$101 05-16-97 03:55:37 ftoxa

canalicular retention as anin vitroassay of tight junctional permeability in isolated hepatocyte...

Documents