hepatobiliary function and toxicity in vitro using isolated hepatocyte couplets

Pergamon 0306-3623(95)00071-2

Gen. Pharmac. Vol. 26, No. 7, pp. 1445-1453, 1995 Copyright © 1995 Elsevier Science Inc.

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REVIEW

Hepatobiliary Function and Toxicity In Vitro

Isolated Hepatocyte Couplets Using

R O G E R C O L E M A N * J O A N N E C. WILTON, VICKI STONE and J. KEVIN C H I P M A N

School o f Biochemistry, University o f Birmingham, Birmingham B15 2TT U.K. [Tel: 0121-414-5447; Faz" 0121-414-3982]

(Received 10 February 1995)

Abstract-1. Hepatocyte couplets can be routinely prepared from rat liver to produce a suitable in vitro model for polarized primary cells.

2. Centrifugal elutriation provides a means of producing enriched subpopulations of periportal and perivenous couplets from the same liver, thus providing a means of studying the influence of zonal heterogeneity on hepatobiliary function.

3. The maintenance of structural and secretory polarity demonstrated by hepatocyte couplets provides a convenient in vitro system for mechanistic studies of factors both regulatory and adversely affecting hepatobiliary functions.

4. Couplets are also uniquely appropriate for specific studies of regulation at the biliary pole, on the performance of junctions and on the maintenance and rate of transcytotic movement.

5. The possibility also exists that effects of an in vivo pre-exposure to agents causing hepatobiliary dysfunction can be assessed in couplets ex vivo.

Key Words: Hepatocyte, couplet, hepatobiliary, toxicity, cholestasis, polarized cell

INTRODUCTION

Hepatocytes are polarized cells, both morphologically and functionally. Their general metabolic functions and nutrient processing are related to interaction with blood at the sinusoidal pole, whereas elaboration of bile oc-

curs at the canalicular pole. The bile canaliculus is a lumen between adjacent hepatocytes, sealed by tight junctions, forming an extracellular tube which delivers bile to the bile ductules. Bile contains bile acids en route

to the intestine and also many of the end products of

xenobiotic metabolism, prior to their excretion from

the body (Coleman, 1987). In some cases the metabolism of xenobiotics results in the formation of reactive intermediates which cause cell toxicity (Monks and Lau,

1988), but in other cases the damage may occur more directly to the processes of bile formation and is then manifested as cholestasis.

Gene expression in hepatocytes is controlled by a combination of hormonal signals from distant and neighbouring cell types, by the interaction of the he-

*To whom all correspondence should be addressed.

patocytes with their supporting matrix and by their position in the acinus relative to the blood supply (which

defines the nature and levels of nutrients and xenobiotics to which they have access). In vivo, all of these factors may be operating simultaneously but, when he-

patocytes are separated from their native environment for a period of time, these influences cease to operate.

The need to study hepatocyte function, especially drug metabolism and toxicity, in a rapid and reproduc- ible manner without the use of large numbers of

animals has encouraged the development of in vitro

hepatocyte models. Long-term cultures of hepatoma,

embryonic or other cell lines have been of limited use in this respect due largely to their modified expression of cytochrome P450 species and conjugation activities (Bridges, 1981).

HEPATOCYTES AS A MODEL OF HEPATOBILIARY FUNCTION

Isolated hepatocytes

Freshly isolated hepatocytes retain, for a short time, gene products which are the "memory" of the influences the genes were exposed to in the original liver. This

1445

1446 Roger Coleman et al.

"memory" declines as specific mRNA molecules, in- eluding those coding for drug metabolism enzymes eta, are degraded without replacement. Isolated (primary) hepatocytes have nevertheless been used successfully for short-term studies on xenobiotic metabolism and toxicity (Bridges, 1981, Berry et al., 1991, 1992).

Isolated hepatocytes are prepared using a collagenase digestion of liver tissue; this can provide a large number of cells which can then be used either in suspension or in monolayer culture. Such cells can be used for uptake, metabolism and excretion studies and can be monitored for evidence of toxicant-induced damage by biochemical and morphological parameters (Berry et al., 1991, 1992).

Isolated hepatocytes are conventionally prepared and used as single cells, "singlets." These cells, which have been separated from their neighbours, cannot maintain their polarity and therefore lack a biliary pole This loss of polarity makes them unsuitable models for

studies of bile secretion, of transcellular transport or of specific pharmacological and toxicological situa- tions.

Isolated hepatocyte couplets

Limited exposure of the liver to collagenase results in a proportion of the cells not separating from one

another. They remain together as pairs ("couplets" or "doublets") or small multiples, joined at their tight junctions (Oshio and Phillips, 1981). During subsequent incubation these tight junctions seal and there is some relocation of junctional and canalicular membrane material. The tube that previously formed the bile canaliculus becomes enclose d, forming a canalicular vacuole into which biliary secretion can occur (Fig. 1). Thus the couplet is a primary bile secretory unit, with the vacuole representing the bile canalicular lumen. The structural and secretory polarities of the cells have been maintained. Isolated hepatocyte couplets are rapidly establishing themselves as a polarized in vitro model of value in studies of hepatobiliary function and mal- function.(Jones and Burwen, 1987; Boyer et al., 1988; Boyer, 1993)

PREPARATION AND MATURATION OF HEPATOCYTE COUPLETS

Preparation

All methods for the isolation of hepatocyte couplets rely upon preper fusion of liver with calcium-free buffer followed by a short term collagenase perfusion. The

method used by this group (Wilton et al., 1991) is based upon those described by Phillips et al. (1982) and Gau-

C Collagenase perfusion and plating

Canalicular vacuole

Singlets Couplet Fig. 1. Hepatocytes in the liver lobule excrete bile components across the canalicular membrane into the bile canalicttlus, which is sealed by contiguous tight junctional contacts between adjacent hepatocytes. Iso- lation of hepatocyte couplets from perfused liver maintains tight junctional contacts. After an incubation period, to allow reseating of the junctions to form a canalicular vacuole, hepatocyte couplas excrete primary bile into this canalicular vacuol~ Singlets have lost both their junctional contacts and their apical polarity.

Hepatobiliary function and toxicity 1447

tam et al. (1987). Initial perfusion with calcium-free Hanks balanced salt solution (Ca2÷-free HBSS) is followed by a 4 min perfusion with 13U collagenase in HBSS. The freed cells are filtered and washed in Lie- bowitz L-15 (L-15) medium (preparation A--typically 25°70 couplets,with a total viabilty of 70°70). The remain- ing tissue is re-incubated in the collagenase solution at 37°C for 5 min, filtered and washed (preparation B - typically up to 40°70 couplets with a viability of 96°70). Preparation B is routinely used.

Purification

If further enrichment is required, centrifugal elutriation of the preparation is necessary. This provides a nondestructive means of separating cells into populations based on size, allowing separation of couplets from singlets. The protocol used (Wilton et al., 1991) maintains the rotational speed of the rotor constant while the flow rate through the separation chamber is increased. From a mixed population, single cells elute first followed sequentially by couplets, triplets and small multiples. An advantage of using this procedure is that viable cells elute later than nonviable cells; the elutri- ated cell preparation therefore has a higher viability than the initial suspension. A typical separation profile is shown in Fig. 2. It is important to verify that the cells fractionated are those of choice as the liver com- prises at least five distinct cell-types. As a preliminary check, morphology (using phase-contrast microscopy) should be used. More stringent assessments involving immuno- or histo-chemical markers of specific cell types can be used; hepatocytes stain for the asialoglycoprotein receptor (Mizuno et al., 1984).

Heterogeneity

The preparations above consist of parenchymal cells from the whole liver lobule; hepatocytes, however, are not a uniform population (Jungermann and Katz, 1989). Periportal cells, khat is, those closest to the por- tal vein, are smaller than perivenous cells which lie adjacent to the hepatic vein (Schmucker et al., 1978). We have exploited this property and by modifying the elutriation protocol outlined above have separated hepatocyte couplets into periportal- and perivenous- enriched populations, without the need to selectively destroy one of the zonal populations during the preparation (Wilton et al., 1993a, Fig. 2). Glutamine syn- thetase, which is only present in perivenous cells, was used as a marker and showed that the larger couplets were of perivenous origin.

Short-term culture

For analyses based on microscopy, the cells are plated, for example, at a density of I x l0 s units/ml, in L-15

medium (a unit is defined as a single cell, a couplet, a triplet, or any other small multiple) (Wilton et ai., 1991). The cells are plated either directly onto plastic petri dishes, or glass cover slips. For biochemical as- says, the number of cells is determined according to requirements. For much microscopic work "preparation B" can be used, but for biochemical analyses, elutriation is essential to provide a more enriched preparation. Supplements to the plating medium are sometimes made. These include serum (e.g., 10% foetal calf serum; Sakisaka et al., 1988), which encourages the cells to settle on the plate. Serum also accelerates the "flat- tening" of the cells, however, and thus restricts the time they are useful for observation of 3-dimensional structure. Antibiotics are sometimes added (Gautam et al.,

1989) and medium supplements such as zinc, growth hormone, epidermal growth factor and prolactin have been used (Spray et ai., 1986).

Maturation

Following the original observations of Oshio and Phillips (1981), Boyer and colleagues (Graf et al., 1984), have pioneered the use of couplets in hepatobiliary phenomena by utilizing the sealing properties of the canalicular vacuole. An incubation at 37°C enables the canalicular vacuole to form and seal within 4 hr (Wil- ton et al., 1993b). Most groups use the preparation between 2-8 hr of incubation during which time there will be a cycle of vacuolar filling and emptying. We routinely use cells between 4-6 hr incubation. The ex- perimental time can be extended by storing plated cells at 4°C for up to 6 hr (Wilton et al., 1993b). During the time in which sealing occurs, canalicular membrane material and supporting cytoskeletal elements reorien- rate to the canalicular vacuole membrane, a process which can be prevented by cytochalasin D, an inhibitor of microfilament function (Gautam et aL, 1987).

100

so

'~ 60

Total sinsl~s

Viable singleet

Couplets

Triplets

MttRiplea

( 0 20 40 60 80

Elutfiation Flow Rate (ml/min) 100

Fig. 2. Purification of rat hepatocytes into separate populations of singlets, couplets (shaded), triplets and small multiples.

1448

L (,,)

Fig. 3. Use of canalicular vacuolar accumulation (CVh,) of the fluorescent bile acid analogue cholyl-lysyl-fluorescein (CLF) by isolated rat hepatocyte couplets to indicate hepatobiliary function and toxicity: a) control couplets; b) couplets

exposed to menadione (100 uM, 15 min).

Couplets after sealing demonstrate a pericanalicular localization of microftlaments, microtubules, lysosomes and Golgi apparatus (Graf and Boyer, 1990).

During this period paracellular permeability to ruthenium red falls, indicating the sealing of the tight

Roger Coleman et al.

junction around the vacuole (Gautam et al., 1987). Elec- trophysiological experiments where microelectrodes have been impaled through one cell into the canalicular vacuole, once sealing is complete have allowed the measurement of gradients of potential difference across the canalicular and basolateral membranes of - 35 mV and - 40 mV, respectively (Boyer et ai., 1988). Such measurements have increased our understanding of the magnitude and role of these gradients in biliary secretory processes.

Techniques f o r assessment o f function

All chemical, spectroscopic and microscopic methods used with singlet cells can be applied to couplets. As polarized secreting cells with a functioning canalicular vacuole, couplets enable further techniques to be used in the study of a variety of hepatobiliary processes. These include: i) video-microscopic optical planimetry, using Nomarski optics, (Gautarn et al., 1989; Boyer, 1993) ii) continuous video recording, using bright-field microscopy (Graf and Boyer, 1990), iii) epifluorescent microscopy of the vacuolar accumulation of fluorescent cholephiles (Graf et al., 1984, Fentem et al., 1990), iv) laser-scanning confocal or image deconvolution high-resolution microscopy of fluorescent cholephiles and indicators (Kitamura et al., 1991, Nathanson and Burgstahler, 1992; Wilton et aL, 1994), v) horseradish peroxidase and analogous materials for transeytosis and paracellular permeability assessment (Sakisaka et aL, 1988), vi) microelectrode and patch-clamping for electrical driving force measurements (Graf et aL, 1984, Weinman et al., 1989), vii) microinjection of dye to as- sess gap-junction activity (Spray et aL, 1986, Guppy et al., 1994).

HEPATOBILIARY FUNCTION AND DYSFUNCTION STUDIES IN COUPLETS

Modelling biliary secretion o f organic anions

The rate of canalicular vacuole filling has been studied as an in vitro indicator of factors affecting bile flow. Filling rate increased when bicarbonate ions, which con- tribute to bile flow in vivo, were available (Gautam et aL, 1989). Rates were achieved which, in proportion, were analogous to those of bile-acid-independent flow in whole liver. The rate of filling was further increased by the presence of taurocholate (10 uM) in the medium (Gautam et al., 1989), indicating that bile-acid-dependent-flow was occurring. Differences in secretory rate between individual couplets were ascribed to the numbers of the transporters in the canalicular membrane

Fluorescein and fluorescein diacetate (Graf et ai., 1984) and 2'-7' bis (carboxy-ethyl)-5-carboxyfluorescein) (Strazzabosco et al., 1988) provided early demonstra-


tions of fluorophores secreted into the canalicular vacuole (as their conjugates) representing organic anion secretion. Other fluorophores have been used subse- quently including carboxy dichlorofluorescein diacetate (the diacetate esters enhance diffusion into the cell), Fura-2 (Takeguchi et aL, 1993), and a considerable number of fluorescent bile acid analogues based on 713- nitroazooxadiol (NBD) or fluorescein (L) moieties, for example, NBD-TC, C-NBD-L, CDC-NBD-L, CGamF (Kitamura et al., 1990a; Weinman et al., 1993; Maglova et al., 1993), and a family of bile acid structures linked to a lysyl-fluorescein moiety, for example, cholyl-lysyl- fluorescein (CLF) (Mills et al., 1991; E1-Seaidy et al., 1993; Wilton et al., 1994).

In experiments using per fused rat liver, CLF was ex- creted into bile with the same first pass uptake and cu- mulative excretion as glycocholate (Mills et al., 1991). In couplets, CLF was shown to be accumulated into the canalicular vacuole, using fluorescence microscopy (Fentem et al., 1990) (Fig. 3). CLF accumulation is rapid, resembling that of native bile acids in isolated livers; substantial canalicular vacuole accumulation (CVA) is observed within 1-2 min. Simultaneous presence of taurocholate reduces the couplet content of CLF with little change in vacuolar size, especially in perivenous couplets (Wilton et al., 1993a). When different bile acids linked to the same fluorescent moiety were studied, different pathways across the cell were observed; cholate and chenodeoxy cholate analogues moved rapidly through the cytoplasm, whereas lithocholate and ursodeoxycholate analogues appeared to involve a slower vesicle-mediated transcytosis. These latter bile acids also brought about a redistribution of annexin II, further indicating the participation of membrane fusion events during their movement across the sinusoidal membrane, and secretion at the canalicular membrane (Wilton et aL, 1994).

CLF has been used, not only as a specific bile acid analogue, but also as a more general indicator of canalicular filling. The introduction of a fluorescent cholephile serves to give a time point from which to study, by fluorescence microscopy, the behaviour of large numbers of canalicular vacuoles. It also allows a representative estimate of the behaviour of a population rather than of individual units. CLF has also allowed the study of canalicular retention (by prefilling the vacuole for 10-15 min and then looking at influences upon the rate of loss). Due to progressive bleaching of the fluorophore with continuous exposure to ultravio- let light, such fluorescence microscopy can, however, only be used as a series of "snap-shots" rather than as continuous recording.

Using flow cytometry, the concentration of CLF specifically in viable couplets has been observed

(Lankester et aL, 1994b). After the initial rapid uptake there was then a time-dependent increase in the accumulation of CLF, reflecting the canalicular accumulation of CLF monitored by fluorescence microscopy.

Bile acids themselves may be regulators of biliary excretion, involving mechanisms beyond their direct secretory activity. Thus, bile acids, especially tauroursodeoxycholate, increase the biliary excretion of cholephiles other than themselves and stimulate vesicular exocy- tosis (Hayakawa et al., 1990, Sakisaka et al., 1994).

The use of couplets from TR- mutant Wistar rats highlights the predictive value of the couplet model for organic ion secretion into bile. These rats are known to be deficient in the ATP-dependent organic anion transporter in the canalicular membrane. Couplets pro- duced from these were defective in canalicular vacuole accumulation of the fluorophore, carboxydichlorofluores- cein diacetate while maintaining normal ability to transport a (fluorescent) bile salt (Kitamura et al., 1990b).

The metabolic fate and disposition of a number of other xenobiotics has also been investigated. With the relatively nonspecific cytochrome P450 substrate, 7-ethoxycoumarin (7-EC), conjugated metabolites increased in the couplets up to 30 min of incubation with 7-EC. These conjugates (mainly glucuronides) were sub- sequently released into the medium. This profile models the predominance of biliary excretion of conjugated 7-hydroxycoumarin in vivo (Lankester et al., 1994a). Preliminary work in our laboratory with paracetamol similarly shows initial retention of paracetamol glucuronide in couplets prior to release into the medium; this characteristic is not shared by the lower molecular weight sulphate conjugate product. This may indicate the operation of a biliary molecular weight excretion threshold in vitro (e.g., by leakage across the tight junction) (Hirom et al., 1972, Coleman, 1987).

Tight junct ion funct ion and paracellular permeabil i ty

Tight junctions restrict the passage of materials between the canalicular lumen and the extracellular fluid in vivo and from the canalicular vacuole in vitro. The protein ZO-1, which is characteristic of tight junctions, is localized at the position of cell-cell contact (Steven- son et al., 1989; Graf and Boyer, 1990). The retention

of (fluorescent) cholephiles within the canalicular vacuole provides evidence that the junctional barriers are intact and relatively impermeable to organic ions. In addition, as canalicular vacuolar accumulation of fluorescent bile acids, etc. is increased, the paracellular penetration of ruthenium red is reduced in paral- lel. Hypertonic raffinose causes the vacuoles to shrink indicating that the tight junctions are permeable to wa- ter but relatively impermeable to noncharged molecules of the size of rafflnose (MW 595). They are even more


impermeable to negatively charged ions as small as bile acids (MW approx 500) and ruthenium red (MW 551).

Tight junctional permeability in couplets appears to be under hormonal control, as in the liver (Lowe et al., 1988, Llopis et al., 1991), since vasopressin caused an

increase in paracellular permeability (to horseradish peroxidase), followed by canalicular vacuole collapse. Increased paracellular permeability to HRP following exposure of couplets to vasopressin was preceded by an elevation in intracellular Ca 2÷ (Nathanson et al.,

1992a). Both of these phenomena were blocked by Ni 2÷, which inhibits movement of Ca 2÷ into the cell from the extracellular fluid; this may indicate that tight junctional function may be influenced by intracellular Ca 2÷ concentrations.

Phorbol dibutyrate also increased paracellular permeability, but without a prior rise in Ca 2+, suggesting that other factors may be operating to control tight junction function. The action of H-7, an inhibitor of PKC activity, in reversing the effects of both vasopressin and phorbol dibutyrate indicates that protein phos- phorylation may be a late factor in this chain of events (Nathanson et al., 1992b).

Dysfunct ion o f canalicular vacuolar accumulation as an indication o f cholestasis

The applicability of CVA as an indicator of canalicular dysfunction and cholestasis has recently been demonstrated in studies with menadione and cyclosporin A.

Menadione (2-methyl-l,4-naphthoquinone, vitamin K3) causes choleresis, cholestasis and hepatocyte death at progressively higher concentrations (Akerboom et

al., 1988). Incubation of hepatocyte couplets with menadione, (up to 100 ~tM) induced a cholestatic response as indicated by a concentration-dependent reduction in CVA (Stone et ak, 1994a) (Fig. 3b). This occurred in the cholestatic concentration range seen for isolated livers (Akerboom et al., 1988) and was not ac- companied by a decrease in ATP concentration, which suggests that the inhibition of hepatobiliary function did not result from a cytotoxic effect (Stone et al., 1994b). The proportion of couplets retaining pre- accumulated CLF in their canalicular vacuole was reduced by 44070 with menadione (40 ~tM) (Stone et al., 1994b). This suggests that the reduction of accumulation might involve an inability to maintain sealing of the vacuole.

It is unlikely that the menadione-induced inhibition of CVA is operating directly via an interference in Ca 2÷ homeostasis. Blocking the entry of extracellular calcium by blocking plasma membrane calcium channels did not prevent the inhibition of CVA by menadione. In addition, release of internal calcium stores by thap-

sigargin was insufficient to inhibit CVA suggesting that neither entry of extracellular Ca 2÷ nor release of intracellular Ca 2÷ are prerequisites for the inhibition of CVA by menadione at sub-cytotoxic concentrations (Stone et al., 1994b).

The concentrations of menadione affecting CVA also

induce depletion of cellular reduced glutathione content and increased production of oxidised glutathione (Stone et al., 1994a). The accumulation of oxidized glutathione accounted for only a small proportion (1007o) of the reduced glutathione which had been depleted, suggesting that oxidative mechanisms are unlikely to be solely responsible for CVA disruption by menadione. In support of this is the observation that 2,3-dimethoxy-l,4-naphthoquinone (a quinone with predominant redox cycling ability) is virtually devoid of effect upon CVA, whereas treatment with the arylating quinone, p-benzoquinone significantly reduced CVA (Stone et al., submitted). Arylation may thus have an important role in the inhibition of hepatobiliary function by menadione.

There is a marked difference in susceptibility of periportal and perivenous couplets to CVA reduction induced by low levels (25 ~tM) of menadione. Peripor- tal cells show both a reduction in CVA (to 20070 of control levels) and a reduction in vacuolar size, that is much greater than those shown by perivenous couplets (to 80070 of control) under identical circumstances (Wil- ton et al., 1993a).

Cyclosporin A is an immunosuppressor commonly used in support of organ transplantation that has com- mon side effects on kidney function and also on the liver, the latter being associated with cholestasis and jaundice (Kahan, 1989). A number of possible causes of this hepatobiliary dysfunction have been put for- ward following chronic and acute studies at a variety of concentrations; these include a study, using rat hepatocyte couplets, in which the multidrug efflux pump was found to show half maximal inhibition by cyclosporin at 600 nM and by FK506 at 10,000 nM (Takeguchi et al., 1993). When incubated with couplets at concentrations in the range of those in the therapeutic trough levels in plasma (30-100 nM), a dose-dependent reduction in CVA of cholyl-lysyl-fluorescein was observed (RomAn and Coleman, 1994). This appeared to involve both a reduction in uptake of fluorescent bile acid, and a reduction in its retention, since pre-accumulated cholephile could not be retained in the canalicular vacuole (RomAn and Coleman, 1994), suggesting tight- junctional dysfunction. At these concentrations of cyclosporin A, no reduction of cytoplasmic glutathione was observed (RomAn and Coleman, 1994); the mo- lecularmechanism(s) involved are likely to be different from those for menadione.


Cytoskeletal structure and funct ion

After several hours incubation in nutritive solution, couplets have a pronounced pericanalicular cytoskeleton, resembling that observed in vivo. The pericanalicular cytoskeleton is composed of F-actin, several actin-associated proteins, and myosin II (Tsukada and Phillips, 1993). These proteins are associated with the canalicular membrane, the pericanalicular cytoplasm, the tight and gap junctions forming a cohesive inter- connected network of membrane and cytoskeleton. In addition, the outer layers of the actin cytoskeleton are surrounded by intermediate filaments, which interact with the desmosomes.

The components of the cytoskeleton can be visualized with appropriate antibodies, but the microfilament cytoskeleton is conveniently visualized by the specific interaction of F-actin with phalloidin (tagged with FITC) (Stone et al., 1994b). The actin cytoskeleton resembles somewhat the terminal web of the intestinal epithelial cell in that it forms a band around the canalicular vacuole below the tight junctions (Mooseker, 1985; Tsukada and Phillips, 1993).

Cytoskeletal integrity appears to determine many of the physiological properties of this area, including vacuolar retention and contractility. The canalicular vacuole can contract, probably due to the presence of myosin and actin in the pericanalicular cytoskeleton (Tsukada and Phillips, 1993); this may be the in vitro manifestation of the in vivo ability to propel materials down the biliary tract by rhythmical peristaltic-like contractions (Watanabe et al., 1991); however, the relative importance of pressure-induced canalicular vacuolar collapse versus an active contraction has yet to be determined.

Contractions of the couplet vacuole and its cytoskeleton can be stimulated by microinjection of ATP (Wata- nabe et al., 1991, Kitamura et aL, 1991) by extracellular ATP (Kitamura et al., 1991), by the entry of extracellular Ca 2* ions (Watanabe and Phillips, 1984) and by increased internal Ca x÷ concentrations (Kitamura et aL,

1991); they can be inhibited by disruption of microfila- ments on treatment with Cytochalasin D (Kitamura et al., 1991) and by a calmodulin inhibitor, trifluopera- zine (Naramoto et al., 1991).

Colchicine, which interferes with microtubular function, does not affect the rate of "maturation" of the canalicular vacuole in short-term culture. It interferes, however, with both the movement of lithocholate analogues (Wilton et al., 1994) (see above) and the transcytosis of horseradish peroxidase in vesicles (Sakisaka et

al., 1988), indicating that an organized, polarized microtubular assembly is present in the matured couplet, and necessary to maintain optimal transport function.

Pericanalicular cytoskeletal alterations associated

with cholestasis

Accompanying the reductions in CVA and CLF retention, menadione and cyclosporin A also induce a depletion of phalloidin-FITC-labelled F-actin from the pericanalicular cytoskeleton of couplets (Stone et al., 1994a, Roman and Coleman, 1994). The cause of this depletion remains to be established. Since the pericanalicular cytoskeleton appears to be involved in canalicular contractility and is also attached to the tight junction, any disturbance in the pericanalicular cytoskeleton is likely to result in dysfunction in bile formation due to a number of causes, for example, increased paracellular permeability (impaired vacuolar retention), reduced contractility and reduced delivery of carriers to the membrane.

Using a different series of cholestatic compounds, (phalloidin, erythromycin estolate and taurolithocho- late) and incubated for a much longer time period (3 hr vs 15 min), cytoskeletal alteration took the form of an increase in pericanalicular F-actin, measured by scanning laser cytometry (Thibault et al., 1992). This increase was not seen with noncholestatic bile acids or the hepatoprotective tauroursodeoxycholate. This increase in pericanalicular cytoskeletal F-actin is unlikely to be an increase in functional material, but rather in polymerized actin, as is very likely in the case of phalloidin. Functional disturbances, for example, the development of cholestasis, are likely to result from accumulation of disorganized cytoskeletal components.

Abnormalities in amount or the polymerization state of cytoskeletal material could be caused by a variety of reactions, for example, alkylation, thiol oxidation, crosslinking, or proteolysis, and it is tempting to spec- ulate that the reductions in CVA accompanying elevation of Ca *÷ (by A23187 exposure) (Stone et al., 1994c) and depression of glutathione (by menadione and DEM) (Stone et al., 1994a) may also involve cytoskeletal dysfunctions due to these changes and in the case of glutathione reduction, to a lowered ability to be pro- tected.

Gap junction function and intercellular communication

Intact gap junctional communication between individual hepatocytes of couplets has been demonstrated both by measurements of electrical coupling (Rever- din and Weingart, 1988; Spray et al., 1986) and by trans- fer of microinjected lucifer yellow (Spray et al., 1986; Guppy et al., 1994). A number of xenobiotics including carbon tetrachloride and phenobarbitone inhibit gap junctional intercellular communication between these cells. These agents appear to operate via a cytochrome P450-dependent mechanism at a post-transcrip-


t ional level. The effect is dose dependent and revers-

ible (Saez et al., 1987, 1989; Guppy et al., 1993, 1994).

C O N C L U S I O N A N D P E R S P E C T I V E S

The maintenance o f structural and secretory polar-

ity demonstrated by hepatocyte couplets provide a con-

venient in vitro system for mechanistic studies of

hepatobiliary function and dysfunction. The recent

availability of perivenous and periportal enriched coup-

let preparations now enables some of these studies to

have a zonal perspective.

Due to their relative novelty, hepatocyte couplets have

not yet been studied from species other than rats,

though we now have preliminary experience in their

production from mice and human liver.

The contr ibution of hepatocyte couplets to our un-

derstanding of hepatobiliary function, dysfunction and

toxicity is still at an early stage; the next few years should

see further exciting contributions from this novel in

vitro model.

Acknowledgments-We are grateful to David Lankester for making available to us some of his preliminary observations on xenobiotic uptake and secretion. We are also grateful for the help and financial support of the Wellcome Trust, Unilever, and SmithKline Beecham.

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hepatobiliary function and toxicity in vitro using isolated hepatocyte couplets

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