Review

The use of isolated rat hepatocyte couplets in hepatobiliary physiology

Undfssoeiated paits of liver cells are tiequently encaun- tered in isolated ceil preparations obtained by various

tecbtdques of rzagenase petfusion. In tissue CtdhJre, these mtdissociated ceUs may spontaneously develop a fbdd-F&d vacuole between them. This obsetvation sug- gested that adjacent cells rearrange their polarity Y) as to express tbeii secretoty epparatvs face to face 1esultiSlg in pmductioo of bile into a clmd space. This bilituy space is

comptxed of two half shells of cattali~~ular surface tnettt-

butte and is sealed born the extmcellulat medium by a

belt of tiebt itmctiottal cell membrane eontact. Thus the

ment more quickly. Tbe techniques wed to obtain cell cnuplets are essentially the same except for a tednction of

the collagenase concettttation wbicb appears to increase the percentage of ceil pairs. After isoMion, cells ate sus- pended in culture medium and aUowed to settk onto glass cover slips where they sponta’~ousty adhere. CovenUps can the” be uanrfetred for tktbet investigatio,, by mkt..,. smpic, bfstochemkl, electnpby&Sogic or eytaphoto- m&c techniques. The formation of wcretoty vacuoles

of c&&c& bile fotmatiotti. vitro (Fig. 1). (1,2). Tb;

following gives a brief dewtiption of the system as ex-

plored so far.

Techniques of liver cell isolation involve perfusion of

the organ with B cakium-free solution in order to loosen

all eotttacts followed by a semnd perfusion with collage-

ttawcmttainbtg medium 13 release the cells from the ex- traceUulat matrix. After the otgao has softened, the cap stde is opened and cells are relspvd into suspettsio,~ that fs filtered to remove remaitdng cotmectjve tissue and law

get cell aggtegates. CeUs ax sedimented eitbet by gravity or by low-speed cetttxifugation. After resuspension of the

c&s sedimetttatioo is repeated in order to retttove mlla-

genase by dilution and to emich for live cells w&b sedi-

388 1. GRAF AND Is.. BOYER

appears not to depend on panicular culture conditions

and is observed in media such as Gibco L-15 or DMEM.

Cell isolation and culture conditions are described in

Refs. 1,2 and 3.

(B) hforphogenesis of hepatocyte ~uplets in cell culture

Freshly isolated cells retain their original polarity, i.e.,

each single cell is surrounded by a band of canalicular cell

membrane, the retained half of a bile canaliculus, the

other half having bee” provided by adjacent cells in intact

tissue. The canalicular membrane can be visualized as a

belt-like structure with histochemical staining for the w.il”. ” .‘. & ( *:: L . marker enzyme Mg-ATPase (2.3) and, at each side of this -,

band, by immunofluorescence staining for the tight junc- 5,

tion-associated protein ZO-1 (4) (Fig. 2). At the retained CI

site of adhesion in a cell pair, remnants cd a complete bide :, ., canaliculus may be found. Immediately after isolation this

remnant represents ” tube like structure which is open to

the external mediun as visualized by peneaation of the electron microscopic extracellular space marker rutheni-

urn red (3). With exception of the cell-cell contact zone,

canalicular membranes are almost completely removed

from the cell surface within the first 2 h in tissue culture, probably by endcqtosis. The tubular canalicular rpace at the contact w”e, on the other hand, becomes sealed at its ends by tight junctions and is no longer awssible to ru-

thenium red. After a few houn in tissue culture, the size

of the canalicular space increases by acquisition of surface

membrane and by secretion of fluid into its lumen. The lu-

minal membrane outlining the vacuole stains for Mg-

ATPase (Fig. 2). Microvilli line its surface bdt smooth

areas may be seen as well, reminiscent of canalicuiar

membranes in cholestatic conditions. The lumen is sealed

from the external medium by a continuous tight junction

around its entire circumference as see” by electron micm-

scopy and localization of ZO-1 protein (Fig. 2). This rear-

ganhation of canalicular membrane requires normal mi-

crailament function a$ indicated by its inhibition by cyto-

chalasins (3). The rearrangement of canalicular and baso-

lateral membranes also includes the polar expression of

membrane transport systems such as lutninal transport of

organic anions and bile acids (see below) and the prcfe:-

enfial IcaIization of NalK-AT&se a-subunit to the bare-

lateral (non-luminal) surface domain (Fig. 3) (5).

Rearrangement of cytoplasmic elements also takes

place. As visualized by electron microscopy and immuno-

fluorescence microtubules (6) and microfilaments (7) be-

cane predominantly localiied to the perihuninai cym-

plasm. In particular, micmtilaments form a dense web un-

derneath the luminal plasma membrane but are also

found around larger vesicular structures mainly in the vi-

cinity of the canalicular space (8). As in intact liver, the

G&i apparatus, lysosomes and autophagic vacuoles be-

umte localized to the secrefory pole ot the liver cell. De-

tails ~&att the development of hepatocyle couplets in cell

cultwe are found in Ref. 3.

Within 3-4 h in cell culture, canalicular spaces enlarge, occasionauy reaching a diameter up to 10 #rn_ Smaller lu- minal *paces are often irregular in shape, sometioles ex-

hibitisg rhon trbular branches, but smaller spherical

spaces may be found as well. Larger spacer are generally

round or ellipsoid in shape, their bmdermg membrane ap-

pearing Smcmth. Larger spaces may z&o appear as flat

disk-shaped gaps ocxpyizg a large zi.d ai the iwaface

between the two dells. This heterogeneity of shape and size is best appreciated by lluorejeence microscopy a&

ter secretion of ckcdephilic fluomchromes such as fluores-

cein. In addition, differential interference contrast optics

enable optical sections to be obtained across tht :r minal

spaces taking advantage of the &allow depth of the focal

plane. Serial se&ons can then be obtained rt diff srent

heights permitt+ rewnstnwdon of the due .-dimersion-

al shape of the It. men (9). As seen by time lapse cinenato-

graphy the shapa of the lumen varies an4 the size of the lu-

men increases ,with time. These dynar,icvariationsprl~ba-

bly represent recruitment of new srembrane and, a$ the

lumen tills utth fluid, represent dte correlate of ph:siw

logic bile seiretion (see below;. After the luminal spaces

reach a certain size by a combinatian of membrane acqui- sitior. and ;luid secretion a sudden reduction of sti ace

curs, sometimes resultirg in virtual disappearance aa’ the

space. Tba event apparently results from the generation

of a high hydrastat+: pressure in the lumen that tea& to

escape o’luminal fiuid through the ti&t junctions into the bathing medium or back inm Ihe cell. This reduction in In-

minal volume may be complete within a few seconds but may &so be more ,,rotracted. Collapse may be fa!Jo’ved

by refilliq and reexpansion of the space. The spent.?-

neous ocsurrence of these events appears to be very ’ ari- able irJm couplet to couplet. Sudden vohnne reducion

ma: oe inhiated by various simatiions. Escape of lun inal

flu2cm bepmducedsimplybymakingaleakin thel Imi-

1181 wall with B microelectrode thus illustrating that i hy-

drostajc pressure difference exists between the Ionen

and thr. external bathing medium. Luminal pressure i.1 ex-

cess 0.’ the compliance of the lumind wall may be gr ner-

ated by fluid secretion that exceeds the capacity of tPs In-

minal space. Fluidrelease may then occur at thesite<.f the

tight junction resulting in mUapse of the space (Fi&. 4).

Collapse may be aka induced by placing couplets irat,> hy

potonic medium which results in rapid accumulatkn of

fluid in the lumen by osmotic water flux. It is thu i en- visaged that, during the enlargement of the fuminal ‘V~EU-

ale in comrol medium or in the presence of bile acids, a phase of physiological fluid secretion occurs but k fd- lowed by P ‘cholestatic’ condition, characterized by ek-

vated luminal pressure, rupture of tight junctions ard al-

terations of the luminal ceU membrane. The latter tncy in-

clude loss of microvilh as -bed above.

Elevated pre?aXe may be also generated by an increase

in the :ensian of the surrounding membrane by contrac-

tribute to bile fomtation, or from studies in isolated cell

membrane preparations, where the physiological signifi-

cance of an identified transport system is less evident. Be-

sides having been affimmtive in character, studies in the

couplet system also provided same new quantitative in-

fommtion. Canalicular bile formation results from the secretion of

osmotically active substances, in patticular bile acids.

which drag water and permeant solutes into the lumen.

Fig. 4 illustrates this concept of osmatic water flow by B

simple experiment: the size of the luminal space was re-

corded by analysis of grey Ievel histograms of consecutive

dieitized cross-sectional images obtained in bricht field il-

ltitinatio”. The expet’hne”t~ltows that rapid shrikage of

I!:: luminal space is obtained by generating an osmotic

gradient through the addition of rafiinose to the external

bathing medium. Although tonicity was only increased by

IS%, the lumen shrank by more than 601, demonswatiq

that osmotic water flow out of the lumen is accompanied

by a flow of permeant solutes. Shrinking thus apparently takes place until the concentrations of impameant or

poorly permeant solutes inside and outride the lumen be-

came equal. This camatic equilibration is quite fart indi-

cating that t!te f!ux of prmeant solutes takes a paracellu- lar route, since the resistance of cell membranes is too high for sufficient rapid ion flux to occur (see berow). The water permeability of cell membranes on the other hand is

hiih enough to accommodate the transepithelial water

flux (14). With continuous fluid secretion and, most like-

ly, by additional slow transport of raffinwe into tb ltt-

men, the canalicttlar space reexpands. The rate of npan-

sion is accelerated by the adjition of taurocholic acid to

the medium finally resulting in a collapse of the space as

fluid accumulation apparently exceeded the capacity af

the canalicular space. These shtdies in the cn~plet system

thus prove two concepts of canalirJlar fluid transport

anticipated from studies in the intact organ: (i) the canali-

c&r epithelium allows for rapid transepithelia! water

flow. Thus, an imposed osmotic gradient is rapidly dissi-

pated by solvent flow and, as consequenti, canaficularse-

cretion of impemteant osmotically active solutes drives

canalicttlar bile formation canfirming the osmotic theory

of bile formation as originally proposed by Sperber (15).

(ii) The paracellular shunt between lumen and bathing

medium allows for passage of small solutes (electrolytes). but restricts the diffusion of larger molecules like rafti-

nax (molecular weight 595), substantiating the concept

that the comparable ion wncentrations that are observed

in bile and blood result from paracellular diffusion ar the

canaticulor level (16). Measurements of tlvid secretion in

hepatocyte couplets are described in more detail in Ref.

9.

tion of the submembraneous microfilament network, thus

representing an active ~+umtenon (Fig. 4). Phillips and

coworker8 have shown that elevation of cytosolic calcium

concentration, either by microinjection (10) or by horma-

nal stimuli (ll), is associated with shrinking of the lumen,

whereas this event is not seen in the presence of cytocha-

lasina (12). It is thus envisaged that nomtal canalicular

bile flow is sustained by peristaltic movements of the ca-

nalicular wall. Dysfunction of these cytoskeletal elements

in the pericanalicutar region may then lead to cholestasis

of either the ‘spastic’ or ‘paralytic’ type. For further infor-

mation on these concepts of bile canalicular contractility

Ref. 13 may be consulted. Time lapse cinematography

also reveals dynamic structure! changes within the cym-

plasm, particularly movement of round vesicular orga-

nelles. sometimes along selected traffic routes, and endo-

cytic events at the luminal cell membrane (1).

(0) Cansticular bile formation in hepatocyte couplets

The hepatocyte couplet system offers the unique possi-

bility to study canalicular bile formation directl? It thus

provides the opportunity to evaluate or substantiate hy_

potheser on secretory mechanisms inferred from studies

in the intact organ. where non-canalicular processes con-

391

bile acid stimulates xc&ion of bicarbonate-rich bile in

the intact liver (Xpj. UDCA also elicits ‘hypercholere- sis‘ in the intact organ. i.e., secreticn of a certain quantity

of UDCA into bile causes a iwger choler&s than the se- cretion of the same quantity of another bite acid, swcb as

TC. However, in the couplet system a larger choleretic re-

sponse to UDCA was not observed (9). Bath these oixer-

vations are in favour of the concept of ‘cholehepatic

shunting’ of UDCA (26). a mechanism which appeas to

initially involve the canalindar secretion of the bile acid

anion. Then, by dissociation of carbonic acid in the lu-

men. HCO; is formed and UDCA is protonated to form

the undisscciated acid. UDCA-H is then reabsorbed,

probably by non-ionic diffusion, and may return to the site

of excretion, whereas HCOj remains in the lumen and is

secreted. The absence of luminal alkaliition in the

hepatcqte couplet system sug.gests that this luminal re-

plaoement of UDCA- by HCOj occurs downstream in

the biliary tree and not at the canalicular level. Isolated hepatcqte couplets have also been used to

study the mechanism of vesicular transport into bile.

Using horseradish peroxidase (HRP) as an electron mi-

crosmpic marker substancq and bistocbemical staining,

endacytic uptake at the basolateral cell membrae and rapid appearance of transqmtic vesicles at the lumioat cell membrane were observed (COmF.Ze Fig. 6). This

transport was p&izdLy inhibited by cokbicine showing it6 dependence on inmct micmtubul~s fuwtion. Application

of taurocholic acid induced elongation and tubular watts-

formation of transport vesicles (6). Extracellular space

Mechanisms of bile acid uptake and secretion have

been studied using a fluorescent bile acid derivative (7@

nitrobenzwxadiazol taurocholate, NBDTC) and cytoflu-

orometric techniques (17). Two components of uptake were identified, one partially dependent on extracellular

sadium and inhibited by taurocholic acid (TC). This ob-

setvation suggests that uptake of NBDTC into liver cells

is accomplished in pan by a Na+ gradient driven carrier

system with bigb affiity for TC and in part by a transport

system with broad substrate specificity. Both transporters

have been identified previously in basolateral membrane

vesicles and have been further characterized by bile acid

phdoaftinity labeling (for references see Refs. 18 and 19).

In amtmst, a carrier systetn for TC has been identified in

canalicular membrane vesicles, which is driven by the

trammembrane electtical potential gradient (20). That

this system actually operates in intact cells to promote ca-

nalicular bile acid secretion could be shown by two sets of

experiments: first, the fate of secretion of the fluorescent

bile acid into the lumen (compare Fig. 5) is reduced after

depolariziog the cell membrane potential with high potas-

sium medium (17). Conversely. application of hyperpo-

k&zing cwient with a,, intracellular microelectrode re-

sults ir; stim”k,tion of fluid secretion when taamchotii acid is present in the medium (21). These experiments provide direct evidence that the bmdnal membrane po- tendal is a major drivbtg force for bile aciddependent bile

secretion. The ability of the hepatwyte couplets to secrete organ

ic anions nwh as fluorescein (222) has also been used to . . I

intrcduce the fltmreSeem pH indicator 2’,7’-bis(c&oxy-

etltyl)-5carboxy8uoreJccin into the. canaiictdar lumen.

Application of ~~~&oxycbolic’aeid (UDCA) exhibited

no signiticant hmtinal alkalinization (23). wlweas this

392

markers such as HRP are secreted into bile of the isolated

perfused :?.er in two phases. The early phase has been

taken to indicate that HRP penetrates into the canalicular

lumen by pssage a- the tight junctions (27,28). How-

ever. the qGck accumulation of HRP in pei-icanalicular

vesicles in the hepatocyte cowlets indicates that a fast

aanscytotic transport component exists. ‘1 his may also BP

count for the early phase of HRP secretion in the intact

organ putting into question whether paracellular pwmea-

tion of Proteins takes place to any significant extent in

liver.

Further applications of the couplet system in studying

canalicular bile formation have appeared in abstract

form. These include the observation that fonkolin, dibu-

tyryl-cyclic-AMP and inhibitors of phosphodiesterase

each have B choleretic effect suggesting a regulatory role

of cyclic AMP in canalicular bile secretion (29). In addi-

tion, 1% has been shown to stimulate the biiary secretion

of inbacelhdar lipids after hepatocyte couplets have been

loaded with the fluorescent lipid derivatives NBD-cera- mide (JO) and NBD-phosphatidylcholine (31).

(E) Electmphysiology of hepaocyte. couplets

Electrophysiological studies have been carried out in order to study the routes, mechanisms, magnitude and driving fores of ion fluxes in the couplet system. For

these experiments this system offers the unique possibility

of applying methods of epithelial elenrophysiology to a

secretory process, since micraelectrodes can be insertco.

into the canalicular lumen. Electrically, the couplet is en-

visaged as an epirhelium separating luminal and base-

lateral fluid compartments. Ion flow occurs across three

barriers, the lumind and basolateral cell membrane and

the paracellular mute comprising the tigltt junction bar-

rier. Accordingly an appropriate equivalent circuit could

be applied to analyze some of the transport properties of

t’lese barriers (32).

The tight ltmctions impose little restriction for electro-

lyte permeation between the lumen and the external me.

dium. Tight junctions have an average resistance of 25

MP as compared to 150 MQ and 780 MS-J for the base-

lateral and lumittal cell membranes, respectively. A lumi-

nal electric potential of -5 mV was measured. In view of

the low tight junction resistance this potential appears to

be generated by the presence of impcrmeant anions in the

lumen causing a transephelial Dotman distribution of

permeant ions. Ionic current flow due to active transport

processes at either cell membrane appear to contribute

little to the luminal potential.

Evidence was obtained that the Nan<-ATPax is pre- dominantly located to the basalateral cell membrane. This pump is electrogenic, btbibited by ouabain and acti- vated by intracellular Na+ and extraeelhdlar K+. When

maximally stimulated, the pump produces a current of ap

p’px. 70 ptVccl1, equivalent to Na’ extntsion and K+ up

take at B tale of Z.l~lO~‘“attd 1.4.10-” m&s, rcspectivc-

ly. Comparable flux rates have been determined in other

liver preparaticnts. The pump generates the transmem-

brane disequilibrium for Na+ and K+, their intracellular

concentrations being 16.3 and 117 mmolll, respectively. oaring to the bigb membrane K+ conductawe, the out-

ward directed Kt mncentration gradient generates the in-

traccllulat negative electric potential of appmx. -40

mV. ?be Na/K-pump thus creates the driving forces for

transport wchaqisms which depend either cm the magni-

tude of the intmcellulsr electric potential or which are

coupledto the ittwdflowof Na’. Cbloddc appears to be

oassivelv distriiuted accordine to the electric wtential at

tion amountittg to 25 mm&l. A more detailed desctiption

of tbc. basic clccttical pmpcrtfcs of the cuuplet system is

given in Ref. 32.

Bvidena obtained in the couplet system that the Na+

mncentration gradient at the basolateral cell membrane

and the electric potential difference at the luminal ECU

mcmbranc are appropriate driving forces for tmnweUular

transport of bile acids (see above) establishes tbc physio-

logical role for the respective carder systems initially identifud in isolated ba%lateral and camalicutar rnem- bmne preparations. Basnlateral Na/H-exchange, also MendBed in the isolated membrane prcparatioa, comdb-

utes to regulation of intrac=eUtdar pH in liver cell couplets.

Using pH-sensitive microelectrc&s it was shown that this

aatupat system operates at a Imv rate under mntm1 cat-

ditiotts, but is .wivzw.d by an btuacellular acid load cott-

tributing to rapid readjustment of itmacelbda~ pH under

biarbaate-free conditicms. From mea.wremcnts of in-

traceflular buffer capacity it is caktdatcd that Nufi-cx-

change can operate at a transport rate of 0.9.1@ mot/s.

Coupling of Na+-uptake to H+-exrmsion at a ratio of 1:l

thus imposes a considerable load ora the NaX-pump (33).

Using the pat&clamp technique an inward retii&iig K*-

channel has been desaibcd and was localirsd to the baw

lateral cell membrane in the couplet system (34).

Liver cells exhibit low resistance intetccllular cnmmu-

nications allowing for the cell-to-cell panage of small mol-

ecules such as xmnd mesxngers. This cell mupliig was

demonstrated electrically in the couplet system using the

double-voltage clamp technique or by cell-to-cell diffu-

sion of Lucifer Yellow injected into one cell (35,36). Cell coupling is reduced by exposure of sells to wtanol and by

393

intracellukw aciditication by cxpatrc to high cottccntra-

tions of CO, (35). Electrical and dye coupling is also re-

duced by application of halcgcnated methans and, for

CQ, this toxic effectwasreduccdby inbibiting the gener-

ation of free radicals through cytochrome P-%50 oxyda- tive metabolism. By these uncoupling mechanisms in-

jured cells appear to fun&n&y detach themselves from

their healthy neighbors (37). With time in tissue culture,

c0upiir.g progressively decreases in association with the

disappearance of the 27-kDe gap junction protein and a

reductionof themRNAencodiogf~thisprotein.Unwn-

pling, and atso flattening of cells in cell culture. is delayed

by application of &bromo-adenosittc-3’.S’~clic mono-

phosphate, suggesting a hormonal regulation of cell corn-

q unication and of gap junction protein htmow (38). Rc-

synthesis of gap junctiott protein and of its mRNA can bc

elicited and cell coupling can be rcstorcd in cell ctdtw,

when certain conlpone* of the extraeellular matrix are

added to the culture medium, panicufarfy t&c proteo-

glycans and gtycwunin~@ycms normally abundant by Liver, indicating a rcgutatmy role of the %tlaceuubu ma-

trix in cell wmmunication (39).

In summary, the hepatayie couplet preparation has

proven to be a unique and pawctftd tool for mtmemus

shtdic.s in hepatobiliary biology. This preparation allows

for application of many different tcchnqucs including

morphometric analysis at the tight and elestmn micro-

scopic level, biiocbemistry and immunc4Iuoresrence

and, more importantly, for amtinuous obligation of

structural and functional changes we, time wing video

image analysis, c ~otometric and ekctraphysi-

ologicat techniques. We have brfetly reviewed studies on

strwtwal and timctiara, modifications of the secretory

apparatus in cell culture, on cytoskeletal functions, warts-

cyiotic vesicular transport, bide acid secretory mecba-

nisms, electrolyte and proton tramport, and on intracellu-

lar communication. Tbcsc examples demonstrate the vcr-

satility in applica:ions of the isolated hepatocyte couplet

system.

Original wwk summarized in this review was supported

by Nlh grantr DK 25636.36854 and 34969. We thack

Dn. Laurent ,%hild and Jon Beck for help in volume mea-

surements, ad Of-Cben Ng, Albert Mennone and Peter

Wyskovsky for preparation of phtiomicragraphs.

374

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