Transcellular offluoresceinin Evidence incultureProc. Natl. Acad. Sci. USA79(1982) 4987 tion ofan...
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Proc. NatL Acad. Sci. USA Vol. 79, pp. 4985-4987, August 1982 Cell Biology Transcellular transport of fluorescein in hepatocyte monolayers: Evidence for functional polarity of cells in culture (bile production/cholestasis/taurolithocholate/fluorescein diacetate/canalicular secretion) C. A. BARTH* AND L. R. SCHWARZt *Forschergruppe Erndhrung, Medizinische Poliklinik der UniversitAt Mfinchen, 8000 Munchen 2, Federal Republic of Germany; and tAbteilung Toxikologie, Gesellschaft fur Strahlen- und Umweltforschung, 8042 Neuherberg, Federal Republic of Germany Communicated by E. H. Ahrens, Jr., May 21, 1982 ABSTRACT The rat liver in vivo transfers bile salts, proteins, and dyes from blood into bile. It is the purpose of this communi- cation to demonstrate the maintenance of this transcellular trans- port in cultured adult rat hepatocytes. Two minutes after adding fluorescein (20 ,ug/ml) to the culture medium, maximal cellular fluorescence was observed through the fluorescence microscope. Subsequently, intercellular clefts showed a steadily increasing flu- orescence with a maximum between 5 and 20 min, resulting in a brightly fluorescent network of intercellular gaps. The following observations are taken as evidence that these findings reflect cel- lular uptake and canalicular secretion of the dye. First, the same sequence of observations was made upon addition of fluorescein diacetate (a nonfluorescent precursor of fluorescein), proving that the compound had been taken up and metabolized in the cells to fluorescein before secretion into intercellular clefts. Second, preincubation of the monolayers with the cholestatic bile salt tau- rolithocholate (100 j.mol/liter) suppressed almost completely in- tercellular but not cellular fluorescence. It is concluded that he- patocytes in culture show a functional polarity permitting the transcellular transport of substances bound for biliary secretion. The hepatocyte is an epithelial cell whose morphology and physiology show a marked polarity. Its cell membrane has two distinct domains: the sinusoidal membrane, which mediates solute exchange with blood, and the canalicular membrane, which faces the bile canaliculus and is involved in bile formation (1, 2). Functional polarity is most clearly demonstrated by trans- cellular transport of bile salts, proteins, and various organic compounds from the blood compartment to bile. This trans- cellular transfer is mediated by two transport systems-one si- nusoidal and one canalicular-operating in sequence (3). Iso- lated hepatocytes obtained by dissociation of the liver after per- fusion with collagenase (4) have been described as reaggregating to organotypic structures in monolayer culture (5-8). These cells in culture maintain the capability to take up bile salts by the sinusoidal transport system (9). Moreover, their ultrastruc- ture displays numerous elements that show the anatomical fea- tures of bile canaliculi (8, 10, 11). At present, it is open to de- bate whether these ultrastructural elements of the cultured he- patocyte are still operative and perform secretory work. Recent reports of functional polarization of epithelial cells in culture by demonstration of asymmetric virus budding (12) and transcellular net movement of ions (13, 14) in MDCK cells led us to study whether this applies to biliary transport in cultured adult rat hepatocytes. For this purpose, we have investigated uptake and secretion of fluorescent dyes that are subject to bil- iary elimination in vivo (15) with fluorescence microscopy. MATERIALS AND METHODS Materials. Fluorescein was obtained from Drobena GmbH, (Berlin), fluorescein diacetate was from Sigma, taurolithocho- late was from Calbiochem, and culture media were from GIBCO (Karlsruhe, Federal Republic of Germany). Cell Culture. Preparation of hepatocyte suspensions and cul- ture conditions have been described in detail elsewhere (16) and are a modification of the method described by Lin and Snod- grass (17). Hepatocytes were cultured on Falcon plastic dishes (no. 3001). For stabilization of pH, 25 mM Hepes was added to Leibowitz L-15 medium containing 25 mM NaHCO3. In ad- dition, the medium was enriched with the following hormones to enhance cell survival: glucagon, growth hormone,; triiodothy- ronine, dexamethasone, estradiol, testosterone, epinephrine (all at 1 ,uM) and insulin (0.4 ,uM). The viability and integrity of the hepatocytes were documented by the homogenous cel- lular fluorescence after addition of fluorescein diacetate (see below). Fluorescence Microscopy. Monolayers that had been cul- tured for more than 6 days were washed four times with culture medium. Then medium containing fluorescein (20 ,g/ml) or fluorescein diacetate (25 Ag/ml) was added. After various in- cubation times, the monolayers were washed three times, cov- ered with a thin layer of medium, and viewed under an Ortho- plan microscope. The primary fluorescence filter was set to 450-490 nm. The cutoff point of the secondary filter was 515 nm. Photomicrographs were made with Ektachrom 400 film. Typical fields were chosen that show most clearly the cellular and intercellular fluorescence, respectively. Every observation presented has been confirmed with more than three cultures. Control cultures (no fluorescein added) showed only negligible autofluorescence. No cellular or intercellular fluorescence was observed when fluorescein or fluorescein diacetate and 2% glu- taraldehyde were added simultaneously (dead cells). In the case of pretreatment of the cultures with taurolitho- cholate, the monolayers were incubated with the cholestatic bile acid (100 Amol/liter) and 0.5% bovine serum albumin for 14 hr prior to the addition of fluorescein. Controls were prein- cubated with albumin AIone. RESULTS Fig. 1 shows the sequence of events observed after adding flu- orescein to hepatocyte monolayers. During the first minute, the cells started to show fluorescence (Fig. la), with a subsequent gradual rise of intensity reaching a maximum after about 2 min of incubation (Fig. lb). At this stage, cellular fluorescence seemed to be more intense at the cell periphery close to the intercellular clefts. About 5 min later, extracellular spaces be- tween cells started to show fluorescence (Fig. ic). Phase-con- trast micrographs proved these extracellular spaces to be iden- 4985 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertise- nmnt" in accordance with 18 U. S. C. §1734 solely to indicate this fact. Downloaded by guest on November 7, 2020
Transcript of Transcellular offluoresceinin Evidence incultureProc. Natl. Acad. Sci. USA79(1982) 4987 tion ofan...
Proc. NatL Acad. Sci. USAVol. 79, pp. 4985-4987, August 1982Cell Biology
Transcellular transport of fluorescein in hepatocyte monolayers:Evidence for functional polarity of cells in culture
(bile production/cholestasis/taurolithocholate/fluorescein diacetate/canalicular secretion)
C. A. BARTH* AND L. R. SCHWARZt*Forschergruppe Erndhrung, Medizinische Poliklinik der UniversitAt Mfinchen, 8000 Munchen 2, Federal Republic of Germany; and tAbteilung Toxikologie,Gesellschaft fur Strahlen- und Umweltforschung, 8042 Neuherberg, Federal Republic of Germany
Communicated by E. H. Ahrens, Jr., May 21, 1982
ABSTRACT The rat liver in vivo transfers bile salts, proteins,and dyes from blood into bile. It is the purpose of this communi-cation to demonstrate the maintenance of this transcellular trans-port in cultured adult rat hepatocytes. Two minutes after addingfluorescein (20 ,ug/ml) to the culture medium, maximal cellularfluorescence was observed through the fluorescence microscope.Subsequently, intercellular clefts showed a steadily increasing flu-orescence with a maximum between 5 and 20 min, resulting in abrightly fluorescent network of intercellular gaps. The followingobservations are taken as evidence that these findings reflect cel-lular uptake and canalicular secretion of the dye. First, the samesequence of observations was made upon addition of fluoresceindiacetate (a nonfluorescent precursor offluorescein), proving thatthe compound had been taken up and metabolized in the cells tofluorescein before secretion into intercellular clefts. Second,preincubation of the monolayers with the cholestatic bile salt tau-rolithocholate (100 j.mol/liter) suppressed almost completely in-tercellular but not cellular fluorescence. It is concluded that he-patocytes in culture show a functional polarity permitting thetranscellular transport of substances bound for biliary secretion.
The hepatocyte is an epithelial cell whose morphology andphysiology show a marked polarity. Its cell membrane has twodistinct domains: the sinusoidal membrane, which mediatessolute exchange with blood, and the canalicular membrane,which faces the bile canaliculus and is involved in bile formation(1, 2). Functional polarity is most clearly demonstrated by trans-cellular transport of bile salts, proteins, and various organiccompounds from the blood compartment to bile. This trans-cellular transfer is mediated by two transport systems-one si-nusoidal and one canalicular-operating in sequence (3). Iso-lated hepatocytes obtained by dissociation ofthe liver after per-fusion with collagenase (4) have been described as reaggregatingto organotypic structures in monolayer culture (5-8). Thesecells in culture maintain the capability to take up bile salts bythe sinusoidal transport system (9). Moreover, their ultrastruc-ture displays numerous elements that show the anatomical fea-tures of bile canaliculi (8, 10, 11). At present, it is open to de-bate whether these ultrastructural elements of the cultured he-patocyte are still operative and perform secretory work.
Recent reports of functional polarization of epithelial cells inculture by demonstration ofasymmetric virus budding (12) andtranscellular net movement of ions (13, 14) in MDCK cells ledus to study whether this applies to biliary transport in culturedadult rat hepatocytes. For this purpose, we have investigateduptake and secretion of fluorescent dyes that are subject to bil-iary elimination in vivo (15) with fluorescence microscopy.
MATERIALS AND METHODSMaterials. Fluorescein was obtained from Drobena GmbH,
(Berlin), fluorescein diacetate was from Sigma, taurolithocho-late was from Calbiochem, and culture media were fromGIBCO (Karlsruhe, Federal Republic of Germany).
Cell Culture. Preparation ofhepatocyte suspensions and cul-ture conditions have been described in detail elsewhere (16) andare a modification of the method described by Lin and Snod-grass (17). Hepatocytes were cultured on Falcon plastic dishes(no. 3001). For stabilization of pH, 25 mM Hepes was addedto Leibowitz L-15 medium containing 25 mM NaHCO3. In ad-dition, the medium was enriched with the following hormonesto enhance cell survival: glucagon, growth hormone,; triiodothy-ronine, dexamethasone, estradiol, testosterone, epinephrine(all at 1 ,uM) and insulin (0.4 ,uM). The viability and integrityof the hepatocytes were documented by the homogenous cel-lular fluorescence after addition of fluorescein diacetate (seebelow).
Fluorescence Microscopy. Monolayers that had been cul-tured for more than 6 days were washed four times with culturemedium. Then medium containing fluorescein (20 ,g/ml) orfluorescein diacetate (25 Ag/ml) was added. After various in-cubation times, the monolayers were washed three times, cov-ered with a thin layer of medium, and viewed under an Ortho-plan microscope. The primary fluorescence filter was set to450-490 nm. The cutoff point of the secondary filter was 515nm. Photomicrographs were made with Ektachrom 400 film.Typical fields were chosen that show most clearly the cellularand intercellular fluorescence, respectively. Every observationpresented has been confirmed with more than three cultures.Control cultures (no fluorescein added) showed only negligibleautofluorescence. No cellular or intercellular fluorescence wasobserved when fluorescein or fluorescein diacetate and 2% glu-taraldehyde were added simultaneously (dead cells).
In the case of pretreatment of the cultures with taurolitho-cholate, the monolayers were incubated with the cholestaticbile acid (100 Amol/liter) and 0.5% bovine serum albumin for14 hr prior to the addition of fluorescein. Controls were prein-cubated with albumin AIone.
RESULTSFig. 1 shows the sequence of events observed after adding flu-orescein to hepatocyte monolayers. During the first minute, thecells started to show fluorescence (Fig. la), with a subsequentgradual rise of intensity reaching a maximum after about 2 minof incubation (Fig. lb). At this stage, cellular fluorescenceseemed to be more intense at the cell periphery close to theintercellular clefts. About 5 min later, extracellular spaces be-tween cells started to show fluorescence (Fig. ic). Phase-con-trast micrographs proved these extracellular spaces to be iden-
4985
The publication costs ofthis article were defrayed in part by page chargepayment. This article must therefore be hereby marked "advertise-nmnt" in accordance with 18 U. S. C. §1734 solely to indicate this fact.
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4986 Cell Biology: Barth and Schwarz
FIG. 1. Fluorescence micrographs (a-d) and phase-contrast micrographs (e-h) of hepatocyte monolayers. The cultures were incubated for 1 (aand e), 2 (b and f), 5 (c and g), and 20 min (d and h) with fluorescein (20 jug/ml). a and e, b and f, c and g, and d and h are pairs of micrographstaken sequentially from identical fields. (x 250.)
FIG. 2. Influence of taurolithocholate on cellular and intercellular fluorescence. Monolayers a-c were preincubated with taurolithocholate (100,4mol/liter) and 0.5% bovine serum albumin for 14 hr. Monolayers d-fwere controls preincubated with albumin alone. Incubation times with flu-orescein were as follows: a and d, 1 min; b and e, 7 min; and c and f, 30 min. (x 250.)
FIG. 3. Fluorescence pattern after fluorescein diacetate. Monolayers were incubated for 1 (a), 5 (b), or 40 (c) min with fluorescein diacetate(25 i.g/ml). (x280.)
tical with intercellular clefts (Fig. 1 g and h). No other fluores- observed. Maximum intercellular fluorescence occurred be-cent structures besides the cells or the intercellular clefts were tween 5 and 20 min and vanished subsequently. The observa-
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Proc. Natl. Acad. Sci. USA 79 (1982) 4987
tion of an intercellular fluorescence after fluorescein additionhas been described previously in fetal hepatocytes from chicken(18) and rats (19).
Further evidence that these observations show transcellulartransport and biliary secretion in culture is provided by the fol-lowing results.
Cultures were preincubated with the cholestatic bile salt tau-rolithocholate before fluorescein transport was studied (Fig. 2).Intercellular fluorescence was not observed at 7 min (Fig. 2b)and was strongly impaired at 30 min (Fig. 2c) in these cultures,whereas the controls showed the usual pattern of fluorescence(Fig. 2 e andf).
Moreover, hepatocyte monolayers were incubated with thefluorogenic substrate, fluorescein diacetate. This nonfluores-cent compound is transformed to fluorescein by cellular ester-ases (20). Hence, uptake of the fluorogenic substrate and sub-sequent intracellular generation of fluorescein are obligatoryintermediary steps before an eventual intercellular fluores-cence can occur. Fig. 3 shows that the same sequence of phe-nomena was observed as with fluorescein itself: 1 min after ad-dition of fluorescein diacetate, a strong cellular fluorescencebecame apparent. This documents the viability of the culturedhepatocytes, as fluorescence emitted by cells after addition ofthe diacetate derivative is generally held to correlate positivelywith their viability (20). At 5 min, a weak intercellular fluores-cence started to appear. This intercellular fluorescence becamegradually more intense, leading finally to an interconnectingnetwork of fluorescent intercellular clefts.
It is noteworthy that, also with fluorescein diacetate, a stronginhibition of the intercellular (without impairment of cellular)fluorescence was observed by a 14-hr preincubation with 100,mol of taurolithocholate per liter (data not shown).
DISCUSSIONWe report herein that hepatocytes from adult rats in monolayerculture accumulate and excrete fluorescein into intercellularclefts.
Hanzon has published a thorough study on fluorescein trans-port by the rat liver in vivo (15). The dye was shown to accu-mulate in the liver cells about 45 sec after intravenous injection,and a bright fluorescent network of bile capillaries appearedbetween 4 and 30 min thereafter (15).A very similar sequence of phenomena occurred in mono-
layer cultures of rat hepatocytes as described herein. In thissystem, cellular fluorescence was maximal about 2 min afteraddition to the culture medium, and the intercellular cleftsstarted to show fluorescence at 5 min, reaching a maximum at10-20 min. At this point, the pattern of fluorescence was verysimilar to the network observed on the superficial cellamina ofthe liver in vivo (figure 18 of ref. 15), which corresponds mostwith the cell arrangement of a monolayer in culture.
It is our contention that the intercellular accumulation of flu-orescein can be considered as an equivalent to biliary secretion.This is not only supported by the above morphological analogyof fluorescence between the cultured cells and the observationsin vivo but also is corroborated by the following results.
First, fluorescein diacetate was observed to cause the samesequential fluorescence of hepatocytes and intercellular clefts.From the known metabolic fate of this fluorogenic substrate(20), it can be concluded that the intercellular fluorescence isconsequential to an intracellular generation of fluorescein. Thisfinding lends strong support to the contention that this dye issecreted across the canalicular membrane into the intercellularclefts.
Second, taurolithocholate suppressed markedly the inter-
cellular fluorescence without reducing cellular fluorescence.This bile salt causes cholestasis in the whole animal (21) and inisolated perfused liver (22). Changes of ultrastructure (23, 24)and of activities ofmembrane-bound enzymes (25) make it veryprobable that taurolithocholate does so by interfering with thefluidity and function of the canalicular membrane. Hence, theinhibitory effect of taurolithocholate demonstrates that an un-impaired function ofthe canalicular membrane is a sine qua nonfor the intercellular accumulation offluorescein and contributesindependent evidence that canalicular transport mediates thisphenomenon.A number of observations have been reported which dem-
onstrate a morphological polarization of hepatocytes in mono-layer culture. Especially, the reappearance of bile canaliculi inculture (8, 10, 11) and the concentration of golgi cisternae intheir vicinity (26) can be cited here. The results reported nowdemonstrate that this morphological polarity of the hepatocytein culture is paralleled by afunctional polarity.We congratulate Professor Helmut Holzer, University of Freiburg,
on the occasion of his 60th birthday. The technical assistance of Mrs.Brigitte Weber and secretarial help of Ms. Uta Rasche are gratefullyacknowledged. We thank Dr. Lutz Gurtler from the Institut fur An-thropologie und Humangenetik for his helpful advice and generouspermission to use the fluorescence microscope. Generous financial sup-port by the Falk Foundation, Freiburg, is gratefully acknowledged.This work was supported by the Deutsche Forschungsgemeinschaft (Zo27/7).
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