Graefe's Arch Clin Exp Ophthalmol (1994) 232: 608-613 © Springer-Verlag 1994

Walter Noske B6atrice Levarlet Klaus Martin Kreusel Michael Fromm Michel Hirsch

Received: 4 January 1994 Accepted: 11 April 1994

The results reported here were presented in part at the 91st Congress of the German Ophthalmological Society, Mannheim, 19-22 September 1993 and published in abstract form [22]. W. Noske - K.M. Kreusel Augenklinik, Klinikum Steglitz, Freie Universit~it Berlin, D-12200 Berlin, Germany B. Levarlet • M. Hirsch ([~) INSERM U86, H6tel Dieu, 1 place du Parvis Notre Dame, F-75181 Paris Cedex 04, France K.M. Kreusel • M. Fromm Institut ftir Klinische Physiologie, Klinikum Steglitz, Freie Universit/it Berlin, D-12200 Berlin, Germany

Tight junctions and paraceUular permeability in cultured bovine corneal endothelial cells

Abstract Intramembrane specializations of cultured bovine corneal endothelial cells were studied with thin section and freeze-fracture electron microscopy and related to the paracellular permeability and the transendothelial resistance (R t) of the monolayers. The following intercellular junctions were found: single and discontinuous networks of tight junctions (TJ) which girdle the apico-lateral cell perimeter incompletely, gap junctions, and membrane undulations suggesting intermediate junctions. The macromolecular tracer ruthenium red penetrated into the lateral intercellular space beyond the level of the incomplete belt of TJ. R t of these monolayers was 20.9 _+ 1.0 f~. cm 2.

Introduction The tight junctions (TJ) are specialized plasma membrane structures that girdle the apical cell perimeter of epithelial and endothelial cells. They are involved in the control of paracellular permeability and in the maintenance of apico-basal polarity of the plasma membrane (see [2] for references). The corneal endothelial monolayer covers the posterior face of the cornea and separates the underhydrated environment of the avascular corneal stroma from the aqueous humor. Despite the incompleteness of its TJ belt [10, 26], the endothelial layer of the cornea functions as a barrier to excessive water flow from the anterior chamber into the corneal stroma and effectively pumps small solutes and water from the stromal side into the

Protamine induced a reversible increase of R t to 118 ± 5% of its control value. We conclude that incomplete belts of TJ may be the morphological counterpart of the high paracellular permeability of this monolayer and functionally and morphologically resemble those of their native endothelium. Cultured corneal endothelial cells are an excellent model for studying the influence of incomplete belts of TJ on paracellular permeability of cells.

aqueous humor, thus controlling corneal hydration and transparency [5, 19, 29]. Cultured corneal endothelial cells can reconstitute a confluent polarized monolayer of polygonal cells which secrete a thick basement membrane and reveal several characteristics of corneal endothelium in vivo [8, 9, 11, 14]. The role of the TJ in regulating the paracellular pathway in this important endothelial layer is not completely understood. Therefore, we studied the morphology of intercellular junctions in cultured bovine corneal endothelium and related it to the barrier function towards ruthenium red and the transendothelial electrical resistance (R t) of the cultures. Furthermore, since it has been shown that protamine, a polycationic protein, increases R t in Necturus gallbladder [6], we investigated its effect on the corneal endothelial monolayer.

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Materials and methods Cell culture Bovine corneas were obtained from the slaughterhouse. The endothelial layer was scraped in phosphate-buffered saline and cultured in Dulbecco's modification of Eagle's minimum essential medium (DMEM) supplemented with 10% fetal calf serum, 100 U/ml of penicillin and 25 gg/ml of gentamycin at 37° C and gassed with 5% CO 2 in air on commercial cell culture dishes with microporous membranes (Millicell-HA filter, area 0.6 cm 2, Millipore, Bedford, Mass.) or on plastic coverslips (Thermanox, Sigma, St. Louis, Mo.). All experiments were performed on 10- to 15-day-old confluent subcultures. Thin-section electron microscopy Monolayers were fixed in 2.5% glutaraldehyde in 0.1 M cacodylate buffer, pH 7.4, at room temperature, rinsed in the buffer and post-fixed in 1% osmium tetroxide in the same buffer. In some cultures tannic acid (1%) or ruthenium red (1%) was added to the fixative at the apical side of the monolayer. After dehydration in ethanol, the cells were embedded in Epon 812. Thin sections were contrasted with lead citrate and uranyl acetate and observed with a Philips EM 300 or EM 410 electron microscope. Freeze-fracture electron microscopy Glutaraldehyde-fixed samples were cryoprotected in 25% glycerol in 0.1 M cacodylate buffer for 1 h. The supports with the attached monolayers were rapidly frozen in solid-liquid nitrogen. Platinum-carbon replicas were produced in a freeze-fracture apparatus (Cryofract CF 250, Leica, France) equipped with electron beam guns and a quartz thickness film monitor at a stage temperature of - 150° C and in a vacuum of at least 10 -7 torr. Cleaned replicas were mounted on 300 mesh copper grids and observed with a Philips EM 300 or EM 410 electron microscope. Measurement of R t In order to monitor R t, the filters with confluent monolayers were mounted into Ussing-type chambers [16]. The hemi-chambers were filled with Ringer's solution (in mmol/l: 151 Na +, 5 K +, 130.4 CI, 1.7 Ca 2+, 0.9 Mg 2+, 1 H2PO4-, 0.9 SO42-, 28 HCO3; pH 7.4, 37° C) and gassed with 95% 02 5% CO2 and stirred by a bubble lift. The monolayers were short-circuited by a computerized automatic voltage clamp device (CVC 6, Hard & Software, O. Fiebig, Berlin, Germany), allowing continuous monitoring of R t. Protamine (Hoffmann-La Roche, Grenzach-Whylen, Switzerland) was used from a 1000 IU/ml stock solution and added to the apical side of the monolayer to give a final concentration of 3 x 10-5 M, and its effect was reversed by 6 x 10 5M heparin (Sigma, Munich, Germany). Data are means+standard errors of the means (SEM), and significances were evaluated using the unpaired t-test.

Results Cultured bovine corneal endothelial cells reconstituted confluent m o n o l a y e r s of polygonal cells. The apico-lateral p l a s m a m e m b r a n e s between adjacent cells interdigitated, and in places the external leaflets of the two inter-

Fig. 1 Apical part of cultured endothelium fixed in the presence of tannic acid. At places (arrows) the external leaflets of the adjacent plasma membranes seem to fuse, indicating the location of apico-lateral TJ. IS = intercellular space. Bar 0.2 gm digitating p l a s m a m e m b r a n e s seemed to "fuse", indicating the presence of TJ (Fig. 1). At these points no tannic acid was revealed between the two opposing m e m branes, and at times the intercellular space seemed to be bridged by tiny white spots. With the freeze-fracture technique, large areas of the different p l a s m a m e m b r a n e regions with their respective m e m b r a n e specializations were exposed. In glutaraldehyde-fixed cultured corneal endothelial cells, the TJ structures a p p e a r e d as s m o o t h strands on the protoplasmic face (P-face) of the fracture (Fig. 2a) and as furrows on the exoplasmic face (E-face) of the fracture (Fig. 2b) in the apico-lateral p l a s m a m e m b r a n e . The geometrical pattern of the TJ network varied considerably, even within short distances along the cell perimeter. TJ often consisted of mostly unbranched P-face strands or E-face furrows, often oriented in an apicobasal direction (Fig. 2a). Therefore, the apical and basolateral p l a s m a m e m b r a n e domains often were in continuity between these strands without interspersed junctional elements. At other positions, TJ strands or furrows formed m o r e or less loose networks (fasciae or maculae occludentes) with varying n u m b e r s of anastomoses (Figs. 2b, 3), forming a local "fence" between the two p l a s m a m e m b r a n e domains. Very elaborated networks of TJ strands or furrows girdling long distances of the apical perimeter of the lateral p l a s m a m e m b r a n e were not revealed. Therefore, when sufficiently large areas of the apical lateral p l a s m a m e m b r a n e were exposed, the apical and lateral m e m b r a n e d o m a i n were

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Fig. 2 Apico-basally oriented TJ strands on a fracture Pface (P) with few anastomoses and many free ends (a) and small anastomosing TJ furrows on the fracture E-face (E) forming maculae occludentes associated with gap junctions (b). Arrows indicate regions of the plasma membrane not barred with any TJ elements. GJ = gap junction. (a) Bar 0.5 p~m;(b) Bar i Ixm

always in continuity at some areas without interposed junctional specializations. Sometimes the exposed plasma membrane displayed linear or curved undulations a r o u n d the apico-lateral cell perimeter. The density of intramembrane particles was lower in these regions than in the surrounding plasma m e m b r a n e (Fig. 4). No typical TJ elements were present at these undulations, but small gap junction-like condensations of intramembranous particles were often associated with these m e m b r a n e areas. These zones may represent intermediate junctions. Frequently, typical gap junctions were intimately associated with TJ, especially with the more elaborated networks (Figs. 2b, 3), whereas other gap junctions were found isolated in the lateral plasma membrane. Clusters of vesicle fusion sites were observed isolated on the apical plasma membrane (Fig. 5a), but were especially numerous on the basal plasma membrane, at times prefer-

Fig. 3 Anastomosing TJ network with gap junctions (GJ) not demonstrating discontinuities in the network. P = Fracture P-face, E=Fracture E-face. Bar 0.5 pm

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Fig. 4 Fracture P-face (P) of the apico-lateral plasma membrane with low membrane undulations (arrowheads) which are relatively poor in intramembrane particles. GJ = gap junction. Bar 0.5 gm

Fig. 6 The tracer ruthenium red applied to the apical side (ap) of the monolayer stains the entire intercellular space (arrowheads), indicating the leakiness of the apical TJ towards this electrondense tracer. Bar 2 gm Fig. 5 Fracture E-face (E) of the apical plasma membrane (a) and fracture P-face (P) of the basal plasma membrane (b) showing numerous vesicle fusion sites (thick arrows). The shallow circular elevations (thin arrow) probably represent coated pits. Bars 1 mm entially oriented in linear rows (Fig. 5b). We did not find increased n u m b e r s of vesicle fusion sites in m e m b r a n e areas where TJ elements are n o r m a l l y located. W h e n the m a c r o m o l e c u l a r tracer ruthenium red was

added to the apical side in order to test the barrier characteristics of the endothelial monolayer, it was often found in the lateral intercellular space b e y o n d the apical region of close m e m b r a n e contacts, indicating the leakiness of the paracellular p a t h w a y for r u t h e n i u m red in this m o n o l a y e r (Fig. 6). R t was used as a measure of small solute permeability of the monolayers. R t of 10 day-old m o n o l a y e r s was

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late of the high permeability of the paracellular pathway in this monolayer. The relatively low R t values measured in this study further support this interpretation. Taking into account the influences on R t of the support on which the cells grow [8, 20] and the additives to the medium, such as glucose and adenosine [5], the R t valP vs control g t (~'~" cm 2) ues of our study correlate well with values found in other studies on cultured corneal endothelium [8] and in Control 20.9 + 1.0 whole mounted corneas [12, 17], but are lower than the Protamine 24.6 _+ 1.0 < 0.05 exceptionally high R t values reported by Narula et al. Heparin 19.8 + 0.9 n.s. [20]. In the leaky epithelium of Necturus gallbladder, pro30 tamine has been shown to cause a rapid increase in R t Protemine Heplrin without affecting apical membrane permeability, selectively altering the function of the TJ [7]. The observed rapid and reversible increase in R t after addition of pro25 tamine in cultured monolayers of corneal endothelial E cells supports the interpretation that the incomplete TJ o belts limit, at least in part, the paracellular permeability in this monolayer. However, the protamine-induced increase in R t (18%) is relatively low compared to the 20 70-90% increase in R t in Necturus gallbladder with complete belts of TJ [1, 6] and the 84% increase in cultured human non-pigmented ciliary epithelial cells with incomplete belts of TJ [21, 27]. Therefore, the contribu15 tion of the incomplete TJ network to the total paracellu40 60 80 100 120 140 lar resistance in corneal endothelial monolayers may be less than in other systems. Furthermore, the relatively time (min) high number of vesicle fusion sites in the plasma memFig. 7 Increase in Rt after addition of protamine (3 × 10-SM), branes may indicate a very active transcellular route for rapidly reversed by the addition of heparin (6 x 10 5M), in cul- bulk transport of charged or uncharged molecules tured corneal endothelial cells across the endothelial layer [24]. This may have to be considered when interpreting transendothelial trans20.9_ 1.0 f 2 - c m 2 and remained stable for at least 2 h port and R t of these monolayers. Incomplete TJ belts have also been encountered in (Table 1). In order to test the regulation of the paracellular permeability by the T J, protamine (3 x 10-5 M) was developing [3] and cultured [21] epithelia that normally added to the monolayer. A rapid rise in R t to 118 _+5% express complete TJ belts and in cultured cerebral enof the control value was recorded. This effect of pro- dothelia in the absence of astrocyte-derived factors (see tamine was reversed by heparin (6 x 10-5 M) (Fig. 7). [25] for review). Interestingly, discontinuous TJ belts are also found in native corneal endothelium [10, 26]. It is interesting to note that tight vascular endothelia may be formed in the anterior chamber in the presence of astroDiscussion cytes [13]. Thus, the abundance of incomplete TJ in the Despite the presence of apico-lateral membrane "fu- absence of complete TJ belts in cultured corneal ension" sites, the apically applied macromolecular tracer dothelial cells is probably a reflection of some inherent ruthenium red penetrated the lateral intercellular space properties of corneal endothelium rather than due to of cultured bovine corneal endothelial cells, indicating the in vitro conditions. The membrane undulations sometimes observed in that the paracellular pathway is permeable to relatively large molecules. Passage of horseradish peroxidase (row the apico-lateral areas of the plasma membrane which 40 000) [15] and ruthenium red (mw 551) [4] across the are poor in intramembranous particles and on which, at apical TJ domain of corneal endothelial cells has also times, aggregates of intramembranous particles may be been found in vivo. This correlates well with the freeze- found may correspond to intermediate junctions [18, 23, fracture images of the apico-lateral T J, since the TJ 28]. These are believed to be a prerequisite of TJ formastrands form incomplete belts around the cell perimeter tion and may indicate continuous formation of TJ in 10in vivo [10, 22, 26] and in cultures [11]. The discontinu- to 15-day-old confluent monolayers of bovine corneal ities in the TJ network may be the morphological corre- endothelial cells. Table 1 Transendothelial resistance (R t) of cultured corneal endothelial monolayers and the effect of protamine. (Protamine (3 x 10-5 M) was added after stabilization of R t. Protamine values were read 20 rain after its addition, just before addition of heparin (6 x 10-s M). Heparin values were read after 10 rain when its effect was complete. Values represent mean_+ SEM of five monolayers.)

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Our results show that cultured corneal endothelial cells morphologically and functionally closely resemble native corneal endothelium. Thus, cultured corneal endothelium represents a model for studying incomplete TJ belts and their influence on the paracellular pathway and cellular polarization.

Acknowledgements We would like to thank Drs. M. Wiederholt, S. Berweck and A. Lepple-Wienhues and Mrs. A. Krolik of the Institut ffir Klinische Physiologie, Universit/itsklinikum Steglitz, Germany, and Jacqueline Tassin of Ul18, INSERM, France, for providing cultures of corneal endothelial cells. We further want to thank Ursula Lempart, Frangoise Dagonet, Dani61e Raison and Genevieve Prenant for skilled technical assistance. This research was supported by grants from the French Institut National de la Sant~ et de la Recherche M6dicale.

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