Cell Tiss. Res. 187, 439-448 (1978) Cell and Tissue Research �9 by Springer-Verlag 1978

The Junctional Connections between the Cells of the Urinary Bladder in the Rat*

Stephan Peter**

Anatomisches Institut der Universit~it Heidelberg, Bundesrepublik Deutschland

Summary. The junctional connections between the cells of the urinary bladder epithelium in rat have been studied by freeze-fracturing. Tight junctions and desmosomes are known as structural features of the epithelium. In addition, gap junctions (nexus) have been found to connect the epithelial cells in an irregular distribution pattern. The junction size ranges f rom few assembled particles up to plaques with irregular forms. This may indicate that the gap junctions are mobile structures. The functional significance of the junctions in comparison with electrophysiological data is discussed.

Key words: Urinary bladder epithelium (Rat) - Gap junctions - Tight junctions - Electron microscopy - Freeze-fracturing.

Introduction

In the last two decades numerous studies have dealt with the structure of the luminal membrane of the transitional epithelium of the urinary bladder in mammalians and man (Walker, 1960; Kurosumi et al., 1961; Leeson, 1962; Richter and Moize, 1963; Wolff, 1963; Petry and Amon, 1966; Hicks, 1966; Monis and Zambrano, 1968; Hicks and Ketterer, 1970; Staehelin et al., 1972; Chlapowski et al., 1972).

The junctional connections between the cells of the mammals urinary bladder have not been investigated so far. They are, however, of interest to understand the permeability and electrophysiological properties of this epithelium. Therefore, we studied the rat urinary bladder by freeze-fracturing.

Send offprint requests to: Dr. Stephan Peter, Anatomisches Institut, Im Neuenheimer Feld 307, D 6900 Heidelberg, Federal Republic of Germany

* Dedicated to Professor Dr. Helmut Ferner on the occasion of his 65th birthday. ** I am indebted to Dr. W. Kriz for reviewing the manuscript. The skillful technical assistance of Mrs. Marlis Kopp is highly appreciated.

0302-766X/78/0187/0439/$02.00

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Materials and Methods

Male Wistar rats (Ivanovas, Kissleg) weighing 180500 g were used. They had free access to water and laboratory chow (Altromin R, Fa. Altromin, Lage/Lippe, Germany).

The animals were anaesthetized with an intraperitoneal injection of inactin (100mg/kg). The abdominal aorta was cannulated in blood flow direction and perfused with oxygenated Ringer's solution. Following the wash-out of blood the urogenital system and the lower extremity were perfused with 3 % glutaraldehyde in 100 mM cacodylate buffer (pH 7.3) for 10 min. Subsequently, the urinary bladder was removed and fixed over-night in the glutaraldehyde-buffer solution. Small pieces of the fixed tissue were treated with 25 % glycerol in 100 mM cacodylate-buffer (pH 7.3) for 2-24 hr prior to freezing. Samples were freeze-fractured at - 100 ~ C in an Denton DFE-3 apparatus according to the method of Steere (1973). Carbon-platinum replicas were cleaned in bleach and water prior to mounting on copper grids. All electron microscopic observations were made with a Philips 301. The recently proposed nomenclature system (Branton et al., 1975) is applied.

Results

The epithelium of the rat ur inary bladder - in the stage o f moderate filling, not collapsed, not distended - cons i s t s o f several layers; one can roughly distinguish a superficial, an intermediate, and a basal cell layer.

The cells are extensively interdigitated th roughout the total thickness o f the epithelium; this may be the reason why it is almost impossible to expose large cell membrane surfaces by freeze-fracturing: the fracture plane splits preferentially th rough the cytoplasm.

The superficial cells are connected by tight junctions (zonulae occludentes) which separate the luminal space f rom the interstitial space o f the epithelium. They consist o f at least four layers o f interconnected strands which characteristically appear as a network o f raised ridges on the P-face o f the fracture plane (Fig. 1). These tight junct ions have often been found somewhat distant f rom the very surface o f the epithelium, deeper within infoldings between two neighbouring superficial cells. Thus, the luminal cell membrane must be considered to cover the luminal side as well as open parts o f the lateral sides down to the tight junct ion barrier. These infoldings between superficial cells may help to provide for the increased epithelial surface necessary during distension.

It has been shown (Staehelin et al., 1972) that the luminal cell membrane regularly contains assymmetric membrane plaques which are found down to the tight junct ion barrier. Beneath this barrier the cell membranes o f superficial intermediate and basal cells are established by the conventional tri laminar unit membrane.

In ultrathin sections the epithelial cells have been found to be connected by few and small desmosomes. N o equivalents o f this type o f junct ion has been found in the freeze-fracture specimens.

In contrast , freeze-fracturing clearly revealed the existence o f gap junctions a m o n g the cells o f this epithelium. They show some specific characteristics concerning the distribution and the internal structure.

The gap junct ions are irregularly distributed. In the upper parts o f the epithelium (immediately beneath the zonula occludens) gap junctions have never been found. They are restricted to the middle and basal parts o f the epithelia. Also

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Pig. 1. Superficial cells o f the urinary bladder epithelium connected by a tight junction (Tar) somewhat distant from the very surface. Note numerous vesicles (arrows) with membrane plaques. Luminal space (L). • ~ 14000. Inset: higher magnification of the same tight junction x ~ 30000

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Fig. 2. Superficial and intermediate cells of the urinary bladder. Asterisk indicates area with gap junctions. In this specimen, they have been found only in the middle part of the epithelium. Nuclei (N), Luminal space (L). x ~ 4000

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Fig. 3. Higher magnification o f the gap junct ion area of Fig. 2 with encircled gap junctions. Two junctions in the lower left comer are on the E-face, the other gap junct ions are on the P-face. • ~ 15000

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Fig. 4. a--d shows gap junctions of different form and size on the P-face, e/f on the E-face. a two densely packed gap junctions; h/c gap junction with densely and loosely packed particles; d a loosely packed gap junction with scattered particles. Arrow indicates scattered pits on the E-face similar to the scattered particles on the P-face. x ~ 80000

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at this site, however, an irregular pattern is apparent: fracture planes of cell membranes with numerous "gaps" (Fig. 3) alternate with those containing none.

On the P-faces the gap junctions characteristically look like an accumulation of membrane-associated particles all of uniform size. On the E-face they are characterized by pits (Fig. 4e/f); as the pits are more difficult to identify smaller gaps may easily be overlooked. The encountered gap junctions range in size from those consisting of only a few particles up to plaques with a diameter of 4 nm. Moreover, they vary in form (Fig. 4) and in the pattern of their membrane associated particles. In addition to the densely packed forms occurring usually, those with loosely distributed particles (all of which, however, are of the same typical size) have been found (Fig. 4d). Frequently, inhomogenous types consisting of a closely and a loosely packed portion (Fig. 4b/c) are evident.

Discussion

The transitional epithelium of the empty rat urinary bladder is composed of three to four cell layers, a surface layer, a middle layer (or layers) and a basal layer (Leeson, 1962; Monis and Zambrano, 1968). As has been shown by Petry and Amon (1966) the cells of all these layers are all anchored on the basement membrane of the epithelium by slender cytoplasmic processes; thus, this epithelium must be regarded to be pseudo-stratified.

Three types of junctions are present between the cells of this epithelium. The superficial cells as has been already found previously by usual transmission electron microscopy (Monis and Zambrano, 1968) are connected by tight junctions (zonulae occludentes). In freeze-fracture specimens they consist of at least four (4-6) interconnected strands. A recent electrophysiological investigation of the rabbit urinary epithelium (Lewis et al., 1976) revealed that the surface layer contains almost all the transepithelial resistance and that the junctional resistance of this layer is extremely high. These electrophysiological properties are in as far in accordance with our findings as tight junctions only exist between superficial cells indicating that the interstitial spaces between the epithelial cells beneath the tight junction barrier are in free communication with each other. There is, however, a discrepancy as to the absolute height of the junctional resistance. According to the classification by Claude and Goodenough (1973) a zonula occludens consisting of 4-6 strands might be expected to be at best "intermediate tight". This discrepancy between the measured value for the junctional resistance which (according to Lewis et al., 1976) in the rabbit urinary bladder appears to represent an upper extreme even among tight epithelia and the morphological finding of moderatly developed tight junctions needs some discussion. Surely there may exist species differences but those are not very probable in this magnitude. A more probable explanation would be that the surface cells are very big cells which, in the region of the zonulae occludentes, are not interdigitated with each other. Thus, the length of the zonula occludens must be regarded to be short in relation to the total epithelial surface as well as in relation to highly interdigitating epithelia. Moreover, the tight junctions are often not situated at the very surface of the epithelium, but somewhat deeper in infoldings between superficial cells. Therefore, it might be difficult in voltage

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scanning experiments to detect a potential inhomogeneity which would indicate the existence of an only medium tight junctional resistance in an overall very tight superficial epithelial layer.

Gap junctions have hitherto not been described to exist in the mammalian urinary epithelium. Gap junctions connect the cytoplasm of adjacent cells; they contain hydrophilic channels by which adjacent cells may be electrically as well as metabolicly coupled (Loewenstein, 1975). Our freeze-fracture specimens clearly prove the existence of gap junctions in the urinary bladder epithelium; they could, however, only be detected in middle and basal parts of the epithelium. In the upper parts of the epithelium, just beneath the tight junction barrier we never encountered a gap junction, what may be due to the fact that at this site, probably on account of the extensive interdigitation, it was not possible to uncover sufficiently large planes of lateral cell membranes. As, however, also the superficial and intermediate cells extend down to the basement membrane (Petry and Amon, 1966), the encountered gap junctions within the more basal parts of the epithelium may well connect superficial and intermediate cells as well.

Recent electrophysiological experiments in the rabbit urinary bladder (Lewis et al., 1976) revealed that there is a radial cell to cell coupling within the superficial cell layer. Radial current spread within this layer proved to be small but symmetrically without preferential directions. A transverse, i.e. a coupling of superficial to intermediate and of intermediate to basal cells could not be detected in these experiments. Comparing these electrophysiological data with our morphological findings, there is agreement that the electrical coupling of the cells is small in magnitude. Comparing, for instance, with pancreas acinus cells (Bieger et al., 1977) the gap junctions in the bladder epithelium are much less frequent. There is, however, a discrepancy as to the question which cells of the epithelium are coupled. From our data we do not believe that only the superficial cells are electrically coupled; gap junctions must be expected also to connect the different cell layers; predominantly the basal cells should be coupled to each other and to other cell layers. To explain the discrepancy we have to point out that Lewis et al. (1976) used a direct and sensitive method to look for the radial coupling, whereas they could apply but an indirect and poorly discriminating method to measure the transverse current spread. Thus, it is supposed that the few gap junctions responsible for the electrical coupling could functionally be detected only by the direct method sensitive for the radial coupling, whereas the transverse coupling has not become apparent. Certainly, also at this point species differences cannot be excluded.

Lewis et al. (1976) reported data from stretch experiments which indicate that the junctional structures providing the cell to cell conductance and those responsible for the transepithelial resistance are different. Stretching the urinary bladder epithelium in vitro uncoupled the cells electrically, the transepithelial junctional resistance, however, was maintained. This may well be interpreted as a further evidence that also in the urinary bladder the tight junctions are responsible for the junctional resistance, the gap junctions for the cell to cell coupling.

The encountered gap junctions of the urinary bladder epithelium frequently exhibit some peculiarities as to their internal structure. In addition to forms which- as typical gap junctions in other organs, too - are characterized by regularly and densely arranged uniformly sized particles on the P-face (and the corresponding pits

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on the E-face), there are forms which in total or in par t o f the junct ional membrane are characterized by a loosely packing o f the particles. Nevertheless, the identification as gap junct ions is possible, as all o f these particles are o f uni form and typical size. As has already been proposed previously (Friend and Gilula, 1972), those junct ions suggest that a minimal gap junct ion might exist which only consists o f one single particle which is matched with a corresponding pit in the adjacent membrane. Moreover , the loosely packed forms of gap junct ions may suggest that they are developing or vanishing indicating that the gap junct ions are mobile structures. The fact that even in the basal parts o f the epithelium large membrane fracture planes have been found wi thout any gap junct ion may speak in favour o f a dynamic concept o f the gap junctions.

Desmosomes have been found to connect the cells o f the bladder epithelium (Richter and Moize, 1963). They do no t appear to be very numerous and have been described to be small and poor ly developed only consisting o f a thickening o f the opposite membranes. We could detect them only in the usual thin EM-sect ions; the freeze-fracture replicas did no t clearly reveal equivalents for this sort o f junction. This is in accordance with the fact, that up to date equivalents for desmosomes in freeze-fracture specimens have only been described for the highly developed desmosomes as they are present, for instance, in the stratified squamous epithelium o f the uterus and skin (McNut t et al., 1971 ; Breathnach et al., 1972). According to these findings, desmosomes in freeze-fracture replicas are characterized by plaques o f particles o f irregular size not only on the P-, but also on the E-face o f a fracture plane. Such particle plaques have not been found in our specimens what may be due to the fact that infolded membrane parts are difficult to split.

References

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Branton, D., Bullivant, S., Gilula, N.B., Karnovsky, MJ., Moor, H., Miihlethaler, K., Northcote, D.H., Packer, L., Satir, B., Satir, P., Speth, V., Staehelin, L.A., Steere, R.L., Weinstein, R.S.: Freeze- etching nomenclatur. Science 190 (4209) 54-56 (1975)

Breathnach, A.S., Stolinski, C., Gross, M.: Ultrastructure of fetal and post-natal human skin as revealed by freeze-fracture replication technique. Micron 3, 278-304 (1972)

Chlapowski, FJ,, Bonneville, M A., Staelielin, L.A.: Luminal plasma membrane of the urinary bladder. II. Isolation and structure of membrane components. J. Cell Biol. 53, 92-104 (1972)

Claude, P., Goodenough, D.A.: Fracture faces of zonulae occludentes from "tight" and "leaky" epithelia. J. Cell Biol. 58, 390400 (1973)

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Hicks, R.M., Ketterer, B.: Isolation of the plasma membrane of the luminal surface of rat bladder epithelium, and the occurrence of a hexagonal lattice of subunits in both negatively stained whole mounts and in sectioned membranes. J. Cell Biol. 45, 542-553 (1970)

Kurosumi, K., Yamagashi, M., Yamamoto, T.Y.: The fine structure of the transitional epithelium of urinary bladder and its functional significance as disclosed by electron microscopy. Arch. Histol. Jap. 21, 155-183 (1961)

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Lewis, S.A., Eaton, D.C., Diamond, J.M.: The mechanism of Na § transport by rabbit urinary bladder. J. Membrane Biol. 28, 41-70 (1976)

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Richter, W.R., Moize, S.M.: Electron microscopic observations on the collapsed and distended mammalian urinary bladder (transitional epithelium). J. Ultrastruct. Res. 9~ 1-9 (1963)

Staehelin, L.A., Chlapowski, F.J., Bonneville, M.A.: Luminal plasma membrane of the urinary bladder. I. Three-dimensional reconstruction from freeze-etch images. J. Cell Biol. 53, 73-91 (1972)

Steere, R.L.: Preparation of high-resolution freeze-etch, freeze-fracture, frozen-surface and freeze-dried replicas in a single freeze-etch module, and the use of stereo electron microscopy to obtain maximum information from them. In: Freeze-etching techniques and applications (E.L. Benedetti and P. Favard, eds.). Paris 1973

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Accepted December 15, 1977