To determine the three-dimensional structure of the lumenal membrane of transitional epithelium, a study was made of sectioned, negatively stained, and freeze-etched specimens from intact epithelium and membrane fractions from rabbit urinary bladder. Particulate membrane components are confined to plaque regions within which the unit membrane is asymmetric, having a thicker outer leaflet . Transversely fractured freeze-etched plaques display a thick (-80 A), particulate lumenal leaflet and a thin (-40 A) cytoplasmic one . Four different faces of the two leaflets can be distinguished : two complementary, split, inner membrane faces exposed by freeze-cleaving the bilayer and two external (lumenal and cytoplasmic) membrane surfaces revealed by deep-etching . On the split, inner face of the lumenal leaflet appear polygonal plaques of hexagonally arranged particles . These fit into holes observed on the complementary, split, innerface of the cytoplasmic leaflet . The particles, which have a center-to-center spacing of -160 A, also seem to protrude from the external surface of the lumenal leaflet, where their subunits ("50 A in diameter) are revealed by freeze-etching and negative staining . The plaques are separated from each other by smoothsurfaced regions, which cleave like simple lipid bilayers . Since the array of plaque particles covers only -73 %o of the membrane surface area, whereas 27 % is taken up by particle-free interplaque regions, the presence of particles cannot in itself entirely account for the permeability barrier of the lumenal membrane . Although no particles are observed protruding from the cytoplasmic surface of the membrane, cytoplasmic filaments are attached to it by short, cross-bridge-like filaments that seem to contact the particles within the membrane . These long cytoplasmic filaments cross-link adjacent plaques . Therefore, we suggest that at least one function of the particles is to serve as anchoring sites for cytoplasmic filaments, which limit the expansion of the lumenal membrane during distention of the bladder, thereby preventing it from rupturing . The particle-free interplaque regions probably function as hinge areas between the stiff plaques, allowing the membrane to fold up when the bladder is contracted .

The flow of membrane between the cytoplasm and the lumenal surface during the expansion-contraction cycle of urinary bladder was estimated by stereological examination of electron micrographs of urothelial cells from guinea pigs, gerbils, hamsters, rabbits, and rats. The quantitative data obtained allowed an approximation of the surface area, volume, and numbers of lumenal membranelike vesicles and infoldings per unit volume of cytoplasm. Depending upon the species, -85 to -94% of the membrane surface area translocated into and out of the cytoplasm was in the form of discoidal vesicles. The remainder was accounted for by infoldings of the lumenal plasma membrane. The density of vesicles involved in transfer of membrane was quite similar in all the species examined, except guinea pigs which yielded lower values. In contrast, the densities of the total cytoplasmic pools of discoidal vesicles potentially available for translocation varied greatly among the different species. In general, species of animals with a highly concentrated urine had a greater density of discoidal vesicles than species with a less concentrated urine. This correlation may indicate an authentic relationship between lumenal membranes and the tonicity of urine, such as increased membrane recycling or turnover with increasingly hypertonic urine; or it may signify the existence of some other, more obscure relationship.KEY WORDS urinary bladder -lumenal plasma membrane discoidal vesicles Discoidal vesicles can be found in the apical cells of the transitional epithelium lining the urinary bladder of a variety of mammals (3,(8)(9)(10). The vesicles, which appear fusiform when sectioned transversely, are composed of two apposing plaques identical to those seen in the plasma membrane covering the cell's lumenal surface. The lumenal membrane is made up of the concave plaques which are interconnected by smooth interplaque regions (8-10). It has been proposed that the hexagonal arrays of particles within the plaques serve as anchors for an underlying filamentous network, thereby preventing rupturing of the specialized membrane during expansion of the bladder (1, 10).J. CELL BIOLOGY 9 The Rockefeller University Press 9

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