The effect of thioglycolate on intermediate filaments and membrane translocation in rat urothelium during the expansion-contraction cycle

Summary The functional role of cytokeratin intermediate filaments in the translocation of asymmetric membrane plaques between cytoplasm and surface of apical urothelial cells was investigated during contraction and expansion of rat urinary bladders. A stereological investigation of electron micrographs provided estimations of surface area, volume, and number of discoidal vesicles and infoldings per unit volume of urothelial apical cell cytoplasm. Contracted and distended bladders incubated in 0.01 M sodium bicarbonate were compared to identical preparations experimentally incubated in 5 mM thioglycolic acid. The latter reagent disrupts the intermediate filament network by reducing sulfhydryl bridges. Densities of discoidal vesicles in cells contracted after incubation in thioglycolate were similar to density estimations in cells expanded under control conditions. Similarly, densities of vesicles in cells expanded after exposure to thioglycolate were comparable in number to those in normally contracted cells. Thus, membrane translocation to and from the luminal surface was blocked by thioglycolate treatment. The lack of normal membrane transfer at the luminal surface induces apical cells exposed to experimental conditions to undergo extraordinary adjustments in response to external pressures of bladder contraction and distension. During contraction, the apical-intermediate cell interface unfolded while the luminal surface ballooned out into the lumen. In distended bladders, large intercellular spaces formed between apical cells along their lateral margins. The results support a model published earlier implicating the filament network as a critical mediator of membrane translocation.     

Morphometric analysis of the translocation of lumenal membrane between cytoplasm and cell surface of transitional epithelial cells during the expansion-contraction cycles of mammalian urinary bladder

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, approximately 85 to approximately 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.     

Electron tomography of fusiform vesicles and their organization in urothelial cells

The formation of fusiform vesicles (FVs) is one of the most distinctive features in the urothelium of the urinary bladder. FVs represent compartments for intracellular transport of urothelial plaques, which modulate the surface area of the superficial urothelial (umbrella) cells during the distension-contraction cycle. We have analysed the three-dimensional (3D) structure of FVs and their organization in umbrella cells of mouse urinary bladders. Compared to chemical fixation, high pressure freezing gave a new insight into the ultrastructure of urothelial cells. Electron tomography on serial sections revealed that mature FVs had a shape of flattened discs, with a diameter of up to 1.2 μm. The lumen between the two opposing asymmetrically thickened membranes was very narrow, ranging from 5 nm to 10 nm. Freeze-fracturing and immunolabelling confirmed that FVs contain two opposing urothelial plaques connected by a hinge region that made an omega shaped curvature. In the central cytoplasm, 4-15 FVs were often organized into stacks. In the subapical cytoplasm, FVs were mainly organized as individual vesicles. Distension-contraction cycles did not affect the shape of mature FVs; however, their orientation changed from parallel in distended to perpendicular in contracted bladder with respect to the apical plasma membrane. In the intermediate cells, shorter and more dilated immature FVs were present. The salient outcome from this research is the first comprehensive, high resolution 3D view of the ultrastructure of FVs and how they are organized differently depending on their location in the cytoplasm of umbrella cells. The shape of mature FVs and their organization into tightly packed stacks makes them a perfect storage compartment, which transports large amounts of urothelial plaques while occupying a small volume of umbrella cell cytoplasm.     

Function of the cytoskeleton in cells with microridges from the oral epithelium of the carp Cyprinus carpio

Superficial cells of the oral mucosal epithelium in the carp and the cytoskeleton of the epithelial cells are examined by scanning and transmission electron microscopy. Microridges are formed on the surface of the epithelium. Epithelial cells contain two types of vesicles: mucous secretory vesicles and coated vesicles. Most of the mucous vesicles are situated in the center of the cell near the Golgi apparatus. In freeze-fracture replicas, intramembranous particles are abundant in the membranes of the secretory vesicles but rare in the apical plasma membrane. Coated vesicles are situated in the apical and subapical cytoplasm. A great number of thick filaments, considered to be keratin filaments, run randomly throughout the cell to form a meshwork. Thick filaments, which are sparse in the central cytoplasm, are connected to the membranes of the secretory vesicles and other membranous organelles. A layer of closely packed thin filaments, considered to be actin filaments, is found just beneath the apical plasma membrane. Microtubules also occur in the apical cytoplasm and run almost parallel to the cell surface. Both kinds of vesicles are connected to the thin and thick filaments. Their functional significance in the regulation of membrane at the free surface is discussed.     

Roles of ATP and cytoskeleton in the regulation of Na+/H+ exchanger along the nephron luminal membrane

Although in LLC-PK cells ATP depletion has been shown to result in alterations of cytoskeleton actin and an inhibition of Na+/H+ exchanger activity, there is little information concerning the regulation of this exchanger in the distal luminal membrane by ATP and actin filaments. The present study examined the direct effect of ATP and cytochalasin B on the Na+/H+ exchanger activity in the proximal and distal tubule luminal membranes. The presence of 100 microM ATP in the luminal membrane vesicles from rabbit proximal tubules did not influence the Ethyl Isopropyl Amiloride sensitive Na+ uptake by these membranes. In contrast, the same treatment of luminal membranes from distal tubules significantly enhanced the exchanger activity from 0.22 +/- 0.04 to 0.39 +/- 0.08 pM/microg/10 sec (P < 0.02). When ATP was replaced by its nonhydrolysable form, ATPgammas, the effect on the distal luminal membrane was strongly diminished suggesting that the action of the nucleotide implicates a phosphorylation step. Confirming this hypothesis, addition of 300-microM-Rp cAMP, a protein kinase A inhibitor, completely abolished the effect of ATP. In view of the fact that a tight relationship has been described between ATP, the cytoskeleton complex and the exchanger activity, we studied the effect of cytochalasin B on this activity. The presence of 20 microM cytochalasin B in the distal luminal membrane vesicles induced, as observed with ATP, a significant increase in the Na+ uptake. However, the actions of ATP and cytochalasin B were not additive. These results suggest that firstly, ATP and short actin filaments of the cytoskeleton regulate the distal luminal isoform through an intramembranous mechanism and secondly, a phosphorylation mechanism is, at least partially, implicated in the action of ATP. In contrast, the proximal tubule exchanger is regulated through different mechanisms.     

ATP directly enhances calcium channels in the luminal membrane of the distal nephron.

Calcium (Ca(2+)) transport by the distal tubule (DT) luminal membrane is regulated by the parathyroid hormone (PTH) and calcitonin (CT) through the action of messengers, protein kinases, and ATP as the phosphate donor. In this study, we questioned whether ATP itself, when directly applied to the cytosolic surface of the membrane could influence the Ca(2+) channels previously detected in this membrane. We purified the luminal membranes of rabbit proximal (PT) and DT separately and measured Ca(2+) uptake by these vesicles loaded with ATP or the carrier. The presence of 100 microM ATP in the DT membrane vesicles significantly enhanced 0.5 mM Ca(2+) uptake from 0.57 +/- 0.02 to 0.71 +/- 0.02 pmol/microg per 10 sec (P < 0. 01) in the absence of Na(+) and from 0.36 +/- 0.03 to 0.59 +/- 0.01 pmol/microg per 10 sec (P < 0.01) in the presence of 100 mM Na(+). This effect was dose dependent with an EC(50) value of approximately 40 microM. ATP action involved the high-affinity component of Ca(2+) transport, decreasing the Km from 0.08 +/- 0.01 to 0.04 +/- 0.01 mM (P< 0.02). Replacement of the nucleotide by the nonhydrolyzable ATPgammas abolished this action. Because ATP has been reported to be necessary for cytoskeleton integrity, we also investigated the effect of intravesicular cytochalasin on Ca(2+) transport. Inclusion of 20 microM cytochalasin B decreased 0.5 mM Ca(2+) uptake from 0.33 +/- 0.01 to 0.15 +/- 0.01 pmol/microg per 10 sec (P< 0.01). However, when both 100 microM ATP and 20 microM cytochalasin were present in the vesicles, the uptake was not different from that observed with ATP alone. Neither ATP nor cytochalasin had any influence on Ca(2+) uptake by the PT luminal membrane. We conclude that the high-affinity Ca(2+) channel of the DT luminal membrane is regulated by ATP and that ATP plays a crucial role in the integrity of the cytoskeleton which is also involved in the control of Ca(2+) channels within this membrane.     

Improved preservation and staining of HeLa cell actin filaments, clathrin-coated membranes, and other cytoplasmic structures by tannic acid-glutaraldehyde-saponin fixation

Fixation of HeLa cells with a mixture of 100 mM glutaraldehyde, 2 mg/ml tannic acid and 0.5 mg/ml saponin allows the tannic acid to penetrate intact cells without disruption of membranes or extraction of the cytoplasmic matrix. After subsequent treatment with OsO4 cytoplasmic structures are stained so densely that fine details are visible even in very thin (dark gray) sections. Actin filaments are protected from disruption by OsO4 so that straight, densely stained filaments are seen in the cell cortex, filopodia, ruffling membranes, and stress fibers. Stress fibers also have 15-18-nm densities similar in appearance to myosin filaments. Tannic acid staining reveals that the coats of coated vesicles, pits, and plaques have a 12-nm layer of amorphous material between the membrane and the clathrin basketwork. HeLa cells have very large clathrin-coated membrane plaques on the basal surface. These coated membrane plaques appear to be a previously unrecognized site of cell-substrate adhesion.     

THE MAMMALIAN URINARY BLADDERAN ACCOMMODATING ORGAN

1. Urinary bladders are found in the amphibia, chelonian reptiles and mammals. In these orders liquid urine is stored in the bladder and eliminated at intervals from the body by micturation. 2. In the amphibia and chelonian reptiles, the urinary bladder is a functional extension of the renal tubules. The composition of the urine in the bladder is modified by the active movement of water and ions across the bladder wall, and these transporting processes are under hormonal control. The bladder acts as a water reservoir which can be drawn upon in times of water shortage. 3. The mammalian bladder separates two widely differing water phases, namely the urine which is frequently hypertonic to the blood and the tissue fluids which are isotonic. Its function is uniquely one of storage, and no adjustment to the composition of the urine is made by active transport of either water or ions across the bladder wall. 4. The epithelium lining the mammalian bladder is the site of the osmotic barrier between urine and tissue fluids. This functional barrier is dependent on the structure of the epithelium and is maintained despite large alterations in the surface area of the epithelium as the bladder rapidly contracts, or slowly dilates. 5. The epithelium is of mixed mesodermal and endodermal origin, is transitional in type and is usually 3 or 4 cell-layers thick. If this urothelium is damaged, it has a high capacity for regeneration and rapidly re-establishes an intact barrier over the luminal surface. 6. The superficial cell layer of this epithelium is composed of large, polyploid, highly differentiated squamous cells which have a long life span. These cells are limited on their free surface by an unusual, angular, semi-rigid luminal membrane. This membrane is assembled in the Golgi complex. 7. The luminal membrane is composed of thickened, discoidal plaques, separated by narrow bands of thinner membrane. When the bladder contracts, the membrane folds along the thinner ‘hinge’ regions, and the rigid discoidal plates invaginate to form fusiform, cytoplasmic vacuoles. The thickened plaques contain a hexagonal lattice of sub-units, spaced at 14 nm centre-to-centre. Each sub-unit in the lattice is itself composed of 12 smaller particles. These particles may be envisaged as small rods 3 nm in diameter and 12 nm long, and are inserted into matrix from which they project on the luminal face by about 3 nm. Each rod has a central hydrophobic portion separating distal hydrophilic ends. 8. The chemical composition of this luminal membrane is unusual. Cerebroside is a major component of the polar lipid fraction and there is an unusually high proline content in the protein fraction. When the mucoproteins are adequately dispersed, and the proteins separated by electrophoresis, a few major proteins are revealed in 33000–80000 dalton range of molecular weight. 9. If the normal structure of the luminal membrane is altered, either by physical damage or by failure of the cells to produce it, the barrier function of the epithelium is lost. 10. The structure and function of this membrane depend ultimately on its chemical composition. Cerebroside is known to decrease the permeability of lipid bi-layers to water, but for maximum impermeability a lipid bi-layer must be maintained in a condensed configuration. The stresses of bladder distension and contraction might be expected to disrupt the bi-layer, and it is suggested that the function of the rigid plaque regions is to reduce mechanical stresses in the membrane to a minimum. The plaque areas occupy between 73 and 90 % of the membrane surface, and only the remaining 10–27% of the membrane is thus subject to bending and distortion when the bladder contracts or expands. The structure of the plaque areas is probably determined by the nature of the complex proteins which form the sub-units. Proline is known to confer rigidity on polypeptide chains, and may play an important rôle in ordering the structure of the plaques. 11. The bladder epithelium, though normally differentiated as a transitional epithelium, has other biologicai potentialities. It can undergo squamous metaplasia to form a stratified cornified epithelium in response to mechanical irritation and/or vitamin A deficiency. If transplanted from its normal location, it can induce other supporting mesenchyme tissues to lay down bone. When regenerating in response to damage, the newly formed transitional cells can act as phagocytes and engulf and digest damaged or dying cells. In the normal animal the epithelium is largely protected from tumour formation by cell-mediated immunological surveillance. The defensive mechanisms are triggered by tissue-type specific antigens which develop in neoplastic bladder epithelial cells.     

Effect of distension on ADH-induced osmotic water flow in toad urinary bladder

Summary We recently described a method by which the resistance to water flow of the luminal membrane of ADH-stimulated toad bladder can be quantitatively distinguished from that of barriers lying in series with it. This method requires estimates of both total bladder water permeability (assessed by transbladder osmotic water flow at constant gradient) and luminal membrane water permeability (assessed by quantitation of the frequency of ADH-induced luminal membrane particle aggregates). In the present study we examined the effect of bladder distension on transepithelial osmotic water flow before and during maximal ADH stimulation. Base-line water flow was unaffected by bladder distension, but hormonally stimulated flow increased systematically as bladders became more distended. Distension had no effect on the frequency of ADH-induced intramembranous particle aggregates. By comparing the relationships between aggregate frequency and hormonally induced water permeability in distended and undistended bladders, we found that distension appeared to enhance ADH-stimulated water flow by decreasing the resistance of the series permeability barrier while the apparent water permeability associated with each single luminal membrane aggregate was unaffected. In that bladder distension causes tissue thinning, the series resistance limiting ADH-stimulated water flow appears to be accounted for by deformable barriers within the bladder tissue itself, probably unstirred layers of water.     

ATP-induced cell contraction in dermal fibroblasts: effects of cAMP and myosin light-chain kinase

When 1 mM ATP is added to human dermal fibroblasts (DF) in monolayer culture permeabilized by glycerol, they undergo a rapid reduction in length and their intracellular actin filaments aggregate. This process is referred to as cell contraction. Treating glycerol-permeabilized DF with alkaline phosphatase before adding 1 mM ATP should cause dephosphorylation. Dephosphorylated preparations do not undergo cell contraction initiated by ATP. When myosin light-chain kinase (MLCK) isolated from turkey gizzard is added with cofactors to cells dephosphorylated by alkaline phosphatase treatment, contraction is restored. DF incubated for 24 h with db cAMP or cholera toxin show elevated intracellular concentrations of cAMP and little cell contraction. Contraction is reestablished when MLCK with cofactors is incubated with these preparations before ATP is added. Fibroblasts from Epidermolysis Bullosa dystrophica recessive patients produce excess cAMP. Those cells show minimal contraction, however; treating them with MLCK and cofactors renews contraction brought about by ATP. When DF are incubated with trifluoperazine to block calmodulin-dependent enzyme reactions, cell contraction is inhibited. Adding cytochalasin B disrupts microfilaments and also inhibits contraction. This work supports the idea that myosin ATPase is critical to cell contraction. Myosin ATPase is dependent on the phosphorylation of the regulatory peptide, myosin light chain. Elevating intracellular concentrations of cAMP or treatment of permeabilized cell preparations with alkaline phosphatase may inhibit myosin ATPase activity. The restoration of phosphorylation by adding MLCK with cofactors served to reestablish cell contraction.     

Regulation of cell pH by Ca+2-mediated exocytotic insertion of H+- ATPases

Exposure to CO2 acidifies the cytosol of mitochondria-rich cells in turtle bladder epithelium. The result of the decrease in pH in these, the acid-secreting cells of the epithelium, is a transient increase in cell calcium, which causes exocytosis of vesicles containing proton-translocating ATPase. Because mitochondria-rich cells have rapid luminal membrane turnover, we were able to identify single mitochondria-rich cells by their endocytosis of rhodamine-tagged albumin. Using fluorescence emission of 5,6-carboxyfluorescein at two excitation wavelengths, we measured cell pH in these identified mitochondria-rich cells and found that although the cell pH fell, it recovered within 5 min despite continuous exposure to CO2. This pH recovery also occurred at the same rate in Na+-free media. However, pH recovery did not occur when luminal pH was 5.5, a condition under which the H+-pump does not function, suggesting that recovery of cell pH is due to the luminally located H+ ATPase. Chelation of extracellular calcium by EGTA prevented the CO2-induced rise in cell calcium measured with the intracellular fluorescent dyes Quin 2 or Fura 2 and also prevented recovery of cell pH. When the change in cell calcium was buffered by loading the cells with high concentrations of Quin 2, the CO2-induced decrease in pH did not return back to basal levels. We had found previously that buffering intracellular calcium transients prevented CO2-stimulated exocytosis. Further, we show here that the increased H+ current in voltage-clamped turtle bladders, which is directly proportional to the number of H+-pump-containing vesicles that fuse with the luminal membrane, was significantly reduced in calcium-depleted bladders. These results suggest that pH regulation in these acid-secreting cells occurs by calcium-dependent exocytosis of vesicles containing proton pumps, whose subsequent turnover restores the cell pH to its initial levels.     

The superficial cells of the transitional epithelium in the expanded and unexpanded rat urinary bladder. Transmission and scanning electron-microscopic study.

The superficial epithelial layer in the urinary bladder of adult rats was examined, in various states, using the transmission and scanning electron microscopes. A good agreement was obtained between the results of the two methods. When the urinary bladder is unexpanded, the superficial cells show marked bulges into the bladder lumen and the contacts between cells (mainly desmosomes) are displaced deep into the epithelium. The luminal surface is bizarrely bent and large parts of the membrane intrude into the cytoplasm, where they give the appearance of discoid and fusiform vesicles. Between neighboring cells, deep interdigitations are observed. In the scanning electron microscope, the surface of the epithelium appears cauliflower-like and has deep grooves, gullys and folds. When the bladder is expanded, the surface becomes smoother and the contacts between cells move to the surface. The stretched cells are angular in form (5-, 6- or 7-sided) and show great variations in surface area (150-500 mum2). The luminal cell membrane consists of an alternation of asymmetrical areas (120 A thick and 0.2-0.4 mum in length) with normal sections which are 80 A thick. In the scanning electron microscope, these thick areas appear as 4-, 5- or 6-sided plaques with a maximal diameter of 0.4 mum. The borders of the plaques are formed of portions of cell membrane which have a normal thickness and extrude as microcristae into the lumen. This produces a honeycomb appearance on the cell surface.     

The maintenance of the permeability barrier of bladder facet cells requires a continuous fusion of discoid vesicles with the apical plasma membrane

The luminal surface of the bladder epithelium is continuously exposed to urine that differs from blood in its ionic composition and osmolality. The apical plasma membrane of facet or umbrella cells, facing the urine, is covered with rigid-looking plaques consisting of hexagonal uroplakin particles. Together with tight junctions these plaques form a specialized membrane compartment that represents one of the tightest and most impermeable barriers in the body. Plaques also occur in the membrane of cytoplasmic discoid vesicles. Here it is shown shown that synaptobrevin, SNAP23 and syntaxin are perfectly colocalized with uroplakin III at the apical plasma membrane as well as with membranes of discoid vesicles. Such a distribution suggests that discoid vesicles in facet cells may gain access to the apical plasma membrane probably by combination of homotypic and heterotypic fusion events. Furthermore, we detected uroplakin III-containing membranes of different sizes in the urine of healthy humans and rats. Probably facet cells maintain their permeability barrier by a process of continuous membrane regeneration that includes the cutting off of areas of the apical membrane and its replacement by newly fused discoid vesicles.     

On the sidedness of membrane phosphorylation by Pi and ATP synthesis during reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles.

The membrane sidedness of Pi interaction in reactions which characterize reversal of the Ca2+ pump of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle was investigated. Vesicles previously loaded with calcium [32P]phosphate were incubated with 0.1 mM ADP and different concentrations of nonradioactive Pi. Alternatively, vesicles loaded with nonradioactive calcium phosphate were incubated in a medium containing 32Pi. The rates of Ca2+ efflux and ATP synthesis were siginficantly activated only when Pi was included in the assay medium. Although the Pi contained by the vesicles crosses the membrane at a rate proportional to the Ca2+ efflux, [gamma-32P]ATP was synthesized only when 32Pi interacted with the outer surface of the membrane. Similarly, ATP in equilibrium 32Pi or ITP in equilibrium 32Pi exchange could be measured only when the external pool of Pi was labeled. Both for ATP synthesis and for the ITP in equilibrium Pi exchange reaction, membrane phosphorylation by 32Pi was negligible unless the external pool of Pi was labeled. The ionophore X-537 A increased the rate of Ca2+ efflux but inhibited the synthesis of ATP. During reversal of the Ca2+ pump, Pi apparently interacts with the membrane only at the outer surface, and at a site different from that where Ca2+ crosses the membrane.     

Calcium and magnesium regulation of phosphorylation by ATP and ITP in sarcoplasmic reticulum vesicles.

Membrane phosphorylation and nucleoside triphosphatase activity of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were studied using ATP and ITP as substrates. The Ca2+ concentration was varied over a range large enough to saturate either the high affinity Ca2+-binding site or both high and low affinity binding sites. In intact vesicles, which are able to accumulate Ca2+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca2+ concentration is increased to 10 mM. Essentially the same pattern of membrane phosphorylation by ATP is observed when leaky vesicles, which are unable to accumulate Ca2+, are used. However, for leaky vesicles, when ITP is used as substrate, the phosphoenzyme level increases 3- to 4-fold when the Ca2+ concentration is raised from 0.02 to 20 mM. When Mg2+ is omitted from the assay medum, the degree of membrane phosphorylation by ATP varies with Ca2+ in the same way as when ITP is used in the presence of Mg2+. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added Mg2+. When these vesicles are incubated in media containing ITP and 0.1 mM Ca2+, addition of Mg2+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When ATP is used, the addition of 10 mM Mg2+ increases both the steady state level of phosphoenzyme and the rate of ATP hydrolysis. When the Ca2+ concentration is raised to 10 or 20 mM, the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg2+. In these conditions the ATPase and ITPase activities are activated by Mg2+, although not to the level observed in 0.1 mM Ca2+. An excess of Mg2+ inhibits both the rate of hydrolysis and membrane phosphorylation by either ATP or ITP.     

Post-stimulation retrieval of luminal surface membrane in parotid acinar cells is calcium-dependent

The Ca²⁺ dependence of surface membrane retrieval (i.e., the process by which the excess surface membrane resulting from exocytosis is recycled to the cytoplasm of secretory cells) has been investigated in rat parotid tissue lobules first incubated for 40 min in the presence of a secretagogue drug (the β-adrenergic agonist isoprenaline) and then in the presence of the β-blocker, 1-propranolol, up to 4 h. The dynamics of the luminal surface membrane was monitored by measuring, in ultrathin sections, the length of the luminal profile of all examined acinar cells abutting to a lumen before and immediately at the end of the stimulation, as well as at various times thereafter. Such a profile doubled during isoprenaline stimulation, concomitantly with the discharge of most secretion granules. After the stimulation was blocked, the luminal profile decreased to reach values even lower than those observed in unstimulated cells. The kinetics of this reduction was apparently first-order, both in the presence and in the absence of extracellular Ca²⁺. However, its rate differed appreciably in these two situations: it was relatively fast (apparent ) in lobules incubated in complete medium (Ca²⁺ concentration, 2 mM), and much slower (apparent ) in lobules incubated in a Ca²⁺-free medium containing 1 mM EGTA. The slowing down of the membrane retrieval occurring in Ca²⁺-free conditions was rapidly reversed by reintroduction of Ca²⁺ into the medium. These findings indicate that the retrieval of the luminal surface membrane in parotid acinar cells is Ca²⁺-dependent.     

Trajectorial organisation of cytokeratins within the subapical region of umbrella cells

The subapical region of umbrella cells in the urinary bladder contains a dense cytokeratin (CK) network. Yet, this network should enable a very intensive traffic of specific fusiform vesicles involved in alterations of the surface area of the apical membrane. Therefore, the cytokeratins should be organised in a way to be both mechanically strong and also passable for fusiform vesicles. The supramolecular organisation of the CKs in the subapical region of umbrella cells in mice was studied by conventional fluorescence, confocal laser microscopy, and TEM. It has been found that the first 150 to 300 nm under the apical membrane is filled with fusiform vesicles and only below that the CK network begins. The CK 7 and CK 20 compose a subapical network, which is created as an array of parallel trajectories pointing to the apical plasma membrane. The network is framed by a strong wall of CK, which is parallely aligned in neighbouring cells. The double labelling of the urothelial-specific membrane proteins, uroplakins, and CKs proved the presence of fusiform vesicles within these trajectories. The measurements proved that the trajectory diameter in the upper half of the network is smaller than in the lower half. The diameters of the trajectories in animals with distended bladders exceeded those in contracted bladders for 70%, which most likely facilitates the transport of fusiform vesicles to the apical membrane. Discovery of the subapical CK network elucidates the until now undescribed supramolecular organisation of CKs in the apical region of urothelial cells.     

Recent observations on the ultrastructure of human urothelium

Summary An electron microscopic study of normal bladder urothelium of elderly patients ranging in age from 61 to 82 years has shown the occurrence of unusually thin regions consisting of either one or two layers of undifferentiated cells interspersed between 3–4 cell layers thick regions. A morphometric study has confirmed the existence of a pattern of cytodifferentiation in cells of the thick region. The generally microvillous nature of the luminal surface is attributed to incompletely differentiated cells that have come to occupy the superficial layer. The lack of thickened and/or asymmetric membrane plaques in luminal plasma as well as the dearth of characteristic precursor vesicles in the cytoplasm are also explicable in terms of a failure of normal cell differentiation. It is suggested that the unusual features noted are consequences of tissue ageing rather than prognostic of cancer. There are indications that the aged urothelium may be prone to increased leakiness and the bladder tissues may therefore be at greater risk from urine-borne chemicals and carcinogens.     

Evidence for the participation of actin microfilaments and bristle coats in the internalization of gap junction membrane

Thin sections of rabbit granulosa, human SW-13 adrenal cortical adenocarcinoma, and mouse B-16 melanoma cells revealed an apparent single-layered basket of 4- to 7-nm filaments surrounding cytoplasmic gap junction vesicles. This interpretation was based upon apparent longitudinal, cross, and en face sections of structures surrounding the vesicle profiles in tissue treated with tannic acid-glutaraldehyde. In granulosa cells incubated with the S-1 fragment of heavy meromyosin, arrowhead-decorated filaments were observed at the periphery of the gap junction vesicles, suggesting that these filaments contained actin. In addition, we found that small gap junctional blebs and vesicles at the cell surface were coated with short electron-dense bristles similar in appearance to the cloathrin-containing coat of coated vesicles of nonjunctional membrane. The role of these and other cytoskeletal elements in the possible endocytosis of gap junction membrane is discussed.     

Very high water permeability in vasopressin-induced endocytic vesicles from toad urinary bladder

The regulation of transepithelial water permeability in toad urinary bladder is believed to involve a cycling of endocytic vesicles containing water transporters between an intracellular compartment and the cell luminal membrane. Endocytic vesicles arising from luminal membrane were labeled selectively in the intact toad bladder with the impermeant fluid-phase markers 6-carboxyfluorescein (6CF) or fluorescein-dextran. A microsomal preparation containing labeled endocytic vesicles was prepared by cell scraping, homogenization, and differential centrifugation. Osmotic water permeability was measured by a stopped-flow fluorescence technique in which microsomes containing 50 mM mannitol, 5 mM K phosphate, pH 8.5 were subject to a 60-mM inwardly directed gradient of sucrose; the time course of endosome volume, representing osmotic water transport, was inferred from the time course of fluorescence self-quenching. Endocytic vesicles were prepared from toad bladders with hypoosmotic lumen solution treated with (group A) or without (group B) serosal vasopressin at 23 degrees C, and bladders in which endocytosis was inhibited by treatment with vasopressin at 0-2 degrees C (group C), or with vasopressin plus sodium azide at 23 degrees C (group D). Stopped-flow results in all four groups showed a slow rate of 6CF fluorescence decrease (time constants 1.0-1.7 s for exponential fit) indicating a component of nonendocytic 6CF entrapment into sealed vesicles. However, in vesicles from group A only, there was a very rapid 6CF fluorescence decrease (time constant 9.6 +/- 0.2 ms, SEM, 18 separate preparations) with an osmotic water permeability coefficient (Pf) of greater than 0.1 cm/s (18 degrees C) and activation energy of 3.9 +/- 0.8 kcal/mol (16 kJ/mol). Pf was inhibited reversibly by greater than 60% by 1 mM HgCl2. The rapid fluorescence decrease was absent in vesicles in groups B, C, and D. These results demonstrate the presence of functional water transporters in vasopressin-induced endocytic vesicles from toad bladder, supporting the hypothesis that water channels are cycled to and from the luminal membrane and providing a functional marker for the vasopressin-sensitive water channel. The calculated Pf in the vasopressin-induced endocytic vesicles is the highest Pf reported for any biological or artificial membrane.