Ultrastructure and elemental composition of frog bladder granular epithelial cells in normal state and upon stimulation of water transport

Changes in the frog urinary bladder granular cell ultrastructure were analysed in parallel with those in element composition of these cells after induction of water transport across the urinary bladder wall. Two ultrastructural (ultrathin section and freeze-fracture) methods were used in addition to two methods of object preparation for electron microprobe analysis--freeze-drying and freeze-substitution. It has been shown that arginin-vasotocin stimulation of osmotic water flow across the urinary bladder wall causes certain morphological changes in the granular cells: decrease in electron density of the cytoplasm, depolymerization of the apical submembrane layer of actin microfilaments, increase in the number of sites of specific granules and apical membrane fusion, emergency of intramembrane particle aggregates in the apical membrane P-face. The quantitative electron microprobe analysis made it possible to reveal a statistically significant increase in sodium and calcium concentration and fall in that of potassium and chlorine in granular cells after water transport stimulation. A concentration gradient of sodium and potassium ions was seen to appear along the apical-basal axis in the cytoplasm of granular cells. Possible association between the obvious morphological transformations in granular cells and changes in their elemental composition has been discussed, in addition to some regulatory significance of calcium concentration increase in granular cells after arginin-vasotocin-induced osmotic water transport.     

Isolation and characterization of specialized regions of toad urinary bladder apical plasma membrane involved in the water permeability response to antidiuretic hormone

Summary Antidiuretic hormone (ADH) increases the apical (external facing) membrane water permeability of granular cells that line the toad urinary bladder. In response to ADH, cytoplasmic vesicles called aggrephores fuse with the apical plasma membrane and insert particle aggregates which are visualized by freeze-fracture electron microscopy. Aggrephores contain particle aggregates within their limiting membranes. It is generally accepted that particle aggregates are or are related to water channels. High rates of transepithelial water flow during ADH stimulation and subsequent hormone removal decrease water permeability and cause the endocytosis of apical membrane and aggrephores which retrieve particle aggregates. We loaded the particle aggregate-rich endocytic vesicles with horseradish peroxidase (HRP) during ADH stimulation and removal. Epithelial cells were isolated and homogenized, and a subcellular fraction was enriched for sequestered HRP obtained. The HRP-enriched membrane fraction was subjected to a density shifting maneuver (Courtoy et al.,J. Cell Biol. 98:870, 1984), which yielded a purified membrane fraction containing vesicles with entrapped HRP. The density shifted vesicles were composed of approximately 20 proteins including prominent species of 55, 17 and 7 kD. Proteins of these molecular weights appear on the apical surface of ADH-stimulated bladders, but not the apical surface of control bladders. Therefore, we believe these density shifted vesicles contain proteins involved in the ADH-stimulated water permeability response, possibly components of particle aggregates and/or water channels.     

Visualization of endocytosed markers in freeze-fracture studies of toad urinary bladder

Antidiuretic hormone (ADH) stimulation increases the apical membrane water permeability of granular cells in toad urinary bladder. This response correlates closely with the fusion of tubular cytoplasmic vesicles with the membrane and delivery of intramembrane particle (IMP) aggregates from the tubules (aggrephores) to the apical membrane. These aggregates are believed to be associated with the channels responsible for the water permeability increase. Removal of ADH triggers apical membrane endocytosis and disappearance of aggregates from the apical membrane. However, it has been unclear whether aggregate disappearance is due to disassembly of aggregates within the apical membrane or to their endocytic retrieval as intact structures. Using colloidal gold and horseradish peroxidase to follow endocytosis from the apical surface after ADH removal, we have directly observed in cross-fractured bladder cells the intramembrane structure of intracellular vesicles that contain these fluid-phase markers. Under these conditions, intact aggregates can be identified in the membrane of tubular endocytosed vesicles. This directly demonstrates that conditions which lower apical membrane water permeability cause the tubular aggrephores to "shuttle" intact aggregates from the apical membrane back into the cytoplasm. An additional population of vesicles with tracer are found which are spherical and display structural features of the apical membrane, as well as occasional aggregates. These vesicles may be responsible for retrieval of aggregates from the surface apical membrane.     

Distribution of actin microfilaments in frog skin epithelial granular cells

Summary— Microfilaments were localised by immunofluorescence and immunogold cytochemistry to examine their distribution in granular cells of the isolated frog skin epithelium. Strongly fluorescent bundles of actin were observed beneath the plasma membrane with little evidence for actin in the central regions. Higher resolution offered by cytochemistry revealed that bundles of actin filaments comprised a substantial portion of the cortical cytoskeleton. Quantitative analysis of the frequency of gold label revealed an extremely rich array of filaments beneath the apical membrane of granular cells, with markedly less babel along the basolateral membrane and in the central cytoplasm. Treating cells with cytochalasin B or arginine vasopressin caused an apparent disruption of the apical actin fibres, concurrent with a decrease in gold label density. Assumably these signs are indicative of depolymerization of the filaments. Although the significance of this distribution is unknown, the apical polarisation of actin is consistent with a role in regulating the Na⁺ permeability of the apical membrane. The data are discussed in relation to possible roles of the cytoskeleton in the regulation of transepithelial sodium transport by vasopressin.     

Membrane structural studies of the action of vasopressin

Freeze-fracture electron microscopy of the toad urinary bladder indicates that distinctive intramembrane particle aggregates are responsible for the increase in apical membrane water permeability that occurs with vasopressin (VP) stimulation. In unstimulated bladders the aggregates occur in the cytoplasm of the cells in tubular membrane structures now called aggrephores. After stimulation by VP, aggrephores are shuttled to the surface and fuse with the apical membrane. It is suggested by structural observations and by measurements of membrane capacitance that the area of aggregates inserted into the apical membrane is much greater than previously suspected because many aggregates remain in the wall of the fused aggrephores. The area of the aggregates in a stimulated bladder is sufficiently large for these structures to represent an organized array of water channels that mediates the change in apical membrane permeability. Work with antibodies supports the concept that these channels are not always resident in the apical membrane but become inserted only after stimulation by the hormone VP.     

On the role of calcium in the mechanism of ADH action

With an increased influx of Ca2+ in the cytoplasm, the response of cells to ADH in the urinary bladder of the frog was lowered by addition of ionophore A23187 from the side of the basolateral cell membrane, but inhibited when it was added from the apical cell membrane. The removal of calcium by EGTA from the serosal surface was accompanied by a sharp increase of osmotic permeability not only to water, but also to inulin; while when calcium was removed from the mucosal surface of the urinary bladder, osmotic permeability was not changed. After being added to the Ringer solution from the outer surface of the apical cell membrane, the inhibitors of Ca2+ channels (verapamil, Ni2+, Mn2+, Co2+) decreased the effect of ADH. These data indicate that Ca2+ applied onto the outer surface of apical plasma membrane plays an important role in the action of ADH.     

Ultrastructure of the apical plasma membrane of the granular cells in the frog bladder during cobalt-ion decrease in the vasopressin effect

Simultaneous studies were performed on changes in water permeability and on the ultrastructural organization of the frog urinary bladder epithelium in the presence of Co-ions under vasopressin-stimulated water flow. A possible inhibition of the vasopressin-stimulated water flows by Co-ions is supposed from the extracellular surface of the apical membrane of granular cells responsible for water permeability of this epithelium. Using the freeze-fracture technique for studying the apical membrane ultrastructure, it was shown that with the maximum water flow the square occupied by intramembrane particle aggregates was as much as 1.8% of the total square of membranes, to reduce to 0.3% with the smaller water flow, the average sizes of aggregates being 0.35 mkm and 0.08 mkm in both these cases, respectively. Application of 1 x 10(-3)-1 x 10(-4) M CoCl2 from the mucose part inhibits the vasopressin-stimulated water flow. In this case no aggregates are actually seen on the P-face of the apical membrane, the number of intramembrane particles of the E-face being similar to that when the water permeability was originally low. It is concluded that Co-ion may influence the structure and function of the apical plasma membrane from its extracellular surface.     

Antidiuretic response in the urinary bladder of Xenopus laevis: Presence of typical aggrephores and apical aggregates

The urinary bladder of the aquatic toad Xenopus laevis is known to exhibit a low permeability to water and a poor sensitivity to antidiuretic hormone. In order to precise the characteristics and the specific cellular mechanisms of this reduced hydroosmotic response we used a sensitive volumetric technique to monitor net water flow and studied the correlation between the anti-diuretic hormone (ADH)-induced net water flow and the fine ultrastructural appearence of the urinary bladder epithelium. Transmural net water flow was entirely dependent on the osmotic gradient across the preparation and not on the hydrostatic pressure difference. We observed the existence of a low but significant hydro-osmotic response to arginine vasopressin. Freeze-fracture electron microscopy demonstrated the presence of typical aggrephores in the subapical cytoplasm. The response to the hormone was accompanied by the appearance of typical intramembrane aggregates into the apical plasma membrane. Water permeability increase and apical aggregate insertion were both slowly but fully reversible. Except for the multilayered structure of the epithelium and the particularly low response to antidiuretic hormone, all the studied permeability and ultrastructural characteristics of the bladder were thus very similar to those observed in other sensitive epithelia such as the amphibian bladder and skin and the mammalian collecting duct which exhibit a high hydro-osmotic response to the hormone.     

Barriers to water flow in vasopressin-treated toad urinary bladder

Summary Unstirred layers of water complicate the measurement of water permeability across epithelia. In the toad urinary bladder, the hormone vasopressin increases the osmotic water permeability of the granular epithelial cell's luminal membrane, and also leads to the appearance of aggregates of particles within this membrane. The aggregates appear to be markers for luminal membrane osmotic water permeability. This report analyzes the relationship between transbladder osmotic water flow and aggregate frequency, and demonstrates that flow across the bladder is significantly attenuated by unstirred layers of water or by structural barriers other than the luminal membrane when the luminal membrane is made permeable by vasopressin. This analysis in addition yields unique values for the permeabilities of both the luminal membrane and the barriers to water flow which lie in series with it.     

Electron microscopic research on the mitochondria-rich bladder cells of the frog

The ultrastructural peculiarities of mitochondria-rich cells of the frog urinary bladder are analysed using three electron microscopic methods: ultrathin sections, scanning electron microscopy, freeze fracture. The mitochondria and tubular and vesicular structures are most abundant in the apical region of cytoplasm. The P-face (PF) of the apical plasma membrane is characterized by the presence of rod-shaped intramembrane particles (IMP), whereas the E-face (EF) possesses complementary pits. Depending on the distribution density of the rod-shaped IMP, three types of cells are described. The apical plasma membrane has an invert distribution of the globular IMP: a great quantity of IMP on the EF and a few particles on the PF. This structure of the apical plasma membrane is supposed to correlate with its very low water permeability. Using filipin as a marker of cholesterol localization, it has been shown that the mitochondria-rich cell apical membrane contains more cholesterol than that of the granular cells. The nature of the rod-shaped IMP and their role in the transmembrane ion transport have been discussed.     

Electron microscopic study of colonic epithelial cells from the grass frog Rana temporaria under different intensity of water absorption

Three cell types have been revealed in the epithelium of the frog large intestine: granular, mitochondria-rich, and mucosal cells. Under a low water permeability (0.12 +/- 0.10 mkl/(min.cm2)) the distribution of intramembrane particles (IMP) in the apical cell membrane was the same as in the most cell plasma membranes studied with freeze-fracture method. Under rising osmotic permeability and water absorption (0.43 +/- 0.05 mkl/(min.cm2)) the IMP distribution did not change. In these conditions, the quantity of fusion sites between granule membranes and the apical membrane increased, and the intercellular spaces in basolateral epithelial region were diluted. A a low water permeability, in addition to usual microtubules, bundles of noncentrosomal microtubules with associated osmiophilic globules were revealed. A comparative analysis has been made of the present evidence and previously obtained data on the frog urinary bladder epithelium.     

Gene cloning and expression of an aquaporin (AQP-h3BL) in the basolateral membrane of water-permeable epithelial cells in osmoregulatory organs of the tree frog

An aquaporin (Hyla AQP-h3BL), consisting of 292 amino acid residues, has been cloned from the urinary bladder of Hyla japonica. In a swelling assay using Xenopus oocytes, AQP-h3BL cRNA-injected oocytes developed a sevenfold and 2.8-fold higher permeability to water and glycerol, respectively, than the water-injected oocytes. This permeability was inhibited by HgCl2. Immunofluorescence revealed that AQP-h3BL is localized in the basolateral plasma membrane of both granular cells in the ventral pelvic and dorsal skins and the secretory cells in the mucous glands. Immunopositive cells were also observed in the basolateral membrane of principal cells in the collecting ducts and in a portion of the late distal tubules in the kidneys, as well as in the principal cells of the urinary bladder. Sequence homology suggests that AQP-h3BL is a homolog to mammalian AQP3. This conclusion is supported by the observed localization of AQP-h3BL to the basolateral membrane in water- and glycerol-permeable epithelial cells. In ventral pelvic skins and urinary bladders, water enters into the cytoplasm through the apical plasma membrane at sites where AQP-h2, sometimes in association with AQP-h3, responds to stimulation by vasotocin; the water exits throughout AQP-h3BL to extracellular spaces. In the mucous glands, on the other hand, water enters throughout this AQP-h3BL and exits through AQP-x5, which is in the apical membrane of secretory cells. Thus, water homeostasis in the frog body is regulated by AQP-h3BL expressed in the basolateral membrane in concert with arginine vasotocin (AVT)-dependent or AVT-independent AQP.     

ADH-induced water permeability: what role for the microtubular network?

1. ADH-induced intramembrane particle aggregates in the apical membrane of the epithelial cells are specifically related to water permeability in the epithelium. 2. Colchicine and nocadozole (both of which bind to tubulin) inhibit ADH-induced osmotic water flow in the amphibian bladder. 3. Microtubules may be involved in the translocation of the aggrephores prior to their insertion into the plasma membrane.     

Water, proton, and urea transport in toad bladder endosomes that contain the vasopressin-sensitive water channel

Vasopressin (VP) increases the water permeability of the toad urinary bladder epithelium by inducing the cycling of vesicles containing water channels to and from the apical membrane of granular cells. In this study, we have measured several functional characteristics of the endosomal vesicles that participate in this biological response to hormonal stimulation. The water, proton, and urea permeabilities of endosomes labeled in the intact bladder with fluorescent fluid-phase markers were measured. The diameter of isolated endosomes labeled with horse-radish peroxidase was 90-120 nm. Osmotic water permeability (Pf) was measured by a stopped-flow fluorescence quenching assay (Shi, L.-B., and A. S. Verkman. 1989. J. Gen. Physiol. 94:1101-1115). The number of endosomes formed when bladders were labeled in the absence of a transepithelial osmotic gradient increased with serosal [VP] (0-50 mU/ml), and endosome Pf was very high and constant (0.08-0.10 cm/s, 18 degrees C). When bladders were labeled in the presence of serosal-to-mucosal osmotic gradient, the number of functional water channels per endosome decreased (at [VP] = 0.5 mU/ml, Pf = 0.09 cm/s, 0 osmotic gradient; Pf = 0.02 cm/s, 180 mosmol gradient). Passive proton permeability was measured from the rate of pH decrease in voltage-clamped endosomes in response to a 1 pH unit gradient (pHin = 7.5, pHout = 6.5). The proton permeability coefficient (PH) was 0.051 cm/s at 18 degrees C in endosomes containing the VP-sensitive water channel; PH was not different from that measured in vesicles not containing water channels. Measurement of urea transport by the fluorescence quenching assay gave a urea reflection coefficient of 0.97 and a permeability coefficient of less than 10(-6) cm/s. These results demonstrate: (a) VP-induced endosomes from toad urinary bladder have extremely high Pf. (b) In states of submaximal bladder Pf, the density of functional water channels in endosomes in constant in the absence of an osmotic gradient, but decreases in the presence of a serosal-to-mucosal gradient, suggesting that the gradient has a direct effect on the efficiency of packaging of water channels into endosomes. (c) The VP-sensitive water channel does not have a high proton permeability. (d) Endosomes that cycle the water channel do not contain urea transporters. These results establish a labeling procedure in which greater than 85% of labeled vesicles from toad urinary bladder are endosomes that contain the VP-sensitive water channel in a functional form.     

Osmotic permeabilities across corneal endothelium and antidiuretic hormone-stimulated toad urinary bladder structures

Osmotic permeabilities of several epithelial structures have been determined with novel optical procedures based on specular microscopy. The osmotic permeabilities of several tissue layers were determined by continuously monitoring the position of the apical tissue borders while an osmotic flow was imposed across those layers. The values found were (in μm/s; mean ± SE): corneal epithelium, 137 ± 30 (n = 5); antidiuretic hormone stimulated toad bladder, 429 ± 64 (n = 6); and corneal endothelium, 711 ± 34 (n = 7). In addition, the osmotically-induced transient change in thickness of the corneal endothelial cells was determined with the help of a computer, and the apparent osmotic permeability measured for the apical membrane was 1420 ± 160 μm/s (n = 5). It is concluded that the osmotic permeability across the endothelial layer is sizably larger than had been previously detected and that osmotic flows across such layer largely traverse the cellular membranes. With osmotic permeability values (per unit of cell membrane area) as large as presently reported, isotonic fluid transport by epithelia can be explained simply on the basis of local osmotic gradients.     

Electron-microscopic and immunohistochemical study of the reorganization of the microtubule system in granular cells of frog urinary bladder after water transport induction]

Structural changes in organization of the microtubule system in granular cells of frog urinary bladder after water transport induction by vasopressin were studied by methods of electron microscopy and immunocytochemistry. It is shown that in steady-state conditions microtubules form a wide network equally distributed in the whole cytoplasm of granular cells. After vasopressin action, the amount of microtubules increases in the apical region of the cytoplasm. A predominant orientation of microtubules, perpendicular to the apical membrane direction, appears. A structural association of microtubules with specific granules and large vacuoles was observed. A supposition is advanced about association of the described microtubule system reorganization with the activation of vectorial intracellular transport occurring after transepithelial water transport induction.     

The role of microtubules and microfilaments in the hydrosmotic response to antidiuretic hormone

To test the effects of colchicine and cytochalasin B on the ADH-induced response, unidirectional and net water fluxes were measured at one or two minutes intervals in frog urinary bladder. The action of these agents on the appearance of intramembrane particles aggregates in the luminal membrane of target cells under oxytocin stimulation and the changes in the tissue ultrastructure induced by cytochalasin B were also studied. It was observed that: the time-course of the response to oxytocin was strongly slowed by colchicine while the washout was not affected; the time-course of the 'on and off' of the response to oxytocin was not modified by cytochalasin B; cytochalasin B pretreatment proportionally reduced unidirectional and net water fluxes measured after glutaraldehyde fixation; the combined action of colchicine and cytochalasin B proportionally reduced the net water flux and the number of intramembrane particles aggregates, observed in freeze-fracture studies; after cytochalasin B action the dilation of intercellular spaces classically observed under oxytocin stimulation is strongly reduced. It is concluded that: microtubules probably play an important role in the water channels plug-in, but not in their removal; microfilaments integrity is necessary for the mechanisms inducing intercellular space dilation and the observed results confirm that water permeability is controlled by the number of permeation units present in the luminal border of granular cells and probably represented by the intramembrane particle aggregates.     

Regulation of membrane permeability by vasopressin; activation of the water permeability pathway in toad urinary bladder by N-ethyl-maleimide

1. Vasopressin induces a rapid increase in water permeability and stimulates net sodium transport in responsive epithelia through the mediation of cAMP. 2. In amphibian urinary bladder, the increase in water permeability is dependent on an intact cytoskeleton and is associated with the exocytotic insertion of tubular vesicles containing particle aggregates (the putative water channels) into the apical membrane of the granular epithelial cells. 3. In the toad bladder, mucosal addition of NEM, 0.1 mM, elicits a slow and irreversible increase in transepithelial water flow, whilst decreasing net sodium transport. 4. The hydrosmotic response to mucosal NEM is inhibited by cellular acidification, by pretreatment with cytoskeleton-disruptive drugs, and by agents that increase cytosolic calcium. 5. Mucosal NEM potentiates the hydrosmotic response to a submaximal, but not a maximal, dose of vasopressin. 6. Mucosal NEM, like vasopressin, induces both vesicle fusion and the appearance of particle aggregates at the granular cell apical surface. 7. NEM, unlike vasopressin, does not increase cellular cAMP content. 8. Mucosal NEM appears to increase transcellular water flow by activating cellular processes normally triggered by vasopressin, at a step beyond cAMP.     

The participation of intracellular membranes in forming highly permeable domains in the plasma membrane of epithelial cells during the vasopressin stimulation of water transport]

Using different electron microscopic techniques, parallel studies of structural alterations in the apical membrane and specific granules of the frog urinary bladder granular cells were made. The results obtained suggest the participation of granule membranes in the formation of highly permeable domains in the apical membranes. After ADH action, the domains with high water permeability are internalized bringing cell membrane retrieval.     

Microfilament-rich cells in the toad bladder epithelium

Summary Basal cells of the bladder epithelium ofBufo marinus have been found heterogenous and consist of microfilament-rich cells (MFR-cell) and undifferentiated cells (Un-cell). The MFR-cell, which represents approximately 20% of the epithelial cell population, lies between the epithelial layer lining the urinary space and the basement membrane; it extends under several epithelial cells by processes of varying widths and lengths which contact, via desmosomes, other MFR-cells, as well as cells in the superficial layer, i.e., granular and mitochondria-rich cells. The cytoplasm of MFR-cell is filled with intermediate filaments arranged in bundles which run parallel to the plane of the epithelium and no dense granules, typical of granular cells, have been detected. Strong immunofluorescence for actin is associated with cells which occupy the same basal position as MFR-cells. Undifferentiated cells have no contact via desmosomes with adjacent cells and their cytoplasm is filled with free ribosomes; they lack bundles of intermediate filaments and posses no specialized organelles.After a 4-hr pulse of³H-thymidine, 1.5% of epithelial cells incorporate thymidine into nuclear DNA, out of which 3/4 are basally 1/4 are apically located. Identification of cell types by electron microscopy reveals that 10% of undifferentiated basal cells are labeled, whereas less than 0.1% of granular cells and no MFR-cells incorporate³H-thymidine into DNA. When dissociated from the epithelium and separated by isopycnic centrifugation, MFR-cells possess a mean buoyant density of approximately 1.025, cosediment with mitochondria-rich cells and exhibit a strong immunofluorescence for actin. The function of MFR-cells remains unknown; however, they may play a role in cell coupling and responses to hormonal and physical factors.