The Mechanism of Isotonic Water Transport

The mechanism by which active solute transport causes water transport in isotonic proportions across epithelial membranes has been investigated. The principle of the experiments was to measure the osmolarity of the transported fluid when the osmolarity of the bathing solution was varied over an eightfold range by varying the NaCl concentration or by adding impermeant non-electrolytes. An in vitro preparation of rabbit gall bladder was suspended in moist oxygen without an outer bathing solution, and the pure transported fluid was collected as it dripped off the serosal surface. Under all conditions the transported fluid was found to approximate an NaCl solution isotonic to whatever bathing solution used. This finding means that the mechanism of isotonic water transport in the gall bladder is neither the double membrane effect nor co-diffusion but rather local osmosis. In other words, active NaCl transport maintains a locally high concentration of solute in some restricted space in the vicinity of the cell membrane, and water follows NaCl in response to this local osmotic gradient. An equation has been derived enabling one to calculate whether the passive water permeability of an organ is high enough to account for complete osmotic equilibration of actively transported solute. By application of this equation, water transport associated with active NaCl transport in the gall bladder cannot go through the channels for water flow under passive conditions, since these channels are grossly too impermeable. Furthermore, solute-linked water transport fails to produce the streaming potentials expected for water flow through these passive channels. Hence solute-linked water transport does not occur in the passive channels but instead involves special structures in the cell membrane, which remain to be identified.     

Transport of Salt and Water in Rabbit and Guinea Pig Gall Bladder

A simple and reproducible method has been developed for following fluid transport by an in vitro preparation of mammalian gall bladder, based upon weighing the organ at 5 minute intervals. Both guinea pig and rabbit gall bladders transport NaCl and water in isotonic proportions from lumen to serosa. In the rabbit bicarbonate stimulates transport, but there is no need for exogenous glucose. The transport rate is not affected by removal of potassium from the bathing solutions. Albumin causes a transient weight loss from the gall bladder wall, apparently by making the serosal smooth muscle fibers contract. Active NaCl transport can carry water against osmotic gradients of up to two atmospheres. Under passive conditions water may also move against its activity gradient in the presence of a permeating solute. The significance of water movement against osmotic gradients during active solute transport is discussed.     

Effects of electrical gradients on volume flows across gall bladder epithelium

A volumetric method has been developed which permits continuous registration of volume flows across epithelial tissues. The method was applied to volume flow measurements across rabbit gall bladder epithelium. The rate of fluid reabsorption measured in this way was twice as high as previously observed in sac preparations of the gall bladder. This is probably due to better aeration and stirring of the mucosal solution. It was demonstrated that electrical gradients across the gall bladder induced volume flows towards the negative electrode. In non-transporting bladders volume flows were linearly related with current between 300 and 900 μA in both directions. However, volume flow rates were three times higher from mucosa to serosa than in the opposite direction. From the magnitude of polarization potentials, observed after switching off the current, the conclusion was reached that all of the current-induced volume flow is an osmotic flow due to salt polarization in the unstirred layers of the tissue. By implication, so-called streaming potentials observed during osmotic flows reflect solely polarization effects. In actively transporting gall bladders a 200 μA current increased or decreased the flow rate twice as much as expected from polarization effects alone. Therefore passage of current interfered directly with the active transport mechanism of gall bladder epithelium.     

Scanning electron microscopy: Identification of mast cells by indirect cathodoluminescence

Summary The epithelial tissues of the rabbit gall bladder reacted for acid mucosaccharides were studied with the electron microscope. A series of acid mucosaccharide-containing ultrastructures of the gall bladder epithelium were observed in specimens treated with dialyzed iron, colloidal thorium and ruthenium red. In the epithelium stained with dialyzed iron, reactive ultrastructures are not only extra- but intracellular; the surface coat of the plasma membrane, pinocytotic vesicles, granules of secretion and certain elements of the Golgi apparatus. In the epithelial tissues stained by colloidal thorium or ruthenium red, the surface coat of the plasma membrane is the only ultrastructure which is reacted positively for the acid mucosaccharide stains. The present images of ultrastructural elements containing acid mucosaccharides are taken to indicate a multiple function of the substances in rabbit gall bladder epithelium and are well correlated with the results of previous light and electron microscopic studies on the gall bladder epithelium of various vertebrate species.     

FLUID TRANSPORT IN THE RABBIT GALLBLADDER : A Combined Physiological and Electron Microscopic Study

The fine structure of the rabbit gallbladder has been studied in specimens whose functional state was undetermined, which were fixed either in situ or directly after removal from the animal; in specimens whose rate of fluid absorption was determined, either in vivo or in vitro, immediately prior to fixation; and in specimens from bladders whose absorptive function was experimentally altered in vitro. Considerable variation was found in the width of the epithelial intercellular spaces in the bladders whose functional state was undefined. In bladders known to be transporting fluid, either in vivo or in vitro, the intercellular spaces were always distended, as were the subepithelial capillaries. This distension was greatest in bladders which had been functioning in vitro. When either Na⁺ or Cl^- was omitted from the bathing media, there was no fluid transport across the wall of the gallbladder studied in vitro. The epithelial intercellular spaces of biopsies taken from several bladders under these conditions were of approximately 200 A width except for minor distension at the crests of mucosal folds. The addition of the missing ion rapidly led to the reestablishment of fluid transport and the distension of the intercellular spaces throughout most of the epithelium of these bladders. Studies of sodium localization (by fixation with a pyroantimonate-OsO₄ mixture) showed high concentrations of this ion in the distended intercellular spaces. Histochemical studies of ATPase activity showed that this enzyme was localized along the lateral plasma membrane of the epithelial cells. The analogy is drawn between the structure of the gallbladder mucosa and a serial membrane model proposed by Curran to account for coupled solute-solvent transport across epithelia. It is concluded that the intercellular compartment fulfills the conditions for the middle compartment of the Curran model and that active transport of solute across the lateral plasma membrane into the intercellular space may be responsible for fluid absorption by the gall bladder.     

Lack of correlation between transepithelial transport capacity and paracellular pathway ultrastructure in Alcian blue-treated rabbit gallbladders

The effects of mucosal application of 1 mg% Alcian blue (a trivalent cationic phthalocyanine dye) on functional and ultrastructural parameters of the isolated rabbit gallbladder have been studied. Apart from minor changes in the shape of the group of central microvilli observed in thin-section electron microscopy and scanning electron microscopy, the major ultrastructural change induced by Alcian blue was an almost complete collapse of intercellular spaces in the region above the tight junctions up to the bases of the marginal microvilli as revealed by thin-section electron microscopy. Freeze-fracture electron microscopy demonstrated a complete disappearance of intramembrane particles of neighboring cell membranes corresponding to the region of interspace collapse. Transepithelial electrical resistance (RT) increased from 44.5 to 58.7 ohm . cm2 upon treatment with Alcian blue. This increase could be well accounted for by the observed structural changes in the paracellular pathway if this pathway determines the low resistance of the rabbit gallbladder epithelium. Despite the increase in RT, net mucosa-to-serosa fluid transport and the spontaneous mucosa- positive potential difference of 3 mV were unaltered by Alcian blue treatment, supporting the hypothesis that the transepithelial transport mechanism per se is electroneutral. A calculation of the maximal paracellular mucosa-to-serosa waterflow in response to a lateral intercellular space hypertonicity of 20 mosM demonstrates that in the Alcian blue-treated gallbladder the resulting figure is about three orders of magnitude too low to keep up with the unaltered spontaneous transepithelial net fluid transport. It is therefore concluded that the tight junction pathway in rabbit gallbladders does not serve as a route for net fluid transport.     

An estimate of the salt concentration in the lateral intercellular spaces of rabbit gall-bladder during maximal fluid transport

Summary The ability of the gall-bladder to transport water between identical bathing solutions depends on active NaCl transport, which is thought to maintain the salt concentration in the lateral intercellular spaces above bathing solution levels and thus to create a local osmotic gradient. The mean value of this gradient has been estimated by an electrical procedure, based on measuring the small diffusion potential resulting from this gradient and from the preferential cation permeability of the gall-bladder. The electrical potential difference (p.d.) in maximally transporting rabbit gall-bladders is 1.4 mV, mucosal-solution positive to serosal solution. This p. d. is reversibly abolished or greatly reduced by six procedures which abolish or greatly reduce fluid transport (low temperature, replacement of Cl^– by SO ₄ ^-- , replacement of Cl^– and HCO ₃ ^– by SO ₄ ^-- , replacement of Na⁺ by choline, removal of HCO ₃ ^– , and metabolic poisoning). The p. d. is increased by symmetrical partial replacement of NaCl by sucrose, which is expected to increase the salt concentration gradient between the lateral spaces and the bathing solutions. Since the transport mechanism of the gall-bladder is a neutral NaCl pump that cannot produce a p. d. directly, it is concluded that the observed p. d. is the expected diffusion potential. From this diffusion potential and from the measured value of a diffusion potential resulting from a known NaCl concentration gradient, the mean concentration of NaCl in the lateral spaces is calculated to be of the order of 10mm above the bathing solution value. Comparison of the external osmotic gradient required to stop water flow with the p. d. recorded under this condition of zero flow supports the validity of interpreting the p.d. in this fashion as a measure of the excess local salt concentration.     

Standing-gradient osmotic flow. A mechanism for coupling of water and solute transport in epithelia

At the ultrastructural level, epithelia performing solute-linked water transport possess long, narrow channels open at one end and closed at the other, which may constitute the fluid transport route (e.g., lateral intercellular spaces, basal infoldings, intracellular canaliculi, and brush-border microvilli). Active solute transport into such folded structures would establish standing osmotic gradients, causing a progressive approach to osmotic equilibrium along the channel's length. The behavior of a simple standing-gradient flow system has therefore been analyzed mathematically because of its potential physiological significance. The osmolarity of the fluid emerging from the channel's open end depends upon five parameters: channel length, radius, and water permeability, and solute transport rate and diffusion coefficient. For ranges of values of these parameters encountered experimentally in epithelia, the emergent osmolarity is found by calculation to range from isotonic to a few times isotonic; i.e., the range encountered in epithelial absorbates and secretions. The transported fluid becomes more isotonic as channel radius or solute diffusion coefficient is decreased, or as channel length or water permeability is increased. Given appropriate parameters, a standing-gradient system can yield hypertonic fluids whose osmolarities are virtually independent of transport rate over a wide range, as in distal tubule and avian salt gland. The results suggest that water-to-solute coupling in epithelia is due to the ultrastructural geometry of the transport route.     

Morphology,fine structure,and functional aspects of the labial gland reservoirs of the subterranean termite Reticulitermes santonensis de Feytaud (Isoptera: Rhinotermitidae)

The morphology and fine structure of the labial gland reservoirs in the subterranean termite Reticulitermes santonensis (Isoptera: Rhinotermitidae) was studied by light and transmission electron microscopy. The reservoir wall consists of a single epithelial cell layer and a cuticular intima. The reservoir ducts are formed by a flat epithelial matrix with cuticular ridges lining the duct lumen. Measurements of the ionic concentrations of reservoir fluids and haemolymph show that the osmolality of reservoir fluid ranges from 7 to 28 mosmol kg⁻¹; the haemolymph osmotic pressure was 201 ± 31 mosmol kg⁻. The reservoir lumen is effectively separated from the haemolymph compartment; a net water flow through the reservoir wall could not be induced in physiological experiments. Moreover, typical epithelial structures associated with a fluid transport against an osmotic gradient are lacking. Thus, our fine structural and physiological data support the view that a water transfer from the haemolymph through the reservoir wall into the reservoir lumen does not occur.     

Asymmetry of canine tracheal epithelium: osmotically induced changes

The symmetry of osmotic conductivity of the canine tracheal epithelial cells was examined in vitro. When an osmotic load of 100 mosM sucrose was added to the serosal bathing solution, no change in the transepithelial potential difference was observed in 15 tissue preparations. In contrast, when the same osmotic load was added to the mucosal bathing solution, there was a rapid decrease in the transepithelial potential difference of 3.9 +/- 0.5 mV (n = 23); ouabain (10(-4) M) eliminated this change. Tissues that had been exposed to the osmotic load added to either the mucosal or serosal side were compared with the control using light and electron microscopy. When the osmotic load was added to the mucosal fluid, there was no change in the nuclear-to-cytoplasmic area ratio of the cell types examined. However, when the same osmotic load was added to the serosal fluid, a marked increase in the nuclear-to-cytoplasmic area ratio of the ciliated cells was observed. This finding indicated cell shrinkage. Dilution potentials measured by substituting NaCl with mannitol also showed asymmetry. The morphological features are probably caused by differences in the osmotic conductivity (Lp) of the basolateral and apical cell membranes, with the Lp of the apical membrane being less than that of the basolateral membrane. The basis for osmotically induced potentials remained undetermined.     

ELECTRON MICROSCOPY OF ABSORPTION OF TRACER MATERIALS BY TOAD URINARY BLADDER EPITHELIUM

The absorption of Thorotrast and saccharated iron oxide by the epithelium of the toad urinary bladder was studied by electron microscopy. Whether the toads were hydrated, dehydrated, or given Pitressin, no significant differences in transport of colloidal particles by epithelial cells were observed. This implies that these physiological factors had little effect on the transport of the tracer particles. Tracer particles were encountered in three types of epithelial cells which line the bladder lumen, but most frequently in the mitochondria-rich cells. Tracer materials were incorporated into the cytoplasm of epithelial cells after being adsorbed to the coating layer covering the luminal surface of the cells. In the intermediate stage (1 to 3 hours after introducing tracer) particles were present in small vesicles, tubules, and multivesicular bodies. In the later stages (up to 65 hours), the particles were more commonly seen to be densely packed within large membrane-bounded bodies which were often found near the Golgi region. These large bodies probably were formed by the fusion of small vesicles. Irrespective of the stages of absorption, no particles were found in the intercellular spaces or in the submucosa. Particles apparently did not penetrate the intercellular spaces of the epithelium beyond the level of the tight junction.     

Energetics of coupled active transport of sodium and chloride

A Clark electrode was used to measure oxygen consumption by the gall bladder, in which there is a direct and one-to-one linkage between active Na and active Cl transport. O2 uptake was reversibly depressed when Cl in the mucosal bathing solution was replaced by a poorly transported anion, such as sulfate. This effect of Cl was abolished by ouabain or in Na-free solutions. When the anion was chloride, treatment with ouabain or replacement of Na by a poorly transported cation depressed QO2 more than did replacement of Cl. However, ouabain or removal of Na also depressed QO2 in Na2SO4 solutions, in which salt transport is minimal. It is concluded that oxygen uptake in the gall bladder consists of three fractions: 9% requires both Na and Cl, is inhibited by ouabain, and is linked to the NaCl pump; 36% requires Na but not Cl, is inhibited by ouabain, and possibly is linked to the cellular K uptake mechanism; and 55% represents basal uptake. If the extra oxygen uptake observed during transport supplies all the energy for transport, then 25 Na + 25 Cl ions are transported actively per O2 consumed; i.e., twice as many ions as in epithelia which transport only Na actively. This extra uptake is more than sufficient to supply the energy for overcoming internal membrane resistance under the experimental conditions used.     

Mucosal surface morphology of the toad urinary bladder. Scanning electron microscope study of the natriferic and hydro-osmotic response to vasopressin

The mucosal cell surface of the toad urinary bladder was examined by scanning electron microscopy, and changes in the structure of the surface of the granular cell were correlated with specific physiological responses to vasopressin. Survey views of the mucosal surface demonstrated that there was no consistent repeating anatomical relationship between the granular cell and the mitochondria-rich cell that would support the concept of cooperativeness in the response to vasopressin. During base-line states of Na+-transport and water flux, the microvilli on the mucosal surface of the granular cell are arranged in a ridge-like network with occasional individual projections. When water flux is increased by exposing the tissue to vasopressin, in the presence of an osmotic gradient across the tissue the microvilli on the granular cell lose the ridge structure and appear, predominantly, as individual projection. Variability-of this appearance points out the necessity of examining large areas and many samples before the significance of any morphological change can be assessed. Blocking the simultaneously occurring natriferic response of the toad urinary bladder with 10(-2)M ouabain does not prevent these changes in the microvilli. When the hydro-osmotic response is blocked by eliminating the osmotic gradient, the granular cell shows no consistent change in mucosal surface morphology even when fixed at the height of the natriferic response. The mitochondria-rich and mucous cells did not show any change in morphology throughout these studies. We conclude that the changes in the mucosal surface morphology of the toad bladder seen after exposure to vasopressin are a result of the increased water flux that occurs when an osmotic gradient exists across the tissue, and are not related to the natriferic response or any specific alteration in the membrane properties.     

Ultrastructure of intestinal and gall-bladder epithelium in the teleost Gasterosteus aculeatus L., as related to their osmoregulatory function

Summary Intestinal and gall-bladder epithelial cells in sticklebacks have been examined in ultrathin sections and freeze-etch replicas. Enterocytes throughout the intestine appear to have a well-developed basal labyrinth similar to that of renal tubular cells, consisting of baso-lateral infoldings closely associated with numerous mitochondria. The lumen inside these intracellular membranes is continuous with the intercellular space via pores. Such a membrane system is also present in the epithelial cells lining the gall bladder, distinguishing them from gall-bladder cells of higher vertebrates. Morphometric analysis indicates that the basal labyrinth of enterocytes in the posterior part of the intestine increases markedly in both sexually mature males and androgen-treated females. This does not occur in the anterior part or gall bladder. In sticklebacks, androgens cause reduced urine excretion and enhanced fluid release via the anus. We conclude that the cells lining the intestine and gall bladder possess an extensive basal labyrinth that may function as a backward channel system, enabling fluid to be produced in the intestine of fish. The androgen-induced increase in the extent of the basal labyrinth in the posterior part of the intestine may be related to the enhanced rate of intestinal fluid excretion observed in sexually mature male sticklebacks.     

The fine structure of the gall bladder epithelium of the sheep

Summary The electron microscopy of the gall bladder epithelium in the sheep shows that the cells are secretory. They have an extensive Golgi apparatus and sparse endoplasmic reticulum with secretory droplets localised in the apical region and have been referred to the group known as mucoid cells. The intercellular spaces which are elaborately developed in those species whose gall bladder is primarily absorptive are poorly developed. Pinocytosis is not a prominent feature. It is believed that the features noted are correlated with the reported absence of absorption in the ungulate gall bladder.     

Type I cell-like morphology in tight alveolar epithelial monolayers

The pulmonary alveolar epithelium separates air spaces from a fluid-filled interstitium and might be expected to exhibit high resistance to fluid and solute movement. Previous studies of alveolar epithelial barrier properties have been limited due to the complex anatomy of adult mammalian lung. In this study, we characterized a model of isolated alveolar epithelium with respect to barrier transport properties and cell morphology. Alveolar epithelial cells were isolated from rat lungs and grown as monolayers on tissue culture-treated Nuclepore filters. On Days 2-6 in primary culture, monolayers were analyzed for transepithelial resistance (Rt) and processed for electron microscopy. Mean cell surface area and arithmetic mean thickness (AMT) were determined using morphometric techniques. By Day 5, alveolar epithelial cells in vitro exhibited morphologic characteristics of type I alveolar pneumocytes, with thin cytoplasmic extensions and protruding nuclei. Morphometric data demonstrated that alveolar pneumocytes in vitro develop increased surface area and decreased cytoplasmic AMT similar to young type I cells in vivo. Concurrent with the appearance of type I cell-like morphology, monolayers exhibited high Rt (greater than 1000 omega.cm2), consistent with the development of tight barrier properties. These monolayers of isolated alveolar epithelial cells may reflect the physiological and morphological properties of the alveolar epithelium in vivo.     

Histological and histochemical studies of the gall bladder of the graylag goose (Anser anser)

ABSTRACT

We investigated the histological structure of the graylag goose (Anser anser) gall bladder. Sections of the gall bladder were stained with hematoxylin and eosin (H & E), Alcian blue (pH 2.5) for acid mucopolysaccharides, Gomori’s method for reticular fibers, Masson’s trichrome, periodic acid-Schiff (PAS) and Verhoeff’s elastin stain. The goose gall bladder was composed of a tunica mucosa, tunica muscularis and tunica adventitia or tunica serosa. The tunica mucosa formed regularly distributed simple isometric folds plus larger, less numerous, branched folds. The luminal surface was lined by tall columnar epithelial cells that stained for both acid and neutral mucopolysaccharides. The epithelial cells formed a discontinuous striated border of interdigitating microvilli on the luminal surface. Neither a lamina muscularis nor goblet cells were observed in the tunica mucosa. Unusual findings included branched mucosal folds, discontinuous microvilli and absence of an outer longitudinal layer in the tunica muscularis. No marked sex-associated differences were found. The general histochemical and histological structures of the graylag goose gall bladder are similar to those of birds such as chukar partridge and quail, but with some unique elements that may reflect differences in organ function.     

Lung edema clearance: 20 years of progress: invited review: role of aquaporin water channels in fluid transport in lung and airways.

Water transport across epithelial and endothelial barriers in bronchopulmonary tissues occurs during airway hydration, alveolar fluid transport, and submucosal gland secretion. Many of the tissues involved in these processes are highly water permeable and express aquaporin (AQP) water channels. AQP1 is expressed in microvascular endothelia throughout the lung and airways, AQP3 in epithelia in large airways, AQP4 in epithelia throughout the airways, and AQP5 in type I alveolar epithelial cells and submucosal gland acinar cells. The expression of some of these AQPs increases near the time of birth and is regulated by growth factors, inflammation, and osmotic stress. Transgenic mouse models of AQP deletion have provided information about their physiological role. In lung, AQP1 and AQP5 provide the principal route for osmotically driven water transport; however, alveolar fluid clearance in the neonatal and adult lung is not affected by AQP deletion nor is lung CO(2) transport or fluid accumulation in experimental models of lung injury. In the airways, AQP3 and AQP4 facilitate water transport; however, airway hydration, regulation of the airway surface liquid layer, and isosmolar fluid absorption are not impaired by AQP deletion. In contrast to these negative findings, AQP5 deletion in submucosal glands in upper airways reduced fluid secretion and increased protein content by greater than twofold. Thus, although AQPs play a major physiological role outside of the airways and lung, AQPs appear to be important mainly in airway submucosal gland function. The substantially slower rates of fluid transport in airways, pleura, and lung compared with renal and some secretory epithelia may account for the apparent lack of functional significance of AQPs at these sites. However, the possibility remains that AQPs may play a role in lung physiology under conditions of stress and/or injury not yet tested or in functions unrelated to transepithelial fluid transport.     

Standing-gradient osmotic flow Examination of its validity using an analytical method

The solutions to the non-linear differential equations governing solute-solvent coupling in the intercellular spaces of epithelial layers have been obtained by using an analytical method, rather than the usual numerical ones. When the present series solution includes second-order correction terms, the concentration and velocity profiles obtained by the analytical method agree very well with those coming from numerical solutions. This method has further allowed us to examine the standing-gradient hypothesis when applied to the backwards fluid transport system of the corneal endothelium. With the information presently available for the relevant parameters (osmotic permeability, rate of transport, radius and length of the spaces, and location of the pumping sites), near-isotonicity of the transported fluid would not be explained by the standing-gradient model.     

Pathways for movement of ions and water across toad urinary bladder

Summary Mucosal hypertonicity, produced by the addition of NaCl, KCl, mannitol, urea, sucrose or raffinose, reduced the electrical resistance of toad urinary bladder and induced bullous deformations (blisters) of the most apical junctions of the mucosal epithelium: the smaller solutes were most effective in eliciting both phenomena. Study of the effect of addition and subsequent removal of mannitol from the mucosal medium indicated that both the electrical and morphologic changes are reversible and follow the same time course. Mucosal hypertonicity induced comparable changes in the tissue in the presence or absence of inhibition of active sodium transport by replacement of sodium by choline, or by addition of ouabain or amiloride. Dilution of the tonicity of the serosal medium similarly reduced the tissue resistance and induced blisters within the epithelium, demonstrating that the osmotic gradient, rather than the mucosal hypertonicity itself is the cause of the osmotically-induced resistance change. The data indicate, therefore, that the osmotic gradient reduces the electrical resistance of the tissue primarily by deforming the apical junctions.The simplest interpretation of the data is that the apical tight junctions are considerably more permeable to water and small solutes than had previously been thought. Addition of solute to the mucosal medium leads to the diffusion of solute into the junctions: the subsequent transfer of water from the lateral intercellular spaces and/or the adjacent cellular cytoplasm, deforms these structures and reduces the resistance to the passage of ions across the tissue. The results suggest that the apical junctions constitute the rate-limiting permeability barrier of the putative parallel shunt pathway across toad bladder.