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.     

The effect of osmotically induced water flows on the permeability and ultrastructure of the rabbit gallbladder

Summary The purpose of these experiments was to determine the effect of osmotic gradients on the permeability of the rabbit gallbladder. Increasing the tonicity of the mucosal solution reduced the permeability of the gallbladder to both ions and nonelectrolytes, whereas there was no significant effect when the serosal solution was made hypertonic. These results cannot be explained by solvent/solute interactions in either the epithelial membranes or the unstirred layers. Associated with the changes in permeability was the appearance of the transport number effect and current induced resistance changes. Morphological studies of the gallbladder under these conditions showed that the extracellular spaces of the epithelium and the rest of the wall dilate in the presence of osmotic flow to the serosa, but that the spaces collapse when the flow is in the opposite direction. Reconstruction of the permeability changes from the dimensions of the tissue show that all the physiological phenomena are accounted for by the changes in morphology, the dominant effect being in the lateral intercellular spaces. These results suggest that the lateral spaces are a common pathway shared by all solutes crossing the epithelium, and that diffusion along these spaces becomes rate limiting as the spaces collapse.     

Structure and permeability of goblet cell tight junctions in rat small intestine

Summary Two major cell types, goblet and absorptive cells, dominate the epithelial lining of small intestinal villi. We used freezefracture replicas of rat ileal mucosa to examine the possibility that tight junction structure, known to relate to transepithelial resistance, might vary with cell type. Tight junctions between absorptive cells were uniform in structure while those associated with villus goblet cells displayed structural variability. In 23% of villus goblet cell tight junctions the strand count was less than 4 and in 30% the depth was less than 200 nm. In contrast, only 4% of absorptive cell tight junctions had less than 4 strands and only 9% had depth measurements less than 200 nm. Other structural features commonly associated with villus goblet cell tight junctions but less commonly with absorptive cell tight junctions were: deficient strand cross-linking, free-ending abluminal strands, and highly fragmented strands. Bothin vivo ileal segments and everted loops were exposed to ionic lanthanum. Dense lanthanum precipitates in tight junctions and paracellular spaces were restricted to a subpopulation of villus goblet cells and were not found between villus absorptive cells. After exposure of prefixed ileal loops to lanthanum for 1 hour, faint precipitates of lanthanum were found in 14% of tight junctions and paracellular spaces between absorptive cells compared to 42% of tight junctions and paracellular spaces adjacent to villus goblet cells. When tested in Ussing chambers, the methods used for lanthanum exposure did not lower transepithelial resistance. Everted loops exposed to ionic barium and examined by light microscopy showed dense barium precipitates in the junctional zone and region of the paracellular space of villus goblet cells but not in these regions between absorptive cells. However, the macromolecular tracers, microperoxidase, cytochromec and horseradish peroxidase, were excluded from both villus goblet cell and absorptive cell paracellular spaces inin vivo segments. These findings suggest that a subpopulation of villus goblet cells may serve as focal sites of high ionic permeability and contribute to the relatively low resistance to ionic flow which characterizes the small intestinal epithelium.     

Fluid transport and the dimensions of cells and interspaces of living Necturus gallbladder

The volume of the cells and lateral intercellular spaces were measured in living Necturus gallbladder epithelium. Under control conditions, the volume of the lateral spaces was 9% of the cell volume. Replacement of mucosal NaCl by sucrose or tetramethylammonium chloride (TMACl) caused intercellular spaces to collapse. During mucosal NaCl replacement, cell volume decreased to 79% of its control value. When NaCl was reintroduced into the mucosal bath, the intercellular spaces reopened and the cells returned to control volume. The NaCl active transport rate, calculated from the rate of cell volume decrease, was 266 pM/cm2.s, close to the observed rate of transepithelial salt transport. It was calculated from the decrease in cell volume that all of the intracellular NaCl was transported out of the cell during removal of mucosal NaCl. The flux of salt across the apical membrane, calculated from the rate of cell volume increase upon reintroducing mucosal NaCl, was 209 pM/cm2.s, in good agreement with estimates by other methods. The electrical resistance of the tight junctions was estimated to be 83.9% of the total tissue resistance in control conditions, suggesting that the lateral intercellular spaces normally offer only a small resistance to electrolyte movement.     

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.     

Effects of raised osmolarity on canine tracheal epithelial ion transport function

Evaporation of water from upper airway surfaces increases surface liquid osmolarity. We studied the effects of raised osmolarity of the solution bathing the luminal surface of excised canine tracheal epithelium. Osmolarity was increased by adding NaCl or mannitol. NaCl addition induced a concentration-dependent fall in short-circuit current and a rise in transepithelial conductance (-33% and +14% per 100 mosM, respectively). Unidirectional isotopic fluxes of 22Na, 36Cl, and [14C]mannitol were measured in short-circuited tissues in the base-line state and after addition of NaCl or mannitol to an isotonic mucosal solution. NaCl addition (75 mM) caused a 50% increase in conductance (G) and a parallel increase in [14C]mannitol permeability (Pmann), indicating an increase in paracellular permeability. Net Cl- secretion was reduced 50%, and net Na+ absorption was unchanged despite an increased chemical gradient for absorption, indicating an inhibition of active ion transport. Mannitol addition (150 mM) abolished net Na+ absorption but did not increase G or Pmann or change net Cl- secretion. These results suggest that responses to increased tracheal surface liquid osmolarity during spontaneous breathing may occur in both the cellular (inhibition of active Na+ and Cl- transport) and paracellular (increased [14C]mannitol permeability) compartments of the mucosa.     

Volume flows across gallbladder epithelium induced by small hydrostatic and osmotic gradients

Summary The hydraulic conductivity of rabbit gallbladder epithelium has been studied using a continuous volumetric method based on capacitance measurements. The time resolution for measuring osmotic flows is in the range of seconds. Volume flows have been induced by osmotic gradients between 0 and 100 mosmol. In this range the flow-force relation is linear and theP _f value is 9.3×10^–3 cm/sec. After correction for solute polarization effects, theP _f value amounts to 0.05 cm/sec. The observed flow is constant between 5 sec up to 20 min after a sudden increase in the osmolarity of the mucosal solution. The wet weight of the gallbladder tissue decreases by 22% and increases by 30% during osmotic flows from serosa to mucosa and from mucosa to serosa, respectively. Volume flows induced by hydrostatic pressure gradients on the mucosal surface are linearly related to the driving forces between 0 and 40 mbar. TheP _f value is 0.15 cm/sec. The volume flows are constant between 2 sec and 15 min after pressure application. The flow-force relation for pressure gradients on the serosal surface is markedly nonlinear for gradients greater than 5 mbar. Below 5 mbar theP _f value is 4.5 cm/sec. From electrical measurements, e.g., resistance and streaming potentials, and from flux studies with inulin and polyethylene glycol 4000, it is concluded that hydrostatic and osmotic gradients are not comparable when they are applied to gallbladder epithelium. They induce volume flows across different pathways, e.g., osmosis predominantly across the cellular route and pressure filtration predominantly across paracellular routes.     

PG-mediated closure of paracellular pathway and not restitution is the primary determinant of barrier recovery in acutely injured porcine ileum

Small bowel epithelium is at the frontline of intestinal barrier function. Restitution is considered to be the major determinant of epithelial repair, because function recovers in parallel with restitution after acute injury. As such, studies of intact mucosa have largely been replaced by migration assays of cultured epithelia. These latter studies fail to account for the simultaneous roles played by villous contraction and paracellular permeability in recovery of barrier function. NSAIDs result in increased intestinal permeability and disease exacerbation in patients with inflammatory bowel disease (IBD). Thus we examined the reparative attributes of endogenous PGs after injury of ileal mucosa by deoxycholate (6 mM) in Ussing chambers. Recovery of transepithelial electrical resistance (TER) from 20-40 Omega.cm2 was abolished by indomethacin (Indo), whereas restitution of 40-100% of the villous surface was unaffected despite concurrent arrest of villous contraction. In the presence of PG, resident crypt and migrating epithelial cells were tightly apposed. In tissues treated with Indo, crypt epithelial cells had dilated intercellular spaces that were accentuated in the migrating epithelium. TER was fully rescued from the effects of Indo by osmotic-driven collapse of the paracellular space, and PG-mediated recovery was significantly impaired by blockade of Cl- secretion. These studies are the first to clearly distinguish the relative contribution of paracellular resistance vs. restitution to acute recovery of epithelial barrier function. Restitution was ineffective in the absence of PG-mediated paracellular space closure. Failure of PG-mediated repair mechanisms may underlie barrier failure resulting from NSAID use in patients with underlying enteropathy.     

The maximum and minimum water content and cell volume of human erythrocytes in vitro

The maximum and minimum water contents of human erythrocytes were measured after exposure to various osmotic pressures. Within a range of osmolarities, at which no haemolysis occurred, the water content reached its maximum, 78.1%, at 180 mosM and its minimum, 54.8%, at 800 mosM. Simultaneously, the mean cell volume increased to 98.5 microns 3 at 180 mosM and decreased to 77.2 microns 3 at 800 mosM.     

Relative humidity affects the intercellular spaces and cell contacts of the kidney epithelium of the terrestrial snail Helix aspersa müller

The effects of relative humidity on hemolymph osmolarity and on kidney ultrastructure are explored in Helix aspersa. The snails are active at 95% relative humidity and less active at 50% relative humidity. The hemolymph osmotic pressure increases with the decrease of relative humidity. Pericardial fluid and hemolymph collected from the heart contain similar amounts of total proteins, and both fluids display hemocyanin molecules in negatively stained preparations. When the snails are kept in an atmosphere of 95% relative humidity, numerous wide intercellular spaces are observed in the single-layered-kidney epithelium. The spaces are almost absent when the snails are kept at 50% relative humidity. It is suggested that prourine is formed through a paracellular junctional pathway across the single-layered kidney epithelium, and that the pericardial cavity is not the site of prourine formation. The septate junctions joining the kidney epithelial cells form a continuous belt of intimate contact in the paracellular pathway of prourine. Long septate junctions with many septa are present in the kidneys of snails from the atmosphere of 50% relative humidity, whereas short septate junctions with fewer septa are found in the kidneys of snails from the atmosphere of 95% relative humidity. It is possible that the longer septate junctions with many septa reduce prourine formation across the kidney sac epithelium.     

Study of Sugar Binding to the Sucrose-specific ScrY Channel of Enteric Bacteria Using Current Noise Analysis

CACO-2 BBE was used to determine the response of a gastrointestinal epithelium to tumor necrosis factor-α (TNF). Incubation of CACO-2 BBE with TNF did not produce any effect on transepithelial resistance (TER) within the first 6 hr but resulted in a 40–50% reduction in TER and a 30% decrease in I _sc (short circuit current) relative to time-matched control at 24 hr. The decrease in TER was sustained up to 1 week following treatment with TNF and was not associated with a significant increase in the transepithelial flux of [¹⁴C]-d-mannitol or the penetration of ruthenium red into the lateral intercellular space. Dilution potential and transepithelial ²²Na⁺ flux studies demonstrated that TNF-treatment of CACO-2 BBE cell sheets increased the paracellular permeability of the epithelium to Na⁺ and Cl⁻. The increased transepithelial permeability did not associate with an increase in the incidence of apoptosis. However, there was a TNF-dependent increase in [³H]-thymidine labeling that was not accompanied by a change in DNA content of the cell sheet. The increase in transepithelial permeability was concluded to be across the tight junction because: (i) 1 mm apical amiloride reduced the basolateral to apical flux of ²²Na⁺, and (ii) dilution potential studies revealed a bidirectionally increased permeability to both Na⁺ and Cl⁻. These data suggest that the increase in transepithelial permeability across TNF-treated CACO-2 BBE cell sheets arises from an alteration in the charge selectivity of the paracellular conductive pathway that is not accompanied by a change in its size selectivity. Received: 4 March 1997/Revised: 3 November 1997     

PI3K signaling is required for prostaglandin-induced mucosal recovery in ischemia-injured porcine ileum

We have previously shown that PGE(2) and PGI(2) induce recovery of transepithelial resistance (TER) in ischemia-injured porcine ileal mucosa, associated with initial increases in Cl(-) secretion. We believe that the latter generates an osmotic gradient that stimulates resealing of tight junctions. Because of evidence implicating phosphatidylinositol 3-kinase (PI3K) in regulating tight junction assembly, we postulated that this signaling pathway is involved in PG-induced mucosal recovery. Porcine ileum was subjected to 45 min of ischemia, after which TER was monitored for a 180-min recovery period. Endogenous PG production was inhibited with indomethacin (5 microM). PGE(2) (1 microM) and PGI(2) (1 microM) stimulated recovery of TER, which was inhibited by serosal application of the osmotic agent urea (300 mosmol/kgH(2)O). The PI3K inhibitor wortmannin (10 nM) blocked recovery of TER in response to PGs or mucosal urea. Immunofluorescence imaging of recovering epithelium revealed that PGs restored occludin and zonula occludens-1 distribution to interepithelial junctions, and this pattern was disrupted by pretreatment with wortmannin. These experiments suggest that PGs stimulate recovery of paracellular resistance via a mechanism involving transepithelial osmotic gradients and PI3K-dependent restoration of tight junction protein distribution.     

Transport pathways in canine lingual epithelium involved in sweet taste

The responses of canine lingual epithelium to D-glucose weremeasured in an Ussing chamber to determine the possible contributionof the osmotic changes of taste cells to the response of saccharides.With the mucosal solution containing 50 mM NaCl, 2 mM HEPES,pH 7.4 (solution A) and the serosal solution containing Krebs—Henseleit(KH) buffer the addition of up to 0.5 M D-glucose in the mucosalsolution increased the short circuit current (I_sc) in a sigmoidalmanner. The D-glucose-stimulated I_sc was inhibited by 0.1 mMamiloride or 1 mM ouabain added to either the mucosal or theserosal solution, and partially inhibited by 5 mM BaCl₂ addedto the serosal solution. The inhibition by these three compoundswas also observed in the presence of 0.5 M NaCl. Ouabain alsoinhibited transport when added to solution A. These experimentssuggest that in canine lingual epithelium the paracellular pathwaypermits molecules as large as ouabain (mol. wt 586) to diffusefrom the mucosal to the serosal solution and vice versa underall osmotic conditions. These results may explain the phenomenonof intravascular taste. Such is not the case in rat tongue whereouabain only inhibited transport when added to the serosal solution.Increasing the osmolality of the serosal KH buffer by additionof relatively membrane-impermeable saccharides such as sucroseor L-glucose did not significantly alter the I_sc, whereas makingthe serosal KH solution hypo-osmotic resulted in a transientdecrease in I_sc. These data suggest that the increase in I_scinduced by saccharides, such as D-glucose, is not simply anosmotic response of the epithelium but more likely the consequenceof saccharides binding weakly to receptors. That the responseto both salts by themselves and in the presence of saccharidesexhibits the same cation selectivity, and that both are inhibitedby amiloride, ouabain, BaCl₂ and LaCl₃ suggest that in caninelingual epithelia, in contrast to rat epithelium, the responsesto hyperosmotic concentrations of salts and saccharides mightoccur via the same transcellular pathways.     

A change in a single gene of Salmonella typhimurium can dramatically change its buoyant density.

The growth rates and buoyant densities of a Salmonella typhimurium mutant, TL126 (proB74A+), with enhanced osmotolerance caused by proline overproduction were measured and compared with the growth rates and buoyant densities of an isogenic (wild-type) strain, TL128 (proB+ A+), with normal control of proline production. Growth rates were determined in a rich medium (Luria broth) with added NaCl to produce various osmotic strengths ranging from 300 to 2,000 mosM. At low concentrations of NaCl, there was little variation in doubling times between the two strains. However, as the osmotic strength of the medium approached and exceeded 1,300 mosM, the doubling times of TL126 (osmotolerant) were 1.5 to 2 times faster than those of TL128 (wild type), confirming the osmotolerance of TL126. Buoyant densities were determined by equilibrium sedimentation in a Percoll gradient of osmotic strength equal to that of the growth medium. The osmolarity of the Percoll gradient was adjusted by the addition of NaCl. At low osmolarities (300 to 500 mosM), the buoyant density of TL126 (osmotolerant) was slightly but consistently lower than that of TL128 (wild type). As the osmotic strength was increased, the buoyant density of TL126 (osmotolerant) increased in proportion to the osmotic strength. In contrast, the buoyant density of strain TL128 (wild type) did not increase as much. At high osmolarities (1,600 to 2,000 mosM), the buoyant density of TL126 (osmotolerant) was consistently higher than that of TL128 (wild type). These results suggest that the intracellular accumulation of proline by TL126, the osmotolerant strain, increases both the growth rates and buoyant densities at osmolarities of 1,300 mosM and above.     

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.     

Effects of ouabain on fluid transport and electrical properties of Necturus gallbladder. Evidence in favor of a neutral basolateral sodium transport mechanism

Net fluid transport (Jv) and electrical properties of the cell membranes and paracellular pathway of Necturus gallbladder epithelium were studied before and after the addition of ouabain (10(-4) M) to the serosal bathing medium. The glycoside inhibited Jv by 70% in 15 min and by 100% in 30 min. In contrast, the potentials across both cell membranes did not decrease significantly until 20 min of exposure to ouabain. At 30 min, the basolateral membrane potential (Vcs) fell only by ca 7 mV. If basolateral Na transport were electrogenic, with a coupling ratio (Na:K) of 3:2, the reductions of Vcs at 15 and 30 min should be 12--15 and 17--21 mV, respectively. Thus, we conclude that the mechanism of Na transport from the cells to the serosal bathing solution is not electrogenic under normal transport conditions. The slow depolarization observed in ouabain is caused by a fall of intracellular K concentration, and by a decrease in basolateral cell membrane K permeability. Prolonged exposure to ouabain results also in an increase in paracellular K selectivity, with no change of P Na/P Cl.     

Effects of changes in osmolarity on isolated human airways

The effects of hypo- and hyperosmolarity on the function of isolated human airways were studied. Changes in osmolarity induced an increasing bronchoconstriction that was proportional to the magnitude of the change in osmolarity. Hypertonicity-induced airway narrowing resulted when buffer was made hypertonic with sodium chloride or mannitol but not with urea. The airways showed no tachyphylaxis to repetitive exposure to hypo- and hypertonic buffer of 200 and 600 mosM, respectively. The bronchoconstriction was not secondary to stimulation of H1 or leukotriene C4/D4 receptors or the release of prostaglandins in the preparation. The bronchoconstriction in hypotonic buffer was totally dependent on extracellular calcium, whereas in hypertonic buffer the bronchoconstriction seemed partially dependent on intracellular calcium release. Isoprenaline prevented the bronchoconstriction in hyper- or hypotonic buffer of 450 and 250 mosM but not in buffer of 600 and 150 mosM. It is concluded that hypo- and hypertonic buffers lead to bronchoconstriction via different mechanisms, which relate to influx of extracellular calcium in hyposmolar buffer and probably to release of calcium from intracellular stores in hypertonic buffer. In strongly hypertonic buffer, part of the bronchoconstriction may be due to osmotic shrinkage. The relevance of our data for the mechanism of bronchoconstriction after inhalation of hypo- or hypertonic saline depends on whether changes in osmolarity around the airway smooth muscle occur in asthmatics but not in normal subjects, and this has not yet been established.     

Ca++ regulation of paracellular permeability in the middle intestine of the eel, Anguilla anguilla

The role of Ca++ on the regulation of the paracellular pathway permeability of the middle intestine of Anguilla anguilla was studied by measuring the transepithelial resistance and the dilution potential, generated when one half of NaCl in the mucosal solution was substituted iso-osmotically with mannitol, in various experimental conditions altering extracellular and/or intracellular calcium levels. We found that removal of Ca++ in the presence of ethylene glycol-bis(beta-aminoethyl ether) (EGTA) from both the mucosal and the serosal side, but not from one side only, reduced both the transepithelial resistance and the magnitude of the dilution potential. The irreversibility of this effect suggests a destruction of the organization of the junction in the nominal absence of Ca++. However a modulatory role of extracellular Ca++ cannot be excluded. The decrease of the intracellular Ca++ activity, produced by using verapamil to block the Ca++ entry into the cell, or by adding 3,4,5-trimethoxybenzoic acid 8-(diethylamino) octyl ester (hydrochloride) (TMB-8), an inhibitor of Ca++ release from the intracellular stores, reduced both the transepithelial resistance and the magnitude of the dilution potential, indicating a role of cytosolic Ca++ in the modulation of the paracellular permeability. However the rise of calcium activity produced by the Ca++ ionophore calcimycin (A23187) evoked an identical effect, suggesting that any change in physiological intracellular Ca++ activity alters the paracellular permeability.     

Passive Electrical Properties of Toad Urinary Bladder Epithelium : Intercellular Electrical Coupling and Transepithelial Cellular and Shunt Conductances

The electrical resistances of the transcellular and paracellular pathways across the toad urinary bladder epithelium (a typical "tight" sodium-transporting epithelium) were determined by two independent sets of electrophysiological measurements: (a) the measurement of the total transepithelial resistance, the ratio of resistance of the apical to the basal cell membrane, and cable analysis of the voltage spread into the epithelium; (b) the measurement of the total transepithelial resistance and the ratio of resistances of both cell membranes before and after replacing all mucosal sodium with potassium (thus, increasing selectively the resistance of the apical membrane). The results obtained with both methods indicate the presence of a finite transepithelial shunt pathway, whose resistance is about 1.8 times the resistance of the transcellular pathway. Appropriate calculations show that the resistance of the shunt pathway is almost exclusively determined by the zonula occludens section of the limiting junctions. The mean resistance of the apical cell membrane is 1.7 times that of the basal cell membrane. The use of nonconducting materials on the mucosal side allowed us to demonstrate that apparently all epithelial cells are electrically coupled, with a mean space constant of 460 µm, and a voltage spread consistent with a thin sheet model.     

Unstirred layer effects in osmotic water flow across gallbladder epithelium

Summary The standard one-dimensional model of the unstirred layer is applied in a re-examination of the experimental results of Wright, Smulders and Tormey (Wright, E.M., Smulders, A.P., Tormey, J. McD., 1972,J. Membrane Biol. 7:198) who reported large transients in the osmotic flux of water from the serosal to the mucosal side of rabbit gallbladder epithelium. They initiated osmosis by the addition of sucrose to the mucosal bathing solution (initially, approximately 300mOsm NaCl) and observed that the initial flux was more than ten times its eventual steady-state value; they interpreted this as a consequence of the piling-up of NaCl in the unstirred tissue layer on the serosal side of the epithelium. The present analysis (both steady-state and unsteady) shows that if measured values of layer thickness are used, together with reasonable values of the reduced diffusivity of NaCl in the tissue and of the fraction of tissue available for water flow, then one would predict a discrepancy of only about 10%, not tenfold, between the initial and final values of the flux. Thus the standard model is inconsistent with the observations. Furthermore, Wright et al's results cannot be used to infer that the osmotic permeability of epithelial cell membranes is much larger than steadystate measurements on whole epithelia would indicate. Mucosal-to-serosal flow is also analyzed, and in this case a considerably greater osmotic permeability is predicted; this result is consistent with the observed changes in structure of the lateral intercellular spaces when the direction of flow is reversed.