Protamine reversibly decreases paracellular cation permeability inNecturus gallbladder

Summary Protamine, a naturally occurring arginine-rich polycationic protein (pI 9.7 to 12), was tested inNecturus gallbladder using a transepithelial AC-impedance technique. Protamine sulfate or hydrochloride (100 g/ml=20 m), dissolved in the mucosal bath, increased transepithelial resistance by 89% without affecting the resistance of subepithelial layers. At the same time, transepithelial voltage ( ^ms ) turned from slightly mucosapositive values to mucosa-negative values of approximately +1 to –5 mV. The effect of protamine on transepithelial resistance was minimal at concentrations below 5 g/ml but a maximum response was achieved between 10 and 20 g/ml. Resistance started to increase within 1 min and was maximal after 10 min. These effects were not inhibited by serosal ouabain (5×10^–4 m) but could be readily reversed by mucosal heparin. The sequence of protamine effect and heparin reversal could be repeated several times in the same gallbladder. Mucosal heparin, a strong negatively charged mucopolysaccharide, or serosal protamine were without effect. Mucosal protamine reversibly decreased the partial ionic conductance of K and Na by a factor of 3, but did not affect Cl conductance. Net water transport from mucosa to serosa was reversibly increased by 60% by protamine. We conclude that protamine reversibly decreases the conductance of the cation-selective pathway through the tight junction. Although this effect is similar to that reported for 2,4,6-triamino-pyrimidinium (TAP), the mechanism of action may differ. We propose that protamine binds to the apical cell membrane and induces a series of intracellular events which leads to a conformational alteration of the tight junction structure resulting in decreased cationic permeability.     

Protamine-induced epithelial barrier disruption involves rearrangement of cytoskeleton and decreased tight junction-associated protein expression in cultured MDCK strains

Natural and synthetic polycationic proteins, such as protamine, have been used to reproduce the tissue injury and changes in epithelial permeability caused by positively charged substances released by polymorphonuclear cells during inflammation. Protamine has diverse and often conflicting effects on epithelial permeability. The effects of this polycation on the distribution and expression of tight junction (TJ)-associated proteins have not yet been investigated. In this work, we examined the influence of protamine on paracellular barrier function and TJ structure using two strains of the epithelial Madin-Darby canine kidney (MDCK) cell line that differed in their TJ properties ("tight" TJ-strain I and "leaky" TJ-strain II). Protamine induced concentration-, time- and strain-dependent alterations in transepithelial electrical resistance (Rt) only when applied to apical or apical+basolateral monolayer surfaces, indicating a polarity of action. In MDCK II cells, protamine (50 microg/ml) caused a significant increase in Rt that returned to control values after 2 h. However, the treatment of this MDCK strain with a higher concentration of protamine (250 microg/ml) significantly decreased the Rt after 30 min. In contrast, treated MDCK I monolayers showed a significant decrease in Rt after apical treatment with protamine at both concentrations. The protamine-induced decrease in Rt was paralleled by an increase in the phenol red basal-to-apical flux in both MDCK strains, suggesting disruption of the paracellular barrier. Marked changes in cytoskeletal F-actin distribution/polymerization and a significant reduction in the junctional expression of the tight junctional proteins occludin and claudin-1 but subtle alterations in ZO-1 were observed following protamine-elicited paracellular barrier disruption. In conclusion, protamine induces alterations in the epithelial barrier function of MDCK monolayers that may involve the cytoskeleton and TJ-associated proteins. The various actions of protamine on epithelial function may reflect different degrees of interaction of protamine with the plasma membrane and different intracellular processes triggered by this polycation.     

Fracture faces of zonulae occludentes from "tight" and "leaky" epithelia

Epithelia vary with respect to transepithelial permeability. In those that are considered "leaky", a large fraction of the passive transepithelial flux appears to follow the paracellular route, passing across the zonulae occludentes and moving down the intercellular clefts. In "tight" epithelia, the resistance of the paracellular pathway to passive flux is greatly increased. To see whether differences in the morphology of the zonula occludens could contribute to this variability in leakiness among epithelia, replicas of zonulae occludentes in freeze-fractured material from a variety of tight and leaky epithelia were examined. The junctions appear as a branching and anastomosing network of strands or grooves on the A and B membrane fracture faces, respectively. It was found that the zonula occludens from a "very leaky" epithelium, the proximal convoluted tubule of the mouse kidney, is extremely shallow in the apical-basal direction, consisting in most places of only one junctional strand. In contrast, the "very tight" frog urinary bladder exhibits a zonula occludens that is relatively deep (>0.5 µm) in the apical-basal direction, and consists of five or more interconnected junctional strands interposed between luminal and lateral membrane surfaces. Epithelia of intermediate permeabilities exhibited junctions with intermediate or variable morphology. Toad urinary bladder, mouse stomach, jejunum, and distal tubule, rabbit gallbladder, and Necturus kidney and gallbladder were also examined, and the morphological data from these epithelia were compared to physiological data from the literature.     

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.     

Electrical properties of the rabbit urinary bladder assessed using gramicidin D

Summary Recently, antibiotics have enjoyed widespread usage as tools in studies of epithelial transport. In the present study we assess the usefulness of the pore-forming antibiotic gramicidin D as a means for probing the electrical properties of the tight epithelium rabbit urinary bladder. Addition of 50 M gramicidin to the mucosal bath (either a NaCl or KCl Ringer's solution) led to a large irreversible increase in the transepithelial conductance (G _T ) within 800 sec.G _T increased by approximately 1200% and 500% in KCl and NaCl Ringer's solutions, respectively. Microelectrode measurements of the resistance ration (the ration of apical membrane resitance to basolateral membrane resistance) showed that apical membrane resistance is dereased by the drug. Measurements of the basolateral membrane resistance (R _bl ) and tight junctional resistance (R _j ) using a new and independent method (based on the perturbation of basolateral membrane electrogenic Na⁺ pump) demonstrated thatR _bl andR _j were unaffected, suggesting that the effects of gramicidin are restricted to the apical membrane for periods of at least 2 hours after drug addition. The selectivity of the gramicidin-induced permeability in the apical membrane was calculated from measurements of the apical membrane potential after ion substitutions using a modified version of the constant field equation. The selectivity sequence for cations was Cs⁺>K⁺>Na⁺>Li⁺>choline. Unlike the commonly used polyene antibiotics nystatin and amphotericin B, gramicidin did not induce a significant Cl^– permeability. In addition, the dose-response curve had a slope of 1. A method is described for calculating membrane resistances directly from transepithelial measurements under some conditions of gramicidin use, without requiring the use of microlectrode measurements.     

Structural basis for physiological regulation of paracellular pathways in intestinal epithelia

Summary Isolated segments of hamster small intestine were perfused with oxygenated salt-fluorocarbon emulsions with or without 10–25mm glucose, alanine or leucine. Resistances of inter-cellular occluding junctions and of lateral spaces and the distributed capacitance of epithelial plasma membranes were estimated from steady-state transepithelial impedances at frequencies from 0.01–10 kHz. The segments were then fixedin situ with isorheic 2.5% glutaraldehyde while continuing to measure impedance. This method of fixation increased the resistance of lateral spaces but had little effect on the resistance of occluding junctions or on membrane capacitance. The large decreases of impedance induced by glucose or amino acids were preserved in fixed tissue and could therefore be correlated with changes in structure. The observed changes of impedance were interpreted as decreased resistance of occluding junctions and lateral spaces together with increased exposed surface of lateral membranes (capacitance). Glucose, alanine or leucine induced expansion of lateral intercellular spaces as seen by light and electron microscopy. Large dilatations within absorptive cell occluding junctions were revealed by electron microscopy. Freeze-fracture analysis revealed that these dilatations consisted of expansions of compartments bounded by strands/grooves. These solute-induced structural alterations were also associated with condensation of microfilaments in the zone of the perijunctional actomyosin ring, typical of enhanced ring tension. Similar anatomical changes were found in epithelia fixedin situ at 38°C during luminal perfusion with glucose in blood-circulated intestinal segments of anesthetized animals. These structural changes support the hypothesis that Na-coupled solute transport triggers contraction of perijunctional actomyosin, thereby increasing junctional permeability and enhancing absorption of nutrients by solvent drag as described in the two accompanying papers.     

Channels in epithelial cell membranes and junctions.

Epithelia may be classified as "tight" or "leaky," depending on whether there is a significant pathway for transepithelial ion permeation via the junctions and bypassing the cells. The resistance of this paracellular channel may depend partly on structures visible in the electron microscope, partly on wall charge. Permeability determinations in the leaky junctions of gallbladder epithelium, using many different organic cations, suggest that the critical barriers barriers to ion permeation are 5--8 A in radius and bind cations by up to four strongly proton-accepting oxygens. The apical cell membrane of tight epithelia contains a Na+-selective channel that is blocked by amiloride and Ca2+, subject to negative feedback control by the Na+ pump in the basolateral membrane, and somehow promoted by aldosterone. To determine the permeabilities of these two channels (the junctional channel of leaky epithelia, and the Na+ channel of tight epithelia) to water and nonelectrolytes remains a major unsolved problem.     

Modulation of the junctional integrity by low or high concentrations of cytochalasin B and dihydrocytochalasin B in associated with distinct changes in F-actin and ZO-1

In a study of Necturus gallbladder epithelium Benzel et al. (Benzel et al., 1980) found that low (0.2–1.2 M) and higher concentrations (1.5 M and more) of cytochalasin B (CB) caused an increase and decrease in the transepithelial electrical resistance (TER), respectively. Moreover, there were slight changes in the height and complexicity of tight junction (TJ) strands, as visualized by freeze-fracture and freeze-etching. To elucidate the mechanisms of these findings, we first demonstrated that the effect is also present in monolayers of Madin-Darby Canine Kidney strain I (MDCK-I) cells. Thus, a low concentration (0.1 ng/ml) cytochalasin B (CB) strengthened the permeability barrier, as evidenced quantitatively by increases in TER on transepithelial electrical measurements. Furthermore, indirect immunofluorescence and confocal microscopy demonstrated that this effect was paralleled with an accumulation of F-actin and the tight junction marker protein, ZO-1, at the level of TJ. Equimolar concentrations of dihydrocytochalasin B (dhCB), on the other hand, did not lead to a tightening of the epithelium. Confirming previous studies, there was a general decrease in epithelial resistance after treatment with high concentrations (1 g/ml) of CB and dhCB, which was accompanied by distinct changes in the F-actin network and distribution of ZO-1. We speculate that the divergent effects of CB and dhCB on the F-actin and ZO-1 organization might be due to specific effects on the transport of monosaccharides across the plasma membrane, or that CB and dhCB in distinct ways involve the turnover of phosphatidylinositols in the membrane, thereby modulating junctional permeability and F-actin structure.     

Morphological factors influencing transepithelial permeability: A model for the resistance of theZonula Occludens

Summary Epithelial cells are joined at their apical surfaces byzonulae occludentes. Claude and Goodenough (1973) demonstrated a correlation between the structure of thezonula occludens as seen in freeze-fracture preparations and the passive electrical permeability of several simple epithelia. In epithelia with high transepithelial resistance, thezonula occludens consisted of many strands. In epithelia with low transepithelial resistance thezonula occludens was much reduced, sometimes consisting of only one strand.Evidence is reviewed here that indicates that in a number of simple epithelia the structure of thezonula occludens is largely responsible for the magnitude of transepithelial conductance. An equation is derived relating transepithelial junctional resistance to the number of junctional strands:R=R _min p ^–n whereR is the transepithelial resistance of thezonula occludens,R _min is the minimum resistance of the junction (as when there areno strands in the zonula occludens),p is the probability a given strand is open andn is the number of strands in the junction. Using published experimental values ofR andn for different epithelia, the calculated value ofp was found to be as high as 0.4, which suggests that the strands in thezonula occludens are remarkably labile.Other morphological parameters relevant to transepithelial permeability are also considered, such as the width and depth of the intercellular spaces, and the size of the epithelial cells themselves.     

Passive ionic properties of frog retinal pigment epithelium

Summary The isolated pigment epithelium and choroid of frog was mounted in a chamber so that the apical surfaces of the epithelial cells and the choroid were exposed to separate solutions. The apical membrane of these cells was penetrated with microelectrodes and the mean apical membrane potential was –88 mV. The basal membrane potential was depolarized by the amount of the transepithelial potential (8–20mV). Changes in apical and basal cell membrane voltage were produced by changing ion concentrations on one or both sides of the tissue. Although these voltage changes were altered by shunting and changes in membrane resistance, it was possible to estimate apical and basal cell membrane and shunt resistance, and the relative ionic conductanceT _i of each membrane. For the apical membrane:T _K0.52,T _{HCO 3}=0.39 andT _Na=0.05, and its specific resistance was estimated to be 6000–7000 cm². From the basalT _K=0.90 and its specific resistance was estimated to be 400–1200 cm². From the basal potassium voltage responses the intracellular potassium concentration was estimated at 110mm. The shunt resistance consisted of two pathways: a paracellular one, due to the junctional complexes and another, around the edge of the tissue, due to the imperfect nature of the mechanical seal. In well-sealed tissues, the specific resistance of the shunt was about ten times the apical plus basal membrane specific resistances. This epithelium, therefore, should be considered tight. The shunt pathway did not distinguish between anions (HCO₃ ^–, Cl^–, methylsulfate, isethionate) but did distinguish between Na⁺ and K⁺.     

Regeneration of resistance and ion transport in rabbit corneal epithelium after induced surface cell exfoliation

Summary Exposure of the rabbit corneal surface to a 20-m digitonin-0.9% NaCl solution leads to permeabilization of the most superficial cells of the stratified epithelium. The devitalized cells exfoliate spontaneously from the corneal surface. Detergent exposure limited to 4–8 min leads to permeabilization and rapid exfoliation of a monolayer of surface cells. Consistent with the presence of the epithelial paracellular permeability barrier in this cell layer, their permeabilization results in complete loss of transepithelial resistance (R _t ). Within minutes after detergent removal an initial recovery ofR _t can be noticed indicating generation of a new paracellular permeability barrier by the viable subsurface cells. This recovery proceeds rapidly andR _t reaches within 70 min a maximum equal to > 90% of the preexfoliation values (=2.43 k·cm²,n=22). TheR _t recovery is fully blocked in a reversible manner by 10 m dihydrocytochalasin B. The recovery is not affected by inhibition of protein synthesis with 5 m cycloheximide. When the ocular surface is treated again with digitonin the permeabilization and exfoliation of a monolayer of cells and loss ofR _t are repeated. After the second detergent exposure an initial recovery ofR _t occurs as before within minutes. However, the pace ofR _t recovery is much slower: 4–5 hr are required to reach a stable maximalR _t values amounting to about 73% of initial control. This recovery can be fully blocked by 5 m cycloheximide indicating that protein synthesis is required for generation of tight junctions by the second subcellular layer. With only a fraction ofR _t recovered, short-circuit currents amounting to, at least, 50% of control values and attributable in part to cell-to-tear movement of Cl^– through the apical surface can be measured. This suggests that apical-type Cl^– channels are either present in the apically facing membrane of subsurface cells or that they are rapidly inserted in it from preexisting intracellular pools immediately following the devitalization of the surface cells by digitonin.     

Maintenance of the macromolecular barrier at cell extrusion sites in intestinal epithelium: Physiological rearrangement of tight junctions

Summary All epithelia slough dying cells but the consequences of this physiological process to epithelial barrier functions is unknown. In mammalian small intestine absorptive cells are known to migrate from the villus base to the villus tip from which they slough. These villus tip extrusion zones are often envisioned as sites at which macromolecules could leak across the epithelium. However, only trace amounts of macromolecules cross this epithelium even though, based on known epithelial turnover rates, extrusion events occur millions of times daily. Here, we examine the characteristics of the epithelial barrier to macromolecular permeation at villus tip extrusion zones in rats and hamsters. Freeze-fracture, light and electron microscope studies reveal that extruding cells do not leave transient holes behind as they lift from the epithelium. Rather, as cells extrude, processes of adjacent cells extend under them. Moreover, tight junction elements proliferate between extruding cells and their neighbors and appear to move down the lateral margin of the extruding cell as it extends into the lumen. These observations suggest that newly formed junctional elements zipper the epithelium closed as extrusion proceeds thus preventing epithelial discontinuities from occurring. Correlative in vivo perfusion experiments using horseradish peroxidase as a macromolecular-tracershow that the above described dynamic alterations in tight junctions at extrusion sites are generally sufficient to prevent transepithelial leaks of macromolecules.     

Voltage- and time dependence of apical membrane conductance during current clamp inNecturus gallbladder epithelium

Summary The effects of short (1 sec) and long (1 min) transepithelial current clamps on membrane voltages and resistances ofNecturus gallbladder were investigated. Transepithelial and cell membrane current-voltage relationships determined from 1-sec clamps revealed that: a) depolarization of the apical membrane voltage (V _mc) results in a marked decrease in apical membrane fractional resistance (fR _a), whereas hyperpolarization ofV _mc results in either no change infR _a or a small increase, and b) the voltage-dependent changes infR _a are essentially complete within 500 msec. Exposure of the tissue to 5mm TEA⁺ on the mucosal side caused no significant change in baselineV _mc (–69±2 mV) and yet virtually abolished the voltage dependence offR _a. A possible interpretation of these results is that two types of K⁺ channels exist in the apical membrane, with different voltage dependencies and TEA⁺ sensitivities. Acidification or Ba²⁺ addition to the mucosal solution also reduced the voltage-dependent changes infR _a. The time courses of the changes infR _a and in the cable properties of the epithelium were assessed during 1-min transepithelial current clamps (±200 A/cm²). No secondary change infR _a was observed with mucosa-to-serosa currents, but a slow TEA⁺-sensitive decrease infR _a (half-time of seconds) was evident with serosa-to-mucosa currents. Cable analysis experiments demonstrated that the initial (<500 msec) voltage-dependent decrease infR _a is due to a fall in apical membrane resistance. The later decrease infR _a is due to changes in both cell membrane resistances attributable to the increase in transcellular current flow resulting from a fall in paracellular conductance. The voltage dependence of the apical membrane conductance is a more significant problem in estimatingfR _a than the current-induced effects on the lateral intercellular spaces. In principle, TEA⁺ can be used to prevent the nonlinear behavior ofR _a during measurements of the voltage divider or membrane resistance ratio.     

Tight junction of sinus endothelial cells of the rat spleen

The fine structure of the tight junctions between sinus endothelial cells of the rat spleen and the permeability of such sinus endothelial cells were examined by transmission electron microscopy, using freeze-fracture, triton extraction, and lanthanum-tracer techniques. In freeze-fracture replicas, the segmented strands and grooves of the tight junctions were frequently observed on the basolateral surfaces of the sinus endothelial cells irrespective of the location of the ring fiber. There were one or two wavy-strands or grooves which were, for the most part, oriented parallel to the long cell axis thus forming networks at places. In addition, some strands or grooves were discontinuous while some networks of the junctional strands were not closed. These strands also occasionally lacked intramembranous particles in the tight junctions. The junctional strands run apicobasically at certain sites. In the vertical sections of the sinus endothelial cells treated with lanthanum nitrate, although no tight junctions were observed wherever the endothelial cells were apposed, most of them were situated on the basal part of the lateral surfaces of the adjacent endothelial cells. Several fusions of the junctional membranes were observed in a vertical section of the lateral surfaces of the adjacent endothelial cells. The intercellular spaces of the adjacent endothelial cells except for the fusion of the junctional membranes, were electron dense and the infiltration of lanthanum nitrate was found not to be interrupted by these tight junctions. Based on these observations, the molecular 'fence' and paracellular 'gate' functions of the tight junctions in the sinus endothelial cells are discussed.     

Uncoupling of gate and fence functions of MDCK cells by the actin-depolymerizing reagent mycalolide B

The tight junction serves as a paracellular gate to seal the paracellular space of apposing cells and as a molecular fence to prevent diffusion of membrane proteins and lipids in epithelial cells. Although involvement of the actin cytoskeleton has been considered to be important in these two functions, it remains to be elucidated whether both functions are regulated in a coupled manner or differentially by actin. Treatment of highly polarized MDCK cells with mycalolide B (MB), a recently developed actin-depolymerizing reagent, induced a decrease of transepithelial resistance in a dose- and time-dependent manner with reversibility when the reagent was washed out. Changes in cytoskeletal actin, such as a reduction of cortical actin, irregularity of stress fibers, and punctated actin aggregates, were observed after MB treatment. However, the fence function, as studied by diffusion of apically labeled sphingomyelin/BSA complex, remained intact in the MB-treated MDCK cells. Localization of junctional molecules and apical marker proteins such as E-cadherin, ZO-1, and 114-kDa protein was shown to be unaffected. Furthermore, freeze-fracture study showed apparent tight junction strands. Collectively, MB treatment abolished the paracellular gate but not the fence function of MDCK cells, suggesting that cytoskeletal actin may play differential roles in the gate and fence functions of the tight junction.     

Modulation of apical Na permeability of the toad urinary bladder by intracellular Na,Ca, and H

Summary The Na conductance of the apical membrane of the toad urinary bladder was measured at different concentrations of Na both in the external medium and in the cell. Bladders were bathed in high K-sucrose medium to reduce basal-lateral resistance and voltage, and the transepithelial currents measured under voltage-clamp conditions. Amiloride was used as a specific blocker of the apical Na channel. At constant external Na, the internal Na concentration was increased by blocking the basallateral Na pump with ouabain. With high Na activity in the mucosal medium (86mm), increases in intracellular Na activity from 10 to over 40mm increased the amiloride-sensitive slope conductance at zero voltage while apical Na permeability, estimated from current-voltage plots using the constant field equation, decreased by less than 20%. Lowering the serosal Ca concentration from 1 to 0.1mm had no effect on the change inP _Na with increasing Na_c, but increasing serosal Ca to 5mm enhanced the reduction inP _Na with increasing Na _c , presumably by increasing Ca influx into the cell.P _Na was also reduced by serosal vanadate (0.5mm), a putative blocker of ATP-dependent Ca extrusion from the cell, and by acute exposure to CO₂, which presumably acidifies the cytoplasm. Current-voltage relationships of the amiloridesensitive transport pathway were also measured in the absence of a Na gradient across the apical membrane. These plots show that outward current passes through the channels somewhat less easily than does inward current. The shape of theI-V relationships was not significantly altered by changes in cellular Na, Ca or H, indicating that the effects of these ions onP _Na are voltage independent.     

Permeable junctional complexes. The movement of lanthanum across rabbit gallbladder and intestine

Ionic lanthanum has been used to study transepithelial ion permeation in in vitro rabbit gallbladder and intestine (ileum) by adding 1 mM La³⁺ to only the mucosal bathing solution. Transepithelial fluid transport electrical potential differences (p.d.), and resistances were measured. During La³⁺ treatment the gallbladder''s rate of active solute-coupled fluid transport remained constant, the resistance increased, and the 2:1 NaCl diffusion p.d. decreased. Mucosa-to-serosa fluxes of ¹⁴⁰La³⁺ were measured and indicate a finite permeability of the gallbladder to La³⁺. La³⁺ also increased the transepithelial resistance and p d. of ileum. Electron microscopic examination of La³⁺-treated gallbladder showed: (a) good preservation of the fine structure, (b) electron-opaque lanthanum precipitates in almost every lateral intercellular space, most frequently near the apical end of the lateral spaces close to or within the junctional complex, (c) lanthanum among the subjacent muscle and connective tissue layers, and (d) lanthanum filling almost the entire length of so-called "tight" junctions. No observations were made which unequivocally showed the penetration of lanthanum into the gallbladder cells. Electron micrographs of similar La³⁺-treated ilea showed lanthanum deposits penetrating the junctional complexes. These results coupled with other physiological studies indicate that the low resistance pathway for transepithelial ion permeation in gallbladder and ileum is through the tight junctions A division of salt-transporting epithelia into two main groups, those with "leaky" junctional complexes and those with tight junctional complexes, has been proposed.     

Basolateral membrane potassium conductance of A6 cells

Summary To study the properties of the basolateral membrane conductance of an amphibian epithelial cell line, we have adapted the technique of apical membrane selective permeabilization (Wills, N.K., Lewis, S.A., Eaton, D.C., 1979b, J. Membrane Biol. 45:81–108). Monolayers of A6 cells cultured on permeable supports were exposed to amphotericin B. The apical membrane was effectively permeabilized, while the high electrical resistance of the tight junctions and the ionic selectivity of the basolateral membrane were preserved. Thus the transepithelial current-voltage relation reflected mostly the properties of the basolateral membrane. Under basal conditions, the basolateral membrane conductance was inward rectifying, highly sensitive to barium but not to quinidine. After the induction of cell swelling either by adding chloride to the apical solution or by lowering the osmolarity of the basolateral solution, a large out-ward-rectifying K⁺ conductance was observed, and addition of barium or quinidine to the basolateral side inhibited, respectively, 82.4±1.9% and 90.9±1.0% of the transepithelial current at 0 mV. Barium block was voltage dependent; the half-inhibition constant (K _i) varied from 1499±97 m at 0 mV to 5.7±0.5 m at –120 mV.Cell swelling induces a large quinidine-sensitive K⁺ conductance, changing the inward-rectifying basolateral membrane conductance observed under basal conditions into a conductance with outward-rectifying properties.     

Electrophysiological studies of forskolin-induced changes in ion transport in the human colon carcinoma cell line HT-29 cl.19A: Lack of evidence for a cAMP-activated basolateral K+ conductance

Summary Forskolin (i.e, cAMP)-modulation of ion transport pathways in filter-grown monolayers of the Cl^–-secreting subclone (19A) of the human colon carcinoma cell line HT29 was studied by combined Ussing chamber and microimpalement experiments.Changes in electrophysiological parameters provoked by serosal addition of 10^–5 m forskolin included: (i) a sustained increase in the transepithelial potential difference (3.9±0.4 mV). (ii) a transient decrease in transepithelial resistance with 26±3 · cm² from a mean value of 138±13 · cm² before forskolin addition, (iii) a depolarization of the cell membrane potential by 24±1 mV from a resting value of –50±1 mV and (iv) a decrease in the fractional resistance of the apical membrane from 0.80±0.02 to 0.22±0.01. Both, the changes in cell potential and the fractional resistance, persisted for at least 10 min and were dependent on the presence of Cl^– in the medium. Subsequent addition of bumetanide (10^–4 m), an inhibitor of Na/K/2Cl cotransport, reduced the transepithelial potential, induced a repolarization of the cell potential and provoked a small increase of the transepithelial resistance and fractional apical resistance. Serosal Ba²⁺ (1mm), a known inhibitor of basolateral K⁺ conductance, strongly reduced the electrical effects of forskolin. No evidence was found for a forskolin (cAMP)-induced modulation of basolateral K⁺ conductance.The results suggest that forskolin-induced Cl^– secretion in the HT-29 cl.19A colonic cell line results mainly from a cAMP-provoked increase in the Cl^– conductance of the apical membrane but does not affect K⁺ or Cl^– conductance pathways at the basolateral pole of the cell. The sustained potential changes indicate that the capacity of the basolateral transport mechanism for Cl^– and the basal Ba²⁺-sensitive K⁺ conductance are sufficiently large to maintain the Cl^– efflux across the apical membrane. Furthermore, evidence is presented for an anomalous inhibitory action of the putative Cl^– channel blockers NPPB and DPC on basolateral conductance rather than apical Cl^– conductance.     

Osmotic reversal induces assembly of tight junction strands at the basal pole of toad bladder epithelial cells but does not reverse cell polarity

Summary This paper reports the effect of reversing the osmotic environment between luminal and serosal compartments of a toad urinary bladder on the polarity of assembly of tight junction strands. Toad bladders were filled with Ringer's solution (220 mOsm) and were immersed in distilled water at room temperature or at 37°C. Within two minutes, new tight junction strands are assembled. The new tight junctional strands unite the basal pole of epithelial cells with the apical side of basal cells. Physiological studies show that oxytocin, a synthetic analog of antidiuretic hormone, is still capable of inducing increases in water transport in epithelia which were osmotically reversed. This capacity decreases significantly for longer periods of osmotic reversal. Osmotic reversal does not alter the original polarity of epithelial cells: 1) the apical tight junction belt, at the apical pole, is not displaced; 2) the freeze-fracture morphology typical of apical plasma membrane (particle-rich E faces; particle-poor P faces) is not altered; 3) oxytocin and cyclic AMP induce aggregates which are observed only at the apical plasma membrane. Massive assembly of junctional elements occurs even in epithelia preincubated in the presence of cycloheximide (an inhibitor of protein synthesis) or of cytoskeleton perturbers. Our experiments show that the polarity of assembly of tight junction strands depends on the vectorial orientation of the osmotic environment of the epithelium.