Low Resistance Epithelium Research Articles

Human Platelet Lysates as a Serum Substitute in Renal Epithelial Cell Culture

Cultured renal epithelia form monolayers of differentiated, polarized cells. On permeable supports, an apical and a basolateral compartment are separated by the cultured epithelium to study epithelial transport in vitro. However, culture conditions and culture media have a significant impact. In this respect, the quality of fetal bovine serum (FBS) seemed to be of major importance. The high lot‐to‐lot variablity of FBS was found to substantially influence the differentiation of epithelial cultures with regard to the generation of a transepithelial electrical resistance (TEER). Thus, batch‐testing of FBS was required. We recently reported on the use of human platelet lysates (PL) as a replacement for FBS. The growth promoting capacity of PL was tested on renal epithelial cell lines, for which differentiation end points are well established. PL support growth, proliferation and differentiation of LLC‐PK1, HK‐ 2 and MDCK cells. In addition, TEER was monitored in filter‐grown LLC‐PK1 and in MDCK II and MDCK I epithelia. Low‐resistance epithelia generated a TEER of 150–250 Ω•cm2 in PL‐media, as seen with FBS, and high‐resistance MDCK I retained their TEER of 8,000 Ω•cm2. PL are a valuable, animal‐derived component‐free substitute for FBS in renal epithelial cell culture with a proven high batch‐to batch uniformity.

Hans Henriksen Ussing. 30 December 1911 — 22 December 2000

Hans Ussing was born on 30 December 1911 at Sorø Academy in Denmark, where his father Dr Henrik Ussing was a lecturer and, as historian, a leading Danish folklorist. After his doctoral thesis in marine biology, Hans Ussing came to August Krogh's laboratory, where he studied protein turnover by using deuterium-labelled amino acids. After World War II, when radioactive isotopes of light elements became available for biological research, Ussing pioneered the development of epithelial physiology by introducing new concepts and theoretical tools, such as unidirectional fluxes, exchange diffusion, the flux-ratio equation, the shortcircuiting technique, solvent drag, anomalous solvent drag and the pre-steady-state flux ratio theorem. In studies on frog skin, combining electrophysiology and radioactive tracer technology, he provided the first unambiguous demonstration of active transport of sodium ions. His two-membrane hypothesis of active transport by frog skin initiated studies of epithelial transport at the cellular level in other organs and of the mechanisms of action of hormones and drugs. His discovery of paracellular ion transports bridged the physiology of high-resistance and low-resistance epithelia. With the Na + recirculation theory of isotonic transport he continued his studies of epithelial physiology until shortly before his death. Ussing's scientific research provided analytical methods and new insights of general applicability for the study of absorbing and secreting epithelia—of equal importance to biology and medicine. Hans Ussing died on 22 December 2000 after a short illness.

Claudins 6, 9, and 13 are developmentally expressed renal tight junction proteins.

The adult proximal tubule is a low-resistance epithelium where there are high rates of both active transcellular and passive paracellular NaCl transport. We have previously demonstrated that the neonatal rabbit and rat proximal tubule have substantively different passive paracellular transport properties than the adult proximal tubule, which results in a maturational change in the paracellular passive flux of ions. Neonatal proximal tubules have a higher P(Na)/P(Cl) ratio and lower chloride and bicarbonate permeabilities than adult proximal tubules. Claudins are a large family of proteins which are the gate keepers of the paracellular pathway, and claudin isoform expression determines the permeability characteristics of the paracellular pathway. Previous studies have shown that claudins 1, 2, 3, 4, 5, 7, 8, 10, 11, 12, 15, and 16 are expressed in the adult mouse kidney. To determine whether there are developmental claudin isoforms, we compared the claudin isoforms present in the neonatal and adult kidney using RT-PCR to detect mRNA of claudin isoforms. Claudin 6, claudin 9, and claudin 13 were either not expressed or barely detectable in the adult mouse kidney using traditional PCR, but were expressed in the neonatal mouse kidney. Using real-time RT-PCR, we were able to detect a low level of claudin 6 mRNA expression in the adult kidney compared with the neonate, but claudin 9 and claudin 13 were only detected in the neonatal kidney. There was the same maturational decrease in these claudin proteins with Western blot analysis. Immunohistochemistry showed high levels of expression of claudin 6 in neonatal proximal tubules, thick ascending limb, distal convoluted tubules, and collecting ducts in a paracellular distribution but there was no expression of claudin 6 in the adult kidney. Using real-time RT-PCR claudin 6 and 9 mRNA were present in 1-day-old proximal convoluted tubules and were virtually undetectable in proximal convoluted tubules from adults. Claudin 13 was not detectable in neonatal or adult proximal convoluted tubules. In summary, we have identified developmentally expressed claudin isoforms, claudin 6, claudin 9, and claudin 13. These paracellular proteins may play a role in the maturational changes in paracellular permeability.

The paracellular pathway in the lepidopteran larval midgut: Modulation by intracellular mediators

The features of the paracellular pathway, an important route for the transfer of ions and molecules in epithelia, are in insects still poorly investigated and it has not yet been elucidated how the septate junction (SJ) acts as a transepithelial barrier. In this study, some properties of the paracellular pathway of Bombyx mori larval midgut, isolated in Ussing chambers, were determined and the modulation of SJ permeability by intracellular events disclosed. Diffusion potentials evoked by transepithelial gradients of different salts indicated that the junction bore weak negative charges and that the paracellular pathway was selective with respect to ion charge and size. In standard conditions, the transepithelial resistance was 28.2 ± 2.1 Ω cm 2, a value indicating that the midgut is a low resistance epithelium. The modulation of midgut SJ by typical enhancers of mammalian tight junction permeability known to act on the cytoskeleton was studied by measuring the shunt resistance and the lumen-to-haemolymph flux of sucrose. An increase of the intracellular level of cAMP and Ca 2+ caused a significant decrease of the shunt resistance and an increase of SJ permeability. The attenuation of Ca 2+ effect in the presence of the calcium channel blocker nifedipine indicated that the influx of external Ca 2+ into the cytoplasm was important for the opening of the SJ, as well as the release of Ca 2+ from the intracellular stores.

Nucleotide regulation of paracellular Cl− permeability in natural rabbit airway epithelium

In this study, we demonstrate a novel regulatory mechanism by which mucosal nucleotides via P2Y receptors decrease paracellular Cl(-) ion permeability in natural rabbit airway epithelium (in addition to a decrease in active Na(+) absorption). In contrast to primary cultures, the natural airway epithelium is a low-resistance epithelium, and an equivalent circuit model predicts that changes of more than approximately 12% in transepithelial conductance (G (t)) must include an effect on paracellular conductance (G (s)). Mucosal P2Y receptor stimulation with uridine triphosphate (UTP; 200 microM) decreased G (t) by up to 50% (average, 24%) and simultaneously decreased the paracellular Cl(-) permeability (mucosa-to-serosa Cl(-) flux) by 16%, but had no effect on mannitol permeability. The G (t) response to UTP was mimicked and attenuated by ionomycin (1 microM), suggesting a dependence on Ca(2+) (i). Amiloride (100 microM) and hyperosmolarity (+75 mM mannitol) also decreased G (t), indicating a role of cell shrinkage. Elevation of cAMP with forskolin (8 microM) or isoproterenol (10 microM) increased G (t) by 55 and 32%, and forskolin increased paracellular Cl(-) permeability by 37% without affecting mannitol permeability. The opposite effects of Ca(2+) (i) and cAMP on G (t) suggest an autocrine nucleotide signaling sequence where P2Y-dependent decrease in passive, paracellular Cl(-) transport is succeeded by a reversion of this effect due to P1-receptor-stimulated cAMP formation by adenosine originating from a time-dependent breakdown of mucosal ATP.

Expression of tight junction proteins in low and high resistance epithelial cell lines MDCK-C7 and -C11

Epithelial tight junctions consist of the transmembranal proteins occludin and several claudins. It is an unresolved puzzle so far, which of these proteins are responsible for forming the paracellular barrier. Expression of the tight junction proteins occludin and claudins 1, 2, 3, 4, and 5 was studied in tight and leaky clones of Madin-Oarby canine kidney (MDCK) cells. As cell adhesion and the paracellular barrier function of epithelia depend on the extracellular calcium concentration, we studied effects of extracellular Ca 2+ on transepithelial electrical resistance (17) and on the expression of occludin and claudins 1, 2, 3, 4, and 5. Methods: Epithelial cell monolayers of Madin-Darby canine kidney (MDCK) cell clones C7 and Cll were grown on porous polycarbonate filters (n = 17-18 / cell strain) at 37 ° C and the resistance was measured on day 7. Ca ~÷ switch experiments were performed employing a 2 or 4 h low extracellular Ca 2+ (2.5/~M) incubation step. Subsequently, tissues were used for membrane protein preparation. The expression of tight junction proteins was detected by immunoblotting. Results: Monolayers of MDCK-Cll revealed features of a low resistance epithelium (52 + 1 g.c--.......~, whereas MDCK-C7 cells exhibited a relatively high resistance (1056 _+ 74 O.cm2). Low extracellular Ca 2+ decreased ? to less than 50 % of initial/7 in monolayers of both clones, without effect of the inhibitor of protein kinases, H7. Occludin and claudin 1, 2, 3, 4, and 5 were detected in all cell lines under investigation. Expression of occludin and claudin 3 did not differ in C7 and Cll. However, claudins 1 and 2 were stronger expressed in Cll cells, whereas the expression of claudin 4 was pronounced in C7. Low Ca 2+ incubation revealed a decrease of claudin 2 in Cll cells whereas other tight junction proteins remained constant. Conclusion: These observations indicate that tight junction contribution by claudins is correlated to barrier properties of mammalian epithelial cell monolayers. In MDCK cells claudins 1 and 2 are strongly expressed in permeable tight junctions, while claudin 4 is predominantly present in tight junctions of low permeability. These observations suggest that barrier properties of mammalian epithelial cells depend on different combinations of claudin proteins. Furthermore, a decrease of claudin 2 during low Ca 2+ incubation was detected, indicating a differential effect of extracellular Ca 2+ on tight junction protein expression.

Junctional proteins and Ca2+ transport in the rat odontoblast-like cell line MRPC-1.

A transcellular bulk flow of Ca2+ ions through the odontoblast layer is of central importance during dentinogenesis. For this, specialized mechanisms may exist, which by a concerted action, gate Ca2+ into the proximal end of the cells and extrude the ions towards the mineralization front. To elucidate these mechanisms, an in vitro model would be useful. Mature odontoblasts are, however, post-mitotic cells and cannot be propagated in cell culture. The aim of the present study was, therefore, to characterize the odontoblast-like rat cell line MRPC-1(1) with regard to transcellular Ca2+ transport, barrier function, and intercellular junctions when cultured on membranes in Transwell chambers. The MRPC-1 cells grew as epithelial-like cells in a continuous bilayer separated by a thin collagenous matrix and with intercellular junctional complexes. They exhibited properties of a low-resistance epithelium, maintained a Ca(2+)-dependent diffusion barrier, and exhibited a functional diversity between the two cell layers. MRPC-1 cells expressed ZO-1, occludin, E-, and N-cadherins in addition to alpha-, beta-, gamma- and p120cat catenins, thereby demonstrating some traits in common with, but also differences from, epithelial cells and major differences from fibroblasts. The transcellular Ca2+ flux was inhibitable by nifedipine unidirectionally, giving evidence for an active intracellular Ca2+ transport through voltage-gated channels of the L-type. Similarities with native odontoblasts indicate that MRPC-1 cells may be useful for in vitro studies of transcellular Ca2+ transport mechanisms of importance for the calcification process.

Comparative Aspects of Actions of a Short-Chain Phospholipid on Epithelial Na + Channels and Tight Junction Conductance

Ion transport in both the frog skin (a high-resistance epithelium) and the rabbit nasal airway epithelium (a low-resistance epithelium) are dominated by electrogenic Na + absorption via apical membrane amiloride-sensitive Na + channels, and short-circuit current (I SC) is essentially a measure of Na + absorption in both epithelia. In both epithelia, mucosal application of the short-chain phospholipid didecanoyl-L- α-phosphatidylcholine (DDPC) dose-dependently inhibited the amiloride-sensitive I SC and caused an initial decrease in epithelial conductance (G t) followed by an increase in G t to steady-state values above control level. The effects were reversible. It is concluded that DDPC (a) inhibits epithelial amiloride-sensitive Na + channels and (b) induces an increase in paracellular tight junction conductance. These effects may involve changes in non-specific lipid-protein interactions at the cell membrane level.

Ultrastructural, histochemical and electrophysiological study of calf gallbladder epithelium

Calf ( Bos taurus) gallbladder, contrary to that of most mammalian, does not concentrate bile. It is to be ascertained whether this phenomenon is the result of a lack of fluid absorption or a balance between the latter and fluid secretion. To this end we began a characterization of bovine gallbladder epithelium by means of electron microscopy, immunocytochemistry, enzymatic activity and electrophysiological measurements. We also partially characterized bile content. The ultrastructural examination showed that surface epithelial cells have the general structure that is observed in absorptive epithelia. Mucous secretory activity is also evident. Moreover, two distinct types of secreting cells line the glands in the lamina propria and contribute to the production of an abundant secretion. The cell surface shows a marked reactivity with the anti-alkaline phosphatase serum. Most of the activity of alkaline phosphatase and L-γ-glutamyltransferase is found at the apical side of the epithelium. Electrophysiological parameters indicate that this is a low resistance epithelium. Therefore, coexistence of features typical of absorptive epithelia and the inability of concentrating bile suggest that, in this organ, fluid absorption and secretion are both present.

A new approach to measuring transepithelial potentials in the bovine lens reveals a chloride‐dependent component

A new approach was used to measure anterior and posterior potentials and resistances of the bovine lens. Segments of the anterior, central and posterior lens were studied by mounting a disc of tissue from either the front or the back of the lens in a double chamber. At 35 degrees C the isolated anterior epithelial preparation exhibited a high potential of 9 mV and a low resistance of 279 omega cm2. The lens epithelium can therefore be classified as a 'leaky' or low resistance epithelium. Amiloride, benzamil and furosemide (frusemide) all reduced the anterior transepithelial potential and increased the resistance, amphotericin B (aqueous side) abolished the potential, whereas SITS (4-acetamido-4'-isothiocyanatostilbene-2,2'-disulphonic acid) had no effect. The posterior lens preparation was found to have a 'transepithelial' of -4 mV. This potential and a proportion of the anterior potential were not sensitive to ouabain, amiloride, furosemide, Na+ removal or raised K+, but were abolished by lowering [Cl-] and reduced by the Cl- channel blocker NPPB. We conclude that chloride plays a significant role in the maintenance of transepithelial potentials at both the anterior and posterior surfaces of the lens.

Voltage-clamp measurements on single split lamellae of posterior gills of the shore crab Carcinus maenas

Single split lamella preparations of posterior gills of low-salt adapted shore crabs Carcinus maenas (collected from the Kiel Bay, Baltic Sea in 1991) were mounted in a micro Ussing-chamber. With NaCl salines on both sides we found an outside positive potential difference (PD) of 6.6±1.3 mV, a short-circuit current (Isc) of-240±65 μAcm-2 and a resistance (Rt) of 25±3 Ωcm2 (n=8). Substitution of Cl- (gluconate) on both sides of the preparation resulted in a decrease of Isc by more than 90% at constant Rt. Isc disappeared and Rt increased after substitution of Na+ (choline). When ouabain (2 mmol l-1) was applied to the internal NaCl-saline, Isc decreased and Rt remained unchanged. Internal addition of 0.1 mmol l-1 acetazolamide left Isc and Rt unaffected. Application of amiloride to the external NaCl saline resulted in a increase of both inward negative Isc and Rt. The dose dependence of the diuretic showed a maximal effect between 50 and 200 μmol l-1 with a half-maximal blocker concentration (KAMI) of ca. 10 μmol l-1. The results show that the split lamella preparation of posterior gills of C. maenas is a low resistance epithelium which is able to effect a massive, electrogenic and coupled absorption of Na+ and Cl-.

Lithium absorption by the rabbit gall‐bladder

Lithium (Li+) absorption across the low-resistance epithelium of the rabbit gall-bladder was studied in order to elucidate possible routes and mechanisms of Li+ transfer. Li+ at a concentration of 0.4 mM in both mucosal and serosal media did not affect isosmotic mucosa-to-serosa fluid absorption. At this low concentration net mucosa-to-serosa Li+ absorption was insignificant when the ambient Na+ concentration was 115 mM, although the gall-bladder had a significant Li+ permeability (2.7 X 10(-5) cm s-1) and a significant mucosa-to-serosa Li+ gradient developed as a result of fluid absorption. Net Li+ absorption was induced at reduced mucosal Na+ concentrations (by lowering the Na+ concentration down to 50 mM with or without substitution with sucrose, or by adding sucrose to the mucosal medium). This Li+ absorption occurred even in the absence of a mucosa-to-serosa Li+ gradient. Na+ and Li+ absorptions occurring at 50 mM Na+ were inhibited to the same degree by mucosal 1 mM amiloride. Substitution of 5-50 mM (44%) Na+ by Li+ in the external medium dose-dependently depressed Na+ absorption by up to 76%, while substitution by 50 mM choline had no significant effect. Li+ inhibition of Na+ absorption was elicited from the mucosal side and was not accounted for by compensatory Li+ absorption; water and Na+ absorption rates decreased nearly in parallel. The effects of 0.4 mM amiloride and of substitution with 20 mM Li+ were only partly additive. It is concluded that Li+ absorption in the rabbit gall-bladder cannot be explained by passive (paracellular) transport, but must be the result of transcellular, active transport. Both at low and at high concentrations Li+ may enter the cell via an Na+/H+ exchanger in the apical cell membrane. At high concentrations Li+ may inhibit Na+ absorption by interference with the exchange mechanism and/or via effects at the cytoplasmic level. The Li+ transfer mechanism across the basolateral cell membrane remains unknown.

Direct demonstration of high transepithelial chloride-conductance in normal human sweat duct which is absent in cystic fibrosis.

The transepithelial electrical potential difference (Vt) and the transepithelial resistance (Rt) were measured across individual microdissected sweat ducts obtained from biopsies of the scapular region of normal human volunteers and of a Cystic Fibrosis (CF) patient. During luminal and contraluminal perfusion with NaCl Ringer solution Vt and Rt were -10.7 +/- 1.1 (mV, lumen negative) and 10.9 +/- 1.7 (omega X cm2). Bilateral substitution of Cl- by gluconate increased Vt and Rt to -42.7 +/- 5.1 (mV) and 98.0 +/- 12.0 (omega X cm2), respectively. Luminal application of amiloride (10(-4) mol/l) collapsed Vt and increased Rt to 11.4 +/- 1.7 and 159.7 +/- 12.0 omega X cm2 in Cl- and gluconate Ringer respectively. These data indicate that 90% of the total transepithelial conductance is attributed to Cl- and 5% to Na+ while the remaining 5% is unaccounted for at present. In CF ducts Vt and Rt were high already in Cl- Ringer solution (-78.9 +/- 9.9 mV and 91.2 +/- 15.6 omega X cm2) and did not change significantly after replacement of Cl- by gluconate. The data demonstrate that normal human sweat duct epithelium, despite its ability to generate high electric and osmotic gradients, is a low resistance epithelium, probably because of a high Cl(-)-permeability of its cell membranes. In addition, the data demonstrate that this Cl(-)-conductance is missing in CF, in conformation of what has been postulated previously from simple potential measurements.

Effect of amiloride on sodium and water reabsorption in the rabbit gall-bladder.

The effects of the Na+-channel-blocking diuretic agent amiloride were assessed in the rabbit gall-bladder epithelium, a low-resistance epithelium with an isosmotic, coupled NaCl transport mechanism. Amiloride caused a rapid, reversible, and dose-dependent decrease in fluid absorption when applied from the mucosal side in concentrations between 8.8 X 10(-5) and 1.76 X 10(-3) M. These concentrations were without effect from the serosal side, suggesting an action of amiloride in the luminal cell membrane as in high-resistance epithelia. Amiloride did not affect the epithelial resistance or the passive serosa-to-mucosa Na+ flux, while net Na+ and water reabsorption were inhibited in parallel. Thus, amiloride did not affect the paracellular tight junction pathway, but inhibited a transcellular, coupled salt and water transport mechanism. The kinetics of the amiloride effect were of a Michaelis-Menten type. The dose of amiloride giving 50% inhibition of fluid absorption (ID50) was 4 X 10(-4) M, a value about three orders of magnitude higher than in high-resistance, Na+-retaining epithelia. The percentage inhibitory effect at each concentration of amiloride increased with increasing rate of spontaneous (control) fluid transport, reaching maximal responses fitting a Michaelis-Menten kinetic with an ID50 of 1.5 X 10(-4) M. No effects of changing the extracellular Na+ concentration between 51 and 145 mequiv/l on the maximal inhibitory effect of amiloride on Na+ and water reabsorption were observed. This suggests a non-competitive type of action of amiloride on a Na+-dependent isosmotic fluid transport mechanism. Removal of mucosal Ca2+ did not alter the effect of amiloride. The implications of these findings are discussed in relation to concepts concerning the mechanism of isosmotic salt and water transport. The data are compatible with the concept that amiloride interferes with a Na+-dependent formation and transcellular transport of isosmotic fluid volumes in a sequestered compartment in the epithelial cells.

Transepithelial migration of human neutrophils: an in vitro model system.

An in vitro model system for studying transepithelial migration of human neutrophils has been developed. Canine kidney epithelial cells grown on micropore filters form a confluent, polarized monolayer with an average transepithelial electrical resistance of 181 ohms.cm2. Neutrophils in a chemotactic chamber are stimulated to undergo random migration, chemokinesis, or chemotaxis through the epithelium. When stimulated by a gradient of the synthetic chemoattractant fMet-Leu-Phe, significantly more neutrophils traverse the low-resistance epithelium than do under conditions of random migration or chemokinesis. Transmission and scanning electron microscopy of this process reveal that neutrophils traverse the epithelium through the intercellular space. After leukocyte emigration, lateral epithelial cell membranes reapproximate. Neutrophils undergoing chemotaxis can also traverse the polarized epithelium from the basal epithelial surface, which suggests that the chemotactic gradient and not the apical-basal polarity of the epithelial cells determines the direction of transepithelial migration. The data further suggest that (i) the in vitro model of leukocyte transepithelial migration morphologically simulates the in vivo process, (ii) neutrophils more readily penetrate the epithelium when attracted by a chemotactic factor, and (iii) neutrophils can traverse a low-resistance epithelium in the absence of serum and connective tissue factors.

Effects of cytochalasin B and dimethylsulphoxide on isosmotic fluid transport by rabbit gall-bladder in vitro.

1. Net fluid transport rate, transepithelial ohmic resistance and potential difference (p.d.), and unidirectional fluxes of Na+ were measured in rabbit gall-bladder preparations in vitro exposed on both sides to Ringer solutions of identical electrolyte composition. 2. Bilateral application of 1% dimethylsulphoxide (DMSO), the solvent for cytochalasin B, rapidly and reversibly depressed net fluid transport rate by 15% and increased the lumen positive p.d. by 1-5-2-0 mV. Resistance did not change significantly. These effects of DMSO were shown to be non-specific osmotic effects. 3. Cytochalasin B (10(-5)M) applied bilaterally caused: (a) a progressive inhibition of net fluid transfer rate to 40-50% of its control value within 60 min; the effect was partly reversible within 60 min and independent of the substrates glucose, glutamate and pyruvate; (b) a progressive depression of the mucosal-to-serosal Na+ flux within the first 30 min with no further change in the flux during the following 30 min of exposure to cytochalasin B; the effect was partly reversible within 70 min; (c) a rapid but moderate increase in the passive serosal-to-mucosal Na+ flux, which continued to increase gradually during the entire 60 min period of exposure to cytochalasin B; the effect was completely reversible within 70 min; (d) a prompt drop in ohmic resistance (30%) and p.d. (40%) with no further changes in these parameters during the following 60 min of exposure to cytochalasin B. The effect on resistance was partly reversible within 90 min; the effect on p.d. was completely reversible within 30 min. 4. The results are interpreted to indicate an early inhibitory action of cytochalasin B on the active transcellular pump mechanism and to suggest a cytochalasin B-mediated progressive increase in cell membrane permeability to sodium resulting ultimately in a highly leaky epithelium. The results are compatible with the concept that a mechanochemical process is involved in isosmotic transcellular transport of fluid across low-resistance epithelia.

A new model for regulation of sodium transport in high resistance epithelia

A new three barrier, four compartment model for sodium transport in high resistance urinary epithelia is presented. This model provides a unified and simplified mechanistic explanation for sodium transport and its quantitative regulation. Sodium enters the epithelial cell by passive diffusion. Active extrusion occurs across the lateral cell membrane into the lateral intercellular space (LICS). Sodium movement from the LICS into the serosal compartment is not free and unobstructed as in the models for low resistance epithelia, but rather occurs through a regulatory channel of the LICS passing through desmosomes and the basilar slit. The exact configuration of this regulatory channel controls the rate of sodium movement from the LICS into the serosal compartment. Thus, the configuration of the regulatory channel controls the afterload on the sodium pump and thus ultimately controls the rate of transepithelial sodium transport. Antidiuretic hormone could act by increasing the effective width of this regulatory channel by contraction of intracellular microtubules or microfilaments. Present theories for regulation of transepithelial sodium transport in high resistance eipithelia invoke a regulatory barrier at the apical cell membrane or at the active sodium pump located in the basolateral cell membrane. The hypothetical model presented here invokes a new alternative: regulation of the active pump rate by the sodium concentration in the LICS serving as an afterload on the pump; sodium escape from the LICS into the serosal compartment thus becomes the regulatory step for transepithelial transport.

The role of the lateral intercellular spaces in the control of ion permeation across the rabbit gall bladder.

Diffusion potentials and conducatance measurements were used to evaluate the changes in permeability of the rabbit gall bladder when the lateral spaces were 1) closed by the addition of sucrose to the mucosal fluid, and 2) dilated by the addition of sucrose to the serosal fluid. The results showed that when the lateral spaces were closed (less than 10 nm/ 1) there was a significant decrease in the conductance of the epithelium, and 2) the ion selectivity of the epithelium moved towards the free solution sequence. The conductance decreased from 31 to 13 mmhos/cm2, and the selectivety changed from Na(1) greater than Li(0.92) greater than Cs(0.85) to Cs(1.27) greater than Na(1) greater than Li(0.84). Neither dilation of the spaces to greater than 1.5 mum nor addition of sucrose to both sides of the gall bladder changed the conductance or the ion selectivity. These results are consistant with the hypothesis that in the gall bladder the major barrier to ion permeation across the epithelium lies in 1) the tight junctions, when the lateral spaces are dilated, 2) the lateral spaces when the spaces are collapsed, and 3) a combination of both the spaces and the junctions when the spaces are reduced much below 0.5 mum. Consequently the status of the lateral intercellular spaces has to be taken into account when assessing the mechanisms of ion permeation across low resistance epithelia.

The effects of electrical and osmotic gradients on lateral intercellular spaces and membrane conductance in a low resistance epithelium.

We have studied the effect of d-c currents and osmotic gradients on the conductance and ultrastructure of the frog gallbladder. Passing current across the epithelium produces three voltage transients which are due to (i) the membrane capacitance, (ii) ion polarization effects, and (iii) changes in membrane resistance. The direction and magnitude of these resistance changes depended on (i) the direction of the current pulse, (ii) the current density, (iii) the ion selectivity of the epithelium, (iv) the thickness of the unstirred layers, and (v) the presence or absence of osmotic gradients across the tissue. Osmotic gradients also change the resistance of the gallbladder. These resistance changes are accompanied by changes in cell structure. Resistance increases are associated with the collapse of the lateral intercellular spaces. We conclude that osmotic and electrical gradients increase tissue resistance by narrowing the spaces and thereby increasing their resistance. On the other hand, we believe that the drop in resistance brought about by both current and osmotic gradients is not fully accounted for by dilation of the spaces, but is related to changes within the epithelial membranes similar to those in high resistance tissues.