Na+ Recirculation and Isosmotic Transport

The Na(+) recirculation theory for solute-coupled fluid absorption is an expansion of the local osmosis concept introduced by Curran and analyzed by Diamond & Bossert. Based on studies on small intestine the theory assumes that the observed recirculation of Na(+) serves regulation of the osmolarity of the absorbate. Mathematical modeling reproducing bioelectric and hydrosmotic properties of small intestine and proximal tubule, respectively, predicts a significant range of observations such as isosmotic transport, hyposmotic transport, solvent drag, anomalous solvent drag, the residual hydraulic permeability in proximal tubule of AQP1 (-/-) mice, and the inverse relationship between hydraulic permeability and the concentration difference needed to reverse transepithelial water flow. The model reproduces the volume responses of cells and lateral intercellular space (lis) following replacement of luminal NaCl by sucrose as well as the linear dependence of volume absorption on luminal NaCl concentration. Analysis of solvent drag on Na(+) in tight junctions provides explanation for the surprisingly high metabolic efficiency of Na(+) reabsorption. The model predicts and explains low metabolic efficiency in diluted external baths. Hyperosmolarity of lis is governed by the hydraulic permeability of the apical plasma membrane and tight junction with 6-7 mOsm in small intestine and < or = 1 mOsm in proximal tubule. Truly isosmotic transport demands a Na(+) recirculation of 50-70% in small intestine but might be barely measurable in proximal tubule. The model fails to reproduce a certain type of observations: The reduced volume absorption at transepithelial osmotic equilibrium in AQP1 knockout mice, and the stimulated water absorption by gallbladder in diluted external solutions. Thus, it indicates cellular regulation of apical Na(+) uptake, which is not included in the mathematical treatment.     

The Chloride Conductance of Tight Junctions of Rat Ileum Can Be Increased by cAMP But Not by Carbachol

It is well known, that in mammalian small intestine, cAMP increases Cl⁻ permeability of the apical membrane of enterocytes as part of its secretory action. Paradoxically, this is usually accompanied by an increase of the transepithelial resistance. In the present study we report that in the presence of bumetanide (to block basolateral Cl⁻ uptake) cAMP always decreased the transepithelial resistance. We examined whether this decrease in resistance was due to a cAMP-dependent increase of the paracellular electrolyte permeability in addition to the increase of the Cl⁻ permeability of the apical cell membrane. We used diffusion potentials induced by serosal replacement of NaCl, and transepithelial current passage to evoke transport number effects. The results revealed that cAMP (but not carbachol) could increase the Cl⁻ permeability of the tight junctions in rat ileum. Moreover, we observed a variation in transepithelial resistance of individual tissue preparations, inversely related to the cation selectivity of the tissue, suggesting that Na⁺ permeability of the tight junctions can vary between preparations. Received: 7 September 1996/Revised: 5 November 1996     

Effects of acute vs. chronic phorbol ester exposure on transepithelial permeability and epithelial morphology.

In previous experiments we have shown that acute (30 minutes) exposure to phorbol esters or other protein kinase C activators causes increased transepithelial permeability, specifically by the increased paracellular permeability through tight junctions. However, the role of protein kinase C activators in carcinogenesis is predicted upon a chronic exposure of an effective dose at frequent intervals for a prolonged period of time. We therefore sought to determine the effect of chronic phorbol ester exposure on transepithelial permeability by exposing cells of the polar renal epithelial cell line, LLC-PK1, to phorbol esters for time periods as long as 16 weeks. The following changes ensued: (1) after the initial drop in transepithelial resistance due to phorbol ester exposure, i.e., an increase in transepithelial permeability (in the acute phase of exposure), an adaptive response occurs as transepithelial resistances in chronically exposed cultures recover to approximately 50% of control values, (2) the cell sheets in chronically exposed cultures lose their acute responsiveness of transepithelial permeability to phorbol ester exposure, (3) cell sheet architecture changes as cells occasionally multilayer and actual polyp-like cell masses appear at high frequency, and (4) cytosolic protein kinase C activity decreases to 50% of control level with acute exposure and then is further decreased to less than 1% of control level in chronically treated cells; membrane-associated PKC activity is not as sharply decreased. The possible role of transepithelial permeability in carcinogenesis and the value of chronically treated epithelial cell cultures as a model for two-stage carcinogenesis are discussed.     

Fetal bovine serum and other sera used in tissue culture increase epithelial permeability

Summary Fetal bovine serum (FBS) or heat-inactivated FBS (56° C for 30 min, HFBS) caused a dose-dependent decrease in the transepithelial electrical resistance of an epithelial monolayer (MDCK). A saturating concentration of HFBS (30%) caused an average fall of 25 ± 2% within 60 min. Upon removal of HFBS, the resistance returned to its starting value within 1 h. Flux studies with [³H]mannitol demonstrate that the fall in resistance is due to an increased permeability of the tight junctions. Thirty percent heat inactivated sera from goat, newborn calf, calf, bovine, and horse caused falls ranging from 26 to 47%. In contrast with the basolateral preference of human and bovine adult sera, fetal bovine and newborn calf sera elicit this response primarily by interacting with the apical surface of the epithelium. HFBS-treated monolayers show a significant increase in the condensation of F-actin at points where ≥3 cells meet. These results demonstrate that FBS and other sera used as nutritional supplements can increase the permeability of the tight junctions of cultured epithelial cells.     

Selective decrease in paracellular conductance of tight junctions: role of the first extracellular domain of claudin-5

Claudin-5 is a protein component of many endothelial tight junctions, including those at the blood-brain barrier, a barrier that limits molecular exchanges between the central nervous system and the circulatory system. To test the contribution of claudin-5 to this barrier function of tight junctions, we expressed murine claudin-5 in Madin-Darby canine kidney II cells. The result was a fivefold increase in transepithelial resistance in claudin-5 transductants and a reduction in conductance of monovalent cations. However, the paracellular flux of neither neutral nor charged monosaccharides was significantly changed in claudin-5 transductants compared to controls. Therefore, expression of claudin-5 selectively decreased the permeability to ions. Additionally, site-directed mutations of particular amino acid residues in the first extracellular domain of claudin-5 altered the properties of the tight junctions formed in response to claudin-5 expression. In particular, the conserved cysteines were crucial: mutation of either cysteine abolishted the ability of claudin-5 to increase transepithelial resistance, and mutation of Cys(64) strikingly increased the paracellular flux of monosaccharides. These new insights into the functions of claudin-5 at the molecular level in tight junctions may account for some aspects of the blood-brain barrier's selective permeability.     

Electrical changes in isolated rat jejunum induced by hypertonicity

Mucosal hypertonicity produces a drop of transepithelial potential difference in isolated rat jejunum with a half time of about 15 s. The same effect is obtained when the osmorality of both bathing solutions is raised simultaneously. Serosal hypertonicity produces an increase of transepithelial potential difference an order of magnitude lower than the drop produced by mucosal hypertonicity. The change in the short circuit current parallels the one in potential difference.When the transepithelial potential difference is varied by adding different concentrations of glucose to the bathing media, the potential drop induced by mucosal hypertonicity is linearly related to the magnitude of the transepithelial potential difference just before the increase in osmolarity.The drop of potential can be explained by a decrease of the electrical resistance of the extracellular shunt pathway due to opening of the tight junctions. The results can be accounted for in terms of an equivalent electrical circuit proposed for small intestine. Using this equivalent circuit model it is possible to obtain estimates of the values of the diffusion potential and the salt gradient across the tight junction.     

Different responses to acute or progressive osmolarity increases in the mIMCD3 cell line

Cells from the kidney medulla are able to survive and function when exposed to high concentrations of NaCl and urea. In vitro, cultured epithelial cells from the kidney medulla are able to survive stronger acute hyperosmotic shocks when both solutes are present. However, in vivo, increases in osmolarity are not acute. In this study, we compared the survival of a murine renal epithelial cell line during acute or progressive (two step) adaptation to hypertonic NaCl and/or urea. Increasing osmolarity to 700 mOsm/l with NaCl or urea in a single step led to massive cell death ( 50% in 24 hours). However, genomic DNA of dying cells was not degraded, and electron microscopy revealed weak condensation of chromatin, absence of membrane blebbing, and no nuclear indentation. Pre-adaptation to permissive concentrations of NaCl (200 mOsm/l giving a final osmolarity of 500 mOsm/l) protected cells against subsequent increases in osmolarity, allowing adaptation to final osmolarities as high as 900 mOsm/l. In contrast, pre-adaptation to permissive concentrations of urea (200 mOsm/l) did not lead to enhanced cell survival after a subsequent 200 mOsm/l step. Cell death was as rapid as after an acute shock, but was more typical of apoptosis (genomic DNA laddering, strong chromatin condensation, nuclear indentation, and blebbing of the membrane giving rise to apoptotic bodies). Thus, acute hyperosmolarity induces cell death with essentially similar responses to NaCl and urea. In contrast, progressive adaptation of mIMCD3 cells to NaCl allows cell survival, whereas progressive adaptation to hyperosmotic urea triggers a cell death pathway different from the one triggered by acute hyperosmotic shocks.     

Different size limitations for increased transepithelial paracellular solute flux across phorbol ester and tumor necrosis factor-treated epithelial cell sheets

By observing increases in the transepithelial paracellular permeability of a range of radiolabeled solutes and electron dense dyes, changes in molecular sieving caused by the cytokine, TNF (tumor necrosis factor), and the phorbol ester, TPA (12-0-tetra-decanoylphorbol-13-acetate), were characterized. Using ¹⁴C-labeled mannitol (mw 182), raffinose (mw 504), PEG (polyethylene glycol; mw 4000), and dextran (mw 10,000, 70,000 and 2,000,000), the transepithelial flux rates of these compounds were determined at the peak of the transepithelial electrical resistance (TER) changes caused by these two agents. TNF treatment resulted in increased permeability across LLC-PK₁ epithelial cell sheets only to relatively small solutes, with an upper limit of approximately 4,000 mw. The low molecular weight “ceiling” for the TNF-treated epithelium is further evidence against TNF increasing transepithelial permeability by means of inducing nonspecific, microscopic “holes” in the epithelium, for which a “ceiling” would not exist. TPA treatment increases transepithelial paracellular permeability to a much broader range of solutes, extending well beyond 2 million mw. Transmission electron micrographs provide evidence that even the electron-dense dye complex, ruthenium red, can cross tight junctions of TPA-treated cell sheets. However, cationic ferritin cannot cross tight junctions of TPA-treated cell sheets. This shows that there is an upper limit to solutes able to cross TPA-treated cell sheets, but that this upper limit will include most proteins, which would then be able to cross tumor promoter-exposed (protein kinase C-activated) epithelial layers at accelerated rates. The biomedical implications for a high molecular weight cutoff in tumor promoter action in epithelial carcinogenesis, and for a low molecular weight cutoff in cytokine-induced epithelial apoptosis in inflammation, are discussed. J. Cell. Physiol. 171:226–233, 1997. © 1997 Wiley-Liss, Inc.     

Biphasic regulation of mammary epithelial resistance by serotonin through activation of multiple pathways

Mammary gland homeostasis and the lactation-to-involution switch are regulated by serotonin (5-hydroxytryptamine (5-HT)). Mammary epithelial tight junctions are physiological targets of 5-HT, and their disruption marks an early stage of mammary gland involution. In these studies, we have identified signal transduction mechanism employed by 5-HT during regulation of mammary gland transepithelial resistance. Transepithelial electrical resistance and tight junction protein architecture were studied in cultures of MCF10A human mammary epithelial cells. Serotonin had biphasic effects on mammary epithelial resistance. At lower concentrations and earlier time points, 5-HT potentiated epithelial transmembrane resistance, whereas at higher concentrations and later time points, 5-HT decreased transepithelial electrical resistance and disrupted tight junctions. Both the early and delayed actions of 5-HT were mediated by the 5-HT7 receptor through activation of G(s)/cAMP. 5-HT induced the activities of both protein kinase A and p38 mitogen-activated protein kinase. Inhibition of p38 mitogen-activated protein kinase abrogated 5-HT-induced disruption of mammary epithelial tight junctions (the delayed effect). In contrast, inhibition of protein kinase A prevented the increased epithelial resistance in response to 5-HT (the transient effect). These studies imply an integrated set of mechanisms whereby transient, modest activation of 5-HT7 promotes tight junction integrity, and sustained 5-HT7 activation drives involution by disrupting tight junctions.     

Knockdown of occludin expression leads to diverse phenotypic alterations in epithelial cells

The function of occludin (Occ) in the tight junction is undefined. To gain insight into its role in epithelial cell biology, occludin levels in Madin-Darby canine kidney II cells were suppressed by stably expressing short interfering RNA. Suppression of occludin was associated with a decrease in claudins-1 and -7 and an increase in claudins-3 and -4. Claudin-2 levels were unaffected. The tight junction "fence" function was not impaired in suppressed Occ (Occ–) clones, as determined by BODIPY-sphingomyelin diffusion in the membrane. The most striking changes were those related to control of the cytoskeleton and the "gate" function of tight junctions. A reduced ability of Occ– clones to extrude apoptotic cells from the monolayers suggested that neighbors of apoptotic cells either failed to sense their presence or were unable to coordinate cytoskeletal activity necessary for their extrusion. To further test the extent to which actin cytoskeletal activity depends on the presence of occludin, Occ– and Occ+ monolayers were depleted of cholesterol. Previous studies showed that cholesterol depletion is associated with reorganization of the actin cytoskeleton and a fall in transepithelial electrical resistance. In contrast to control Occ (Occ+) cells, transepithelial electrical resistance did not fall significantly in cholesterol-depleted Occ– monolayers and they failed to generate Rho-GTP, one of the signaling molecules involved in regulating the actin cytoskeleton. While steady-state transepithelial electrical resistance was similar in all clones, tight junction permeability to mono- and divalent inorganic cations was increased in Occ– monolayers. In addition, there was a disproportionately large increase in permeability to monovalent organic cations, up to 6.96 Å in diameter. Chloride permeability was unaffected and there was little change in mannitol flux. The data suggest that occludin transduces external (apoptotic cells) and intramembrane (rapid cholesterol depletion) signals via a Rho signaling pathway that, in turn, elicits reorganization of the actin cytoskeleton. Impaired signaling in the absence of occludin may also alter the dynamic behavior of tight junction strands, as reflected by an increase in permeability to large organic cations; the permeability of ion pores formed of claudins, however, is less affected. tight junction; occludin; Rho-GTP     

Resistance to osmotic stress of horse spermatozoa: the role of ionic pumps and their relationship to cryopreservation success

To study the resistance of horse spermatozoa against hyperosmotic stress, cells were incubated in solutions of 600 to 4000 mOsm(undisturbed media). Then, semen was immediately placed into an iso-osmotic solution (disrupted media). Incubation in undisturbed media decreased sperm viability in an osmolarity- and temperature-dependent manner. Viability was further decreased in disrupted media, with the effect dependent upon the initial osmolarity of the media and on the temperature. Treatment with ouabain or amiloride impaired the resistance of horse spermatozoa to hyperosmotic stress. Very few correlations were strong between viability after hyperosomotic stress and quality parameters of fresh and frozen-thawed horse semen. The results indicate that the usefulness of resistance to hyperosmotic stress in assessing frozen-thawed semen quality is compromised, since other factors are involved in the resistance to freezing-thawing. Both Na (+)K (+) ATP-ase and the Na (+)H (+) antiporter act in the resistance to hyperosmotic stress in horse spermatozoa.     

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.     

Changes in extracellular osmolality initiate sperm motility in freshwater teleost rosy barb Puntius conchonius

The objective was to investigate the effects of extracellular osmolality and membrane osmotic-sensitive channels on the initiation of sperm motility and to explore mechanisms of sperm initiation in rosy barb (Puntius conchonius). We found that (1) sperm were immotile in seminal plasma and remained quiescent in electrolyte or nonelectrolyte solutions isotonic to seminal plasma; (2) sperm movement was initiated when the sperm were exposed to hypo-osmotic electrolyte or hypo-osmotic nonelectrolyte solutions, and that the responsiveness of sperm to changes in the extracellular osmolalities (100, 200, 250, 270, and 300 mOsm/kg) differed among sperm cells (P < 0.05); (3) sperm movement could be initiated and terminated repeatedly by decreasing and increasing the osmolality (in increments of 100 and 300 mOsm/kg) of a nonelectrolyte mannitol solution, respectively (P < 0.05); (4) gadolinium (20, 40, and 80 μM) inhibited the initiation of sperm motility and abolished the sperm activation caused by the hypo-osmotic media treatment in dose- and time-dependent manners (P < 0.05); and (5) sperm activation in a hypo-osmotic medium and inhibition in an isotonic solution were associated with swelling and shrinkage of the sperm sleeves, respectively. Therefore, we concluded that osmolality was a critical physiologic signal in regulating the initiation and termination of sperm motility in freshwater teleost rosy barb. Furthermore, we inferred that rosy barb sperm were hypo-osmotic–dependent conformers, and the osmotic-sensitive channel could be involved in the mechanism of sperm initiation.     

Opening of blood-brain barrier in Triturus cristatus carnifex by hyperosmolar mannitol solutions

The permeability of the newt cerebral capillaries to lanthanum ion has been studied after perfusion with mannitol solutions of increasing molarity. In the control specimens lanthanum deposits were limited to the luminal side of the capillaries and tracer did not spread to the pericapillary spaces due to the tight junctions. Treatment with hypertonic solutions of mannitol (0.25M, 0.5M, 1M) caused opening of the blood brain barrier with a progressive increase in lanthanum between the endothelial cell edges, in the basal lamina and in the extracellular spaces of the nervous parenchyma in relation to the molarity of the mannitol solution. The spread of lanthanum is probably due to opening of the tight junctions between the endothelial cells, since pinocytotic vesicles labelled with tracer were not evident.     

Contribution of claudin-5 to barrier properties in tight junctions of epithelial cells

Claudin-5 is a transmembrane protein reported to be primarily present in tight junctions of endothelia. Unexpectedly, we found expression of claudin-5 in HT-29/B6 cells, an epithelial cell line derived from human colon. Confocal microscopy showed colocalization of claudin-5 with occludin, indicating its presence in the tight junctions. By contrast, claudin-5 was absent in the human colonic cell line Caco-2 and in Madin-Darby canine kidney cells (MDCK sub-clones C7 and C11), an epithelial cell line derived from the collecting duct. To determine the contribution of claudin-5 to tight junctional permeability in cells of human origin, stable transfection of Caco-2 with FLAG-claudin-5 cDNA was performed. In addition, clone MDCK-C7 was transfected. Synthesis of the exogenous FLAG-claudin-5 was verified by Western blot analysis and confocal fluorescent imaging by employing FLAG-specific antibody. FLAG-claudin-5 was detected in transfected cells in colocalization with occludin, whereas cells transfected with the vector alone did not exhibit specific signals. Resistance measurements and mannitol fluxes after stable transfection with claudin-5 cDNA revealed a marked increase of barrier function in cells of low genuine transepithelial resistance (Caco-2). By contrast, no changes of barrier properties were detected in cells with a high transepithelial resistance (MDCK-C7) after stable transfection with claudin-5 cDNA. We conclude that claudin-5 is present in epithelial cells of colonic origin and that it contributes to some extent to the paracellular seal. Claudin-5 may thus be classified as a tight-junctional protein capable of contributing to the "sealing" of the tight junction.     

Occludin modulates transepithelial migration of neutrophils

Neutrophils cross epithelial sheets to reach inflamed mucosal surfaces by migrating along the paracellular route. To avoid breakdown of the epithelial barrier, this process requires coordinated opening and closing of tight junctions, the most apical intercellular junctions in epithelia. To determine the function of epithelial tight junction proteins in this process, we analyzed neutrophil migration across monolayers formed by stably transfected epithelial cells expressing wild-type and mutant occludin, a membrane protein of tight junctions with four transmembrane domains and both termini in the cytosol. We found that expression of mutants with a modified N-terminal cytoplasmic domain up-regulated migration, whereas deletion of the C-terminal cytoplasmic domain did not have an effect. The N-terminal cytosolic domain was also found to be important for the linear arrangement of occludin within tight junctions but not for the permeability barrier. Moreover, expression of mutant occludin bearing a mutation in one of the two extracellular domains inhibited neutrophil migration. The effects of transfected occludin mutants on neutrophil migration did not correlate with their effects on selective paracellular permeability and transepithelial electrical resistance. Hence, specific domains and functional properties of occludin modulate transepithelial migration of neutrophils.     

Hyperosmotic stress contributes to mouse colonic inflammation through the methylation of protein phosphatase 2A

There are several reports suggesting hyperosmotic contents in the feces of patients suffering from inflammatory bowel disease (IBD). Previous works have documented that hyperosmolarity can cause inflammation attributable to methylation of the catalytic subunit of protein phosphatase 2A (PP2A) and subsequent NF-kappaB activation resulting in cytokine secretion. In this study, we demonstrate that dextran sulfate sodium (DSS) induces colitis due to hyperosmolarity and subsequent PP2A activation. Mice were randomized and fed with increased concentrations of DSS (0 mOsm, 175 mOsm, 300 mOsm, and 627 mOsm) for a duration of 3 wk or with hyperosmotic concentrations of DSS (627 mOsm) or mannitol (450 mOsm) for a duration of 12 wk. Long-term oral administration of hyposmotic DSS or mannitol had no demonstrable effect. Hyperosmotic DSS or mannitol produced a significant increase in colonic inflammation, as well as an increase in the weight of sacral lymph nodes and in serum amyloid A protein levels. Similar results were obtained through the ingestion of comparable osmolarities of mannitol. Hyperosmolarity induces the methylation of PP2A, nuclear p65 NF-kappaB activation. and cytokine secretion. The rectal instillation of okadaic acid, a well-known PP2A inhibitor, reverses the IBD. Short inhibiting RNAs (siRNAs) targeted toward PP2Ac reverse the effect of hyperosmotic DSS. The present study strongly suggests that DSS-induced chronic colitis is a consequence of the methylation of PP2Ac induced by hyperosmolarity.     

Serum Opens Tight Junctions and Reduces ZO-1 Protein in Retinal Epithelial Cells

Abstract: We have shown previously that serum inhibits tight junction formation in a retinal epithelial cell culture model for the blood-brain barrier. We have now examined in detail the effects of serum on the tight junctions. Our data show that serum induces a breakdown in tight junction function as indicated by decreased transepithelial electrical resistance and increased permeability. Rat serum had effects similar to those of bovine serum, indicating that the activity is species-independent. The effect is concentration-dependent, reversible, and specific for the apical surface, suggesting the involvement of a specific receptor-ligand interaction. Differences in the time course, response magnitude, and structural manifestations between the serum-induced breakdown and that induced by switching the cultures to a low-calcium medium suggest fundamental differences in their mechanisms. The calcium switch results in an immediate and complete junctional breakdown with cell retraction and perinuclear translocation of both actin and the tight junction protein zonula occludens-1. The serum-induced breakdown occurs slowly, is incomplete, and is manifested structurally by decreases in zonula occludens-1 protein, whereas actin organization is unchanged. Thus, serum induces a specific breakdown in retinal epithelial cell tight junctions that may be mediated by effects on the expression of zonula occludens-1.     

Ras mutation impairs epithelial barrier function to a wide range of nonelectrolytes

Although ras mutations have been shown to affect epithelial architecture and polarity, their role in altering tight junctions remains unclear. Transfection of a valine-12 mutated ras construct into LLC-PK1 renal epithelia produces leakiness of tight junctions to certain types of solutes. Transepithelial permeability of D-mannitol increases sixfold but transepithelial electrical resistance increases >40%. This indicates decreased paracellular permeability to NaCl but increased permeability to nonelectrolytes. Permeability increases to D-mannitol (Mr 182), polyethylene glycol (Mr 4000), and 10,000-Mr methylated dextran but not to 2,000,000-Mr methylated dextran. This implies a "ceiling" on the size of solutes that can cross a ras-mutated epithelial barrier and therefore that the increased permeability is not due to loss of cells or junctions. Although the abundance of claudin-2 declined to undetectable levels in the ras-overexpressing cells compared with vector controls, levels of occludin and claudins 1, 4, and 7 increased. The abundance of claudins-3 and -5 remained unchanged. An increase in extracellular signal-regulated kinase-2 phosphorylation suggests that the downstream effects on the tight junction may be due to changes in the mitogen-activated protein kinase signaling pathway. These selective changes in permeability may influence tumorigenesis by the types of solutes now able to cross the epithelial barrier.     

Hyperosmolarity and the Net Transport of Nonelectrolytes in Frog Skin

The permeability of frog skin to a series of nonelectrolytes (thiourea, urea, mannitol, and sucrose) under the influence of 2.5 times normal osmolarity in the outer bathing solution has been investigated. Although the flux of the tracer nonelectrolytes across the skin in either direction is greatly increased by hyperosmolarity, the influx is found to be increased to a significantly greater extent than the outflux. Flux ratios as high as 3:1 can be observed. The net inward movement of the nonelectrolyte proceeds in spite of a sizeable bulk flow of water in the opposite direction. Possible driving forces for this phenomenon are discussed.