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.     

Derivation of unstirred-layer transport number equations from the Nernst-Planck flux equations.

Since the late 1960s it has been known that the passage of current across a membrane can give rise to local changes in salt concentration in unstirred layers or regions adjacent to that membrane, which in turn give rise to the development of slow transient diffusion potentials and osmotic flows across those membranes. These effects have been successfully explained in terms of transport number discontinuities at the membrane-solution interface, the transport number of an ion reflecting the proportion of current carried by that ion. Using the standard definitions for transport numbers and the regular diffusion equations, these polarization or transport number effects have been analyzed and modeled in a number of papers. Recently, the validity of these equations has been questioned. This paper has demonstrated that, by going back to the Nernst-Planck flux equations, exactly the same resultant equations can be derived and therefore that the equations derived directly from the transport number definitions and standard diffusion equations are indeed valid.     

Phloem transport,solute flux and the kinetics of sap exudation in Ricinus communis L.

The osmotic characteristics of phloem-sap exudation were examined in soil-grown and watercultured plants of Ricinus communis L. Prolonged exudation occurred from bark incisions in water-cultured plants. Fresh incisions caused large alterations in solute flux, but phloem-sap solute potential _s changed by less than ±8% over a period of 7 h. This was associated with a constancy in the levels of sucrose and K⁺, the principal solutes in the sap. Studies with foliar-applied tracers and leaf-excision experiments suggested that exudation was maintained by solute loading from mature leaves. A wide range of mass transfer values through the phloem was found, these being a function of exudation rate. We consider that the exudation process possesses essentially similar characteristics to phloem transport in the intact plant. The way in which bark incisions bring about large changes in solute flux is discussed in terms of the physical properties of the sieve-tube system.Abbreviations water potential - _s solute potential - _p pressure potential     

Influence of cellular and paracellular conductance patterns on epithelial transport and metabolism

Theoretical analysis of transepithelial active Na transport is often based on equivalent electrical circuits comprising discrete parallel active and passive pathways. Recent findings show, however, that Na+ pumps are distributed over the entire basal lateral surface of epithelial cells. This suggests that Na+ that has been actively transported into paracellular channels may to some extent return to the apical (mucosal) bathing solution, depending on the relative conductances of the pathways via the tight junctions and the lateral intercellular spaces. Such circulation, as well as the relative conductance of cellular and paracellular pathways, may have an important influence on the relationships between parameters of transcellular and transepithelial active transport and metabolism. These relationships were examined by equivalent circuit analysis of active Na transport, Na conductance, the electromotive force of Na transport, the "stoichiometry" of transport, and the degree of coupling of transport to metabolism. Although the model is too crude to permit precise quantification, important qualitative differences are predicted between "loose" and "tight" epithelia in the absence and presence of circulation. In contrast, there is no effect on the free energy of metabolic reaction estimated from a linear thermodynamic formalism. Also of interest are implications concerning the experimental evaluation of passive paracellular conductance following abolition of active transport, and the use of the cellular voltage-divider ratio to estimate the relative conductances of apical and basal lateral plasma membranes.     

Autocrine production of insulin-like growth factor-I (IGF-I) affects paracellular transport across epithelial cells in vitro

Autocrine production of growth factors can have significant effects on cell activity. We report for the first time that autocrine production of insulin-like growth factor-I (IGF-I) alters paracellular transport across bovine mammary epithelial cells in vitro. Paracellular transport was assessed by measuring phenol red transport across mammary alveolar cells-large T antigen (MAC-T cells) derived from parental mammary epithelial cells, cultured on porous membranes and compared with two different transfected MAC-T cell lines that constitutively secrete IGF-I. Phenol red transport was essentially blocked in parental cell culture after six days, while IGF-I secreting cells provided essentially no barrier. Surprisingly, neither co-culture studies between parental and IGF-I-secreting cells nor addition of exogenous IGF-I or IGF-binding protein-3 reversed the phenol red transport properties. IGF-I-secreting cells did however express lower levels of the junction components occludin and E-cadherin than parental cells, suggesting that localized autocrine IGF-I activity might lead to increased permeability via changes in both the tight and adherens junction protein levels.     

Proton-coupled solute transport in the animal cell plasma membrane.

This review focuses primarily on the progress made in the last couple of years in the understanding of the intestinal peptide transporter, a prototype for H(+)-coupled solute transport systems in the animal cell plasma membrane. The impressive number of transport systems currently known to be energized by the components of the proton-motive force indicates that the role of H+ as the coupling ion for active transport has not been lost during evolution.     

Direct interaction between linear electron transfer chains and solute transport systems in bacteria

Abstract In studies on alanine and lactose transport in Rhodopseudomonas sphaeroides we have demonstrated that the rate of solute uptake in this phototrophic bacterium is regulated by the rate of lightinduced cyclic electron transfer.
In the present paper the interaction between linear electron transfer chains and solute transport systems was studied in Rhodopseudomonas sphaeroides and Escherichia coli .
The results demonstrate that the activities of alanine transport in Rps. sphaeroides and lactose and proline transport in E. coli are directly controlled by the electron transfer activity in the respiratory chain, under conditions that the proton-motive force remains constant.     

Estimation of paracellular conductance of primary rat alveolar epithelial cell monolayers.

Freshly isolated rat type II pneumocytes, when grown on permeable tissue culture-treated polycarbonate filters, form confluent alveolar epithelial cell monolayers (RAECM). Cells in RAECM undergo transdifferentiation, exhibiting over time morphological and phenotypic characteristics of type I pneumocytes in vivo. We recently reported that transforming growth factor-beta(1) (TGF-beta(1)) decreases overall monolayer resistance (R(te)) and stimulates short-circuit current in a dose-dependent manner. In this study, we investigated the effects of TGF-beta(1) (50 pM) or 10% newborn bovine serum (NBS) on modulation of paracellular passive ion conductance and its contribution to total passive ion conductance across RAECM. On days 5-7 in culture, tight-junctional resistance (R(tj), kOmegacm(2)) of RAECM, cultured in minimally defined serum-free medium (MDSF) with or without TGF-beta(1) or NBS, was estimated from the relationship between observed transmonolayer voltage and resistance after addition of gramicidin D to apical potassium isethionate Ringer solution under open-circuit conditions. NaCl Ringer solution bathed the basolateral side throughout the experimental period. Results showed that transmonolayer conductance (1/R(te)) and tight-junctional conductance (1/R(tj)) are 0.59 and 0.14 mS/cm(2) for control monolayers in MDSF, 1.59 and 0.38 mS/cm(2) for monolayers exposed to TGF-beta(1), and 0.38 and 0.18 mS/cm(2) for monolayers grown in the presence of NBS. The contributions to total transepithelial ion conductance by the paracellular pathway are estimated to be 23, 23, and 47% for control, TGF-beta(1)-exposed, and newborn bovine serum (NBS)-treated RAECM, respectively.     

Regulation of solute flux through plasmodesmata in the root meristem

Plasmodesmata permit solutes to move between cells nonspecifically and without having to cross a membrane. This symplastic connectivity, while straightforward to observe using fluorescent tracers, has proven difficult to quantify. We use fluorescence recovery after photobleaching, combined with a mathematical model of symplastic diffusion, to assay plasmodesmata-mediated permeability in the Arabidopsis (Arabidopsis thaliana) root meristem in wild-type and transgenic lines, and under selected chemical treatments. The permeability measured for the wild type is nearly 10-times greater than previously reported. Plamodesmal permeability remains constant in seedlings treated with auxin (30 mM indoleacetic acid for 2 and 24 h; 100 nm indoleacetic acid for 2 h); however, permeability is diminished in two lines previously reported to have impaired plasmodesmal function as well as in wild-type seedlings treated for 24 h with 0.6 mM tryptophan. Moreover, plasmodesmal permeability is strongly altered by applied hydrogen peroxide within 2 h of treatment, being approximately doubled at a low concentration (0.6 mM) and nearly eliminated at a higher one (6 mM). These results reveal that the plasmodesmata in the root meristem carry a substantial flux of small molecules and that this flux is subject to rapid regulation.     

The interaction between electron transfer,proton motive force and solute transport in bacteria

The properties of proton solute symport have been studied inStreptococcus cremoris, Rhodopseudomonas sphaeroides andEscherichia coli. In the homolactic fermentative organismS. cremoris the efflux of lactate is a membrane proteinmediated process, which can lead to the generation of a proton motive force. These observations support the energy-recycling model that postulates the generation of metabolic energy by end-product efflux. Studies with oxidants and reductants and specific dithiol reagents inE. coli membrane vesicles demonstrated the presence of two redox-sensitive dithiol-disulphide groups in the transport proteins of proline and lactose. The redox state of these groups is controlled by the redox potential of the environment and by the proton motive force. One redox-sensitive group is located at the inner surface, the other at the outer surface of the membrane. InRps. sphaeroides andE. coli the activity of several transport proteins depends on the activity of the electron transfer systems. On the basis of these results a redox model for proton solute transport coupled in parallel to the electron transfer system is postulated.     

Claudins create charge-selective channels in the paracellular pathway between epithelial cells

Epithelia separate tissuespaces by regulating the passage of ions, solutes, and water throughboth the transcellular and paracellular pathways. Paracellularpermeability is defined by intercellular tight junctions, which varywidely among tissues with respect to solute flux, electricalresistance, and ionic charge selectivity. To test the hypothesis thatmembers of the claudin family of tight junction proteins create chargeselectivity, we assessed the effect of reversing the charge of selectedextracellular amino acids in two claudins using site-directedmutagenesis. Claudins were expressed in cultured Madin-Darby caninekidney cell monolayers under an inducible promoter, and clones werecompared with and without induction for transmonolayer electricalresistance and dilution potentials. Expression and localization ofclaudins were determined by immunoblotting, immunofluorescencemicroscopy, and freeze-fracture electron microscopy. We observed thatsubstituting a negative for a positive charge at position 65 in thefirst extracellular domain of claudin-4 increased paracellularNa⁺ permeability. Conversely, substituting positive fornegative charges at three positions in the first extracellular domainof claudin-15, singly and in combination, reversed paracellular charge selectivity from a preference for Na⁺ to Cl.These results support a model where claudins create charge-selective channels in the paracellular space.

     

A mathematical model of solute coupled water transport in toad intestine incorporating recirculation of the actively transported solute

A mathematical model of an absorbing leaky epithelium is developed for analysis of solute coupled water transport. The non-charged driving solute diffuses into cells and is pumped from cells into the lateral intercellular space (lis). All membranes contain water channels with the solute passing those of tight junction and interspace basement membrane by convection-diffusion. With solute permeability of paracellular pathway large relative to paracellular water flow, the paracellular flux ratio of the solute (influx/outflux) is small (2-4) in agreement with experiments. The virtual solute concentration of fluid emerging from lis is then significantly larger than the concentration in lis. Thus, in absence of external driving forces the model generates isotonic transport provided a component of the solute flux emerging downstream lis is taken up by cells through the serosal membrane and pumped back into lis, i.e., the solute would have to be recirculated. With input variables from toad intestine (Nedergaard, S., E.H. Larsen, and H.H. Ussing, J. Membr. Biol. 168:241-251), computations predict that 60-80% of the pumped flux stems from serosal bath in agreement with the experimental estimate of the recirculation flux. Robust solutions are obtained with realistic concentrations and pressures of lis, and with the following features. Rate of fluid absorption is governed by the solute permeability of mucosal membrane. Maximum fluid flow is governed by density of pumps on lis-membranes. Energetic efficiency increases with hydraulic conductance of the pathway carrying water from mucosal solution into lis. Uphill water transport is accomplished, but with high hydraulic conductance of cell membranes strength of transport is obscured by water flow through cells. Anomalous solvent drag occurs when back flux of water through cells exceeds inward water flux between cells. Molecules moving along the paracellular pathway are driven by a translateral flow of water, i.e., the model generates pseudo-solvent drag. The associated flux-ratio equation is derived.     

Global flow equations for membrane transport from local equations of motion—II. The case of a single nonelectrolyte solute plus water

The global flow equations of nonequilibrium thermodynamics for a single nonelectrolyte solute and water passing through a membrane are obtained by solving the local equations of motion. The method follows that developed for the general,n-solute case in the previous paper (Mikulecky, 1978). It is easily seen in this simple case that the passage from local interactions, formulated as position dependent frictional interactions in the equations of motion, to ghe global result involves a loss of any simple way of identifying particulars about local information. Two particular cases are analyzed in further detail: the case of no interaction within the pore and the case of constant interaction for both solute and solvent across the pore. In the former case, Onsager reciprocity survives in the global result if a self-consistent definition of the partial viscosity coefficients is used, while in the latter case, reciprocity is lost. Since, in many biologically interesting cases, the presence of interaction of the type considered here is likely to occur, the reciprocity condition should not automatically be assumed to hold.     

Single-file diffusion multi-ion mechanism of permeation in paracellular epithelial channels

Summary The transepithelial fluxes, conductances and permeabilities of Li⁺, Na⁺, K⁺, Cs⁺, NH ₄ ⁺ and H₃CNH ₃ ⁺ were studied under ionic concentrations ranging from 12 to 250mm inBufo arenarum gallbladders. When these measurements are carefully corrected in order to get only the component due to the paracellular cation channels, the following results are obtained: (1) The permeability ratios (cationic/anionic) are a decreasing function of salt concentration. (2) The partial conductances through paracellular cationic channels show nonlinear saturable concentration kinetics. (3) Moreover, partial conductance kinetics of K⁺, Cs⁺ and NH ₄ ⁺ present a maximum followed, at higher concentratons, by a negative-slope region. (4) The selectivity sequences obtained from biionic potentials do not agree with those obtained from partial conductance measurements. (5) The unidirectional²²Na tracer flux (serosal to mucosal) is inhibited by 63% when the K⁺ symmetrical concentration in the bathing solutions is raised from 25 to 200mm. (6) When the unidirectional⁴²K fluxes (serosal to mucosal) at 200mm KCl Na-free solutions are compared with K⁺ partial conductance by means of the Hodgkin and Keynes (Hodgkin, A.L., Keynes, R.D. 1955.J. Physiol London 128:61–88) expression, then factor is 2.0. These results indicate that cations do not follow the independence principle and behave as in single-file diffusion multi-ion pores when crossing the paracellular cation channels ofBufo arenarum gallbladder epithelium.