NaCl flux between apical and basolateral side recruits claudin-1 to tight junction strands and regulates paracellular transport

In multicellular organisms, epithelia separate and divide the internal environment maintaining appropriate conditions in each compartment. To maintain homeostasis in these compartments, claudins, major cell adhesion molecules in tight junctions (TJs), regulate movements of several substances through the paracellular pathway (barrier function). In this study, we investigated effects of the flux of several substances between apical and basolateral side on paracellular transport and TJ protein localization. NaCl flux from apical to basolateral side increased paracellular conductance (Gp) and recruited claudin-1 from lateral cell membrane to the apical end with the colocalization with occludin, one of the TJ proteins concentrated at TJ strands. Oppositely-directed flux of sucrose against NaCl flux inhibited these reactions and same directional flux of sucrose with NaCl enhanced the increase of Gp, whereas 10-kDa dextran inhibited these reactions regardless of the side of administration. Our present findings indicated that TJ protein localization and barrier function are regulated depending on the environmental differences between apical and basolateral side.     

Lack of correlation between transepithelial transport capacity and paracellular pathway ultrastructure in Alcian blue-treated rabbit gallbladders

The effects of mucosal application of 1 mg% Alcian blue (a trivalent cationic phthalocyanine dye) on functional and ultrastructural parameters of the isolated rabbit gallbladder have been studied. Apart from minor changes in the shape of the group of central microvilli observed in thin-section electron microscopy and scanning electron microscopy, the major ultrastructural change induced by Alcian blue was an almost complete collapse of intercellular spaces in the region above the tight junctions up to the bases of the marginal microvilli as revealed by thin-section electron microscopy. Freeze-fracture electron microscopy demonstrated a complete disappearance of intramembrane particles of neighboring cell membranes corresponding to the region of interspace collapse. Transepithelial electrical resistance (RT) increased from 44.5 to 58.7 ohm . cm2 upon treatment with Alcian blue. This increase could be well accounted for by the observed structural changes in the paracellular pathway if this pathway determines the low resistance of the rabbit gallbladder epithelium. Despite the increase in RT, net mucosa-to-serosa fluid transport and the spontaneous mucosa- positive potential difference of 3 mV were unaltered by Alcian blue treatment, supporting the hypothesis that the transepithelial transport mechanism per se is electroneutral. A calculation of the maximal paracellular mucosa-to-serosa waterflow in response to a lateral intercellular space hypertonicity of 20 mosM demonstrates that in the Alcian blue-treated gallbladder the resulting figure is about three orders of magnitude too low to keep up with the unaltered spontaneous transepithelial net fluid transport. It is therefore concluded that the tight junction pathway in rabbit gallbladders does not serve as a route for net fluid transport.     

Polar protein transport between apical and basal cells during tobacco early embryogenesis

Key message

We found that protein trafficking between apical and basal cell can be unidirectional, which reveals the different roles of the two cells in the cell-to-cell communication between them during early embryogenesis.

Abstract

In most angiosperm species, asymmetric zygote division results in an apical cell and a basal cell that have distinct cell fates. Much has been speculated about possible communication between these cell types in relation to their cell fate determination. Here, we report on the use of photoactivatable green fluorescent protein (PA-GFP) in tobacco to trace intercellular communication between apical and basal cells during early embryogenesis. We found that PA-GFP was transported between apical and basal cells of a two-celled proembryo, and that protein trafficking was unidirectional toward the apical cell, highlighting different cell communication roles. Further ultrastructural analysis showed numerous plasmodesmata in the walls connecting the apical and basal cells, which may provide channels for protein trafficking. Our data show a possible unique method of cell-to-cell communication between apical and basal cells during early embryogenesis.     

Energetics of active transport processes.

Active sodium transport across epithelial membranes has been analyzed by means of linear nonequilibirium thermodynamics. In this formulation the rates of active sodium transport JNa and the associated metabolic reaction Jr are postulated to be linear functions of both the electrochemical potential difference of sodium--XNa and the affinity A (negative free energy) of the metabolic reaction of driving transport. Experimental studies in various epithelia demonstrate that both JNa and Jr (oxygen consumption) are indeed linear functions of XNa. Theoretical considerations and experimental studies in other systems suggest that likelihood of linearity in A as well. If so, A may be evaluated. Several observations indicate that the quantity A evaluated from the thermodynamic formalism does in fact reflect the substrate-product ratio of the metabolic reaction which supports transport. This is in contrast to measurements of mean cellular concentrations, which may not reflect conditions at the site of transport. Associated studies of isotope kinetics permit the distinction between effects on the permeability of the active and passive transport pathways. With these combined approaches, it may prove possible to characterize both the energetic and permeability factors which regulate transport. The formulation has been applied to an analysis of the mechanism of action of the hormone aldosterone.     

Modeling transport processes in sterilization-in-place.

SIP (sterilization-in-place) of equipment using saturated steam is limited by transport processes that restrict the distribution of sterilizing steam. The following are two crucial operations: the removal of air prior to sterilization, and the removal of condensate during the sterilization. Using simple model systems of pipes and tanks, characteristic operating parameters were examined and steady-state models were analyzed. The results were used to evaluate design aspects of SIP, including heat insulation, spacing of steam traps, sloping of lines, steam velocities and consumption, placement of temperature sensors, and scale factors in piping. A more reliable SIP design is achievable by insulating equipment, spacing steam traps to limit condensate buildup, providing an effective air removal operation, and providing reliable, high-quality steam.     

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.     

Interrelations/cross talk between transcellular transport function and paracellular tight junctional properties in lung epithelial and endothelial barriers

In this synopsis of a symposium at EB2007, we start with an overview of noise and impedance analyses that have been applied to various epithelial barriers. Noise analysis yields specific information about ion channels and their regulation in epithelial and endothelial barriers. Impedance analysis can yield information about apical and basolateral membrane conductances and paracellular conductance of both epithelial and endothelial barriers. Using a morphologically based model, impedance analysis has been used to assess changes in apical and basolateral membrane surface areas and dimensions of the lateral intercellular space. Impedance analysis of an in vitro airway epithelial barrier under normal, nucleotide-stimulated, and cigarette smoke-exposed conditions yielded information on how activation and inhibition of secretion occur in airway epithelial cells. Similarly, impedance analysis of primary rat alveolar epithelial cell monolayer model under control and EGTA exposure conditions indicate that EGTA causes decreases in resistances of tight junctional routes as well as apical and basolateral cell membranes without causing much change in cell capacitances. In a stretch-caused injury model of alveolar epithelium, transcellular ion transport function and paracellular permeability of solute transport appear to be differentially regulated. Finally, inhibition of caveolae-mediated transcytosis in lung endothelium led to disruption of paracellular routes, increasing the physical dimension and permeability of tight junctional region. These data together demonstrate the cross talk between transcellular and paracellular transport (function and routes) of lung epithelial and endothelial barriers. Mechanistic (e.g., signaling cascades) information on such cross talk remain to be determined.     

Coupling between transmembrane calcium transport and membrane potential in retinal rod discs

Changes in the transmembrane potential of bovine rod discs were studied by use of the potential-sensitive fluorescence probes diS-C3-(5) and diBA-C4-(5). The disc membrane was shown to be impermeable to potassium ions. Their concentration in the disc is as high as 2.1 +/- 0.3 mM. The permeability of the disc membrane to Ca2+ was shown to be selective. The accumulation and release of Ca2+ were found to be accompanied by the generation of inside positive and inside negative transmembrane potentials, respectively. The uptake of Ca2+ in the discs may operate against the concentration gradient of the ion. The value of the potential developed is directly proportional to the logarithm of free Ca2+ concentration in the medium (delta phi m = 11.2 +/- 1.6 mV at 4.85 microM Ca2+fr). The accumulation of Ca2+ is decreased by sodium ions and totally inhibited by monensin. This indicates that a Na-Ca exchange process participates in Ca2+ uptake of photoreceptor discs.     

Convective paracellular solute flux. A source of ion-ion interaction in the epithelial transport equations

An electrolyte model of an epithelium (a cell and a tight junction in parallel, both in series with a lateral interspace basement membrane) is analyzed using the formalism of nonequilibrium thermodynamics. It is shown that if the parallel structures are heteroporous (i.e., reflection coefficients for two ion species differ between the components), then a cross-term will appear in the overall transport equations of the epithelium. Formally, this cross-term represents an ion-ion interaction. With respect to the rat proximal tubule, data indicating epithelial ionic reflection coefficients less than unity, together with the assumption of no transcellular solvent drag, imply the presence of convective paracellular solute flux. This means that a model applicable to a heteroporous structure must be used to represent the tubule, and, in particular, the cross-terms for ion-ion interaction must also be evaluated in permeability determinations. A series of calculations is presented that permits the estimation of the Na-Cl interaction for rat proximal tubule from available experimental data. One consequence of tubule heteroporosity is that an electrical potential may be substantially less effective than an equivalent concentration gradient in driving reabsorptive ion fluxes.     

Fatty acids alter monolayer integrity, paracellular transport, and iron uptake and transport in Caco-2 cells

The Caco-2 cell line was used as a model to determine if the type of fatty acid, unsaturated versus saturated, differentially altered the uptake and transport of iron in the human intestine and if the changes were the result of alterations in monolayer integrity and paracellular transport. Cells were cultured in either a lower-iron or normal-iron medium and incubated with a bovine serum albumin control, linoleate, oleate, palmatate, or stearate. Oleate, palmatate, and stearate enhanced (p<0.05) iron uptake in cells grown in lower iron. The fatty acid effect on iron uptake by cells grown in normal iron was not as pronounced. Iron transport was not affected (p>0.05) by an interaction between the type of medium (iron concentration) and the type of fatty acid. Iron transport was enhanced (p<0.05) with palmatate and stearate. Various indicators of monolayer integrity and paracellular transport were also affected by the fatty acids, thus impacting iron uptake and transport. These results indicate that oleate, palmatate, and stearic can enhance iron uptake and transport; however, this enhancement may be the result of alterations in the integrity of the intestine. A portion of the data was presented at Experimental Biology 96 as a poster session. E. A. Droke, L. K. Johnson, and H. C. Lukaski. Fatty acids affect iron uptake and transport in Caco-2 cells. FASEB J. 10, 1431 (1996).     

Intracellular processes associated with glycoprotein transport and processing.

The intracellular transport of mucus glycoprotein precursor (apomucin) from endoplasmic reticulum (ER) to Golgi was quantitated by the immunoprecipitation with 3G12 antimucin monoclonal antibody and by estimation of the apomucin glycosylation using UDP-[3H]galactose. The assembly of the entities carrying apomucin to Golgi was assessed by electron microscopy and by quantitation of the incorporation of [14C]choline, [14C]ethanolamine, and [14C]oleic acid into their lipids. The microscopic image of the isolated transport components revealed a population of 80- to 100-nm vesicles with occasional membranes of the ER used for their synthesis. On the average, the vesicles contained 82 ng apomucin/microgram of protein and 80-90% of the total incorporated lipid precursors. From that, 91% of [14C]choline was detected in phosphatidylcholine, and 9% in phosphatidylethanolamine, lysophosphatidylcholine, and sphingomyelin. With [14C]oleate, 54% of the label was incorporated into ceramide, diglyceride, and phosphatidic acid, 35% to phosphatidylcholine, 7% in phosphatidylethanolamine, and 2% in sphingomyelin. After incubation of the vesicles with Golgi, the apomucin was found glycosylated and the lipids of the transport vesicles incorporated into Golgi membranes. The fusion of the vesicular membranes was accompanied by the synthesis of sphingomyelin. In the Golgi, 39-55% of the radiolabeled phosphatidylcholine of transport vesicles was converted to sphingomyelin. The results indicate that the newly synthesized membranes of apomucin transporting vesicles are enriched in phosphoglycerides and ceramides. Upon fusion with the Golgi, the membranes of the vesicles are replenished with sphingomyelin by exchange reaction between phosphatidylcholine and ceramide.     

Carbohydrate-mediated Golgi to cell surface transport and apical targeting of membrane proteins.

Polarized expression of most epithelial plasma membrane proteins is achieved by selective transport from the Golgi apparatus or from endosomes to a specific cell surface domain. In Madin-Darby canine kidney (MDCK) cells, basolateral sorting generally depends on distinct cytoplasmic targeting determinants. Inactivation of these signals often resulted in apical expression, suggesting that apical transport of transmembrane proteins occurs either by default or is mediated by widely distributed characteristics of membrane glycoproteins. We tested the hypothesis of N-linked carbohydrates acting as apical targeting signals using three different membrane proteins. The first two are normally not glycosylated and the third one is a glycoprotein. In all three cases, N-linked carbohydrates were clearly able to mediate apical targeting and transport. Cell surface transport of proteins containing cytoplasmic basolateral targeting determinants was not significantly affected by N-linked sugars. In the absence of glycosylation and a basolateral sorting signal, the reporter proteins accumulated in the Golgi complex of MDCK as well as CHO cells, indicating that efficient transport from the Golgi apparatus to the cell surface is signal-mediated in polarized and non-polarized cells.     

Cytoplasmic dynein regulation by subunit heterogeneity and its role in apical transport.

Despite the existence of multiple subunit isoforms for the microtubule motor cytoplasmic dynein, it has not yet been directly shown that dynein complexes with different compositions exhibit different properties. The 14-kD dynein light chain Tctex-1, but not its homologue RP3, binds directly to rhodopsin's cytoplasmic COOH-terminal tail, which encodes an apical targeting determinant in polarized epithelial Madin-Darby canine kidney (MDCK) cells. We demonstrate that Tctex-1 and RP3 compete for binding to dynein intermediate chain and that overexpressed RP3 displaces endogenous Tctex-1 from dynein complexes in MDCK cells. Furthermore, replacement of Tctex-1 by RP3 selectively disrupts the translocation of rhodopsin to the MDCK apical surface. These results directly show that cytoplasmic dynein function can be regulated by its subunit composition and that cytoplasmic dynein is essential for at least one mode of apical transport in polarized epithelia.     

Regulation of apical constriction via microtubule- and Rab11-dependent apical transport during tissue invagination

The formation of an epithelial tube is a fundamental process for organogenesis. During Drosophila embryonic salivary gland (SG) invagination, Folded gastrulation (Fog)-dependent Rho-associated kinase (Rok) promotes contractile apical myosin formation to drive apical constriction. Microtubules (MTs) are also crucial for this process and are required for forming and maintaining apicomedial myosin. However, the underlying mechanism that coordinates actomyosin and MT networks still remains elusive. Here, we show that MT-dependent intracellular trafficking regulates apical constriction during SG invagination. Key components involved in protein trafficking, such as Rab11 and Nuclear fallout (Nuf), are apically enriched near the SG invagination pit in a MT-dependent manner. Disruption of the MT networks or knockdown of Rab11 impairs apicomedial myosin formation and apical constriction. We show that MTs and Rab11 are required for apical enrichment of the Fog ligand and the continuous distribution of the apical determinant protein Crumbs (Crb) and the key adherens junction protein E-Cadherin (E-Cad) along junctions. Targeted knockdown of crb or E-Cad in the SG disrupts apical myosin networks and results in apical constriction defects. Our data suggest a role of MT- and Rab11-dependent intracellular trafficking in regulating actomyosin networks and cell junctions to coordinate cell behaviors during tubular organ formation.     

Indole-3-acetic acid transport in apical dominance: a quantitative approach. Influence of endogenous and exogenous IAA apical source on inhibitory power of IAA transport

The relationship between the amount of indole-3-acetic acid transported (IAA transport) through the second node of 7-day-old pea seedlings and the degree of inhibition of axillary bud outgrowth at the same node was studied. For both the endogenous apical IAA source (leaves of apical bud) and the exogenous one (lanolin paste containing 0.25–1.0 mg mL^–1 IAA) the slope of linear dependence between inhibition and IAA transport was similar. However, the same IAA transport induced different inhibitions, which were higher for the endogenous source. Moreover, the apical bud induced higher inhibition at the same level of IAA transport when the 4th leaf was present than when it was absent. Apparently, the source of IAA also may regulate the inhibitory power of IAA transported from it. IAA transport appears to consists of active and slightly active one moving along different pathways.Abbreviations a and b coefficients of linear regression of the type y = a+bx; - confidence level of t-test - ELISA enzyme linked immunosorbent assay - GR_1,2 ^e/d growth rate of the lateral bud of experimental/decapitated (control) pea plants at the first and second days after treatment or decapitation - I degree of inhibition of lateral bud outgrowth - IAA indole-3-acetic acid - L_1,2,3 the lengths of lateral bud at 1, 2 or 3rd day after treatment or decapitation of pea plants - n data number - r correlation coefficient - T amount of IAA transported through the second node of pea plant for 3 hours - TIBA 2, 3, 5-triiodobenzoic acid - t-test statistical test used here to compare slopes of linear regressions (y = a+bx) calculated as % MathType!MTEF!2!1!+-% feaafiart1ev1aaatCvAUfeBSjuyZL2yd9gzLbvyNv2CaerbuLwBLn% hiov2DGi1BTfMBaeXatLxBI9gBaerbd9wDYLwzYbItLDharqqtubsr% 4rNCHbGeaGqiVu0Je9sqqrpepC0xbbL8F4rqqrFfpeea0xe9Lq-Jc9% vqaqpepm0xbba9pwe9Q8fs0-yqaqpepae9pg0FirpepeKkFr0xfr-x% fr-xb9adbaqaaeGaciGaaiaabeqaamaabaabaaGcbaGaaeiDaiaabc% cacaqG9aGaaeiiaiaadkgadaWgaaWcbaGaaGymaaqabaGccaqGGaGa% aeylaiaabccacaWGIbWaaSbaaSqaaiaaikdaaeqaaOGaaeiiaiaab+% cacaqGGaWaaOaaaeaacaqGBbaaleqaaOGaaeikaiaabohacaqGLbGa% aeiiaiaadkgadaWgaaWcbaGaaGymaaqabaGccaqGPaWaaWbaaSqabe% aacaqGYaaaaOGaaeiiaiaabUcacaqGGaGaaeikaiaabohacaqGLbGa% aeiiaiaadkgadaWgaaWcbaGaaGOmaaqabaGccaqGPaWaaWbaaSqabe% aacaqGYaaaaOGaaeyxaiaab6caaaa!524A!\[{\text{t = }}b_1 {\text{ - }}b_2 {\text{ / }}\sqrt {\text{[}} {\text{(se }}b_1 {\text{)}}^{\text{2}} {\text{ + (se }}b_2 {\text{)}}^{\text{2}} {\text{]}}{\text{.}}\]     

Calcium regulation in neurons: transport processes.

The ionic stoichiometry of the major Ca2+ transport mechanisms in neurons is still a matter for debate. The past year has seen some particularly interesting developments in this field, not least the finding that the neuronal Na(+)-Ca2+ exchange may be able to transport K+.