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  • 1
    ISSN: 1432-1424
    Keywords: epithelia ; tight junctions ; G-protein ; Ca2+-MDCK phospholipase C ; protein kinase C ; calmodulin ; exocytic fusion
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary The making and sealing of a tight junction (TJ) requires cell-cell contacts and Ca2−, and can be gauged through the development of transepithelial electrical resistance (TER) and the accumulation of ZO-1 peptide at the cell borders. We observe that pertussis toxin increases TER, while AIF3 and carbamil choline (carbachol) inhibit it, and 5-guanylylimidodiphosphate (GTPΓs) blocks the development of a cell border pattern of ZO-1, suggesting that G-proteins are involved. Phospholipase C (PLC) and protein kinase C (PKC) probably participate in these processes since (i) activation of PLC by thyrotropin-1 releasing hormone increases TER, and its inhibition by neomycin blocks the development of this resistance; (ii) 1,2-dioctanoylglycerol, an activator of PKC, stimulates TER development, while polymyxin B and 1-(5-isoquinoline sulfonyl)-2-methyl-piperazine dihydrochloride (H7), which inhibit this enzyme, abolish TER. Addition of 3-isobutyl-1-methyl-xanthine, dB-cAMP or forskolin do not enhance the value of TER, but have just the opposite effect. Trifluoperazine and calmidazoline inhibit TER development, suggesting that calmodulin (CaM) also plays a role in junction formation. These results indicate that junction formation may be controlled by a network of reactions where G-proteins, phospholipase C, adenylate cyclase, protein kinase C and CaM are involved.
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  • 2
    ISSN: 1432-1424
    Keywords: Key words: Epithelia — Cell/cell junctions — Tight junctions — Apical/basolateral polarity — Transepithelial electrical resistance — Lipid composition — Occludin — Paracellular
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract. Tight junctions (TJs) are cell-to-cell contacts made of strands, which appear as ridges on P faces and complementary furrows on E faces on freeze fracture replicas. Evidences and opinions on whether these strands are composed of either membrane-bound proteins or lipid micelles are somewhat varied. In the present work we alter the lipid composition of Madin-Darby canine kidney monolayers using a novel approach, while studying (i) their transepithelial electrical resistance, a parameter that depends on the degree of sealing of the TJs; (ii) the apical-to-basolateral flux of 4 kD fluorescent dextran (JDEX), that reflects the permeability of the intercellular spaces; (iii) the ability of TJs to restrict apical-to-basolateral diffusion of membrane lipids; and (iv) the pattern of distribution of endogenous and transfected occludin, the sole membrane protein presently known to form part of the TJs. We show that changing the total composition of phospholipids, sphingolipids, cholesterol and the content of fatty acids, does not alter TER nor the structure of the strands. Interestingly, enrichment with linoleic acid increases the JDEX by 631%. The fact that this increase is not reflected in a decrease of TER, suggests that junctional strands do not act as simple resistive elements but may contain mobile translocating mechanisms.
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  • 3
    ISSN: 1432-1424
    Keywords: epithelial monolayers ; MDCK cells ; tight junctions ; calcium ; biosynthesis of junctions ; junctional assembly ; apical/basolateral polarization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Synthesis and assembly of tight junctions are studied in monolayers of MDCK cells plated at a density sufficient for confluence, allowed to attach for 1 hr, and transferred to fresh media without cells containing or not Ca2+, 20 hr later, while monolayers with Ca2+ have fully developed junctions that confer an electrical resistance across of 346±51 Ω cm2, those without Ca2+ have a negligible resistance. If at this time Ca2+ is added, junctions assemble and seal with a fast kinetics, that can be followed through the development of electrical resistance, penetration of ruthenium red, and electron microscopy. Drugs that impair synthesis, maturation and transport of proteins (cycloheximide, tunicamycin, monensin) indicate that protein components are synthesized early upon plating, do not seem to require N-glycosylation, and are stored in the Golgi compartment. Upon addition of Ca2+ they are transferred to the membrane with the participation of microfilaments but not of microtubules. These components seem to insert directly in the position they occupy in the strands, and the cell circles its perimeter with one strand as early as 15 min, even if in some segments it only consists of a row of particles. New strands develop in association with previous ones, and the pattern completes in 4 to 6 hr. Ca2+ is required for the maintenance of the assembly and also for the sealing with neighboring cells. These processes cannot occur below 25°C. Serum is not required. Polarized distribution of intramembrane particles (IMP) in apical and basolateral regions follows the same time course as junction formation, in spite of the fence constituted by those strands that are already assembled. This suggests that IMP do not redistribute by lateral displacements in the plane of the membrane, but by removal and insertion in the apical and basolateral domains.
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  • 4
    ISSN: 1432-1424
    Keywords: tight junctions ; cell attachment ; epithelia ; cultured monolayers ; freeze fracture ; ruthenium red ; paracellular route
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary Epithelial cells establish tight junctions (TJs) that offer an ample range of transepithelial electrical resistances (TER), in adjustment to physiological requirements. In the present work, we demonstrate that cells from different animal origins, co-cultured in monolayers, can make sealed TJs, suggesting that this structure has a basic universal structure. TJs cannot be established, however, if one of the partners does not normally express TJs, indicating that each neighbor has to contribute its moiety. Furthermore, we observe that clones of the same cell line, with widely different values of TER, do not differ, in the number and length of their junctional trands, suggesting that the difference is due to their ability to express ionic channels traversing their strands. The value of TER achieved in mixed monolayers of cells of the same or different lines is the one that may be expected by taking into account the proportion of each type in the mixture and adding in parallel the electrical resistance that they exhibit in pure monolayers. Therefore, epithelial TJs appear to behave as parallel resistances.
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  • 5
    ISSN: 1432-1424
    Keywords: Epithelia ; K+ channels ; Expression of ion channels ; Ca2+ ; Membrane area ; Cell-cell contacts
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Harvesting MDCK cells with trypsin-EDTA reduces potassium currents (I K) to a mere 10%, presumably by hydrolysis of K+ channels, but replating at confluence restores them in 12–18 hr, through a process that requires transcription, translation and exocytic fusion of intracellular membrane vesicles to the plasma membrane (Ponce & Cereijido, 1991; Ponce et al., 1991a). In the present work we find that this restoration of I K also requires cell-cell contacts and the presence of 1.8 mm Ca2+. The role of extracellular Ca2+ may be substituted by 2.0 μm TRH, 10 nm PMA or 200 μg/ml DiC8, drags that stimulate the system of phospholipase C (PLC) and protein kinase C (PKC). Conversely, the recovery of I K triggered by Ca-dependent contacts can be blocked by 110 μm neomycin, 2.0 μm H7, and 250 nm staurosporine, inhibitors of PLC and PKC. These results suggest that the expression of new K+ channels depends on Ca2+-activated contacts with neighboring cells and that the information is conveyed through PLC and PKC, a process in keeping with changes in its enzymatic activity and cellular distribution of PKC. Plasma membrane is also reduced and restored upon harvesting and replating, and depends on Ca2+-activated contracts. However, the effects of the chemicals tested on I K differ from the ones they elicit on the recovery of plasma membrane, suggesting that cells can independently regulate their population of K+ channels and the surface of their membrane.
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  • 6
    ISSN: 1432-1424
    Keywords: epithelial monolayers ; MDCK cells ; occluding junctions ; intramembrane particles ; electrical resistance ; temperature
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Summary In previous works it was demonstrated that the monolayer of MDCK cells behaves as a leaky epithelium where the electrical resistance across reflects the sealing capacity of the occluding junction. In the present work we study whether this sealing capacity can be modified by temperature and whether this is accompanied by changes in the structure of the occluding junction. Monolayers were prepared on disks of nylon cloth coated with collagen and mounted as a flat sheet between two Lucite chambers. The changes in resistance elicited by temperature were large (306% between 3 and 37°C), fast (less than 2 sec), and reversible. An Arrhenius plot of conductance versus the inverse of temperature shows a broken curve (between 22 and 31°C), and the activation energies calculated (3.2 and 4.0 kcal·mol−1) fall within the expected values for processes of simple diffusion. The morphology of the occuluding the number of evaluated in freeze-fracture replicas by counting the number of strands and the width of the band occupied by the junction every 133 nm. In spite of the change by 306% of the electrical resistance and the phase transition, we were unable to detect any appreciable modification of the morphology of the occluding junction. Since the freeze-fracture replicas also show a density of intramembrane particles (IMP) different in the apical from that in the basolateral regions of the plasma membrane, as well as differences between faceE and faceP, we also investigated whether this is modified by temperature. Cold increases the population of IMP, but does not affect their polarization with the incubation time it takes to elicit changes in electrical resistance.
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  • 7
    ISSN: 1432-1424
    Keywords: Na+, K+-pump ; Na+, K+-ATPase ; K+ content ; Epithelial cells ; Proliferation ; Attachment ; Ouabain ; Chloroquine ; Strophanthidin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Na+, K+-pumps of most eukaryotic animal cells bind ouabain with high affinity, stop pumping, and consequently loose K+, detach from each other and from the substrate, and die. Lack of affinity for the drug results in ouabain resistance. In this work, we report that Ma104 cells (epithelial from Rhesus monkey kidney) have a novel form of ouabain-resistance: they bind the drug with high affinity (Km about 4×10−8 m), they loose their K+ and stop proliferating but, in spite of these, up to 100% of the cells remain attached in 1.0 μm ouabain, and 53% in 1.0 mm. When 4 days later ouabain is removed from the culture medium, cells regain K+ and resume proliferation. Strophanthidin, a drug that attaches less firmly than ouabain, produces a similar phenomenon, but allows a considerably faster recovery. This reversal may be associated to the fact that, while in ouabain-sensitive MDCK cells Na+, K+-ATPases blocked by the drug are retrieved from the plasma membrane, those in Ma104 cells remain at the cell-cell border, as if they were cell-cell attaching molecules. Cycloheximide (10 μg/ml) and chloroquine (10 μm) impair this recovery, suggesting that it also depends on the synthesis and insertion of a crucial protein component, that may be different from the pump itself. Therefore ouabain resistance of Ma104 cells is not due to a lack of affinity for the drug, but to a failure of its Na+, K+-ATPases to detach from the plasma membrane in spite of being blocked by ouabain.
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  • 8
    ISSN: 1432-1424
    Keywords: Ouabain-resistance ; Epithelia ; Ma104 cells ; MDCK cells ; Tight junctions ; Na+, K+-ATPase ; K+ content ; Metabolic cooperation ; Cell attachment
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Ma104 cells (renal, epithelial) have a peculiar way of resisting ouabain: their Na+,K+-pumps bind the drug with high affinity, cellular K+ is lost and cell division arrested, but cells do not detach as most cell types do. Then, if up to 4 days later the drug is removed, Ma104 cells recover K+ and resume proliferation (Contreras et al., 1994). In the present work, we investigate whether Ma104 cells are able to protect ouabain-sensitive MDCK cells in co-culture. The main finding is that they do, but in this case protection is not elicited by the usual mechanism of maintaining the K+ content of neighboring cells through cell-cell communications. Ma104 cells treated with ouabain simply remain attached to the substrate and to their MDCK neighbors, and both cells lose K+. This attachment includes tight junctions, because the transepithelial electrical resistance of the monolayers is not abolished by ouabain. Although the β-subunit of the Na+,K+-ATPase is known to possess molecular characteristics of cell-cell attachment molecules, attachment between Ma104-MDCK cells does not seem to be mediated by this enzyme, as immunofluorescence analysis reveals that Na+,K+-ATPase is only inserted in the plasma membrane facing a neighboring cell of the same type.
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  • 9
    Electronic Resource
    Electronic Resource
    Palo Alto, Calif. : Annual Reviews
    Annual Review of Physiology 51 (1989), S. 785-795 
    ISSN: 0066-4278
    Source: Annual Reviews Electronic Back Volume Collection 1932-2001ff
    Topics: Medicine , Biology
    Type of Medium: Electronic Resource
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