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Tight junctions, the most apical of the intercellular junctions that connect individual cells in a epithelial sheet, are thought to form a seal that restricts paracellular and intramembrane diffusion. To analyze the functioning of tight junctions, we generated stable MDCK strain 2 cell lines expressing either full-length or COOH-terminally truncated chicken(More)
An epithelial cell line (MDCK) was used to prepare monolayers which, in vitro, develop properties of transporting epithelia. Monolayers were formed by plating cells at high densities (10(6) cells/cm2) on collagen-coated nylon cloth disks to saturate the area available for attachment, thus avoiding the need for cell division. An electrical resistance(More)
Extracellular Ca2+ triggers assembly and sealing of tight junctions (TJs) in MDCK cells. These events are modulated by G-proteins, phospholipase C, protein kinase C (PKC), and calmodulin. In the present work we observed that 1,2-dioctanoylglycerol (diC8) promotes the assembly of TJ in low extracellular Ca2+, as evidenced by translocation of the(More)
The experimental opening and resealing of occluding junctions in monolayers of cultured MDCK cells (epithelioid of renal origin) was explored by measuring changes in the electrical resistance across the monolayer and by freeze-fracture electron microscopy. As in natural epithelia, the function of occluding junctions as permeability barriers specifically(More)
The tight junction (TJ) is not randomly located on the cell membrane, but occupies a precise position at the outermost edge of the intercellular space and, therefore, is itself considered a polarized structure. This article reviews the most common experimental approaches for studying this relationship. We then discuss three main topics. (a) The mechanisms(More)
Rho family GTPases are important regulators of epithelial tight junctions (TJs); however, little is known about how the GTPases themselves are controlled during TJ assembly and function. We have identified and cloned a canine guanine nucleotide exchange factor (GEF) of the Dbl family of proto-oncogenes that activates Rho and associates with TJs. Based on(More)
Epithelia can adjust the permeability of their paracellular permeation route to physiological requirements, pathological conditions, and pharmacological challenges. This is reflected by a transepithelial electrical resistance (TER) ranging from a few tenth to several thousands Omega.cm(2), depending on the degree of sealing of the tight junction (TJ). The(More)
Patch-clamp techniques were used to study a K channel in the cell membrane of MDCK cells. This cell line derives from the kidney of a normal dog, presumably from the distal nephron, a region involved in potassium secretion. The cells were cultured in confluent monolayers and approached from the apical side. The K channel we describe is Ca2+ and voltage(More)
The outflux of chloride through the isolated skin (JCl31) of the South American frog Leptodactylus ocellatus (L.) is carried by a mechanism that saturates at high concentration of chloride on the inside, and is stimulated by the presence of Cl- in the outer solution (trans side). The presence of Na+ on the outside, by itself, does not increase JCl31.(More)
Madin-Darby canine kidney (MDCK) I and Fisher rat thyroid (FRT) cells exhibit transepithelial electrical resistance (TER) values in excess of 5,000 Omega. cm(2). When these cells were incubated in the presence of various inhibitors of sphingolipid biosynthesis, a >5-fold reduction of TER was observed without changes in the gate function for uncharged(More)