Effects of T cell-induced colonic inflammation on epithelial barrier function.

Background: Epithelial barrier disturbance is thought to contribute to the pathogenesis of inflammatory bowel diseases; however, it remains unclear whether it is a primary defect participating to the onset of inflammation or only a consequence of sustained inflammation.

Methods: A time course study of epithelial barrier functions and immune mediators was performed in the CD4(+)CD45RB(hi) T cell transfer model of colitis using Ussing chambers.

Results: In nonreconstituted severe combined immunodeficiency (SCID) mice, no epithelial dysfunction was observed.

Human ClC-6 is a late endosomal glycoprotein that associates with detergent-resistant lipid domains.

Background: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7.

Principal Findings: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M.

Opposite effects of Ni2+ on Xenopus and rat ENaCs expressed in Xenopus oocytes.

The epithelial Na+ channel (ENaC) is modulated by various extracellular factors, including Na+, organic or inorganic cations, and serine proteases. To identify the effect of the divalent Ni2+ cation on ENaCs, we compared the Na+ permeability and amiloride kinetics of Xenopus ENaCs (xENaCs) and rat ENaCs (rENaCs) heterologously expressed in Xenopus oocytes. We found that the channel cloned from the kidney of the clawed toad Xenopus laevis [wild-type (WT) xENaC] was stimulated by external Ni2+, whereas the divalent cation inhibited the channel cloned from the rat colon (WT rENaC).

Extracellular Ca2+ regulates the stimulation of Na+ transport in A6 renal epithelia.

We investigated the involvement of intracellular and extracellular Ca2+ in the stimulation of Na+ transport during hyposmotic treatment of A6 renal epithelia. A sudden osmotic decrease elicits a biphasic stimulation of Na+ transport, recorded as increase in amiloride-sensitive short-circuit current (Isc) from 3.4 +/- 0.

The transoocyte voltage clamp: a non-invasive technique for electrophysiological experiments with Xenopus laevis oocytes.

We developed a non-invasive technique for electrophysiological investigations of ion transport proteins endogenously or heterologously expressed in Xenopus laevis oocytes. We named this technique the transoocyte voltage clamp (TOVC). Whereas in the classical two-microelectrode voltage-clamp (TEVC) technique, the oocyte is impaled with two glass microelectrodes, we mount the egg in a modified Ussing chamber as used for transepithelial electrophysiological studies.

Zinc is a voltage-dependent blocker of native and heterologously expressed epithelial Na+ channels.

Zn(2+) (1-1,000 microM) applied to the apical side of polarized A6 epithelia inhibits Na(+) transport, as reflected in short-circuit current and conductance measurements. The Menten equilibrium constant for Zn(2+) inhibition was 45 microM. Varying the apical Na(+) concentration, we determined the equilibrium constant of the short-circuit current saturation (34.

Hypotonic treatment evokes biphasic ATP release across the basolateral membrane of cultured renal epithelia (A6).

In renal A6 epithelia, an acute hypotonic shock evokes a transient increase in the intracellular Ca(2+) concentration ([Ca(2+)](i)) through a mechanism that is sensitive to the P2 receptor antagonist suramin, applied to the basolateral border only. This finding has been further characterized by examining ATP release across the basolateral membrane with luciferin-luciferase (LL) luminescence. Polarized epithelial monolayers, cultured on permeable supports were mounted in an Ussing-type chamber.

Stimulatory effects on Na+ transport in renal epithelia induced by extracts of Nigella arvensis are caused by adenosine.

Effects of the extract of Nigella arvensis (NA) seeds on transepithelial Na(+) transport were studied in cultured A6 toad kidney cells by recording short-circuit current (I(sc)), transepithelial conductance (G(T)), transepithelial capacitance (C(T)) and fluctuation in I(sc). Apical application of NA extract had merely a small stimulatory effect on Na(+) transport, whereas basolateral administration markedly increased I(sc), G(T) and C(T). A maximal effect was obtained at 500 microll(-1) of lyophilized NA extract.

Mg(2+)-sensitive non-capacitative basolateral Ca(2+) entry secondary to cell swelling in the polarized renal A6 epithelium.

Polarized renal A6 epithelia respond to hyposmotic shock with an increase in transepithelial capacitance (C(T)) that is inhibited by extracellular Mg(2+). Elevation of free cytosolic [Ca(2+)] ([Ca(2+)](i)) is known to increase C(T). Therefore, we examined [Ca(2+)](i) dynamics and their sensitivity to extracellular Mg(2+) during hyposmotic conditions.