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Modulation of epithelial [Na.sup.+] channel activity by long-chain n-3 fatty acids. Am J Physiol Renal Physiol 287: F850-F855, 2004. First published June 15, 2004; 10.1152/ajprenal. 00078.2004.--The epithelial sodium channel is found in apical membranes of a variety of native epithelial tissues, where it regulates sodium and fluid balance. In vivo, a number of hormones and other endogenous factors, including polyunsaturated fatty acids (PUFAs), regulate these channels. We tested the effects of essential n-3 and n-6 PUFAs on amiloride-sensitive sodium transport in A6 epithelial cells. Eicosapentaenoic acid [EPA: C20:5(n-3)] transiently stimulated amiloride-sensitive open-circuit current ([I.sub.Na]) from 4.0 [+ or -] 0.3 to 7.7 [+ or -] 0.3 [micro]A/[cm.sup.2] within 30 min (P < 0.001). No activation was seen in the presence of 10 [micro]M amiloride. In cell-attached but not in cell-excised patches, EPA acutely increased the open probability of sodium channels from 0.45 [+ or -] 0.08 to 0.63 [+ or -] 0.10 (P = 0.02, paired t-test), n-6 PUFAs, including linoleic acid (C18:2), eicosatetraynoic acid (C20: 4), and docosapentanoic acid (C22:5) had no effect, whereas n-3 docosahexanoic acid (C22:6) activated amiloride-sensitive [I.sub.Na] in a manner similar to EPA. Activation of [I.sub.Na] by EPA was prevented by H-89, a PKA inhibitor. Similarly, PKA activity was stimulated by EPA. Nonspecific stimulation of phosphodiesterase activity by Co[Cl.sub.2] completely prevented the effect of EPA on sodium transport. We conclude that n-3 PUFAs activate epithelial sodium channels downstream of cAMP in a cAMP-dependent pathway also involving PKA. patch clamp: epithelial sodium channel; PKA; A6 cells; amiloride

The epithelial sodium channel is found in apical membranes of a variety of native epithelial tissues, where it regulates sodium and fluid balance. In vivo, a number of hormones and other endogenous factors, including polyunsaturated fatty acids (PUFAs), regulate these channels. We tested the effects of essential n-3 and n-6 PUFAs on amiloride-sensitive sodium transport in A6 epithelial cells. Eicosapentaenoic acid [EPA; C20:5(n-3)] transiently stimulated amiloride-sensitive open-circuit current (INa) from 4.0 ± 0.3 to 7.7 ± 0.3 µA/cm² within 30 min (P < 0.001). No activation was seen in the presence of 10 µM amiloride. In cell-attached but not in cell-excised patches, EPA acutely increased the open probability of sodium channels from 0.45 ± 0.08 to 0.63 ± 0.10 (P = 0.02, paired t-test), n-6 PUFAs, including linoleic acid (C18:2), eicosatetraynoic acid (C20: 4), and docosapentanoic acid (C22:5) had no effect, whereas n-3 docosahexanoic acid (C22:6) activated amiloride-sensitive INa in a manner similar to EPA. Activation of INa by EPA was prevented by H-89, a PKA inhibitor. Similarly, PKA activity was stimulated by EPA. Nonspecific stimulation of phosphodiesterase activity by COCl2 completely prevented the effect of EPA on sodium transport. We conclude that n-3 PUFAs activate epithelial sodium channels downstream of cAMP in a cAMP-dependent pathway also involving PKA. [ABSTRACT FROM AUTHOR]

The activity of epithelial Na[sup +] selective channels is modulated by various factors, with growing evidence that membrane lipids also participate in the regulation. In the present study, Triton X-100 extracts of whole cells and of apical membrane-enriched preparations from cultured A6 renal epithelial cells were floated on continuous-sucrose-density gradients. Na[sup +] channel protein, probed by immunostaining of Western blots, was detected in the high-density fractions of the gradients (between 18 and 30% sucrose), which contain the detergentsoluble material but also in the lighter, detergent-resistant 16% sucrose fraction. Single amiloride-sensitive Na[sup +] channel activity, recorded after incorporation of reconstituted proteoliposomes into lipid bilayers, was exclusively localized in the 16% sucrose fraction. In accordance with other studies, highand low-density fractions of sucrose gradients likely represent membrane domains with different lipid contents. However, exposure of the cells to cholesterol-depleting or sphingomyelin-depleting agents did not affect transepithelial Na[sup +] current, single-Na[sup +] channel activity, or the expression of Na[sup +] channel protein. This is the first reconstitution study of native epithelial Na[sup +] channels, which suggests that functional channels are compartmentalized in discrete domains within the plane of the apical cell membrane. [ABSTRACT FROM AUTHOR]

Presents information on a study that examined the guanosine triphosphate (GTP)-binding proteins from cultured A6 epithelia in isolated membrane preparations. Materials and methods; Results and discussion.

Discusses aldosterone stimulation of guanosine triphosphate (GTP) hydrolysis in cell membranes in renal epithelia. Stimulation of sodium transport across target epithelial cells by increasing apical sodium permeability; GTPPase activity in homogenates and apical membranes; Expression of G proteins.

Methylation Increases Sodium Transport into A6 Apical Membrane Vesicles: Possible Mode of Aldosterone Action Aldosterone increases sodium transport across responsive epithelia, including the epithelia formed in tissue culture by the [...]

Serotonin is released in the respiratory track under various physiological conditions such as hypoxia and epithelial stretch, and may participate to the inflammatory response associated with these pathological conditions. However, the effect of serotonin on alveolar epithelial fluid transport is unknown. In this study, we found serotonin causes a dose-dependent decrease in the amiloride-sensitive current across A549, rat ATII and human ATII cells with an IC50 of 1mM and kinetics suggesting a direct inhibition of ENaC. We established a dose-response curve in A549 cells for amiloride in the presence of 2mM serotonin using whole cell patch-clamp technique. The IC50 of amiloride under control conditions or under 2mM serotonin are comparable, indicating that serotonin does not compete with amiloride and interacts with another site of ENaC. Furthermore, pre-treatment with Bapta-AM and the non selective antagonist to the 5-HT2 receptor that have been identified in both rat and human did not impair the effect of serotonin on Isc, indicating that this effect is not receptor-mediated. Finally, our data show that alveolar instillation of serotonin causes a dose-dependent decrease of the alveolar fluid transport in mice. All together our results indicate that serotonin is a potential endogenous inhibitor of the ENaCs in ATII cells by direct interaction with the channel. [ABSTRACT FROM AUTHOR]

Essential polyunsatured fatty acids have been shown to modulate enzymes, channels and transporters, to interact with lipid bilayers and to affect metabolic pathways. We have previously shown that eicosapentanoic acid (EPA, C20:5, n-3) activates epithelial sodium channels (ENaCs) in a cAMP-dependent manner involving stimulation of cAMP-dependent protein kinase (PKA). In the present study, we explored further the mechanism of EPA stimulation of ENaC in A6 cells. Fluorescence resonance energy transfer experiments confirmed activation of PKA by EPA. Consistent with our previous studies, EPA had no further stimulatory effect on amiloride-sensitive transepithelial current (INa) in the presence of CPT-cAMP. Thus, we investigated the effect of EPA on cellular pathways which produce cAMP. EPA did not stimulate adenylate cyclase activity or total cellular cAMP accumulation. However, membrane-bound phosphodiesterase activity was inhibited by EPA from 2.46 pmol/mg of protein/mm to 1.3 pmol/mg of protein/mm. To investigate the potential role of an A-kinase-anchoring protein (AKAP), we used HT31, an inhibitor of the binding between PKA and AKAPs as well as cerulenin, an inhibitor of myristoylation and palmitoylation. Both agents prevented the stimulatory effect of EPA and CPT-cAMP on INa and drastically decreased the amount of PKA in the apical membrane. Colocalization experiments in A6 cells cotransfected with fluorescently labeled ENaC β subunit and PKA regulatory subunit confirmed the close proximity of the two proteins and the membrane anchorage of PKA. Last, in A6 cells transfected with a dead mutant of Sgk, an enzyme which up-regulates ENaCs, EPA did not stimulate Na+ current. Our results suggest that stimulation of ENaCs by EPA occurs via SGK in membrane-bound compartments containing an AKAP, activated PKA, and a phosphodiesterase. [ABSTRACT FROM AUTHOR]

Confluent monolayers of epithelial cells grown on nonporous support form fluid-filled hemicysts called domes, which reflect active ion transport across the epithelium. Clara-like H441 lung adenocarcinoma cells grown on glass supports and exposed to 50 nM dexamethasone developed domes in a time-dependent fashion. Uplifting of small groups of cells occurred within 6-12 h, well formed domes appeared between 24 and 48 h, and after 7 days, individual domes started to merge. Cells inside of domes compared with those outside domes, or with monolayers not exposed to dexamethasone, differed by higher surfactant production, an increased cytokeratin expression, and the localization of claudin-4 proteins to the plasma membrane. In patch clamp studies, amiloride-blockable sodium currents were detected exclusively in cells inside domes, whereas in cells outside of domes, sodium crossed the membrane through La3+-sensitive nonspecific cation channels. Cells grown on permeable support without dexamethasone expressed amiloride-sensitive currents only after tight electrical coupling was achieved (transepithelial electrical resistance (Rt) > 1 kilohm). In real-time quantitative PCR experiments, the addition of dexamethasone increased the content of claudin-4, occludin, and Na+ channel γ-subunit (γ-ENaC) mRNAs by 1.34-, 1.32-, and 1.80-fold, respectively, after 1 h and was followed by an increase at 6 h in the content of mRNA of α- and β-ENaC and of α1- and β1-Na,K-ATPase. In the absence of dexamethasone, neither change in gene expression nor cell uplifting was observed. Our data suggest that during epithelial differentiation, coordinated expression of tight junction proteins precedes the development of vectorial transport of sodium, which in turn leads to the fluid accumulation in basolateral spaces that is responsible for dome formation. [ABSTRACT FROM AUTHOR]

During inhalational anesthesia, halogenated gases are in direct contact with the alveolar epithelium, in which they may affect transepithelial ion and fluid transport. The effects of halogenated gases in vivo on epithelial Na+ and K+ channels, which participate in alveolar liquid clearance, remain unclear. In the present study, the effects of halothane (1, 2, and 4% atm) on ion-channel function in cultured human alveolar cells were investigated using the patch-clamp technique. After exposure to 4% halothane, amiloride-sensitive whole-cell inward currents increased by 84+/-22%, whereas tetraethylammonium-sensitive outward currents decreased by 63+/-7%. These effects, which occurred within 30 s, remained for 30-min periods of exposure to the gas, were concentration-dependent, and were reversible upon washout. Pretreatment with amiloride prevented 90+/-7% of the increase in inward currents without change in outward currents, consistent with an activation of amiloride-sensitive epithelial sodium channels. Tetraethylammonium obliterated 90+/-9% of the effect of halothane on outward currents, without change in inward currents, indicating inhibition of Ca2+-activated K+ channels. These channels were identified in excised patches to be small-conductance Ca2+-activated K+ channels. These effects of halothane were not modified after the inhibition of cytosolic phospholipase A2 by aristolochic acid. Exposure of the cells to either trypsin or to low Na+ completely prevented the increase in amiloride-sensitive currents induced by halothane, suggesting a release of Na+ channels self-inhibition. Thus, halothane modifies differentially and independently Na+ and K+ permeabilities in human alveolar cells.