Sarah Sariban-Sohraby

Differentiation of Epithelial Na+ Channel Function AN IN VITRO MODEL

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.

Isoprenylcysteine-O-carboxyl methyltransferase regulates aldosterone- sensitive Na+ reabsorption

The Xenopus laevis distal tubule epithelial cell line A6 was used as a model epithelia to study the role of isoprenylcysteine-O-carboxyl methyltransferase (pcMTase) in aldosterone-mediated stimulation of Na+transport. Polyclonal antibodies raised against X. laevispcMTase were immunoreactive with a 33-kDa protein in whole cell lysate. These antibodies were also reactive with a 33-kDa product from in vitro translation of the pcMTase cDNA. Aldosterone application increased pcMTase activity resulting in elevation of total protein methyl esterification in vivo, but pcMTase protein levels were not affected by steroid, suggesting that aldosterone increased activity independent of enzyme number. Inhibition of pcMTase resulted in a reduction of aldosterone-induced Na+transport demonstrating the necessity of pcMTase-mediated transmethylation for steroid induced Na+ reabsorption. Transfection with an eukaryotic expression construct containing pcMTase cDNA increased pcMTase protein level and activity. This resulted in potentiation of the natriferic actions of aldosterone. However, overexpression did not change Na+ reabsorption in the absence of steroid, suggesting that pcMTase activity is not limiting Na+ transport in the absence of steroid, but that subsequent to aldosterone addition, pcMTase activity becomes limiting. These results suggest that a critical transmethylation is necessary for aldosterone-induction of Na+ transport. It is likely that the protein catalyzing this methylation is isoprenylcysteine-O-carboxyl methyltransferase and that aldosterone activates pcMTase without affecting transferase expression.

Epithelial Na+ channel stimulation by n-3 fatty acids requires proximity to a membrane-bound A-kinase-anchoring protein complexed with protein kinase A and phosphodiesterase

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/min to 1.3 pmol/mg of protein/min. 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.

Halothane Directly Modifies Na+ and K+ Channel Activities in Cultured Human Alveolar Epithelial Cells

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.

Guanine nucleotide binding proteins in cultured renal epithelia: studies with pertussis toxin and aldosterone

The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [35S]GTPγS revealed a class of binding sites with an apparent K d value of 100 nM and a Bmax of 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX) decreased Bmax by 50%. The mRNA levels for Gαi-3, Gα0, Gαs, and Gαq were not increased after aldosterone. The patterns of small M r G proteins and of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of [α-32P]GTP, in control membranes, showed either no labeling or a faint band of M r 59.5 kDa. This protein became prominent after aldosterone, and its labeling decreased with spironolactone. Thus short-term aldosterone does not promote increased expression of known heterotrimeric G proteins in epithelial membranes but activates resident PTX-sensitive Gi proteins and stimulates the expression of a specific GTP-binding protein of M r 59.5 kDa.The GTP-binding proteins from cultured A6 epithelia were examined in isolated membrane preparations. Binding of [35S]GTPgammaS revealed a class of binding sites with an apparent Kd value of 100 nM and a Bmax of 220 pmol/mg protein. Short-term aldosterone treatment of the cells did not modify the binding kinetics, whereas pertussis toxin (PTX) decreased Bmax by 50%. The mRNA levels for Galphai-3, Galpha0, Galphas, and Galphaq were not increased after aldosterone. The patterns of small Mr G proteins and of PTX-ribosylated proteins were identical in membranes of both control and aldosterone-treated cells. Cross-linking of [alpha-32P]GTP, in control membranes, showed either no labeling or a faint band of Mr 59.5 kDa. This protein became prominent after aldosterone, and its labeling decreased with spironolactone. Thus short-term aldosterone does not promote increased expression of known heterotrimeric G proteins in epithelial membranes but activates resident PTX-sensitive Gi proteins and stimulates the expression of a specific GTP-binding protein of Mr 59.5 kDa.

Chapter 6 Role of G Proteins in the Regulation of Apical Membrane Sodium Permeability by Aldosterone in Epithelia

Publisher Summary The chapter discusses a study on the role of G proteins in the regulation of apical membrane sodium permeability by aldosterone in epithelia. A number of distinct biochemical pathways are involved in aldosterones action on Na + permeability—namely, the activation of phospholipid fatty acid metabolism and phospholipase A 2 , the methylation of apical proteins and lipids, and the activation of GTP-binding proteins. Some or possibly all of these pathways, not mutually exclusive, ultimately increase the transepithelial Na + transport rate. The chapter discusses GTP-dependent carboxymethylation and activation of GTPase activity, two pathways that are tightly linked, with regard to aldosterone stimulation of Na + transport. Guanine nucleotides are important regulators of a number of ion channels. In epithelia, the G-protein-mediated pathways controlling Na + channels are different from the ones involved with membrane-bound G-protein-coupled receptors and diffusible second messengers. Guanine nucleotides stimulate protein carboxymethylation in a variety of mammalian cell membranes such as macrophages, brain, liver, and buffy-coat cells. Several classes of carboxyl methyltransferase catalyze methyl esterification at different amino acid residues.