AICAR decreases the activity of two distinct amiloride-sensitive Na+-permeable channels in H441 human lung epithelial cell monolayers

Transepithelial transport of Na(+) across the lung epithelium via amiloride-sensitive Na(+) channels (ENaC) regulates fluid volume in the lung lumen. Activators of AMP-activated protein kinase (AMPK), the adenosine monophosphate mimetic AICAR, and the biguanide metformin decreased amiloride-sensitive apical Na(+) conductance (G(Na+)) in human H441 airway epithelial cell monolayers. Cell-attached patch-clamp recordings identified two distinct constitutively active cation channels in the apical membrane that were likely to contribute to G(Na+): a 5-pS highly Na(+) selective ENaC-like channel (HSC) and an 18-pS nonselective cation channel (NSC). Substituting NaCl with NMDG-Cl in the patch pipette solution shifted the reversal potentials of HSC and NSC, respectively, from +23 mV to -38 mV and 0 mV to -35 mV. Amiloride at 1 microM inhibited HSC activity and 56% of short-circuit current (I(sc)), whereas 10 microM amiloride partially reduced NSC activity and inhibited a further 30% of I(sc). Neither conductance was associated with CNG channels as there was no effect of 10 microM pimoside on I(sc), HSC, or NSC activity, and 8-bromo-cGMP (0.3-0.1 mM) did not induce or increase HSC or NSC activity. Pretreatment of H441 monolayers with 2 mM AICAR inhibited HSC/NSC activity by 90%, and this effect was reversed by the AMPK inhibitor Compound C. All three ENaC proteins were identified in the apical membrane of H441 monolayers, but no change in their abundance was detected after treatment with AICAR. In conclusion, activation of AMPK with AICAR in H441 cell monolayers is associated with inhibition of two distinct amiloride-sensitive Na(+)-permeable channels by a mechanism that likely reduces channel open probability.     

Dopamine activates amiloride-sensitive sodium channels in alveolar type I cells in lung slice preparations

Active Na+ reabsorption by alveolar epithelial cells generates the driving force used to clear fluids from the air space. Using single-channel methods, we examined epithelial Na+ channel (ENaC) activity of alveolar type I (AT1) cells from live 250- to 300-microm sections of lung tissue, circumventing concerns that protracted cell isolation procedures might compromise the innate transport properties of native lung cells. We used fluorescein-labeled Erythrina crystagalli lectin to positively identify AT1 cells for single-channel patch-clamp analysis. We demonstrated, for the first time, single-channel recordings of highly selective and nonselective amiloride-sensitive ENaC channels (HSC and NSC, respectively) from AT1 cells in situ, with mean conductances of 8.2+/-2.5 and 22+/-3.2 pS, respectively. Additionally, 25 nM amiloride in the patch electrode blocked Na+ channel activity in AT1 cells. Immunohistochemical studies demonstrated the presence of dopamine D1 and D2 receptors on the surface of AT1 cells, and single-channel recordings showed that 10 microM dopamine increased Na+ channel activity [product of the number of channels and single-channel open probability (NPo)] from 0.31+/-0.19 to 0.60+/-0.21 (P<0.001). The D1 receptor antagonist SCH-23390 (10 microM) blocked the stimulatory effect of dopamine on AT1 cells, but the D2 receptor antagonist sulpiride did not.     

Oxygen induction of epithelial Na(+) transport requires heme proteins

Fetal distal lung epithelial (FDLE) cells exposed to a postnatal O(2) concentration of 21% have higher epithelial Na(+) channel (ENaC) mRNA levels and Na(+) transport relative to FDLE cells grown in a fetal O(2) concentration of 3%. To investigate the mechanism of this process, FDLE monolayers were initially cultured in 3% O(2), and then some were switched to a 21% O(2) environment. Incubation of FDLE cells with the iron chelator deferoxamine, CoCl(2), NiCl(2), or an inhibitor of heme synthesis prevented or diminished the O(2) induction of amiloride-sensitive short-circuit current in FDLE cells. Similarly, defer- oxamine and cobalt prevented O(2)-induced ENaC mRNA expression. Exposure of FDLE cells grown under hypoxic conditions to carbon monoxide increased both ENaC mRNA expression and amiloride-sensitive short-circuit current. We therefore concluded that induction of ENaC mRNA expression and amiloride-sensitive Na(+) transport in FDLE cells by a physiological increase in O(2) concentration seen at birth requires iron and heme proteins.     

Inhibitory non-genomic effects of progesterone on Na+ absorption in epithelial cells from Xenopus kidney (A6)

The effect of the steroid hormone progesterone on transepithelial sodium transport was measured in confluent monolayers of the A6-cell line from Xenopus kidney. Apical permeabilization with Amphotericin B enabled us to measure the Na+/K+-pump current, and current-fluctuation analysis was used to analyze changes in apical channel density and gating characteristics. Basolateral progesterone (22.2 microM) had a rapid inhibitory effect on the Na+/K+-pump current, and a corresponding decrease in Na+ channel density. The effect occurred within some minutes and took about 50 min to reach a new steady state, in which 45% of the macroscopic current (ISC) was inhibited. Progesterone also inhibits the hypo-osmotic stimulation of Na+ channels which occurs in untreated monolayers. Compared with the known effects of adrenal steroids, our results show a rapid inhibitory action of a steroid hormone on Na+ absorption. The time profile of the progesterone effect suggests, at least in the first minutes, a non-genomic action of progesterone.     

Involvement of ion channels in the induction of proenkephalin A gene expression by nicotine and cAMP in bovine chromaffin cells

The involvement of Na+ and Ca2+ channels in the stimulatory effect of nicotine and cAMP upon proenkephalin A mRNA (mRNA ENK) levels in primary cultures of bovine adrenal chromaffin cells was analyzed. Nicotine (10 microM) caused about a 2-3-fold increase in mRNA ENK which was abolished by the nicotinic receptor antagonist tubocurarine (4 X 10(-7) M), inhibited by the Ca2+ channel antagonist nifedipine (100 nM) abolished by the Ca2+ channel blocker D600 (10 microM), and augmented by the Ca2+ channel agonist BayK 8644 (100 nM). In contrast, blockade of the Na+ channel by tetrodotoxin (1 microM) did not modulate the nicotine-induced increase in mRNA ENK. Incubation of the cells with forskolin (25 microM) and 8-bromo-cAMP (1 mM) also resulted in an increase in mRNA ENK levels that was inhibited by the Ca2+ channel blocker verapamil (50 microM) and nifedipine (100 nM), whereas it was enhanced by BayK 8644 (100 nM). In addition, the effect of forskolin and 8-bromo-cAMP was decreased by the Na+ channel blocker tetrodotoxin (1 microM). These results suggest that the induction of proenkephalin A gene expression by cAMP and nicotine involves the modulation of ion channels. It appears that changes in Ca2+ flux are involved in mediating this induction. The dihydropyridines nifedipine and BayK 8644 and the Ca2+ channel blockers verapamil and D600 all modulate 45Ca uptake. In addition, we show that incubation of the cells with A23187 (10(-7) M), a Ca2+ ionophore, resulted in an increase in mRNA ENK, indicating that changes in intracellular Ca2+ levels may indeed modulate proenkephalin A gene expression. Although it appears that an elevation of mRNA ENK upon nicotinic receptor activation occurs rapidly (an increase could be detected after 2 h incubation), the findings that the rise in mRNA ENK could be abolished by the Ca2+ channel blocker D600 but not affected by tetrodotoxin (1 microM), and that agents such as KCl (20 mM) and veratridine (5 microM) that increase mRNA ENK by activation of voltage-dependent Ca2+ channels do not result in an increase in intracellular cAMP, provide no evidence for a major role of the adenylate cyclase system in the inducing effect of nicotine upon proenkephalin A gene expression.     

Fatty acid modulation and sequence identity of fetal guinea pig alveolar type II cell amiloride-sensitive Na+ channel.

Removal of fetal lung fluid at birth is crucial to survival. In vivo, a reversal in the direction of vectorial, amiloride-sensitive Na+) transport can be stimulated by ETYA, a nonmetabolizable analogue of the naturally occurring unsaturated fatty acid, arachidonate. Using the patch-clamp technique, fetal guinea pig alveolar type II pneumocyte single Na+ channel activity was robustly activated by 10 microM arachidonate, ETYA, oleate and stearate; this was unaffected by cyclooxygenase and 5'lipoxygenase inhibitors. The Na+ channel expressed in fetal guinea pig alveolar epithelial type II pneumocytes has biophysical properties compatible with species-specific coexpression of a novel variant of alphaENaC with betaENaC. gammaENaC is either not expressed in this tissue or shares very little homology with the rat and human gamma subunit. Thus, dramatic stimulation of this channel by arachidonate explains the in vivo observation of gestation-dependent reversal of fetal transepithelial driving force and may, therefore, be of physiological significance during the transition to breathing air at birth.     

Molecular cloning, functional expression and chromosomal localization of an amiloride-sensitive Na(+) channel from human small intestine

Amiloride-sensitive Na(+) channels belonging to the recently discovered NaC/DEG family of genes have been found in several human tissues including epithelia and central and peripheral neurons. We describe here the molecular cloning of a cDNA encoding a novel human amiloride-sensitive Na(+) channel subunit that is principally expressed in the small intestine and has been called hINaC (human intestine Na(+) channel). This protein is similar to the recently identified rodent channel BLINaC and is relatively close to the acid sensing ion channels (ASICs) (79 and 29% amino acid identity, respectively). ASICs are activated by extracellular protons and probably participate in sensory neurons to nociception linked to tissue acidosis. hINaC is not activated by lowering the external pH but gain-of-function mutations can be introduced and reveal when expressed in Xenopus oocytes, an important Na(+) channel activity which is blocked by amiloride (IC(50)=0.5 microM). These results suggest the existence of a still unknown physiological activator for hINaC (e.g. an extracellular ligand). The presence of this new amiloride-sensitive Na(+) channel in human small intestine probably has interesting physiological as well as physiopathological implications that remain to be clarified. The large activation of this channel by point mutations may be associated with a degenerin-like behavior as previously observed for channels expressed in nematode mechanosensitive neurons. The hINaC gene has been mapped on the 4q31.3-q32 region of the human genome.     

Glucocorticoids stimulate the activity of large-conductance Ca2+ -activated K+ channels in pituitary GH3 and AtT-20 cells via a non-genomic mechanism

The effects of glucocorticoids on ion currents were investigated in pituitary GH3 and AtT-20 cells. In whole-cell configuration, dexamethasone, a synthetic glucocorticoid, reversibly increased the density of Ca2+ -activated K+ current (IK(Ca)) with an EC50 value of 21 +/- 5 microM. Dexamethasone-induced increase in IK(Ca) density was suppressed by paxilline (1 microM), yet not by glibenclamide (10 microM), pandinotoxin-Kalpha (1 microM) or mifepristone (10 microM). Paxilline is a blocker of large-conductance Ca2+ -activated K+ (BKCa) channels, while glibenclamide and pandinotoxin-Kalpha are blockers of ATP-sensitive and A-type K+ channels, respectively. Mifepristone can block cytosolic glucocorticoid receptors. In inside-out configuration, the application of dexamethasone (30 microM) into the intracellular surface caused no change in single-channel conductance; however, it did increase BKCa -channel activity. Its effect was associated with a negative shift of the activation curve. However, no Ca2+ -sensitiviy of these channels was altered by dexamethasone. Dexamethasone-stimulated channel activity involves an increase in mean open time and a decrease in mean closed time. Under current-clamp configuration, dexamethasone decreased the firing frequency of action potentials. In pituitary AtT-20 cells, dexamethasone (30 microM) also increased BKCa -channel activity. Dexamethasone-mediated stimulation of IK(Ca) presented here that is likely pharmacological, seems to be not linked to a genomic mechanism. The non-genomic, channel-stimulating properties of dexamethasone may partly contribute to the underlying mechanisms by which glucocorticoids affect neuroendocrine function.     

Cyclic AMP-dependent protein kinase modulation of the glucocorticoid-induced cytolytic response in murine T-lymphoma cells

The antiproliferative effect of glucocorticoid hormones on lymphoid tissue serves as the basis for their use in chemotherapy of lymphomas and leukemias. The effectiveness of the steroid-mediated response is potentially contingent upon a variety of factors, including the cellular level of glucocorticoid receptors. This report demonstrates that differences in the expression of the glucocorticoid receptor gene can modulate steroid sensitivity of individuals within a population of lymphoma cells. We have also found that loss of cAMP-dependent protein kinase activity caused a measurable decrease of steroid sensitivity in the murine T-lymphoma WEHI-7 without producing a significant change in steroid binding capacity. However, the extent of this change in sensitivity was dependent upon the level of glucocorticoid receptor expression. Lymphoma cells containing few spare steroid receptors became significantly resistant to glucocorticoids through loss of cAMP-dependent kinase function. On the other hand, elevated levels of cAMP were found to cause an increase in glucocorticoid receptor mRNA concentrations. Thus, cAMP-dependent protein kinase activity has the potential to modulate a lymphoma cell's steroid sensitivity by affecting the level of glucocorticoid receptor expression as well as the receptor's efficiency in producing a cytolytic response.     

Modulation of alpha-ENaC and alpha1-Na+-K+-ATPase by cAMP and dexamethasone in alveolar epithelial cells

cAMP and dexamethasone are known to modulate Na+ transport in epithelial cells. We investigated whether dibutyryl cAMP (DBcAMP) and dexamethasone modulate the mRNA expression of two key elements of the Na+ transport system in isolated rat alveolar epithelial cells: alpha-, beta-, and gamma-subunits of the epithelial Na+ channel (ENaC) and the alpha1- and beta1-subunits of Na+-K+-ATPase. The cells were treated for up to 48 h with DBcAMP or dexamethasone to assess their long-term impact on the steady-state level of ENaC and Na+-K+-ATPase mRNA. DBcAMP induced a twofold transient increase of alpha-ENaC and alpha1-Na+-K+-ATPase mRNA that peaked after 8 h of treatment. It also upregulated beta- and gamma-ENaC mRNA but not beta1-Na+-K+-ATPase mRNA. Dexamethasone augmented alpha-ENaC mRNA expression 4.4-fold in cells treated for 24 h and also upregulated beta- and gamma-ENaC mRNA. There was a 1.6-fold increase at 8 h of beta1-Na+-K+-ATPase mRNA but no significant modulation of alpha1-Na+-K+-ATPase mRNA expression. Because DBcAMP and dexamethasone did not increase the stability of alpha-ENaC mRNA, we cloned 3.2 kb of the 5' sequences flanking the mouse alpha-ENaC gene to study the impact of DBcAMP and dexamethasone on alpha-ENaC promoter activity. The promoter was able to drive basal expression of the chloramphenicol acetyltransferase (CAT) reporter gene in A549 cells. Dexamethasone increased the activity of the promoter by a factor of 5.9. To complete the study, the physiological effects of DBcAMP and dexamethasone were investigated by measuring transepithelial current in treated and control cells. DBcAMP and dexamethasone modulated transepithelial current with a time course reminiscent of the profile observed for alpha-ENaC mRNA expression. DBcAMP had a greater impact on transepithelial current (2.5-fold increase at 8 h) than dexamethasone (1.8-fold increase at 24 h). These results suggest that modulation of alpha-ENaC and Na+-K+-ATPase gene expression is one of the mechanisms that regulates Na+ transport in alveolar epithelial cells.     

Downregulation of ENaC activity and expression by TNF-alpha in alveolar epithelial cells

Sodium absorption by an amiloride-sensitive channel is the main driving force of lung liquid clearance at birth and lung edema clearance in adulthood. In this study, we tested whether tumor necrosis factor-alpha (TNF-alpha), a proinflammatory cytokine involved in several lung pathologies, could modulate sodium absorption in cultured alveolar epithelial cells. We found that TNF-alpha decreased the expression of the alpha-, beta-, and gamma-subunits of epithelial sodium channel (ENaC) mRNA to 36, 43, and 16% of the controls after 24-h treatment and reduced to 50% the amount of alpha-ENaC protein in these cells. There was no impact, however, on alpha(1) and beta(1) Na(+)-K(+)-ATPase mRNA expression. Amiloride-sensitive current and ouabain-sensitive Rb(+) uptake were reduced, respectively, to 28 and 39% of the controls. A strong correlation was found at different TNF-alpha concentrations between the decrease of amiloride-sensitive current and alpha-ENaC mRNA expression. All these data show that TNF-alpha, a proinflammatory cytokine present during lung infection, has a profound influence on the capacity of alveolar epithelial cells to transport sodium.     

Oxidative stress disrupts glucocorticoid hormone-dependent transcription of the amiloride-sensitive epithelial sodium channel alpha-subunit in lung epithelial cells through ERK-dependent and thioredoxin-sensitive pathways

The amiloride-sensitive epithelial Na(+) channel (ENaC) plays a critical role in the maintenance of alveolar fluid balance. It is generally accepted that reactive oxygen and nitrogen species can inhibit ENaC activity and aggravate acute lung injury; however, the molecular mechanism for free radical-mediated ENaC inhibition is unclear. Previously, we showed that the expression of the alpha-subunit of ENaC, alpha-ENaC, which is indispensable for ENaC activity, is repressed by Ras activation in salivary epithelial cells. Here, we investigated whether exogenous H(2)O(2) modulates alpha-ENaC gene expression in lung epithelial cells through a similar molecular mechanism. Utilizing transient transfection reporter assays and site-directed mutagenesis analyses, we found that the glucocorticoid response element (GRE), located at -1334 to -1306 base pairs of the alpha-ENaC 5'-flanking region, is the major enhancer for the stimulated alpha-ENaC expression in A549 lung epithelial cells. We further demonstrate that the presence of an intact GRE is necessary and sufficient for oxidants to repress alpha-ENaC expression. Consistent with our hypothesis, exogenous H(2)O(2)-mediated repression of alpha-ENaC GRE activity is partially blocked by either a specific inhibitor for extracellular signal-regulated kinase (ERK) pathway activation, U0126, or dominant negative ERK, suggesting that, in part, activated ERK may mediate the repressive effects of H(2)O(2) on alpha-ENaC expression. In addition, overexpression of thioredoxin restored glucocorticoid receptor action on the alpha-ENaC GRE in the presence of exogenous H(2)O(2). Taken together, we hypothesize that oxidative stress impairs Na(+) transport activity by inhibiting dexamethasone-dependent alpha-ENaC GRE activation via both ERK-dependent and thioredoxin-sensitive pathways. These results suggest a putative mechanism whereby cellular redox potentials modulate the glucocorticoid receptor/dexamethasone effect on alpha-ENaC expression in lung and other tight epithelia.     

The amiloride-sensitive Na+/H+ exchange system in skeletal muscle cells in culture

Chick skeletal muscle cells in culture have an amiloride-sensitive Na+-transporting system that has the following properties. Na+ uptake is dependent on the extracellular Na+ concentration. The Km value for Na+ is 25 mM and remains constant between pH 7.5 and 8.5. The maximal rate of Na+ transport is higher at alkaline pH. An ionizable group with a pK of 7.6 is essential for the system to be functional. The activity of the amiloride-sensitive Na+ uptake system is controlled by internal Na+ and H+ concentrations. Amiloride inhibition of Na+ uptake is competitively antagonized by increasing Na+ concentration. The dissociation constant for amiloride is 5 microM in Na+-free conditions and is constant between pH 7.5 and 8.5. The Km value for Na+ found from competition experiments is 13 mM. The amiloride-sensitive Na+ influx occurs in parallel with an amiloride-sensitive H+ efflux. This H+ efflux is stimulated by increasing external Na+ concentrations, the Km for Na+ being 15 mM. It is inhibited by amiloride with the same concentration dependence as Na+ influx.     

Riluzole-induced block of voltage-gated Na+ current and activation of BKCa channels in cultured differentiated human skeletal muscle cells

Riluzole is known to be of therapeutic use in the management of amyotrophic lateral sclerosis. In this study, we investigated the effects of riluzole on ion currents in cultured differentiated human skeletal muscle cells (dHSkMCs). Western blotting revealed the protein expression of alpha-subunits for both large-conductance Ca2+-activated K+ (BK(Ca)) channel and Na+ channel (Na(v)1.5) in these cells. Riluzole could reduce the frequency of spontaneous beating in dHSkMCs. In whole-cell configuration, riluzole suppressed voltage-gated Na+ current (I(Na)) in a concentration-dependent manner with an IC50 value of 2.3 microM. Riluzole (10 microM) also effectively increased Ca2+-activated K+ current (I(K(Ca))) which could be reversed by iberiotoxin (200 nM) and paxilline (1 microM), but not by apamin (200 nM). In inside-out patches, when applied to the inside of the cell membrane, riluzole (10 microM) increased BK(Ca)-channel activity with a decrease in mean closed time. Simulation studies also unraveled that both decreased conductance of I(Na) and increased conductance of I(K(Ca)) utilized to mimic riluzole actions in skeletal muscle cells could combine to decrease the amplitude of action potentials and increase the repolarization of action potentials. Taken together, inhibition of I(Na) and stimulation of BK(Ca)-channel activity caused by this drug are partly, if not entirely, responsible for its muscle relaxant actions in clinical setting.     

Mineralocorticoid concentrations in unstressed female rabbits and embryonic sodium transport

Plasma aldosterone, corticosterone, and cortisol were measured during the first week of pseudopregnancy or pregnancy in New Zealand White rabbits to determine whether any sustained elevations of adrenal steroids occur. There were no pregnancy-specific alterations in circulating adrenal steroid concentrations during the preimplantation stages of embryonic development. Elevation of plasma aldosterone in vivo did not induce amiloride-sensitive Na+ transport across the embryonic trophectoderm. It therefore seems unlikely that an increase in maternal adrenal steroid concentrations is necessary for the development of amiloride-sensitive Na+ transport in rabbit blastocysts. Sodium efflux from Day 6 post coitum (p.c.) blastocysts was lower than Na+ influx. By day 7 p.c. Na+ efflux was equivalent in magnitude to the component of Na+ influx not inhibited by amiloride. This suggests that between Days 6 and 7 p.c. the amiloride-sensitive component of Na+ influx becomes essential for blastocyst expansion.     

Expression of epithelial Na channels in Xenopus oocytes

Epithelial Na channel activity was expressed in oocytes from Xenopus laevis after injection of mRNA from A6 cells, derived from Xenopus kidney. Poly A(+) RNA was extracted from confluent cell monolayers grown on either plastic or permeable supports. 1-50 ng RNA was injected into stage 5-6 oocytes. Na channel activity was assayed as amiloride- sensitive current (INa) under voltage-clamp conditions 1-3 d after injection. INa was not detectable in noninjected or water-injected oocytes. This amiloride-sensitive pathway induced by the mRNA had a number of characteristics in common with that in epithelial cells, including (a) high selectivity for Na over K, (b) high sensitivity to amiloride with an apparent K1 of approximately 100 nM, (c) saturation with respect to external Na with an apparent Km of approximately 10 mM, and (d) a time-dependent activation of current with hyperpolarization of the oocyte membrane. Expression of channel activity was temperature dependent, being slow at 19 degrees C but much more rapid at 25 degrees C. Fractionation of mRNA on a sucrose density gradient revealed that the species of RNA inducing channel activity had a sedimentation coefficient of approximately 17 S. Treatment of filter-grown cells with 300 nM aldosterone for 24 h increased Na transport in the A6 cells by up to fivefold but did not increase the ability of mRNA isolated from those cells to induce channel activity in oocytes. The apparent abundance of mRNA coding for channel activity was 10-fold less in cells grown on plastic than in those grown on filters, but was increased two- to threefold by aldosterone.