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.     

Hypoxia decreases active Na transport across primary rat alveolar epithelial cell monolayers

Hypoxia has been reported to inhibit activity and expression of ion transporters of alveolar epithelial cells. This study extended those observations by investigating the mechanisms underlying inhibition of active Na transport across primary cultured adult rat alveolar epithelial cell monolayers grown on polycarbonate filters. Cell monolayers were exposed to normoxia and hypoxia (1.5% and 5% O(2), 5% CO(2)), and resultant changes in bioelectric properties [i.e., short-circuit current (I(sc)) and transepithelial resistance (R(t))] were measured in Ussing chambers. Results showed that I(sc) decreased with duration of exposure to hypoxia, while relatively little change was observed for R(t). In normoxia, amiloride inhibited approximately 70% of I(sc). The amiloride-sensitive portion of I(sc) decreased over time of exposure to hypoxia, whereas the magnitude of the amiloride-insensitive portion of I(sc) was not affected. Na pump capacity measured after permeabilization of the apical plasma membrane with amphotericin B decreased in monolayers exposed to 1.5% O(2) for 24 h, as did the capacity of amiloride-sensitive Na uptake measured after imposing an apical to basolateral Na gradient and permeabilization of the basolateral membrane. These results demonstrate that exposure to hypoxia inhibits alveolar epithelial Na reabsorption by reducing the rates of both apical amiloride-sensitive Na entry and basolateral Na extrusion.     

cAMP-induced changes of apical membrane potentials of confluent H441 monolayers

We recorded apical membrane potentials (Va) of H441 cells [a human lung cell line exhibiting both epithelial Na+ (ENaC) and CFTR-type channels] grown as confluent monolayers, using the microelectrode technique in current-clamp mode before, during, and after perfusion of the apical membranes with 10 microM forskolin. When perfused with normal Ringer solution, the cells had a Va of -43 +/- 10 mV (means +/- SD; n = 31). Perfusion with forskolin resulted in sustained depolarization by 25.0 +/- 3.5 mV (means +/- SD; n = 23) and increased the number, open time, and the open probability of a 4.2-pS ENaC. In contrast to a previous report (Jiang J, Song C, Koller BH, Matthay MA, and Verkman AS. Am J Physiol Cell Physiol 275: C1610-C1620, 1998), no transient hyperpolarization was observed. The forskolin-induced depolarization of Va was almost totally prevented by pretreatment of monolayers with 10 microM amiloride or by substitution of Na+ ions in the bath solution with N-methyl-d-glucamine. These findings indicate that cAMP stimulation of Na+ influx across H441 confluent monolayers results from activation of an amiloride-sensitive apical Na+ conductance and not from Va hyperpolarization due to Cl- influx through CFTR-type channels.     

Effects of PKA inhibitors, H-compounds, on epithelial Na+ channels via PKA-independent mechanisms.

The Na+ transport in alveolar type II epithelial cells of rat fetal lung was stimulated by cAMP, which is generally thought to act through activation of protein kinase A (PKA). PKA inhibitors (H8, H89 and H7) stimulated amiloride-sensitive Na+ transport in the alveolar type II epithelial cells. H85, an inactive form of H89 as a PKA inhibitor, had also mimicked the stimulatory action of H89 on the Na+ transport. On the other hand, another type of PKA inhibitor, KT5720 or myristoylated PKA inhibitory peptide [14-22] amide, did not stimulate the Na+ transport, but inhibited the Na+ transport unlike H-compounds. These observations suggest that H-compounds act on the Na+ transport depending on the structure.     

Effects of Purinergic Stimulation,CFTR and Osmotic Stress on Amiloride-sensitive Na<Superscript>+</Superscript> Transport in Epithelia and <Emphasis Type="Italic">Xenopus</Emphasis> Oocytes

Both stimulation of purinergic receptors by ATP and activation of the cystic fibrosis transmembrane conductance regulator (CFTR) inhibit amiloride-sensitive Na+ transport and activate Cl- secretion. These changes in ion transport may well affect cell volume. We therefore examined whether cell shrinkage or cell swelling do affect amiloride-sensitive Na+ transport in epithelial tissues or Xenopus oocytes and whether osmotic stress interferes with regulation of Na+ transport by ATP or CFTR. Stimulation of purinergic receptors by ATP/UTP or activation of CFTR by IBMX and forskolin inhibited amiloride-sensitive transport in mouse trachea and colon, respectively, by a mechanism that was Cl- dependent. When exposed to a hypertonic but not hypotonic bath solution, amiloride-sensitive Na+ transport was inhibited in mouse trachea and colon, independent of the extracellular Cl- concentration. Both inhibition of Na+ transport by hypertonic bath solution and ATP were additive. When coexpressed in Xenopus oocytes, activation of CFTR by IBMX and forskolin inhibited the epithelial Na+ channel (ENaC) in a Cl- dependent fashion. However, both hypertonic and hypotonic bath solutions showed only minor effects on amiloride-sensitive conductance, independent of the bath Cl- concentration. Moreover, CFTR-induced inhibition of ENaC could be detected in oocytes even after exposure to hypertonic or hypotonic bath solutions. We conclude that amiloride-sensitive Na+ absorption in mouse airways and colon is inhibited by cell shrinkage by a mechanism that does not interfere with purinergic and CFTR-mediated inhibition of ENaC.     

cAMP regulation of Cl(-) and HCO(-)(3) secretion across rat fetal distal lung epithelial cells

We isolated and cultured fetal distal lung epithelial (FDLE) cells from 17- to 19-day rat fetuses and assayed for anion secretion in Ussing chambers. With symmetrical Ringer solutions, basal short-circuit currents (I(sc)) and transepithelial resistances were 7.9 +/- 0.5 microA/cm(2) and 1,018 +/- 73 Omega.cm(2), respectively (means +/- SE; n = 12). Apical amiloride (10 microM) inhibited basal I(sc) by approximately 50%. Subsequent addition of forskolin (10 microM) increased I(sc) from 3.9 +/- 0.63 microA/cm(2) to 7.51 +/- 0.2 microA/cm(2) (n = 12). Basolateral bumetanide (100 microM) decreased forskolin-stimulated I(sc) from 7.51 +/- 0.2 microA/cm(2) to 5.62 +/- 0.53, whereas basolateral 4,4'-dinitrostilbene-2,2'-disulfonate (5 mM), an inhibitor of HCO secretion, blocked the remaining I(sc). Forskolin addition evoked currents of similar fractional magnitudes in symmetrical Cl(-)- or HCO(-)(3)-free solutions; however, no response was seen using HCO(-)(3)- and Cl(-)-free solutions. The forskolin-stimulated I(sc) was inhibited by glibenclamide but not apical DIDS. Glibenclamide also blocked forskolin-induced I(sc) across monolayers having nystatin-permeablized basolateral membranes. Immunolocalization studies were consistent with the expression of cystic fibrosis transmembrane conductance regulator (CFTR) protein in FDLE cells. In aggregate, these findings indicate the presence of cAMP-activated Cl(-) and HCO(-)(3) secretion across rat FDLE cells mediated via CFTR.     

Biophysical properties and molecular characterization of amiloride-sensitive sodium channels in A549 cells

Amiloride-sensitive Na(+) channels, present in fetal and adult alveolar epithelial type II (ATII) cells, play a critical role in the reabsorption of fetal fluid shortly after birth and in limiting the extent of alveolar edema across the adult lung. Because of the difficulty in isolating and culturing ATII cells, there is considerable interest in characterizing the properties of ion channels and their response to injury of ATII cell-like cell lines such as A549 that derive from a human alveolar cell carcinoma. A549 cells were shown to contain alpha-, beta-, and gamma-epithelial Na(+) channel mRNAs. In the whole cell mode of the patch-clamp technique (bath, 145 mM Na(+); pipette, 145 mM K(+)), A549 cells exhibited inward Na(+) currents reversibly inhibited by amiloride, with an inhibition constant of 0.83 microM. Ion substitution studies showed that these channels were moderately selective for Na(+) (Na(+)-to-K(+) permeability ratio = 6:1). Inward Na(+) currents were activated by forskolin (10 microM) and inhibited by nitric oxide (300 nM) and cGMP. Recordings in cell-attached mode revealed the presence of an amiloride-sensitive Na(+) channel with a unitary conductance of 8.6 +/- 0.04 (SE) pS. Channel activity was increased by forskolin and decreased by nitric oxide and the cGMP analog 8-bromo-cGMP. These data demonstrate that A549 cells contain amiloride-sensitive Na(+) channels with biophysical properties similar to those of ATII cells.     

Transforming growth factor-beta1 decreases expression of the epithelial sodium channel alphaENaC and alveolar epithelial vectorial sodium and fluid transport via an ERK1/2-dependent mechanism

Acute lung injury (ALI) is characterized by the flooding of the alveolar airspaces with protein-rich edema fluid and diffuse alveolar damage. We have previously reported that transforming growth factor-beta1 (TGF-beta1) is a critical mediator of ALI after intratracheal administration of bleomycin or Escherichia coli endotoxin, at least in part due to effects on lung endothelial and alveolar epithelial permeability. In the present study, we hypothesized that TGF-beta1 would also decrease vectorial ion and water transport across the distal lung epithelium. Therefore, we studied the effect of active TGF-beta1 on 22Na+ uptake across monolayers of primary rat and human alveolar type II (ATII) cells. TGF-beta1 significantly reduced the amiloride-sensitive fraction of 22Na+ uptake and fluid transport across monolayers of both rat and human ATII cells. TGF-beta1 also significantly decreased alphaENaC mRNA and protein expression and inhibited expression of a luciferase reporter downstream of the alphaENaC promoter in lung epithelial cells. The inhibitory effect of TGF-beta1 on sodium uptake and alphaENaC expression in ATII cells was mediated by activation of the MAPK, ERK1/2. Consistent with the in vitro results, TGF-beta1 inhibited the amiloride-sensitive fraction of the distal airway epithelial fluid transport in an in vivo rat model at a dose that was not associated with any change in epithelial protein permeability. These data indicate that increased TGF-beta1 activity in the distal airspaces during ALI promotes alveolar edema by reducing distal airway epithelial sodium and fluid clearance. This reduction in sodium and fluid transport is attributable in large part to a reduction in apical membrane alphaENaC expression mediated through an ERK1/2-dependent inhibition of the alphaENaC promoter activity.     

Cholinergic inhibition of electrogenic sodium absorption in the guinea pig distal colon

Submucosal cholinergic and noncholinergic neurons in intestines have been shown to be involved in regulating epithelial transport functions, particularly stimulating Cl(-) secretion. This study investigates the role of submucosal cholinergic neurons in regulating electrogenic Na(+) absorption in distal colon. Amiloride-sensitive short-circuit current (I(sc)) and (22)Na(+) flux were measured in mucosal and mucosal-submucosal preparations mounted in Ussing chambers. In the mucosal preparation, carbachol (CCh) added to the serosal side inhibited amiloride-sensitive I(sc) and amiloride-sensitive (22)Na(+) absorption. The inhibitory effect of CCh was observed at approximately 0.1 microM, and maximum inhibition of approximately 70% was attained at approximately 30 microM (IC(50) = approximately 1 microM). CCh-induced inhibition of amiloride-sensitive I(sc) was almost totally abolished by 10 microM atropine. Treatment of the tissue with ionomycin markedly reduced amiloride-sensitive I(sc), but a subsequent addition of CCh further decreased it. Also, CCh still had an inhibitory effect, although significantly attenuated, after the tissue had been incubated with a low-Ca(2+) solution containing ionomycin and BAPTA-AM. Applying electrical field stimulation to submucosal neurons in the mucosal-submucosal preparation resulted in inhibition of amiloride-sensitive I(sc), approximately 33% of this inhibition being atropine sensitive. Physostigmine inhibited amiloride-sensitive I(sc), this effect being abolished by atropine. In conclusion, submucosal cholinergic and noncholinergic neurons were involved in inhibiting electrogenic Na(+) absorption in colon. This inhibition by cholinergic neurons was mediated by muscarinic receptor activation.     

Physiological modulation of CFTR activity by AMP-activated protein kinase in polarized T84 cells

The cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated, ATP-gated Cl(-) channel and cellular conductance regulator, but the detailed mechanisms of CFTR regulation and its regulation of other transport proteins remain obscure. We previously identified the metabolic sensor AMP-activated protein kinase (AMPK) as a novel protein interacting with CFTR and found that AMPK phosphorylated CFTR and inhibited CFTR-dependent whole cell conductances when coexpressed with CFTR in Xenopus oocytes. To address the physiological relevance of the CFTR-AMPK interaction, we have now studied polarized epithelia and have evaluated the localization of endogenous AMPK and CFTR and measured CFTR activity with modulation of AMPK activity. By immunofluorescent imaging, AMPK and CFTR share an overlapping apical distribution in several rat epithelial tissues, including nasopharynx, submandibular gland, pancreas, and ileum. CFTR-dependent short-circuit currents (I(sc)) were measured in polarized T84 cells grown on permeable supports, and several independent methods were used to modulate endogenous AMPK activity. Activation of endogenous AMPK with the cell-permeant adenosine analog 5-amino-4-imidazolecarboxamide-1-beta-d-ribofuranoside (AICAR) inhibited forskolin-stimulated CFTR-dependent I(sc) in nonpermeabilized monolayers and monolayers with nystatin permeabilization of the basolateral membrane. Raising intracellular AMP concentration in monolayers with basolateral membranes permeabilized with alpha-toxin also inhibited CFTR, an effect that was unrelated to adenosine receptors. Finally, overexpression of a kinase-dead mutant AMPK-alpha1 subunit (alpha1-K45R) enhanced forskolin-stimulated I(sc) in polarized T84 monolayers, consistent with a dominant-negative reduction in the inhibition of CFTR by endogenous AMPK. These results indicate that AMPK plays a physiological role in modulating CFTR activity in polarized epithelia and suggest a novel paradigm for the coupling of ion transport to cellular metabolism.     

Cultured monolayers of the dog jejunum with the structural and functional properties resembling the normal epithelium

The development of a culture of the normal mammalian jejunum motivated this work. Isolated crypt cells of the dog jejunum were induced to form primary cultures on Snapwell filters. Up to seven subcultures were studied under the electron microscope and in Ussing chambers. Epithelial markers were identified by RT-PCR, Western blot, and immunofluorescent staining. Confluent monolayers exhibit a dense apical brush border, basolateral membrane infoldings, desmosomes, and tight junctions expressing zonula occludens-1, occludin-1, and claudin-3 and -4. In OptiMEM medium fortified with epidermal growth factor, hydrocortisone, and insulin, monolayer transepithelial voltage was -6.8 mV (apical side), transepithelial resistance was 1,050 Omega.cm(2), and short-circuit current (I(sc)) was 8.1 microA/cm(2). Transcellular and paracellular resistances were estimated as 14.8 and 1.1 kOmega.cm(2), respectively. Serosal ouabain reduced voltage and current toward zero, as did apical amiloride. The presence of mRNA of alpha-epithelial Na(+) channel (ENaC) was confirmed. Na-d-glucose cotransport was identified with an antibody to Na(+)-glucose cotransporter (SGLT) 1. The unidirectional mucosa-to-serosa Na(+) flux (19 nmol.min(-1).cm(-2)) was two times as large as the reverse flux, and net transepithelial Na(+) flux was nearly double the amiloride-sensitive I(sc). In plain Ringer solution, the amiloride-sensitive I(sc) went toward zero. Under these conditions plus mucosal amiloride, serosal dibutyryl-cAMP elicited a Cl(-)-dependent I(sc) consistent with the stimulation of transepithelial Cl(-) secretion. In conclusion, primary cultures and subcultures of the normal mammalian jejunum form polarized epithelial monolayers with 1) the properties of a leaky epithelium, 2) claudins specific to the jejunal tight junction, 3) transepithelial Na(+) absorption mediated in part by SGLT1 and ENaC, and 4) electrogenic Cl(-) secretion activated by cAMP.     

Transepithelial bioelectrical properties of rabbit acinar cell monolayers on polyester membrane scaffolds

In our quest to develop a tissue-engineered tear secretory system, we have tried to demonstrate active transepithelial ion fluxes across rabbit lacrimal acinar cell monolayers on polyester membrane scaffolds to evaluate the bioelectrical properties of the cultured cells. Purified lacrimal gland acinar cells were seeded onto polyester membrane inserts and cultured to confluency. Morphological properties of the cell monolayers were evaluated by transmission electron microscopy and immunofluorescence staining for Na(+),K(+)-ATPase and the tight junction-associated protein occludin. Sections revealed cell monolayers with well-maintained epithelial cell polarity, i.e., presence of apical (AP) secretory granules, microvilli, and junctional complexes. Na(+),K(+)-ATPase was localized on both the basal-lateral and apical plasma membranes. The presence of tight cell junctions was demonstrated by a positive circumferential stain for occludin. Bioelectrical properties of the cell monolayers were studied in Ussing chambers under short-circuit conditions. Active ion fluxes were evaluated by inhibiting the short-circuit current (I(sc)) with a Na(+),K(+)-ATPase inhibitor, ouabain (100 microM; basal-lateral, BL), and under Cl(-)-free buffer conditions after carbachol stimulation (CCh; 100 microM). The directional apical secretion of Cl(-) was demonstrated through pharmacological analysis, using amiloride (1 mM; BL) and bumetanide (0.1 mM; BL), respectively. Regulated protein secretion was evaluated by measuring the beta-hexosaminidase catalytic activity in the AP culture medium in response to 100 microM basal CCh. In summary, rabbit lacrimal acinar cell monolayers generate a Cl(-)-dependent, ouabain-sensitive AP --> BL I(sc) in response to CCh, consistent with current models for Na(+)-dependent Cl(-) secretion.     

Role of luminal ATP in regulating electrogenic Na(+) absorption in guinea pig distal colon

Extracellular ATP regulates a variety of functions in epithelial tissues by activating the membrane P2-receptor. The purpose of this study was to investigate the autocrine/paracrine regulation by luminal ATP of electrogenic amiloride-sensitive Na(+) absorption in the distal colon from guinea pigs treated with aldosterone by measuring the amiloride-sensitive short-circuit current (I(sc)) and (22)Na(+) flux in vitro with the Ussing chamber technique. ATP added to the luminal side inhibited the amiloride-sensitive I(sc) and (22)Na(+) absorption to a similar degree. The concentration dependence of the inhibitory effect of ATP on amiloride-sensitive I(sc) had an IC(50) value of 20-30 microM, with the maximum inhibition being approximately 50%. The effects of different nucleotides and of a nucleoside were also studied, the order of potency being ATP = UTP > ADP > adenosine. The effects of ATP were slightly, but significantly, reduced in the presence of suramin in the luminal solution. The inhibitory effect of luminal ATP was more potent in the absence of both Mg2+ and Ca2+ from the luminal solution. Pretreatment of the tissue with ionomycin or thapsigargin in the absence of serosal Ca2+ did not affect the percent inhibition of amiloride-sensitive I(sc) induced by ATP. Mechanical perturbation with a hypotonic luminal solution caused a reduction in amiloride-sensitive I(sc), this effect being prevented by the presence of hexokinase, an ATP-scavenging enzyme. These results suggest that ATP released into the luminal side by hypotonic stimulation could exert an inhibitory effect on the electrogenic Na(+) absorption. This effect was probably mediated by a P2Y(2) receptor on the apical membrane of colonic epithelial cells, and a change in the intracellular Ca2+ concentration may not be necessary for this process.     

Beta-adrenoceptor-mediated control of apical membrane conductive properties in fetal distal lung epithelia

Distal lung epithelial cells isolated from fetal rats were cultured (48 h) on permeable supports so that transepithelial ion transport could be quantified electrometrically. Unstimulated cells generated a short-circuit current (I(sc)) that was inhibited (~80%) by apical amiloride. The current is thus due, predominantly, to the absorption of Na(+) from the apical solution. Isoprenaline increased the amiloride-sensitive I(sc) about twofold. Experiments in which apical membrane Na(+) currents were monitored in basolaterally permeabilized cells showed that this was accompanied by a rise in apical Na(+) conductance (G(Na(+))). Isoprenaline also increased apical Cl- conductance (G(Cl-)) by activating an anion channel species sensitive to glibenclamide but unaffected by 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS). The isoprenaline-evoked changes in G(Na(+)) and G(Cl(minus sign)) could account for the changes in I(sc) observed in intact cells. Glibenclamide had no effect upon the isoprenaline-evoked stimulation of I(sc) or G(Na(+)) demonstrating that the rise in G(Cl-) is not essential to the stimulation of Na(+) transport.     

Induction of differentiation of HL-60 cells by protein kinase C inhibitor, K252a

To clarify the role of protein kinase C and protein kinase A in cell proliferation and differentiation, the effects of K252a and its derivatives (K252b, KT5720), which have different inhibitory activity to these protein kinases, on the proliferation and differentiation of HL-60 cells were investigated. The proliferation and DNA synthesis of the HL-60 cells were inhibited by K252a in a dose dependent manner. However, K252b and KT5720 which are more specific inhibitors of protein kinase C or protein kinase A, respectively, had no observable effect on cell proliferation. K252a (40nM) enhanced the differentiation of HL-60 cells induced by 1,25(OH)2D3, retinoic acid and DMSO. K252b and KT5720 did not affect 1,25(OH)2D3-induced differentiation. K252a significantly inhibited the differentiation induced by PMA. These results demonstrate that K252a but not its derivatives can function as an antitumor drug and enhancer of the differentiation induced by various inducers.     

DNA microarray analysis of neonatal mouse lung connects regulation of KDR with dexamethasone-induced inhibition of alveolar formation

Treating H441 cells with dexamethasone raised the abundance of mRNA encoding the epithelial Na(+) channel alpha- and beta-subunits and increased transepithelial ion transport (measured as short-circuit current, I(sc)) from <4 microA.cm(-2) to 10-20 microA.cm(-2). This dexamethasone-stimulated ion transport was blocked by amiloride analogs with a rank order of potency of benzamil >or= amiloride > EIPA and can thus be attributed to active Na(+) absorption. Studies of apically permeabilized cells showed that this increased transport activity did not reflect a rise in Na(+) pump capacity, whereas studies of basolateral permeabilized cells demonstrated that dexamethasone increased apical Na(+) conductance (G(Na)) from a negligible value to 100-200 microS.cm(-2). Experiments that explored the ionic selectivity of this dexamethasone-induced conductance showed that it was equally permeable to Na(+) and Li(+) and that the permeability to these cations was approximately fourfold greater than to K(+). There was also a small permeability to N-methyl-d-glucammonium, a nominally impermeant cation. Forskolin, an agent that increases cellular cAMP content, caused an approximately 60% increase in I(sc), and measurements made after these cells had been basolaterally permeabilized demonstrated that this response was associated with a rise in G(Na). This cAMP-dependent control over G(Na) was disrupted by brefeldin A, an inhibitor of vesicular trafficking. Dexamethasone thus stimulates Na(+) transport in H441 cells by evoking expression of an amiloride-sensitive apical conductance that displays moderate ionic selectivity and is subject to acute control via a cAMP-dependent pathway.     

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.