Selective regulation of acid-sensing ion channel 1 by serine proteases

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that belong to the epithelial Na(+) channel/degenerin family. ASICs are transiently activated by a rapid drop in extracellular pH. Conditions of low extracellular pH, such as ischemia and inflammation in which ASICs are thought to be active, are accompanied by increased protease activity. We show here that serine proteases modulate the function of ASIC1a and ASIC1b but not of ASIC2a and ASIC3. We show that protease exposure shifts the pH dependence of ASIC1a activation and steady-state inactivation to more acidic pH. As a consequence, protease exposure leads to a decrease in current response if ASIC1a is activated by a pH drop from pH 7.4. If, however, acidification occurs from a basal pH of approximately 7, protease-exposed ASIC1a shows higher activity than untreated ASIC1a. We provide evidence that this bi-directional regulation of ASIC1a function also occurs in neurons. Thus, we have identified a mechanism that modulates ASIC function and may allow ASIC1a to adapt its gating to situations of persistent extracellular acidification.     

Genistein improves regulatory interactions between G551D-cystic fibrosis transmembrane conductance regulator and the epithelial sodium channel in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR) in addition to its well defined Cl(-) channel properties regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and non-epithelial cells, whereas the presence of ENaC increases CFTR functional expression. This interregulation is reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl(-) channels are increased when CFTR is co-expressed with alphabetagamma-mouse ENaC (mENaC) and conversely when the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, different functional regulatory interactions were observed between G551D-CFTR and alphabetagamma-mENaC. The co-expression of G551D-CFTR and alphabetagamma-mENaC resulted in a 5-fold increase in G551D-CFTR Cl(-) current compared with oocytes expressing G551D-CFTR alone. Oocytes co-injected with both G551D-CFTR and ENaC expressed an amiloride-sensitive whole cell current that was similar to that observed before and after G551D-CFTR activation with forskolin/isobutylmethylxanthine. Treatment with genistein both enhanced the functional expression of G551D-CFTR and improved regulatory interactions between G551D-CFTR and ENaC. These data suggest that genistein may be useful in patients with cystic fibrosis and the G551D-CFTR mutation.     

Genistein restores functional interactions between Delta F508-CFTR and ENaC in Xenopus oocytes.

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its Cl(-) channel properties, has regulatory interactions with other epithelial ion channels including the epithelial Na(+) channel (ENaC). Both the open probability and surface expression of wild type CFTR Cl(-) channels are increased significantly when CFTR is co-expressed in Xenopus oocytes with alphabetagamma-ENaC, and conversely, the activity of ENaC is inhibited following wild type CFTR activation. Using the Xenopus oocyte expression system, a lack of functional regulatory interactions between DeltaF508-CFTR and ENaC was observed following activation of DeltaF508-CFTR by forskolin and isobutylmethylxanthine (IBMX). Whole cell currents in oocytes expressing ENaC alone decreased in response to genistein but increased in response to a combination of forskolin and IBMX followed by genistein. In contrast, ENaC currents in oocytes co-expressing ENaC and DeltaF508-CFTR remained stable following stimulation with forskolin/IBMX/genistein. Furthermore, co-expression of DeltaF508-CFTR with ENaC enhanced the forskolin/IBMX/genistein-mediated activation of DeltaF508-CFTR. Our data suggest that genistein restores regulatory interactions between DeltaF508-CFTR and ENaC and that combinations of protein repair agents, such as 4-phenylbutyrate and genistein, may be necessary to restore DeltaF508-CFTR function in vivo.     

Trypsin cleaves acid-sensing ion channel 1a in a domain that is critical for channel gating

Acid-sensing ion channels (ASICs) are neuronal Na(+) channels that are members of the epithelial Na(+) channel/degenerin family and are transiently activated by extracellular acidification. ASICs in the central nervous system have a modulatory role in synaptic transmission and are involved in cell injury induced by acidosis. We have recently demonstrated that ASIC function is regulated by serine proteases. We provide here evidence that this regulation of ASIC function is tightly linked to channel cleavage. Trypsin cleaves ASIC1a with a similar time course as it changes ASIC1a function, whereas ASIC1b, whose function is not modified by trypsin, is not cleaved. Trypsin cleaves ASIC1a at Arg-145, in the N-terminal part of the extracellular loop, between a highly conserved sequence and a sequence that is critical for ASIC1a inhibition by the venom of the tarantula Psalmopoeus cambridgei. This channel domain controls the inactivation kinetics and co-determines the pH dependence of ASIC gating. It undergoes a conformational change during inactivation, which renders the cleavage site inaccessible to trypsin in inactivated channels.     

Stomatin modulates gating of acid-sensing ion channels

Acid-sensing ion channels (ASICs) are H(+)-gated members of the degenerin/epithelial Na(+) channel (DEG/ENaC) family in vertebrate neurons. Several ASICs are expressed in sensory neurons, where they play a role in responses to nociceptive, taste, and mechanical stimuli; others are expressed in central neurons, where they participate in synaptic plasticity and some forms of learning. Stomatin is an integral membrane protein found in lipid/protein-rich microdomains, and it is believed to regulate the function of ion channels and transporters. In Caenorhabditis elegans, stomatin homologs interact with DEG/ENaC channels, which together are necessary for normal mechanosensation in the worm. Therefore, we asked whether stomatin interacts with and modulates the function of ASICs. We found that stomatin co-immunoprecipitated and co-localized with ASIC proteins in heterologous cells. Moreover, stomatin altered the function of ASIC channels. Stomatin potently reduced acid-evoked currents generated by ASIC3 without changing steady state protein levels or the amount of ASIC3 expressed at the cell surface. In contrast, stomatin accelerated the desensitization rate of ASIC2 and heteromeric ASICs, whereas current amplitude was unaffected. These data suggest that stomatin binds to and alters the gating of ASICs. Our findings indicate that modulation of DEG/ENaC channels by stomatin-like proteins is evolutionarily conserved and may have important implications for mammalian nociception and mechanosensation.     

The cytosolic termini of the beta- and gamma-ENaC subunits are involved in the functional interactions between cystic fibrosis transmembrane conductance regulator and epithelial sodium channel

Epithelial sodium channel (ENaC) and cystic fibrosis transmembrane conductance regulator (CFTR) are co-localized in the apical membrane of many epithelia. These channels are essential for electrolyte and water secretion and/or reabsorption. In cystic fibrosis airway epithelia, a hyperactivated epithelial Na(+) conductance operates in parallel with defective Cl(-) secretion. Several groups have shown that CFTR down-regulates ENaC activity, but the mechanisms and the regulation of CFTR by ENaC are unknown. To test the hypothesis that ENaC and CFTR regulate each other, and to identify the region(s) of ENaC involved in the interaction between CFTR and ENaC, rENaC and its mutants were co-expressed with CFTR in Xenopus oocytes. Whole cell macroscopic sodium currents revealed that wild type (wt) alphabetagamma-rENaC-induced Na(+) current was inhibited by co-expression of CFTR, and further inhibited when CFTR was activated with a cAMP-raising mixture (CKT). Conversely, alphabetagamma-rENaC stimulated CFTR-mediated Cl(-) currents up to approximately 6-fold. Deletion mutations in the intracellular tails of the three rENaC subunits suggested that the carboxyl terminus of the beta subunit was required both for the down-regulation of ENaC by activated CFTR and the up-regulation of CFTR by ENaC. However, both the carboxyl terminus of the beta subunit and the amino terminus of the gamma subunit were essential for the down-regulation of rENaC by unstimulated CFTR. Interestingly, down-regulation of rENaC by activated CFTR was Cl(-)-dependent, while stimulation of CFTR by rENaC was not dependent on either cytoplasmic Na(+) or a depolarized membrane potential. In summary, there appear to be at least two different sites in ENaC involved in the intermolecular interaction between CFTR and ENaC.     

Epithelial sodium channels regulate cystic fibrosis transmembrane conductance regulator chloride channels in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl(-) channel properties, regulates other ion channels. CFTR inhibits epithelial Na(+) channel (ENaC) currents in many epithelial and nonepithelial cells. Because modulation of net NaCl reabsorption has important implications in extracellular fluid volume homeostasis and airway fluid volume and composition, we investigated whether this regulation was reciprocal by examining whether ENaC regulates CFTR. Co-expression of human (h) CFTR and mouse (m) alphabetagammaENaC in Xenopus oocytes resulted in a significant, 3.7-fold increase in whole-cell hCFTR Cl(-) conductance compared with oocytes expressing hCFTR alone. The forskolin/3-isobutyl-1-methylxanthine-stimulated whole-cell conductance in hCFTR-mENaC co-injected oocytes was amiloride-insensitive, indicating an inhibition of mENaC following hCFTR activation, and it was blocked by DPC (diphenylamine-2-carboxylic acid) and was DIDS (4, 4'-diisothiocyanatostilbene-2,2'-disulfonic acid)-insensitive. Enhanced hCFTR Cl(-) conductance was also observed when either the alpha- or beta-subunit of mENaC was co-expressed with hCFTR, but this was not seen when CFTR was co-expressed with the gamma-subunit of mENaC. Single Cl(-) channel analyses showed that both CFTR Cl(-) channel open probability and the number of CFTR Cl(-) channels detected per patch increased when hCFTR was co-expressed with alphabetagammamENaC. We conclude that in addition to acting as a regulator of ENaC, CFTR activity is regulated by ENaC.     

Acid-sensing ion channels contribute to the effect of acidosis on the function of dendritic cells

As an H(+)-gated subgroup of the degenerin/epithelial Na(+) channel family, acid-sensing ion channels (ASICs) were reported to be involved in various physiological and pathological processes in neurons. However, little is known about the role of ASICs in the function of dendritic cells (DCs). In this study, we investigated the expression of ASICs in mouse bone marrow-derived DCs and their possible role in the function of DCs. We found that ASIC1, ASIC2, and ASIC3 are expressed in DCs at the mRNA and protein levels, and extracellular acid can evoke ASIC-like currents in DCs. We also demonstrated that acidosis upregulated the expression of CD11c, MHC class II, CD80, and CD86 and enhanced the Ag-presenting ability of DCs via ASICs. Moreover, the effect of acidosis on DCs can be abolished by the nonsteroidal anti-inflammatory drugs ibuprofen and diclofenac. These results suggest that ASICs are involved in the acidosis-mediated effect on DC function.     

Regulation of ENaC and CFTR expression with K+ channel modulators and effect on fluid absorption across alveolar epithelial cells

In a recent study (Leroy C, Dagenais A, Berthiaume Y, and Brochiero E. Am J Physiol Lung Cell Mol Physiol 286: L1027-L1037, 2004), we identified an ATP-sensitive K(+) (K(ATP)) channel in alveolar epithelial cells, formed by inwardly rectifying K(+) channel Kir6.1/sulfonylurea receptor (SUR)2B subunits. We found that short applications of K(ATP), voltage-dependent K(+) channel KvLQT1, and calcium-activated K(+) (K(Ca)) channel modulators modified Na(+) and Cl(-) currents in alveolar monolayers. In addition, it was shown previously that a K(ATP) opener increased alveolar liquid clearance in human lungs by a mechanism possibly related to epithelial sodium channels (ENaC). We therefore hypothesized that prolonged treatment with K(+) channel modulators could induce a sustained regulation of ENaC activity and/or expression. Alveolar monolayers were treated for 24 h with inhibitors of K(ATP), KvLQT1, and K(Ca) channels identified by PCR. Glibenclamide and clofilium (K(ATP) and KvLQT1 inhibitors) strongly reduced basal transepithelial current, amiloride-sensitive Na(+) current, and forskolin-activated Cl(-) currents, whereas pinacidil, a K(ATP) activator, increased them. Interestingly, K(+) inhibitors or membrane depolarization (induced by valinomycin in high-K(+) medium) decreased alpha-, beta-, and gamma-ENaC and CFTR mRNA. alpha-ENaC and CFTR proteins also declined after glibenclamide or clofilium treatment. Conversely, pinacidil augmented ENaC and CFTR mRNAs and proteins. Since alveolar fluid transport was found to be driven, at least in part, by Na(+) transport through ENaC, we tested the impact of K(+) channel modulators on fluid absorption across alveolar monolayers. We found that glibenclamide and clofilium reduced fluid absorption to a level similar to that seen in the presence of amiloride, whereas pinacidil slightly enhanced it. Long-term regulation of ENaC and CFTR expression by K(+) channel activity could benefit patients with pulmonary diseases affecting ion transport and fluid clearance.     

Ion transport mechanisms linked to bicarbonate secretion in the esophageal submucosal glands

The esophageal submucosal glands (SMG) secrete HCO(3)(-) and mucus into the esophageal lumen, where they contribute to acid clearance and epithelial protection. This study characterized the ion transport mechanisms linked to HCO(3)(-) secretion in SMG. We localized ion transporters using immunofluorescence, and we examined their expression by RT-PCR and in situ hybridization. We measured HCO(3)(-) secretion by using pH stat and the isolated perfused esophagus. Using double labeling with Na(+)-K(+)-ATPase as a marker, we localized Na(+)-coupled bicarbonate transporter (NBCe1) and Cl(-)-HCO(3)(-) exchanger (SLC4A2/AE2) to the basolateral membrane of duct cells. Expression of cystic fibrosis transmembrane regulator channel (CFTR) was confirmed by immunofluorescence, RT-PCR, and in situ hybridization. We identified anion exchanger SLC26A6 at the ducts' luminal membrane and Na(+)-K(+)-2Cl(-) (NKCC1) at the basolateral membrane of mucous and duct cells. pH stat experiments showed that elevations in cAMP induced by forskolin or IBMX increased HCO(3)(-) secretion. Genistein, an activator of CFTR, which does not increase intracellular cAMP, also stimulated HCO(3)(-) secretion, whereas glibenclamide, a Cl(-) channel blocker, and bumetanide, a Na(+)-K(+)-2Cl(-) blocker, decreased it. CFTR(inh)-172, a specific CFTR channel blocker, inhibited basal HCO(3)(-) secretion as well as stimulation of HCO(3)(-) secretion by IBMX. This is the first report on the presence of CFTR channels in the esophagus. The role of CFTR in manifestations of esophageal disease in cystic fibrosis patients remains to be determined.     

Cyclic AMP-induced K⁺ secretion occurs independently of Cl⁻ secretion in rat distal colon

cAMP induces both active Cl(-) and active K(+) secretion in mammalian colon. It is generally assumed that a mechanism for K(+) exit is essential to maintain cells in the hyperpolarized state, thus favoring a sustained Cl(-) secretion. Both Kcnn4c and Kcnma1 channels are located in colon, and this study addressed the questions of whether Kcnn4c and/or Kcnma1 channels mediate cAMP-induced K(+) secretion and whether cAMP-induced K(+) secretion provides the driving force for Cl(-) secretion. Forskolin (FSK)-enhanced short-circuit current (indicator of net electrogenic ion transport) and K(+) fluxes were measured simultaneously in colonic mucosa under voltage-clamp conditions. Mucosal Na(+) orthovanadate (P-type ATPase inhibitor) inhibited active K(+) absorption normally present in rat distal colon. In the presence of mucosal Na(+) orthovanadate, serosal FSK induced both K(+) and Cl(-) secretion. FSK-induced K(+) secretion was 1) not inhibited by either mucosal or serosal 1-[(2-chlorophenyl) diphenylmethyl]-1H-pyrazole (TRAM-34; a Kcnn4 channel blocker), 2) inhibited (92%) by mucosal iberiotoxin (Kcnma1 channel blocker), and 3) not affected by mucosal cystic fibrosis transmembrane conductance regulator inhibitor (CFTR(inh)-172). By contrast, FSK-induced Cl(-) secretion was 1) completely inhibited by serosal TRAM-34, 2) not inhibited by either mucosal or serosal iberiotoxin, and 3) completely inhibited by mucosal CFTR(inh)-172. These results indicate that cAMP-induced colonic K(+) secretion is mediated via Kcnma1 channels located in the apical membrane and most likely contributes to stool K(+) losses in secretory diarrhea. On the other hand, cAMP-induced colonic Cl(-) secretion requires the activity of Kcnn4b channels located in the basolateral membrane and is not dependent on the concurrent activation of apical Kcnma1 channels.     

No evidence for inhibition of ENaC through CFTR-mediated release of ATP

Both purinergic stimulation and activation of cystic fibrosis transmembrane conductance regulator (CFTR) increases Cl(-) secretion and inhibit amiloride-sensitive Na(+) transport. CFTR has been suggested to conduct adenosine 5'-triphosphate (ATP) or to control ATP release to the luminal side of epithelial tissues. Therefore, a possible mechanism on how CFTR controls the activity of epithelial Na(+) channels (ENaC) could be by release of ATP or uridine 5'-triphosphate (UTP), which would then bind to P2Y receptors and inhibit ENaC. We examined this question in native tissues from airways and colon and in Xenopus oocytes. Inhibition of amiloride-sensitive transport by both CFTR and extracellular nucleotides was observed in colon and trachea. However, nucleotides did not inhibit ENaC in Xenopus oocytes, even after coexpression of P2Y(2) receptors. Using different tools such as hexokinase, the P2Y inhibitor suramin or the Cl(-) channel blocker 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid (DIDS), we did not detect any role of a putative ATP secretion in activation of Cl(-) transport or inhibition of amiloride sensitive short circuit currents by CFTR. In addition, N(2),2'-O-dibutyrylguanosine 3',5'-cyclic monophosphate (cGMP) and protein kinase G (PKG)-dependent phosphorylation or the nucleoside diphosphate kinase (NDPK) do not seem to play a role for the inhibition of ENaC by CFTR, which, however, requires the presence of extracellular Cl(-).     

Effect of [Cl(-)]i on ENaC activity from mouse cortical collecting duct cells

Na(+) transport via epithelial Na(+) channel (ENaC) occurs across many epithelial surfaces and plays a key role in regulating salt and water absorption. In this study, we have examined the effects of cytosolic Na(+) and Cl(-) on ENaC activity by patch clamping single channel recording method in mouse cortical collecting duct cells (M1). Cytosolic Na(+) exerts its effect in change of ENaC open probability (Po). High cytosolic Na(+) significantly reduces ENaC Po. No change in channel conductance by cytosolic Na(+) is observed. However, decrease of cytosolic Cl(-) concentration significantly increases channel conductance and ENaC Po. This effect is due to the right shift of ENaC I-V curve to positive membrane potential. The virtue of ENaC conductance remains the same. Cl(-) channels like CFTR and VRAC are unlikely to be involved in this regulation. The results suggest that cytosolic Cl(-) could serve as a mediator to regulate ENaC activity, in accordance with the activities of Cl(-) channels.     

Aquaporin 3 cloned from Xenopus laevis is regulated by the cystic fibrosis transmembrane conductance regulator

The cystic fibrosis transmembrane conductance regulator (CFTR) is essential for epithelial electrolyte transport and has been shown to be a regulator of epithelial Na(+), K(+), and Cl(-) channels. CFTR also enhances osmotic water permeability when activated by cAMP. This was detected initially in Xenopus oocytes and is also present in human airway epithelial cells, however, the mechanisms remain obscure. Here, we show that CFTR activates aquaporin 3 expressed endogenously and exogenously in oocytes of Xenopus laevis. The interaction requires stimulation of wild type CFTR by cAMP and an intact first nucleotide binding domain as demonstrated for other CFTR-protein interactions.     

Functional modifications of acid-sensing ion channels by ligand-gated chloride channels

Together, acid-sensing ion channels (ASICs) and epithelial sodium channels (ENaC) constitute the majority of voltage-independent sodium channels in mammals. ENaC is regulated by a chloride channel, the cystic fibrosis transmembrane conductance regulator (CFTR). Here we show that ASICs were reversibly inhibited by activation of GABA(A) receptors in murine hippocampal neurons. This inhibition of ASICs required opening of the chloride channels but occurred with both outward and inward GABA(A) receptor-mediated currents. Moreover, activation of the GABA(A) receptors modified the pharmacological features and kinetic properties of the ASIC currents, including the time course of activation, desensitization and deactivation. Modification of ASICs by open GABA(A) receptors was also observed in both nucleated patches and outside-out patches excised from hippocampal neurons. Interestingly, ASICs and GABA(A) receptors interacted to regulate synaptic plasticity in CA1 hippocampal slices. The activation of glycine receptors, which are similar to GABA(A) receptors, also modified ASICs in spinal neurons. We conclude that GABA(A) receptors and glycine receptors modify ASICs in neurons through mechanisms that require the opening of chloride channels.     

Cystic fibrosis transmembrane conductance regulator differentially regulates human and mouse epithelial sodium channels in Xenopus oocytes

The cystic fibrosis transmembrane conductance regulator (CFTR), in addition to its well defined Cl- channel properties, regulates other ion channels. CFTR inhibits murine or rat epithelial Na+ channel (mENaC or rENaC) currents in many epithelial and non-epithelial cells, whereas murine or rat ENaC increases CFTR functional expression. These regulatory interactions are reproduced in Xenopus oocytes where both the open probability and surface expression of wild type CFTR Cl- channels are increased when CFTR is co-expressed with alphabetagamma mENaC, and conversely the activity of mENaC is inhibited after wild type CFTR activation. Using the Xenopus oocyte expression system, differences in functional regulatory interactions were observed when CFTR was co-expressed with either alphabetagamma mENaC or alphabetagamma human ENaC (hENaC). Co-expression of CFTR and alphabetagamma mENaC or hENaC resulted in an approximately 3-fold increase in CFTR Cl- current compared with oocytes expressing CFTR alone. Oocytes co-injected with both CFTR and mENaC or hENaC expressed an amiloride-sensitive whole cell current that was decreased compared with that observed with the injection of mENaC or hENaC alone before CFTR activation with forskolin/3-isobutyl-1-methylxanthine. CFTR activation resulted in a further 50% decrease in mENaC-mediated currents, an approximately 20% decrease in alpha-T663-hENaC-mediated currents, and essentially no change in alpha-A663-hENaC-mediated currents. Changes in ENaC functional expression correlated with ENaC surface expression by oocyte surface biotinylation experiments. Assessment of regulatory interactions between CFTR and chimeric mouse/human ENaCs suggest that the 20 C-terminal amino acid residues of alpha ENaC confer species specificity regarding ENaC inhibition by activated CFTR.     

Electrolyte Transport in the Mouse Trachea: No Evidence for a Contribution of Luminal K+ Conductance

Recent studies on frog skin acini have challenged the question whether Cl(-) secretion or Na(+) absorption in the airways is driven by luminal K(+) channels in series to a basolateral K(+) conductance. We examined the possible role of luminal K(+) channels in electrolyte transport in mouse trachea in Ussing-chamber experiments. Tracheas of both normal and CFTR (-/-) mice showed a dominant amiloride-sensitive Na+ absorption under both, control conditions and after cAMP-dependent stimulation. The lumen-negative transepithelial voltage was enhanced after application of IBMX and forskolin and Cl(-) secretion was activated. Electrolyte secretion induced by IBMX and forskolin was inhibited by luminal glibenclamide and the blocker of basolateral Na(+2)Cl(-)K(+) cotransporter azosemide. Similarly, the compound 293B, a blocker of basolateral KCNQ1/KCNE3 K(+) channels effectively blocked Cl(-) secretion when applied to either the luminal or basolateral side of the epithelium. RT-PCR analysis suggested expression of additional K(+) channels in tracheal epithelial cells such as Slo1 and Kir6.2. However, we did not detect any functional evidence for expression of luminal K(+) channels in mouse airways, using luminal 293B, clotrimazole and Ba(2+) or different K(+) channel toxins such as charybdotoxin, apamin and a-dendrotoxin. Thus, the present study demonstrates Cl(-) secretion in mouse airways, which depends on basolateral Na(+2)Cl(-)K(+) cotransport and luminal CFTR and non-CFTR Cl(-) channels. Cl(-) secretion is maintained by the activity of basolateral K(+) channels, while no clear evidence was found for the presence of a luminal K(+) conductance.     

IL-4 is a potent modulator of ion transport in the human bronchial epithelium in vitro.

Recent data show that proinflammatory stimuli may modify significantly ion transport in the airway epithelium and therefore the properties of the airway surface fluid. We have studied the effect of IL-4, a cytokine involved in the pathogenesis of asthma, on transepithelial ion transport in the human bronchial epithelium in vitro. Incubation of polarized bronchial epithelial cells with IL-4 for 6-48 h causes a marked inhibition of the amiloride-sensitive Na(+) channel as measured in short circuit current experiments. On the other hand, IL-4 evokes a 2-fold increase in the current activated by a cAMP analog, which reflects the activity of the cystic fibrosis transmembrane conductance regulator (CFTR). Similarly, IL-4 enhances the response to apical UTP, an agonist that activates Ca(2+)-dependent Cl(-) channels. These effects are mimicked by IL-13 and blocked by an antagonist of IL-4Ralpha. RT-PCR experiments show that IL-4 elicits a 7-fold decrease in the level of the gamma amiloride-sensitive Na(+) channel mRNA, one of the subunits of the amiloride-sensitive Na(+) channel, and an increase in CFTR mRNA. Our data suggest that IL-4 may favor the hydration of the airway surface by decreasing Na(+) absorption and increasing Cl(-) secretion. This could be required to fluidify the mucus, which is hypersecreted during inflammatory conditions. On the other hand, the modifications of ion transport could also affect the ion composition of airway surface fluid.     

Acid-induced modulation of airway basal tone and contractility: role of acid-sensing ion channels (ASICs) and TRPV1 receptor

The role of extracellular acidosis in inflammatory airway diseases is not well known. One consequence of tissue acidification is the stimulation of sensory nerves via the polymodal H(+)-gated transmembrane channels ASICs and TRPV1 receptor. The present study investigated the effect of acidosis on airway basal tone and responsiveness in the guinea pig. Acidosis (pH 6.8, 10 min, 37 degrees C) significantly decreased the basal tone of tracheal rings (p<0.01 vs. paired control). Moreover, pH fall raised the maximal contraction of tracheal rings to acetylcholine (p<0.05 vs. paired control). The pH-induced relaxation of airway basal tone was inhibited by pretreatments with ASIC1a or ASIC3/ASIC2a inhibitors (0.5 mM ibuprofen, 0.1 mM gadolinium), nitric oxide synthase inhibitor (1 mM L-NAME), and guanylate cyclase inhibitor (1 microM ODQ). In contrast, the pH-induced relaxation of airway basal tone was not modified by epithelium removal or pretreatments with a TRPV1 antagonist (1 microM capsazepine), a combination of NK(1,2,3) receptor antagonists (0.1 microM each), a blocker of voltage-sensitive Na(+) channels (1 microM tetrodotoxin), a cyclooxygenase inhibitor with no activity on ASICs (1 microM indomethacin) or ASIC3 and ASIC3/ASIC2b inhibitors (10 nM diclofenac, 1 microM aspirin). Furthermore, acid-induced hyperresponsiveness to acetylcholine was inhibited by epithelium removal, capsazepine, NK(1,2,3) receptor antagonists, tetrodotoxin, amiloride, ibuprofen and diclofenac. In summary, the initial pH-induced airway relaxation seems to be independent of sensory nerves, suggesting a regulation of airway basal tone mediated by smooth muscle ASICs. Conversely, the pH-induced hyperresponsiveness involves sensory nerves-dependent ASICs and TRPV1, and an unknown epithelial component in response to acidosis.