Dual Oxidase 2 (Duox2) Regulates Pannexin 1-mediated ATP Release in Primary Human Airway Epithelial Cells via Changes in Intracellular pH and Not H2O2 Production*

Human airway epithelial cells express pannexin 1 (Panx1) channels to release ATP, which regulates mucociliary clearance. Airway inflammation causes mucociliary dysfunction. Exposure of primary human airway epithelial cell cultures to IFN-γ for 48 h did not alter Panx1 protein expression but significantly decreased ATP release in response to hypotonic stress. The IFN-γ-induced functional down-regulation of Panx1 was due to the up-regulation of dual oxidase 2 (Duox2). Duox2 suppression by siRNA led to an increase in ATP release in control cells and restoration of ATP release in cells treated with IFN-γ. Both effects were reduced by the pannexin inhibitor probenecid. Duox2 up-regulation stoichiometrically increases H2O2 and proton production. H2O2 inhibited Panx1 function temporarily by formation of disulfide bonds at the thiol group of its terminal cysteine. Long-term exposure to H2O2, however, had no inhibitory effect. To assess the role of cellular acidification upon IFN-γ treatment, fully differentiated airway epithelial cells were exposed to ammonium chloride to alkalinize the cytosol. This led to a 2-fold increase in ATP release in cells treated with IFN-γ that was also inhibited by probenecid. Duox2 knockdown also partially corrected IFN-γ-mediated acidification. The direct correlation between intracellular pH and Panx1 open probability was shown in oocytes. Therefore, airway epithelial cells release less ATP in response to hypotonic stress in an inflammatory environment (IFN-γ exposure). Decreased Panx1 function is a response to cell acidification mediated by IFN-γ-induced up-regulation of Duox2, representing a novel mechanism for mucociliary dysfunction in inflammatory airway diseases.

ATP is a key regulator of the innate pulmonary host defense by activating purinergic P2Y2 receptors, which promote chloride secretion by calcium-activated Cl Ϫ channels, inhibit Na ϩ absorption by epithelial Na ϩ channels, increase ciliary beat fre-quency and airway surface liquid volume, and induce mucin release, thereby activating mucociliary clearance (1)(2)(3)(4).
Although the effects of ATP on airway epithelial cells have been studied widely, Panx1 channels have only been recognized recently to be involved in ATP release in these tissues (5,6). Mechanical stress has been shown to be one of the prime stimuli to increase the ATP concentration on the airway surface to a concentration sufficient to activate P2Y2 receptors (7). This ATP release was neither dependent on the intracellular calcium concentration, excluding an exocytotic release mechanism, nor caused by the cystic fibrosis transmembrane conductance regulator. In previous studies, we were the first to show that Panx1, an ortholog of the invertebrate innexin, is expressed at the apical membrane of airway epithelia, contributing to ATP release (5).
Pannexin proteins form pannexons, which are channels that open at resting membrane potential because of mechanical stress and in response to extracellular ATP when co-expressed with P2Y2 receptors (8,9). In addition to stimulated ATP release, resting ATP levels on the apical surface reflect a steady state that, depending on its range, can help autoregulate the release or metabolism of ATP.
Currently little is known about the physiological regulation of pannexin channels in the airway epithelium. Most studies have been performed on oocytes or transfected HEK cells. These studies demonstrated that ATP itself is a potent regulator by increasing the permeability of Panx1 initially when coexpressed with P2Y2 receptors but, consequently, causing inhibition as a feedback loop (10,11).
The cytokine IFN-␥ is produced in inflammatory airway diseases such as severe asthma (12,13) and chronic bronchitis with and without airflow obstruction (14). We have shown that IFN-␥ can increase H 2 O 2 production via increased expression of Duox2, a member of the NADPH oxidase gene family, in ALI 2 cultures (15,16). In addition to H 2 O 2 , Duox also releases cytosolic H ϩ (17), which could contribute to intracellular acidification. Consequently, a decrease in intracellular pH could decrease mucociliary function by direct inhibition of the ciliary beat frequency (18). In fact, Duox is up-regulated in chronic bronchitis and patients with smoke exposure (15,19,20). Preliminary data also suggested that intracellular pH is essential in * This work was supported by National Institutes of Health Grants R01 HL-89399 and R01 HL-60644, the Flight Attendant Medical Research Institute (CIA103027 and CIA13033), the Cystic Fibrosis Foundation (SALATH14G0; to M. S.), and Chilean Grants 1120802 and ACT1104 and Millennium Initiative P09 -022-F (to C. G.). The authors declare that they have no conflicts of interest with the contents of this article. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health. 1  regulating pannexin channels, pointing to an important regulatory role of pannexin channels in the host defense of airway epithelia. The data presented here reveal novel regulation mechanisms demonstrating that IFN-␥ exposure of airway epithelial cells decreases ATP release by inhibition of Panx1 channels. The inhibition is mediated by Duox2 and caused by intracellular acidification. H 2 O 2 causes a temporary inhibition via oxidation of a terminal cysteine.

Experimental Procedures
Chemicals and Solutions-Unless stated otherwise, all materials were purchased from Sigma-Aldrich (St. Louis, MO).
ALI Cell Culture-Human airways were obtained from organ donors whose lungs were rejected for transplant. Institutional review board-approved consent for research was obtained by the Life Alliance Organ Recovery Agency of the University of Miami or the LifeCenter Northwest and conformed to the Declaration of Helsinki. Airway epithelial cells were isolated and dedifferentiated through expansion. Passage 1 cells were redifferentiated at an ALI on collagen-coated 12-or 24-mm-diameter inserts (Costar, Corning, NY) as described previously (21)(22)(23)(24).
RNA Extraction and Quantitative RT-PCR-Total RNA was extracted from ALI-cultured human airway epithelial cells using the RNeasy Protect mini kit (Qiagen, Valencia, CA). Reverse transcription was done using the iScript cDNA synthesis kit (Bio-Rad) with 1 g of RNA according to the instructions of the manufacturer. Real-time quantitative PCR was performed using the following TaqMan probes: Hs00209790_m1 for Panx1, Hs00213694_m1 for Duox1, Hs00204187_m1 for Duox2, Hs00167309_m1 for SOD2, and Hs02758991_g1 for GAPDH.
Hypotonic Stress-induced ATP Release Assay-Culture media in the basolateral compartment were replaced with media containing IFN-␥ (100 ng/ml) or vehicle (distilled H 2 O), and the cultures were incubated for 48 h at 37°C. Media were removed, and the filters were transferred to a Turner TD 20/20 luminometer (Turner Biosystems, Sunnyvale, CA), where 26 l of a luciferin (150 mM) and luciferase (2.0 mg) solution was added gently to the apical compartment. The culture was allowed to equilibrate for 100 s to re-establish basal conditions, and then (time 0), 15 l of a hypotonic solution (H 2 O with 1 mM CaCl 2 and 1 mM MgCl 2 ) was added to the apical compartment, causing hypotonic stress for an additional 200 s, as described previously (5,7). ATP release was recorded in arbitrary light units every 0.2 s. ATP release was reported as peak ATP response upon hypotonic stress, with data given as fold increases (or decreases) over the appropriate control increase (measured as the difference between baseline and peak arbitrary light units).
Voltage Clamp Experiments in Oocytes-An mRNA for mouse Panx1 was prepared using the mMessage in vitro transcription kit (Invitrogen) (20). Oocytes were injected with 20 -40 nl of mRNA (1 mg/ml) and incubated for 18 -48 h at 18°C. Oocytes were tested using a two-electrode voltage clamp (model OC725C, Warner Instruments, Hamden, CT) under constant perfusion with solutions containing sodium acetate for acidification. Oocytes expressing Panx1 were held at Ϫ60 mV, and pulses to ϩ10 mV were applied at a rate of 0.1 Hz to transiently open the channels.
Patch Clamp Experiments in ALI Cultures-The currents were recorded in intact, fully differentiated normal human bronchial epithelial (NHBE) cells in a cell-attached patch configuration using glass patch pipettes of 1-3 M diameter and of 2.5-4 M⍀ resistance. The bath and pipette solution contained 1 mM KCl, 10 mM HEPES, 2 mM CaCl 2 and 99 mM NaCl (pH 7.1). The currents were elicited by a symmetric voltage ramp between Ϫ60 to ϩ140 mV and returned to Ϫ60 mV. The speed of the ramp was 0.4 mV/s. The analog signal was filtered before digitalization with an eight-pole low-pass Bessel filter with a cutoff frequency of 1/5 of the conversion frequency. Experiments were performed at room temperature (25°C). Data were collected with an Axopatch 200B (Molecular Devices, Sunnyvale, CA) and sampled with a Digidata 1322A interface. The acquisition and basic analysis of data were performed with pClamp 9.2 (Axon Instruments, Inc.) Intracellular pH Measurement-Intracellular pH (pH i ) was measured as described previously (18). Briefly, ALI cultures were loaded with 2.5 M 2Ј,7Ј-bis(2-carboxyethyl)-5,6-carboxyfluorescein acetoxymethyl ester (BCECF)-AM for 45 min at 37°C and 5% CO 2 and rinsed three times. Then filters were cut out of the inserts and mounted in a closed bath imaging perfusion chamber (Warner Instruments, large diamond, RC-21B). All solutions were perfused at a rate of 250 l/min. For fluorescence measurements, a Lambda DG4 excitation system (Sutter, Novato, CA) was used, with 10-nm-wide excitation filters centered on 495 and 440 nm (Chroma Technology Corp., Brattleboro, VT). At the end of these experiments, the system was perfused with a high K ϩ HEPES solution with 15 M of nigericin equilibrated at pH 7.2. The calibration of pH measurements was done according to methods described previously (18).
Knockdown of Duox2 Using Lentiviruses-pLKO.1-Puro plasmids encoding non-targeting or Duox2 shRNA (catalog nos. SHC002 and TRCN0000045963, respectively, Sigma-Al-drich) were amplified. The virus was produced as described previously (15). Briefly, HEK293T cells were co-transfected using third-generation replication-defective lentiviral vectors and packaging plasmids. The viral yield was quantified using a p24 ELISA assay (PerkinElmer Life Sciences). Dedifferentiated NHBE cells from four lung donors were infected with 40 ng/2 ϫ 10 5 cells of Duox2 shRNA or non-targeting lentiviruses in suspension and selected with 1 g/ml puromycin. After growth to confluency, cultures were redifferentiated at the ALI for 4 -6 weeks, at which point cells exhibited mucus production and beating cilia. mRNA levels were assayed by real-time PCR using TaqMan kits as described previously (15), and Western blotting was done as above. We have been successful in knocking down several proteins in fully differentiated airway epithelial cells (5,25,26).
Statistics-Statistical analysis was performed by Student's t test and analysis of variance or Kruskal Wallis test with appro-priate post tests for at least three independent experiments, with significance accepted at p Ͻ 0.05.

Results
Patch Clamp Confirmation of Panx1 in ALI Cultures-Patch clamp recordings of macroscopic currents of fully differentiated ALI cultures in the cell-attached configuration confirmed Panx1 currents by stimulation with positive membrane potentials ( Fig. 1, A and A1). The high single-channel conductance recorded is typical for Panx1. Furthermore, probenecid, an inhibitor of Panx1, caused significant inhibition of the currents (Fig. 1, A and B1). Probenecid discriminates pannexins from connexins. Other known targets include transporters. Although we cannot rule out that probenecid inhibits other channels, it represents a useful tool for studying Panx1 function.    IFN-␥ Reduces Hypotonic Stress-induced ATP Release in Normal Bronchial Epithelial Cells-ALI cultures were exposed basolaterally to 100 ng/ml IFN-␥ for 48 h and subjected to hypotonic stress (200 mosmol), then ATP release was assessed as described previously (5,7). Our results demonstrated that ALI cultures exposed to IFN-␥ showed a statistically significant decrease in ATP release compared with control cells (Fig. 1B). INF-␥ exposure (100 ng/ml for 48 h) was related to an increase in Duox2 mRNA and protein expression (Fig. 1, C-E). On the other hand, INF-␥ exposure did not change Duox1 mRNA or protein expression. Although Panx1 mRNA increased with INF-␥, Panx1 protein did not change (Fig. 1, C-E). In addition, we have shown before that IFN-␥ does not change the mRNA levels of the NAPDH oxidases Nox1, 2, 4, and 5, possible offtarget oxidases (15).
To show the specificity of INF-␥-induced Duox2 up-regulation, we used other proinflammatory stimuli. TNF-␣ (10 ng/ml for 48 h), as shown before (19), did not change Duox1 or Duox2 mRNA or protein expression ( Fig. 1, C-E). Even though there was a trend for an increased ATP release (Fig. 1B, right panel), it did not reach statistical significance. An increase in superoxide dismutase 2 (SOD2) mRNA ( Fig. 1C) was not only seen with TNF-␣ but also with IFN-␥. Because TNF-␣ increased ATP release and IFN-␥ decreased it (Fig. 1B, right panel), SOD2 was not related to changes in ATP release. IL-4 (10 ng/ml for 48 h) increased expression of Duox1 mRNA and protein (Fig. 1, C-E), as shown previously (19), but did not change ATP release (Fig. 1B, right panel).
Suppression of Duox2 by shRNA Results in an Increase in ATP Release in ALI Cultures with and without IFN-␥ Pretreatment-ALI cultures were infected with Duox2 shRNA or non-targeting control lentiviruses and selected with puromycin as described previously (15). After full differentiation (4 -6 weeks at ALI), cultures were stimulated with IFN-␥ for 48 h. ATP release was then assessed at baseline and after hypotonic challenge. Cells infected with siDuox2 showed significantly decreased protein ( Fig. 2A) and mRNA levels of Duox2 (Fig. 2B) but not SOD2 (Fig. 2C). As shown before, IFN-␥ caused a decrease in ATP release in non-targeting, shRNA-infected control cells. Cells infected with siDuox2, however, showed a significant increase in ATP release even at baseline. More importantly, siDuox2 attenuated the decrease in ATP release after IFN-␥ pretreatment (Fig. 2D). The increased ATP release was blocked by probenecid, indicating the involvement of Panx1 and not another mechanism (Fig. 2E) (28 -30). The reason is most likely the lower aquaporin expression in oocytes. Aquaporins are considered the main pathway for bringing H 2 O 2 through membranes (31,32). Control plasmids were used by us before, with no effects on the results (e.g. Ref. 33).
H 2 O 2 (10 mM) affected pannexons in a complex way (Fig.  3A). Initially, the currents were attenuated after extracellular application of H 2 O 2 . While H 2 O 2 was still present, the currents increased, sometimes exceeding the pretreatment levels. After washout, the currents declined to the levels of maximal inhibition to return to pretreatment levels. Reaction of the (cytoplasmic) terminal cysteine (Cys-426) of Panx1 with the thiol reagent maleimidobutyryl-biocytin prevented the attenuation of the pannexon currents by H 2 O 2 but did not affect the current increase (Fig. 3, B and C). The increase occurred with a similar delay of minutes after the application of H 2 O 2 and led to similar increases of the currents above the original current levels observed for the recovery from H 2 O 2 -induced inhibition.
An extensive cysteine scan has been performed by us before (33). The terminal cysteine Cys-426 was the only one in wildtype Panx1 to react with thiol reagents, including maleimidobutyryl-biocytin. Therefore, replacement of the terminal cysteine by serine was examined. H 2 O 2 did not inhibit pannexon currents but increased them with a delay after H 2 O 2 application (Fig. 3, A-C). Double cysteine replacement, combining C426S with either C40S, C136S, C215S, C227S, or C346S, eliminated the attenuation but not the enhancement of pannexon currents (Fig. 3D). Neither inhibition nor enhancement of the pannexon currents were affected by staurosporine, a nonspecific inhibitor of protein kinases (Fig. 3E).
Because Panx1 is constitutively expressed in NHBE cells, experiments with mutants were limited to oocytes. However, similar results were obtained in NHBE cells using H 2 O 2 . Duox2 can create at least a concentration of 150 M H 2 O 2 in the apical fluid above airway cells, and 0.5 mM is a reasonable maximum (16). Therefore, we used 0.5 mM H 2 O 2 for our human cell experiments. A short-term (Ͻ10 min) exposure to 0.5 mM H 2 O 2 (preincubation with H 2 O 2 for 10 min and consecutive exposure to hypotonic stress) led to a decrease in ATP release that could be partially rescued by catalase (30 units in 100 l of PBS apically, proven to be the most effective dose, Fig. 4A). A 60-min preincubation with H 2 O 2 did not affect hypotonic stress-induced ATP release (Fig. 4B). Interestingly, even the control cultures revealed higher ATP release after 60 min of rest, which might be due to less perturbation of the apical membrane for this period of time. On the other hand, catalase had no effect on the IFN-␥-mediated inhibition of ATP release (Fig.  4C).
In summary, short-term H 2 O 2 exposures of oocytes inhibited the currents of expressed Panx1 via oxidation of a terminal cysteine residue. ATP release from NHBE cells was also inhibited by short-term exposure with H 2 O 2 . However, longer-term exposures did not inhibit ATP release. In addition, catalase could not reverse IFN-␥-mediated inhibition of ATP release. pH i Changes Affect the Function of Panx1 Channels-To evaluate the role of intracellular pH on pannexin channels, the Panx1 protein was again expressed in Xenopus oocytes. To show a possible regulation by varying intracellular pH, the oocytes were immersed in sodium acetate for intracellular acidification. Oocytes contain an endogenous pH-sensitive channel that was activated by extracellular acetate (Fig. 5A). In contrast, acetate attenuated the membrane currents in Panx1-expressing oocytes (Fig. 5A). The degree of Panx1 channel inhibition was 28.2% Ϯ 1.8% (n ϭ 8), with correction for the average current increase because of the endogenous channels.
Treatment with IFN-␥ Leads to Intracellular Acidification of NHBE Cells-ALI cultures were exposed to 100 ng/ml IFN-␥ for 48 h. Then the pH level was measured as described previously (18). Ciliated cells exposed to IFN-␥ revealed significantly lower pH i compared with control cells (Fig. 5B).
Intracellular Alkalization with NH 4 Cl Increases ATP Release in ALI Cultures Exposed to IFN-␥-To further demonstrate that pH i plays a role in the regulation of ATP release, ALI cultures were preincubated basolaterally with IFN-␥ for 48 h and then incubated with NH 4 Cl or vehicle before hypotonic stress. Treatment of cells with NH 4 Cl increased the pH in control cells as well as in IFN-␥-treated cells (Fig. 5, C and D). ALI cultures exposed to IFN-␥ and NH 4 Cl showed a significant increase in ATP release compared with cells exposed to IFN-␥ alone (Fig. 5, E and F), an effect blocked by probenecid (Fig. 5F). This showed that alkalization of the cytosol led to attenuation of the IFN-␥ effect. Furthermore, cells infected with siDuox2 also revealed an increased pH i in the presence of IFN-␥ compared with non-infected cells, tying together pH changes and ATP release modified by Duox2 (Fig. 5G).

Discussion
For the first time, we showed the electrophysiological presence and activity of Panx1 in primary normal human bronchial epithelial cells. Our data also depict novel regulation mechanisms of Panx1 channels. Airway epithelial cells release less  ATP in response to hypotonic stress in an environment expected during airway inflammation (IFN-␥ exposure). This was due to decreased Panx1 function with preserved expression levels in response to cell acidification mediated by increased Duox2 activity (15). Initially we thought that Duox2 may affect pannexons through the production of H 2 O 2 . Indeed, H 2 O 2 was found to affect pannexon currents in oocytes sequentially in opposite ways: a primary inhibition reverted to an enhancement of currents. The inhibitory effect is attributable to the terminal cysteine of Panx1, whereas reaction of its thiol group with maleimidobutyryl-biocytin (33) or its replacement by serine abolished the inhibitory effect of H 2 O 2 . The effects of H 2 O 2 on thiol groups can lead to disulfides (reversible) or the formation of sulfinic or sulfonic acid (both irreversible). The reversibility of the effect of H 2 O 2 on pannexon currents therefore indicates a formation of disulfide bonds between the terminal cysteine C426S with other thiol groups. These thiol groups could be on other proteins, or H 2 O 2 may induce disulfide bonding between Panx1 subunits within the same pannexon.
These results exclude thiol reactions to be responsible for the delayed enhancement of pannexon currents. Because this phenomenon also remained unaffected by staurosporine, a phosphorylation event appears to be an unlikely cause. The mechanisms leading to current enhancement could include a reactive alkalization of the cytoplasm or an increase in cytoplasmic calcium ion concentration (9,34).
Similarly, short-term exposure of NHBE cells to H 2 O 2 also decreased ATP release, whereas longer exposures (60 min) had no effect. However, this adaptive response is blunted in an inflammatory environment because Duox2 up-regulation not only leads to production of H 2 O 2 but also intracellular acidification. Intracellular acidification indeed inhibited both Panx1 currents in oocytes and ATP release in NHBE cells. By changing 10   , where an initial attenuation was followed by recovery to base levels or even a small increase of the currents. Following washout of H 2 O 2 , the currents dropped to the level of maximal inhibition but then recovered to the level seen before the interference. With both Panx1 mutants, only an increase in current amplitude was observed that reversed upon washout of H 2 O 2 . B and C, quantitative analysis of the inhibitory (B) and enhancing (C) components of the H 2 O 2 effect on currents in oocytes expressing WT Panx1 or Panx1C426S. The inhibitory component was attenuated by pretreatment of the oocytes with 1 mM maleimidobutylyl-biocytin (MBB), known to react with the terminal cysteine Cys-426 of Panx1 (33). Replacement of this cysteine by serine abolished (C426S) the inhibitory component, revealing the full extent of amplification of the currents by H 2 O 2 . Percent inhibition or enhancement was determined on the basis of current measurements indicated by the dotted lines in A. Numbers indicate the number of oocytes used. D, series of double cysteine mutants. Because no inhibition was seen, only the enhancement is shown. *, p Ͻ 0.05. In C and D, no statistically significant differences were found. E, inhibition of phosphorylation by staurosporine does not affect inhibition or enhancement of pannexin currents in oocytes expressing WT Panx1 or the mutant Panx1C426S. pH i to a more alkaline environment, IFN-␥-mediated decreases in ATP release were reversed. This indicates that acidification is the main reason of a reduced ATP release under inflammatory conditions. It has been widely recognized that ATP release from NHBE cells is an essential regulator of airway host defense mechanisms, mainly because of preservation of mucociliary clearance (7,35). Nevertheless, only a few reports exist that evaluate the exact mechanism. In previous papers, it has been shown that pannexins are proteins and orthologs of the invertebrate innexins that can form "hemichannels" in addition to complete gap junction channels. ATP release in erythrocytes has been found to be regulated by Panx1 channels (36), and our group has shown previously that Panx1 is expressed in the apex of airway epithelial cells (5). Hypotonic stress-induced ATP release in these cultures could be inhibited by carbenoxolone and probenecid but not flufenamic acid, speaking for Panx1 as the main contributor to ATP release (5). Furthermore, fully differentiated NHBE cells infected with an shRNA-expressing lentivirus to Panx1 also led to attenuation of ATP release upon hypotonic stress (5).
A threshold ATP concentration of about 1 M has to be reached for induction of signaling pathways via P2Y2 receptors (37). Furthermore, other reports illustrated the important role of ATP as a paracrine mediator of airway epithelial cell function (38). Previous studies have shown that the contribution of nonexocytosis-mediated ATP release of ALI cultures is sufficient for the induction of ATP signaling pathways (7), consistent with ATP release through Panx1 (5). Consequently, inhibition of these channels would impair ATP-induced effects. Our studies show that exposure to IFN-␥ causes inhibition of hypotonic stress-induced ATP release by ϳ78%, which would impair proper mucociliary clearance. Our data fit well with data from a recent publication showing that smokers and patients with chronic bronchitis have not only decreased ATP concentrations in their airway surface liquid but also ATP levels below the ones needed for signaling (39).
In contrast to connexins, pannexins mainly form homomeric membrane channels rather than gap junctions (36,40,41). There are also significant differences between connexons and pannexins regarding their regulation. In voltage clamp studies, it has been shown that Panx1 channels can be opened by positive transmembrane potentials (8,9,42) and that they are highly mechanosensitive, resulting in increased open probability by mechanical stress (8,43,44). Intracellular acidification correlates negatively with the open probability of many connexin channels (45,46) via identified intracellular regions shown to participate in pH gating as well as pH-induced changes in the conformation of Cx43 hemichannels by atomic force microscopy (47,48). Interestingly, CO 2 perfusion also abolished the conductance of Panx1 currents in oocytes (9), which is in accordance with our findings on hand showing that lowering of pH i leads to an inhibition of Panx1 currents (Fig. 5). Whether pannexon gating by protons is as direct as that shown for connexin channels contained in isolated membrane patches (49,50) has not been determined.
Connexin channels are also regulated by phosphorylation, and because Panx channels have predicted phosphorylation sites, their regulation could be similar (51). Others have demonstrated a direct inverse correlation between gap junctional permeability and the phosphorylation state of Cx43 (52). However, because staurosporine did not affect the modulation of Panx1 channel activity, phosphorylation events are not likely involved in H 2 O 2 -mediated regulation of these channels, at least in oocytes.
We have shown previously that IFN-␥-mediated up-regulation of Duox2 increases H 2 O 2 levels, creating oxidative stress (15). This process by itself can negatively affect pannexon function, at least transiently. Adaptive rebound alkalization is blocked by lowering pH i related to Duox2 activity and, thereby, inhibiting the permeability of Panx1 channels.
It is interesting to note that increases in ATP release were significantly greater in the absence of Duox2 than the change in whole cell pH i (Figs. 2D and 5G). This could indicate that some additional regulation mechanisms are at work that are not addressed here. On the other hand, changes in pH close to the apical membrane, where Duox makes protons, may be larger than changes in whole cell pH i in analogy to calcium influx into a cell (53). Therefore, whole cell pH change measurements may underestimate the pH changes at the apical membrane that are ultimately responsible for Panx1 regulation.
In summary, our data show that IFN-␥ induces intracellular acidification by Duox2 up-regulation. Both intracellular acidification and increased H 2 O 2 production by Duox2 synergistically inhibit Panx1 channels, leading to decreased ATP release from the airway (Fig. 6). These are new mechanisms how the loss of pannexin function contributes to mucociliary dysfunction in airway inflammation.
Author Contributions-S. K. contributed to the overall conception of the article, partially designed, performed, and analyzed experiments shown in all figures, and wrote the first draft of the paper. J. W. and G. D. designed, performed, and analyzed all experiments in oocytes. M. S. P. performed and analyzed the pilot experiments and some experiments shown in Fig. 5. C. G. was responsible for the patch clamp experiments. M. S. conceived and supervised the work in airway epithelial cells, finalized the writing of the paper, and contributed to the preparation of the figures. All authors reviewed the results, edited the paper, and approved the final version of the manuscript.