NADPH Alters DUOX1 Calcium Responsiveness.

Hydrogen peroxide is a key element in redox signaling and in setting cellular redox tone. DUOX1 and DUOX2, that directly synthesize hydrogen peroxide, are the most abundant NADPH oxidase transcripts in most epithelia. DUOX1 and DUOX2 hydrogen peroxide synthesis is regulated by intracellular calcium transients and thus cells can respond to signals and initiate responses by increasing cellular hydrogen peroxide synthesis. Nevertheless, many details of their enzymatic regulation are still unexplored. DUOX1 and DUOXA1 were expressed in HEK293T cells and activity was studied in homogenates and membrane fractions. When DUOX1 homogenates or membranes were pre-incubated in NADPH and started with addition of Ca2+, to mimic intracellular activation, progress curves were distinctly different from those pre-incubated in Ca2+ and started with NADPH. The Ca2+ EC50 for DUOX1's initial rate when pre-incubated in Ca2+, was three orders of magnitude lower (EC50 âˆ¼ 10-6 M) than with preincubation in NADPH (EC50 âˆ¼ 10-3 M). In addition, activity was several fold lower with Ca2+ start. Identical results were obtained using homogenates and membrane fractions. The data suggested that DUOX1 Ca2+ binding in expected physiological signaling conditions only slowly leads to maximal hydrogen peroxide synthesis and that full hydrogen peroxide synthesis activity in vivo only can occur when encountering extremely high concentration Ca2+ signals. Thus, a complex interplay of intracellular NADPH and Ca2+ concentrations regulate DUOX1 over a wide extent and may limit DUOX1 activity to a restricted range and spatial distribution.

Regulation of dual oxidase hydrogen peroxide synthesis results in an epithelial respiratory burst.

Redox status is a central determinant of cellular activities and redox imbalance is correlated with numerous diseases. NADPH oxidase activity results in formation of H2O2, that, in turn, sets cellular redox status, a key regulator of cellular homeostasis and responses to external stimuli. Hydrogen peroxide metabolism regulates cell redox status by driving changes in protein cysteine oxidation often via cycling of thioredoxin/peroxiredoxin and glutathione; however, regulation of enzymes controlling synthesis and utilization of H2O2 is not understood beyond broad outlines. The data presented here show that calcium-stimulated epithelial Duox H2O2 synthesis is transient, independent of intracellular calcium renormalization, H2O2 scavenging by antioxidant enzymes, or substrate depletion. The data support existence of a separate mechanism that restricts epithelial H2O2 synthesis to a burst and prevents harmful changes in redox tone following continuous stimulation. Elucidation of this H2O2 synthesis tempering mechanism is key to understanding cellular redox regulation and control of downstream effectors, and this observation provides a starting point for investigation of the mechanism that controls H2O2-mediated increases in redox tone.

Epithelial TLR4 Signaling Activates DUOX2 to Induce Microbiota-Driven Tumorigenesis.

BACKGROUND & AIMS: Chronic colonic inflammation leads to dysplasia and cancer in patients with inflammatory bowel disease. We have described the critical role of innate immune signaling via Toll-like receptor 4 (TLR4) in the pathogenesis of dysplasia and cancer. In the current study, we interrogate the intersection of TLR4 signaling, epithelial redox activity, and the microbiota in colitis-associated neoplasia. METHODS: Inflammatory bowel disease and colorectal cancer data sets were analyzed for expression of TLR4, dual oxidase 2 (DUOX2), and NADPH oxidase 1 (NOX1). Epithelial production of hydrogen peroxide (H2O2) was analyzed in murine colonic epithelial cells and colonoid cultures. Colorectal cancer models were carried out in villin-TLR4 mice, carrying a constitutively active form of TLR4, their littermates, and villin-TLR4 mice backcrossed to DUOXA-knockout mice. The role of the TLR4-shaped microbiota in tumor development was tested in wild-type germ-free mice. RESULTS: Activation of epithelial TLR4 was associated with up-regulation of DUOX2 and NOX1 in inflammatory bowel disease and colorectal cancer. DUOX2 was exquisitely dependent on TLR4 signaling and mediated the production of epithelial H2O2. Epithelial H2O2 was significantly increased in villin-TLR4 mice; TLR4-dependent tumorigenesis required the presence of DUOX2 and a microbiota. Mucosa-associated microbiota transferred from villin-TLR4 mice to wild-type germ-free mice caused increased H2O2 production and tumorigenesis. CONCLUSIONS: Increased TLR4 signaling in colitis drives expression of DUOX2 and epithelial production of H2O2. The local milieu imprints the mucosal microbiota and imbues it with pathogenic properties demonstrated by enhanced epithelial reactive oxygen species and increased development of colitis-associated tumors. The inter-relationship between epithelial reactive oxygen species and tumor-promoting microbiota requires a 2-pronged strategy to reduce the risk of dysplasia in colitis patients.

Proton channel blockers inhibit Duox activity independent of Hv1 effects.

The NADPH oxidase reaction produces protons. In the case of the NADPH oxidase, NOX2, activity depends on secretion of these protons and is inhibited by blockade of the voltage-gated proton channel (Hv1). Duox1 and Duox2 activities similarly produce intracellular protons but synthesize hydrogen peroxide directly instead of superoxide. Hv1 contributes to acid secretion in some epithelia that express Duox. To test the hypothesis that Duox activity is also sensitive to Hv1 channel blockers, Duox was assayed in the presence of either Zn2+ or 5-chloro-2-guanidinobenzimidazole (ClGBI). Both compounds inhibited Duox activity in normal human bronchial epithelial cells but with an IC50 over 10-fold higher than that reported for Hv1 (IC50 Zn2+ = 0.68 mM; IC50 ClGBI = 0.07-0.14 mM). Homogenized HEK293T cells expressing either Duox1 or Duox2 showed similar IC50 values for ClGBI suggesting these compounds inhibit the enzymes through alternate mechanisms independent of Hv1 proton secretion. Inclusion of superoxide dismutase did not restore Duox hydrogen peroxide synthesis. Addition of nigericin to eliminate any possible transmembrane pH gradients in intracellular membrane-localized Duox did not alter activity in HEK293T homogenates. Extracellular Zn2+ blocked intracellular Ca2+ increases needed for Duox activity. Together the data suggest that Duox enzyme activities in epithelia are inhibited by compounds that block Hv1 but inhibition occurs through Hv1-independent mechanisms and support the idea that Hv1 is not required for Duox activity.

Intestinal Epithelial Cells Respond to Chronic Inflammation and Dysbiosis by Synthesizing H2O2.

The microbes in the gastrointestinal tract are separated from the host by a single layer of intestinal epithelial cells (IECs) that plays pivotal roles in maintaining homeostasis by absorbing nutrients and providing a physical and immunological barrier to potential pathogens. Preservation of homeostasis requires the crosstalk between the epithelium and the microbial environment. One epithelial-driven innate immune mechanism that participates in host-microbe communication involves the release of reactive oxygen species (ROS), such as hydrogen peroxide (H2O2), toward the lumen. Phagocytes produce high amounts of ROS which is critical for microbicidal functions; the functional contribution of epithelial ROS, however, has been hindered by the lack of methodologies to reliably quantify extracellular release of ROS. Here, we used a modified Amplex Red assay to investigate the inflammatory and microbial regulation of IEC-generated H2O2 and the potential role of Duox2, a NADPH oxidase that is an important source of H2O2. We found that colonoids respond to interferon-γ and flagellin by enhancing production of H2O2 in a Duox2-mediated fashion. To extend these findings, we analyzed ex vivo production of H2O2 by IECs after acute and chronic inflammation, as well as after exposure to dysbiotic microbiota. While acute inflammation did not induce a significant increase in epithelial-driven H2O2, chronic inflammation caused IECs to release higher levels of H2O2. Furthermore, colonization of germ-free mice with dysbiotic microbiota from mice or patients with IBD resulted in increased H2O2 production compared with healthy controls. Collectively, these data suggest that IECs are capable of H2O2 production during chronic inflammation and dysbiotic states. Our results provide insight into luminal production of H2O2 by IECs as a read-out of innate defense by the mucosa.

Human Lung Cell Pyroptosis Following Traumatic Brain Injury.

Approximately 30% of traumatic brain injured patients suffer from acute lung injury or acute respiratory distress syndrome. Our previous work revealed that extracellular vesicle (EV)-mediated inflammasome signaling plays a crucial role in the pathophysiology of traumatic brain injury (TBI)-induced lung injury. Here, serum-derived EVs from severe TBI patients were analyzed for particle size, concentration, origin, and levels of the inflammasome component, an apoptosis-associated speck-like protein containing a caspase-recruiting domain (ASC). Serum ASC levels were analyzed from EV obtained from patients that presented lung injury after TBI and compared them to EV obtained from patients that did not show any signs of lung injury. EVs were co-cultured with lung human microvascular endothelial cells (HMVEC-L) to evaluate inflammasome activation and endothelial cell pyroptosis. TBI patients had a significant increase in the number of serum-derived EVs and levels of ASC. Severe TBI patients with lung injury had a significantly higher level of ASC in serum and serum-derived EVs compared to individuals without lung injury. Only EVs isolated from head trauma patients with gunshot wounds were of neural origin. Delivery of serum-derived EVs to HMVEC-L activated the inflammasome and resulted in endothelial cell pyroptosis. Thus, serum-derived EVs and inflammasome proteins play a critical role in the pathogenesis of TBI-induced lung injury, supporting activation of an EV-mediated neural-respiratory inflammasome axis in TBI-induced lung injury.

Gefitinib, an EGFR Tyrosine Kinase inhibitor, Prevents Smoke-Mediated Ciliated Airway Epithelial Cell Loss and Promotes Their Recovery.

Cigarette smoke exposure is a major health hazard. Ciliated cells in the epithelium of the airway play a critical role in protection against the noxious effects of inhaled cigarette smoke. Ciliated cell numbers are reduced in smokers which weakens host defense and leads to disease. The mechanisms for the loss of ciliated cells are not well understood. The effects of whole cigarette smoke exposure on human airway ciliated ciliated cells were examined using in vitro cultures of normal human bronchial epithelial cells and a Vitrocell® VC 10® Smoking Robot. These experiments showed that whole cigarette smoke causes the loss of differentiated ciliated cells and inhibits differentiation of ciliated cells from undifferentiated basal cells. Furthermore, treatment with the epidermal growth factor receptor (EGFR) tyrosine kinase inhibitor, Gefitinib, during smoke exposure prevents ciliated cell loss and promotes ciliated cell differentiation from basal cells. Finally, restoration of ciliated cells was inhibited after smoke exposure was ceased but was enhanced by Gefitinib treatment. These data suggest that inhibition of EGFR activity may provide therapeutic benefit for treating smoke related diseases.

High affinity complexes of pannexin channels and L-type calcium channel splice-variants in human lung: Possible role in clevidipine-induced dyspnea relief in acute heart failure.

Clevidipine, a dihydropyridine (DHP) analogue, lowers blood pressure (BP) by inhibiting l-type calcium channels (CaV1.2; gene CACNA1C) predominantly located in vascular smooth muscle (VSM). However, clinical observations suggest that clevidipine acts by a more complex mechanism. Clevidipine more potently reduces pulmonary vascular resistance (PVR) than systemic vascular resistance and its spectrum of effects on PVR are not shared by other DHPs. Clevidipine has potent spasmolytic effects in peripheral arteries at doses that are sub-clinical for BP lowering and, in hypertensive acute heart failure, clevidipine, but not other DHPs, provides dyspnea relief, partially independent of BP reduction. These observations suggest that a molecular variation in CaV1.2 may exist which confers unique pharmacology to different DHPs. We sequenced CACNA1C transcripts from human lungs and measured their affinity for clevidipine. Human lung tissue contains CACNA1C mRNA with many different splice variations. CaV1.2 channels with a specific combination of variable exons showed higher affinity for clevidipine, well below the concentration associated with BP reduction. Co-expression with pannexin 1 further increased the clevidipine affinity for this CaV1.2 splice variant. A high-affinity splice variant of CaV1.2 in combination with pannexin 1 could underlie the selective effects of clevidipine on pulmonary arterial pressure and on dyspnea. RESEARCH IN CONTEXT: Clevidipine lowers blood pressure by inhibiting calcium channels in vascular smooth muscle. In patients with acute heart failure, clevidipine was shown to relieve breathing problems. This was only partially related to the blood pressure lowering actions of clevidipine and not conferred by another calcium channel inhibitor. We here found calcium channel variants in human lung that are more selectively inhibited by clevidipine, especially when associated with pannexin channels. This study gives a possible mechanism for clevidipine's relief of breathing problems and supports future clinical trials testing the role of clevidipine in the treatment of acute heart failure.

Soluble adenylyl cyclase mediates hydrogen peroxide-induced changes in epithelial barrier function.

BACKGROUND: Elevated H2O2 levels are associated with inflammatory diseases and H2O2 exposure is known to disrupt epithelial barrier function, leading to increased permeability and decreased electrical resistance. In normal human bronchial epithelial (NHBE) cells, fully differentiated at the air liquid interface (ALI), H2O2 activates an autocrine prostaglandin pathway that stimulates transmembrane adenylyl cyclase (tmAC) as well as soluble adenylyl cyclase (sAC), but the role of this autocrine pathway in H2O2-mediated barrier disruption is not entirely clear. METHODS: To further characterize the mechanism of H2O2-induced barrier disruption, NHBE cultures were treated with H2O2 and evaluated for changes in transepithelial resistance and mannitol permeability using agonist and inhibitors to dissect the pathway. RESULTS: A short (<10 min) H2O2 treatment was sufficient to induce resistance and permeability changes that occurred 40 min to 1 h later and the changes were partially sensitive to EP1 but not EP4 receptor antagonists. EP1 receptors were localized to the apical compartment of NHBE. Resistance and permeability changes were sensitive to inhibition of sAC but not tmAC and were partially blocked by PKA inhibition. Pretreatment with a PLC inhibitor or an IP3 receptor antagonist reduced changes in resistance and permeability suggesting activation of sAC occurred through increased intracellular calcium. CONCLUSION: The data support an important role for prostaglandin activation of sAC and PKA in H2O2-induced barrier disruption.

Transforming growth factor-ß1 and cigarette smoke inhibit the ability of ß2-agonists to enhance epithelial permeability.

Chronic bronchitis, caused by cigarette smoke exposure, is characterized by mucus hypersecretion and reduced mucociliary clearance (MCC). Effective MCC depends, in part, on adequate airway surface liquid. Cystic fibrosis transmembrane conductance regulator (CFTR) provides the necessary osmotic gradient for serosal to mucosal fluid transport through its ability to both secrete Cl(-) and regulate paracellular permeability, but CFTR activity is attenuated in chronic bronchitis and in smokers. ß2-adrenergic receptor (ß2-AR) agonists are widely used for managing chronic obstructive pulmonary disease, and can activate CFTR, stimulate ciliary beat frequency, and increase epithelial permeability, thereby stimulating MCC. Patients with chronic airway diseases and cigarette smokers demonstrate increased transforming growth factor (TGF)-ß1 signaling, which suppresses ß2-agonist-mediated CFTR activation and epithelial permeability increases. Restoring CFTR function in these diseases can restore the ability of ß2-agonists to enhance epithelial permeability. Human bronchial epithelial cells, fully redifferentiated at the air-liquid interface, were used for (14)C mannitol flux measurements, Ussing chamber experiments, and quantitative RT-PCR. ß2-agonists enhance epithelial permeability by activating CFTR via the ß2-AR/adenylyl cyclase/cAMP/protein kinase A pathway. TGF-ß1 inhibits ß2-agonist-mediated CFTR activation and epithelial permeability enhancement. Although TGF-ß1 down-regulates both ß2-AR and CFTR mRNA, functionally it only decreases CFTR activity. Cigarette smoke exposure inhibits ß2-agonist-mediated epithelial permeability increases, an effect reversed by blocking TGF-ß signaling. ß2-agonists enhance epithelial permeability via CFTR activation. TGF-ß1 signaling inhibits ß2-agonist-mediated CFTR activation and subsequent increased epithelial permeability, potentially limiting the ability of ß2-agonists to facilitate paracellular transport in disease states unless TGF-ß1 signaling is inhibited.

IL-17A induces Pendrin expression and chloride-bicarbonate exchange in human bronchial epithelial cells.

The epithelium plays an active role in the response to inhaled pathogens in part by responding to signals from the immune system. Epithelial responses may include changes in chemokine expression, increased mucin production and antimicrobial peptide secretion, and changes in ion transport. We previously demonstrated that interleukin-17A (IL-17A), which is critical for lung host defense against extracellular bacteria, significantly raised airway surface pH in vitro, a finding that is common to a number of inflammatory diseases. Using microarray analysis of normal human bronchial epithelial (HBE) cells treated with IL-17A, we identified the electroneutral chloride-bicarbonate exchanger Pendrin (SLC26A4) as a potential mediator of this effect. These data were verified by real-time, quantitative PCR that demonstrated a time-dependent increase in Pendrin mRNA expression in HBE cells treated with IL-17A up to 48 h. Using immunoblotting and immunofluorescence, we confirmed that Pendrin protein expression is increased in IL-17 treated HBE cells and that it is primarily localized to the mucosal surface of the cells. Functional studies using live-cell fluorescence to measure intracellular pH demonstrated that IL-17A induced chloride-bicarbonate exchange in HBE cells that was not present in the absence of IL-17A. Furthermore, HBE cells treated with short interfering RNA against Pendrin showed substantially reduced chloride-bicarbonate exchange. These data suggest that Pendrin is part of IL-17A-dependent epithelial changes and that Pendrin may therefore be a therapeutic target in IL-17A-dependent lung disease.

Submersion and hypoxia inhibit ciliated cell differentiation in a notch-dependent manner.

The epithelium that lines the conducting airways is composed of several distinct cell types that differentiate from common progenitor cells. The signals that control fate selection and differentiation of ciliated cells, a major component of the epithelium, are not completely understood. Ciliated cell differentiation can be accomplished in vitro when primary normal human bronchial epithelial (NHBE) cells are cultured at an air-liquid interface, but is inhibited when NHBE cells are cultured under submerged conditions. The mechanism by which submersion prevents ciliogenesis is not understood, but may provide clues to in vivo regulation of ciliated cell differentiation. We hypothesized that submersion creates a hypoxic environment that prevents ciliated cell differentiation by blocking the gene expression program required for ciliogenesis. This was confirmed by showing that expression of multicilin and Forkhead box J1, key factors needed for ciliated cell differentiation, was inhibited when NHBE cells were cultured in submerged and hypoxic conditions. Multicilin and Forkhead box J1 expression and ciliated cell differentiation were restored in submerged and hypoxic cells upon treatment with the γ-secretase inhibitor, N-[(3,5-difluorophenyl)acetyl]-L-alanyl-2-phenyl]glycine-1,1-dimethylethyl ester (DAPT), which suggested that Notch signaling was involved. Overexpression of Notch intracellular domain inhibited differentiation in the presence of DAPT, confirming the role of Notch signaling. These results indicate that submersion and hypoxia prevent ciliated cell differentiation by maintaining Notch signaling, which represses genes necessary for ciliogenesis. These data provide new insights into the molecular mechanisms that control human bronchial differentiation.

IFN-γ-mediated reduction of large-conductance, Ca2+-activated, voltage-dependent K+ (BK) channel activity in airway epithelial cells leads to mucociliary dysfunction.

Effective mucociliary clearance (MCC) depends in part on adequate airway surface liquid (ASL) volume to maintain an appropriate periciliary fluid height that allows normal ciliary activity. Apically expressed large-conductance, Ca(2+)-activated, and voltage-dependent K(+) (BK) channels provide an electrochemical gradient for Cl(-) secretion and thus play an important role for adequate airway hydration. Here we show that IFN-γ decreases ATP-mediated apical BK activation in normal human airway epithelial cells cultured at the air-liquid interface. IFN-γ decreased mRNA levels of KCNMA1 but did not affect total protein levels. Because IFN-γ upregulates dual oxidase (DUOX)2 and therefore H2O2 production, we hypothesized that BK inactivation could be mediated by BK oxidation. However, DUOX2 knockdown did not affect the IFN-γ effect on BK activity. IFN-γ changed mRNA levels of the BK ß-modulatory proteins KCNMB2 (increased) and KCNMB4 (decreased) as well as leucine-rich repeat-containing protein (LRRC)26 (decreased). Mallotoxin, a BK opener only in the absence of LRRC26, showed that BK channels lost their association with LRRC26 after IFN-γ treatment. Finally, IFN-γ caused a decrease in ciliary beating frequency that was immediately rescued by apical fluid addition, suggesting that it was due to ASL volume depletion. These data were confirmed with direct ASL measurements using meniscus scanning. Overexpression of KCNMA1, the pore-forming subunit of BK, overcame the reduction of ASL volume induced by IFN-γ. Key experiments were repeated in cystic fibrosis cells and showed the same results. Therefore, IFN-γ induces mucociliary dysfunction through BK inactivation.

H2O2 stimulates cystic fibrosis transmembrane conductance regulator through an autocrine prostaglandin pathway, using multidrug-resistant protein-4.

Cystic fibrosis transmembrane conductance regulator (CFTR) activity is essential for the maintenance of airway surface liquid depth, and therefore mucociliary clearance. Reactive oxygen species, increased during inflammatory airway diseases, alter CFTR activity. Here, H2O2 levels in the surface liquid of normal human bronchial epithelial cultures differentiated at the air-liquid interface were estimated, and H2O2-mediated changes in CFTR activity were examined. In Ussing chambers, H2O2-induced anion currents were sensitive to the CFTR inhibitors CFTRinh172 and GlyH-101. These currents were absent in cells from patients with cystic fibrosis. Responses to greater than 500 µM H2O2 were transient. Cyclooxygenase inhibitors blocked the H2O2 response, as did EP1 and EP4 receptor antagonists. A multidrug-resistant protein (MRP) inhibitor and short hairpin RNA directed against MRP4 blocked H2O2 responses. EP1 and EP4 agonists mimicked H2O2 in both control and MRP4 knockdown cells. Thus, H2O2 activates the synthesis, export, and binding of prostanoids via EP4 and, interestingly, EP1 receptors in normal, differentiated human airway epithelial cells to activate cyclic adenosine monophosphate pathways that in turn activate CFTR channels in the apical membrane.

Albuterol modulates its own transepithelial flux via changes in paracellular permeability.

Although inhaled bronchodilators are commonly used in the treatment of airway disease to dilate airway smooth muscle, little is known regarding the mechanisms that regulate albuterol movement across the epithelium to reach its target, the airway smooth muscle. Because the rate of onset depends on the transepithelial transport of albuterol, to determine the mechanisms that regulate the transepithelial movement of albuterol is essential. Human bronchial epithelial cells, fully redifferentiated in culture at the air-liquid interface, were used to study the cellular uptake and total transepithelial flux of (3)H-albuterol from the apical to the basolateral surfaces. (3)H-mannitol and transepithelial electrical resistance were used to quantify changes in paracellular permeability. The majority of albuterol flux across the epithelium occurred via the paracellular route. The cellular uptake of albuterol was found to be saturable, whereas transepithelial flux was not. Cellular uptake could be inhibited by the amino acids lysine and histidine, with no effect on net transepithelial flux. Transepithelial flux was altered by maneuvers that collapsed or disrupted intercellular junctions. Acidification, usually seen in exacerbations of airway disease, decreased albuterol flux. In addition, albuterol increased its own paracellular permeability. The ability of albuterol to modulate paracellular permeability was blocked by the ß(2)-adrenergic receptor-selective antagonist ICI 118551. Albuterol mainly crosses the epithelium via the paracellular pathway, but has the ability to modulate its own permeability through changes in the leakiness of tight junctions, which is modulated through the signaling of the ß(2)-adrenergic receptor.

Rapid nongenomic actions of inhaled corticosteroids on long-acting ß(2)-agonist transport in the airway.

Corticosteroids inhibit organic cation transporters (OCTs) that play an important role in drug absorption, tissue distribution and elimination. Corticosteroid sensitivity of bronchodilator trafficking in the airway tissue, however, is poorly understood. To assess the effects of inhaled corticosteroids on airway absorption and disposal mechanisms of long-acting ß(2)-agonists, human airway epithelial and smooth muscle cell uptake of tritiated formoterol and salmeterol was measured in vitro. Corticosteroids caused a rapid, concentration-dependent inhibition of uptake of the cationic formoterol by airway smooth muscle cells, but not airway epithelial cells. Uptake of the non-charged lipophilic salmeterol was corticosteroid-insensitive in both cell types. In smooth muscle cells, inhaled corticosteroids inhibited formoterol uptake with a novel potency rank order: des-ciclesonide > budesonide > beclomethasone 17-monopropionate > beclomethasone dipropionate > ciclesonide > fluticasone. The inhibitory action was rapidly reversible, and was not enhanced by prolonged corticosteroid exposure or sensitive to a transcription inhibitor. Suppression of OCT3 expression using lentivirus-mediated production of shRNA reduced corticosteroid sensitivity of formoterol uptake by smooth muscle cells. Our data support a corticosteroid insensitive absorption and a corticosteroid-sensitive disposition mechanism for cationic long-acting ß(2)-agonist bronchodilators in the airway. Potency rank order and other 'classical' features of anti-inflammatory effects do not apply to inhaled corticosteroids' rapid drug transport actions.

Mucous solids and liquid secretion by airways: studies with normal pig, cystic fibrosis human, and non-cystic fibrosis human bronchi.

To better understand how airways produce thick airway mucus, nonvolatile solids were measured in liquid secreted by bronchi from normal pig, cystic fibrosis (CF) human, and non-CF human lungs. Bronchi were exposed to various secretagogues and anion secretion inhibitors to induce a range of liquid volume secretion rates. In all three groups, the relationship of solids concentration (percent nonvolatile solids) to liquid volume secretion rate was curvilinear, with higher solids concentration associated with lower rates of liquid volume secretion. In contrast, the secretion rates of solids mass and water mass as functions of liquid volume secretion rates exhibited positive linear correlations. The y-intercepts of the solids mass-liquid volume secretion relationships for all three groups were positive, thus accounting for the higher solids concentrations in airway liquid at low rates of secretion. Predictive models derived from the solids mass and water mass linear equations fit the experimental percent solids data for the three groups. The ratio of solids mass secretion to liquid volume secretion was 5.2 and 2.4 times higher for CF bronchi than for pig and non-CF bronchi, respectively. These results indicate that normal pig, non-CF human, and CF human bronchi produce a high-percent-solids mucus (>8%) at low rates of liquid volume secretion (≤1.0 µl·cm(-2)·h(-1)). However, CF bronchi produce mucus with twice the percent solids (~8%) of pig or non-CF human bronchi at liquid volume secretion rates ≥4.0 µl·cm(-2)·h(-1).

Functional apical large conductance, Ca2+-activated, and voltage-dependent K+ channels are required for maintenance of airway surface liquid volume.

Large conductance, Ca(2+)-activated, and voltage-dependent K(+) (BK) channels control a variety of physiological processes in nervous, muscular, and renal epithelial tissues. In bronchial airway epithelia, extracellular ATP-mediated, apical increases in intracellular Ca(2+) are important signals for ion movement through the apical membrane and regulation of water secretion. Although other, mainly basolaterally expressed K(+) channels are recognized as modulators of ion transport in airway epithelial cells, the role of BK in this process, especially as a regulator of airway surface liquid volume, has not been examined. Using patch clamp and Ussing chamber approaches, this study reveals that BK channels are present and functional at the apical membrane of airway epithelial cells. BK channels open in response to ATP stimulation at the apical membrane and allow K(+) flux to the airway surface liquid, whereas no functional BK channels were found basolaterally. Ion transport modeling supports the notion that apically expressed BK channels are part of an apical loop current, favoring apical Cl(-) efflux. Importantly, apical BK channels were found to be critical for the maintenance of adequate airway surface liquid volume because continuous inhibition of BK channels or knockdown of KCNMA1, the gene coding for the BK α subunit (KCNMA1), lead to airway surface dehydration and thus periciliary fluid height collapse revealed by low ciliary beat frequency that could be fully rescued by addition of apical fluid. Thus, apical BK channels play an important, previously unrecognized role in maintaining adequate airway surface hydration.

Oxidative epithelial host defense is regulated by infectious and inflammatory stimuli.

Epithelia express oxidative antimicrobial protection that uses lactoperoxidase (LPO), hydrogen peroxide (H(2)O(2)), and thiocyanate to generate the reactive hypothiocyanite. Duox1 and Duox2, found in epithelia, are hypothesized to provide H(2)O(2) for use by LPO. To investigate the regulation of oxidative LPO-mediated host defense by bacterial and inflammatory stimuli, LPO and Duox mRNA were followed in differentiated primary human airway epithelial cells challenged with Pseudomonas aeruginosa flagellin or IFN-gamma. Flagellin upregulated Duox2 mRNA 20-fold, but upregulated LPO mRNA only 2.5-fold. IFN-gamma increased Duox2 mRNA 127-fold and upregulated LPO mRNA 10-fold. DuoxA2, needed for Duox2 activity, was also upregulated by flagellin and IFN-gamma. Both stimuli increased H(2)O(2) synthesis and LPO-dependent killing of P. aeruginosa. Reduction of Duox1 by siRNA showed little effect on basal H(2)O(2) production, whereas Duox2 siRNA markedly reduced basal H(2)O(2) production and resulted in an 8-fold increase in Nox4 mRNA. In conclusion, large increases in Duox2-mediated H(2)O(2) production seem to be coordinated with increases in LPO mRNA and, without increased LPO, H(2)O(2) levels in airway secretion are expected to increase substantially. The data suggest that Duox2 is the major contributor to basal H(2)O(2) synthesis despite the presence of greater amounts of Duox1.

Pannexin 1 contributes to ATP release in airway epithelia.

ATP is a paracrine regulator of critical airway epithelial cell functions, but the mechanism of its release is poorly understood. Pannexin (Panx) proteins, related to invertebrate innexins, form channels (called pannexons) that are able to release ATP from several cell types. Thus, ATP release via pannexons was examined in airway epithelial cells. Quantitative RT-PCR showed Panx1 expression in normal human airway epithelial cells during redifferentiation at the air-liquid interface (ALI), at a level comparable to that of alveolar macrophages; Panx3 was not expressed. Immunohistochemistry showed Panx1 expression at the apical pole of airway epithelia. ALI cultures exposed to hypotonic stress released ATP to an estimated maximum of 255 (+/-64) nM within 1 minute after challenge (n = 6 cultures from three different lungs) or to approximately 1.5 (+/-0.4) microM, recalculated to a normal airway surface liquid volume. Using date- and culture-matched cells (each n > or = 16 from 4 different lungs), the pannexon inhibitors carbenoxolone (10 microM) and probenecid (1 mM), but not the connexon inhibitor flufenamic acid (100 microM), inhibited ATP release by approximately 60%. The drugs affected Panx1 currents in Xenopus oocytes expressing exogenous Panx1 correspondingly. In addition, suppression of Panx1 expression using lentivirus-mediated production of shRNA in differentiated airway epithelial cells inhibited ATP release upon hypotonic stress by approximately 60% as well. These data not only show that Panx1 is expressed apically in differentiated airway epithelial cells but also that it contributes to ATP release in these cells.