Matthias Salathe

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.

Hyaluronan serves a novel role in airway mucosal host defense

Enzymes secreted onto epithelial surfaces play a vital role in innate mucosal defense, but are believed to be steadily removed from the surface by mechanical actions. Thus, the amount and availability of enzymes on the surface are thought to be maintained by secretion. In contrast to this paradigm, we show here that enzymes are retained at the apical surface of the airway epithelium by binding to surface‐associated hya‐luronan, providing an apical enzyme pool ‘ready for use’ and protected from ciliary clearance. We have studied lactoperoxidase, which prevents bacterial colonization of the airway, and kallikrein, which mediates allergic bronchoconstriction that limits the inhalation of noxious substances. Binding to hyaluronan inhibits kallikrein, which is needed only in certain situations, whereas lactoperoxidase, useful at all times, does not change its activity. Hyaluronan itself interacts with the receptor for hyaluronic acid‐mediated motility (RHAMM or CD168) that is expressed at the apex of ciliated airway epithelial cells. Functionally, hyaluronan binding to RHAMM stimulates ciliary beating. Thus, hyaluronan plays a previouslyunrecognized pivotal role in mucosal host defense by stimulating ciliaryclearance of foreign material while simultaneously retaining enzymes important for homeostasis at the apical surface so that they cannot be removed by ciliary action.—Forteza, R., Lieb, T., Aoki, T., Savani, R. C., Conner, G. E., Salathe, M. Hyaluronan serves a novel role in airway mucosal host defense. FASEB J. 15, 2179–2186 (2001)

Soluble adenylyl cyclase is localized to cilia and contributes to ciliary beat frequency regulation via production of cAMP

Ciliated airway epithelial cells are subject to sustained changes in intracellular CO2/HCO3 − during exacerbations of airway diseases, but the role of CO2/HCO3 −-sensitive soluble adenylyl cyclase (sAC) in ciliary beat regulation is unknown. We now show not only sAC expression in human airway epithelia (by RT-PCR, Western blotting, and immunofluorescence) but also its specific localization to the axoneme (Western blotting and immunofluorescence). Real time estimations of [cAMP] changes in ciliated cells, using FRET between fluorescently tagged PKA subunits (expressed under the foxj1 promoter solely in ciliated cells), revealed CO2/HCO3 −-mediated cAMP production. This cAMP production was specifically blocked by sAC inhibitors but not by transmembrane adenylyl cyclase (tmAC) inhibitors. In addition, this cAMP production stimulated ciliary beat frequency (CBF) independently of intracellular pH because PKA and sAC inhibitors were uniquely able to block CO2/HCO3 −-mediated changes in CBF (while tmAC inhibitors had no effect). Thus, sAC is localized to motile airway cilia and it contributes to the regulation of human airway CBF. In addition, CO2/HCO3 − increases indeed reversibly stimulate intracellular cAMP production by sAC in intact cells.

The Lactoperoxidase System Links Anion Transport To Host Defense in Cystic Fibrosis

Chronic respiratory infections in cystic fibrosis result from CFTR channel mutations but how these impair antibacterial defense is less clear. Airway host defense depends on lactoperoxidase (LPO) that requires thiocyanate (SCN−) to function and epithelia use CFTR to concentrate SCN− at the apical surface. To test whether CFTR mutations result in impaired LPO‐mediated host defense, CF epithelial SCN− transport was measured. CF epithelia had significantly lower transport rates and did not accumulate SCN− in the apical compartment. The lower CF [SCN−] did not support LPO antibacterial activity. Modeling of airway LPO activity suggested that reduced transport impairs LPO‐mediated defense and cannot be compensated by LPO or H2O2 upregulation.

Prolonged increase in ciliary beat frequency after short‐term purinergic stimulation in human airway epithelial cells

Stimulation of ovine airway epithelial cells with 10 μm ATP for 1 min at 25 °C transiently increased both cytoplasmic calcium (fura‐2 epifluorescence microscopy) and ciliary beat frequency (CBF; differential interference contrast microscopy) with a similar time course. Identical purinergic stimulation of human airway epithelial cells at 25 or 35 °C, however, lead to an increase in CBF that outlasted the calcium transient at least 20 min. While a nitric oxide synthase inhibitor had no effect, pre‐treatment of human cells with inhibitors of cAMP‐dependent kinase (PKA), 10 μm myristoylated PKA‐inhibitory peptide and 1 μm KT‐5720, as well as an inhibitor of adenylyl cyclase, 1 mm SQ22536, blocked the prolonged, but not calcium‐coupled CBF increase. Addition of PKA inhibitors after purinergic stimulation only partially reduced CBF from its elevated plateau. Prolonged CBF increases did not depend on adenosine production as 10 μm UTP had an effect similar to ATP and 8‐sulphophenyl‐theophylline did not block them. After increasing human CBF in a PKA‐dependent manner to a stable plateau with forskolin (10 μm), ATP caused only a transient, calcium‐coupled CBF increase. Calcium transients were necessary for both short‐term and prolonged CBF changes as ATP failed to produce CBF increases after emptying calcium stores with 1 μm thapsigargin. These data suggest that in human, but not ovine airway epithelial cells, ATP‐induced calcium transients activate a signalling cascade including adenylyl cyclase and PKA. The resulting prolonged CBF stimulation does not rely only on PKA activity, suggesting that the decay of CBF is influenced by ciliary phosphatase activity.

Mode of Ca2+ action on ciliary beat frequency in single ovine airway epithelial cells

1 We analysed the kinetics of coupling between cytoplasmic calcium ([Ca2+]i) and ciliary beat frequency (CBF) using simultaneous single cilium recording and single cell [Ca2+]i measurements from cultured ovine tracheal epithelial cells. 2 CBF and [Ca2+]i (indicated by fura‐2) were measured at rest and in response to activation of the G‐protein coupled M3 muscarinic receptor by 10 μM acetylcholine (ACh). 3 Fourier transform analysis of 3 s data segments of light intensity from phase‐contrast microscopy showed no significant delay between changes in [Ca2+]i and CBF during a 2 min exposure to ACh and subsequent washout. 4 CBF time resolution was improved by computing instantaneous beat frequency. This revealed that CBF lagged the rapid increase in [Ca2+]i in response to ACh with a delay of less than 1 beat cycle (143 ms at 7 Hz). When CBF was estimated by an improved Fourier method, this delay was observed to be 70 ± 30 ms (mean ± s.e.m.; n= 20 cilia). During the slower return to baseline, a lag of 8 ± 3.2 s was observed, indicative of hysteresis. 5 While calmodulin inhibitors (calmidazolium and W‐7; each n= 5) decreased baseline CBF by an average of 1.1 ± 0.1 Hz, they did not alter the kinetic relationship between [Ca2+]i and CBF. Similarly, phosphatase inhibitors (okadaic acid and cyclosporin A; each n= 5), changed neither baseline CBF nor the kinetic coupling between [Ca2+]i and CBF. 6 These data suggest that the timing of Ca2+ action on CBF in ovine airway epithelial cells, is unlikely to be determined by phosphorylation reactions involving calmodulin or kinase/phosphatase reactions. 7 A simple model for Ca2+ stimulation of CBF is presented. Fits of the model to the data suggest four or more Ca2+ ions bind cooperatively to speed up CBF.

Transcellular thiocyanate transport by human airway epithelia

Human airway mucosa synthesizes and secretes lactoperoxidase (LPO). As H2O2 and thiocyanate (SCN−) are also present, a functional LPO antibacterial defence system exists in the airways. SCN− concentrations in several epithelial secretions are higher than in serum, although the mechanisms of transepithelial transport and accumulation in these secretions are unknown. To examine SCN− accumulation in secretions, human airway epithelial cells, re‐differentiated at the air–liquid interface, were used in open‐circuit conditions. [14C]SCN−, in the basolateral medium, was transported across the epithelium and concentrated tenfold at the apical surface. Measurement of the transepithelial potential showed that the basolateral compartment was positive relative to the apical surface (13.7 ± 1.8 mV) and therefore unfavourable for passive movement of SCN−. Transport was dependent on basolateral [SCN−] and saturable (Km,app= 69 ± 25 μm); was inhibited by increased apical [SCN−]; and was dependent on the presence of basolateral Na+. Perchlorate (Ki,app= 0.6 ± 0.05 μm) and iodide (Ki,app= 9 ± 8 μm) in the basolateral medium reversibly inhibited transport, but furosemide did not. Iodide was also transported (Km,app= 111 ± 69 μm). RT‐PCR and immunohistochemistry confirmed expression of Na+−I− symporter (NIS) in the airways. SCN− transport was insensitive to apical disulphonic acid Cl− channel blockers, but sensitive to apical glibenclamide and arylaminobenzoates. Forskolin and dibutyryl cAMP increased transport. These data suggest SCN− transport may occur through basolateral NIS‐mediated SCN− concentration inside cells, followed by release through an apical channel, perhaps cystic fibrosis transmembrane conductance regulator.

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.

Real-time analysis of cAMP-mediated regulation of ciliary motility in single primary human airway epithelial cells

Airway ciliary beat frequency regulation is complex but in part influenced by cyclic adenosine monophosphate (cAMP)-mediated changes in cAMP-dependent kinase activity, yet the cAMP concentration required for increases in ciliary beat frequency and the temporal relationship between ciliary beat frequency and cAMP changes are unknown. A lentiviral gene transfer system was developed to express a fluorescence resonance energy transfer (FRET)-based cAMP sensor in ciliated cells. Expression of fluorescently tagged cAMP-dependent kinase subunits from the ciliated-cell-specific foxj1 promoter enhanced expression in fully differentiated ciliated human airway epithelial cells, and permitted simultaneous measurements of ciliary beat frequency and cAMP (represented by the FRET ratio). Apical application of forskolin (1 μM, 10 μM, 20 μM) and, in permeabilized cells, basolateral cAMP (20 μM, 50 μM, 100 μM) caused dose-dependent, albeit similar and simultaneous–increases in cAMP and ciliary beat frequency. However, decreases in cAMP preceded decreases in ciliary beat frequency, suggesting that either cellular cAMP decreases before ciliary cAMP or the dephosphorylation of target proteins by phosphatases occur at a rate slower than the rate of cAMP hydrolysis.

Rapid reversal of heart failure in a patient with phaeochromocytoma and catecholamine-induced cardiomyopathy who was treated with captopril

A patient with a phaeochromocytoma and severe left ventricular heart failure caused by a catecholamine-induced cardiomyopathy is described. The clinical signs of congestive heart failure resolved rapidly on treatment with captopril and myocardial performance became normal within two weeks of medical treatment with captopril for one week and with captopril in combination with phenoxybenzamine for another week.