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Soluble Adenylyl Cyclase Mediates Hydrogen Peroxide-Induced Changes in Epithelial Barrier Function Pedro Ivonnet1, Hoshang Unwalla2, Matthias Salathe1 and Gregory E

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Soluble Adenylyl Cyclase Mediates Hydrogen Peroxide-Induced Changes in Epithelial Barrier Function Pedro Ivonnet1, Hoshang Unwalla2, Matthias Salathe1 and Gregory E Ivonnet et al. Respiratory Research (2016) 17:15 DOI 10.1186/s12931-016-0329-4 RESEARCH Open Access Soluble adenylyl cyclase mediates hydrogen peroxide-induced changes in epithelial barrier function Pedro Ivonnet1, Hoshang Unwalla2, Matthias Salathe1 and Gregory E. Conner3* Abstract 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. Keywords: Soluble adenylyl cyclase, Hydrogen peroxide, Airway epithelium, EP1 Background [1]. A large number of studies have shown that H2O2, Junctional complexes are composed of an assortment of frequently elevated in inflammatory diseases, revers- proteins that anchor cells to each other and their base- ibly alters paracellular epithelial permeability and re- ment membranes, thereby forming a stable tissue that sistance (e.g., [2–4]). H2O2 on epithelial surfaces can serves to regulate passage of materials across the mu- result from the respiratory burst of invading phago- cosa. Regulation of the apical junctional complex is key cytes or from epithelial cells themselves that produce to epithelial barrier function. Numerous studies have H2O2 through the enzymatic action of the NADPH shown changes in transepithelial permeability and elec- oxidases Duox 1 & 2 [5–7]. trical resistance can occur rapidly and reversibly and The mechanism by which H2O2 alters permeability mirror changes in intercellular junction structure. Loss and transepithelial resistance is multifactorial and differs of barrier function is often associated with inflammation between differentiated epithelia, endothelia and cell lines (e.g., [8]), but uniformly entails junctional protein re- distribution (e.g., [2, 9–13]). Occludin, ZO1 and claudins * Correspondence: [email protected] 3Department of Cell Biology, Miller School of Medicine, University of Miami, are released from junctions after H2O2 exposure. H2O2 1600 NW 10th Ave, Miami 33136 FL, USA alteration of the epithelial barrier is known to rely on Full list of author information is available at the end of the article © 2016 Ivonnet et al. Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Ivonnet et al. Respiratory Research (2016) 17:15 Page 2 of 8 increased protein tyrosine phosphorylation by inhibition Ussing chambers of protein tyrosine phosphatase [8, 11], p38 MAP kinase Snapwell filters containing differentiated NHBE cells activity [14] and dephosphorylation of occludin by PP2A were rinsed with Krebs-Henseleit (KH), and then in a Src kinase-dependent fashion [15]. Involvement of mounted in Ussing chambers (EasyMount Chamber; protein kinase C has been reported in some cases [16] Physiologic Instruments; San Diego, CA) with KH in ap- but ruled out in others [3]. ical and basolateral chambers. KH consisted of: 118 mM Studies in bovine tracheal epithelia [17], in human air- NaCl, 25 mM NaHCO3, 4.7 mM KCl, 1.2 mM MgSO4, way epithelial cell lines [18, 19] and more recently in 1.2 mM NaH2PO4, 1.2 mM CaCl2, 5.5 mM glucose, pH fully differentiated normal human bronchial epithelial 7.35 when gassed with 95 % O2–5%CO2. Solutions (NHBE) cells [20] show that acute exposure to H2O2 were maintained at 37 °C by heated water jackets and stimulates an autocrine prostanoid signaling pathway continuously bubbled with a 95 % O2–5%CO2 mixture. that elicits an increase in CFTR-mediated anion secre- To monitor ISC and resistance, the transepithelial mem- tion, which can be seen in Ussing chamber experiments brane potential was clamped at 0 mV with a 6-channel as short circuit currents (Isc). The autocrine EP1 and voltage clamp (model VCC MC6, Physiologic Instru- EP4 pathways operate through G-proteins that indirectly ments) using Ag/AgCl electrodes in agar bridges. Signals stimulate sAC through increases in intracellular Ca2+ were digitized and recorded with DAQplot software 2+ ([Ca ]i), thereby amplifying the cAMP signal to increase (VVI Software, College Station, PA) via a LabJack A/D CFTR conductance [21]. More prolonged exposures to converter (LabJack Corp, Lakewood, CO). The input re- H2O2 induces a decrease in resistance with concomitant sistance of each filter was measured by application of increase in permeability. These changes are believed to 1 mV bipolar pulses of 2 s duration. After addition of represent alteration of epithelial barrier function. Thus, amiloride, ISC was allowed to stabilize and then H2O2 to better understand the mechanism underlying the was included in the apical perfusate. Time scales were H2O2-induced junctional disruption, we explored the initialized at the time of mounting in the chamber. To role of the H2O2-mediated decreases in resistance and explore the mechanisms responsible for this change in increases in permeability using primary NHBE cell cul- ISC, cultures were incubated apically and basolaterally tures re-differentiated at the air liquid interface. These with pharmacological agents for 20–50 min before and experiments showed that the H2O2-induced effects on during apical exposure to H2O2. resistance and permeability depended not only on direct inhibition of tyrosine protein phosphatases by H2O2, but Permeability measurements also on a G-protein coupled receptor (GPCR) transduc- NHBE ALI cultures were preincubated for 10 min at tion path that involves the Ca2+-mediated stimulation of 37 °C in KH-buffer (pH 7.35). 14C mannitol was added sAC activity and PKA. apically and initial apical to basolateral mannitol flux was determined [24]. Cells were washed apically and Methods basolaterally three times to remove 14C mannitol, treated 14 Cell culture apically with H2O2 or inhibitors/activators, fresh C Human airway epithelial cells were obtained from organ mannitol added apically and final mannitol flux was de- donors whose lungs were rejected for transplant. termined. Apparent permeability Papp was calculated for Consent was obtained through the Life Alliance Organ initial and final mannitol flux using the following equa- . Recovery Agency of the University of Miami and the tion: Papp = dQ/dT/A C0 where dQ/dt is the flux deter- LifeCenter Northwest in WA according to IRB approved mined from the amount transported (Q) over time (T) protocols. Epithelial cells from the lower trachea and of the experiment, A is the surface area of the filter, and bronchi were isolated as previously described [22, 23]. C0 is the initial concentration on the donor side. Results Air-liquid interface (ALI) cultures were allowed to differ- were expressed as mannitol Papp normalized to initial entiate for at least 2 weeks prior to experiments. All ex- Papp. periments were performed with date, passage and lung matched control cultures. Intracellular Ca2+ measurements NHBE ALI cultures were loaded at room temperature Chemicals for 2 h by gentle rocking of cultures in 10 μM fura- DMEM, Ham’s nutrient F-12 and Hank’s balanced salt 2 AM (ThermoFisher Scientific, Waltham, MA) in solution were purchased from Gibco, Life Technologies Dulbecco’s PBS containing 1 % glucose and 10 % FBS. (Grand Island, NY). Gly-H 101 was from Calbiochem. Cultures were washed and mounted in a perfusion Prostanoid receptor antagonists were from Cayman chamber in Krebs-Henseleit buffer at room temperature Chemicals. All other chemicals, unless otherwise stated, and ratiometric images captured and quantified as de- were purchased from Sigma Aldrich (St. Louis, MO). scribed previously [25]. Ivonnet et al. Respiratory Research (2016) 17:15 Page 3 of 8 Immunocytochemistry Fully differentiated NHBE cells in ALI cultures were fixed in 4 % paraformaldehyde in PBS, pH 7.4 for 15 min and permeabilized with 1 % Triton X-100
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