Am J Physiol Lung Cell Mol Physiol 309: L1430 –L1437, 2015. First published October 16, 2015; doi:10.1152/ajplung.00067.2015.

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Lipopolysaccharide-induced pulmonary endothelial barrier disruption and lung edema: critical role for bicarbonate stimulation of AC10 Jordan Nickols,1 Boniface Obiako,2,3 K. C. Ramila,1 Kevin Putinta,1 Sarah Schilling,4 and Sarah L. Sayner1,3 1

Department of Physiology and Cell Biology, University South Alabama, Mobile, Alabama; 2Department of Pharmacology, University South Alabama, Mobile, Alabama; 3Center for Lung Biology, University South Alabama, Mobile, Alabama; and 4 University of Applied Sciences Bingen, Bingen am Rhein, Germany Submitted 3 March 2015; accepted in final form 14 October 2015

Nickols J, Obiako B, Ramila KC, Putinta K, Schilling S, Sayner SL. Lipopolysaccharide-induced pulmonary endothelial barrier disruption and lung edema: critical role for bicarbonate stimulation of AC10. Am J Physiol Lung Cell Mol Physiol 309: L1430 –L1437, 2015. First published October 16, 2015; doi:10.1152/ajplung.00067.2015.—Bacteria-induced sepsis is a common cause of pulmonary endothelial barrier dysfunction and can progress toward acute respiratory distress syndrome. Elevations in intracellular cAMP tightly regulate pulmonary endothelial barrier integrity; however, cAMP signals are highly compartmentalized: whether cAMP is barrier-protective or -disruptive depends on the compartment (plasma membrane or cytosol, respectively) in which the signal is generated. The mammalian soluble adenylyl cyclase isoform 10 (AC10) is uniquely stimulated by bicarbonate and is expressed in pulmonary microvascular endothelial cells (PMVECs). Elevated extracellular bicarbonate increases cAMP in PMVECs to disrupt the endothelial barrier and increase the filtration coefficient (Kf) in the isolated lung. We tested the hypothesis that sepsis-induced endothelial barrier disruption and increased permeability are dependent on extracellular bicarbonate and activation of AC10. Our findings reveal that LPS-induced endothelial barrier disruption is dependent on extracellular bicarbonate: LPS-induced barrier failure and increased permeability are exacerbated in elevated bicarbonate compared with low extracellular bicarbonate. The AC10 inhibitor KH7 attenuated the bicarbonate-dependent LPS-induced barrier disruption. In the isolated lung, LPS failed to increase Kf in the presence of minimal perfusate bicarbonate. An increase in perfusate bicarbonate to the physiological range (24 mM) revealed the LPS-induced increase in Kf, which was attenuated by KH7. Furthermore, in PMVECs treated with LPS for 6 h, there was a dose-dependent increase in AC10 expression. Thus these findings reveal that LPS-induced pulmonary endothelial barrier failure requires bicarbonate activation of AC10. compartmentalized cAMP; lipopolysaccharide; lung edema; soluble adenylate cyclase; bicarbonate ACUTE RESPIRATORY DISTRESS syndrome (ARDS) is a life-threatening condition characterized by acute-onset, arterial hypoxemia and diffuse bilateral infiltrates accompanied by noncardiogenic pulmonary edema (29, 30, 54). Clinically, ARDS can be attributed to pulmonary (direct) causes, such as pneumonia,

Address for reprint requests and other correspondence: S. L. Sayner, Dept. of Physiology and Cell Biology, Center for Lung Biology, College of Medicine, Univ. of South Alabama, Mobile, AL 36688 (e-mail: [email protected]). L1430

aspiration, or mechanical ventilation. Alternatively, ARDS can be attributed to extrapulmonary (indirect) causes, such as sepsis, severe burns, or pancreatitis. Irrespective of the insult, a key characteristic of the pathogenesis of ARDS (mild, moderate, and severe) is endothelial barrier dysfunction. In particular, sepsis-induced vascular damage increases endothelial permeability at the level of the alveolar capillaries, but the mechanism of this barrier disruption is not completely understood. Advances in mechanical ventilation and fluid conservative management have improved ARDS outcomes, yet no pharmacological treatments have proven effective at reducing mortality. Thus a better understanding of the mechanisms associated with the development of the syndrome and new drug targets to reverse the damage is required. cAMP signaling has long been known to regulate the integrity of the endothelial barrier. For example, cAMP signals, generated through indirect activation of transmembrane adenylyl cyclase (AC) through G␣S protein signaling or direct activation using forskolin, stabilize the cortical actin rim, strengthen cell-cell adhesions, and increase endothelial barrier integrity (7–10, 15, 19, 31, 33, 43). However, cAMP signals are highly compartmentalized, and emerging evidence demonstrates that the origin of the cAMP signal, plasma membrane or cytosol, is critical to its influence on endothelial barrier integrity (3, 16, 36, 38, 42, 45, 46). For example, the bacterium Pseudomonas aeruginosa has a type 3 secretion system, which encodes an injection-type needle capable of inserting toxins into the cytosol of eukaryotic cells. One of these toxins is a soluble AC, exotoxin Y (ExoY), which generates cAMP in the cytosol, rather than the plasma membrane, to disrupt the pulmonary microvascular endothelial barrier (3, 34, 36, 46, 49). A soluble AC (sAC) chimera derived from transmembrane AC I and II, sACI/II, is stimulated by forskolin; thus, forskolin activation of pulmonary microvascular endothelial cells (PMVECs) expressing sACI/II generates cAMP in the plasma membrane and cytosol, yet the increased cytosolic cAMP is sufficient to disrupt the barrier (38, 44). While expression of these exogenous sACs leads to endothelial barrier disruption, the effects of the endogenous bicarbonate-stimulated sAC AC10, which localizes to the cytosol, are just emerging. Our previous studies revealed that PMVECs and pulmonary artery endothelial cells express AC10; however, protein levels are much higher in PMVECs (35). Furthermore, addition of extra-

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LPS-MEDIATED ENDOTHELIAL PERMEABILITY: BICARBONATE AND AC10

cellular bicarbonate to confluent endothelial monolayers generated a phosphodiesterase-sensitive cAMP pool in PMVECs that was not observed in pulmonary artery endothelial cells. In PMVECs, increasing bicarbonate in the culture medium not only increased cAMP; it also disrupted the endothelial barrier. Furthermore, increasing bicarbonate in the perfusate of the isolated lung led to edema. Thus, bicarbonate stimulation of AC10 disrupts the endothelial barrier, but it is not known how bicarbonate regulates pulmonary endothelial barrier integrity in a model of acute lung injury. Gram-negative sepsis is a common cause of ARDS (28 –30, 32). Endotoxin, which, in its purified form, is generally known as lipopolysaccharide or LPS, is derived from gram-negative bacteria and is associated with pulmonary endothelial barrier disruption (4, 5, 8, 9, 14, 17, 24, 50, 52, 53). In these studies we sought to determine whether media bicarbonate concentrations alter the extent of endothelial barrier disruption when combined with an LPS challenge. Our findings suggest that bicarbonate plays a key role in regulating LPS-induced endothelial barrier disruption: while low bicarbonate attenuates LPS-induced injury, elevated bicarbonate augments LPS-induced pulmonary endothelial injury. Furthermore, we show that LPS treatment of PMVEC monolayers increases AC10 expression over time. METHODS

Reagents. DMEM (with and without bicarbonate) and penicillinstreptomycin were obtained from Gibco; LPS from Escherichia coli (serotype O55:B5, catalog no. L2880, lot no. 102M4017V), KrebsHenseleit buffer solution, FITC-labeled dextran (10 kDa), HEPES, sodium bicarbonate, and Lowry protein assay from Sigma-Aldrich (St. Louis, MO); radioimmunoprecipitation buffer from Boston Bio Products (Boston, MA); and Halt protease and phosphatase inhibitor cocktail from Thermo Scientific. AC10 antibody (R21, 1:500 dilution) was a gift from Drs. Levin and Buck (Weill Medical College, New York, New York). GAPDH antibody was obtained from Cell Signaling Technology (Danvers, MA); secondary antibodies from Santa Cruz Technologies (Dallas, TX); and the AC10 inhibitor KH7 from Tocris Bioscience (Minneapolis, MN). Isolation and culture of rat pulmonary endothelial cells. PMVECs were obtained from the Cell Culture Core at the University of South Alabama Center for Lung Biology. Isolation and characterization of these cells under the approval of the Animal Care and Use Committee of the University of South Alabama are described elsewhere in detail (25, 48). Cells were cultured in endothelial cell medium (high-glucose DMEM, 10% fetal bovine serum, and 1% penicillin-streptomycin) at 37°C in 21% O2 and 5% CO2. Transendothelial electrical resistance. Transendothelial electrical resistance (TER) across PMVECs was measured in response to LPS and increasing bicarbonate concentrations, as described previously (35). Briefly, PMVECs were seeded onto polycarbonate wells containing small evaporated gold microelectrodes in series with a large gold counterelectrode (10⫺3 cm2, 8W10E⫹, Applied Biophysics, Troy, NY). Resistance across the monolayer was measured using an electrical cell-substrate impedance-sensing (ECIS) system (Applied Biophysics). Once cells had achieved confluence, the medium was exchanged for serum-free medium, and a stable baseline TER (1,200 –1,500 ⍀) was recorded over several hours (data not shown). TER was monitored every 5 min for the duration of the experiment. After baseline recordings, LPS was added at the appropriate concentration in the presence or absence of the AC10 inhibitor KH7 (30 ␮M). For low-bicarbonate (0 mM) experiments, cells were placed in HEPES (20 mM)buffered bicarbonate-free DMEM and 0% CO2 to maintain phys-

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iological pH (7.4). An aliquot of the medium was removed during and at the end of the experiment for determination of CO2, pH, and bicarbonate using a blood gas analyzer (model ABL5, Radiometer, Copenhagen, Denmark), and the average bicarbonate concentration during the experiment was reported. Data from each microelectrode were normalized to the initial baseline resistance and plotted vs. time. Permeability assays. In vitro endothelial permeability was measured by diffusion of FITC-labeled dextran (10 kDa) across the confluent PMVEC monolayer seeded onto Transwell permeability supports (0.4-␮m polyester membrane, 12-mm insert, Costar, Cambridge, MA). Once PMVECs reached confluence, medium was removed from the lower chamber and replaced with 600 ␮l of serumfree DMEM. After the cells were rinsed in PBS, medium in the upper chamber was replaced with fresh serum-free medium containing FITC-dextran, and cells were returned to the incubator. After a 10-min equilibration period, FITC-dextran in the upper chamber was replaced with serum-free FITC-dextran medium containing the appropriate concentration of LPS. Cells were returned to the incubator, and, at appropriate time points (5, 30, 60, 120, 180 and 240 min), 10 ␮l of medium were removed from the lower chamber of each Transwell support. Fluorescence of the sample was measured using a fluorospectrometer (model 3300, NanoDrop Technologies). For low-bicarbonate (0 mM) experiments, bicarbonate-free DMEM contained HEPES (20 mM), and cells were incubated at 0% CO2 to maintain pH at 7.4. For high-bicarbonate (40 mM) experiments, bicarbonate was added to bicarbonate-free medium, and cells were incubated at 9% CO2 to maintain pH at 7.4. An aliquot of medium was removed during and at the end of the experiment for determination of CO2, pH, and bicarbonate using a blood gas analyzer (model ABL5, Radiometer), and the average bicarbonate concentration during the experiment was reported. Isolation of total cellular proteins and Western analysis following LPS treatment. Confluent PMVECs were rinsed in PBS, and fresh serum-free DMEM was added at 24 or 0 mM bicarbonate. After 30 min of incubation at 37°C and 5% CO2 for 24 mM bicarbonate or 0% CO2 for 0 mM bicarbonate, increasing concentrations of LPS or appropriate vehicle control was added, and cells were returned to the incubator. After 4 or 6 h, LPS was removed, and cells were rinsed in ice-cold PBS and lysed in ice-cold radioimmunoprecipitation buffer (10 mM sodium phosphate, 150 mM NaCl, 1% Triton X-100, 0.5% sodium deoxycholate, and 0.1% SDS, pH 7.2; Boston Bio Products) with Halt protease and phosphatase inhibitor cocktail (Thermo Scientific). Then all samples were rotated at 4°C for 15 min and centrifuged at 13,000 rpm at 4°C for 10 min (Sorvall Legend Micro 17, Thermo Scientific). The supernatant was collected and stored at ⫺80°C for protein concentration analysis (Lowry protein assay, Sigma-Aldrich). Samples were adjusted to equal protein concentration, sample buffer (62.4 mM Tris·HCl, pH 6.4, 10% glycerol, 1.8% ␤-mercaptoethanol, and 70 mM SDS) was added, and samples were heated to 37°C for 30 min. Proteins were resolved by SDS-PAGE (Nu-PAGE, Invitrogen) and transferred to nitrocellulose membranes. Nitrocellulose membranes were incubated with blocking buffer [Tris-buffered saline (TBS: 137 mM NaCl and 25 mM Tris, pH 7.40) supplemented with 0.5% Tween 20 (TTBS) and 5% nonfat dry milk]. After they were rinsed in TTBS, membranes were rocked with anti-AC10 antibody (55) or GAPDH in TTBS containing 1% nonfat dry milk overnight at 4°C. The membranes were washed (3 times for 10 min each) in TTBS with rocking and then incubated with secondary antibody (IgG conjugated to horseradish peroxidase) in TTBS containing 1% nonfat dry milk for 1 h. Finally, membranes were rinsed (3 times for 10 min each) in TTBS, and chemiluminescence of proteins was visualized with enhanced chemiluminescence reagent (ECL, Amersham Biosciences) and detected with the Fujifilm LAS-1000 Image Reader system. Western blots were performed at least four times, and a representative blot is shown. Densitometry was

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00067.2015 • www.ajplung.org

LPS-MEDIATED ENDOTHELIAL PERMEABILITY: BICARBONATE AND AC10

performed on immunoblots using Fujifilm Science Lab ImageGauge V4.0 software. Animals. All animal experiments were performed using adult male Sprague-Dawley rats (250 –350 g body wt; Charles River, Wilmington, MA) according to a protocol approved by the Animal Care and Use Committee of the University of South Alabama and in accordance with the National Institutes of Health guidelines for the care and use of laboratory animals. Isolated lung studies. Isolated lung studies were performed as described in detail previously (13, 35). Briefly, animals were anesthetized with pentobarbital sodium (60 mg/kg ip), and a tracheotomy catheter was inserted. After cannulation of the heart, the lungs were ventilated with 21% O2-5% CO2-74% N2. Heparin (200 U) was administered into the right ventricle and allowed to circulate. The lungs were perfused at constant flow with Krebs-Henseleit salt solution containing (in mM) 11.1 D-glucose, 1.2 MgSO4, 1.2 KH2PO4, 4.7 KCl, 118.1 NaCl, 1.5 CaCl2, and 20 HEPES, sodium bicarbonate depending on experimental conditions, and 4% BSA for osmotic stabilization. The lungs and heart were removed en bloc. After the baseline filtration coefficient (Kf) was recorded, bicarbonate, LPS (250 ␮g/ml), and KH7 (30 ␮M) were added to the perfusate as indicated, and Kf was measured at 30 and 60 min. Kf was calculated as the rate of weight gain after a 10-cmH2O increase in pulmonary venous pressure and normalized to lung dry weight (g). A perfusate sample was withdrawn during the experiment for blood gas analysis (ABL5 blood gas analyzer, Radiometer). Graphing and statistics. Values are means ⫾ SE. Comparison between data groups was accomplished using an unpaired Student’s t-test or one-way analysis of variance in conjunction with post hoc multiple-comparison test as needed. In all cases, P ⬍ 0.05 was considered statistically significant. Data were graphed using GraphPad Prism 6 (version 6.0f, GraphPad Software).

elevated media bicarbonate at a concentration that was sufficient to disrupt the endothelial barrier as determined from our previous studies would affect LPS-induced barrier disruption. We performed ECIS experiments with confluent PMVECs in medium with physiological (24 mM) bicarbonate concentration (Fig. 1A) or in bicarbonate-free (0 mM) medium (Fig. 1B). LPS induced a dose-dependent decrease in resistance across the monolayer over 8 h at 0 and 24 mM bicarbonate. While 10 ␮g/ml LPS was sufficient to decrease resistance at a media bicarbonate concentration of 24 mM (Fig. 1C), removal of bicarbonate from the medium prevented the LPS-induced PMVEC barrier disruption. At 50 ␮g/ml LPS, the decrease in resistance across the PMVEC monolayer was attenuated when bicarbonate was removed from the medium (Fig. 1D). Thus, 24 vs. 0 mM bicarbonate was sufficient to promote a LPS-induced decrease in resistance over 8 h, which becomes significant at 50 ␮g/ml LPS (Fig. 2A). The AC10 inhibitor KH7 attenuated the LPS-induced decrease in resistance, implicating AC10 in bicarbonate-stimulated and LPS-induced PMVEC barrier disruption (Fig. 2B). Bicarbonate is required and augments LPS-induced increase in PMVEC permeability. FITC-dextran transfer across PMVEC monolayers grown on Transwell supports was performed to determine whether bicarbonate affects LPSinduced PMVEC permeability. Once PMVEC monolayers achieved confluence, growth medium in the upper chamber was replaced with serum-free medium at 0, 24, or 40 mM bicarbonate and FITC-dextran. Bicarbonate concentration in medium was the same in the lower and upper chambers. After 10 min of equilibration, medium in the upper chamber was replaced with LPS- or vehicle control-containing FITCdextran. At the physiological (24 mM) bicarbonate concentration, 50 ␮g/ml, but not 25 ␮g/ml, LPS was sufficient to increase permeability across the PMVEC monolayer over time (Fig. 3A). In contrast, in the absence of bicarbonate, LPSinduced permeability was ablated at both concentrations (Fig. 3B); however, 40 mM bicarbonate revealed an LPS-induced permeability at 25 and 50 ␮g/ml LPS (Fig. 3C). Thus, at 4 h

LPS-induced decrease in PMVEC barrier resistance is dependent on media bicarbonate. Our previous studies revealed that increasing media bicarbonate to 40 mM led to an increase in PMVEC whole cell cAMP. Furthermore, this elevated bicarbonate was sufficient to decrease TER across the PMVEC monolayer and increase Kf in the isolated lung (35). Thus, in our current studies we were interested to determine whether

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Fig. 1. Bicarbonate exacerbates the LPS decrease in resistance across pulmonary microvascular endothelial cell (PMVEC) monolayers. A: once a stable baseline resistance was established, LPS was added (at minute 15) to PMVEC monolayers grown on electrical cellsubstrate impedance-sensing (ECIS) arrays, and resistance was recorded every 5 min for 8 h. At 24 mM HCO⫺ 3 , LPS dose-dependently decreased resistance across the PMVEC monolayer. B: reducing HCO⫺ 3 concentration to 0 mM attenuated the LPS-induced decrease in resistance. C and D: for each LPS dose (10 or 50 ␮g/ml), low HCO⫺ 3 (0 mM) attenuated the decrease in resistance compared with the physiological HCO⫺ 3 concentration (24 mM). Results are representative of ⱖ3 independent experiments.

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permeability is not only dose-dependent; it is also dependent on extracellular bicarbonate concentration. LPS increases AC10 protein expression. At 24 mM bicarbonate, LPS dose-dependently decreased resistance, which became significant at 6 h; therefore, we examined the change in AC10 protein expression at 6 h and at an earlier time point (4 h). Western analysis was performed on PMVEC lysates following 4 or 6 h of incubation with increasing LPS concentrations at 24 mM bicarbonate or at a bicarbonate concentration that does not activate AC10 (0 mM bicarbonate). In the absence of bicarbonate (0 mM), neither 4 nor 6 h of LPS treatment increased AC10 expression (data not shown). Furthermore, there was no significant increase in AC10 expression at the 4-h time point in 24 mM bicarbonate (data not shown); however, after 6 h of LPS (25 and 50 ␮g/ml) treatment at 24 mM bicarbonate, AC10 expression increased significantly above baseline (Fig. 4). As described previously (35), 48-kDa (lower band) and 50-kDa (upper band) protein bands were observed. Thus, bicarbonate is required for the LPS-induced increase in AC10 expression over time. Bicarbonate is required for the LPS-induced increase in Kf and is dependent on AC10. To address whether perfusate bicarbonate concentrations affect LPS-induced changes in endothelial permeability in situ, we performed isolated perfused studies. After the initial baseline recording in lowbicarbonate (5 mM) medium, LPS was added to the perfusate. At this low perfusate bicarbonate concentration, there was no increase in Kf at 30 and 60 min after the addition of LPS (Fig. 5). However, when LPS was added in the presence of 25 mM bicarbonate, Kf began to increase at 30 min and increased sixfold by 60 min. This increase in permeability was blocked when the AC10 inhibitor KH7 was added to the perfusate with LPS. Thus, bicarbonate is critical for LPS to increase Kf in the isolated lung and requires AC10.

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Fig. 2. LPS-induced decrease in transendothelial electrical resistance (TER) is augmented with increasing bicarbonate concentration. A: at 8 h after its addition, LPS decreased baseline TER across PMVEC monolayers grown to confluence on ECIS arrays at 0 or 24 mM extracellular HCO⫺ 3 . At 8 h after addition of 50 ␮g/ml LPS, percent decrease in TER was attenuated in the absence of extracellular HCO⫺ 3 . B: the AC10 inhibitor KH7 attenuated the LPS-induced decrease in resistance at 24 mM HCO⫺ 3 . Results are representative of 3–4 independent experiments. *P ⬍ 0.05, 24 vs. 0 mM bicarbonate (A) or absence vs. presence of KH7 (B).

after its addition, 25 ␮g/ml LPS increased permeability when bicarbonate was increased to 40 mM, while 50 ␮g/ml LPS significantly increased permeability at 24 and 40 mM bicarbonate (Fig. 3D). Thus, LPS-induced increase in PMVEC

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Fig. 3. LPS-induced permeability across PMVEC monolayers requires and is exacerbated by extracellular bicarbonate. A: at 24 mM extracellular HCO⫺ 3 , LPS dose-dependently increased FITC-dextran transfer across the PMVEC monolayer. B: in the absence of HCO⫺ 3 , LPS did not increase FITC-dextran transfer across the PMVEC monolayer. C: increasing HCO⫺ 3 to 40 mM exacerbated LPS-induced dextran transfer across the monolayer. *P ⬍ 0.05 vs. vehicle control. Results are representative of 3–5 independent experiments. D: at 4 h after addition of 25 ␮g/ml LPS, 40 mM extracellular bicarbonate was required to increase dextran flux across the PMVEC monolayer. At 50 ␮g/ml LPS, 24 mM extracellular HCO⫺ 3 was required for LPS-induced increase in dextran flux, which was exacerbated when extracellular HCO⫺ 3 was increased to 40 mM. *P ⬍ 0.05. Results are representative of 3–5 independent experiments. RFU, relative fluorescence units.

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Bicarbonate is a ubiquitous molecule that can become elevated in respiratory disorders or pulmonary edema or following infusion (i.e., given to correct plasma pH, when it falls below 7.2–7.25 as a result of hypercapnic acidosis associated with low-tidal volume ventilation) (1, 2, 27, 51). Within the cellular environment, bicarbonate does more than increase intracellular pH. Bicarbonate is also a signaling molecule that activates soluble AC10 to generate cytosolic cAMP (11, 12); however, while the pulmonary endothelium is in direct contact with plasma bicarbonate, the independent effects of bicarbonate in the face of existing lung injury are unknown. We have shown that elevated LPS-induced lung endothelial permeability is dependent on extracellular bicarbonate and AC10. It is well recognized that bacterial products, such as LPS, increase pulmonary endothelial permeability. These studies reveal that LPS barrier disruption and permeability are dependent on and exacerbated by bicarbonate. Indeed, removal of bicarbonate from the culture medium essentially prevented LPS-induced PMVEC barrier disruption. Once bicarbonate reached a normal physiological concentration (24 mM), LPS barrier disruption was revealed. Furthermore, increasing bicarbonate beyond the normal physiological range permitted PMVEC barrier disruption at a lower LPS concentration. The bicarbonate- and LPS-induced barrier disruption was dependent on AC10, as revealed by the AC10 inhibitor KH7. In the isolated lung, the LPS-induced increase in Kf was dependent on perfusate bicarbonate, and without bicarbonate, the LPS-induced edema was absent. The AC10 inhibitor KH7 attenuated the LPS- and bicarbonate-induced increase in Kf, thus demonstrating the role of AC10 in this response. Thus it appears that while activation of endogenous transmembrane ACs with agonists such as prostacyclin or iloprost protects against LPSinduced endothelial barrier disruption (8, 9), activation of the endogenous bicarbonate-stimulated soluble AC (AC10) exacerbates LPS-induced endothelial barrier disruption and further supports the disparate roles of compartmentalized cAMP in regulation of endothelial barrier integrity. ARDS is a common complication associated with high morbidity and mortality (41). In the clinical setting, ARDS

patients often have elevated CO2, reaching levels ⬎65–70 mmHg, and an associated increase in bicarbonate (ⱖ30 mM); however, the effects of this elevated bicarbonate are only beginning to be appreciated. Our previous studies revealed that elevated media bicarbonate (30 – 40 mM) increased intracellular cAMP and was associated with a decrease in PMVEC barrier integrity and an increase in Kf. In the current studies we investigated the role of this elevated bicarbonate in the setting of sepsis-induced ARDS. Indeed, infection is one of the leading causes of ARDS, yet even within the category of gram-negative-associated infection, the syndrome is heterogeneous and associated with broad clinical phenotypes (29). Experimentally, endotoxin-mediated lung injury is also heterogeneous (52). LPS or purified endotoxin is the characteristic component of the cell wall of gram-negative bacteria and is not found on gram-positive bacteria. Typically, LPS is made up of three components: a hydrophobic lipid A responsible for the toxic properties of the molecule, a hydrophilic core polysaccharide chain, and a hydrophilic O-antigenic polysaccharide side chain (39). Because of their biological origin and despite a common architecture, lipid A molecules have intrinsic heterogeneity in their fine structure that can dramatically change biological activity (47). In addition, different purification methods yield LPS containing various levels of protein and/or RNA contaminants. In the current studies we used phenol-extracted LPS from E. coli, which induced a rapid disruption of the PMVEC monolayer and a significant increase in Kf. While these responses seem more rapid and dramatic than those reported previously, we also paid close attention to the buffer bicarbonate levels throughout the experiments. Indeed, once bicarbonate levels began to fall below the normal range (24 mM), the ability to induce endothelial injury was attenuated. Thus a source of discrepancy in the literature with regard to the ability of LPS to induce injury could be associated with variability in buffer bicarbonate concentrations. Another significant and novel finding of our study was the induction of AC10 expression following 6 h of LPS treat-

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LPS-MEDIATED ENDOTHELIAL PERMEABILITY: BICARBONATE AND AC10

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Fig. 5. Bicarbonate is required for AC10-mediated, LPS-induced lung permeability. Perfusate HCO⫺ 3 (25 mM) was required for LPS (250 ␮g/ml) to increase the filtration coefficient (Kf). HCO⫺ 3 - and LPS-induced increase in Kf was attenuated when the AC10 inhibitor KH7 (30 ␮M) was added to the perfusate. *P ⬍ 0.001 vs. baseline (BL). #P ⬍ 0.05 vs. 25 mM HCO⫺ 3 ⫹ KH7. Results are representative of 5 independent experiments.

AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00067.2015 • www.ajplung.org

LPS-MEDIATED ENDOTHELIAL PERMEABILITY: BICARBONATE AND AC10

ment. While LPS has been shown to alter protein expression (5, 21, 37, 50), such as inflammatory cytokines, chemokines, and adhesion molecules, this is the first study to show LPS induction of AC10. In an animal model of endotoxemia, LPS attenuated AC5, AC6, AC7, and AC9 mRNA in the lung, spleen, heart, kidney, and liver, while AC4 mRNA was elevated in the lung and heart (40). Since AC6 is the predominant transmembrane AC in PMVECs, LPS-induced reduction of AC6 expression could decrease the barrierprotective effects of subplasma membrane cAMP while simultaneously increasing the barrier-disruptive effects of cytosolic cAMP. However, the subtle decrease in membrane-localized cAMP vs. the subtle increase in cytosolic cAMP could be difficult to detect with whole cell cAMP measurements. We do not know whether the increase in AC10 expression is linked to increased gene expression, increased stability of the mRNA, or reduced protein degradation. The AC10 promoter has not been fully characterized; therefore, future studies are required to determine the mechanism of LPS-induced AC10 protein expression. The crystal structure of human AC10, which has recently been described, confirms that AC10 more closely resembles cyclases of bacterial origin than the mammalian transmembrane isoforms (26). Soluble AC10 lacks transmembrane domains and has been detected in various intracellular locations, such as nucleus, mitochondria, and centrioles (18, 20, 55, 56). It is regulated by bicarbonate and calcium in certain cell types, although calcium sensitivity was not detected in PMVECs (35). Unlike transmembrane ACs, which have only AC activity to generate cAMP (23), AC10 is a promiscuous cyclase. Indeed, only cAMP is detected following forskolin activation of transmembrane AC1-8, yet addition of bicarbonate (40 mM) to HEK 293 or B103 neuroblastoma cells expressing endogenous AC10 leads to an increase not only in intracellular cAMP, but also in cUMP, cCMP, and cGMP (23). The AC10 inhibitor KH7 reduced the bicarbonate-stimulated increase in cNMPs in a dose-dependent manner, and cNMP levels returned to baseline upon removal of bicarbonate from the medium. This ability to synthesize cNMPs is common to other bacteriasoluble cyclases. Indeed, ExoY, which is part of the type 3 secretion system of the common lung pathogen P. aeruginosa, synthesizes not only cAMP, but also cCMP and high levels of cGMP and cUMP (6, 34). Other bacterial cyclases, such as edema factor of Bacillus anthracis and the AC toxin (CyaA) of Bordetella pertussis, also exhibit broad substrate specificity and demonstrate both cytidylyl and uridylyl activity to synthesize cCMP and cUMP, respectively (22). The contribution of these other cNMPs to the pathogenicity of these toxins is not clear. While the barrier-disruptive effects of ExoY are primarily attributed to cAMP and, to a lesser extent, cGMP, the role of cUMP and cCMP in pulmonary endothelial barrier disruption has not been determined (36). Therefore, it appears that, similar to bacteria-soluble ACs, AC10 is a nucleotidyl cyclase capable of generating other cyclic nucleotides (cNMPs), yet this bicarbonate-sensitive cNMP profile has not been characterized in PMVECs. Furthermore, the role of these cNMPs in regulation of the pulmonary endothelial barrier is unclear. In summary, our data reveal that bicarbonate levels are critical in determining the ability and extent of LPS pulmo-

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nary endothelial barrier disruption, such that low bicarbonate attenuates LPS-induced barrier disruption. In the isolated lung, bicarbonate was required to promote the LPSinduced increase in permeability and was dependent on AC10. Thus, while the normal physiological function of AC10 in the endothelium has not been established, bicarbonate stimulation of AC10 increases LPS-induced permeability, and the LPS-induced increase in AC10 protein expression could further increase a cytosolic cAMP signal and exacerbate lung injury. ACKNOWLEDGMENTS The authors thank Dr. Troy Stevens for careful review of the manuscript and Linn Ayers and Anna Buford (Center for Lung Biology Tissue Culture Core) for help with cell seeding. GRANTS This work was supported by a Parker B. Francis Fellowship Foundation Award (S. L. Sayner) and National Heart, Lung, and Blood Institute Grant HL-121513 (S. L. Sayner). DISCLOSURES No conflicts of interest, financial or otherwise, are declared by the authors. AUTHOR CONTRIBUTIONS J.N., B.O.O., and S.L.S. developed the concept and designed the research; J.N., B.O.O., K.R., K.P., and S.S. performed the experiments; J.N., B.O.O., K.R., K.P., S.S., and S.L.S. analyzed the data; J.N., B.O.O., K.R., and S.L.S. interpreted the results of the experiments; J.N., B.O.O., K.R., and S.L.S. prepared the figures; J.N., B.O.O., K.R., K.P., S.S., and S.L.S. approved the final version of the manuscript; S.L.S. drafted the manuscript; S.L.S. edited and revised the manuscript. REFERENCES 1. Amato MB, Barbas CS, Medeiros DM, Magaldi RB, Schettino GP, Lorenzi-Filho G, Kairalla RA, Deheinzelin D, Munoz C, Oliveira R, Takagaki TY, Carvalho CR. Effect of a protective-ventilation strategy on mortality in the acute respiratory distress syndrome. N Engl J Med 338: 347–354, 1998. 2. Anonymous. Ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome. The Acute Respiratory Distress Syndrome Network. N Engl J Med 342: 1301–1308, 2000. 3. Balczon R, Prasain N, Ochoa C, Prater J, Zhu B, Alexeyev M, Sayner S, Frank DW, Stevens T. Pseudomonas aeruginosa exotoxin Y-mediated ␶ hyperphosphorylation impairs microtubule assembly in pulmonary microvascular endothelial cells. PLos One 8: e74343, 2013. 4. Bannerman DD, Goldblum SE. Mechanisms of bacterial lipopolysaccharide-induced endothelial apoptosis. Am J Physiol Lung Cell Mol Physiol 284: L899 –L914, 2003. 5. Barabutis N, Dimitropoulou C, Birmpas C, Joshi A, Thangjam G, Catravas JD. p53 protects against LPS-induced lung endothelial barrier dysfunction. Am J Physiol Lung Cell Mol Physiol 308: L776 –L787, 2015. 6. Beckert U, Wolter S, Hartwig C, Bahre H, Kaever V, Ladant D, Frank DW, Seifert R. ExoY from Pseudomonas aeruginosa is a nucleotidyl cyclase with preference for cGMP and cUMP formation. Biochem Biophys Res Commun 450: 870 –874, 2014. 7. Birukova AA, Burdette D, Moldobaeva N, Xing J, Fu P, Birukov KG. Rac GTPase is a hub for protein kinase A and Epac signaling in endothelial barrier protection by cAMP. Microvasc Res 79: 128 –138, 2010. 8. Birukova AA, Meng F, Tian Y, Meliton A, Sarich N, Quilliam LA, Birukov KG. Prostacyclin post-treatment improves LPS-induced acute lung injury and endothelial barrier recovery via Rap1. Biochim Biophys Acta 1852: 778 –791, 2014. 9. Birukova AA, Wu T, Tian Y, Meliton A, Sarich N, Tian X, Leff A, Birukov KG. Iloprost improves endothelial barrier function in lipopolysaccharide-induced lung injury. Eur Respir J 41: 165–176, 2013.

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AJP-Lung Cell Mol Physiol • doi:10.1152/ajplung.00067.2015 • www.ajplung.org

Lipopolysaccharide-induced pulmonary endothelial barrier disruption and lung edema: critical role for bicarbonate stimulation of AC10.

Bacteria-induced sepsis is a common cause of pulmonary endothelial barrier dysfunction and can progress toward acute respiratory distress syndrome. El...
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