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• W1987751707 abstract "Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin (∼7 μm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nm. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of ∼250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached ∼1 μm independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation. Extracellular ATP and its metabolite adenosine regulate mucociliary clearance in airway epithelia. Little has been known, however, regarding the actual ATP and adenosine concentrations in the thin (∼7 μm) liquid layer lining native airway surfaces and the link between ATP release/metabolism and autocrine/paracrine regulation of epithelial function. In this study, chimeric Staphylococcus aureus protein A-luciferase (SPA-luc) was bound to endogenous antigens on primary human bronchial epithelial (HBE) cell surface and ATP concentrations assessed in real-time in the thin airway surface liquid (ASL). ATP concentrations on resting cells were 1-10 nm. Inhibition of ecto-nucleotidases resulted in ATP accumulation at a rate of ∼250 fmol/min/cm2, reflecting the basal ATP release rate. Following hypotonic challenge to promote cell swelling, cell-surface ATP concentration measured by SPA-luc transiently reached ∼1 μm independent of ASL volume, reflecting a transient 3-log increase in ATP release rates. In contrast, peak ATP concentrations measured in bulk ASL by soluble luciferase inversely correlated with volume. ATP release rates were intracellular calcium-independent, suggesting that non-exocytotic ATP release from ciliated cells, which dominate our cultures, mediated hypotonicity-induced nucleotide release. However, the cystic fibrosis transmembrane conductance regulator (CFTR) did not participate in this function. Following the acute swelling phase, HBE cells exhibited regulatory volume decrease which was impaired by apyrase and facilitated by ATP or UTP. Our data provide the first evidence that ATP concentrations at the airway epithelial surface reach the range for P2Y2 receptor activation by physiological stimuli and identify a role for mucosal ATP release in airway epithelial cell volume regulation. ATP regulates the airway epithelial mucociliary clearance activities that are critical for pulmonary host defense against bacteria which deposit on airway surfaces. ATP activates the Gq/phospholipase C-coupled P2Y2 receptors (P2Y2-R), 2The abbreviations used are: P2Y2-R, P2Y2 receptor; ADO, adenosine; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; ASL, airway surface liquid; Ca2+i, intracellular calcium; SPA-luc, Staphylococcus aureus protein A-luciferase; HBE, human bronchial epithelial; WD, well differentiated; KS, keratan sulfate; WGA, wheat germ agglutinin; ELISA, enzyme-linked immunosorbent assay; HRP, horseradish peroxidase; PBS, phosphate-buffered saline; BSA, bovine serum albumin; HBSS, Hanks' balanced salt solution; ALU, arbitrary light unit; BAPTA, 1,2-bis(2-aminopheoxy)ethane-N,N,N′,N′-tetraacetic acid; AM, acetoxymethyl ester; RVD, regulatory volume decrease; 8-SPT, 8-(p-sulfophenyl)theophylline; ATPγS, adenosine 5′-O-(thiotriphosphate). which in turn promotes Cl− secretion via calcium-activated Cl− channels (CaCC) (1Clarke L.L. Boucher R.C. Am. J. Physiol. 1992; 263: C348-C356Crossref PubMed Google Scholar), inhibits Na+ absorption mediated by epithelial sodium channels (2Mall M. Wissner A. Gonska T. Calenborn D. Kuehr J. Brandis M. Kunzelmann K. Am. J. Respir. Cell Mol. Biol. 2000; 23: 755-761Crossref PubMed Scopus (93) Google Scholar), increases ciliary beat frequency (3Geary C.A. Davis C.W. Paradiso A.M. Boucher R.C. Am. J. Physiol. 1995; 268: L1021-L1028PubMed Google Scholar), and triggers mucin release (4Lethem M.I. Dowell M.L. Van Scott M. Yankaskas J.R. Egan T. Boucher R.C. Davis C.W. Am. J. Respir. Cell Mol. Biol. 1993; 9: 315-322Crossref PubMed Scopus (108) Google Scholar, 5Davis C.W. Dowell M.L. Lethem M. Van Scott M. Am. J. Physiol. 1992; 262: C1313-C1323Crossref PubMed Google Scholar). Released ATP is rapidly hydrolyzed to ADP, AMP, and adenosine (ADO) by cell-surface nucleotidases. ADO activates Gs/adenylyl cyclase-coupled A2b receptor to promote cyclic AMP-dependent cystic fibrosis transmembrane conductance regulator (CFTR) activation and Cl− secretion (6Huang P. Lazarowski E.R. Tarran R. Milgram S.L. Boucher R.C. Stutts M.J. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 14120-14125Crossref PubMed Scopus (177) Google Scholar, 7Cobb B.R. Ruiz F. King C.M. Fortenberry J. Greer H. Kovacs T. Sorscher E.J. Clancy J.P. Am. J. Physiol. 2002; 282: L12-L25Crossref PubMed Google Scholar). Functional and biochemical evidence indicates that release and subsequent metabolism of ATP on the airway surface contribute to P2Y2 and A2b receptor-regulated electrolyte transport and airway surface liquid (ASL) volume homeostasis (8Lazarowski E.R. Tarran R. Grubb B.R. van Heusden C.A. Okada S. Boucher R.C. J. Biol. Chem. 2004; 279: 36855-36864Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 9Tarran R. Button B. Picher M. Paradiso A.M. Ribeiro C.M. Lazarowski E.R. Zhang L. Collins P.L. Pickles R.J. Fredburg J.J. Boucher R.C. J. Biol. Chem. 2005; 280: 35751-35759Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar). Whereas the roles of ATP and ADO in modulating mucociliary clearance and associated airway epithelial activities have been intensively investigated, it is largely unknown how ATP concentrations in the thin (∼7 μm) periciliary liquid lining airway surfaces are regulated. Airway epithelia release ATP into ASL basally (10Lazarowski E.R. Boucher R.C. Harden T.K. J. Biol. Chem. 2000; 275: 31061-31068Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar, 11Donaldson S.H. Lazarowski E.R. Picher M. Knowles M.R. Stutts M.J. Boucher R.C. Mol. Med. 2000; 6: 969-982Crossref PubMed Google Scholar) and in response to mechanical stresses (12Grygorczyk R. Hanrahan J.W. Am. J. Physiol. 1997; 272: C1058-C1066Crossref PubMed Google Scholar, 13Guyot A. Hanrahan J.W. J. Physiol. (Lond.). 2002; 545: 199-206Crossref Scopus (56) Google Scholar, 14Rich P.B. Douillet C.D. Mahler S.A. Husain S.A. Boucher R.C. J. Trauma. 2003; 55: 290-297Crossref PubMed Scopus (53) Google Scholar). ATP concentrations measured in media covering primary or immortalized airway cells (<100 nm, (12Grygorczyk R. Hanrahan J.W. Am. J. Physiol. 1997; 272: C1058-C1066Crossref PubMed Google Scholar, 15Okada S.F. O'Neal W.K. Huang P. Nicholas R.A. Ostrowski L.E. Craigen W.J. Lazarowski E.R. Boucher R.C. J. Gen. Physiol. 2004; 124: 513-526Crossref PubMed Scopus (157) Google Scholar)) are below EC50 values for ATP-promoted P2Y2-R-mediated intracellular calcium (Ca2+i) mobilization and Cl− secretion in airway epithelial cells (∼1 μm (16Mason S.J. Paradiso A.M. Boucher R.C. Br. J. Pharmacol. 1991; 103: 1649-1656Crossref PubMed Scopus (273) Google Scholar, 17Paradiso A.M. Ribeiro C.M. Boucher R.C. J. Gen. Physiol. 2001; 117: 53-67Crossref PubMed Scopus (97) Google Scholar)) or second messenger formation in P2Y2-R-transfected cells (230 nm (18Lazarowski E.R. Watt W.C. Stutts M.J. Boucher R.C. Harden T.K. Br. J. Pharmacol. 1995; 116: 1619-1627Crossref PubMed Scopus (222) Google Scholar)). However, functional studies suggested that autocrine activation of airway epithelial P2Y2-R occurs in response to physiological stimuli (9Tarran R. Button B. Picher M. Paradiso A.M. Ribeiro C.M. Lazarowski E.R. Zhang L. Collins P.L. Pickles R.J. Fredburg J.J. Boucher R.C. J. Biol. Chem. 2005; 280: 35751-35759Abstract Full Text Full Text PDF PubMed Scopus (293) Google Scholar), raising the possibility that ATP concentrations measured in the bulk extracellular medium, even with our micro-sampling technique from small volumes (12Grygorczyk R. Hanrahan J.W. Am. J. Physiol. 1997; 272: C1058-C1066Crossref PubMed Google Scholar, 15Okada S.F. O'Neal W.K. Huang P. Nicholas R.A. Ostrowski L.E. Craigen W.J. Lazarowski E.R. Boucher R.C. J. Gen. Physiol. 2004; 124: 513-526Crossref PubMed Scopus (157) Google Scholar), significantly underestimate the concentrations at the cell surface, as suspected for other cell types (19Joseph S.M. Buchakjian M.R. Dubyak G.R. J. Biol. Chem. 2003; 278: 23331-23342Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar, 20Hayashi S. Hazama A. Dutta A.K. Sabirov R.Z. Okada Y. Sci. STKE. 2004; 2004: pl14Crossref PubMed Scopus (55) Google Scholar). Several approaches to measure cell-surface ATP concentrations in situ have recently been developed. These include the use of an atomic force microscopy probe coated with myosin fragments (21Schneider S.W. Egan M.E. Jena B.P. Guggino W.B. Oberleithner H. Geibel J.P. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 12180-12185Crossref PubMed Scopus (68) Google Scholar), biosensors comprised of P2X receptor-expressing cells to measure ATP-activated P2X receptor-mediated changes in currents and Ca2+i (20Hayashi S. Hazama A. Dutta A.K. Sabirov R.Z. Okada Y. Sci. STKE. 2004; 2004: pl14Crossref PubMed Scopus (55) Google Scholar, 22Hazama A. Hayashi S. Okada Y. Pfluegers Arch. 1998; 437: 31-35Crossref PubMed Scopus (131) Google Scholar, 23Bell P.D. Lapointe J.Y. Sabirov R. Hayashi S. Peti-Peterdi J. Manabe K. Kovacs G. Okada Y. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4322-4327Crossref PubMed Scopus (250) Google Scholar), luciferin fluorescence to visualize ATP release from single cells (24Sorensen C.E. Novak I. J. Biol. Chem. 2001; 276: 32925-32932Abstract Full Text Full Text PDF PubMed Scopus (157) Google Scholar), and a chimeric luciferase with a leader sequence and a glycosylphosphatidylinositol (GPI) anchor expressed at the plasma membrane of cells (25Pellegatti P. Falzoni S. Pinton P. Rizzuto R. Di Virgilio F. Mol. Biol. Cell. 2005; 16: 3659-3665Crossref PubMed Scopus (236) Google Scholar). The ATP sensitivity of most of these methods is from semiquantitative to the micromolar range, suitable for the studies of ATP release from secretory cells, whereas nanomolar range sensitivity might be necessary for studies of airway epithelial ATP release. Joseph et al. utilized a recombinant luciferase fused to the IgG-binding domain of Staphylococcus aureus protein A (SPA-luc) to position luciferase on the cell surface (19Joseph S.M. Buchakjian M.R. Dubyak G.R. J. Biol. Chem. 2003; 278: 23331-23342Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). SPA-luc was attached to 1321N1 astrocytoma cells via antibodies against CD14 heterogeneously expressed in these cells and exhibited sensitivity for ATP in the 10-10,000 nm range. Stimulation of 1321N1 cells with thrombin resulted in enhanced release of ATP, which reached submicromolar levels as assessed by the cell-attached SPA-luc. In contrast, thrombin-induced changes in ATP concentrations in the bulk extracellular compartment as measured by soluble luciferase were negligible (19Joseph S.M. Buchakjian M.R. Dubyak G.R. J. Biol. Chem. 2003; 278: 23331-23342Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar). In the present study, we examined the physiological regulation of ATP concentrations in the micro-environment at the airway luminal surface. A well differentiated (WD) primary human bronchial epithelial (HBE) culture system was utilized as a model for native human airway epithelia. SPA-luc (19Joseph S.M. Buchakjian M.R. Dubyak G.R. J. Biol. Chem. 2003; 278: 23331-23342Abstract Full Text Full Text PDF PubMed Scopus (182) Google Scholar) was re-engineered and purified to exhibit 100-fold increase in luciferase activity. Real-time measurements of ATP concentration in the thin film of ASL on HBE cells were performed with cell-attached SPA-luc under resting and luminal hypotonic challenge conditions. Results from this technique were compared with those obtained with real-time measurements by luciferase dissolved in ASL and with pipette-sampling and off-line luminometry approaches (8Lazarowski E.R. Tarran R. Grubb B.R. van Heusden C.A. Okada S. Boucher R.C. J. Biol. Chem. 2004; 279: 36855-36864Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 15Okada S.F. O'Neal W.K. Huang P. Nicholas R.A. Ostrowski L.E. Craigen W.J. Lazarowski E.R. Boucher R.C. J. Gen. Physiol. 2004; 124: 513-526Crossref PubMed Scopus (157) Google Scholar). The physiological role for released ATP in airway epithelial volume regulation in response to hypotonic challenge was also investigated. Cell Culture—WD primary HBE cultures were established from surgical specimens of main stem or lobar bronchi from healthy or cystic fibrosis (CF) donors on 12 or 6.5 mm diameter Transwell supports (Corning) as described (26Fulcher M.L. Gabriel S. Burns K.A. Yankaskas J.R. Randell S.H. Methods Mol. Med. 2005; 107: 183-206PubMed Google Scholar). Cultures typically became fully differentiated in 20-30 days. Purification of SPA-luc—pMALU5, a cDNA construct encoding SPA-luc, was kindly provided by Dr. George Dubyak (Case Western Reserve University). The SPA-luc sequence was amplified from pMALU5 by PCR with up and down primers harboring at their 5′ ends BamHI and KpnI restriction sites, respectively. The PCR product was digested with BamHI and KpnI and ligated into similarly digested pT7-HTb, thus introducing a His6 tag and tobacco etch virus cleavage site at the N terminus of SPA-luc. pT7-HTb/SPA-luc was transformed into Escherichia coli (BL21-*; Invitrogen) and SPA-luc was expressed by overnight induction with 0.2 mm isopropyl β-d-thiogalactopyranoside at 20 °C. The cells were lysed by an Emulsiflex homogenizer (Avestin, Ontario, Canada). Soluble proteins were precipitated with 55% ammonium sulfate, resuspended, and purified by two passages over a Ni2+-chelating column (GE Healthcare). The His6 tag was cleaved from the fusion protein using His6-tobacco etch virus protease (3 h at 30 °C), and the digestion mixture was run over a Ni2+-chelating column a third time and SPA-luc eluted in the flow-through. The protein concentration and luciferase activity of fractions obtained through the purification process were assayed using the BCA protein assay kit (Pierce) and the LB953 AutoLumat luminometer (15Okada S.F. O'Neal W.K. Huang P. Nicholas R.A. Ostrowski L.E. Craigen W.J. Lazarowski E.R. Boucher R.C. J. Gen. Physiol. 2004; 124: 513-526Crossref PubMed Scopus (157) Google Scholar), respectively. SPA Binding Antigens—SPA-luc binding to endogenous antigens on the apical surface of isolated WD HBE cells was investigated with a Leica SP2 AOBS confocal microscope as described previously (27Kreda S.M. Mall M. Mengos A. Rochelle L. Yankaskas J. Riordan J.R. Boucher R.C. Mol. Biol. Cell. 2005; 16: 2154-2167Crossref PubMed Scopus (222) Google Scholar). Immunostaining was performed on fixed, non-permeabilized cultures with primary antibodies against keratan sulfate (KS) or MUC1 followed by fluorescein isothiocyatate-labeled secondary antibodies, or with fluorescein isothiocyatate-labeled wheat germ agglutinin (WGA). Cell ELISA—The concentrations of the selected antibodies/lectins required to achieve maximal SPA binding to WD HBE culture surfaces were optimized by quantitating cell-surface-bound protein A-horseradish peroxidase conjugates (SPA-HRP) by a cell ELISA. The apical surface of WD HBE cultures was first incubated with 50 μl of phosphate-buffered saline (PBS) containing 1% bovine serum albumin (BSA) for 30 min. The solution was replaced with 50 μl of PBS/BSA containing various concentrations of an anti-MUC1 antibody (mouse IgG2b, ab8323, Abcam, Cambridge, MA), an anti-KS antibody (mouse IgG2b, Chemicon, Temecula, CA), or biotinylated WGA (Vector Laboratories, Burlingame, CA). In the latter instance, an additional incubation (1 h) with 2 μg/ml anti-biotin antibody (rabbit, Bethyl Laboratories, Montgomery, TX) in 50 μl of PBS/BSA was performed. The apical surface of the cultures was washed three times with PBS (10 min/wash) and incubated with 1 μg/ml SPA-HRP (Pierce) in 50 μl of PBS for 1 h. The basolateral surfaces of cultures were bathed in 1 ml of Hank's balanced salt solution buffered with 10 mm HEPES (HBSS/HEPES). All incubation procedures were performed at 4 °C. After three apical washes with PBS (10 min each), an ELISA with o-phenylenediamine was performed according to the manufacturer's instruction (Pierce) and the intensity of the colorimetric reaction assessed at 490 nm by a microplate spectrophotometer (Molecular Devices, Sunnyvale, CA) with SOFTmax Pro software. ATP Sensitivity of Cell-attached SPA-luc—Attachment of SPA-luc was carried out using the optimal antibody/lectin concentrations determined as above. Briefly, after blocking with PBS/BSA for 30 min, the apical surface of WD HBE cultures (on 12-mm Transwells) was incubated with 20 μg/ml anti-MUC1 antibody, 10 μg/ml anti-KS antibody, or 4 μg/ml biotinylated WGA followed by 2 μg/ml anti-biotin antibody for 1 h at 4°C. After three washes with PBS, the apical HBE surface was incubated with 0.5 mg/ml purified SPA-luc for 1 h at 4°C, washed with PBS, and the mucosal surface replenished with a specified volume of HBSS/HEPES. SPA-luc-attached cultures were equilibrated at room temperature for 30 min to re-establish basal ATP concentrations. Cultures were subsequently transferred to a 35 mm polyacetal assay chamber, which contained 1 ml of HBSS/HEPES in a basolateral reservoir, and placed gently in a Turner TD-20/20 luminometer (Turner Biosystems, Sunnyvale, CA). Luciferin (150 μm) was added to the ASL and arbitrary light units (ALU) integrated for 10 s and recorded. Known concentrations of ATP were added in a stepwise manner (e.g. 1 nm added twice, 10 nm added twice, then 100 nm added twice). The ATP sensitivity of SPA-luc dissolved in bulk solution (100 μl of HBSS/HEPES) was obtained in parallel, using a Transwell without cells. ATP Concentration and Release Rates on Resting HBE Cells— For real-time ATP measurements, HBE cells, with either cell-attached SPA-luc or soluble luciferase (Sigma L9506; 0.5-2.0 μg/culture), were placed in the Turner TD-20/20 luminometer and luciferin (150 μm) added to the mucosal liquid. Luminescence was measured every minute until the ALU values (i.e. basal ATP concentration) reached steady state (i.e. less than ± 10% variability over 10 min). At the end of each assay, an ATP-luminescence relationship was obtained as above. For off-line ATP measurements, the ASL on resting WD HBE cultures was sampled by gently pipetting (from volume >100 μl) or micro-sampling (from volume ≤100 μl) and ATP concentrations measured as described previously (8Lazarowski E.R. Tarran R. Grubb B.R. van Heusden C.A. Okada S. Boucher R.C. J. Biol. Chem. 2004; 279: 36855-36864Abstract Full Text Full Text PDF PubMed Scopus (198) Google Scholar, 15Okada S.F. O'Neal W.K. Huang P. Nicholas R.A. Ostrowski L.E. Craigen W.J. Lazarowski E.R. Boucher R.C. J. Gen. Physiol. 2004; 124: 513-526Crossref PubMed Scopus (157) Google Scholar). ATP release rates from resting cells were determined by monitoring ATP accumulation in real-time under conditions resulting in maximal inhibition of cell-surface nucleotidase activities. The nucleotidase inhibitor mixture contained 300 μm β,γ-methylene-ATP, 30 μm ebselen, and 10 mm levamisole, to inhibit ecto-nucleotide pyrophosphatase/phosphodiesterases (eNPPs) (28Joseph S.M. Pifer M.A. Przybylski R.J. Dubyak G.R. Br. J. Pharmacol. 2004; 142: 1002-1014Crossref PubMed Scopus (56) Google Scholar), ecto-nucleotide triphosphate diphosphohydrolases (eNTPDases) (29Furstenau C.R. Spier A.P. Rucker B. Luisa Berti S. Battastini A.M. Sarkis J.J. Chem. Biol. Interact. 2004; 148: 93-99Crossref PubMed Scopus (15) Google Scholar, 30Picher M. Button B. Boucher R.C. Pediatr. Pulmon. 2005; 28: 274Google Scholar), and nonspecific alkaline phosphatases (31Picher M. Burch L.H. Hirsh A.J. Spychala J. Boucher R.C. J. Biol. Chem. 2003; 278: 13468-13479Abstract Full Text Full Text PDF PubMed Scopus (163) Google Scholar), respectively. After recording the basal ATP concentration as described above, the inhibitor mixture was added to the mucosal liquid and ATP concentrations measured every minute. The efficacy of the nucleotidase inhibitors was tested on separate HBE cultures in the presence of 500 nm ATP as described previously (10Lazarowski E.R. Boucher R.C. Harden T.K. J. Biol. Chem. 2000; 275: 31061-31068Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). Hypotonic Challenge—After recording basal ATP concentrations in real-time, H2O (half the initial volume, containing 1 mm CaCl2 and 1 mm MgCl2) was added gently to the ASL. Luminescence was subsequently recorded for 5 min: every 0.2 s for the first minute, then every 10 s for the next 4 min. For an isosmotic control, the same volume of mannitol solution (300 mm, containing 1 mm CaCl2 and 1 mm MgCl2) was added as above. At the end of each assay, an ATP-luminescence relationship was obtained as above. Changes in luciferin/luciferase activity generated by addition of hypotonic or isosmotic solutions were tested on wells without cells containing ATP standards and confirmed to be <10%. ATP concentrations were alternatively measured by off-line luminometry of sampled ASL as described previously (15Okada S.F. O'Neal W.K. Huang P. Nicholas R.A. Ostrowski L.E. Craigen W.J. Lazarowski E.R. Boucher R.C. J. Gen. Physiol. 2004; 124: 513-526Crossref PubMed Scopus (157) Google Scholar). The initial rate of ATP release following hypotonic challenge was assessed in the presence of the nucleotidase inhibitor mixture in 50 μl of mucosal liquid with soluble luciferase. Cell Volume Regulation—Changes in cell height were used as a parameter to estimate cell volume changes following a hypotonic challenge, as described previously (15Okada S.F. O'Neal W.K. Huang P. Nicholas R.A. Ostrowski L.E. Craigen W.J. Lazarowski E.R. Boucher R.C. J. Gen. Physiol. 2004; 124: 513-526Crossref PubMed Scopus (157) Google Scholar). In brief, WD HBE cells were loaded with 5 μm calcein-AM (Molecular Probes, Eugene, Oregon) for 30 min at 37 °C. The apical surface of cultures was equilibrated for 10 min with 33 μl of HBSS/HEPES before study. The cultures were then positioned on a Zeiss 510 confocal microscope. H2O (17 μl) was added to apical surface to generate a 200 mosm solution, and xz-scanning images were obtained every second for initial 15 s, then every 5 s for next 75 s. Experiments were also performed on cultures pretreated with apyrase (ATP diphosphohydrolase, 10 units/ml) for 5 min, 8-(p-sulfophenyl)theophylline (8-SPT, an ADO receptor antagonist, 100 μm) for 30 min or ATP, UTP, or ATPγS (100 μm) on the luminal surface. Ca2+i Dependence of Hypotonicity-induced ATP Release— BAPTA-AM loading (100 μm for 30 min at 37 °C) of WD HBE cells and Ca2+i mobilization studies with Fura-2-AM were performed as described previously (32Ribeiro C.M. Paradiso A.M. Carew M.A. Shears S.B. Boucher R.C. J. Biol. Chem. 2005; 280: 10202-10209Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). ATP release from BAPTA- and vehicle-loaded cultures was measured with soluble luciferase in 50 μl of mucosal liquid. Statistical Analysis—All experiments were performed on cultures established from at least three donors. Data were expressed as mean values ± S.E. of observations. Where appropriate, data were analyzed by Student's t test or analysis of variance with GraphPad Prism software. Unpaired t tests or analysis of variance were used to obtain the p value for differences between means; a repeated-measures analysis of variance was used when comparing series of ATP values at each time point among different conditions. Statistical significance was defined as p < 0.05. Optimization of SPA-luc Attachment to HBE Cell Surfaces— Partially purified His6-SPA-luc exhibited a specific activity of 1.6 × 105 ALU/μg when assessed in the presence of 1 μm ATP. This specific activity was 100-fold higher than that initially detected in E. coli homogenate and represented 67% of the initial activity in the crude supernatant starting material (Fig. 1 and Table 1). Indeed, the specific activity of purified SPA-luc was similar to that of commercially available luciferase (Sigma catalog number L9506, 1.3 × 105 ALU/μg).TABLE 1Purification of SPA-luc Soluble proteins from SPA-luc transfected E.coli lysates (“Crude sup”) were precipitated with 55% ammonium sulfate (“AmSO4 55% pellet”), resuspended, and purified by three passages over a Ni2+-chelating column. 1-4 correspond to lanes 1-4 in Fig. 1.1: crude sup2: AmSO455% pellet3: 1st column fractions 13–314: 3rd column flow-throughVolume, ml200404.53.9Protein concentration, mg/ml2060197.1Activity, ALU·μl−13.18 × 1043.56 × 1041.06 × 1061.09 × 106Specific activity, ALU·μg−11.59 × 1035.93 × 1025.58 × 1041.60 × 105Relative total activity, % recovery10022.475.066.7 Open table in a new tab Anti-KS and anti-MUC1 antibodies, as well as WGA, bound avidly to the apical surface of WD HBE cultures as revealed by confocal microscopy (Figs. 2, A-C) suggesting that these reagents would bind SPA-luc to endogenous HBE cell-surface antigens. Subsequently, the concentration of the antibody or lectin required for attachment of maximal amounts of SPA to HBE cell surfaces was determined by a cell ELISA as 10, 20, and 4 μg/ml for anti-KS antibody, anti-MUC1 antibody, and biotinylated WGA, respectively (Fig. 2D). Next, we examined the luminescence generated by cell-attached SPA-luc in response to exogenously added ATP. Luminescence was linear in the range of 10-10,000 nm exogenous ATP and was similar for the three different methods of anchoring SPA-luc to the cell surface (Fig. 3A). Nonspecific binding of SPA-luc occurred but generated ∼20-fold less luminescence than SPA-luc attachment via antibodies or lectin (Fig. 3A). Notably, the ATP-luminescence curves were flatter in the 0-10 nm range, which likely reflected the contribution of endogenous ATP release by HBE cells (compare the curves with those made in wells without cells (Fig. 3B)). The ATP sensitivity of SPA-luc complexed to HBE cells was compared with that of known concentrations of SPA-luc in solution. The ATP-luminescence relationship of SPA-luc bound to the cells (Fig. 3A) aligned best with that of 10 μg/ml SPA-luc dissolved in 100 μl of buffer (Fig. 3B). Thus, bound SPA-luc was equivalent to ∼1 μg/culture or ∼0.88 μg/cm2 of culture surface. Basal ATP Concentrations—To accurately assess basal ATP concentrations on HBE cell surfaces, we investigated the effect of ASL volume on ATP concentrations on resting cells utilizing a spectrum of techniques. The ATP concentrations in varied ASL volumes on resting WD HBE cultures were accordingly measured by: 1) pipette- or micro-sampling and off-line luminometry, 2) real-time luminometry with soluble luciferase (Sigma), and 3) real-time luminometry with cell-attached SPA-luc. Basal ATP concentrations were similar (1-10 nm) over a range of ASL volumes as measured by the three techniques (Fig. 4). However, micro-sampling generated more variability than real-time luminometry, likely reflecting shear stress artifacts induced by the aspiration of liquid. Basal ATP Release Rates—ATP concentrations on resting HBE cells reflect the balance between basal ATP release and hydrolysis (10Lazarowski E.R. Boucher R.C. Harden T.K. J. Biol. Chem. 2000; 275: 31061-31068Abstract Full Text Full Text PDF PubMed Scopus (277) Google Scholar). For example, ATP (500 nm) added to 300 μl of ASL on HBE cells exhibited a half-life of ∼0.5 min, illustrating the activity of ecto-nucleotidases on HBE cell surfaces (Fig. 5A). Addition of the nucleotidase inhibitors, β,γ-methylene-ATP, ebselen, or levamisole, each of which blocks a different ecto-nucleotidase enzyme family expressed on WD HBE surface, partially inhibited ATP hydrolysis (Fig. 5A). When combined together, the three inhibitors exhibited an additive effect, producing a >50-fold reduction in the rate of ATP hydrolysis. Therefore, the combination of the three enzyme inhibitors was utilized to assess ATP release rates from resting cells. Under conditions where nucleotidases were maximally inhibited, endogenous ATP accumulation was measured by cell-attached SPA-luc and by luciferase dissolved in the mucosal liquid. Addition of the inhibitor mixture to the ASL resulted in ATP accumulation (Fig. 5B). Changes in ATP concentrations were negligible when the inhibitor mixture was add" @default.
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