Nucleotide Release Provides a Mechanism for Airway Surface Liquid Homeostasis

Abstract

Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca2+ -and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A2b adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N6-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolndogenayers that eously express a luminal A2b adenosine receptor, we found that basal as well asforskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A2b receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis. Nucleotides within the airway surface liquid (ASL) regulate airway epithelial ion transport rates by Ca2+ -and protein kinase C-dependent mechanisms via activation of specific P2Y receptors. Extracellular adenine nucleotides also serve as precursors for adenosine, which promotes cyclic AMP-mediated activation of the cystic fibrosis transmembrane regulator chloride channel via A2b adenosine receptors. A biological role for extracellular ATP in ASL volume homeostasis has been suggested by the demonstration of regulated ATP release from airway epithelia. However, nucleotide hydrolysis at the airway surface makes it difficult to assess the magnitude of ATP release and the relative abundance of adenyl purines and, hence, to define their biological functions. We have combined ASL microsampling and high performance liquid chromatography analysis of fluorescent 1,N6-ethenoadenine derivatives to measure adenyl purines in ASL. We found that adenosine, AMP, and ADP accumulated in high concentrations relative to ATP within the ASL covering polarized primary human normal or cystic fibrosis airway epithelial cells. By using immortalized epithelial cell monolndogenayers that eously express a luminal A2b adenosine receptor, we found that basal as well asforskolin-promoted cyclic AMP production was reduced by exogenous adenosine deaminase, suggesting that A2b receptors sense endogenous adenosine within the ASL. The physiological role of adenosine was further established by illustrating that adenosine removal or inhibition of adenosine receptors in primary cultures impaired ASL volume regulation. Our data reveal a complex pattern of nucleotides/nucleosides in ASL under resting conditions and suggest that adenosine may play a key role in regulating ASL volume homeostasis. Airways continuously remove noxious materials through a mucus clearance process that requires a complex regulation of airway surface liquid (ASL) 1The abbreviations used are: ASL, airway surface liquid; MCC, mucociliary clearance; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; HBSS, Hanks' balanced salt solution; HPLC, high performance liquid chromatography; ϵ, etheno; ADO, adenosine; HNE, human nasal epithelial cells; HBE, human bronchial epithelial; 8-SPT, 8-(-sulfophenyl)theophylline; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid.1The abbreviations used are: ASL, airway surface liquid; MCC, mucociliary clearance; CF, cystic fibrosis; CFTR, cystic fibrosis transmembrane conductance regulator; HBSS, Hanks' balanced salt solution; HPLC, high performance liquid chromatography; ϵ, etheno; ADO, adenosine; HNE, human nasal epithelial cells; HBE, human bronchial epithelial; 8-SPT, 8-(-sulfophenyl)theophylline; TES, 2-{[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]amino}ethanesulfonic acid. volume by active ion transport and coordinated ciliary beating. In normal airways, the cystic fibrosis transmembrane conductance regulator (CFTR) protein mediates liquid balance on airway surfaces by regulating Na+ absorption and by acting as a cyclic AMP-regulated Cl– channel. Proper regulation of salt and water movement across epithelial cell surfaces is required in both ASL compartments: (i) sufficient volume in the periciliary liquid ensures efficient cilia movement; and (ii) sufficient volume optimizes the viscoelastic properties of the mucus layer for transport (1Knowles M.R. Boucher R.C. J. Clin. Investig. 2002; 109: 571-577Crossref PubMed Scopus (957) Google Scholar). Defective CFTR activity causes the syndrome of cystic fibrosis (CF), which is characterized by accelerated Na+-dependent volume absorption and failure to secrete Cl–, leading to ASL volume depletion (2Boucher R.C. Adv. Drug Delivery Rev. 2002; 54: 1359-1371Crossref PubMed Scopus (190) Google Scholar). An alternative Ca2+-regulated Cl– channel that is functional in CF airway epithelial cells is also present on the mucosal surface but is relatively inactive in nonstimulated cells (3Gabriel S.E. Fuller C.M. Current Topics in Membranes: Calcium-activated Chloride Channels. Academic Press, San Diego2002: 193-207Google Scholar). The expression of purinergic receptors in airway epithelia that couple via different mechanisms to salt (and water) transport suggests a role of purinergic agonists in the autocrine regulation of ASL volume and mucociliary clearance (MCC). Indeed, the accumulation of physiologically active concentrations of ATP in human airway secretions in vivo (4Donaldson 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 the recognition that airway epithelial cells release ATP constitutively in vitro (4Donaldson 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, 5Lazarowski E.R. Boucher R.C. Harden T.K. J. Biol. Chem. 2000; 275: 31061-31068Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, 6Lazarowski E.R. Boucher R.C. Harden T.K. Drug Dev. Res. 2001; 53: 66-71Crossref Scopus (9) Google Scholar) suggest that release and metabolism of nucleotides may control the basal activity of ion transport and, hence, ASL volume homeostasis. Functional data suggest that ATP mediates acute responses via P2Y2 receptor stimulation (7Homolya L. Steinberg A.D. Boucher R.C. J. Cell Biol. 2000; 150: 1349-1360Crossref PubMed Scopus (223) Google Scholar, 8Paradiso A.M. Ribeiro C.M.P. Boucher R.C. Pediatr. Pulmonol. 2001; and that ATP metabolism a of adenosine for stimulation of the A2b adenosine receptor Lazarowski E.R. Boucher R.C. Stutts M.J. 2001; PubMed Scopus Google Scholar). However, of the and removal of purinergic it has been to the relative concentrations of agonists for the purinergic receptors in airway epithelia and the relative of the A2b and P2Y receptors to the control of ASL volume and By the for the of adenyl purines PubMed Scopus Google we to measure the of adenine nucleotides and adenosine in ASL. We cell to the of of ATP release and of nucleotides in cell By using well primary human airway epithelia as a of the epithelia in we ASL volume regulation is by ASL adenosine in autocrine and the of adenyl purines in ASL on CFTR By using cells as a of a polarized epithelial we cell surface for nucleotide ASL adenosine control cyclic AMP and cyclic AMP and CFTR promotes nucleotide Cell and human nasal epithelial and human bronchial epithelial cultures from CF or by the of on under conditions and as as Boucher R.C. J. Clin. Investig. 2000; PubMed Scopus Google Scholar). airway epithelial cells as polarized as Lazarowski E.R. Boucher R.C. Stutts M.J. 2001; PubMed Scopus Google Scholar). by the to the luminal surface of or of Hanks' balanced salt for and microsampling the for and for nucleotide analysis or at at in a the cells and of and was of was used to the of a to the of the of luminal surface as S.H. Boucher R.C. Full Text Full Text PDF PubMed Scopus Google Scholar). A of was from and as has been in E.R. Boucher R.C. Harden T.K. J. Biol. Chem. 2000; 275: 31061-31068Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). a was to a and the volume was to The was to a the of was and ATP in was and of and have and the by PubMed Scopus Google to conditions as under In a of from or from for at in the of and in a volume of to of and by within to in a and of a to and at in the A was a The from to the at for The was for in and to the conditions for of as and cyclic AMP of AMP from was by using a from to for and from to and AMP at and In using and a to a was and the ion under conditions to of from and from and through the and E.R. Boucher R.C. Harden T.K. L. and Scholar). of was in of to the mucosal surface of The volume was and the cultures at in a the cultures to the of the a of Boucher R.C. J. 2001; PubMed Scopus Google Scholar, Boucher R.C. Mol. Med. 2002; Google Scholar). was to of the ASL and cultures as Boucher R.C. J. 2001; PubMed Scopus Google Scholar, Boucher R.C. Mol. Med. 2002; Google Scholar). and ATP from AMP, adenosine, cyclic AMP, and adenosine from and from and from of the was and by using from concentrations of AMP, and adenosine, which and in at the and at the of to was used for the by and at for the of adenyl purines as fluorescent PubMed Scopus Google Scholar). and ATP and adenyl purines within the ASL, we the conditions by in and fluorescent the of of of ATP was within at in the of as by the in of that was by of fluorescent of to and of fluorescent of or ATP The at at or in at conditions for by ion and to a of An of a in the of adenosine, AMP, and ATP is in The of the as a of ATP is in and adenosine and AMP, and cyclic AMP By we the fluorescent pattern from the resting primary airway epithelial cells as that have been used for nucleotide (4Donaldson 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, 5Lazarowski E.R. Boucher R.C. Harden T.K. J. Biol. Chem. 2000; 275: 31061-31068Abstract Full Text Full Text PDF PubMed Scopus (275) Google but in to that in cultures of nasal epithelial cells from normal for the of of to the mucosal to the cells to from that in ATP release E.R. Boucher R.C. Harden T.K. Mol. PubMed Scopus Google Scholar). A was from the mucosal and to and A is in and further the of the fluorescent to to a depletion of as and E.R. A.M. Harden T.K. Boucher R.C. PubMed Scopus Google Scholar) in of the to and of of the or the as and and that of as data primary cultures of cells and the airway epithelial cell Lazarowski E.R. Boucher R.C. Stutts M.J. 2001; PubMed Scopus Google Scholar). in of in that the in the extracellular of resting airway epithelial cells endogenous nucleotides and their from cultures of well primary nasal and bronchial epithelial cells. ATP concentrations in the in the mucosal of control and suggesting that epithelia of ATP on the luminal The that ATP was the in and cells suggested that the ATP metabolism is to ATP release on both cell However, and in M. Boucher R.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. Boucher R.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) rates of hydrolysis of and AMP on bronchial cells relative to nasal bronchial and nasal cells accumulation also suggests that luminal accumulation is impaired in CF cells. in of the of CF of ATP release rates and nucleotide accumulation on nasal cultures and in nasal secretions from CF (4Donaldson 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, Lazarowski E.R. Boucher R.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). analysis of from to cultures from normal and CF also in the accumulation of mucosal purines In in accumulation of adenyl was cells from suggesting that release of ATP to the of the airway epithelia is impaired in airway was at the of adenosine and AMP on CF and cells relative to control suggesting that activity is in normal cells. ATP in CF relative to control ATP the normal and CF bronchial cultures and of the adenyl by ADP and AMP and adenosine further the of nucleotide and at from to a on and cells A and a to the of also cells a epithelial cell as a polarized Lazarowski E.R. Boucher R.C. Stutts M.J. 2001; PubMed Scopus Google Scholar, J. Cell Mol. Biol. PubMed Scopus Google Scholar, A.M. M. J. Biol. PubMed Scopus Google Scholar). the of airway epithelia to ATP release (4Donaldson 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, 6Lazarowski E.R. 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The data also the and of the nucleotide hydrolysis on the epithelial cell which ATP to AMP and to adenosine, and the data also suggest in nucleotide release in the the epithelial cells. accelerated metabolism in cells for the of purines in of and to and the present in the at was by was mucosal at rates on both surfaces of cells that the of in the of resting cell was a of clearance but or nucleotide release through the may the nucleotide in the physiologically ASL volume the and volume of ASL airway epithelial surfaces is and airway epithelial cells to luminal a in the concentrations of nucleotides in ASL that in vivo is to the airway surface liquid ATP we a to measure resting of nucleotides and adenosine in primary cultures ASL that in we the clearance rates of of and we a to ASL on resting cells for cultures of cells of luminal for by the of for the the of the was in the In at to assess ATP concentrations by the of the the of ATP in the volume in the that extracellular ATP hydrolysis was balanced by ATP the of basal ATP release E.R. Boucher R.C. Harden T.K. J. Biol. Chem. 2000; 275: 31061-31068Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar) was as to of mucosal ATP release rates covering airway epithelial cells (4Donaldson 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). also relatively on the mucosal cell surface ASL that was of within the ASL cells suggested the of on the mucosal surface of cells. of the to the of cells in the extracellular suggesting that mucosal has By that mucosal ATP and adenosine under we endogenous adenyl purines in to ATP in physiological ASL volume covering resting cells. nasal and bronchial epithelial cells and cells for in of mucosal HBSS, and from and that the of ATP and adenosine E.R. Boucher R.C. Harden T.K. J. Biol. Chem. 2000; 275: 31061-31068Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar, M. J. Boucher R.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M. Boucher Drug Dev. Res. 2001; Scopus Google analysis of nucleotide hydrolysis on cultures that in of ASL, and AMP accumulation of purines in volume ASL a pattern to that in ATP was the in cell in ASL and in control ASL. in accumulation control and CF cells In the relative of in ASL volume also was that in However, of purines in of ASL relative to and suggesting that in volume ASL in the physiological of the adenosine and the in and in adenosine accumulation on control and CF illustrating high on the mucosal surface of cells. suggest that accumulation of mucosal purines a in which the rates of hydrolysis by rates of of nucleotides/nucleosides in of ASL the in ASL under physiologically and concentrations at the cell luminal adenosine concentrations on resting cells under volume ASL conditions may in a to adenosine receptors. the role of A2b adenosine receptors in the control of Cl– and ASL volume the of to the endogenous luminal adenosine was of receptor to in cyclic and endogenous adenosine A2b ASL volume regulation. We used airway epithelial cell which express a well characterized luminal A2b receptor to and Lazarowski E.R. Boucher R.C. Stutts M.J. 2001; PubMed Scopus Google Scholar, C.M. J. J. 2002; PubMed Google to the of cyclic AMP as a of endogenous luminal we have that exogenous adenosine cyclic AMP in and airway epithelial cells Lazarowski E.R. Boucher R.C. Stutts M.J. 2001; PubMed Scopus Google Scholar, E.R. L. Harden T.K. Boucher R.C. J. PubMed Scopus Google Scholar) and that of cyclic AMP in resting airway epithelial cells in the of cyclic E.R. L. Harden T.K. Boucher R.C. J. PubMed Scopus Google Scholar). was a accumulation of AMP in cells However, of the to resting cells in accumulation of of cyclic AMP cells in cyclic AMP for and receptors J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, PubMed Scopus Google the of adenosine was by We both basal and of cyclic AMP in cells activation of the A2b receptor by endogenous adenosine within the ASL. In adenosine a but of basal cyclic AMP suggesting that endogenous adenosine has activation to the A2b receptor on cyclic AMP in the of also reduced cyclic AMP in to further suggesting that the of on the the A2b is that in the of luminal adenosine or adenosine nucleotides data suggest that extracellular adenosine concentrations regulated by cyclic AMP-mediated but a process in resting cells from cyclic AMP CFTR Cl– channel activity a role in epithelial transport and ASL By that endogenous adenosine basal of cyclic AMP in cell we used well cells from normal to ASL adenosine ASL airway epithelia regulate the ASL to the of cilia (2Boucher R.C. Adv. Drug Delivery Rev. 2002; 54: 1359-1371Crossref PubMed Scopus (190) Google Scholar, Boucher R.C. Mol. Med. 2002; Google Scholar). we to the adenosine is a key regulator in physiological ASL of adenosine deaminase, or the adenosine receptor 8-SPT, was to the mucosal The liquid was to Boucher R.C. Mol. Med. 2002; Google Scholar) and in ASL by that ASL in in control cells but to in cells in the of or adenosine suggest that endogenous adenosine mediates physiological ASL volume in the that cyclic CFTR chloride regulate periciliary liquid volume by Cl– ion (and water) and Na+-dependent volume absorption (2Boucher R.C. Adv. Drug Delivery Rev. 2002; 54: 1359-1371Crossref PubMed Scopus (190) Google Scholar). By of airway epithelial cultures physiologically of ASL and we have that physiologically of nucleotides and adenosine within the ASL of well primary and immortalized cultures of airway epithelial cells. Our that ATP and adenosine concentrations in ASL a epithelia a release of ATP that ATP which in a for of Indeed, the in that basal ATP and adenosine concentrations in ASL that metabolism However, the ATP is and a the purines that within the ASL covering resting airway epithelial cells. in the the and of adenosine and ATP on primary airway and cells suggests that ATP is the primary of extracellular is by functional and that but is on airway epithelial cells M. J. Boucher R.C. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar) and by cells illustrating that reduced ion transport that of CFTR, to under the control of the A2b receptor Lazarowski E.R. Boucher R.C. Stutts M.J. 2001; PubMed Scopus Google Scholar). of the is that of ATP concentrations may in of the magnitude of ATP is by by and M. J. PubMed Google Scholar, M.R. J. Biol. Chem. Full Text Full Text PDF Scopus Google ATP concentrations by to the cell surface by using a protein of the of protein A the ATP concentrations at the surface of cells found to at ATP concentrations in M. J. PubMed Google Scholar, M.R. J. Biol. Chem. Full Text Full Text PDF Scopus Google Scholar). ATP in their was by that ATP release and hydrolysis may The concentrations of ATP and adenosine within the ASL have physiological for airway Proper of cyclic and Ca2+ epithelial ion and water transport and cilia for efficient mucus cyclic A2b adenosine receptors and Ca2+ P2Y2 receptors by the the human airway is and to we that rates under autocrine control by extracellular Indeed, airway epithelial cells release nucleotides both constitutively and in to stimulation (4Donaldson 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, 5Lazarowski E.R. Boucher R.C. Harden T.K. J. Biol. 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PubMed Google Scholar) and may Cl– transport via protein kinase of CFTR activation J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar). airway epithelial cells to the P2Y2 receptor role and protein kinase C-dependent in airways, and as suggested by the of ATP release and P2Y2 ion transport responses in airway cultures to A.M. Ribeiro C.M.P. Boucher R.C. Pediatr. Pulmonol. 2001; Scholar). ATP accumulation in CF airway epithelia may for the of cyclic CFTR activity via P2Y2 Ca2+ Cl– channel Our data suggest that ASL adenosine may of the epithelial A2b receptor on resting cells. Our microsampling that and removal of adenosine in physiologically ASL in of adenosine at which for the A2b of adenosine and removal may airway as suggested by in nasal and bronchial epithelial cells The of mucosal adenosine accumulation was by that the basal CFTR Cl– channel activity of cells was to A2b receptor Lazarowski E.R. Boucher R.C. Stutts M.J. 2001; PubMed Scopus Google Scholar). is by present cells of endogenous adenosine in ASL, which basal and cyclic AMP accumulation is that cells as polarized well for the of ASL adenosine to cyclic AMP a difficult to primary epithelia. the in ASL volume in primary airway epithelia that endogenous adenosine in physiologically concentrations in ASL. The ASL adenosine the regulation of the ion transport Cl– which ASL a key of the mechanisms in the as is a activation of the A2b receptor by endogenous adenosine, receptors in resting airway epithelial The basal of ASL adenosine to and may sufficient to the A2b receptor However, from that cyclic AMP accumulation in airway epithelial cells was in the of adenosine E.R. L. Harden T.K. Boucher R.C. J. PubMed Scopus Google suggesting that the A2b receptor may by endogenous An by the present was that well primary epithelial cells accumulated of nucleotides on both luminal nucleotide accumulation was in cell in the mucosal by a of metabolism of extracellular purines at the surface of the of was that of mucosal and at rates on both cell However, well primary epithelia and immortalized cells for primary cultures of bronchial epithelial cells a cells resting on or of basal cells Boucher R.C. J. Clin. Investig. 2000; PubMed Scopus Google Scholar). In cell cultures the of Lazarowski E.R. Boucher R.C. Stutts M.J. 2001; PubMed Scopus Google Scholar). it is that the nucleotide in the of primary cultures nucleotide release by basal cells. suggests that ATP release through that to the cell cells to a of cells of the airway J. Google Scholar, C.M. J. PubMed Google Scholar, J. 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In present that purines in physiologically concentrations on and surfaces of resting Our that ATP release and metabolism within the ASL a primary for the accumulation of adenosine, which basal activity to the A2b receptor that CFTR ASL volume regulation. We to for primary cultures and and to for We Picher for and