Free Radical Biology & Medicine 41 (2006) 29–40www.elsevier.com/locate/freeradbiomed
Serial Review: Redox Signaling in Immune Function and Cellular Responses in Lung Injury and DiseasesSerial Review Editors: Victor Darley–Usmar, Lin Mantell
Purinergic signaling and kinase activation for survival in pulmonaryoxidative stress and disease☆
Shama Ahmad ⁎, Aftab Ahmad, Carl W. White
Department of Pediatrics, National Jewish Medical and Research Center, 1400 Jackson Street, Denver, CO 80206, USA
Received 11 November 2005; revised 27 February 2006; accepted 2 March 2006Available online 31 March 2006
Abbreviations: BPD, bronchopulmonary dysplasia; CFTR, cystic fibrosis transmembrane conductance regulator; FKBP12, FK506-binding protein; ERK,extracellular signal-regulated kinase; HLMVEC, human lung microvascular endothelial cells; HKII, hexokinase II; IP3, inositol trisphosphate; LDH, lactatedehydrogenase; MAPK, mitogen-activated protein kinases; MEK, mitogen-activated protein kinase kinase or ERK kinase; MDR, multidrug resistance; mTOR,mammalian target of rapamycin; PI3K, phosphatidylinositol 3-kinase; PLA2, phospholipase A2; PLD, phospholipase D; PLC, phospholipase C; PKC, protein kinaseC; P70S6K, ribosomal p70 S6 kinase; PGE2, prostaglandin E2; RTPCR, reverse-transcriptase polymerase chain reaction; ROS, reactive oxygen species; RVD,regulatory volume decrease; VDAC, voltage-dependent anion channel.☆ This article is part of a series of reviews on “Redox Signaling in Immune Function and Cellular Responses in Lung Injury and Diseases.” The full list of papers maybe found on the home page of the journal.⁎ Corresponding author. Fax: +1 303 398 1851.E-mail address: [email protected] (S. Ahmad).
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Introduction
ATP is the principal intracellular energy source. Recentattention has been focused on ATP's functions outside the cell.Cytosolic ATP and that packed into secretory granules andvesicles can be released from cells either under basal conditionsor upon stimulation by cellular stress via regulated nonlyticmechanisms [1,2]. Released ATP and its metabolites ADP andadenosine can mediate a diverse spectrum of effects in manycell types upon binding to their distinct and specific receptors.P1 receptors are activated by adenosine, whereas ATP and itsanalogs activate P2 receptors. P2 receptors are of two types: theionotropic P2X and the metabotropic P2Y. Cells expressmultiple isoforms of P2X and P2Y receptors (Fig. 1) [2,3].Once released, ATP interacts with these purinergic receptorsand activates rapid signaling events that are both autocrine andparacrine [4]. Thus, ATP is an important signaling molecule thatparticipates in intercellular communication and also regulates abroad range of physiological responses like (i) increases inintracellular calcium; (ii) activation of phospholipases; (iii)modulation of cell volume; (iv) inhibition of platelet aggrega-tion; (v) alteration of vascular tone; (vi) neurotransmission; (vii)elevation of cardiac/skeletal muscle contractility; (viii) immunecell activation; (ix) increased ciliary beat frequency and mucussecretion; (x) pulmonary surfactant release from alveolar type IIepithelial cells; (xi) bronchoconstriction in individuals withhyperreactive airways; and (xii) enhanced cell growth andproliferation [5]. Extracellular ATP and adenosine may alsocause cell death/apoptosis depending on their extracellularconcentration and cell type so exposed [6].
ATP release and ATP-mediated signaling during oxidativestress have been less thoroughly investigated in the lung than inthe cardiovascular system. Several studies indicate thatpurinergic receptors are involved in the cellular response tooxidative stress [7–13]. However, the role of ATP release in
Fig. 1. Mammalian purine receptors. There are two main families of purine receptorsand UDP. P1 receptors comprise A1, A2A, A2B, and A3 receptors, and P2 receptors a(GPCR). Their structure consists of typical features of GPCRs including 7 transmembamino acid residues in their transmembrane region determine the ligand binding and sA3 receptors. P2Y receptors are further subclassified into two phylogenetically dist(P2Y1, P2Y2, P2Y4, P2Y6, and P2Y11), and the other containing receptors that couphighly diverse in both their amino acid sequences (<18% sequence homology) and punrelated in amino acid sequence to other ion channels and other proteins, but sharsubtypes P2X1–P2X7 which often oligomerize to form heteromers and homomers. T
oxidative stress has not been altogether clear. Severe oxidativestress can diminish intracellular ATP [14,15]. Delayed catab-olism of extracellular nucleotides due to diminished ecto-nucleotidase activity in the presence of reactive oxygen species(ROS) also has been suggested [15]. Further, extracellular ATPitself could lead to oxidative stress in some cell types [16,17].For example, extracellular ATP can induce H2O2 production byincreasing Ca2+-mediated Duox, an NADPH oxidase, in airwayepithelium [18]. Stimulation of P2X receptors also can inducesuperoxide production and contraction of mouse aorta [19].Thus, ATP not only responds to redox changes but also canitself trigger redox changes.
Oxidative stress like that in hyperoxia also signals throughvarious pathways. Previously we and others have shown thatmicrovascular endothelial cells are primary targets of hyperoxiclung injury and in other forms of acute respiratory failure[20,21]. Hypoxic preexposure of human lung microvascularendothelial cells (HLMVEC) attenuates cell death caused bysubsequent hyperoxic exposure [21]. This is partially attributedto upregulation of PI 3-kinase (PI3K), a component of anestablished cell survival pathway, during the hyperoxicexposure. Hypoxic (1 or 3% O2) exposure also induces ATPrelease in HLMVEC and fetal lung fibroblasts [22,23]. There,hypoxia and extracellular ATP acted synergistically to upregu-late common proliferation-inducing pathways in lung adventi-tial fibroblasts. Extracellular ATP and purinergic receptoractivation play a critical role in determining the intracellulareffects of key growth-regulating factors [4,24,25]. In addition toATP release, hypoxic conditions also favor proliferation inendothelial cells [22,26]. In primary HLMVEC, we have alsoshown previously that ATP is released in hyperoxia and that thepresence of extracellular ATP is essential for survival of thesecells [27]. Enhanced glucose uptake and upregulation ofhexokinase II (HKII) are other potential contributing survivalfactors in hyperoxia [28,29]. A possible link between these
: adenosine or P1 receptors and P2 receptors, which recognize ATP, ADP, UTP,re of two types the P2X and P2Y. P1 and P2Y are G protein-coupled receptorsrane regions connected by 3 extracellular and 3 intracellular loops. The conservedpecificity. P1 receptors share <50% homology among cloned A1, A2A, A2B, andinct groups, one encompassing the receptors that mainly couple to Gq proteinsle to Gi proteins (P2Y12, P2Y13, and P2Y14). The mammalian P2Y subtypes areharmacological profiles. P2X receptors are ligand-gated ion channels. They aree striking topological similarity to epithelial sodium channels. There are sevenhe P2X receptor proteins share ∼30–50% sequence identity.
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events and ATP signaling remains under investigation. In sum,deviations from usual oxygen tensions, both high and low, cancause ATP release from endothelial and other lung cell types.
Another common pulmonary oxidant is ozone. It is animportant component of ambient air pollution and exposure toozone can cause exacerbation of asthma and other chronic lungdiseases [30–32]. Extracellular ATP protects lung epithelialcells against ozone toxicity [33]. P2Y receptor-mediatedincreases in ERK and Akt phosphorylations were importantsignaling events in this protection. Therefore, identification oflinks between ATP release and activation of survival pathwayswill illuminate a novel pathway in cellular defense againstoxidative stress. In this Review we highlight the unique role ofextracellular ATP as a danger signal, an “SOS” for host defenseduring pulmonary disorders caused by oxidative stress such asbronchopulmonary dysplasia and cystic fibrosis.
Purinoreceptor expression in pulmonary cells
Both P2Y and P2X receptors are ubiquitous in lung. Airwayepithelium has been one of the most heavily studied tissues foreffects of purinergic agonists [34–36]. Airway epithelial cellsrelease ATP and possess both P2X and P2Y receptors, as doendothelial cells [2,5,25,37]. Localization and distribution ofP2X receptor subtypes in lungs, along with methods ofdetection used, are summarized in Table 1. In general, themammalian pulmonary vasculature contains most P2X receptorsubtypes except for P2X6. P2X receptors are found in lungepithelium as well as endothelium. P2X4, P2X5, and P2X7 are
commonly present in bronchial and alveolar epithelial cells. Theapical and luminal surfaces contain one or more of thesereceptors that are often present as homomultimers andheteromultimers. Pulmonary artery endothelial cells containP2X1, P2X2, P2X4, P2X5, and P2X7 subtypes, whereas lungmicrovascular endothelial cells are reported to have P2X1,P2X4, P2X5, and P2X7 receptors.
P2Y receptors were identified in human lungs following thefinding of Knowles et al. [38] that extracellular nucleotidesstimulated chloride secretion in the lungs of cystic fibrosispatients. As for P2X receptors, the rank order potency ofagonists was initially the method of choice for defining thepresence of P2Y receptors and their subtypes (Table 2).Molecular cloning led to identification of several P2Y receptors,some of which are verified to be nucleotide receptors. P2Yreceptors have been identified and characterized in whole lungs,lung vasculature, parenchyma, smooth muscle cells, andendothelial and epithelial cells. P2Y receptors are present
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along with P2X receptors, and a combination of their subtypesmay exist on the same cell. Novel P2Y receptors specific fordinucleotides have also been identified in human lung [39].
Endogenous ATP release in lung and its effects
Stimuli which cause ATP release by regulated mechanismsinclude mechanical stress/trauma, shear stress (increased bloodflow and or lung ventilation; volutrauma) [40], hypotonicmedium [41,42], inflammation, cAMP, and ATP itself [43]. Themechanisms responsible for the appearance of extracellular ATPare likely multiple and probably differ by cell type. First, it mayexit the cell via ATP-permeable release channels. Second, ATPmay be conducted down favorable concentration gradients bysuch channels, e.g., plasma membrane forms of the voltage-dependent anion channel (VDAC) [44], a mitochondrialchannel capable of conducting ATP and ADP. Third, adeninenucleotide transporters, such as the ABC (ATP-bindingcassette) family of transporters like the multidrug resistancechannel (MDR or Mrp) [45], may serve as transporters of ATPand many other substrates [46,47]. Another member of thisfamily, the cystic fibrosis transport regulator (CFTR), generallyis acknowledged to modulate extracellular transport of ATPthrough such pathways, but also is conceded not to be an ATPrelease channel itself [48]. ATP exchange with ADP, as inmitochondria, also is possible through ABC transporters.Fourth, ATP release may occur through vesicular exocytosis[49]. There is considerable support for such a mechanism ofrelease from various epithelia. Fifth, two or more such processescould act in combination. For example, ATP channels ortransporters could load ATP into vesicles which then fuse withthe plasma membrane and release their contents (exocytosis)(Fig. 2).
Fig. 2. Schematic representation of mechanisms of ATP release from cells. Upon stimand transporters. Cell death and cellular injury may also cause direct ATP release intobe metabolized to its degradation products (ADP, AMP, adenosine) by the action ofspecific receptors or be converted back to ATP by the reverse action of these enzym
Once released from the cell, extracellular adenine nucleotideconcentrations can be further dynamically controlled by (a)receptor interaction and/or uptake, and (b) cell surface enzymescalled ecto-enzymes. Hence, ATP can be broken down orconverted into lower energy forms, including ADP, AMP,cAMP, and adenosine. Ecto-apyrases break down ATP to ADPand 5′-AMP. Ecto-ATPases and ADPases also can act to breakdown ATP. Ecto-5′-nucleotidases convert 5′-AMP to adenosine[50,51]. These enzymes also can act in reverse to resynthesizeATP. In addition, more elaborate complexes may exist tosynthesize ATP extracellularly [52] (Fig. 2). Changes inexpression or activity of these ecto-enzymes can modifyphysiologic responses because the different metabolites gener-ated have maximal actions on different receptors. For example,the breakdown product adenosine triggers a host of physiologicactions through P1 receptors [53]. Adenosine may be ofparticular interest and importance in lung diseases like asthma[54–56] and cystic fibrosis [57]. Not only can ATP actindirectly via breakdown to adenosine, but, in addition, ATPcan upregulate or activate adenosine receptors to enhance theresponse [58,59].
The effect of ATP and its metabolites in the pulmonarycirculation is variable and depends on the stimulus, the effectormetabolite ,and the endpoint being studied. In the pulmonarycirculation ATP is released primarily from red blood cells. It isalso released by platelets and endothelial cells lining the vesselwalls. For example, collagen injection caused release of ADPand ATP from platelets in the coronary and pulmonarymicrocirculations and elsewhere [60]. Released ADP inducedplatelets to form aggregates that lodged in the vessels resultingin thrombocytopenia and cardiopulmonary dysfunction. In-creased adenosine and its degradation products in thepulmonary circulation are also observed upon anoxic exposure
ulation ATP is released either exocytotically or through transmembrane channelsthe extracellular milieu. Once released ATP may bind to the P2 receptor, or it canthe enzymes called ecto-nucleotidases. ADP, AMP, and adenosine may bind toes.
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[61]. Efflux of [3H]purines from pulmonary artery induced bypotassium or calcium ionophore A23187 has also beeninvestigated [62]. Endogenous ATP released during increasesin flow rate dilates pulmonary vessels [63]. Mechanical stress-induced release of ATP also has been described in humanairway epithelial cells [64]. Recent studies indicate that ATPrelease following mechanical injury also promotes cellmigration, a critical event in epithelial wound closure, invarious epithelial cell types [65,66] including airway epithelialcells (A van der Vliet, personal communication). The role ofextracellular ATP and mechanical deformation-induced stimu-lation of alveolar type II cells to secrete surfactant has beencarefully explored [67–72]. ATP release and extracellular ATPalso can play a major modulatory role in histamine release fromhuman lung mast cells, and therefore, it may be mechanisticallyinvolved in human allergic/asthmatic reactions [73]. Thus, ATPrelease from one tissue/cell type can trigger a response inanother tissue/cell type, thereby establishing interaction.
Pulmonary epithelium is the primary barrier against varioustoxic chemical and mechanical environmental stresses includingoxidant gases and particulates. Preservation of airway andalveolar homeostasis is a necessary component of the host–defense function in lungs. ATP release upon stimulation of theluminal surface of the epithelium is known to cause enhancedfluid secretion and increased ciliary beat frequency which helpsclear the epithelial surface and restore alveolar homeostasis[74,75]. Extracellular ATP acts by triggering purinergicreceptors which, in turn, cause elevation of intracellular calciumconcentration [74]. Nucleotides within the airway surface liquid(ASL) also regulate airway epithelial ion transport rates by Ca2+-and protein kinase C-dependent mechanisms [57]. For thesereasons, a tremendous effort has been focused on developmentof purine analogs as therapeutic agents for treatment of cysticfibrosis. ATP signaling may regulate or modulate numerouscritical cell functions relevant to cystic fibrosis includingpromotion of: (1) volume homeostatic responses to alteredtonicity of the extracellular milieu [2,76,77], increased ciliarybeat frequency [4,78], epithelial cell secretion of ions [76,79–82], and (2) fluid [5,57,83], mucin secretion [6,84], and releaseof inflammatory cytokines [85–87]. Some studies suggest thatATP release from CF epithelial cells is deficient [37,88].However, others do not [89]. Studies regarding the potential ofCF airways to release ATP under basal conditions or in responseto stress appear to differ based on, among other things, thestressful stimulus. CF epithelial cells appear deficient in ATPrelease responses to osmotic stress, while they are reported tohave normal ATP release responses to shear. Braunstein et al.[77] recently reported that transformed non-CF human airwayepithelial cells and CF airway cells transfected with wild-typeCFTR could both undergo regulatory volume decrease (RVD) inresponse to hypotonic stress, and that these responses wereinhibited by blockade of purinergic signaling. By contrast, CFcell line IB3-1 or IB3-1 transfected with mutant CFTRs (DF508,G551D, or S1445X) could not produce RVDs. Nonetheless,IB3-1 cells could produce RVD if their P2Y G protein-coupledreceptors and/or P2X receptors were stimulated. This supportedprevious work by others that cell volume regulation is dependent
on extracellular ATP and is impaired in CF [37,48,90].Lazarowski et al. [57] recently reported that primary CF andcontrol, fully differentiated human bronchial epithelial cellsgrown on supports showed similar levels of apical ATP release,both at rest and following stimulation due to shear stress. Thereare numerous possibilities for different conclusions reached inthese two studies. The latter study utilized fully differentiatedprimary CF versus control airway epithelial cell cultures,whereas the former study used a CF cell line with its stablytransfected, CFTR-corrected control cell, as well as a number ofother transiently transfected CF cells bearing other CFTRmutants or empty vector. The study in primary cells focused onappearance of ATP and its breakdown products in theextracellular space in response to shear or under basalconditions, while the focus in the CF cell line and transfectionwas on RVD response to hypotonic stress and the potency ofvarious agonists/antagonists to correct or inhibit the deficient CFRVD responses. Nonetheless, in each study, CF cells differedfrom controls. In one, in RVD responses, and, in the other, in thatthey had lower levels of extracellular adenosine. Mechanicallyinduced ATP release and its effect in enhancing clearance ofsecretions from CF airways also have recently been described[91]. Taken together, these data indicate that cystic fibrosisairway epithelium, itself a target of profound oxidative stress,may also have altered signaling responses due to extracellularATP and/or adenosine.
P2 receptor knockouts in general are not embryonically lethaland survive with no apparent phenotypic abnormality. BecauseATP-dependent signaling is often more relevant to physiologicstress adaptation, it is conceivable that the important stressorsthat might cause difficulty for these various mice may not yethave been discovered. Moreover, the complexity of distributionand ligand-binding specificity of the P2 receptor family havemade it extremely difficult to assign a particular receptor tospecific physiological response. Knockout mice have, however,uncovered tissue-specific roles of P2Y receptor, as P2Y2−/−mice have decreased ATP-regulated chloride secretion intracheal epithelium [92]. P2Y4 knockout mice, on the otherhand, have complete loss of ATP-and UTP-induced chloridesecretory response in the jejunum as compared to the wild type[93,94]. Inhibition of platelet aggregation to ADP and increasedbleeding time in P2Y1 (−/−) and P2Y12 (−/−) mice have alsobeen reported [95]. Double knockout P2Y1 and P2Y2 (P2Y1/P2Y2−/−) mice have enhanced susceptibility to Pseudomonasaeruginosa-induced death, suggesting the involvement of thesereceptors in innate host defense of the lung [96]. P2X receptorknockout mice reveal important roles of these receptors in theneurogenic control of smooth muscle contraction, pain, boneformation, and inmacrophage function [97]. P2X3−/−, P2X2−/−,and P2X2/P2X3(Dbl−/−) mice present reduced urinary bladderreflexes and decreased pelvic afferent nerve activity in responseto bladder distension [98]. P2X2−/− and P2X2/P2X3(Dbl−/−)and P2X7−/− mice also exhibit reduced pain-related behaviors[98,99]. Genetic elimination of inotropic purinergic receptors(P2X2 and P2X3) eliminates taste responses in taste nerves[100]. In summary, a wide spectrum of physiologic abnormal-ities is revealed in P2 receptor knockout animals. Because of the
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large numbers of receptors of both classes, P2Y and P2X, thelack of abnormal phenotype in many knockouts may be morerelated to redundancies in these systems than to overall lack ofimportance of function.
ATP-mediated protection against oxidative stress
As described above, in lungs, mechanical stress orintroduction of foreign particles on the luminal surface cantrigger nucleotide release. Subsequently, P2Y2 receptor-mediated secretion can be stimulated and larger amounts offluid generated, potentially contributing to airway particleclearance [101]. In addition to clearing the epithelial surfaceand restoring alveolar homeostasis, extracellular ATP alsocould protect pulmonary epithelium and endothelium againstoxidant stresses caused by exposure to hyperoxia and ozone[27,33]. We showed previously that hypoxic exposure protectslung endothelial cells against subsequent hyperoxic exposuretoxicity [21]. Hypoxic exposure, besides causing upregulationof important survival pathways, caused enhanced accumula-tion of ATP in the extracellular medium [22]. A similaraccumulation of ATP in extracellular medium was observed inhyperoxia-exposed cells. Removal of extracellular ATP orblockade of P2 receptors increased hyperoxia-induced toxicity[27]. This suggested that extracellular ATP and its signalingresponses are crucial for survival during hyperoxic exposures.Increased blood plasma ATP level in the pulmonarycirculation of enhanced oxygen-exposed fetal lambs also hasbeen demonstrated [102], indicating that these observations incell culture could be pertinent to in vivo exposure tohyperoxia.
Early enhancement of extracellular ATP content upon ozoneexposure also was observed in lung epithelial cells derived fromall levels of human and rat airways. Interestingly, ozone-mediated toxicity to lung epithelial cells was abolished bysupplemental extracellular ATP or stable ATP analog ATP-γ-S[33]. The inflammatory response of pulmonary endothelium cancause increased transendothelial permeability, contributing toextrusion of fluid and blood cells and resulting in lung edema.In addition to enhancing survival, extracellular ATP and itsanalogs enhance barrier function in pulmonary endothelial cells[103]. Barrier-protective properties of ATP have been describedin endothelial cells from other organs as well [104].Extracellular ATP stimulates airway epithelial cells to produceH2O2, a necessary substrate for the lactoperoxidase (LPO) anti-infection system, thus contributing to airway host defense [18].In another study, extracellular supplementation with ATP alsoprotected astrocytes against hydrogen peroxide-mediated celldeath [105]. Similarly, ATP supplementation may also protecthippocampal neurons from excitotoxic cell death caused byoverstimulation by glutamate [106]. Moreover, ATP releaseddue to brain injury mediates rapid recruitment of microglialcells, principal immune cells of brain, thus signaling neuronaltissue repair responses [107]. Cell death induced by hypoxia inrat cardiomyocyte was significantly reduced by extracellularUTP [108]. Extracellular ATP and adenosine are also known toplay complementary roles in ischemic preconditioning of heart
[109]. Administration of 2-methylthio-ATP, an ATP analog, inmice protected them against subsequent endotoxin-induceddeath [110]. Thus, ATP signaling can mediate a variety ofprotective responses relevant to oxidative stress in lung andother organs.
ATP is also released in response to other oxidants likehydrogen peroxide [111], and this may be pertinent inprotection of cells against hydrogen peroxide-induced death[112]. Extracellular ATP as well as 2-methylthio-ATP and ATP-γ-S (P2Y purinoceptor agonists) can inhibit ischemia-inducedlactate dehydrogenase (LDH) release [113]. Thus, it appearsthat endogenously released ATP or exogenously added ATP canprotect cells against a variety of oxidants.
In addition to ATP mediating tissue protection through its P2receptors, downstream metabolites of ATP like adenosine alsocan protect against oxidants. Adenosine also has beenimplicated in protection of pulmonary endothelial cells againstH2O2-mediated cytotoxicity [114]. Recently, A2A adenosinereceptors were found to be important in limiting andterminating both tissue-specific and systemic inflammatoryresponses [115,116]. Thus, adenosine and ATP may beimportant autocrine/paracrine regulators mediating cellularprotection and regeneration after ischemia [109,117], and P1and P2 receptors may cooperate to limit tissue damage due tooxidants.
ATP analogs and P2Y2 receptor agonists are a new class ofcompounds being developed for the treatment of CF lungdisease. A number of P2Y2 agonist compounds have beenevaluated in healthy subjects and patients with CF. Mostrecently, INS37217, a metabolically stable and potent P2Y2
agonist has been developed, and studies have shown it to bewell-tolerated when given via inhalation. This compound iscurrently being evaluated in children and adults with CF lungdisease [118]. Results on preliminary trials with oral andintravenously administered ATP, as an analgesic, also areencouraging for the treatment of patients with postherpeticneuralgia and low back pain [119,120]. ATP release andsubsequent signaling via P2X receptors mediate excitation ofsensory neurons and evoke muscle contraction in humanurinary bladder. Functional defects in the P2X receptorsignaling are associated with a variety of urologic diseases[121]. Studies on ATP-mediated signaling present a potentialapplication in relation to the diagnosis and treatment of urinarydysfunction. ATP may also inhibit pathological renal cystgrowth through P2X7 signaling [122]. Because of potentialcardiovascular effects of extracellular ATP itself, analogstargeted to one or more receptor(s) may offer greater specificityof effect(s) in therapy.
Signaling events associated with ATP-mediated protection
P2X receptors mediate “rapid: nonselective passage ofcations (Na+, K+, Ca2+) across cell membranes resulting inincreased intracellular Ca2+ and depolarization [123,124].Conductance of extracellular Ca2+ through such channels is asignificant source of increased intracellular Ca2+. Suchresponses are very rapid and do not involve production of
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second messengers. These signals are important for rapidneuronal signaling and regulation of muscle contractility. P2Yreceptors are G-protein-coupled receptors and most of them actvia G-protein coupling to activate phospholipase C (PLC),leading to formation of inositol triphosphate (IP3) andmobilization of intracellular Ca2+. Coupling to adenylatecyclase by some P2Y receptors (e.g., P2Y12, P2Y13, andP2Y14) has also been described [125]. The response time ofP2Y receptors is longer than the rapid responses mediated byP2X receptors, because P2Y-mediated responses involveformation of second messengers. IP3 formation and Ca2+
mobilization can stimulate a variety of signaling pathwaysincluding protein kinase C (PKC), phospholipase A2 (PLA2),etc. PKC, in turn, may activate PLC, PLD, and MAP kinasepathways [126]. These responses, as well as secondaryformation of various eicosanoids, could be important insignaling P2Y-mediated tissue protective responses.
Extracellular ATP can mediate increases in intracellularcalcium via P2Y receptors in alveolar type II epithelial cells,bronchial epithelial cells, and lung fibroblasts [127–129]. Ourunderstanding of ATP-mediated signaling events and theirmodulation following oxidative stress are beginning to unfold.A better understanding of these processes could be helpful indevising protection strategies.
Fig. 3. Survival signaling responses activated by oxidant gases via extracellular ATPby niflumic acid (chloride channel inhibitor), colchicine (a microtubule disrupting agePI3K) suggested involvement of a complex mechanism in hyperoxia-induced ATPU0126 (a MEK specific inhibitor), Y27637 (a rho kinase inhibitor) brefeldin A (a veindicating calcium-dependent vesicular exocytosis as the primary mechanism. ExtraMEK. Activation of PI3K and MEK was accompanied by increased activity of theirfor PI3K, and ERK 1/2 for MEK). Action of apyrase (an ATP scavenger), suramin (signaling and caused increased cell death in both hyperoxia and ozone. Extracellularlysignaling pathways and their metabolic effects.
Oxidative stress due to hyperoxic exposure caused accumu-lation of ATP in the extracellular milieu of HLMVECs by aPI3K-dependent mechanism. ATP release could be modulated inpart by inhibition of CFTR or of microtubular function.Scavenging of extracellular ATP before and during hyperoxicexposure caused enhanced toxicity, indicating the significanceof extracellular nucleotide for cellular protection. Similarly,exposure to lung epithelial-like A549 cells to ozone causedincreases in extracellular ATP (Fig. 3). Therein, a complex-regulated mechanism involving vesicular exocytosis wasobserved for ATP release. Here too, we found that extracellularATP was indispensable for survival in ozone. Protective effectsof extracellular ATP against hyperoxia-and ozone-mediatedtoxicity were due to two key pathways: extracellular signal-regulated protein kinase (ERK) and PI3K. These protein kinasesare critical regulators of cell survival and proliferation[130,131]. Extracellular ATP induced activation of ERK,PI3K, and mTOR (mammalian target of rapamycin), primarilythrough activation of P2Y2 and P2Y6 receptors in HLMVECs(Fig. 3). mTOR mediates growth and survival by regulatingcellular metabolism. Rapamycin (a triene macrolide antibiotic)binds to a receptor protein (FKBP12), and the rapamycin/FKBP12 complex then binds to mTOR and prevents interactionof mTOR with target proteins [132]. Rapamycin also caused
release. Cellular exposure to ozone and hyperoxia causes ATP release. Inhibitionnt), LY 294002 (a specific PI3K inhibitor), and wortmannin (another inhibitor ofrelease. Ozone-mediated ATP release was attenuated by colchicine, LY294002,sicular transport inhibitor), and BAPTA-AM (an intracellular calcium chelator),cellular ATP acted via P2Y receptors to activate survival kinases like PI3K anddownstream target protein kinases (Akt or protein kinase B, mTOR and p70S6KP2 receptor inhibitor), and Cibacron Blue (P2Y receptor inhibitor) blocked thisreleased ATP signals to prevent oxidant gas-induced death via Akt and /or ERK
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enhanced cell death in hyperoxia, further supporting acontributing role for mTOR. Moreover, enhanced P2Y recep-tor-mediated glucose uptake, also inhibited by rapamycin,provided additional evidence for a protective role for mTOR.ERK activation due to ATP release in hyperoxia likelycontributed to enhanced survival. ATP release increasedarachidonic acid release and enhanced prostaglandin (PGE2)synthesis, which may also have contributed to cellularadaptation and resistance to oxidative damage. Activation ofERK and Akt was also critical for survival during ozoneexposure. Activation of these kinases was linked to increasedglucose metabolism, since ATP supplementation enhancedglucose uptake during ozone exposure. These studies demon-strate a novel survival-enhancing function for ATP release andextracellular ATP in the response of cells against oxidativestress. Despite evidence that stimulus-induced, regulated ATPrelease can inhibit oxidative damage, unregulated or unrestrictedATP release in severe tissue injury can exacerbate or enhancetissue damage [133].
Activation of nucleotide receptors in pulmonary endothelialand epithelial cells is coupled to ERK and PI3K pathways thatregulate survival during oxidant stress. Deficits in these signalsdue to impaired nucleotide production or release couldcontribute to disease pathogenesis or exacerbate existingconditions. Because ATP analogs already are in clinical trialsfor certain lung diseases, these findings could have applicationin prevention or treatment of respiratory disorders such asasthma, BPD, and air pollution-related morbidity in vulnerablepopulations. An improved understanding of ATP release,extracellular ATP metabolism, and relevant downstreamsignaling mechanisms also could lead to the development ofnovel therapeutic agents.
Acknowledgments
This publication was made possible by Grant ES014448-01from the National Institute of Environmental Health Sciences(NIEHS), NIH. The authors are also grateful to GabrieleCheatham for the preparation of the manuscript.
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