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ATP released by LPS increases nitric oxideproduction in raw 264.7 macrophage cell line viaP2Z⧹P2X7 receptorsfn1
NEUROCHEMISTRY International Neurochem[ Int[ 22 "0887# 198Ð104
ATP released by LPS increases nitric oxide production in raw 153[6 macrophage cell line via P1Z:P1X6 receptors Beata Sperlagh$%\ Gyorgy Hasko$\ Zoltan Nemeth\ E[ Sylvester Vizi Department of Pharmacolo`y\ Institute of Experimental Medicine\ Hun`arian Academy of Sciences\ Budapest\ Hun`ary Received 0 April 0887\ accepted 5 April 0887
Abstract P1Z:P1X6 receptor is a particular type of purinoceptor\ which is selectively expressed on the surface of immune cells in neuronal and non!neuronal tissues[ Despite intensive research on its involvement in the immune response\ the exact mechanism whereby it a}ects intercellular signaling is far from clear yet[ In this study\ the e}ect of activation P1Z:P1X6 receptor was investigated on the bacterial lipopolysaccharide induced nitric oxide production in RAW 153[6 macrophage call line using the nitrite:nitrate assay[ The P1Z:P1X6 receptor agonist 2?!O!"3!benzoylbenzoyl#!adenosine 4?triphosphate increased concentration!dependency the lipo! plysaccharide "09mg:ml# induced nitric oxide production between 09 mM and 149 mM[ ATP also increased nitric oxide production in response to lipopolysaccharide\ while ADP\ 1!methylo!thio!adenosine 4?!triphosphate and adenosine 4?triphosphate!g!S was without e}ect[ Pretreatment with oxidized adenosine triphosphate\ the selective P1Z:P1X6 receptor antagonise "299 mMÐ0 mM# strongly decreased lipopolysaccaride induced nitric oxide production[ Furthermore\ on macrophages\ pretreated with oxidized adenosine 4?! triphosphate "299 mM −0 mM#\ 2?!O!"3!benzoylbenzoyl#!adenosine 4?!triphosphate and ATP did not a}ect lipopolysaccharide induced nitric oxide production[ 04 min lipopolysaccharide treatment induced a transient and reversible release of endogenous ATP from RAW 153[6 cells\ measured by the luciferinÐluciferase assay[ The e}ect of lipopolysaccharide to promote ATP release was concentration!dependent between 0Ð09 mg:ml[ In summary\ our results show that P1Z:P1X6 receptor activation results in an increase in nitric oxide production in response to lipopolysaccharide challenge[ Since the P1Z:P1X6 receptor antagonist oxidized adenosine triphosphate decreased lipopolysaccharide induced nitric oxide production\ and lipopolysaccharide was able to promote ATP release from macrophage cells\ it seems likely that endogenous ATP is involved in nitric oxide formation during endotoxin challenge[ Þ 0887 Elsevier Science Ltd[ All rights reserved[
0[ Introduction ATP and its metabolic degradation products\ ADP\ AMP and adenosine are accumulated and may reach high con! centration extracellularly during any kind of cell death occurring frequently during in~ammation\ ischemia\ and cell injury[ Since ATP and adenosine receptors are pre! sent on the surface of immune cells\ a possible immun! modulatory role of purine nucleotides and nucleosides has been postulated "Dubyak and El!Moatassim\ 0884^ Di Virgilio\ 0884^ Di Virgilio et al[\ 0885#[ The action of adenosine on immune cells via A0\ A1a and A2 receptors is well documented regulating phagocytosis\ chemotaxis\ cytokine "cf Cronstein\ 0883\ Di Virgilio et al\ 0885#\ and Due to circumstances beyond the Publisher|s control\ this article appears without the authors| corrections[ $ These authors contributed equally to the study[ % Corresponding author[ Tel[]9925!0!109!9708^ fax] 9925!0!109!9702^ e!mail] SPERLAGHÝKOKI[HU 9086Ð9075:87 ,08[99 Þ 0887 Elsevier Science Ltd[ All rights reserved PII] S 9 0 8 6 Ð 9 0 7 5 " 8 7 # 9 9 9 1 4 Ð 3
nitric oxide "NO# production "Hasko et al[\ 0885^ Hon et al[\ 0884#\ acting either as an endogenous pro! or anti! in~ammatory agent "cf Cronstein\ 0883\ Hasko et at[\ 0885#[ A similar function of extracellular ATP is now also being recognized] ATP release has been shown in response to antigen stimulus from di}erent kind of immune cells "Osipchuk and Cahalan\ 0881\ Filippini et al[\ 0889\ Ferrari et at[\ 0886c# which express both ionotropic and metabotropic ATP receptors "Dubyak and El!Moatassim\ 0884#[ Among the number of P1 purinoceptor subtypes\ the function of the so!called P1Z receptors appears to be particularly intriguing\ due to their distinguished expre! ssed in the immune cells of neuronal and non!neuronal tissues "Illes et al[\ 0885^ Chessell et al[\ 0886\ Rassendren et al[\ 0886^ Collo et al[\ 0886#[ These receptors have recently been identi_ed as the seventh member of the ion! channel type P1X receptor family "Suprenant et al[\ 0885# and their expression is upregulated by the induction of immune system by interferon!g and bacterial lipo!
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polysaccharide "LPS#\ suggesting that they are speci_! cally involved in the macrophage response "Blanchard et al[\ 0880\ Humphreys and Dubyak\ 0885^ Falzoni et al[\ 0884#[ Activation of the P1Z:P1X6 receptor results in a pore formation "Steinberg et al[\ 0876^ Falzoni et al[\ 0884\ Virginio et al[\ 0886# which makes the membrane permeable to higher molecular weight substances up to several hundred Da and sustained activation of the chan! nel therefore causes cell death "Murgia et al[\ 0881\ Fal! zoni et al[\ 0884^ Chiozzi et al[\ 0885\ Lammas et al[\ 0886^ Ferrari et al[\ 0886a#[ Nevertheless\ their function may not be restricted to citotoxicity] a line of a recent studies suggest that they also play a role in intercellular signaling during in~ammation process\ i[e[\ they regulate the pro! duction of interleukin*0b "0LÐ0b# in mouse and human macrophages "Ferrari et at[\ 0886b^ Ferrari et al[\ 0886c# and in microglial cells "Ferrari et al[\ 0886c#\ as well as phospholipase D activity "Humphreys and Dubyak\ 0885^ El!Moatassim and Dubyak\ 0882# in response to bacterial lipopolysaccharide "LPS# challenge[ On the con! trary\ it is less clear\ whether P1Z:P1X6 receptors are involved in the production of other in~ammatory mess! engers\ such as NO production[ Although earlier studies indicated that extracellular ATP is a potent co!stimulator of NO synthase expression during LPS induction in mac! rophages "Tonetti et al[\ 0884^ Tonetti et al[\ 0883^ Den! linger et al[\ 0885#\ P1Z:P1X6 selective probes have not been tested\ and the receptor identity remains unidenti! _ed so far[ We therefore investigated the e}ect of di}erent P1 receptor ligands on LPS!induced NO production in the RAW 153[6 macrophage cell line[ Since speci_c inhibition of P1Z:P1X6 receptors by oxidized adenosine tri! phosphate "oxyATP# dramatically reduced LPS!induced NO production\ indicating the regulatory action of endogenously released APT[ The ability of LPS to release ATP from macrophages was also examined[
in 4) phosphoric acid# to 099 ml samples of medium\ respectively[ The OD at 449 nm "OD449# was measured using a Spectramax 149 microplate reader "Molecular Devices\ Sunnyvale\ CA#[ 1[2[ Measurement of mitochondrial respiration Mitochondrial respiration\ an2 indicator of cell viability\ was assessed by the mitochondria!dependent reduction of 2!"3\4!dimethylthiazol!1!yl#!1\4!diphenyltetrazolium bromide "MTT# to formazan "Szabo et al[\ 0882a#[ Cells in 85!well plates were incubated with MTT "9[1 mg:ml# for 59 min at 26>C[ Culture medium was removed by aspiration\ and cells were solubilized in DMSO "099 ml#[ The extent of reduction of MTT to formazan within cells was quantitated by measurement of OD449 using a Spectramax microplate reader[ 1[3[ ATP release experiments 3 day!old cultures of RAW 153[6 cells were used for release experiments[ Cells were gently removed from cul! ture ~asks and suspended in DMEM preheated to 26>C[ After cell counting and two periods of centrifugation at 1999 rpm for 09 min at 09>C\ the cells were resuspended in Gey|s balanced salt solution "GBSS\ from SIGMA\ containing] CaCl1 9[1140\ MgCl1 9[10\ MgSO3 9[9231\ KCl\ 9[26\ KH1PO3 9[92\ NaHCO2 1[16\ NaCl 6[9\ NaH1PO3 9[0085\ D!Glucose\ 3[9\ phenol red\ 9[9095 pH 6[3# obtaining 49 million cell:ml concentration[ 49 ml of this cell suspension were injected into poly! propylene tissue chambers\ separated by Millipore _lters of 9[1 mm diameter[ Cells were superfused at a rate of 9[1 ml:min with GBSS for 59 min at 26>C and subsequently 2 min perfusate samples were collected[ LPS "09 mg:ml# was applied by perfusion\ for 04 min[ ATP content of the samples were measured by the luciferinÐluciferase assay\ as previously described "Sperlagh et al[\ 0884# and were expressed in nmol:l[
1[ Experimental procedures 1[4[ Statistical evaluation 1[0[ Cell Culture The mouse macrophage cell line RAW 153[6 was cultured in Dulbecco|s modi_ed Eagle|s medium "DMEM#[ Cells were exposed to LPS "09 mg:ml# for 13 h[ BzATP and other ATP analogues was applied for a 04 min exposure after LPS challenge\ oxyATP was applied 1 h before LPS treatment[ 1[1[ Measurement of nitrite concentration Nitrite in culture supernatants at 13 h "after conversion of nitrate to nitrite# was measured at previously described "Szabo et al[\ 0882a#\ by adding 099 ml of Griess reagent "0) sulfanilaminde and 9[0) naphthylethylenediamide
Values in the _gures and text are expressed as mean2S[E[M[ of n observations[ Statistical analysis of the data was performed by one or two!way analysis of variance followed by Dunnett|s test\ as appropriate[ P ³ 9[94 was considered statistically signi_cant[ 1[5[ Materials 2?!O!"3!benzoylbenzoyl#!adenosine 4?!triphosphate "BzATP#\ adenosine 4?triphosphate "ATP#\ adenosine 4?diphosphate "ADP#\ adenosine 4?triphosphate!g!S "ATP!g!S#\ 1!methyl!thio!adenosine 4?triphosphate "1! methyl!thioATP#\ oxidized adenosine triphosphate "oxy! ATP#\ and suramin were used[
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DMEM was purchased from Life Technologies "Grand Island\ NY\ U[S[A[#\ 1!methyl!thioATP was obtained from Research Biochemicals Inc[ "Natick\ MA\ U[S[A[#[ Suramin was the gift of Bayer "Leverkusen\ Ger! many#[ LPS from Escherichia coli 944]B4 and all other drugs were obtained from Sigma "St[ Louis\ MO\ U[S[A[#[
2[ Results 2[0[ NO release study In the absence of drugs\ the basal nitrite production of the RAW 153[6 culture was 1[6529[09 mM "n 5#[ In response to 13 h exposure of LPS "09 mg:ml# stimulation\ the nitrite level increased to 55[4421[37 "n 5\ P ³ 9[990# indicating the activation of inducible nitric oxide synthase enzyme[ Neither of the applied drugs a}ected signi_cantly the unstimulated nitrite level "data not shown#[ The P1Z:P1X6 receptor agonist 2?!O!"3!benzoylbenzoyl#! adenosine 4?!triphosphate "BzATP#\ when added to the cells 04 min after the onset of LPS treatment and kept in the ~uid throughout the experiments\ increased LPS induced NO production between 0 mM and 149 mM "Fig[ 0A#[ Its e}ect reached the level of signi_cance at 49 mM concentration\ and increased NO production con! centration!dependently up to 149 mM\ and no further stimulation of NO production was observed above this concentration[ At 499 mM concentration\ its e}ect declined and turned back to control level[ To assess cell viability and possible cytotoxic e}ect of BzATP\ mito! chondrial respiration was monitored concomitantly] apart from a subtle reduction observed at 149 mM BzATP "from 6720[24Ð6120[3) n 5\ P ³ 9[94#\ no sig! ni_cant inhibition of mitochondrial respiration was detected in the concentration range used in the study showing the facilitatory action of BzATP on LPS induced NO production is not related to cytotoxicity[ The e}ect of BzATP "099 mM# was also examined using di}erent LPS concentrations\ ranging from 9[90Ð 09 mg:ml "Fig[ 0C#[ At lower LPS concentrations "9[90Ð 9[0 mg:ml#\ BzATP did not promote NO production\ while at the higher range "0Ð09 mg:ml# its action became manifest\ and powerfully enhanced the e}ect of LPS "Fig[ 0C#[ ATP\ the endogenous agonist of P1Z:P1X6 receptors\ had a biphasic e}ect] between 29Ð299 mM it also increased NO production^ however the maximal response obtained by ATP was considerably less than in case of BzATP\ suggesting that it might act as a partial agonist "Fig[ 0B#[ In contrast\ APT\ when applied at 0999 mM\ strongly inhibited LPS induced NO production\ and also mark! edly reduced mitochondrial respiration[ Since the stimu! latory e}ect of BzATP reached the level of signi_cance at lower concentration "49 mM#\ than APT "299 mM#\
Fig[ 0[ E}ect of BzATP "A# and ATP "B# on LPS induced nitrite production and measured in RAW 153[6 macrophage cells[ Cells were stimulated with LPS "09 mg:ml# for 13 h in the presence of various concentrations of BzATP "0Ð499 mM# and ATP "29Ð0999 mM#\ and nitrite levels measured in the supernatants[ BzAPT and ATP was applied 04 min after LPS treatment[ "C# shows the e}ect of 099 mM BzATP on LPS induced nitrite production at di}erent concentrations of LPS stimuli "9[90Ð09 mg:ml#[ Nitrite production was expressed as the percentage of LPS stimulated control "A\ B# or in mM "C#[ Data show the mean2S[E[M[ of 5Ð04 wells[ Asterisks indicate signi_cant di}er! ences from respective controls[
BzATP appears to be a more potent agonist promoting LPS induced NO formation[ Other ATP analogues\ such as ADP\ 1!methyl!thioATP and ATP!g!S were without e}ect up to 0999 mM "data not shown#[
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Table 0 Inhibition of LPS induced nitrite production and reversal of the fac! ilitatory action of BzATP and ATP by oxyATP "29Ð0999 mM# in RAW 153[6 macrophages *
BzATP
ATP
* oxyATP 299 mM oxyATP 0999 mM
55[421[68 26[2822[50 2[4429[35
70[9420[50 35[9220[40 2[4729[31
65[1620[23 33[2729[71 3[8929[30
Cells were stimulated by 09 mg:ml LPS after 1 h preincubation with di}erent concentrations of oxyATP[ BzATP "099 mM# and ATP "299 mM# were applied 04 min after LPS treatment[ Nitrite concentrations were measured 13 h after LPS administration in the supernatants and expressed in mM[ Data show the mean2S[E[M[ of 5 wells[ Shows signi_cant di}erences between P1Z:P1X6 receptor agonist tre! ated cells and drug free controls "p³9[990#[ Shows signi_cant di}erences between oxyATP treated and non!tre! ated cells "p³9[990#[
Two h preincubation of the cells with oxidized aden! osine triphosphate "oxyATP\ 299Ð0999 mM#\ the selective and irreversible antagonist of P1Z:P1X6 receptors\ decreased dose!dependently LPS induced NO production "Table 0#[ Furthermore\ neither BzATP nor ATP were able to a}ect LPS induced NO production on macro! phages\ pretreated with oxyATP 8299 mMÐ0 mM "Table 0#[ OxyATP\ applied at 299 mM did not a}ect signi_cantly cell viability\ while at 0999 mM concentrations\ impair! ment of mitochondrial respiration was observed[ Another P1 receptor antagonist\ suramin had no e}ect on LPS induced NO release "data not shown#[ 2[1[ ATP release study The basal out~ow of ATP measured by the luciferinÐ luciferase assay from cultured RAW 153[6 cells during 2 min collection period was 9[2429[97 nmol:l "n 4#\ and remained constant throughout the sample collecting per! iod "Fig[ 1#[ To test the ability of LPS to release ATP\ macrophage cells were perfused with medium containing 09 mg:ml LPS for 04 min[ This treatment induced a tran! sient and reversible release of endogenous ATP\ which increased the ATP out~ow to 0[4729[24 nmol:l at the peak of the response "n 4#[ The total ATP release evoked by LPS was 2[2729[75 nmol "n 4#[ The e}ect of LPS was concentration!dependent between 0Ð09 mg:ml\ 0mg:ml LPS elicited a release of 9[4029[02 nmol ATP "n 5\ P ³ 9[94#[
3[ Discussion The question addressed in this study was to show whether LPS induced NO production in RAW 153[6 macrophages is regulated by P1Z:P1X6 receptors[ The following data
support this hypothesis] "0# BzATP\ the selective R1Z:P1X6 receptor agonist and ATP increased nitrite production evoked by endotoxin challenge in a dose dependent fashion\ BzATP being more potent that ATP\ while ADP\ 1!methyl!thioATP and ATP!g!S were with! out e}ect[ This pro_le is consistent with the typical P1Z:P1X6 receptor pro_le "Humphreys and Dubyak\ 0885^ Lammas et at[\ 0886\ Markwandt et al[\ 0886^ Ras! sendren et al[\ 0886^ Jamieson et al[\ 0885^ Chessell at al[\ 0886^ Falzoni et al[\ 0884# and is similar to the cloned P1X6 receptor pro_le "Suprenant et al[\ 0885# with the di}erence that the cloned receptor exhibits some binding activity for ADP\ 1!methyl!thioATP and ATP!g!S\ and were inactive in our study[ A possible explanation of this dissimilarity is that RAW 153[6 macrophages*in contract to HEK 182 cells\ in which the cloned P1Z:P1X6 receptor was studied*express not only P1Z:P1X6 recep! tors\ but also the metabotropic P1Y type receptors "Dubyak and El!Moatassim\ 0884\ Denlinger et al\ 0885#\ and NO production could thereby be also regulated[ Therefore the e}ect of analogues acting not exclusively on P1Z:P1X6 receptors might interfere with their action on other P1 receptors "1#[ Both BzATP and ATP were active at the high micromolar range\ which also _ts well to the usual P1Z:P1X6 receptor pro_le[ The other charac! teristic feature of this receptor is that the fully ionized species "ATP3−# is the active ligand which is recognized "Steiberg et al[\ 0876^ Markwandt et al[\ 0886^ Falzoni et al[\ 0884#[ "2# Since ATP3− represents a small fraction of the total extracellular ATP content under physiological pH\ generally at least one order of magnitude higher concentration of ATP is necessary to activate P1Z:P1X6 receptors and obtain measurable e}ects\ than for other subtypes of P1 receptors[ "3# Prolonged incubation with the irreversible P1Z:P1X6 receptor antagonist\ OxyATP abolished the stimulatory e}ects of both BzATP and ATP on nitrite production\ which also strongly indicates the involvement of P1Z:P1X6 receptors in this e}ect[ Oxy! ATP might be active on other type of P1 receptors as well "Wiley et al[\ 0883^ Ferrari et al[\ 0883#[ However\ conclusive evidence is missing in this respect[ In addition to oxyATP\ pyridoxal!phosphate!5!azophenyl!1?\3?!dis! ulphonic acid tetrasodium "PPADS# is another antag! onist acting presumably on P1Z:P1X6 receptors "Suprenant et al[\ 0885^ Rassendren et al[\ 0886#^ however it was unfortunately unsuitable in our model\ due to its interference with photometry[ Suramin\ a non!selective P1 antagonist proved to be ine}ective in this study\ in agreement with the P1Z:P1X6 receptor character "Ras! sendren et al[\ 0886^ Chessell et al[\ 0886#[ Therefore\ taken together the _ndings\ the most likely receptor responsible for the synergizing action of ATP analogues on LPS induced nitrite production is the P1Z:P1X6 subtype[ Concerning the e}ect of ATP\ our results are in cor! roboration with the _ndings of Tonetti et al[ "0883\ 0884#\
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Fig[ 1[ E}ect of LPS on ATP release measured from superfused RAW 153[6 cells[ Cells were perfused with GBSS solution with 9[1 ml:min and after a 59 min preperfusion\ 2 min samples were collected and assayed for ATP[ ATP levels were measured by the luciferin luciferase assay and expressed in nmol:l\ as a function of time[ LPS was applied into the perfusion solution in 09 mg:ml for 04 min\ as indicated[ Open circles represent the results in the absence of LPS\ _lled circles show values obtained in experiments with LPS treatment[ Data show the mean2S[E[M[ of 4!4 separate experiments[ Asterisks indicate signi_cant di}erences between LPS treated and control cells calculated by two way ANOVA[
who found that high concentration of extracellular ATP enhances nitric oxide synthase expression and nitrite pro! duction induced by LPS in RAW 153[6 macrophages[ More recently\ Denlinger et al[ "0885# examined a series of P1 receptor ligands on LPS induced nitrite production in RAW 153[6 cells\ and all three studies identi_ed puta! tively the receptors regulating NO production in mac! rophages as P1Y purinoceptors[ However\ speci_c ligands of P1Z:P1X6 receptors\ such as BzATP or oxy! ATP has not been tested in these studies[ Our _ndings do not reject the assumption that P1Y receptors are present on macrophages\ but show that in addition to P1Y recep! tors\ NO production is also regulated by P1Z:P1X6 receptors[ At millimolar concentrations\ ATP caused a marked inhibition of LPS induced nitrite production\ and the e}ect of BzATP was also declined at the high micromolar range[ Since mitochondrial respiration was impaired\ when such a high concentration of ATP analogues were applied these e}ects are most likely non!speci_c and related to the known cytotoxic e}ect of these ligands "Falzoni et al[\ 0884^ Ferrari et al[\ 0886b^ Murgia et al[\ 0881^ Chiozzi et al[\ 0885^ Ferrari et al[\ 0886a#\ and also suggest a di}erential action of extracellular ATP\ when it is present at lower or higher concentration[ The question arises\ which subcellular pathway is
responsible for the initiation of P1Z:P1X6 receptor acti! vated NO production in macrophages during endotoxin challenge< There are several alternative possibilities[ On one hand\ it is known that P1Z:P1X6 receptor activation promotes Ca1¦ in~ux in monocytes\ and the Ca1¦ signal may potentiate LPS induced activation of inducible NO synthase enzyme[ However\ studies showing that mac! rophage iNOS activity proved to be Ca1¦ independent argue against this route "Szabo\ 0882a\ 0884#[ On the other hand\ as it has been reported that P1X:P1X6 recep! tor stimulation is involved in LPS induced phospholipase D activation "Humphreys and Dubyak\ 0885^ El!Moat! assim and Dubyak\ 0882^ Fisette et at[\ 0885#\ yet another possible pathway is the activation of NF!kB or other transcription factors by phospholipase D and the sub! sequent stimulation of iNOS enzyme\ which is favored by a very recent study showing that extracellular ATP activates a particular subunit of NF!kB in mouse mic! ro~ial cells via P1A purinoceptors "Ferrari et al[\ 0886d#[ A third possibility is that NO production of macrophages by P1Z:P1X6 receptor is an indirect mechanism] recent evidence suggest that extracellular ATP triggers IL!0b secretion activating P1Z:P1X6 receptors in LPS primed macrophages "Ferrari et al[\ 0886b] Ferrari et al[\ 0886c# and IL!0b could initiate iNOS expression\ as described by Szabo et al[ "0882b#[ This explanation seems to be
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particularly attractive\ because oxyATP proved to be a strong inhibitor LPS induced IL!0b secretion in micro! glial cells and macrophages "Ferrari et al[\ 0886c# as well as LPS induced NO production in our experiments[ How! ever\ ADP which is a powerful stimulator of IL!0b secretion\ was ine}ective in this study[ An important observation of this study in the powerful action of the irreversible P1Z:P1X6 receptor antagonist OxyATP to inhibit LPS induced nitrite production[ Apart from at 0999 mM concentration\ when oxyATP impaired mitochondrial respiration\ at 299 mM concentration\ it reduced by more than 39) LPS!induced nitrite production\ without any signi_cant action on cell viability[ Therefore\ at least at this lower concentration its e}ect can be attributed to the speci_c blockade of P1Z:P1X6 receptors\ and displacing the action of any endogenous ligand on this receptor[ To prove this assumption the ability to LPS exposition to promote endogenous ATP release from RAW 153[6 cells was tested] indeed it induced ATP out~ow in the time frame "04 min# when P1Z:P1X6 receptors were activated[ Although the amount of ATP out~ow evoked by LPS was only moderate\ taken into account its dilution in the superfusate and the possibility of rapid breakdown in the extracellular ~uid\ its biophase concentration might be higher than we detected[ Furthermore under in vivo con! ditions\ there are several other sources from where ATP can be released at the in~ammation site and reach P1Z:P1X6 receptors] it can be released together with noradrenaline from catecholaminergic nerves "cf Sper! lagh and Vizi\ 0885# innervating the immune system such as spleen "Elenkov and Vizi\ 0880# and thymus "Vizi et al\ 0884#^ and it can be released from endothelial cells and platelets inside the circulation "Boeynaems and Pearson\ 0889#[ Furthermore\ it can be the result of P1Z:P1X6 receptor activation itself] interestingly\ Ballerini et al[ "0885#\ reported purine release from astrocytes in response to BzATP and other P1 receptor agonists[ ATP out~ow via P1Z:P1X6 receptor channel may even act as an amplifying signal reinforming other actions such as IL!0b secretion on the same receptors[ Finally\ an impor! tant source of extracellular ATP accumulation during in~ammation might be cell leakage] although ATP release demonstrated by us occurred below cytotoxic LPS con! centrations\ depending on the severity of in~ammation\ cytolytic ATP might be also important factor to in~uence LPS induced activation of immune system[ In summary\ we report that NO production to LPS challenge in RAW 153[6 macrophages is regulated by P1Z:P1X6 receptors[ These _ndings support the view that P1 purinoceptors serve as an important control of the macrophage response during in~ammation and immune response[ Due to the generation of toxic peroxynitrite radicals\ NO production in Macrophages in considered a key event during endotoxin shock "cf Szabo\ 0884\ 0885^ Szabo et al\ 0885#\ and any subcellular target\ which
selectively modulates this pathway might be of clinical interest[ Since P1Z:P1X6 receptors are expressed activity! dependently in immune cells\ they o}er an attractive therapeutic target\ intervening in septic shock and other in~ammatory diseases[
Acknowledgements This study was supported by the grants of the Hungarian Science Foundation "OTKA T914503# and Hungarian Medical Research Council "ETT086#[ The authors are grateful to Ms Julianna Benko½ for excellent technical assistance and to Ms E łva Vass for cell culture assistance[
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B[ Sperla`h et al[:Neurochem[ Int[ 22 "0887# 198Ð104 Baricordi O[R[\ 0884[ The purinergic P1Z receptor of human mac! rophage cells[ Characterization and possible physiological role[ J[ Clin[ Invest[ 84\ 0196Ð0105[ Ferrari\ D[\ Munerati\ M[\ Melchiorri\ L[\ Hanau\ D[\ Di Virgilio\ F[\ Baricordi O[R[\ 0883[ Responses to extracellular ATP of lym! phoblastoid cell lines from Duchenne muscular dystrophy patients[ Am[ J[ Physiol[ 156\ C775ÐC781[ Ferrari\ D[\ Chiozzi\ P[\ Falzoni S[\ dal Susino\ M[\ Collo\ G[\ Buell\ G[\ Di Virgilio\ F[\ 0886a[ ATP!mediated cytotoxicity in microglial cells[ Neuropharmacol[ 25\ 0184Ð0292[ Ferrari\ D[\ Chiozzi\ P[\ Falzoni\ S[\ dal Susino\ M[\ Melchiorri\ L[\ Daricordi\ O[R[\ Di Virgilio\ F[\ 0886b[ Extracellular ATP triggers IL!0beta release by activating the purinergic P1Z receptor of human macrophages[ J[ Immunol[ 048\ 0340Ð0347[ Ferrari\ D[\ Chiozzi\ P[\ Falzoni\ S[\ Hanau S[\ Di Virgilio\ F[\ 0886c[ Purinergic modulation of interleukin!0beta release from microglial cells stimulated with bacterial endotoxin[ J[ Exp[ Med[ 074\ 468Ð 471[ Ferrari\ D[\ Wesselborg\ S[\ Bauer\ M[K[A[\ Schulze!Ostho}\ K[\ 0886d[ Extracellular ATP activates transcription factor NF!kB through the P1Z purinoceptor by selectively targeting NF!kB p54 "RelA#[\ J[ Cell[ Biol[ 0524Ð0532[ Filippini\ A[\ Ta}s\ R[F[\ Sitkvsky\ M[V[\ 0889[ Extracellular ATP in T!lymphocyte activation] possible role in e}ector functions[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 76\ 7156Ð7160[ Fisette\ P[L[\ Denlinger\ L[C[\ Proctor\ R[A[\ Bertics\ P[J[\ 0885[ Modu! lation of macrophage function by P1Y!purinergic receptors[ Drug Dev[ Res[ 28\ 266Ð276[ Hasko Gy[\ Szabo Cs[\ Nemeth\ Z[\ Kvetan\ V[\ McCarthy!Pastores\ S[\ Vizi\ E[S[\ 0885[ Adenosine agonists di}erentially regulate IL! 09\ TNF!alpha\ and nitric oxide production in RAW 153[6 macro! phages and in endotoxemic mice\ J[ Immunol[ 046\ 3523Ð3539[ Hon\ W[M[\ Khoo\ H[E[\ Ngoi\ S[S[\ Moochala\ S[\ 0884[ E}ects of adenosine receptor agonists on nitric oxide release in mouse during endotoxaemia[ Biochem[ Pharmacol[ 49\ 34!36[ Humphreys\ B[D[\ Dubyak\ G[R[\ 0885[ Induction of the P1Z:P1X6 nucleotide receptor and associated phospholipase D activity by lipopolysaccharide and IFN!gamma in the human THP!0 mon! ocytic cell line[ J[ Immunol[ 046\ 4516Ð4526[ Illes\ P[\ Nieber\ K[\ Frohich\ R[\ Norenberg\ W[\ 0885[ P1 pur! inoceptors and pyrimidinoceptors of catecholamine!producing cells and immunocytes[ In] Chadwick\ D[J[\ Goode\ J[A[\ "Eds[#\ P1 purinoceptors] localization\ function and transduction mechanisms[ John Wiley and Sons\ Chichester\ 0885[ pp[ 00Ð018[ Jamieson\ G[P[\ Snook\ M[B[\ Thurlow\ P[J[\ Wiley J[S[\ 0885[ Extra! cellular ATP causes of loss of L!selectin from human lymphocytes via occupancy of P1Z purinoceptors[ J[ Cell[ Physiol[ 055\ 526Ð531[ Lammas\ D[A[\ Stober\ C[\ Harvey\ C[J[\ Kendrick\ N[\ Panchalingam\ S[\ Kumaratne D[S[\ 0886[ ATP!induced killing of mycobacteria by human macrophages is mediated by purinergic P1Z"P1X6# recep! tors[ Immunity 6\ 322Ð333[ Markwandt\ F[\ Lohn\ M[\ Bohm\ T[\ Klapperstuck\ M[\ 0886[ Pur! inoceptor!operated cationic channels in human B lymphocytes[ J[ Physiol[ "Lond# 387\ 032Ð040[
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Murgia\ M[\ Pizzo\ P[\ Steinberg\ T[H[\ Di Virgilio\ F[\ 0881[ Charac! terization of the cytotoxic e}ect of extracellular ATP in J663 mac! rophages[ Biochem[ J[ 177\ 786Ð890[ Osipchuk\ Y[\ Cahalan\ M[\ 0881[ Cell!to!cell spread of calcium signals mediated by ATP receptors in mast cells[ Nature 248\ 130Ð133[ Rassendren\ F[\ Buell\ G[N[\ Virginio\ C[\ Collo\ G[\ North\ R[A[\ Suprenant\ A[\ 0886[ The permeabilizing ATP receptor P1X6[ J[ Biol[ Chem[ 161\ 4371Ð4375[ Sperlagh\ B[\ Kittel\ A[\ Lajtha\ A[\ Vizi\ E[S[\ 0884[ ATP acts as fast neurotransmitter in rat habenula] neurochemical and enzyme! cytochemical evidence[ Neurosci[ 55\ 804Ð819[ Sperlagh\ B[\ Vizi\ E[S[\ 0885[ Neuronal synthesis\ storage and release of ATP[ Semin[ Neurosci[ 7\ 064Ð075[ Steinberg\ T[H[\ Newman\ A[S[\ Swanson\ J[A[\ Silverstein\ S[C[\ 0876[ ATP3!permeabilizes the plasma membrane of mouse macrophages to ~uorescent dyes[ J[ Biol[ Chem[ 151\ 7773Ð7777[ Suprenant\ A[\ Rassendren\ F[\ Kawashima\ E[\ North\ R[A[\ Buell\ G[\ 0885[ The cytolytic P1Z receptor for extracellular ATP identi_ed as a P1X receptor[ Science 161\ 624Ð627[ Szabo C[\ Thiemermann\ C[\ Vane\ J[R[\ 0882a[ Dihydropyridine antag! onists and agonists of calcium channels inhibit the induction of nitric oxide synthase by endotoxin in cultured macrophages[ Diochem[ Biophys[ Res[ Comm[ 085\ 714Ð729[ Szabo C[\ Wu\ C[C[\ Gross\ S[S[\ Thiemermann\ C[\ Perretti\ M[\ Vane\ J[R[\ 0882b[ Interleukin!0 contributes to the induction of nitric oxide synthase by endotoxin in vivo[ Eur[ J[ Pharmacol[ 149\ 046Ð059[ Szabo C[\ 0884[ Alterations in the production of nitric oxide in various forms of circulatory shock[ New Horiz[ 2\ 1Ð21[ Szabo C[\ 0885[ Physiological and pathophysiological roles of nitric oxide in the central nervous system[ Brain Res[ Bull[ 30\ 020Ð030[ Szabo C[\ Zingarelli\ B[\ O|Connor\ M[\ Salzman\ A[L[\ 0885[ DNA strand breakage\ activation of poly!ADP ribosyl synthetase and cellular energy depletion are involved in the cytotoxicity in mac! rophages and smooth muscle cells exposed to peroxynitrite[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 82\ 0642Ð0647[ Tonetti\ M[\ Sturla\ M[\ Bistol_\ T[\ Benatti\ U[\ DeFlora\ A[\ 0883[ Extracellular ATP potentiates nitric oxide synthase expression induced by lipopoysaccharide in RAW 153[6 murine macrophages[ Biochem[ Biophys[ Res[ Comm[ 192\ 329Ð324[ Tonetti\ M[\ Sturla\ L[\ Giovina\ M[\ Benattia\ U[\ De Flora\ A[\ 0884[ Extracellular ATP enhances mRNA levels of nitric oxide synthase and TNF!alpha in lipopolisaccharide!treated RAW 153[6 murine macrophages[ Biochem[ Biophys[ Res[ Comm[ 103\ 014Ð029[ Virginio\ C[\ Church\ D[\ North\ R[A[\ Suprenant\ A[\ 0886[ E}ect of divalent cations\ protons and calmidazolium at the rat P1X6 recep! tor[ Neuropharmacol[ 25\0174Ð0184[ Vizi\ E[S[\ Orso\ E[\ Osipenko\ O[N[\ Hasko G[\ Elenkov\ I[J[\ 0884[Neurochemical\ electrophysiological and immu! nocytochemical evidence for a noradrenergic link between the sym! pathetic nervous system and thymocytes[ Neurosci[ 57\ 0152Ð0165[ Wiley\ J[S[\ Chen\ J[R[\ Snook\ M[B[\ Jamieson\ G[P[\ 0883[ The P1Z! purinoceptor of human lymphocytes] actions of nucleotide agonists and irreversible inhibition by oxidized ATP[ Br[ J[ Pharmacol[ 001\ 835Ð849[
ATP released by LPS increases nitric oxide production in raw 153[6 macrophage cell line via P1Z:P1X6 receptors Beata Sperlagh$%\ Gyorgy Hasko$\ Zoltan Nemeth\ E[ Sylvester Vizi Department of Pharmacolo`y\ Institute of Experimental Medicine\ Hun`arian Academy of Sciences\ Budapest\ Hun`ary Received 0 April 0887\ accepted 5 April 0887
Abstract P1Z:P1X6 receptor is a particular type of purinoceptor\ which is selectively expressed on the surface of immune cells in neuronal and non!neuronal tissues[ Despite intensive research on its involvement in the immune response\ the exact mechanism whereby it a}ects intercellular signaling is far from clear yet[ In this study\ the e}ect of activation P1Z:P1X6 receptor was investigated on the bacterial lipopolysaccharide induced nitric oxide production in RAW 153[6 macrophage call line using the nitrite:nitrate assay[ The P1Z:P1X6 receptor agonist 2?!O!"3!benzoylbenzoyl#!adenosine 4?triphosphate increased concentration!dependency the lipo! plysaccharide "09mg:ml# induced nitric oxide production between 09 mM and 149 mM[ ATP also increased nitric oxide production in response to lipopolysaccharide\ while ADP\ 1!methylo!thio!adenosine 4?!triphosphate and adenosine 4?triphosphate!g!S was without e}ect[ Pretreatment with oxidized adenosine triphosphate\ the selective P1Z:P1X6 receptor antagonise "299 mMÐ0 mM# strongly decreased lipopolysaccaride induced nitric oxide production[ Furthermore\ on macrophages\ pretreated with oxidized adenosine 4?! triphosphate "299 mM −0 mM#\ 2?!O!"3!benzoylbenzoyl#!adenosine 4?!triphosphate and ATP did not a}ect lipopolysaccharide induced nitric oxide production[ 04 min lipopolysaccharide treatment induced a transient and reversible release of endogenous ATP from RAW 153[6 cells\ measured by the luciferinÐluciferase assay[ The e}ect of lipopolysaccharide to promote ATP release was concentration!dependent between 0Ð09 mg:ml[ In summary\ our results show that P1Z:P1X6 receptor activation results in an increase in nitric oxide production in response to lipopolysaccharide challenge[ Since the P1Z:P1X6 receptor antagonist oxidized adenosine triphosphate decreased lipopolysaccharide induced nitric oxide production\ and lipopolysaccharide was able to promote ATP release from macrophage cells\ it seems likely that endogenous ATP is involved in nitric oxide formation during endotoxin challenge[ Þ 0887 Elsevier Science Ltd[ All rights reserved[
0[ Introduction ATP and its metabolic degradation products\ ADP\ AMP and adenosine are accumulated and may reach high con! centration extracellularly during any kind of cell death occurring frequently during in~ammation\ ischemia\ and cell injury[ Since ATP and adenosine receptors are pre! sent on the surface of immune cells\ a possible immun! modulatory role of purine nucleotides and nucleosides has been postulated "Dubyak and El!Moatassim\ 0884^ Di Virgilio\ 0884^ Di Virgilio et al[\ 0885#[ The action of adenosine on immune cells via A0\ A1a and A2 receptors is well documented regulating phagocytosis\ chemotaxis\ cytokine "cf Cronstein\ 0883\ Di Virgilio et al\ 0885#\ and Due to circumstances beyond the Publisher|s control\ this article appears without the authors| corrections[ $ These authors contributed equally to the study[ % Corresponding author[ Tel[]9925!0!109!9708^ fax] 9925!0!109!9702^ e!mail] SPERLAGHÝKOKI[HU 9086Ð9075:87 ,08[99 Þ 0887 Elsevier Science Ltd[ All rights reserved PII] S 9 0 8 6 Ð 9 0 7 5 " 8 7 # 9 9 9 1 4 Ð 3
nitric oxide "NO# production "Hasko et al[\ 0885^ Hon et al[\ 0884#\ acting either as an endogenous pro! or anti! in~ammatory agent "cf Cronstein\ 0883\ Hasko et at[\ 0885#[ A similar function of extracellular ATP is now also being recognized] ATP release has been shown in response to antigen stimulus from di}erent kind of immune cells "Osipchuk and Cahalan\ 0881\ Filippini et al[\ 0889\ Ferrari et at[\ 0886c# which express both ionotropic and metabotropic ATP receptors "Dubyak and El!Moatassim\ 0884#[ Among the number of P1 purinoceptor subtypes\ the function of the so!called P1Z receptors appears to be particularly intriguing\ due to their distinguished expre! ssed in the immune cells of neuronal and non!neuronal tissues "Illes et al[\ 0885^ Chessell et al[\ 0886\ Rassendren et al[\ 0886^ Collo et al[\ 0886#[ These receptors have recently been identi_ed as the seventh member of the ion! channel type P1X receptor family "Suprenant et al[\ 0885# and their expression is upregulated by the induction of immune system by interferon!g and bacterial lipo!
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polysaccharide "LPS#\ suggesting that they are speci_! cally involved in the macrophage response "Blanchard et al[\ 0880\ Humphreys and Dubyak\ 0885^ Falzoni et al[\ 0884#[ Activation of the P1Z:P1X6 receptor results in a pore formation "Steinberg et al[\ 0876^ Falzoni et al[\ 0884\ Virginio et al[\ 0886# which makes the membrane permeable to higher molecular weight substances up to several hundred Da and sustained activation of the chan! nel therefore causes cell death "Murgia et al[\ 0881\ Fal! zoni et al[\ 0884^ Chiozzi et al[\ 0885\ Lammas et al[\ 0886^ Ferrari et al[\ 0886a#[ Nevertheless\ their function may not be restricted to citotoxicity] a line of a recent studies suggest that they also play a role in intercellular signaling during in~ammation process\ i[e[\ they regulate the pro! duction of interleukin*0b "0LÐ0b# in mouse and human macrophages "Ferrari et at[\ 0886b^ Ferrari et al[\ 0886c# and in microglial cells "Ferrari et al[\ 0886c#\ as well as phospholipase D activity "Humphreys and Dubyak\ 0885^ El!Moatassim and Dubyak\ 0882# in response to bacterial lipopolysaccharide "LPS# challenge[ On the con! trary\ it is less clear\ whether P1Z:P1X6 receptors are involved in the production of other in~ammatory mess! engers\ such as NO production[ Although earlier studies indicated that extracellular ATP is a potent co!stimulator of NO synthase expression during LPS induction in mac! rophages "Tonetti et al[\ 0884^ Tonetti et al[\ 0883^ Den! linger et al[\ 0885#\ P1Z:P1X6 selective probes have not been tested\ and the receptor identity remains unidenti! _ed so far[ We therefore investigated the e}ect of di}erent P1 receptor ligands on LPS!induced NO production in the RAW 153[6 macrophage cell line[ Since speci_c inhibition of P1Z:P1X6 receptors by oxidized adenosine tri! phosphate "oxyATP# dramatically reduced LPS!induced NO production\ indicating the regulatory action of endogenously released APT[ The ability of LPS to release ATP from macrophages was also examined[
in 4) phosphoric acid# to 099 ml samples of medium\ respectively[ The OD at 449 nm "OD449# was measured using a Spectramax 149 microplate reader "Molecular Devices\ Sunnyvale\ CA#[ 1[2[ Measurement of mitochondrial respiration Mitochondrial respiration\ an2 indicator of cell viability\ was assessed by the mitochondria!dependent reduction of 2!"3\4!dimethylthiazol!1!yl#!1\4!diphenyltetrazolium bromide "MTT# to formazan "Szabo et al[\ 0882a#[ Cells in 85!well plates were incubated with MTT "9[1 mg:ml# for 59 min at 26>C[ Culture medium was removed by aspiration\ and cells were solubilized in DMSO "099 ml#[ The extent of reduction of MTT to formazan within cells was quantitated by measurement of OD449 using a Spectramax microplate reader[ 1[3[ ATP release experiments 3 day!old cultures of RAW 153[6 cells were used for release experiments[ Cells were gently removed from cul! ture ~asks and suspended in DMEM preheated to 26>C[ After cell counting and two periods of centrifugation at 1999 rpm for 09 min at 09>C\ the cells were resuspended in Gey|s balanced salt solution "GBSS\ from SIGMA\ containing] CaCl1 9[1140\ MgCl1 9[10\ MgSO3 9[9231\ KCl\ 9[26\ KH1PO3 9[92\ NaHCO2 1[16\ NaCl 6[9\ NaH1PO3 9[0085\ D!Glucose\ 3[9\ phenol red\ 9[9095 pH 6[3# obtaining 49 million cell:ml concentration[ 49 ml of this cell suspension were injected into poly! propylene tissue chambers\ separated by Millipore _lters of 9[1 mm diameter[ Cells were superfused at a rate of 9[1 ml:min with GBSS for 59 min at 26>C and subsequently 2 min perfusate samples were collected[ LPS "09 mg:ml# was applied by perfusion\ for 04 min[ ATP content of the samples were measured by the luciferinÐluciferase assay\ as previously described "Sperlagh et al[\ 0884# and were expressed in nmol:l[
1[ Experimental procedures 1[4[ Statistical evaluation 1[0[ Cell Culture The mouse macrophage cell line RAW 153[6 was cultured in Dulbecco|s modi_ed Eagle|s medium "DMEM#[ Cells were exposed to LPS "09 mg:ml# for 13 h[ BzATP and other ATP analogues was applied for a 04 min exposure after LPS challenge\ oxyATP was applied 1 h before LPS treatment[ 1[1[ Measurement of nitrite concentration Nitrite in culture supernatants at 13 h "after conversion of nitrate to nitrite# was measured at previously described "Szabo et al[\ 0882a#\ by adding 099 ml of Griess reagent "0) sulfanilaminde and 9[0) naphthylethylenediamide
Values in the _gures and text are expressed as mean2S[E[M[ of n observations[ Statistical analysis of the data was performed by one or two!way analysis of variance followed by Dunnett|s test\ as appropriate[ P ³ 9[94 was considered statistically signi_cant[ 1[5[ Materials 2?!O!"3!benzoylbenzoyl#!adenosine 4?!triphosphate "BzATP#\ adenosine 4?triphosphate "ATP#\ adenosine 4?diphosphate "ADP#\ adenosine 4?triphosphate!g!S "ATP!g!S#\ 1!methyl!thio!adenosine 4?triphosphate "1! methyl!thioATP#\ oxidized adenosine triphosphate "oxy! ATP#\ and suramin were used[
B[ Sperla`h et al[:Neurochem[ Int[ 22 "0887# 198Ð104
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DMEM was purchased from Life Technologies "Grand Island\ NY\ U[S[A[#\ 1!methyl!thioATP was obtained from Research Biochemicals Inc[ "Natick\ MA\ U[S[A[#[ Suramin was the gift of Bayer "Leverkusen\ Ger! many#[ LPS from Escherichia coli 944]B4 and all other drugs were obtained from Sigma "St[ Louis\ MO\ U[S[A[#[
2[ Results 2[0[ NO release study In the absence of drugs\ the basal nitrite production of the RAW 153[6 culture was 1[6529[09 mM "n 5#[ In response to 13 h exposure of LPS "09 mg:ml# stimulation\ the nitrite level increased to 55[4421[37 "n 5\ P ³ 9[990# indicating the activation of inducible nitric oxide synthase enzyme[ Neither of the applied drugs a}ected signi_cantly the unstimulated nitrite level "data not shown#[ The P1Z:P1X6 receptor agonist 2?!O!"3!benzoylbenzoyl#! adenosine 4?!triphosphate "BzATP#\ when added to the cells 04 min after the onset of LPS treatment and kept in the ~uid throughout the experiments\ increased LPS induced NO production between 0 mM and 149 mM "Fig[ 0A#[ Its e}ect reached the level of signi_cance at 49 mM concentration\ and increased NO production con! centration!dependently up to 149 mM\ and no further stimulation of NO production was observed above this concentration[ At 499 mM concentration\ its e}ect declined and turned back to control level[ To assess cell viability and possible cytotoxic e}ect of BzATP\ mito! chondrial respiration was monitored concomitantly] apart from a subtle reduction observed at 149 mM BzATP "from 6720[24Ð6120[3) n 5\ P ³ 9[94#\ no sig! ni_cant inhibition of mitochondrial respiration was detected in the concentration range used in the study showing the facilitatory action of BzATP on LPS induced NO production is not related to cytotoxicity[ The e}ect of BzATP "099 mM# was also examined using di}erent LPS concentrations\ ranging from 9[90Ð 09 mg:ml "Fig[ 0C#[ At lower LPS concentrations "9[90Ð 9[0 mg:ml#\ BzATP did not promote NO production\ while at the higher range "0Ð09 mg:ml# its action became manifest\ and powerfully enhanced the e}ect of LPS "Fig[ 0C#[ ATP\ the endogenous agonist of P1Z:P1X6 receptors\ had a biphasic e}ect] between 29Ð299 mM it also increased NO production^ however the maximal response obtained by ATP was considerably less than in case of BzATP\ suggesting that it might act as a partial agonist "Fig[ 0B#[ In contrast\ APT\ when applied at 0999 mM\ strongly inhibited LPS induced NO production\ and also mark! edly reduced mitochondrial respiration[ Since the stimu! latory e}ect of BzATP reached the level of signi_cance at lower concentration "49 mM#\ than APT "299 mM#\
Fig[ 0[ E}ect of BzATP "A# and ATP "B# on LPS induced nitrite production and measured in RAW 153[6 macrophage cells[ Cells were stimulated with LPS "09 mg:ml# for 13 h in the presence of various concentrations of BzATP "0Ð499 mM# and ATP "29Ð0999 mM#\ and nitrite levels measured in the supernatants[ BzAPT and ATP was applied 04 min after LPS treatment[ "C# shows the e}ect of 099 mM BzATP on LPS induced nitrite production at di}erent concentrations of LPS stimuli "9[90Ð09 mg:ml#[ Nitrite production was expressed as the percentage of LPS stimulated control "A\ B# or in mM "C#[ Data show the mean2S[E[M[ of 5Ð04 wells[ Asterisks indicate signi_cant di}er! ences from respective controls[
BzATP appears to be a more potent agonist promoting LPS induced NO formation[ Other ATP analogues\ such as ADP\ 1!methyl!thioATP and ATP!g!S were without e}ect up to 0999 mM "data not shown#[
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Table 0 Inhibition of LPS induced nitrite production and reversal of the fac! ilitatory action of BzATP and ATP by oxyATP "29Ð0999 mM# in RAW 153[6 macrophages *
BzATP
ATP
* oxyATP 299 mM oxyATP 0999 mM
55[421[68 26[2822[50 2[4429[35
70[9420[50 35[9220[40 2[4729[31
65[1620[23 33[2729[71 3[8929[30
Cells were stimulated by 09 mg:ml LPS after 1 h preincubation with di}erent concentrations of oxyATP[ BzATP "099 mM# and ATP "299 mM# were applied 04 min after LPS treatment[ Nitrite concentrations were measured 13 h after LPS administration in the supernatants and expressed in mM[ Data show the mean2S[E[M[ of 5 wells[ Shows signi_cant di}erences between P1Z:P1X6 receptor agonist tre! ated cells and drug free controls "p³9[990#[ Shows signi_cant di}erences between oxyATP treated and non!tre! ated cells "p³9[990#[
Two h preincubation of the cells with oxidized aden! osine triphosphate "oxyATP\ 299Ð0999 mM#\ the selective and irreversible antagonist of P1Z:P1X6 receptors\ decreased dose!dependently LPS induced NO production "Table 0#[ Furthermore\ neither BzATP nor ATP were able to a}ect LPS induced NO production on macro! phages\ pretreated with oxyATP 8299 mMÐ0 mM "Table 0#[ OxyATP\ applied at 299 mM did not a}ect signi_cantly cell viability\ while at 0999 mM concentrations\ impair! ment of mitochondrial respiration was observed[ Another P1 receptor antagonist\ suramin had no e}ect on LPS induced NO release "data not shown#[ 2[1[ ATP release study The basal out~ow of ATP measured by the luciferinÐ luciferase assay from cultured RAW 153[6 cells during 2 min collection period was 9[2429[97 nmol:l "n 4#\ and remained constant throughout the sample collecting per! iod "Fig[ 1#[ To test the ability of LPS to release ATP\ macrophage cells were perfused with medium containing 09 mg:ml LPS for 04 min[ This treatment induced a tran! sient and reversible release of endogenous ATP\ which increased the ATP out~ow to 0[4729[24 nmol:l at the peak of the response "n 4#[ The total ATP release evoked by LPS was 2[2729[75 nmol "n 4#[ The e}ect of LPS was concentration!dependent between 0Ð09 mg:ml\ 0mg:ml LPS elicited a release of 9[4029[02 nmol ATP "n 5\ P ³ 9[94#[
3[ Discussion The question addressed in this study was to show whether LPS induced NO production in RAW 153[6 macrophages is regulated by P1Z:P1X6 receptors[ The following data
support this hypothesis] "0# BzATP\ the selective R1Z:P1X6 receptor agonist and ATP increased nitrite production evoked by endotoxin challenge in a dose dependent fashion\ BzATP being more potent that ATP\ while ADP\ 1!methyl!thioATP and ATP!g!S were with! out e}ect[ This pro_le is consistent with the typical P1Z:P1X6 receptor pro_le "Humphreys and Dubyak\ 0885^ Lammas et at[\ 0886\ Markwandt et al[\ 0886^ Ras! sendren et al[\ 0886^ Jamieson et al[\ 0885^ Chessell at al[\ 0886^ Falzoni et al[\ 0884# and is similar to the cloned P1X6 receptor pro_le "Suprenant et al[\ 0885# with the di}erence that the cloned receptor exhibits some binding activity for ADP\ 1!methyl!thioATP and ATP!g!S\ and were inactive in our study[ A possible explanation of this dissimilarity is that RAW 153[6 macrophages*in contract to HEK 182 cells\ in which the cloned P1Z:P1X6 receptor was studied*express not only P1Z:P1X6 recep! tors\ but also the metabotropic P1Y type receptors "Dubyak and El!Moatassim\ 0884\ Denlinger et al\ 0885#\ and NO production could thereby be also regulated[ Therefore the e}ect of analogues acting not exclusively on P1Z:P1X6 receptors might interfere with their action on other P1 receptors "1#[ Both BzATP and ATP were active at the high micromolar range\ which also _ts well to the usual P1Z:P1X6 receptor pro_le[ The other charac! teristic feature of this receptor is that the fully ionized species "ATP3−# is the active ligand which is recognized "Steiberg et al[\ 0876^ Markwandt et al[\ 0886^ Falzoni et al[\ 0884#[ "2# Since ATP3− represents a small fraction of the total extracellular ATP content under physiological pH\ generally at least one order of magnitude higher concentration of ATP is necessary to activate P1Z:P1X6 receptors and obtain measurable e}ects\ than for other subtypes of P1 receptors[ "3# Prolonged incubation with the irreversible P1Z:P1X6 receptor antagonist\ OxyATP abolished the stimulatory e}ects of both BzATP and ATP on nitrite production\ which also strongly indicates the involvement of P1Z:P1X6 receptors in this e}ect[ Oxy! ATP might be active on other type of P1 receptors as well "Wiley et al[\ 0883^ Ferrari et al[\ 0883#[ However\ conclusive evidence is missing in this respect[ In addition to oxyATP\ pyridoxal!phosphate!5!azophenyl!1?\3?!dis! ulphonic acid tetrasodium "PPADS# is another antag! onist acting presumably on P1Z:P1X6 receptors "Suprenant et al[\ 0885^ Rassendren et al[\ 0886#^ however it was unfortunately unsuitable in our model\ due to its interference with photometry[ Suramin\ a non!selective P1 antagonist proved to be ine}ective in this study\ in agreement with the P1Z:P1X6 receptor character "Ras! sendren et al[\ 0886^ Chessell et al[\ 0886#[ Therefore\ taken together the _ndings\ the most likely receptor responsible for the synergizing action of ATP analogues on LPS induced nitrite production is the P1Z:P1X6 subtype[ Concerning the e}ect of ATP\ our results are in cor! roboration with the _ndings of Tonetti et al[ "0883\ 0884#\
B[ Sperla`h et al[:Neurochem[ Int[ 22 "0887# 198Ð104
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Fig[ 1[ E}ect of LPS on ATP release measured from superfused RAW 153[6 cells[ Cells were perfused with GBSS solution with 9[1 ml:min and after a 59 min preperfusion\ 2 min samples were collected and assayed for ATP[ ATP levels were measured by the luciferin luciferase assay and expressed in nmol:l\ as a function of time[ LPS was applied into the perfusion solution in 09 mg:ml for 04 min\ as indicated[ Open circles represent the results in the absence of LPS\ _lled circles show values obtained in experiments with LPS treatment[ Data show the mean2S[E[M[ of 4!4 separate experiments[ Asterisks indicate signi_cant di}erences between LPS treated and control cells calculated by two way ANOVA[
who found that high concentration of extracellular ATP enhances nitric oxide synthase expression and nitrite pro! duction induced by LPS in RAW 153[6 macrophages[ More recently\ Denlinger et al[ "0885# examined a series of P1 receptor ligands on LPS induced nitrite production in RAW 153[6 cells\ and all three studies identi_ed puta! tively the receptors regulating NO production in mac! rophages as P1Y purinoceptors[ However\ speci_c ligands of P1Z:P1X6 receptors\ such as BzATP or oxy! ATP has not been tested in these studies[ Our _ndings do not reject the assumption that P1Y receptors are present on macrophages\ but show that in addition to P1Y recep! tors\ NO production is also regulated by P1Z:P1X6 receptors[ At millimolar concentrations\ ATP caused a marked inhibition of LPS induced nitrite production\ and the e}ect of BzATP was also declined at the high micromolar range[ Since mitochondrial respiration was impaired\ when such a high concentration of ATP analogues were applied these e}ects are most likely non!speci_c and related to the known cytotoxic e}ect of these ligands "Falzoni et al[\ 0884^ Ferrari et al[\ 0886b^ Murgia et al[\ 0881^ Chiozzi et al[\ 0885^ Ferrari et al[\ 0886a#\ and also suggest a di}erential action of extracellular ATP\ when it is present at lower or higher concentration[ The question arises\ which subcellular pathway is
responsible for the initiation of P1Z:P1X6 receptor acti! vated NO production in macrophages during endotoxin challenge< There are several alternative possibilities[ On one hand\ it is known that P1Z:P1X6 receptor activation promotes Ca1¦ in~ux in monocytes\ and the Ca1¦ signal may potentiate LPS induced activation of inducible NO synthase enzyme[ However\ studies showing that mac! rophage iNOS activity proved to be Ca1¦ independent argue against this route "Szabo\ 0882a\ 0884#[ On the other hand\ as it has been reported that P1X:P1X6 recep! tor stimulation is involved in LPS induced phospholipase D activation "Humphreys and Dubyak\ 0885^ El!Moat! assim and Dubyak\ 0882^ Fisette et at[\ 0885#\ yet another possible pathway is the activation of NF!kB or other transcription factors by phospholipase D and the sub! sequent stimulation of iNOS enzyme\ which is favored by a very recent study showing that extracellular ATP activates a particular subunit of NF!kB in mouse mic! ro~ial cells via P1A purinoceptors "Ferrari et al[\ 0886d#[ A third possibility is that NO production of macrophages by P1Z:P1X6 receptor is an indirect mechanism] recent evidence suggest that extracellular ATP triggers IL!0b secretion activating P1Z:P1X6 receptors in LPS primed macrophages "Ferrari et al[\ 0886b] Ferrari et al[\ 0886c# and IL!0b could initiate iNOS expression\ as described by Szabo et al[ "0882b#[ This explanation seems to be
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particularly attractive\ because oxyATP proved to be a strong inhibitor LPS induced IL!0b secretion in micro! glial cells and macrophages "Ferrari et al[\ 0886c# as well as LPS induced NO production in our experiments[ How! ever\ ADP which is a powerful stimulator of IL!0b secretion\ was ine}ective in this study[ An important observation of this study in the powerful action of the irreversible P1Z:P1X6 receptor antagonist OxyATP to inhibit LPS induced nitrite production[ Apart from at 0999 mM concentration\ when oxyATP impaired mitochondrial respiration\ at 299 mM concentration\ it reduced by more than 39) LPS!induced nitrite production\ without any signi_cant action on cell viability[ Therefore\ at least at this lower concentration its e}ect can be attributed to the speci_c blockade of P1Z:P1X6 receptors\ and displacing the action of any endogenous ligand on this receptor[ To prove this assumption the ability to LPS exposition to promote endogenous ATP release from RAW 153[6 cells was tested] indeed it induced ATP out~ow in the time frame "04 min# when P1Z:P1X6 receptors were activated[ Although the amount of ATP out~ow evoked by LPS was only moderate\ taken into account its dilution in the superfusate and the possibility of rapid breakdown in the extracellular ~uid\ its biophase concentration might be higher than we detected[ Furthermore under in vivo con! ditions\ there are several other sources from where ATP can be released at the in~ammation site and reach P1Z:P1X6 receptors] it can be released together with noradrenaline from catecholaminergic nerves "cf Sper! lagh and Vizi\ 0885# innervating the immune system such as spleen "Elenkov and Vizi\ 0880# and thymus "Vizi et al\ 0884#^ and it can be released from endothelial cells and platelets inside the circulation "Boeynaems and Pearson\ 0889#[ Furthermore\ it can be the result of P1Z:P1X6 receptor activation itself] interestingly\ Ballerini et al[ "0885#\ reported purine release from astrocytes in response to BzATP and other P1 receptor agonists[ ATP out~ow via P1Z:P1X6 receptor channel may even act as an amplifying signal reinforming other actions such as IL!0b secretion on the same receptors[ Finally\ an impor! tant source of extracellular ATP accumulation during in~ammation might be cell leakage] although ATP release demonstrated by us occurred below cytotoxic LPS con! centrations\ depending on the severity of in~ammation\ cytolytic ATP might be also important factor to in~uence LPS induced activation of immune system[ In summary\ we report that NO production to LPS challenge in RAW 153[6 macrophages is regulated by P1Z:P1X6 receptors[ These _ndings support the view that P1 purinoceptors serve as an important control of the macrophage response during in~ammation and immune response[ Due to the generation of toxic peroxynitrite radicals\ NO production in Macrophages in considered a key event during endotoxin shock "cf Szabo\ 0884\ 0885^ Szabo et al\ 0885#\ and any subcellular target\ which
selectively modulates this pathway might be of clinical interest[ Since P1Z:P1X6 receptors are expressed activity! dependently in immune cells\ they o}er an attractive therapeutic target\ intervening in septic shock and other in~ammatory diseases[
Acknowledgements This study was supported by the grants of the Hungarian Science Foundation "OTKA T914503# and Hungarian Medical Research Council "ETT086#[ The authors are grateful to Ms Julianna Benko½ for excellent technical assistance and to Ms E łva Vass for cell culture assistance[
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Murgia\ M[\ Pizzo\ P[\ Steinberg\ T[H[\ Di Virgilio\ F[\ 0881[ Charac! terization of the cytotoxic e}ect of extracellular ATP in J663 mac! rophages[ Biochem[ J[ 177\ 786Ð890[ Osipchuk\ Y[\ Cahalan\ M[\ 0881[ Cell!to!cell spread of calcium signals mediated by ATP receptors in mast cells[ Nature 248\ 130Ð133[ Rassendren\ F[\ Buell\ G[N[\ Virginio\ C[\ Collo\ G[\ North\ R[A[\ Suprenant\ A[\ 0886[ The permeabilizing ATP receptor P1X6[ J[ Biol[ Chem[ 161\ 4371Ð4375[ Sperlagh\ B[\ Kittel\ A[\ Lajtha\ A[\ Vizi\ E[S[\ 0884[ ATP acts as fast neurotransmitter in rat habenula] neurochemical and enzyme! cytochemical evidence[ Neurosci[ 55\ 804Ð819[ Sperlagh\ B[\ Vizi\ E[S[\ 0885[ Neuronal synthesis\ storage and release of ATP[ Semin[ Neurosci[ 7\ 064Ð075[ Steinberg\ T[H[\ Newman\ A[S[\ Swanson\ J[A[\ Silverstein\ S[C[\ 0876[ ATP3!permeabilizes the plasma membrane of mouse macrophages to ~uorescent dyes[ J[ Biol[ Chem[ 151\ 7773Ð7777[ Suprenant\ A[\ Rassendren\ F[\ Kawashima\ E[\ North\ R[A[\ Buell\ G[\ 0885[ The cytolytic P1Z receptor for extracellular ATP identi_ed as a P1X receptor[ Science 161\ 624Ð627[ Szabo C[\ Thiemermann\ C[\ Vane\ J[R[\ 0882a[ Dihydropyridine antag! onists and agonists of calcium channels inhibit the induction of nitric oxide synthase by endotoxin in cultured macrophages[ Diochem[ Biophys[ Res[ Comm[ 085\ 714Ð729[ Szabo C[\ Wu\ C[C[\ Gross\ S[S[\ Thiemermann\ C[\ Perretti\ M[\ Vane\ J[R[\ 0882b[ Interleukin!0 contributes to the induction of nitric oxide synthase by endotoxin in vivo[ Eur[ J[ Pharmacol[ 149\ 046Ð059[ Szabo C[\ 0884[ Alterations in the production of nitric oxide in various forms of circulatory shock[ New Horiz[ 2\ 1Ð21[ Szabo C[\ 0885[ Physiological and pathophysiological roles of nitric oxide in the central nervous system[ Brain Res[ Bull[ 30\ 020Ð030[ Szabo C[\ Zingarelli\ B[\ O|Connor\ M[\ Salzman\ A[L[\ 0885[ DNA strand breakage\ activation of poly!ADP ribosyl synthetase and cellular energy depletion are involved in the cytotoxicity in mac! rophages and smooth muscle cells exposed to peroxynitrite[ Proc[ Natl[ Acad[ Sci[ U[S[A[ 82\ 0642Ð0647[ Tonetti\ M[\ Sturla\ M[\ Bistol_\ T[\ Benatti\ U[\ DeFlora\ A[\ 0883[ Extracellular ATP potentiates nitric oxide synthase expression induced by lipopoysaccharide in RAW 153[6 murine macrophages[ Biochem[ Biophys[ Res[ Comm[ 192\ 329Ð324[ Tonetti\ M[\ Sturla\ L[\ Giovina\ M[\ Benattia\ U[\ De Flora\ A[\ 0884[ Extracellular ATP enhances mRNA levels of nitric oxide synthase and TNF!alpha in lipopolisaccharide!treated RAW 153[6 murine macrophages[ Biochem[ Biophys[ Res[ Comm[ 103\ 014Ð029[ Virginio\ C[\ Church\ D[\ North\ R[A[\ Suprenant\ A[\ 0886[ E}ect of divalent cations\ protons and calmidazolium at the rat P1X6 recep! tor[ Neuropharmacol[ 25\0174Ð0184[ Vizi\ E[S[\ Orso\ E[\ Osipenko\ O[N[\ Hasko G[\ Elenkov\ I[J[\ 0884[Neurochemical\ electrophysiological and immu! nocytochemical evidence for a noradrenergic link between the sym! pathetic nervous system and thymocytes[ Neurosci[ 57\ 0152Ð0165[ Wiley\ J[S[\ Chen\ J[R[\ Snook\ M[B[\ Jamieson\ G[P[\ 0883[ The P1Z! purinoceptor of human lymphocytes] actions of nucleotide agonists and irreversible inhibition by oxidized ATP[ Br[ J[ Pharmacol[ 001\ 835Ð849[