Stress-activated Protein Kinase/JNK Activation and Apoptotic Induction by the Macrophage P2X7 Nucleotide Receptor

Abstract

In human and rodent macrophages, activation of the P2X7 nucleotide receptor stimulates interleukin-1β processing and release, apoptosis, and killing of intracellular Mycobacterium tuberculosis. Signaling pathways downstream of this ionotropic ATP receptor are poorly understood. Here we describe the rapid activation of the stress-activated protein kinase (SAPK)/JNK pathway in BAC1 murine macrophages stimulated by extracellular ATP. Brief exposure of the cells to ATP (10–30 min) was sufficient to trigger a rapid accumulation of activated SAPK that was then sustained for >120 min. Several observations indicated that the P2X7 receptor mediated this effect. 1) ATP and 3′-O-(4-benzoyl)benzoyl-ATP were the only agonistic nucleotides. 2) The effect was inhibited by oxidized ATP and the isoquinoline KN-62, two known P2X7 receptor antagonists. 3) ATP-induced SAPK activation could be recapitulated in P2X7 receptor-transfected HEK293 cells, but not in wild-type HEK293 cells. Because P2X7 receptor stimulation can rapidly activate caspase family proteases that have been implicated in the induction of the SAPK pathway, we investigated whether ATP-dependent SAPK activation involved such proteases. Brief exposure of BAC1 macrophages to extracellular ATP induced DNA fragmentation, α-fodrin breakdown, and elevated levels of caspase-3-type activity. Asp-Glu-Val-Asp-cho, a caspase-3 inhibitor, inhibited ATP-induced DNA fragmentation and α-fodrin proteolysis, but had no effect on ATP-induced SAPK activation. Tyr-Val-Ala-Asp-chloromethyl ketone, a caspase-1 inhibitor, prevented ATP-induced release of processed interleukin-1β, but not ATP-dependent SAPK activity. We conclude that activation of ionotropic P2X7 nucleotide receptors triggers a strong activation of SAPK via a pathway independent of caspase-1- or caspase-3-like proteases. In human and rodent macrophages, activation of the P2X7 nucleotide receptor stimulates interleukin-1β processing and release, apoptosis, and killing of intracellular Mycobacterium tuberculosis. Signaling pathways downstream of this ionotropic ATP receptor are poorly understood. Here we describe the rapid activation of the stress-activated protein kinase (SAPK)/JNK pathway in BAC1 murine macrophages stimulated by extracellular ATP. Brief exposure of the cells to ATP (10–30 min) was sufficient to trigger a rapid accumulation of activated SAPK that was then sustained for >120 min. Several observations indicated that the P2X7 receptor mediated this effect. 1) ATP and 3′-O-(4-benzoyl)benzoyl-ATP were the only agonistic nucleotides. 2) The effect was inhibited by oxidized ATP and the isoquinoline KN-62, two known P2X7 receptor antagonists. 3) ATP-induced SAPK activation could be recapitulated in P2X7 receptor-transfected HEK293 cells, but not in wild-type HEK293 cells. Because P2X7 receptor stimulation can rapidly activate caspase family proteases that have been implicated in the induction of the SAPK pathway, we investigated whether ATP-dependent SAPK activation involved such proteases. Brief exposure of BAC1 macrophages to extracellular ATP induced DNA fragmentation, α-fodrin breakdown, and elevated levels of caspase-3-type activity. Asp-Glu-Val-Asp-cho, a caspase-3 inhibitor, inhibited ATP-induced DNA fragmentation and α-fodrin proteolysis, but had no effect on ATP-induced SAPK activation. Tyr-Val-Ala-Asp-chloromethyl ketone, a caspase-1 inhibitor, prevented ATP-induced release of processed interleukin-1β, but not ATP-dependent SAPK activity. We conclude that activation of ionotropic P2X7 nucleotide receptors triggers a strong activation of SAPK via a pathway independent of caspase-1- or caspase-3-like proteases. lipopolysaccharide interleukin-1β stress-activated protein kinase c-Jun N-terminal kinase tumor necrosis factor-α mitogen-activated protein kinase/extracellular signal-regulated kinase kinase MEK kinase glutathioneS-transferase enzyme-linked immunosorbent assay Asp-Glu-Val-Asp-cho Tyr-Val-Ala-Asp-chloromethyl ketone human embryonic kidney dithiothreitol aldehyde nuclear factor of activated T cells tumor necrosis factor receptor-associated factor The P2X7 nucleotide receptor belongs to the P2X family of ATP-gated ion channels. This family comprises seven distinct gene products, each possessing two putative membrane-spanning domains with intracellular N and C termini (for review, see Refs. 1North R.A. Barnard E.A. Curr. Opin. Neurobiol. 1997; 7: 346-357Crossref PubMed Scopus (426) Google Scholar and 2Evans R.J. Surprenant A. North R.A. Turner J.T. Weisman G.A. Fedan J.S. The P2 Nucleotide Receptors. Humana Press Inc., Totowa, NJ1998: 43-61Crossref Google Scholar). The P2X7 receptor is identical to the functionally defined P2Z receptor and is expressed primarily in hematopoietic cells and a limited number of other cell types including parotid acinar cells, testis, and fibroblasts (3Collo G. Neidhart S. Kawashima E. Kosco-Vilbois M. North R.A. Buell G. Neuropharmacology. 1997; 36: 1277-1283Crossref PubMed Scopus (439) Google Scholar, 4Tenneti L. Gibbons S.J. Talamo B.R. J. Biol. Chem. 1998; 273: 26799-26808Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar, 5Solini A. Chiozzi P. Morelli A. Fellin R. Di Virgilio F. J. Cell Sci. 1999; 112: 297-305Crossref PubMed Google Scholar). As a non-desensitizing, nonselective cation channel with low affinity for ATP, P2X7 receptor activation requires millimolar extracellular ATP in the presence of divalent cations. Channel opening triggers rapid depolarization, calcium influx, and equilibration of sodium and potassium gradients. Because individual P2X7 receptor proteins contain only two transmembrane-spanning segments, it is assumed that the functional channels are oligomeric complexes composed of several individual subunits. Recent studies indicate that recombinant P2X7 receptor subunits can self-assemble during in vitro translation and processing into stable, detergent-resistant complexes (6Torres G.E. Egan T.M. Voigt M.M. J. Biol. Chem. 1999; 274: 6653-6659Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar). P2X7 receptor activation additionally induces a nonselective pore able to pass molecules up to 800 Da, a characteristic shared to a lesser degree by other P2X members (7Virginio C. Mackenzie A. Rassendren F.A. North A. Surprenant A. Nat. New Biol. 1999; 2: 315-321Google Scholar, 8Khakh B.S. Lester H.A. Neuron. 1999; 23: 653-658Abstract Full Text Full Text PDF PubMed Scopus (83) Google Scholar). Pore structure remains uncharacterized. The ability of heterologously expressed P2X7 receptors to reconstitute ATP-dependent channel/pore formation has been interpreted as evidence that the pore reflects either further multimerization of the P2X7 channels or a dynamic change in the selectivity filter of P2X7 channels (9Khakh B.S. Bao X.R. Labarca C. Lester H.A. Nat. Neurosci. 1999; 2: 322-330Crossref PubMed Scopus (317) Google Scholar). Other data suggest that the channel and the pore are separate entities (10Schilling W.P. Waslyna T. Dubyak G.R. Humphreys B.D. Sinkins W.G. Am. J. Physiol. 1999; 277: C766-C776Crossref PubMed Google Scholar). Depending on cell background, activation of the P2X7 receptor triggers diverse physiologic processes. For example, human monocytes primed by bacterial endotoxin/lipopolysaccharide (LPS)1 respond to extracellular ATP with the caspase-1-dependent proteolytic maturation and externalization of IL-1β (11Hogquist K.A. Nett M.A. Unanue E.R. Chaplin D.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8485-8491Crossref PubMed Scopus (429) Google Scholar, 12Perregaux D. Gabel C.A. J. Biol. Chem. 1994; 269: 15195-15203Abstract Full Text PDF PubMed Google Scholar, 13Ferrari D. Chiozzi P. Falzoni S. Dal Susino M. Melchiorri L. Baricordi O.R. Di Virgilio F. J. Immunol. 1997; 159: 1451-1458PubMed Google Scholar). Apoptosis is another prominent consequence of P2X7 receptor activation in various types of leukocytes. Zanovello et al. (14Zanovello P. Bronte V. Rosato A. Pizzo P. Di Virgilio F. J. Immunol. 1990; 145: 1545-1556PubMed Google Scholar) first demonstrated ATP-dependent apoptosis in a lymphocyte cell line, and Zheng et al. (15Zheng L.M. Zychlinsky A. Liu C.C. Ojcius D.M. Young J.D. J. Cell Biol. 1991; 112: 279-288Crossref PubMed Scopus (291) Google Scholar) confirmed this observation in murine thymocytes. Hogquist et al. (11Hogquist K.A. Nett M.A. Unanue E.R. Chaplin D.D. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 8485-8491Crossref PubMed Scopus (429) Google Scholar) later showed that brief (30 min) exposure of thioglycolate-elicited murine peritoneal macrophages to ATP was sufficient to initiate the signaling cascade that leads to apoptotic death. Subsequent studies have specifically implicated the P2X7 receptor in mediating ATP-induced apoptosis of human macrophages, mesangial cells, dendritic cells, and microglial cells (16Lammas D.A. Stober C. Harvey C.J. Kendrick N. Panchalingam S. Kumararatne D.S. Immunity. 1997; 7: 433-444Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 17Schulze-Lohoff, E., Hugo, C., Rost S., Arnold, S., Gruber, A., Brune, B., and Sterzel, R. B. (1998) Am. J. Physiol.F962–F971Google Scholar, 18Coutinho-Silva R. Persechini P.M. Da Cunha Bisaggio R. Perfettini J. Torres de Santo A.C. Kanellopoulos J.M. Mottaly I. Dautry-Varsat A. Ojcius D.M. Am. J. Physiol. 1999; 276: C1139-C1147Crossref PubMed Google Scholar, 19Ferrari D. Los M. Bauer M.K. Vandenabeele P. Wesselborg S. Schulze-Osthoff K. FEBS Lett. 1999; 447: 71-75Crossref PubMed Scopus (243) Google Scholar). As with most examples of apoptosis, the P2X7 receptor-initiated cascade involves a defined sequence of phenotypic changes that culminate in death only several hours after the transient exposure to ATP. Agonist-occupied P2X7 receptors also drive signals that induce nuclear accumulation of various activated transcription factors, such as NFAT within minutes or NF-κB within hours (20Ferrari D. Wesselborg S. Bauer M. Schulze-Osthoff K. J. Cell Biol. 1997; 139: 1635-1643Crossref PubMed Scopus (259) Google Scholar, 21Ferrari D. Stroh C. Schulze-Osthoff K. J. Biol. Chem. 1999; 274: 13205-13210Abstract Full Text Full Text PDF PubMed Scopus (137) Google Scholar). The P2X7 receptor-induced proteolytic processing and release of IL-1β from LPS-primed macrophages also precede cell death (13Ferrari D. Chiozzi P. Falzoni S. Dal Susino M. Melchiorri L. Baricordi O.R. Di Virgilio F. J. Immunol. 1997; 159: 1451-1458PubMed Google Scholar). These observations suggest that in the time period between commitment to apoptosis and actual cell death, P2X7 receptor activation triggers additional signals, such as NFAT activation or IL-1β release, that modulate the overall inflammatory response of macrophages. This possibility is supported by the observation that brief ATP pulses trigger not only macrophage apoptosis, but also killing of intracellular mycobacteria, including virulent Mycobacteria tuberculosis (22Kusner D.J. Adams J. J. Immunol. 2000; 164: 379-388Crossref PubMed Scopus (147) Google Scholar). Because other inducers of macrophage apoptosis do not kill M. tuberculosis, an as yet unidentified P2X7-specific signal presumably induces killing of internalizedM. tuberculosis before the macrophage itself dies (16Lammas D.A. Stober C. Harvey C.J. Kendrick N. Panchalingam S. Kumararatne D.S. Immunity. 1997; 7: 433-444Abstract Full Text Full Text PDF PubMed Scopus (346) Google Scholar, 23Molloy A. Laochumroonvorapong P. Kaplan G. J. Exp. Med. 1994; 180: 1499-1509Crossref PubMed Scopus (459) Google Scholar). The biochemical steps linking channel/pore activation to IL-1β release, accumulation of pro-inflammatory transcription factors,M. tuberculosis killing, and macrophage apoptosis are poorly understood. The stress-activated protein kinases (SAPKs; also known as JNKs) phosphorylate and activate transcription factors such as ATF2, Ets, and c-Jun in response to diverse cell stressors. These include UV radiation, osmotic shock, inflammatory cytokines, and endoplasmic reticulum stress (for review, see Refs. 24Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1379) Google Scholar and 25Kyriakis J.M. Avruch J. J. Biol. Chem. 1996; 271: 24313-24316Abstract Full Text Full Text PDF PubMed Scopus (1025) Google Scholar). Many of the downstream effectors of SAPK signaling contribute to the inflammatory response, including the TNF-α-dependent induction of E-selectin, NF-κB induction in T cells, and both pro-apoptotic and anti-apoptotic effects in a variety of cell types (24Ip Y.T. Davis R.J. Curr. Opin. Cell Biol. 1998; 10: 205-219Crossref PubMed Scopus (1379) Google Scholar). A necessary role of SAPK in apoptotic induction by UV irradiation, but not Fas receptor ligation, was demonstrated in a recent study using embryonic fibroblasts derived from double-knockout mice that lack expression of both the Jnk1 and Jnk2 genes (55Tournier C. Hess P. Yang D.D. Xu J. Turner T.K. Nimnual A. Bar-Sagi D. Jones S.N Flavell R.A. Davis R.J. Science. 2000; 288: 870-874Crossref PubMed Scopus (1540) Google Scholar). Proximally, SAPKs are activated by the dual-specificity mitogen-activated protein kinase kinases (MEKs), which in turn are activated by the MEK kinases (MEKKs). Upstream regulators of MEKK are incompletely characterized. TNF-α-dependent SAPK activation is best described and involves recruitment of the adaptor protein TRAF2 to the cytosolic portion of the ligated TNF-α receptor. TRAF2 mediates the activation of a series of downstream kinases that lead to phosphorylation of SAPK itself (26Shi C.S Kehrl J.H. J. Biol. Chem. 1997; 272: 32102-32107Abstract Full Text Full Text PDF PubMed Scopus (99) Google Scholar). Other intermediates have been proposed to play a role in different models of SAPK activation, including oxidative stress, DNA damage, and caspase proteases. Altered ion fluxes have also been associated with SAPK activation in several systems. Kuroki et al. (29Kuroki D.W. Bignami G.S. Wattenberg E.V. Cancer Res. 1996; 56: 637-644PubMed Google Scholar) described activation of SAPK by palytoxin, a natural marine toxin fromPalythoa tuberculosa that acts as a skin tumor promoter and modulator of the Na+,K+-ATPase. Moreover, palytoxin-induced SAPK activity requires sodium flux (30Kuroki D.W. Minden A. Sanchez I. Wattenberg E.V. J. Biol. Chem. 1997; 272: 23905-23911Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar). Similarly, UV irradiation of myeloblastic leukemia cells induces a prominent K+ channel activation that, in turn, induces SAPK-dependent apoptosis (31Wang L. Xu D. Dai W. Lu L. J. Biol. Chem. 1999; 274: 3678-3685Abstract Full Text Full Text PDF PubMed Scopus (92) Google Scholar). Since a major consequence of P2X7 receptor activation is bulk movement of both sodium and potassium, we hypothesized that P2X7 receptor activation would activate SAPK. We found that exposure of murine macrophages to short pulses of extracellular ATP can rapidly induce a sustained activation of SAPK. Pharmacological selectivity and molecular evidence indicated that the P2X7 receptor mediates this ATP-induced kinase activity. The P2X7 receptor can activate caspases involved in either cytokine processing or apoptotic induction (18Coutinho-Silva R. Persechini P.M. Da Cunha Bisaggio R. Perfettini J. Torres de Santo A.C. Kanellopoulos J.M. Mottaly I. Dautry-Varsat A. Ojcius D.M. Am. J. Physiol. 1999; 276: C1139-C1147Crossref PubMed Google Scholar, 19Ferrari D. Los M. Bauer M.K. Vandenabeele P. Wesselborg S. Schulze-Osthoff K. FEBS Lett. 1999; 447: 71-75Crossref PubMed Scopus (243) Google Scholar). Thus, we also evaluated the possible involvement of caspase-1 and caspase-3 in this P2X7 receptor-induced pathway of SAPK activation. The results indicate that although both caspase-1 and caspase-3 were activated by the pulsed ATP protocol, neither protease plays a role in coupling the P2X7 receptor to the SAPK signaling cascade. All nucleotides were from Sigma, except for 2′-methylthio-ATP, which was from Research Biochemicals Inc. (Natick, MA). Anisomycin and ouabain was from Sigma. The GST-Jun-(1–79) plasmid was from Dr. J. Woodgett (Woods Hole Biological Laboratory). [γ-32P]ATP was from NEN Life Science Products. Anti-SAPK and anti-phospho-specific SAPK antibodies were from Santa Cruz Biotechnology (Santa Cruz, CA). The anti-α-fodrin antibody was from Chemicon International, Inc. KN-62 (1-(N,O-bis[5-isoquinolinesulfonyl]-N-methyl-l-tyrosyl)-4-phenylpiperazine) and the caspase-1 fluorogenic substrate peptide were from BIOMOL Research Labs Inc. (Plymouth Meeting, PA). The capture and detecting antibodies used for the murine IL-1β ELISA were from Endogen, Inc. (Woburn, MA). DEVD-cho and YVAD-cmk were from Bachem California. The caspase-3 fluorogenic substrate peptide was from Calbiochem. Recombinant murine TNF-α and a neutralizing antibody against murine TNF-α were from R&D Systems, Inc. The BAC1.2F5 macrophage cell line, a clone of the SV40-transformed murine macrophage cell line BAC1, was maintained using previously described protocols (32el-Moatassim C. Dubyak G.R. J. Biol. Chem. 1992; 267: 23664-23673Abstract Full Text PDF PubMed Google Scholar). Wild-type HEK293 cells and HEK cells stably transfected with the human P2X7 receptor were maintained as described previously (33Humphreys B.D. Virginio C. Surprenant A. Rice J. Dubyak G.R. Mol. Pharmacol. 1998; 54: 22-32Crossref PubMed Scopus (183) Google Scholar). JNK activation was measured according to Kurokiet al. (29Kuroki D.W. Bignami G.S. Wattenberg E.V. Cancer Res. 1996; 56: 637-644PubMed Google Scholar, 30Kuroki D.W. Minden A. Sanchez I. Wattenberg E.V. J. Biol. Chem. 1997; 272: 23905-23911Abstract Full Text Full Text PDF PubMed Scopus (41) Google Scholar) with minor modifications. BAC1 cells (3 × 106/ml) were plated in six-well dishes. Cultures were incubated with test agents dissolved in Iscove's Dulbecco's modified Eagle's medium supplemented with 0.1% bovine serum albumin at 37 °C. Samples were then washed once with cold phosphate-buffered saline and lysed with lysis buffer (25 mm HEPES (pH 7.7), 300 mm NaCl, 1.5 mm MgCl2, 0.2 mm EDTA (pH 8.0), 0.1% Triton X-100, 0.5 mmDTT, 20 mm β-glycerophosphate, 0.1 mmNa3VO4, 2 μg/ml leupeptin, and 100 μg/ml phenylmethylsulfonyl fluoride). Whole cell lysates were rotated for 30 min at 4 °C, followed by centrifugation at 10,000 ×g for 10 min. Supernatant protein concentration was determined by the Bradford assay (57Bradford M.M. Anal. Biochem. 1976; 72: 248-254Crossref PubMed Scopus (215653) Google Scholar). 50 μg of lysate was diluted to contain 20 mm HEPES (pH 7.7), 75 mmNaCl, 2.5 mm MgCl2, 0.1 mm EDTA, 0.05% Triton X-100, 0.5 mm DTT, 20 mmβ-glycerophosphate, 0.1 mmNa3VO4, 2 μg/ml leupeptin, and 100 μg/ml phenylmethylsulfonyl fluoride and mixed with 10 μl of GSH-agarose beads (Sigma) bound to the GST-Jun fusion protein. The mixture was rotated for 2 h at 4 °C, and the beads were then washed three times with HEPES binding buffer (20 mm HEPES (pH 7.7), 50 mm NaCl, 2.5 mm MgCl2, 0.1 mm EDTA, 0.05% Triton X-100, 0.1 mmNa3VO4, 2 μg/ml leupeptin, and 100 μg/ml phenylmethylsulfonyl fluoride). Beads were resuspended in 40 μl of kinase buffer (20 mm HEPES (pH 7.6), 20 mmMgCl2, 20 mm β-glycerophosphate, 20 mm p-nitrophenyl phosphate, 0.1 mmNa3VO4, 2 mm DTT, 20 μm ATP, and 5 μCi of [γ-32P]ATP) and incubated for 15 min at 25 °C. Proteins were eluted in SDS buffer, boiled for 3 min, and separated by SDS-polyacrylamide gel electrophoresis (12%). After Coomassie Blue staining, the gel was dried and exposed to Eastman Kodak x-ray film. GST-Jun phosphorylation was quantitated with a Bio-Rad PhosphorImager. Supernatant lysates were separated by SDS-polyacrylamide gel electrophoresis (12%) and electrophoretically transferred to polyvinylidene difluoride membranes for 15 h at 30 mV. Polyvinylidene difluoride membranes were rinsed in immunoblot buffer (10 mmol/liter Tris (pH 7.4), 0.9% NaCl, 0.05% Tween 20, and 1 mmol/liter EDTA) and blocked with milk buffer (4% nonfat dried milk (Sigma) in immunoblot buffer). After washing (1 × 15 min, 2 × 5 min) with immunoblot buffer, the polyvinylidene difluoride membranes were incubated for 1 h at room temperature with primary antibodies dissolved in milk buffer. Both anti-JNK antiserum and anti-phospho-specific JNK monoclonal antibody were used at 1 μg/ml. Membranes were then washed and incubated for 1 h with 1:5000 dilutions of horseradish peroxidase-conjugated donkey anti-rabbit (for anti-JNK antiserum) or anti-mouse (for anti-phospho-specific JNK antibody) antibody (Amersham Pharmacia Biotech). Membranes were washed and developed using chemiluminescent reagents (SuperSignal from Pierce) for min and exposed to Eastman Kodak x-ray film. time 1 × cells and were at × for 30 resuspended in 0.5 of lysis buffer mm 20 mm EDTA, and Triton (pH and on for 15 min. Samples were then at ×g for 20 min, and the DNA was using were resuspended in and with 0.2 and 1 A for 30 min at 37 °C. DNA were separated on a with and After with cell were washed once with mmNaCl, 20 mm and 1 mm EDTA (pH were for 30 at × and were resuspended in lysis buffer (20 mm mm NaCl, 1 mm DTT, 5 mm EDTA, 5 mm and Triton (pH incubated for 15 min at 37 °C, and at × for 20 min. The were then at for caspase-3-like of lysate was with buffer mm 1 mm EDTA, and 5 mm (pH and fluorogenic caspase-3 substrate to a concentration of was measured at an of and an of at using a were and were the portion of the separate are as the in were plated in at 5 × 1 to the of the cells were stimulated with (1 for 4 h to induce expression of In the YVAD-cmk was for the 30 min of In the presence of Dulbecco's modified Eagle's medium and cells were with 3 mm ATP with or the YVAD-cmk After min, of the were for IL-1β release by a a was with 1 μg/ml primary IL-1β antibody and then blocked with bovine serum albumin in phosphate-buffered saline for 1 were washed three times with buffer (pH and Tween of or murine IL-1β diluted with μl of Dulbecco's modified Eagle's were to the blocked with 50 μl of a IL-1β antibody 0.2 The were incubated at room temperature for 2 h and then washed three The complexes were by with and substrate for horseradish of the effects of P2X7 receptor activation is equilibration of the K+ and Since can trigger SAPK signaling in cell we investigated the ability of P2X7 receptors to to SAPK. As an BAC1 murine macrophages were with extracellular ATP for brief from 10 to 30 min. Brief ATP pulses be a macrophages are to be exposed to extracellular ATP for only short times to the expression of which extracellular ATP. of extracellular divalent can P2X7 receptor affinity for ATP, macrophages are exposed to millimolar levels of extracellular and For this medium Dulbecco's modified Eagle's levels of divalent was in In the in BAC1 cells were pulsed with 5 mm ATP. After a the medium was and with Iscove's the indicated time and cell lysates were for SAPK activity. SAPK activity was low during or the ATP but rapidly after ATP to a of The kinase activated 2 h after ATP Since ATP exposure can trigger lysis in cells, that the ATP itself not cell death. The cytosolic was used as a for in was in the extracellular medium at during ATP pulses not were in at min the ATP the induced SAPK activity was at this further the SAPK response, BAC1 were with ATP for different times min) to that the of SAPK activation at min was on the of the ATP A was sufficient to a accumulation of and activation was within 20 between ATP and induction of sustained SAPK activity. BAC1 macrophages were with or with 5 mm ATP for the indicated the cells were then washed and medium was After a at 37 °C, cells were and SAPK activity was as described SAPK activation is as a of time for the BAC1 macrophages receptors in to receptors B.D. Dubyak G.R. J. Immunol. 1996; Google and the concentration of ATP used to activate we determined whether the ATP-induced SAPK activity was mediated by the P2X7 receptor or by another P2 receptor. A that of macrophages with ATP blocked ATP-induced SAPK activity. In was no of SAPK that the SAPK signaling pathway after oxidized ATP using an antibody that the assay not overall SAPK protein and phosphorylation of SAPK is for and we that kinase activity in with accumulation of 3 oxidized ATP the P2X7 for the P2X7 receptor other P2X receptors is we that the isoquinoline KN-62, a that also the P2X7 receptor in BAC1 macrophages (33Humphreys B.D. Virginio C. Surprenant A. Rice J. Dubyak G.R. Mol. Pharmacol. 1998; 54: 22-32Crossref PubMed Scopus (183) Google blocked ATP-induced SAPK activity 3 These data the P2X7 receptor as the P2 receptor for SAPK activation. The ability of nucleotides other ATP to activate SAPK was also 3 C that only 3′-O-(4-benzoyl)benzoyl-ATP and ATP were SAPK the was a of P2 receptors expressed in BAC1 macrophages. The millimolar of ATP used to the P2X7 receptor strong of extracellular divalent and this could be contribute to the activation of SAPK. Since also divalent but not activate P2X7 the of 5 mm to SAPK activity that of extracellular divalent ATP-dependent SAPK activity The and and the were also at SAPK activity. This nucleotide selectivity is with the of both and expressed P2X7 can release various inflammatory cytokines, such as IL-1β and which also activate SAPK signaling and can as of macrophage activation R.J. Immunol. PubMed Scopus Google Scholar). The but activation of SAPK that followed ATP an that could the release and extracellular accumulation of TNF-α the IL-1β and TNF-α genes are