QUICK SEARCH:   [advanced] Author: Keyword(s): Year:  Vol:  Page:

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.059600v1 308/3/1053    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Donnelly-Roberts, D. L. Articles by Jarvis, M. F. Search for Related Content PubMed PubMed Citation Articles by Donnelly-Roberts, D. L. Articles by Jarvis, M. F. Pubmed/NCBI databases Compound via MeSH Substance via MeSH

CELLULAR AND MOLECULAR

Mitogen-Activated Protein Kinase and Caspase Signaling Pathways Are Required for P2X₇ Receptor (P2X₇R)-Induced Pore Formation in Human THP-1 Cells

Neuroscience Research, Global Pharmaceutical Research and Development Abbott Laboratories, Abbott Park, Illinois

Received for publication September 4, 2003
Accepted November 10, 2003.

	Abstract

Top Abstract Materials and Methods Results Discussion References

 
Brief activation of the ATP-sensitive P2X₇ receptor (P2X₇R) stimulates the maturation and release of interleukin 1 (IL-1)in macrophages, whereas prolonged agonist activation induces the formation of cytolytic pores in cell membranes. The present study investigated potential downstream mechanisms associated with native human P2X₇R activation in lipopolysaccharide and interferon- differentiated THP-1 cells. 2,3-O-(4-Benzoylbenzoyl)-ATP (BzATP)-induced pore formation (EC₅₀ = 35 µM) was blocked by a selective P2X₇R antagonist, 1[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine (KN-62) (IC₅₀ = 44 nM) and by pyridoxal phosphate-6-azophenyl-2-4-disulfonic acid (PPADS) (IC₅₀ = 344 nM). KN-62 and PPADS also blocked BzATP-induced IL-1 release (EC₅₀ = 617 µM) with IC₅₀ values of 75 and 3500 nM, respectively. The selective p38 mitogen-activated protein kinase (MAPK) inhibitor, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole (SB 202190), potently inhibited BzATP-induced pore formation (IC₅₀ = 75 nM) but did not alter P2X₇-mediated calcium influx or IL-1 release. SB 202190 and KN-62 also attenuated BzATP-mediated activation of phosphorylated p38 MAPK (pp38 MAPK). Two caspase inhibitors, YVAD (caspase 1) and DEVD (caspase 3), attenuated both BzATP-induced pore formation and IL-1 release in a concentration-dependent fashion. Neither DEVD nor p38-MAPK inhibitors blocked cell membrane pore formation evoked by maitotoxin or by activation of human P2X_2a receptors. These results indicate that P2X₇R-mediated pore formation results from a coordinated cascade involving both the p38 MAPK and caspase pathways that is distinct from other cytolytic pore-forming mechanisms. In contrast, P2X₇R-mediated IL-1 release is dependent on caspase activity but not p38 MAPK. Taken together, these results support the hypothesis that downstream cellular signaling mechanisms, rather than channel dilation, mediate cytolytic pore formation after prolonged agonist activation, which underlies P2X₇ receptors.

The P2X₇ receptor is also distinguished pharmacologically from other P2X receptors by the fact that ATP has weak affinity (EC₅₀ > 100 µM) for this receptor, whereas 2,3-O-(4-benzoylbenzoyl)-ATP (BzATP) is approximately 30-fold more potent than ATP (Surprenant et al., 1996; Bianchi et al., 1999). P2X₇Rs are uniquely expressed on macrophages, epidermal Langerhans cells, and other antigen-presenting cells (Surprenant et al., 1996). Within the central nervous system, P2X₇R expression has been reported on microglia (Ferrari et al., 1996), astrocytes, Schwann cells (Irnich et al., 2001), and presynaptic neuronal terminals in hippocampus and spinal cord (Duechars et al., 2001).

Another unique function of the P2X₇R is its contribution to a variety of proinflammatory events, including fusion of macrophages to form multinucleated giant cells (Falzoni et al., 1995). Activation of P2X₇ receptors stimulates the release of the proinflammatory cytokine interleukin-1 (IL-1) in immune cells (Surprenant et al., 1996). IL-1 is processed from a precursor to a mature form by the enzyme, caspase 1. ATP has been shown to both time- and dose-dependently induce the release of IL-1 from LPS-primed THP-1 cells (Grahames et al., 1999). This ATP-induced cytokine release appears to be specifically modulated by P2X₇Rs since IL-1 release can be blocked not only by the nonselective P2X antagonists, PPADS and oxidized ATP, but also by KN-62, which is a selective P2X₇R antagonist (Grahames et al., 1999). It has also been shown that the release of IL-1 is independent of cytolysis and appears not to require P2X₇-mediated pore formation (Chessell et al., 2001). P2X₇R activation also results in cell membrane blebbing (Ferrari et al., 1997a) and changes in cellular morphology, which eventually lead to cell death by both necrotic and apoptotic mechanisms (Ferrari et al., 1999).

The present study evaluates the signaling pathways that contribute to two of the hallmark functional consequences of P2X₇R activation, pore formation and IL-1 release, in differentiated human THP-1 macrophage cells. By using pharmacologically selective inhibitors of intracellular signaling pathways, we show that P2X₇R-induced pore formation and IL-1 release are mediated via some common but also some distinct mechanistic pathways. The mechanism of P2X₇R-mediated pore formation was also compared with pore formation induced by non-P2X₇Rs such as the P2X_2aR (Khakh et al., 1999) or via a toxin from marine dinoflagellates, maitotoxin (MTX) (Schilling et al., 1999a,b). The exact mechanism by which prolonged activation of the P2X₇R leads to pore formation remains unknown. Either dilation of the channel or recruitment of ancillary cellular mechanisms has been proposed (North, 2002). The present data provide evidence that P2X₇R-mediated pore formation is likely mediated by intracellular events rather than simply from an agonist-induced conformational change intrinsic to the channel.

	Materials and Methods

Top Abstract Materials and Methods Results Discussion References

 
Materials. Cell culture medium and phosphate-buffered saline, pH 7.4, were obtained from Invitrogen (Carlsbad, CA). BzATP, ATP, UTP, PPADS, protease inhibitor cocktail, and LPS were obtained from Sigma-Aldrich (St. Louis, MO). Maitotoxin was purchased from Wako Bioproducts (Richmond, VA). Fluo-4 AM was purchased from TefLabs (Austin, TX), and Yo-Pro-1 (4-[(3-methyl-2(3H)-benzoxazolylidene) methyl]-1[3-(trimethylammonio)propyl]-diiodide was purchased from Molecular Probes (Eugene, OR). PD 98059, U0126, U0124, SB 202190-HCl, SB 203580, SB 202474, PD 169316, JNK I and JNK II inhibitors, and control analogs, caspase 1 inhibitor (YVAD), caspase 3 inhibitor (DEVD), and cathepsin B, a caspase-negative control inhibitor, were obtained from Calbiochem (San Diego, CA). The synthetic peptide dynorphin A was obtained from Bachem Biosciences (King of Prussia, PA). Human interferon- (IFN) and the IL-1 ELISA kit were obtained from R&D Systems (Minneapolis, MN) or Pierce Endogen (Rockford, IL). KN-62 was purchased from BIOMOL Research Laboratories (Plymouth Meeting, PA). ELISA kits for the measurement of p38 MAPK [pTpY180/182] and total p38 were purchased from BioSource International (Camarillo, CA).

Tissue Culture. Cells of the THP-1 monocytic cell line (American Type Culture Collection, Manassas, VA) were maintained in the log phase of growth in RPMI 1640 medium containing high glucose and 10% fetal calf serum (Invitrogen) according to established procedures (Humphreys and Dubyak, 1996). Fresh vials of frozen THP-1 cells were initiated for growth every 8 weeks. To differentiate THP-1 cells into a macrophage phenotype, a final concentration of 25 ng/ml of LPS and 10 ng/ml of IFN were added to the cells (Humphreys and Dubyak, 1996) either for 3 h for IL-1 release assays or overnight (16 h) for pore formation studies. 1321N1 cells stably expressing the recombinant human P2X_2a receptor were grown and used according to previously published protocols (Bianchi et al., 1999; Lynch et al., 1999). For both the pore formation and IL-1 release assays, cell density and viability were routinely assessed before each experiment by trypan dye exclusion, and cells were found to be >90% viable after differentiation.

Calcium Influx Assay. The Fluorometric Imaging Plate Reader (FLIPR; Molecular Devices, Sunnyvale, CA) was used to measure agonist-induced Ca²⁺ influx using the calcium-chelating dye Fluo-4 as previously described by Bianchi et al. (1999) with the following modifications. One to two hours before the calcium flux assay, the differentiated THP-1 cells were washed in DPBS using a conical tube and loaded with Fluo-4 AM (2.28 µM) in DPBS before centrifugation onto poly-lysine-coated black-walled 96-well plates. Cells were then maintained in a dark environment at room temperature. Immediately before the assay, each plate was washed three times with 250 µl of DPBS per well to remove extracellular Fluo-4 AM. Two 50-µl additions of test compounds (prepared in DPBS) were made to the cells during each experiment. The first compound addition was made after a 30-s baseline determination. Test compounds were incubated for 3 min before the addition of BzATP (final concentration of 30 µM), followed by incubation for an additional 3 min. Fluorescence data were collected at 1 s and then at 5-s intervals throughout the course of each experiment. Data shown are based on the maximal fluorescence response expressed in arbitrary relative fluorescence units (Bianchi et al., 1999). Concentration-response data were analyzed using GraphPad Prism (GraphPad Software Inc., San Diego, CA); the EC₅₀ or IC₅₀ values were derived from a single curve fit to the mean data of n = 6 in duplicates.

Yo-Pro Uptake Assay. Agonist-induced pore formation was also determined using the FLIPR by measuring cellular uptake of Yo-Pro (mol. wt. = 629 Da) in differentiated THP-1 cells with the modifications below. Differentiated THP-1 cells were rinsed once in PBS without Mg²⁺ or Ca²⁺ ions, which have been shown to inhibit pore formation (Michel et al., 1999). The Yo-Pro iodide dye (1 mM in dimethyl sulfoxide) was diluted to a final concentration of 2 µM in PBS (without Mg²⁺ or Ca²⁺) and then placed on the cells immediately before agonist addition. After the addition of various agonist concentrations (0.001-100 µM), Yo-Pro dye uptake was observed in the FLIPR equipped with an argon laser (wavelength = 488 nm) and a charge-coupled device camera. The exposure setting of the camera was 0.25 s with an f-stop setting of 2. Unless otherwise indicated, data points from each experiment were collected over a 60-min period after exposure to appropriate concentrations of BzATP. The intensity of the fluorescence was captured by the charge-coupled device camera every 15 s for the first 10 min of agonist exposure, followed by every 20 s for an additional 50 min. For antagonist activity measurements, the maximal intensity was expressed as a percentage of that induced by the EC₇₀ value for agonist activation (50 µM BzATP for THP-1 cells) and plotted against the concentration of compound to determine IC₅₀ values. For each experiment, differentiated control cells were also measured over the 60-min time course to assess background levels of fluorescence. This nonspecific background Yo-Pro uptake, averaging 6 to 10% of the maximum BzATP response (see Fig. 1), was subtracted from the maximum BzATP-induced fluorescence.

View larger version (39K): [in this window] [in a new window]   Fig. 1. A, kinetics of BzATP-stimulated Yo-Pro uptake in LPS/IFN-treated and untreated human THP-1 cells. Traces represent responses from individual wells of confluent THP-1 cells. B, BzATP (300 µM) stimulated Yo-Pro uptake in LPS/IFN treated (gray bar) and untreated (black bar) human THP-1 cells. Data represent means ± S.E.M. of three to five experiments.

For experiments measuring P2X_2aR-mediated pore formation, a dose-response curve was generated for the agonist ATP under the same conditions used for the P2X₇R Yo-Pro uptake assay. EC₇₀ value was determined and used to examine antagonist effects. For MTX-mediated pore formation studies, various concentrations of MTX were examined under the conditions described above, except that the buffer, DPBS (Invitrogen) contained 0.9 mM CaCl₂ and 0.5 mM MgCl₂, as the presence of extracellular divalent cations are required for MTX activity (Schilling et al., 1999a,b) and inhibition studies were against the EC₇₀ value.

pp38 MAPK ELISA Measurements. Undifferentiated THP-1 cells were plated into a T75 flask (Costar, Cambridge, MA) at a density of 5 x 10⁶ cells/ml/well. The day before the experiment, the cells were differentiated with 25 ng/ml of LPS and 10 ng/ml of IFN for 16 h at 37°C. In the presence of the differentiation media, the cells were incubated with inhibitors for 30 min at 37°C followed by stimulation with 100 µM BzATP for an additional 60 min at 37°C. Pelleted cell lysates were rinsed in PBS, repelleted, and flash-frozen. On the day of the ELISA, cell lysates were lysed and protein extracted in a 10 mM Tris buffer, pH 7.4, containing: 100 mM NaCl, 1 mM EDTA, 1 mM EGTA, 1 mM NaF, 20 mM Na₄P₂O₇, 2 mM Na₃VO₄, 1% Triton X-100, 10% glycerol, 0.1% sodium dodecyl sulfate, 0.5% deoxycholate, 1 mM phenylmethylsulfonyl fluoride, and a 5% final concentration of a protease inhibitor cocktail. Lysates were clarified by centrifugation at 13,000 rpm for 10 min at 4°C before the detection of pp38 MAPK or total p38 MAPK activity using an ELISA, following the manufacturer's instructions.

IL-1 ELISA Measurements. Undifferentiated THP-1 cells were plated in 24-well plates at a density of 1 x 10⁶ cells/ml/well. On the day of the experiment, cells were differentiated with 25 ng/ml of LPS and 10 ng/ml of IFN for 3 h at 37°C. In the presence of the differentiation media, the cells were incubated with inhibitors for 30 min at 37°C followed by a challenge with 1 mM BzATP for an additional 30 min at 37°C. Supernatants were collected after a 5-min centrifugation in microfuge tubes and assayed for the presence of mature IL-1 by ELISA, following the manufacturer's instructions. For each experiment, differentiated control cells were also measured over the 60-min time course of the assay to assess background IL-1 accumulation. This nonspecific background IL-1 release, typically averaged 3 to 8% of the maximum BzATP response, was subtracted from the maximum BzATP-induced release.

	Results

Top Abstract Materials and Methods Results Discussion References

 
BzATP-Mediated Yo-Pro Uptake in THP-1 Cells. Yo-Pro incorporation into differentiated THP-1 cells was detectable 15 min after the addition of BzATP (300 µM) and continued to increase over a 60-min period. These results are consistent with the effects previously described using cells expressing the stably transfected recombinant, human P2X₇R (Rassendren et al., 1997). Exposure of differentiated THP-1 cells to BzATP for 60 min resulted in a 15-fold increase in fluorescence (Fig. 1, A and B). In contrast, ATP (at concentrations up to 1 mM) resulted in only a slight increase in Yo-Pro uptake, and UTP (1 mM) was also inactive (Fig. 2A). BzATP-mediated Yo-Pro uptake (EC₅₀ value of 35 ± 7 µM; Fig. 2A) was attenuated by pretreatment with the P2X₇ receptor antagonists KN-62 and PPADS (Humphreys et al., 1998). The IC₅₀ values obtained for KN-62 and PPADS were 44 ± 2 nM and 334 ± 17 nM, respectively (Fig. 2B).

View larger version (21K): [in this window] [in a new window]   Fig. 2. A, concentration-effect curves for P2 receptor agonists to stimulate Yo-Pro uptake in LPS/IFN-differentiated THP-1 cells. Data were normalized to the peak BzATP response (percentage of maximum response). B, concentration-effect curves for P2X7R receptor antagonists to block 50 µM BzATP-stimulated Yo-Pro uptake. Data represent mean ± S.E.M. of four to six experiments.

Blockade of Pore Formation with MAPK Inhibitors. Differentiated THP-1 cells were pretreated with various MAPK inhibitors for 30 min before the addition of 50 µM BzATP. SB 202190, a selective p38 MAPK inhibitor (Lee et al., 1994), potently inhibited BzATP-stimulated Yo-Pro uptake with an IC₅₀ value of 75 ± 6 nM (Fig. 3). Two other p38 MAPK inhibitors, 4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)1H-imidazole (PD 169316) and SB 203580, also blocked pore formation but with lower potencies than was observed for SB 202190 (Table 1). SB 202474, which is structurally similar to SB 202190 but lacks p38 MAPK inhibitory activity (Lee et al., 1994), exhibited no effect on Yo-Pro uptake (Table 1). The inhibition of BzATP-induced pore formation by SB 202190 was not due to antagonism of the activation of the P2X₇R by BzATP since SB 202190 had no significant effect in the P2X₇R Ca²⁺ influx assay (IC₅₀ value >100 µM) using a 1321 astrocytoma cell line transfected with human P2X₇R (Bianchi, et a., 1999). Using this same cell line for pore formation measurements, it was determined that SB 202190 had an IC₅₀ value of 1500 ± 180 nM.

View larger version (14K): [in this window] [in a new window]   Fig. 3. Concentration-effect curves for two MAPK inhibitors, SB 202190 and PD 98059, to inhibit 50 µM BzATP-induced Yo-Pro uptake. Data represent mean ± S.E.M. of three to five experiments.

PD 98059, a MEK kinase inhibitor (Pang et al., 1995), exhibited only weak inhibition of P2X₇R-mediated pore formation (IC₅₀ 100 µM; Fig. 3). In addition, in the 1321 astrocytoma cell line transfected with human P2X₇R, PD 98059 (IC₅₀ value >100 µM) did not significantly block pore formation. Another MEK kinase inhibitor U0126 (Pang et al., 1995) did not significantly inhibit BzATP-stimulated Yo-Pro uptake (Table 1). In addition, 5-iodotubercidin, a potent inhibitor of the ERK2 and adenosine kinases (Fox et al., 1998), also had no effect on P2X₇R-mediated pore formation (Table 1).

Inhibition of BzATP-Mediated pp38 MAPK Activation. Differentiation of THP-1 cells produced a significant increase in the level of pp38 MAPK relative to that observed for undifferentiated cells (Fig. 4). The level of pp38 MAPK in differentiated THP-1 cells was further enhanced by exposure to 100 µM BzATP. BzATP-induced pp38 MAPK activation was significantly attenuated (P < 0.05) by the p38 MAPK-specific inhibitor SB 202190 (10 µM) and not by the inert analog, SB 247474 (10 µM). This BzATP-induced pp38 MAPK activation was also significantly attenuated (P < 0.05) by the P2X₇-selective antagonist, KN-62 (10 µM), to a level commensurate to that observed for differentiated control THP-1 cells. BzATP stimulation had no effect on the total p38 MAPK detected (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 4. Activation of pp38 MAPK in differentiated (16 h) and nondifferentiated (control) THP-1 cells. The 100 µM BzATP-induced pp38 MAPK activation was attenuated by SB 202190 and KN-62 and not by SB 247474. Data were normalized to the peak BzATP response of pp38 MAPK over total p38 (percentage of maximum response), which ranged from 2000 to 3000 ng/ml of pp38 MAPK product. Data represent means ± S.E.M. of three experiments. *, P < 0.05 versus differentiated THP-1 cells; **, P < 0.05 versus BzATP-treated differentiated THP-1 cells.

Blockade of Pore Formation with Caspase Inhibitors. YVAD, a caspase 1 inhibitor, inhibited BzATP-evoked Yo-Pro uptake in a concentration-dependent fashion (IC₅₀ value of 8 ± 0.8 µM) (Fig. 5). DEVD, a caspase 3 inhibitor, was slightly more potent in inhibiting BzATP-evoked Yo-Pro uptake with an IC₅₀ value of 3.2 ± 0.3 µM (Fig. 5). Cathepsin B, which serves as a negative control for caspase inhibitors as well as two synthetic peptides, oxytocin and dynorphin A, which have comparable molecular weights to the caspase inhibitors and are linked to an inert, longer peptide chain to increase cell permeability (Garcia-Calvo et al., 1998), were also examined for pore-forming inhibitory activity. None of these controls exhibited any significant effect on BzATP-evoked Yo-Pro uptake (Table 1).

View larger version (14K): [in this window] [in a new window]   Fig. 5. Concentration-effect curves for a caspase 1 inhibitor, YVAD (black square), and a caspase 3 inhibitor, DEVD (white square), to attenuate 50 µM BzATP-induced Yo-Pro uptake. Data represent mean ± S.E.M. of three to five experiments.

Effects of Other Signaling Pathway Inhibitors on P2X₇R-Mediated Pore Formation. Inhibitors of other signaling pathways were also assessed for effects on BzATP-induced Yo-Pro uptake. As shown in Table 2, pretreatment with thapsigargin (Won and Orth, 1995) or xestospongin C (Gafni et al., 1997) had no effect on BzATP-mediated Yo-Pro uptake, suggesting that neither mobilization of Ca²⁺ from internal stores nor inositol 1,4,5-triphosphosphate-mediated Ca²⁺ release is involved in pore formation (Table 2). In contrast, three PKC selective inhibitors, Gö-6976, Ro-31-7549, and Ro-31-8220, exhibited concentration-dependent inhibition of BzATP-evoked Yo-Pro uptake with IC₅₀ values of 4.8, 2.5, and 2.4 µM, respectively (Table 2). Two of these PKC inhibitors, Ro-31-7549 and Ro-31-8220, were equally effective in blocking P2X₇R-mediated calcium influx with IC₅₀ values of 5.2 ± 0.5 and 3.5 ± 0.4 µM, respectively.

Inhibition of BzATP-Mediated IL-1 Release. BzATP evoked a concentration-dependent increase in IL-1 release from differentiated THP-1 cells with an EC₅₀ value of 617 ± 57 µM (Fig. 6A). As shown in Fig. 5B, the selective P2X₇R antagonist, KN-62, potently blocked the BzATP-mediated release of IL-1 with an IC₅₀ value of 66 ± 3 nM. The nonselective P2XR antagonist PPADS also inhibited P2X₇R-mediated IL-1 release with an IC₅₀ value of 5.8 ± 0.35 µM (Fig. 6B). BzATP-stimulated IL-1 release was not significantly inhibited by various MAPK inhibitors at concentrations up to 10 µM (Fig. 7). In contrast, both caspase inhibitors, YVAD and DEVD, inhibited the BzATP-induced IL-1 release (Fig. 7). Concentration-response determinations for the caspase 1 inhibitor, YVAD, and the caspase 3 inhibitor, DEVD yielded IC₅₀ values of 9 ± 1 and 23 ± 3 µM, respectively (Fig. 8). Two of the PKC inhibitors, Ro-31-7549 and Ro-31-8220, also blocked P2X₇R-mediated IL-1 release with IC₅₀ values of 2.8 ± 0.6 and 5.0 ± 0.5 µM, respectively.

View larger version (16K): [in this window] [in a new window]   Fig. 6. A, concentration-effect curve for BzATP stimulated release of mature IL-1 in LPS/IFN-differentiated THP-1 cells. B, concentration-dependent inhibition of 1 mM BzATP-stimulated IL-1 release by KN-62 and PPADS. Inhibition data were normalized to the peak BzATP response (percentage of maximum response), which ranged from 200 to 600 pg/ml of IL-1 released. Data represent means ± S.E.M. of three to five experiments.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Effect of MAPK and caspase inhibitors on BzATP-induced IL-1 release. The differentiated THP-1 cells were pretreated for 30 min with 10 µM listed MAPK inhibitors, caspase inhibitors, and control compounds before stimulation of IL-1 release with 1 mM BzATP. Data are shown as a percentage of BzATP-induced IL-1 release in the absence of inhibitors (which usually ranged from 200-600 pg/ml of IL-1). Values shown represent the means ± S.E.M. of three to five experiments in duplicates. Statistical significance (*) represents a difference between control and experimental values with the use of analysis of variance. *, P < 0.05; **, P < 0.01.

View larger version (13K): [in this window] [in a new window]   Fig. 8. Concentration-effect curves for a caspase 1 inhibitor, YVAD, and a caspase 3 inhibitor, DEVD, to attenuate 1 mM BzATP-induced IL-1 release (which ranged from 200-600 pg/ml) Results are shown as a percentage of BzATP-induced IL-1 release in the absence of inhibitors. Data represent mean ± S.E.M. of three to five experiments.

Non-P2X₇-Mediated Pore Formation. MTX, which produces cytolytic membrane pores via a non-P2X₇R-related mechanism (Schilling et al., 1999a), induced pore formation in differentiated THP-1 cells with an EC₅₀ value of 6.8 ± 0.4 pM (Fig. 9A). This effect was blocked by calmidazolium (Fig. 9B, Table 3) consistent with previous reports (Schilling et al., 1999a).

View larger version (23K): [in this window] [in a new window]   Fig. 9. Yo-Pro uptake stimulated by MTX in LPS/IFN-differentiated THP-1 cells or ATP in P2X2aR-expressing 1321N1 cells. A, concentration-effect curve for MTX to induce Yo-Pro uptake in LPS/IFN-differentiated THP-1 cells. Data were normalized to the peak response (percentage response). B, concentration-effect curve for inhibition of 10 pM MTX-induced Yo-Pro uptake by calmidazolium. C, concentration-effect curve for ATP to induce Yo-Pro uptake in P2X2aR expressing 1321N1 cells (Lynch et al., 1999). Data were normalized to the peak ATP response (percentage of maximum response). D, concentration-effect curve for inhibition of ATP (10 µM)-induced Yo-Pro uptake by PPADS. All data represent mean ± S.E.M. of three to five experiments.

Prolonged agonist-evoked activation of the P2X₂ receptors can also lead to the formation of cell membrane pores (Khakh et al., 1999). In 1321N1 cells stably expressing the recombinant human P2X_2a receptor (Lynch et al., 1999), ATP induced Yo-Pro uptake with an EC₅₀ value of 3 ± 0.1 µM (Fig. 9C). This ATP-induced P2X_2a pore formation was blocked by PPADS with an IC₅₀ value of 477 ± 12 nM (Fig. 9D, Table 3).

Table 3 summarizes the effects of several antagonists and inhibitors on pore formation mediated by P2X₇ receptors as well as by other mechanisms. The nonselective P2X receptor antagonist PPADS potently blocked agonist-evoked Yo-Pro uptake mediated by activated P2X₇ and P2X_2a receptors but was ineffective in blocking the effects of MTX. The P2X₇ receptor-selective antagonist, KN-62, only blocked P2X₇-mediated Yo-Pro uptake. Calmidazolium blocked MTX-induced pore formation (IC₅₀ = 8 µM) consistent with previous reports (Schilling et al., 1999b) but had no effect on P2X₇R- and P2X_2aR-mediated Yo-Pro uptake. U73122 [GenBank] [1-[6-[[17-methoxyestra-1,3,5(10)-trien-17-yl]amino]hexyl]-1H-pyrrole-2,5-dione], a phospholipase C inhibitor (Smith et al., 1996) that has been shown to block MTX-mediated cell death (Estacion and Schilling, 2002), only blocked MTX-induced Yo-Pro uptake with an IC₅₀ value of 1800 ± 400 nM.

The caspase 3 inhibitor, DEVD, significantly inhibited both P2X₇- and P2X_2a-mediated Yo-Pro uptake in a concentration-dependent fashion but did not block the MTX-induced pore formation (Table 3). Interestingly, SB 202190, the specific and potent p38 MAPK inhibitor, blocked P2X₇-mediated pore formation but had no effect on Yo-Pro uptake mediated through either P2X_2a receptors or MTX treatment.

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
In the present study, we examined putative signaling mechanisms for two functional endpoints that are associated with P2X₇R activation, agonist-evoked pore formation (Yo-Pro uptake) and IL-1 release. BzATP-stimulated Yo-Pro uptake provided a pharmacologically specific assay for native human P2X₇R function on differentiated THP-1 cells based on the order of agonist (BzATP >> ATP and UTP) and antagonist (KN-62 >> PPADS) potency (Jacobson et al., 2002; North, 2002). Inhibition of BzATP-evoked IL-1 release showed a similar order of antagonist block. Although the precise signaling mechanisms governing each of these events is not completely understood, activation of P2X₇Rs has been associated with a number of downstream pathways, including phospholipase D (Humphreys and Dubyak 1996), phospholipase A₂, nuclear factor kappa B (NF-B) (Ferrari et al., 1997b; Aga et al., 2002), pro-caspase 1 (Verhoef et al., 2003), and MAPK (Aga et al., 2002; Armstrong et al., 2002; Bradford and Soltoff, 2002).

Agonist-evoked P2X₇R pore formation is a property shared by other nondesensitizing P2XRs (e.g., P2X_2, P2X_2/3, and P2X₄), but the time course and pore size formed by these receptors differ from that observed for the P2X₇R (Khakh et al., 1999; Virginio et al., 1999; North, 2002). MTX also induces pore formation via a non-P2X₇R-mediated mechanism (Schilling et al., 1999a,b). Both intrinsic P2X₇R channel dilation and P2X₇R-dependent recruitment of accessory proteins have been proposed as candidate pore-forming mechanisms (North, 2002). Data demonstrating that P2X₇R pore formation is progressive, occurs in multiple cell types, and has an identical pharmacology for the opening of the cation channel support the intrinsic receptor hypothesis.

The present data demonstrate that natively expressed human P2X₇R-mediated pore formation in differentiated THP-1 cells can be blocked by selective inhibitors of p38 MAPK and caspase activity. The present data also show that BzATP stimulated the activation of pp38 MAPK above the levels induced by THP-1 cell differentiation with LPS and IFN (Ono and Han, 2000). The p38 MAPK inhibitor, SB 202190, as well as the P2X₇-selective antagonist, KN-62, both significantly attenuated the BzATP-mediated production of pp38 MAPK, providing evidence for a direct interaction between P2X₇R activation and the p38 MAPK pathway. The p38 MAPK-dependent pore formation was also demonstrated in 1321N1 cells expressing recombinant human P2X₇Rs. The differential potency of SB 202190 to block agonist-evoked pore formation in differentiated THP-1 cells and in the recombinant receptor cell line likely reflects differences in receptor expression as assessed by differences in agonist-evoked Yo-Pro fluorescence (D. L. Donnelly-Roberts, unpublished observations). This finding provides the first evidence that these downstream intracellular events play a role in P2X₇R-mediated pore formation. The involvement of the p38 MAPK pathway may be mechanistically selective for P2X₇R-mediated pore formation since inhibitors of this pathway were ineffective in blocking P2X₇R-mediated IL-1 release and did not alter pore formation mediated by MTX or by activation of P2X_2a receptors. The present demonstration that BzATP-evoked Yo-Pro uptake is modulated by both MAPK and caspase inhibition, whereas BzATP-stimulated IL-1 release is sensitive to only caspase inhibition, is consistent with previous work, suggesting that these P2X₇ receptor-mediated events are at least in part mechanistically distinct (Schilling et al., 1999b; MacKenzie et al., 2001).

MAPKs are a family of serine and threonine kinases that are activated by various external stimuli to regulate a variety of intracellular events (English and Cobb, 2002). To date, there are three major signaling protein families involved in the MAPK cascades: ERK1/2, which is mainly involved in cellular growth and proliferation; JNK/stress-activated protein kinase, which is involved in cellular stress from ultraviolet irradiation, heat shock, or exposure to inflammatory cytokines; and the newest member, p38 kinase, which is activated upon exposure to inflammatory cytokines, endotoxins, or osmotic shock and often leads to apoptotic cell death (Su and Karin, 1996).

In contrast to the inhibitory effects of p38 MAPK inhibitors on BzATP-stimulated Yo-Pro uptake, no significant attenuation of BzATP-evoked pore formation was observed by inhibitors of MEK, ERK1/2, or by inhibitors of intracellular Ca²⁺ mobilization and inositol 1,4,5-triphosphosphate-mediated-Ca²⁺ release. Cell-permeable JNK I and JNK II inhibitors, as well as negative control (JNK-inactive) analogs were found to produce a nonspecific inhibition of BzATP-induced pore formation (D. L. Donnelly-Roberts, unpublished observations), thus limiting a mechanistic interpretation of these findings. Inhibitors of PKC were moderately effective in reducing BzATP-stimulated pore formation, consistent with a recent report demonstrating that PKC activation may be upstream from activation of p38 MAPK in some pathways (Bradford and Soltoff, 2002). However, representative PKC inhibitors are equally effective in blocking both Ca²⁺ influx and IL-1 release, indicating that this pathway may not be unique to pore formation but more generally involved in events mediated by P2X₇ activation. Alternatively, it cannot be ruled out that the effects of PKC and JNK inhibitors may be mediated simply by blocking P2X₇R activation (i.e., receptor antagonism). Taken together, the present data suggest that a P2X₇-linked PKC-p38 kinase pathway is involved in the cytolytic effects of prolonged P2X₇R activation in differentiated THP-1 cells. Supportive evidence for the involvement of p38 MAPK activity in P2X₇R-induced depression of mossy fiber synaptic activity has been previously reported (Armstrong et al., 2002).

Another pathway triggered by P2X₇R activation is the caspase pathway. The caspases are a family of cysteine proteases involved in both apoptosis and cytokine release (Thornberry and Lazebnik, 1995). Caspase 1 is involved in the release of mature IL-1 from leaderless pro-IL-1, whereas caspase 3 activity has been shown to be involved in P2X₇ receptor-mediated apoptotic cell death (Perregaux et al., 2001). In the present study, the caspase 1 and 3 inhibitors, YVAD and DEVD, exhibited concentration-dependent inhibition of P2X₇-mediated IL-1 release and Yo-Pro uptake. Interestingly, these compounds showed inverted potency orders in blocking these two P2X₇ receptor-mediated events. The caspase 1 inhibitor, YVAD, was more potent than the caspase 3 inhibitor, DEVD, in attenuating BzATP-stimulated IL-1 release, whereas the reverse was true for pore formation. These differences suggest some degree of mechanistic divergence in the generation of these two P2X₇ receptor-mediated events. This idea is further supported by the inability of p38 MAPK inhibitors to attenuate P2X₇R-mediated IL-1 release, whereas they effectively block P2X₇R-mediated pore formation. It should be noted that this apparent difference in mechanism cannot be explained by potential differences in cell death due to differentiation conditions, since similar levels of lactate dehydrogenase released were observed under both assay conditions (D. L. Donnelly-Roberts, unpublished observations). Although it has previously been shown that P2X₇R-mediated stress-activated protein kinase-MAPK activity is independent of caspase 1 or caspase 3 activity (Humphreys et al., 2000), this does not rule out the possibility that the caspase isozymes may participate in P2X₇R-stimulated pore formation along a parallel pathway. These two events are also temporally differentiated, since it has been shown that IL-1 containing microvesicle shedding occurs within seconds of P2X₇R activation (MacKenzie et al., 2001), whereas P2X₇R-mediated pore formation requires minutes of agonist exposure.

It was of interest to further investigate whether the involvement of the MAPK and caspase pathways were unique to P2X₇-mediated pore formation or common to other pore formation mechanisms such as those caused by MTX (Schilling et al., 1999a) or P2X_2aR activation (Khakh et al., 1999). In contrast to the effects on P2X₇R-mediated pore formation, the p38 MAPK inhibitor, SB 202190, did not block pore formation induced by MTX or P2X_2aR activation. Both P2X₇R- and P2X_2aR-induced pore formation were attenuated by the caspase 3 inhibitor, DEVD, suggesting an apoptotic cell death mechanism induced by prolonged activation of both P2X receptors (Thornberry and Lazebnik, 1995). Although not sensitive to MAPK or caspase inhibition, MTX-induced pore formation was significantly attenuated by calmidazolium and U73122 [GenBank] , a putative inhibitor of phospholipase C (Estacion and Schilling 2002). Taken together, these data demonstrate that pore formation mediated by activation of P2X₇R is mechanistically distinct from that produced by MTX or agonist activation of P2X_2aRs.

In conclusion, the present data demonstrate that both p38 MAPK and caspase pathways are involved in P2X₇R-mediated pore formation, whereas the caspase pathway but not p38 MAPK is mechanistically linked to agonist-evoked IL-1 release. These data are consistent with other recent reports (MacKenzie et al., 2001; Verhoef et al., 2003), indicating that many of the functional sequelae engendered by P2X₇R activation including IL-1 release, membrane blebbing, and pore formation may be mediated by parallel, rather than convergent, intracellular signal transduction pathways. These data also demonstrate that the involvement of p38 MAPK in pore formation is specific to P2X₇R and does not contribute to the pore-forming mechanisms associated with activation of P2X_2aR or by exposure to MTX. Collectively, the data support the hypothesis that pore formation is a consequence of P2X₇R activation but dependent on the involvement of downstream signaling interactions due to the mechanistic difference between pore formation and channel opening.

	Footnotes

 
DOI: 10.1124/jpet.103.059600.

ABBREVIATIONS: LPS, lipopolysaccharide; P2XR, purinergic receptor; BzATP, 2,3-O-(4-benzoylbenzoyl)-ATP; IL-1, interleukin-1; MTX, maitotoxin; PD 98059, 2'-amino-3'-methoxyflavone; SB 203580, 4-(4-fluorophenyl)-2-(4-methylsulfinylphenyl)-5-(4-pyridyl)1H-imidazole; PD 169316, 4-(4-fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)1H-imidazole; JNK, c-Jun NH₂-terminal kinase; IFN, interferon-; ELISA, enzyme-linked immunosorbent assay; MAPK, mitogen-activated protein kinase; pp38 MAPK, phosphorylated p38 MAPK; FLIPR, Fluorometric Imaging Plate Reader; DPBS, Dulbecco's phosphate-buffered saline; PKC, protein kinase C; MEK, mitogen-activated protein kinase kinase; ERK, extracellular signal-regulated kinase; PPADS, pyridoxal phosphate-6-azophenyl-2-4-disulfonic acid; KN-62, 1-[N,O-bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpiperazine; U0126, 1,4-diamino-2,3-dicyano-1,4-bis(methylthio)butadiene; U0124, 1,4-diamino-2,3-dicyano-1,4-bis(aminophenylthio)butadiene; SB 202190, 4-(4-fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)-1H-imidazole; SB 202474, 4-(ethyl)-2-(4-methoxyphenyl)-5-(4-pyridyl)-1H-imidazole; Ro-31-7549, bisindolylmaleimide VIII acetate, 2-[1-3-(aminopropyl)indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide; Gö-6976, 12-(2-cyanoethyl)-6,7,12,13-tetrahydro-13-methyl-5-oxo-5H-indolo(2,3-)pyrrolo(3,4-c)-carbazole; Ro-31-8220, bisindolylmaleimide IX, 3-[1-[3-(amidinothio)propyl-1H-indol-3-yl]-3-(1-methyl-1H-indol-3-yl) maleimide.

Address correspondence to: Diana L. Donnelly-Roberts, Abbott Laboratories, 100 Abbott Park Rd., Bldg. AP9A, Dept. R4PM, Abbott Park, IL 60064-6123. E-mail: diana.l.donnelly-roberts{at}abbott.com

	References

Top Abstract Materials and Methods Results Discussion References

 

Aga M, Johnson CJ, Hart AP, Guadarrama AG, Suresh M, Svaren J, Bertics PJ, and Darien BJ (2002) Modulation of monocyte signaling and pore formation in response to agonists of the nucleotide receptor P2X₇. J Leukoc Biol 72: 222-232.[Abstract/Free Full Text]
Armstrong JN, Brust TB, Lewis RG, and MacViar BA (2002) Activation of presynaptic P2X7-like receptors depresses mossy fiber-CA3 synaptic transmission through p38 mitogen-activated protein kinase. J Neurosci 22: 5938-5945.[Abstract/Free Full Text]
Bianchi BR, Lynch KJ, Touma E, Niforatos W, Burgard EC, Alexander KM, Park HS, Yu H, Metzger R, Kowaluk E, et al. (1999) Pharmacological characterization of recombinant human and rat P2X receptor subtypes. Eur J Pharmacol 376: 127-138.[CrossRef][Medline]
Bradford MD and Soltoff SP (2002) P2X₇ receptors activate protein kinase D and p42/p44 mitogen-activated protein kinase (MAPK) downstream of protein kinase C. Biochem J 366: 745-755.[CrossRef][Medline]
Chessell IP, Grahames CBA, Michel AD, and Humphrey PPA (2001) Dynamics of P2X7 receptor pore dilation: pharmacological and functional consequences. Drug Dev Res 53: 60-65.[CrossRef]
Denlinger LC, Fisette PL, Sommer JA, Watters JJ, Prabhu U, Dubyak GR, Proctor RA, and Bertics PJ (2001) The nucleotide receptor P2X₇ contains multiple protein- and lipid-interaction motifs including a potential binding site for bacterial lipopolysaccharide. J Immunol 167: 1871-1876.[Abstract/Free Full Text]
Duechars SA, Atkinson L, Brooke RE, Musa H, Milligan CJ, Batten TFC, Buckley NJ, Parson SH, and Deuchars J (2001) Neuronal P2X7 receptors are targeted to presynaptic terminals in the central and peripheral nervous system. J Neurosci 21: 7143-7152.[Abstract/Free Full Text]
English JM and Cobb MH (2002) Pharmacological inhibitors of MAPK pathways. Trends Pharmacol Sci 23: 40-45.[CrossRef][Medline]
Estacion M and Schilling WP (2002) Blockade of maitotoxin-induced oncotic cell death reveals zeiosis. BMC Physiol 2: 2-14.[CrossRef][Medline]
Falzoni S, Munerati M, Ferrari D, Spisani S, Moretti S, and DiVirgilio F (1995) The purinergic P2Z receptor of human macrophage cells. Characterization and possible physiological role. J Clin Investig 95: 1207-1216.
Ferrari D, Chiozzi P, Falzoni S, Dal Susino M, Melchiorri L, Baricordi OR, and DiVirgilio F (1997a) Extracellular ATP triggers IL-1 release by activating the purinergic P2Z receptor of human macrophages. J Immunol 159: 1451-1458.[Abstract]
Ferrari D, Los M, Bauer MKA, Vandenabeele P, Wesselborg S, and Schulze-Osthoff K (1999) P2Z Purinoreceptor ligation induces activation of caspases with distinct roles in apoptotic and necrotic alterations of cell death. FEBS Lett 447: 71-75.[CrossRef][Medline]
Ferrari D, Villalba M, Chiozzi P, Falzoni S, Ricciardi-Castagnoli P, and DiVirgilio F (1996) Mouse microglia cells express a plasma membrane pore gated by extracellular ATP. J Immunol 156: 1531-1539.[Abstract]
Ferrari D, Wesselborg S, Bauer M, and Schulze-Osthoff K (1997b) Extracellular ATP activates transcription factor NF-[]B through the P2Z purinoreceptor by selectively targeting NF-[]b p65. J Cell Biol 139: 1635-1643.[Abstract/Free Full Text]
Fox T, Coll JT, Xie X, Ford PJ, Germann UA, Porter MD, Pazhanisamy S, Flemimg MA, Galullo V, Su MSS, and Wilson KP (1998) A single amino acid substitution makes ERK2 susceptible to pyridinyl imidazole inhibitors of p38 MAP kinase. Protein Sci (Wash DC) 7: 2249-2255.
Gafni J, Munsch JA, Lam TH, Catlin MC, Costa LG, Molinski TF, and Pessah IN (1997) Xestospongins: potent membrane permeable blockers of the inositol 1,4,5-trisphosphate receptor. Neuron 19: 723-733.[CrossRef][Medline]
Garcia-Calvo M, Peterson EP, Leiting B, Ruel R, Nicholson DW, and Thornberry NA (1998) Inhibition of human caspases by peptide-based and macromolecular inhibitors. J Biol Chem 273: 32603-32613.
Grahames GBA, Michel AD, Chessell IP, and Humphrey PPA (1999) Pharmacological characterization of ATP-and LPS-induced IL-1 release in human monocytes. Br J Pharmacol 127: 1915-1921.[CrossRef][Medline]
Humphreys BD and Dubyak GR (1996) Induction of the P2z/P2X7 nucleotide receptor and associated phospholipase D activity by lipopolysaccharide and IFN- in the human THP-1 monocytic cell line. J Immunol 157: 5627-5637.[Abstract]
Humphreys BD, Rice J, Kertesy SB, and Dubyak GR (2000) Stress-activated protein kinase/JNK activation and apoptotic induction by the macrophage P2X7 nucleotide receptor. J Biol Chem 275: 26792-26798.[Abstract/Free Full Text]
Humphreys BD, Virginio C, Surprenant A, Rice J, and Dubyak GR (1998) Isoquinolines as antagonists of the P2X7 nucleotide receptor: high selectivity for the human versus rat receptor. Mol Pharmacol 54: 22-32.[Abstract/Free Full Text]
Irnich D, Burgstahler R, and Grafe P (2001) P2 Nucleotide receptors in peripheral nerve trunk. Drug Dev Res 52: 83-88.[CrossRef]
Jacobson KA, Jarvis MF, and Williams M (2002) Purine and pyrimidine (P2) receptors as drug targets. J Med Chem 45: 4057-4093.[CrossRef][Medline]
Khakh BS, Bao XR, Labarca C, and Lester HA (1999) Neuronal P2X transmitter-gated cation channels change their ion selectivity in seconds. Nat Neurosci 2: 322-330.[CrossRef][Medline]
Kim M, Jiang LH, Wilson HL, North RA, and Surprenant A (2001) Proteomic and functional evidence for a P2X₇ receptor signaling complex. EMBO (Eur Mol Biol Organ) J 20: 6347-6358.[CrossRef][Medline]
Lee JC, Laydon T, McDonnell PC, Gallagher TF, Kumar S, Green D, McNulty D, Blumenthal MJ, Heys JR, Landvatter SW, et al. (1994) A protein kinase involved in the regulation of inflammatory cytokine biosynthesis. Nature (Lond) 372: 739-746.[CrossRef][Medline]
Lynch KJ, Touma E, Niforatos W, Kage KL, Burgard EC, van Biessen T, Kowaluk EA, and Jarvis MF (1999) Molecular and functional characterization of human P2X(2) receptors. Mol Pharmacol 56: 1171-1181.[Abstract/Free Full Text]
MacKenzie A, Wilson HL, Kiss-Toth E, Dower SK, North RA, and Surprenant A (2001) Rapid secretion of interleukin-1 by microvesicle shedding. Immunity 8: 825-835.
Michel AD, Chessell IP, and Humphrey PPA (1999) Ionic effects on human recombinant P2X₇ receptor function. Naunyn-Schmiedeberg's Arch Pharmacol 359: 102-109.[CrossRef][Medline]
North RA (2002) Molecular physiology of P2X receptors. Physiol Rev 82: 1013-1067.[Abstract/Free Full Text]
Ono K and Han J (2000) The p38 signal transduction pathway: activation and function. Cell Signal 12: 1-13.[CrossRef][Medline]
Pang L, Sawada T, Decker SJ, and Saltiel AR (1995) Inhibition of MAP kinase kinase blocks the differentiation of PC-12 cells induced by nerve growth factor. J Biol Chem 270: 13585-13588.[Abstract/Free Full Text]
Perregaux DG, Labasi J, Laliberte R, Stam E, Solle M, Koller B, Griffiths R, and Gabel CA (2001) Interleukin-1 posttranslational processing-exploration of P2X₇ receptor involvement. Drug Dev Res 53: 83-90.[CrossRef]
Rassendren F, Buell GN, Virginio C, Collo G, North RA, and Surprenant A (1997) The permeabilizing ATP receptor, P2X₇. J Biol Chem 272: 5482-5486.[Abstract/Free Full Text]
Schilling WP, Sinkins WG, and Estacion M (1999a) Maitotoxin activates a nonselective cation channel and a P2Z/P2X7-like cytolytic pore in human skin fibroblasts. Am J Physiol 277: C755-C765.
Schilling WP, Wasylyna T, Dubyak GR, Humphreys BD, and Sinkins WG (1999b) Maitotoxin and P2Z/P2X₇ purinergic receptors stimulation activate a common cytolytic pore. Am J Physiol 277: C766-C776.
Smith RJ, Justen JM, McNab AR, Rosenbloom CL, Steele AN, Detmers PA, Anderson DC, and Manning AM (1996) U-73122: a potent inhibitor of human polymorphonuclear neutrophil adhesion on biological surfaces and adhesion-related effector functions. J Pharmacol Exp Ther 278: 320-329.[Abstract/Free Full Text]
Su B and Karin M (1996) Mitogen-activated protein kinase cascades and regulation of gene expression. Curr Opin Immunol 8: 402-411.[CrossRef][Medline]
Surprenant A, Rassendren F, Kawashima E, North RA, and Buell G (1996) The cytolytic P2z receptor for extracellular ATP identified as a P2X receptor (P2X7). Science (Wash DC) 272: 735-738.[Abstract]
Thornberry NA and Lazebnik Y (1995) Caspases: enemies within. Science (Wash DC) 267: 1312-1316.
Torres GE, Haines WR, Egan TM, and Voigt MM (1998) Co-expression of P2X₁ and P2X₅ receptor subunits reveals a novel ATP-gated ion channel. Mol Pharmacol 54: 989-993.[Abstract/Free Full Text]
Verhoef PA, Estacion M, Schilling W, and Dubyak GR (2003) P2X7 receptor-dependent blebbing and the activation of rho-effector kinases, caspases, and IL-1 release. J Immunol 170: 5728-5738.[Abstract/Free Full Text]
Virginio C, MacKenzie A, North RA, and Surprenant A (1999) Kinetics of cell lysis, dye uptake and permeability changes in cells expressing the rat P2X7 receptor. J Physiol 519: 335-346.[Abstract/Free Full Text]
Won JGS and Orth DN (1995) Role of inositol trisphosphate-sensitive calcium stores in the regulation of adrenocorticotropin secretion by perifused rat anterior pituitary cells. Endocrinology 136: 5399-5408. Erratum in:[Abstract]Endocrinology (1996) 137: 1041.

This article has been cited by other articles:

				T. Tomita, T. Yamada, L. M. Weiss, and A. Orlofsky Externally Triggered Egress Is the Major Fate of Toxoplasma gondii during Acute Infection J. Immunol., November 15, 2009; 183(10): 6667 - 6680. [Abstract] [Full Text] [PDF]

				R.X. Faria, R.A.M. Reis, C.M. Casabulho, A.V.P. Alberto, F.P. de Farias, A. Henriques-Pons, and L.A. Alves Pharmacological properties of a pore induced by raising intracellular Ca2+ Am J Physiol Cell Physiol, July 1, 2009; 297(1): C28 - C42. [Abstract] [Full Text] [PDF]

				T. Noguchi, K. Ishii, H. Fukutomi, I. Naguro, A. Matsuzawa, K. Takeda, and H. Ichijo Requirement of Reactive Oxygen Species-dependent Activation of ASK1-p38 MAPK Pathway for Extracellular ATP-induced Apoptosis in Macrophage J. Biol. Chem., March 21, 2008; 283(12): 7657 - 7665. [Abstract] [Full Text] [PDF]

				C. Guo, M. Masin, O. S. Qureshi, and R. D. Murrell-Lagnado Evidence for Functional P2X4/P2X7 Heteromeric Receptors Mol. Pharmacol., December 1, 2007; 72(6): 1447 - 1456. [Abstract] [Full Text] [PDF]

				P. Honore, D. Donnelly-Roberts, M. T. Namovic, G. Hsieh, C. Z. Zhu, J. P. Mikusa, G. Hernandez, C. Zhong, D. M. Gauvin, P. Chandran, et al. A-740003 [N-(1-{[(Cyanoimino)(5-quinolinylamino) methyl]amino}-2,2-dimethylpropyl)-2-(3,4-dimethoxyphenyl)acetamide], a Novel and Selective P2X7 Receptor Antagonist, Dose-Dependently Reduces Neuropathic Pain in the Rat J. Pharmacol. Exp. Ther., December 1, 2006; 319(3): 1376 - 1385. [Abstract] [Full Text] [PDF]

				L.-H. Jiang, F. Rassendren, A. Mackenzie, Y.-H. Zhang, A. Surprenant, and R. A. North N-methyl-D-glucamine and propidium dyes utilize different permeation pathways at rat P2X7 receptors Am J Physiol Cell Physiol, November 1, 2005; 289(5): C1295 - C1302. [Abstract] [Full Text] [PDF]

				R. Auger, I. Motta, K. Benihoud, D. M. Ojcius, and J. M. Kanellopoulos A Role for Mitogen-activated Protein KinaseErk1/2 Activation and Non-selective Pore Formation in P2X7 Receptor-mediated Thymocyte Death J. Biol. Chem., July 29, 2005; 280(30): 28142 - 28151. [Abstract] [Full Text] [PDF]

				L. Liang and E. M. Schwiebert Large pore formation uniquely associated with P2X7 purinergic receptor channels. Focus on "Are second messengers crucial for opening the pore associated with P2X7 receptor?" Am J Physiol Cell Physiol, February 1, 2005; 288(2): C240 - C242. [Full Text] [PDF]

				R. X. Faria, F. P. DeFarias, and L. A. Alves Are second messengers crucial for opening the pore associated with P2X7 receptor? Am J Physiol Cell Physiol, February 1, 2005; 288(2): C260 - C271. [Abstract] [Full Text] [PDF]

This Article Abstract Full Text (PDF) All Versions of this Article: jpet.103.059600v1 308/3/1053    most recent Submit a response Alert me when this article is cited Alert me when eLetters are posted Alert me if a correction is posted Citation Map Services Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Donnelly-Roberts, D. L. Articles by Jarvis, M. F. Search for Related Content PubMed PubMed Citation Articles by Donnelly-Roberts, D. L. Articles by Jarvis, M. F. Pubmed/NCBI databases Compound via MeSH Substance via MeSH