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CELLULAR AND MOLECULAR

Cross Talk between P2Y₂ Nucleotide Receptors and CXC Chemokine Receptor 2 Resulting in Enhanced Ca²⁺ Signaling Involves Enhancement of Phospholipase C Activity and Is Enabled by Incremental Ca²⁺ Release in Human Embryonic Kidney Cells

Department of Cell Physiology and Pharmacology, Maurice Shock Medical Sciences Building, University of Leicester, Leicester, United Kingdom (T.D.W., G.B.W.); and Molecular Pharmacology, Enabling Science and Technology, AstraZeneca, Cheshire, United Kingdom (G.F.W.)

Received June 12, 2003; accepted August 8, 2003.

	Abstract

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We have shown previously that activation of endogenously expressed, G_q/11-coupled P2Y₂ nucleotide receptors with UTP reveals an intracellular Ca²⁺ response to activation of recombinant, G_i-coupled CXC chemokine receptor 2 (CXCR2) in human embryonic kidney cells. Here, we characterize further this cross talk and demonstrate that phospholipase C (PLC) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]-dependent Ca²⁺ release underlies this potentiation. The putative Ins(1,4,5)P₃ receptor antagonist 2-aminoethoxydiphenyl borane reduced the response to CXCR2 activation by interleukin-8, as did sustained inhibition of phosphatidylinositol 4-kinase with wortmannin, suggesting the involvement of phosphoinositides in the potentiation. Against a Li⁺ block of inositol monophosphatase activity, costimulation of P2Y₂ nucleotide receptors and CXCR2 caused phosphoinositide accumulation that was significantly greater than that after activation of P2Y₂ nucleotide receptors or CXCR2 alone, and was more than additive. Thus, PLC activity, as well as Ca²⁺ release, was enhanced. In these cells, agonist-mediated Ca²⁺ release was incremental in nature, suggesting that a potentiation of Ins(1,4,5)P₃ generation in the presence of coactivation of P2Y₂ nucleotide receptors and CXCR2 would be sufficient for additional Ca²⁺ release. Potentiated Ca²⁺ signaling by CXCR2 was markedly attenuated by expression of either regulator of G protein signaling 2 or the G-scavenger G_t1 (transducin subunit), indicating the involvement of G_q and G subunits, respectively.

A phenomenon of particular interest in terms of positive GPCR cross talk is the enhancement of intracellular Ca²⁺ release arising as a consequence of the concomitant or sequential stimulation of two types of GPCR that are preferentially coupled to different G proteins. For example, G_i/o-or G_s-coupled receptors can markedly enhance the Ca²⁺ signaling of simultaneously activated G_q/11-coupled receptors in clonal cell lines (Dickenson and Hill, 1994; Yeo et al., 2001). Such cross talk has also been demonstrated in cells derived from both the central nervous system (Jimenez et al., 1999; Hirono et al., 2001) and peripheral tissues (Shah et al., 1999; Cilluffo et al., 2000; Buckley et al., 2001) where potentiated cellular Ca²⁺ signaling may have important physiological consequences (Buckley et al., 2001; Hirono et al., 2001).

A number of studies have demonstrated an involvement of G_q/11-coupled P2Y nucleotide receptors in potentiated Ca²⁺ signaling (Jimenez et al., 1999; Quitterer and Lohse, 1999). We have also recently demonstrated in human embryonic kidney (HEK) cells that activation of endogenously expressed P2Y₂ nucleotide receptors reveals a Ca²⁺ signal to stimulation of recombinantly expressed G_i-coupled human CXC chemokine receptor 2 (CXCR2) (Werry et al., 2002). The ability of CXCR2 to mediate a Ca²⁺ response is absolutely dependent upon the presence of P2Y₂ nucleotide receptor agonists. Although the Ca²⁺ response to activation of P2Y₂ nucleotide receptors is insensitive to pertussis toxin (PTX), the potentiated CXCR2-mediated response is PTX-sensitive, showing the involvement of G_i/o. Furthermore, the potentiated CXCR2 response is independent of extracellular Ca²⁺, demonstrating that, after the release of Ca²⁺ by P2Y₂ nucleotide receptors, activated CXCR2 is able to play a role in the mobilization of an additional or discrete intracellular store of Ca²⁺ that is inaccessible after the stimulation of either CXCR2 or P2Y₂ nucleotide receptors alone. The identity of this store and the mechanism through which Ca²⁺ is mobilized from it remain to be identified and provide the focus of the current study. A large variety of mechanisms exist that have the potential to mediate such cross talk (Werry et al., 2003), but specific pathways have rarely been identified and have proved difficult to interrogate. In this study, we demonstrate that phospholipase C (PLC) activity and inositol 1,4,5-trisphosphate [Ins(1,4,5)P₃]-mediated Ca²⁺ release are crucial to the enhanced release of Ca²⁺ resulting from cross talk and that it is dependent upon both G_q and G subunits. Furthermore, we demonstrate that activation of G_q/ ¹¹-coupled receptors in these cells results in incremental Ca²⁺ release. As a consequence of this, we suggest that potentiated inositol phosphate production is sufficient to account for the cross talk at the level of Ca²⁺ release and discuss mechanisms through which this may occur.

	Materials and Methods

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Materials. Cell culture reagents were obtained from Invitrogen (Paisley, Scotland). Cell culture plastics were obtained from Nalgene (Europe) Ltd. (Hereford, UK). Genejuice transfection reagent was from Novagen (through CN Biosciences, Nottingham, UK), and pTracer expression vector was supplied by Invitrogen (Inchannan, Scotland). Fura-2 acetoxymethyl ester (fura-2/AM), UTP, thapsigargin, ryanodine, and caffeine were obtained from Sigma Chemical (Poole, Dorset, UK). Fluo-3/AM was from TEF Labs (Austin, TX). Interleukin-8 (IL-8) was supplied by R&D Systems (Abingdon, UK). Staurosporine, 2-aminoethoxydiphenyl borane (2-APB), cyclosporin A, and okadaic acid were from Calbiochem (through CN Biosciences). All other reagents were of analytical grade and were obtained from Sigma Chemical or Fisher Scientific (Loughborough, UK).

Cloned cDNA encoding either the bovine transducin G subunit (G_t1) or human G_q-specific regulator of G protein signaling 2 (RGS2) were kind gifts from Prof. G. Milligan (University of Glasgow, Glasgow, UK) and Dr. C. Doupnik (University of South Florida, Tampa, FL), respectively.

Assay buffer used in all experiments was a balanced salts solution (BSS) composed of 130 mM NaCl, 5.4 mM KCl, 16 mM NaHCO₃, 1.3 mM NaH₂PO₄, 0.8 mM MgCl₂, 1.8 mM CaCl₂, 10 mM HEPES, and 5.5 mM D-glucose, pH 7.4.

Cell Culture: HEK-CXCR2 Cell Line. The HEK cell line expressing recombinant human CXCR2 at approximately 50,000 sites/cell was generated and selected as described previously (Werry et al., 2002). This cell line (HEK-CXCR2) was maintained in Dulbecco's modified Eagle's medium (containing 25 mM D-glucose, 4 mM L-alanyl-l-glutamine, and 1 mM sodium pyruvate) supplemented with 10% fetal calf serum, 1% nonessential amino acids, 50 µg/ml gentamicin, and 400 µg/ml G-418 at 37°C in a 5% CO₂ humidified atmosphere.

Generation of HEK-CXCR2 Cells Expressing Either G_t1 or RGS2. The coding sequence for G_t1 was ligated into pTracer using EcoRI and XhoI restriction sites. The cDNA encoding RGS2 was ligated into pTracer using BstX1 restriction sites. HEK-CXCR2 cells were transfected with one of these pTracer constructs or were control-transfected (using pTracer containing neither G_t1 nor RGS2 cDNA) as a control. Transfection was performed using Genejuice transfection reagent according to the manufacturer's instructions. Nonclonally selected cell lines containing either G_t1, RGS2 or pTracer were created using the blasticidin-resistance property of pTracer, incubating for 3 weeks with growth medium containing 5 µg/ml blasticidin. Untransfected HEK-CXCR2 cells were treated identically as a positive control for the ability of blasticidin to kill nontransfected cells. The pTracer control cell line was used as the control for all experiments using either RGS2- or G_t1-transfected cells.

Measurement of the Intracellular Ca²⁺ Concentration ([Ca²⁺]_i). HEK-CXCR2 cells were seeded onto 22-mm-diameter poly-d-lysine-coated glass coverslips and cultured for 48 h. Cells were then loaded with fluo-3/AM or fura-2/AM (5 µM, 1 h, room temperature) and the coverslips mounted in a perfusion chamber on the stage of an IX70-S1F inverted microscope (Olympus, Tokyo, Japan). The chamber was perfused at a rate of 5 ml/min with BSS or drug solutions and the temperature maintained at 37°C using a Peltier unit. Using a monochromator, cells were excited at 488 nm (fluo-3) or at 340 and 380 nm (1-s intervals; fura-2) by light from a xenon lamp (PerkinElmer Life Sciences, Cambridge, UK). Fluorescence emissions at 510 nm (fluo-3) or above 510 nm (fura-2) were detected by a charge-coupled device camera at a rate of 0.75 frames/s (fluo-3) or 0.5 frames/s (fura-2) and converted into on-screen images by UltraVIEW imaging software (PerkinElmer Life Sciences). Fluo-3 was used preferentially, but fura-2 was used in experiments using cells expressing green fluorescent protein (GFP) from pTracer because there was significant "bleed-through" of fluorescence in the emission spectra of GFP and fluo-3. This bleed-through was substantially reduced when fura-2 was used. Measurements were made by averaging fluorophore fluorescence levels across a field of 10 to 20 cells. All responses were internally controlled, being normalized against the response to a high concentration of nucleotide (100 µM UTP or similar, as indicated in individual figures) in the presence of extracellular Ca²⁺.

Total [³H]Inositol Phosphate Generation. Cells were grown for 48 h in the presence of 3 µCi/ml [³H]myo-inositol. After washing and preincubation (20 min, 37°C) with BSS containing 10 mM Li⁺ to inhibit inositol monophosphatase activity, cells were stimulated for the required time before the reaction was stopped with an equivalent volume of ice-cold, 1 M trichloroacetic acid. The reaction mix (1-ml final volume) was added to 250 µl of 10 mM EDTA together with 1 ml of a freshly prepared 1:1 (v/v) mixture of tri-n-octyl-amine and 1,1,2-trichloro-trifluoroethane and mixed thoroughly by vortexing. A 700-µl aliquot of the upper aqueous layer was removed and added to 50 µl of 250 mM NaHCO₃. Soluble inositol phosphates in this aqueous fraction were subsequently isolated using strongly basic Dowex chloride anion exchange columns (8% cross linkage, 100-200 dry mesh; Sigma 1 x 8-200) by adding the sample to the column, and washing firstly with water and then with 25 mM ammonium formate. [³H]Inositol phosphates ([³H]InsP_x) were eluted from the columns using 1 M HCl and quantified using liquid scintillation counting.

	Results

Top Abstract Materials and Methods Results Discussion References

 
Potentiation of CXCR2-Mediated Ca²⁺ Signaling by P2Y₂ Nucleotide Receptor Activation. Repeated short exposure of HEK-CXCR2 cells to 100 µM UTP (which activates P2Y₂ nucleotide receptors in these cells; Werry et al., 2002) in the absence of extracellular Ca²⁺ resulted in the gradual loss of UTP-mediated [Ca²⁺]_i elevation (Fig. 1a). A similar protocol performed in the presence of extracellular Ca²⁺ did not result in diminished responses to UTP (Fig. 1b), indicating an absence of P2Y₂ nucleotide receptor desensitization to repetitive short exposures to UTP over this time frame. Thus, in the absence of extracellular Ca²⁺, UTP is able to fully drain the intracellular Ca²⁺ store to which it has access. After drainage of the UTP-sensitive store, 10 nM IL-8 is unable to elevate [Ca²⁺]_i unless it is added with UTP (Fig. 1a). The magnitude of this Ca²⁺ response relative to that of the addition of UTP to naive cells is comparable with that seen in our previous studies using a fluorescent light imaging plate reader in which IL-8 was added after, but in the continued presence of UTP (Werry et al., 2002). These data demonstrate that a store of Ca²⁺ is accessed after coaddition of UTP and IL-8 that is inaccessible to UTP or IL-8 alone.

View larger version (20K): [in this window] [in a new window]   Fig. 1. Potentiation of IL-8-mediated elevation of [Ca2+]i in HEK-CXCR2 cells after nucleotide-mediated Ca2+ store depletion. a, protocol used to demonstrate the potentiation of Ca2+ signaling. Using a Ca2+ imager with a perfusion system as described under Materials and Methods, fluo-3-loaded cells were initially challenged with 100 µM UTP as a reference response. Subsequently, cells were repeatedly stimulated with 100 µM UTP for the times indicated (black bars) in the absence of extracellular Ca2+ until no Ca2+ response was observed. Cells were then stimulated with 10 nM IL-8 (to demonstrate the lack of response) followed by a coaddition of 100 µM UTP and 10 nM IL-8. Shown is a representative trace tracking changes in fluo-3 fluorescence in a small population of cells (<20) as an index of [Ca2+]i. Arrow A indicates the point at which some of the inhibitors were added (see text and other figures for details). b, fluo-3-loaded cells were prepared as in a and perfused with 100 µM UTP as indicated (black bars). Shown is a representative trace tracking changes in fluo-3 fluorescence in a small population of cells (<20) as an index of [Ca2+]i. In contrast to a, this experiment was carried out in the presence of extracellular Ca2+ to demonstrate that short, repetitive exposure to 100 µM UTP does not result in a desensitization of the response.

Potentiated Ca²⁺ Responses after Coaddition of UTP and IL-8 Require a Thapsigargin-Sensitive Ca²⁺ Store but Not Ryanodine Receptors. Cells were treated with the sarcoendoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin (2 µM), before stimulating with a single addition of either 100 µM UTP, or a coaddition of 100 µM UTP and 10 nM IL-8. Thapsigargin abolished the Ca²⁺ responses to both of these stimulations (Fig. 2a).

View larger version (19K): [in this window] [in a new window]   Fig. 2. The Ca2+ store accessed by IL-8 in the presence of UTP is thapsigargin-sensitive (a), but is not ryanodine-sensitive (b). a, HEKCXCR2 cells were incubated with or without 2 µM thapsigargin for 5 min before assay. Cells were then stimulated with a single addition of either 100 µM UTP or a coaddition of 100 µM UTP and 10 nM IL-8. Using a Ca2+ imaging system, changes in fluo-3 fluorescence were measured as an index of [Ca2+]i. Responses are expressed as a percentage of the maximal response to 100 µM UTP in the absence of thapsigargin pretreatment. Data are mean ± S.E.M., n = 4, with *, P < 0.05 and **, P < 0.01 determined by unpaired Student's t test. b, using the store depletion protocol and Ca2+ imaging (Fig. 1), cells were incubated with or without 30 µM ryanodine after store depletion in the absence of extracellular Ca2+ (point A, Fig. 1) before stimulating with a coaddition of 100 µM UTP and 10 nM IL-8. Responses to this coaddition were measured as changes in fluo-3 fluorescence and are expressed as a percentage of the maximal response to 100 µM UTP before store drainage. Data are mean ± S.E.M., n = 4. c, as a positive control for the effect of ryanodine, fluo-3-loaded cells were stimulated with 10 mM caffeine in the absence or presence of 30 µM ryanodine (with a 5-min preincubation). Responses to caffeine stimulation are expressed as fluo-3 fluorescence. Data are mean ± S.E.M., n = 4, with *, P < 0.05 determined by unpaired Student's t test.

To test whether Ca²⁺ stores gated by activation of ryanodine receptors are involved in the Ca²⁺ response to coactivation of CXCR2 and P2Y₂ nucleotide receptors, store-depleted cells were incubated for 5 min (from point A, Fig. 1) in the presence or absence of 30 µM ryanodine before a coaddition of 100 µM UTP and 10 nM IL-8. Ryanodine had no significant effect on the response to this coaddition (Fig. 2b). This concentration of ryanodine was shown to be effective at blocking ryanodine receptors because it inhibited the Ca²⁺ elevation seen after stimulation with 10 mM caffeine (Fig. 2c).

Inositol Phosphate Generation Is Enhanced by Costimulation of P2Y₂ Nucleotide Receptors and CXCR2. Agonist-mediated accumulation of [³H]InsP_x against a Li⁺ block of inositol monophosphatase was determined as an index of PLC activity. HEK-CXCR2 cells loaded with [³H]myo-inositol were stimulated in the presence of Li⁺ with either 100 nM IL-8, 1 mM UTP, or a coaddition of both agonists for varying durations (range 0-30 min). IL-8 alone evoked little or no accumulation of [³H]InsP_x (Fig. 3). In contrast, UTP caused an accumulation of [³H]InsP_x to a maximum of 1.6 ± 0.09-fold of basal, whereas coaddition of both agonists elicited a maximum accumulation of 2.99 ± 0.11-fold of basal. Furthermore, in the presence of both agonists, accumulation continued for approximately 15 min compared with an accumulation that only continued for approximately 5 min in the presence of UTP alone (Fig. 3). We also measured mass levels of Ins(1,4,5)P₃ using a radioreceptor assay exactly as described previously (Willars et al., 1998). Basal levels were 33 ± 5 (n = 4) pmol/mg protein but were not consistently elevated by either 1 mM UTP alone or coaddition of 1 mM UTP and 10 nM IL-8, suggesting small, localized production and/or rapid metabolism of Ins(1,4,5)P₃.

View larger version (11K): [in this window] [in a new window]   Fig. 3. Costimulation of P2Y2 nucleotide receptors and CXCR2 also potentiates phosphoinositide hydrolysis. HEK-CXCR2 cells loaded with [3H]myo-inositol were stimulated with the indicated agonist additions for varying periods in the presence of 10 mM Li+. Accumulated [3H]InsPx were isolated by phase separation and quantified by liquid scintillation counting. Shown are the accumulations of [3H]InsPx for each stimulation at each time point, expressed as fold of basal. Data are mean ± S.E.M., n = 5. The responses were compared using two-way analysis of variance (P << 0.001) followed by a comparison of the maximal accumulations by Student's t test (***, P < 0.001).

The role of PLC was also explored using the putative PLC inhibitor U73122 [GenBank] . After store drainage, a 5-min incubation with 10 µM U73122 [GenBank] (from point A, Fig. 1) reduced the Ca²⁺ response to coaddition of 100 µM UTP and 10 nM IL-8 from 64 ± 3% (n = 5) (of the response to 100 µM UTP in naive cells) to 9 ± 4% (n = 5). In contrast 10 µM U73343 [GenBank] (the aminosteroid negative control to U73122 [GenBank] ) did not affect the response to coaddition (64 ± 8%; n = 5). Qualitatively similar results were obtained using a coaddition of 3 µM UTP and 10 nM IL-8 (control 38 ± 6% of the response to 100 µM UTP in naive cells, U73122 [GenBank] -treated 0%, U73343 [GenBank] -treated 38 ± 2%; n = 5 for each). Despite this, a variety of data were obtained that were not consistent with the specific inhibition of PLC by 10 µM U73122 [GenBank] in these cells. As an example, U73122 [GenBank] had no effect on the accumulation of [³H]InsP_x after stimulation of muscarinic M₃ receptors, yet inhibited muscarinic receptor-mediated Ca²⁺ responses, as did U73343 [GenBank] . These data confirm previous observations that U73122 [GenBank] is unreliable as a specific inhibitor of PLC (Taylor and Broad, 1998; Walker et al., 1998) and emphasize caution in the interpretation of experiments in which it is used.

Potentiated Ca²⁺ Responses after Coaddition of UTP and IL-8 Are Inhibited by 2-APB. To examine the involvement of Ins(1,4,5)P₃ receptors in the potentiated response to CXCR2 activation, the effects of the putative Ins(1,4,5)P₃ receptor inhibitor 2-APB were investigated. Responses to 100 µM UTP in the presence and absence of 100 µM 2-APB (5-min preincubation) in nonstore-depleted cells were measured as a positive control to the action of 2-APB. Under these circumstances, 2-APB markedly reduced the Ca²⁺ responses to 100 µM UTP (Fig. 4b). After Ca²⁺ store depletion using 100 µM UTP (Fig. 1), cells were exposed to either 100 µM 2-APB or buffer alone for 5 min (from point A, Fig. 1) and subsequently, in the continued presence or absence of 2-APB, stimulated sequentially with 100 µM UTP, 10 nM IL-8, and a coaddition of these agonists, removing each agonist before addition of the next. Consistent with the data mentioned above, coaddition resulted in a robust Ca²⁺ response, but this was significantly reduced in the presence of 2-APB (Fig. 4a). Neither agonist alone elevated [Ca²⁺]_i in the presence or absence of 2-APB, ruling out the possibility that any Ca²⁺ store refilling had occurred during incubation with 2-APB (data not shown).

View larger version (14K): [in this window] [in a new window]   Fig. 4. Antagonism of Ins(1,4,5)P3 receptors with 2-APB inhibits potentiation. a, after depletion of the UTP-sensitive Ca2+ store (Fig. 1), cells were incubated with or without 2-APB (100 µM; 5 min) before resuming stimulations as in Fig. 1, in the continued presence (or absence) of 2-APB. Shown are responses to coaddition of 100 µM UTP and 10 nM IL-8, using fluo-3 fluorescence as an index of [Ca2+]i. Data are expressed as a percentage of the initial response to 100 µM UTP, mean ± S.E.M., n = 4, with *, P < 0.05 determined by unpaired Student's t test. b, as a positive control for the effect of 2-APB, cells were stimulated with 100 µM UTP by perfusion for 30s, followed by washout for 90 s with buffer containing Ca2+ to allow refilling of intracellular Ca2+ stores. Cells were then restimulated twice more with 100 µM UTP, first in the absence of 2-APB (to demonstrate reproducibility of responses) and subsequently after a 5-min incubation with 100 µM 2-APB (during which time extracellular Ca2+ was present to facilitate store refilling). Data are expressed and analyzed as in main figure; n = 4 (***, P < 0.001).

Chronic, but Not Acute, Inhibition of Phosphatidylinositol 4-Kinase Inhibits Potentiation of Ca²⁺ Responses after Coaddition of UTP and IL-8. Activation of phosphatidylinositol 3-kinase (PI 3-kinase) in HEK cells is blocked by wortmannin at a concentration of 100 to 300 nM (Meier et al., 1997; van der Kaay et al., 1997; Sweeney et al., 2001). Preincubation of our HEK-CXCR2 cells for 20 min with 300 nM wortmannin inhibited the PI 3-kinase-mediated activation of extracellular signal-regulated kinase 1/2 by fetal bovine serum by 73% as assessed using an in vitro kinase assay exactly as described previously (Wylie et al., 1999). However, 300 nM wortmannin had no effect on the Ca²⁺ responses to 10 nM IL-8 when IL-8 was added with either 3 µM UTP (42 ± 3 versus 38 ± 5% in controls, where responses are expressed as a percentage of the response to addition of 100 µM UTP alone in naive cells) or 100 µM UTP (56 ± 8 versus 65 ± 10% in controls), indicating the lack of any involvement of PI 3-kinase.

At higher concentrations than those used to block PI 3-kinase, wortmannin inhibits phosphatidylinositol 4-kinase (PI 4-kinase) (Nakanishi et al., 1995; Willars et al., 1998), a crucial enzyme in maintaining the supply of the PLC substrate, phosphatidylinositol 4,5-bisphosphate (PIP₂). As inhibition of PI 3-kinase had no impact on the potentiated Ca²⁺ response to IL-8 in the presence of UTP (see above), we used wortmannin at 10 µM to inhibit PI 4-kinase activity and limit the supply of PIP₂ (Nakanishi et al., 1995; Willars et al., 1998). A 20-min preincubation with 10 µM wortmannin had no effect on the response to IL-8 when this agonist was added after 150-s prestimulation with 100 µM UTP (Fig. 5a). When the prestimulation with UTP was extended to 7 min in the absence of wortmannin, the Ca²⁺ response seen after the subsequent addition of IL-8 was similar to that seen after 150-s prestimulation with UTP (Fig. 5a), but if cells were preincubated with wortmannin for 20 min and then stimulated with UTP for 7 min in the continued presence of wortmannin, the subsequent Ca²⁺ response after IL-8 addition was significantly reduced (Fig. 5a). This suggests that the response observed after coaddition of UTP and IL-8 is dependent upon phosphoinositides but that significant depletion of PIP₂ requires exposure to wortmannin and sustained UTP signaling and is unlikely to be relevant over the usually short time course of our experiments.

View larger version (19K): [in this window] [in a new window]   Fig. 5. Effects of the inhibition of PI 4-kinase on cross talk between P2Y2 nucleotide receptors and CXCR2. a, cells were preincubated for 20 min with 10 µM wortmannin or vehicle (0.1% DMSO). Using a protocol that maintained the presence of UTP (Werry et al., 2002), cells were stimulated (in the continued presence of 10 µM wortmannin, or vehicle) by perfusion of 100 µM UTP for either 150 s or 7 min. After this, cells were then stimulated with 10 nM IL-8 in the continued presence of UTP. Responses shown are those to the addition of IL-8, expressed as a percentage of the maximal response to 100 µM UTP, using changes in fluo-3 fluorescence as an index of [Ca2+]i. Data are mean ± S.E.M., n = 4, with *, P < 0.05 by Student's unpaired t test versus the response in the absence of wortmannin. b, cells were preincubated for 20 min with 10 µM wortmannin, then stimulated (in the continued presence of wortmannin) with 100 µM UTP (black bars) and 100 µM carbachol (CCh; dark gray bars), separated by periods of agonist washout with buffer. Shown is a representative trace of three experiments. Data are expressed as changes in fluo-3 "gray levels" as an index of [Ca2+]i.

To confirm that PIP₂ levels were not limiting to acute UTP signaling in these cells, repeated short stimulations with UTP were performed in the presence or absence of 10 µM wortmannin. Responses to these stimulations with UTP (in the presence of extracellular Ca²⁺) did not progressively reduce in the presence of wortmannin, even when cells were additionally stimulated for 200 s by carbachol to activate an endogenously expressed G_q-coupled muscarinic M₃ receptor (thus further depleting the cellular PIP₂ pool but without sustained activation and potential desensitization of the P2Y₂ nucleotide receptors) (Fig. 5b).

Incremental Ca²⁺ Release Occurs in HEK-CXCR2 Cells. Cells were repetitively stimulated (in the absence of extracellular Ca²⁺, to prevent store refilling) with progressively increasing concentrations of UTP (Fig. 6). The aim of this protocol was to progressively increase the generation of Ins(1,4,5)P₃ to investigate whether incremental increases in Ins(1,4,5)P₃ resulted in a corresponding fractional release of Ca²⁺ from intracellular stores. Stimulation with a train of 20-s pulses of 1 µM UTP (with 20-s perfusion of nominally Ca²⁺-free buffer separating each) caused progressively diminished responses until no further significant Ca²⁺ release was observed (Fig. 6). These cells were then stimulated with 20-s pulses of progressively higher concentrations of UTP (10 µM then 100 µM), until again no further response was seen to either concentration. Finally, the cells were costimulated with 100 µM UTP and 10 nM IL-8. At the point at which 1 µM UTP could cause no further increase in [Ca²⁺]_i, a robust elevation was seen after subsequent stimulation with 10 µM UTP. At the point at which no Ca²⁺ response was seen to 10 µM UTP, there was little response to stimulation with 100 µM UTP (Fig. 6). However, after depletion of the UTP-sensitive Ca²⁺ store using this protocol, coaddition of 100 µM UTP and 10 nM IL-8 evoked a further robust elevation of [Ca²⁺]_i (Fig. 6).

View larger version (12K): [in this window] [in a new window]   Fig. 6. HEK cells exhibit incremental Ca2+ release. Using a Ca2+ imaging system that allowed continuous perfusion of the cell chamber, cells were initially stimulated (in the absence of extracellular Ca2+) with repeated 20-s pulses of 1 µM UTP until no further Ca2+ response was observed. UTP concentration was then increased to 10 µM, and the same cells were stimulated until there was again no further Ca2+ response, and then likewise with 100 µM UTP. Finally, cells were challenged with a coaddition of 100 µM UTP and 10 nM IL-8. Values shown are the sequential responses to each pulse of agonist, measured as changes in fluo-3 fluorescence. Data are mean ± S.E.M., n = 3.

Expression of Recombinant RGS2 or G_t1 Inhibits the P2Y₂ Nucleotide Receptor-Dependent Ca²⁺ Response to CXCR2 Activation. To assess the participation of G_q subunits in the potentiated response to CXCR2, HEKCXCR2 cells were transfected with RGS2, a GTPase-activating protein that attenuates G_q signaling by selectively accelerating the intrinsic GTPase activity of G_q (Heximer et al., 1997). HEK-CXCR2/RGS2 cells (or cells transfected with the empty pTracer vector) were stimulated with 100 µM UTP followed, 150 s later, with 10 nM IL-8 in the continued presence of UTP. In cells expressing RGS2, the response to 10 nM IL-8 in the presence of 100 µM UTP was reduced by approximately 65% compared with control cells (Fig. 7).

View larger version (17K): [in this window] [in a new window]   Fig. 7. Expression of RGS2 or Gt1 inhibits potentiation of Ca2+ signaling. HEK-CXCR2 cells were transfected with cDNA encoding Gt1 or RGS2 in pTracer vector, or mock-transfected with "empty" pTracer vector. Cells were then selected using blasticidin but clones were not isolated. Using a Ca2+ imager and perfusion system, cells were stimulated with 100 µM UTP followed (150 s later) by 10 nM IL-8 (in the continued presence of UTP). Shown are responses to IL-8, expressed as a percentage of the initial maximal response to UTP. Data are mean ± S.E.M., n = 5, with *, P < 0.05 by Duncan's multiple range test after one-way analysis of variance that gave P < 0.05.

To test the involvement of G subunits in the potentiated response to CXCR2, HEK-CXCR2 cells were transfected with the G-scavenger G_t1. Expression of G_t1 caused a significant reduction in the magnitude of the response to 10 nM IL-8 in the presence of 100 µM UTP (Fig. 7).

Potentiated Ca²⁺ Responses after Coaddition of UTP and IL-8 Are Not a Simple Consequence of the Pooling of G-Subunits. In our HEK cells, ongoing activation of P2Y₂ nucleotide receptors also reveals a robust Ca²⁺ response to activation of endogenously expressed G_s-coupled ₂-adrenoceptors (Werry et al., 2002). Despite this, coactivation of CXCR2 with 10 nM IL-8 and ₂-adrenoceptors with 10 µM isoproterenol did not influence [Ca²⁺]_i (Fig. 8).

View larger version (13K): [in this window] [in a new window]   Fig. 8. Coactivation of Gi- and Gs-coupled receptors does not result in Ca2+ signaling. Ca2+ stores were drained as in Fig. 1a. After this pretreatment, and after UTP was removed, cells were stimulated with either 10 µM isoproterenol (Isop) or 10 nM IL-8, or a combination of both. Fluo-3 fluorescence changes were measured using a Ca2+ imaging system as an index of [Ca2+]i elevation. Values shown are the responses to additions made after store drainage compared with the maximal response to 100 µM UTP ("control"). Data are mean ± S.E.M., n = 4.

Potentiated Ca²⁺ Responses after Coaddition of UTP and IL-8 Are Unaffected by Inhibition of Protein Kinase C, Tyrosine Kinase, or Phosphatase Activities. Cells were treated with either the protein kinase C (PKC) and tyrosine kinase inhibitor staurosporine (3 µM), or with vehicle (0.1% DMSO) for 20 min before assay. This treatment with staurosporine blocks PKC activity in these cells (Ferrari et al., 1999). The UTP-sensitive intracellular Ca²⁺ store was then drained by repeated stimulation with 100 µM UTP in the absence of extracellular Ca²⁺ (Fig. 1) in the continued presence of staurosporine or vehicle control. Consistent with all the data mentioned above, after store drainage the addition of either 100 µM UTP or 10 nM IL-8 alone did not elevate [Ca²⁺]_i, and in four experiments this was unaffected by staurosporine. Coaddition of UTP and IL-8 evoked a Ca²⁺ response that was equivalent in the presence and absence of treatment with staurosporine (Fig. 9).

View larger version (23K): [in this window] [in a new window]   Fig. 9. Ca2+ responses to coactivation of P2Y2 nucleotide receptors and CXCR2 are unaffected by inhibition of either protein kinase C, tyrosine kinase, or phosphatase activities. Cells were pretreated for 20 min with 3 µM staurosporine (Staur). In its continued presence, the UTP-sensitive Ca2+ store was then depleted using the Ca2+ imager perfusion protocol (Fig. 1) in the absence of extracellular Ca2+. Cells were then stimulated with a coaddition of 100 µM UTP and 10 nM IL-8. Alternatively, cells were Ca2+ store-depleted and then incubated with either 10 µM okadaic acid (OA) or 10 µM cyclosporin A (CsA) for 5 min (from point A shown in Fig. 1). Cells were subsequently stimulated with a coaddition of 100 µM UTP and 10 nM IL-8. In all cases, the response to coaddition of UTP and IL-8 is shown expressed as a percentage of the maximal Ca2+ response to an addition of 100 µM UTP before store drainage. Control cells were treated with vehicle only (0.01% DMSO) for the same duration as each inhibitor. Shown is the control response to staurosporine (0.01% DMSO for 20 min before and throughout assay). Controls to OA and CsA are not shown but were identical to the staurosporine control. Data are mean ± S.E.M., n = 4.

Using the protocol described above (Fig. 1), the UTP-sensitive store was drained, and cells were exposed to either 10 µM okadaic acid (an inhibitor of protein phosphatases 1 and 2A) or 10 µM cyclosporin A (an inhibitor of protein phosphatase 2B) for 5 min (from point A, Fig. 1). These concentrations (or lower) have been shown previously to effectively inhibit the activity of these phosphatases (Groblewski et al., 1994; Otero et al., 2000). Neither compound had any effect on the response to a subsequent coaddition of 100 µM UTP and 10 nM IL-8 (Fig. 9).

	Discussion

Top Abstract Materials and Methods Results Discussion References

 
There are many reports of cross talk between G_i/o- and G_q/11-coupled receptors that results in enhanced Ca²⁺ signaling. A diversity of mechanisms have been suggested to account for such cross talk (Werry et al., 2003), but in general they are poorly defined and often not easily tested. Furthermore, it is likely that multiple mechanisms exist and precisely which are involved may depend on factors such as the receptors studied and the cellular background used. In any particular instance of cross talk, determining the source of Ca²⁺ and whether the enhanced Ca²⁺ signaling is associated with increased PLC activity may give some indication of the mechanism involved.

We have shown previously that cross talk between P2Y₂ nucleotide receptors and CXCR2 requires costimulation of the receptors, is PTX-sensitive, and is independent of extracellular Ca²⁺ (Werry et al., 2002). Furthermore, our previous work demonstrates that the ability of P2Y₂ nucleotide receptors to potentiate Ca²⁺ signaling by CXCR2 is dependent upon the concentration of UTP with an EC₅₀ value of approximately 10 µM. In the present study, we have used either maximal (100 µM) or submaximal (3 µM) concentrations of UTP with maximal concentrations of IL-8 (10 nM) to further investigate this cross talk. Our data show that the potentiated CXCR2-mediated Ca²⁺ response is dependent upon a thapsigargin-sensitive intracellular Ca²⁺ store but independent of ryanodine receptors, suggesting that this cross talk may be Ins(1,4,5)P₃-dependent. Inhibition of the potentiated Ca²⁺ response by the putative Ins(1,4,5)P₃ receptor antagonist 2-APB supports this conclusion. Although 2-APB can have nonselective effects on Ca²⁺ handling, particularly the block of Ca²⁺ channels other than Ins(1,4,5)P₃ receptors (Bootman et al., 2002), our experiments were conducted in the absence of extracellular Ca²⁺ to negate any impact on Ca²⁺ entry. Furthermore, the enhanced accumulation of [³H]InsP_x during costimulation with UTP and IL-8 (compared with UTP alone) indicates that potentiated PLC activity is associated with coactivation of P2Y₂ nucleotide receptors and CXCR2. It is of interest that after longer stimulation with UTP in the presence of 10 µM wortmannin (to reduce PIP₂ levels in the plasma membrane), the Ca²⁺ responses to receptor coactivation were reduced. This confirms that PIP₂ is required, presumably for the generation of Ins(1,4,5)P₃.

If levels of PIP₂ are rate-limiting for PLC activity, then an increase in its supply could account for enhanced PLC activity and Ca²⁺ signaling. In this respect, an increase in PIP₂ levels mediated via PTX-sensitive G proteins enhances muscarinic receptor-mediated Ca²⁺ signaling in HEK cells (Schmidt et al., 1996). Thus, an increased supply of PIP₂ in response to activation of CXCR2 could result in enhanced P2Y₂ nucleotide receptor-mediated Ins(1,4,5)P₃ generation. PI 4-kinase may limit the supply of PIP₂ (Willars et al., 1998), and we therefore determined the effect on the cross talk of PI 4-kinase inhibition. We found that stimulation of PI 4-kinase by CXCR2 is unlikely to be involved in potentiation given the lack of effect of PI 4-kinase inhibition on Ca²⁺ responses to coaddition after a relatively short prestimulation with UTP. However, other phosphoinositide kinases may play a role in the maintenance of PIP₂ levels and thus a role of, for example, phosphatidylinositol 4-phosphate 5-kinase, cannot be excluded.

In the absence of Ca²⁺ store refilling, consecutive increases in the concentration of UTP caused further release of intracellular Ca²⁺. This is consistent with quantal Ca²⁺ release (Bootman, 1994) or incremental detection (Meyer and Stryer, 1990). Thus, the intracellular Ca²⁺ store seems to be functionally divided into fractions that are released incrementally according to the concentration of Ins(1,4,5)P₃. The presence of incremental Ca²⁺ release in these cells suggests that other mechanisms such as agonist-dependent shifting of Ca²⁺ between stores (Short and Taylor, 2000) and sensitization of the Ins(1,4,5)P₃ receptor (Tovey et al., 2003) are not required to mediate cross talk, resulting in enhanced Ca²⁺ mobilization when PLC activity and Ins(1,4,5)P₃ generation are potentiated. It is also possible that Ca²⁺ is released from stores that are accessed according to the locality of Ins(1,4,5)P₃ generation. However, at the resolution of our imaging equipment, we were unable to distinguish any spatial differences in the Ca²⁺ signaling mediated by either UTP alone or a coaddition of UTP and IL-8.

Cross talk between CXCR2 and P2Y₂ nucleotide receptors is abolished by PTX treatment (Werry et al., 2002). Given that CXCR2 is coupled to G_i, this may explain the PTX sensitivity of the cross talk, although there remains the possibility that coupling of P2Y₂ nucleotide receptors to G_i/o may be important. However, inhibition of cross talk by the G_q-specific RGS protein RGS2 indicates a role for G_q. One possible mechanism is that coincident receptor stimulation enhances activation of G_q through mechanisms such as heterodimerization and/or G protein switching (Lawler et al., 2001; Mellado et al., 2001). We attempted to measure the activation of G_q directly in cell membranes using the binding of [³⁵S]guanosine 5'-O-(3-thio)triphosphate and specific immunoprecipitation. However, this was not possible as UTP competed effectively with the radiolabeled compound for binding to G_q.

The majority of PLC-coupled GPCRs undergo either full or partial desensitization within seconds of agonist addition, and this may occur at the receptor and/or postreceptor level (Ferguson, 2001). A reduction or reversal of desensitization could account for potentiated signaling. Indeed, the accumulation of [³H]InsP_x during costimulation with UTP and IL-8 was more prolonged than during stimulation with UTP alone (Fig. 3), suggesting that cross talk may protect PLC activity from desensitization. Independent of the molecular site, the common denominator in desensitization is often a change in the phosphorylation state of proteins, and we therefore disrupted pathways by which phosphorylation states can be altered. First, staurosporine was used under conditions shown to inhibit PKC in these cells (Ferrari et al., 1999). PKC is key in the feedback inhibition of signaling by many PLC-coupled GPCRs (Chuang et al., 1996), and the lack of effect of staurosporine demonstrates that inhibition of PKC activity does not mediate cross talk between CXCR2 and P2Y₂ nucleotide receptors. Staurosporine also inhibits tyrosine kinases (Ohmichi et al., 1992), indicating that CXCR2 does not act through, or via a reversal of, a tyrosine kinase-dependent phosphorylation event.

Rapid desensitization is predominantly through receptor phosphorylation via one or more of a family of GPCR kinases, and dephosphorylation is required for receptor resensitization (Ferguson, 2001). For many GPCRs, dephosphorylation is mediated by phosphatases such as the protein phosphatase (PP)-2A family (Pitcher et al., 1995). Phosphorylation of the C terminus of the P2Y₂ nucleotide receptor by a kinase other than PKC mediates agonist-induced receptor desensitization, and inhibition of PP1/PP2A with okadaic acid inhibits resensitization (Otero et al., 2000). Here, we show that inhibition of either PP1/PP2A with okadaic acid, or PP2B with cyclosporin A, does not influence the potentiated Ca²⁺ responses, suggesting that P2Y₂ nucleotide receptor dephosphorylation and any associated resensitization is not the mechanism of cross talk.

Inhibition of potentiated Ca²⁺ signaling by expression of G_t indicates a role for G subunits, consistent with their role in other examples of cross talk (Selbie et al., 1997; Chan et al., 2000). We believe that the facilitated CXCR2 Ca²⁺ response is not, however, a simple consequence of pooling G subunits from two coactivated receptor populations. Thus, although G_s-coupled ₂-adrenoceptors are able to elevate [Ca²⁺]_i if P2Y₂ nucleotide receptors are also activated in these cells (Werry et al., 2002), coactivation of CXCR2 and ₂-adrenoceptors did not influence [Ca²⁺]_i.

The requirement for both G_q and G may be at distinct sites. Alternatively, they could converge to enhance PLC activity directly. The G-sensitive isoforms of PLC (1-3) have distinct binding sites for G_q and G, and stimulation of PLC by PTX-insensitive G proteins and G can be additive (Smrcka and Sternweis, 1993) or even synergistic (Zhu and Birnbaumer, 1996), providing a mechanism for receptor cross talk. Indeed, G_q may prime PLC to subsequent activation by G subunits derived from -opioid receptors in NG108-15 cells (Yoon et al., 1999). The precise mechanism of sensitization is not clear but could involve a conformational change in PLC after G_q binding that relieves a steric hindrance to G binding. Such a mechanism would account for the need for ongoing activation of P2Y₂ nucleotide receptors for this potentiation and also for the dependence on G_q and G.

This study demonstrates that cross talk between P2Y₂ nucleotide receptors and CXCR2 results in the release of Ca²⁺ from a thapsigargin-sensitive, Ins(1,4,5)P₃-dependent intracellular store. Furthermore, cross talk results in the potentiation of PLC activity, and our data suggest that the enhanced generation of Ins(1,4,5)P₃ may be sufficient to account for potentiated Ca²⁺ release. Our data are entirely consistent with enhanced PLC activity through synergistic actions of G_q and G-subunits derived from G_i. The most straightforward interpretation of this is that G_q and G-subunits are derived from activated P2Y₂ nucleotide receptors and CXCR2, respectively. There are alternatives and indeed a large and expanding array of mechanisms that could account for cross talk have been described (for review, see Werry et al., 2003). Further investigation of these is required to define whether they are able to contribute to cross talk under this or any other example of cross talk.

	Footnotes

 
This work was jointly funded by the Biotechnology and Biological Sciences Research Council and AstraZeneca R&D Charnwood (Loughborough, UK).

DOI: 10.1124/jpet.103.055632.

ABBREVIATIONS: GPCR, G protein-coupled receptor; HEK, human embryonic kidney; HEK-CXCR2, human embryonic kidney cell with stable expression of recombinant human CXCR2; CXCR2, CXC chemokine receptor 2 or IL-8 receptor B; PTX, pertussis toxin; PLC, phospholipase C; Ins(1,4,5)P₃, inositol 1,4,5-trisphosphate; AM, acetoxymethyl ester; IL-8, interleukin-8; 2-APB, 2-aminoethoxydiphenyl borane; RGS, regulator of G protein signaling; BSS, balanced salts solution; [Ca²⁺]_i, intracellular Ca²⁺ concentration; GFP, green fluorescent protein; InsP_x, inositol phosphates; PI 3-kinase, phosphatidylinositol 3-kinase; PI 4-kinase, phosphatidylinositol 4-kinase; PIP₂, phosphatidylinositol 4,5-bisphosphate; PKC, protein kinase C; DMSO, dimethyl sulfoxide; PP, protein phosphatase; U73122 [GenBank] , 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl) amino)hexyl]-1H-pyrrole-2,5-dione; U73343 [GenBank] , 1-[6-((17beta-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione.

Address correspondence to: Dr. Gary B. Willars, Department of Cell Physiology and Pharmacology, University of Leicester, University Rd., Leicester LE1 9HN, UK. E-mail: gbw2{at}le.ac.uk

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