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Originally published In Press as doi:10.1074/jbc.M607713200 on September 15, 2006

J. Biol. Chem., Vol. 281, Issue 46, 35147-35155, November 17, 2006
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Knock-out Mice Reveal the Contributions of P2Y and P2X Receptors to Nucleotide-induced Ca2+ Signaling in Macrophages*Formula

Adriana del Rey{ddagger}, Vijay Renigunta{ddagger}, Alexander H. Dalpke§, Jens Leipziger, Joana E. Matos, Bernard Robaye||, Marylou Zuzarte{ddagger}, Annemieke Kavelaars**, and Peter J. Hanley{ddagger}1

From the {ddagger}Institute of Physiology, Marburg University, Deutschhausstrasse 2, 35037 Marburg, Germany, the §Department of Hygiene and Medical Microbiology, University of Heidelberg, Im Neuenheimer Feld 324, 69120 Heidelberg, Germany, Institute of Physiology and Biophysics, University of Aarhus, 8000 Aarhus, Denmark, ||Institute of Interdisciplinary Research, Institut de Biologie et de Médecine Moléculaires, Université Libre de Bruxelles, 6041 Charleroi (Gosselies), Belgium, and **Laboratory for Psychoneuroimmunology, University Medical Center Utrecht, 3584 EA Utrecht, The Netherlands

Received for publication, August 11, 2006


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+-free buffer, the potent (K0.5 <1 µM) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y2/P2Y4 double knock-out mice. Moreover, Formula, but not Formula, macrophages responded to UTP. In Formula macrophages, we could elicit Ca2+ responses to "pure" P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after "activation" with lipopolysaccharide and interferon-{gamma}. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 min, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions. When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only ~40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2+/- and GRK6-/- macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y2, the sole Gq-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y6 receptor expression) increases following macrophage activation.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Antigen-presenting cells such as monocytes, macrophages, and dendritic cells express two families of nucleotide receptors as follows: ATP-gated cation channels (P2X receptors) and P2Y receptors, a subset of the G protein-coupled receptor superfamily (1). In mouse, ATP and UTP are equipotent agonists at the Gq-coupled receptors P2Y2 and P2Y4, whereas human P2Y4 is selectively activated by UTP and competitively antagonized by ATP (2). Moreover, in human, ATP additionally activates the dual Gs- and Gq-coupled P2Y11 receptor, absent in the mouse genome (3). The concentration of ATP or UTP in the local extracellular milieu is estimated to be around 10 nM (4, 5), which may be just sufficient to evoke local inositol 1,4,5-trisphosphate (IP3)2-induced Ca2+ puffs (6). However, mechanical stress, cellular injury, inflammation, degranulation of mast cells, and other factors may increase ATP and UTP to levels sufficient to evoke larger and longer lasting global Ca2+ signals (5, 7, 8). ATP is also released as a cotransmitter from the sympathetic nervous system, a potentially important neuroimmune interaction (9, 10).

Local increases in ATP or UTP levels are transient because of both diffusion and the activity of ecto-nucleotidases such as CD39 (NTPDase 1), which catalyzes the sequential hydrolysis of ATP and UTP to their respective monophosphates (8, 11). A subset of P2Y receptors is selectively activated by nucleotide diphosphates. In particular, P2Y1 and P2Y12 receptors are preferentially activated by ADP and play important roles in platelet function (12). UTP degradation by CD39 yields transiently the intermediate UDP, which is a specific agonist for P2Y6 receptors (13). During prolonged agonist stimulation, the response of P2Y and P2X receptors is typically switched off within minutes (14-16). In the case of G protein-coupled receptors, the activity of the activated G protein is terminated by GTPase-activating proteins, which catalyze the hydrolysis of GTP bound to the {alpha}-subunit (17). Agonist-induced "desensitization" is thought to involve phosphorylation of the C terminus (or intracellular loops) by G protein-coupled receptor kinases (GRKs) or second messenger-induced protein kinase C (PKC). Phosphorylation by GRK promotes the rapid binding of beta-arrestin, which blocks further G protein activation (18, 19).

Because of the lack of specific agonists and antagonists, it is difficult to assign unequivocally P2Y and P2X receptor subtypes to a particular cell type. In this study, by using P2Y2- and/or P2Y4-deficient mice, as well as RT-PCR analyses, we show that P2Y2 is the dominant receptor in resident peritoneal macrophages. Furthermore, we dissected the interplay of P2Y2 receptors, P2X receptors, Ca2+ release-activated Ca2+ channels, and Ca2+-ATPases (Ca2+ pumps) in nucleotide-induced Ca2+ signaling. Finally, we assessed the role of PKC, calmodulin-dependent kinase II (CaMKII), GRK2, and GRK6 in the rapid desensitization of P2Y2 receptors using inhibitors and GRK-deficient mice.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Knock-out (KO) Mice—Formula, Formula, and P2Y2/P2Y4 double KO mice were generated as described recently (20). Heterozygous P2Y2 mice originally were kindly donated by Dr. Beverly H. Koller (University of North Carolina, Chapel Hill), and the inactivation of the P2Y4 gene, which is located on the X chromosome, has been described previously (21). GRK2+/- mice were kindly provided by Dr. Marc G. Caron (Duke University Medical Center, Durham, NC), and GRK6-deficient mice were generously provided by Dr. Richard T. Premont and Dr. Robert J. Lefkowitz (Duke University Medical Center). The genetic backgrounds of the male littermate wild-type (WT) and KO mice used in this study were as follows: Formula (mixed B6D2 x CD-1 x SV129); Formula (mixed CD-1 x SV129); P2Y2/P2Y4 double KO (mixed B6D2 x CD-1 x SV129); GRK2+/- (C57BL/6); and GRK6-/- (C57BL/6). Heterozygous GRK2 mice were used because homozygous mutations are embryonically lethal.

Isolation of Peritoneal Macrophages—Mice were killed by cervical dislocation, and resident peritoneal cells were harvested by lavage with 10 ml of ice-cold Hanks' physiological salt solution. After centrifugation, cells were resuspended in RPMI medium containing 10% heat-inactivated fetal calf serum, 100 units/ml penicillin, and 100 µg/ml streptomycin. Cells were seeded onto glass coverslips and incubated at 37 °C in air with 5% CO2. After 2 h, nonadherent cells were removed by washing the coverslip. In selected experiments, macrophages were activated by incubation for >24 h in RPMI medium (with 10% calf serum) containing 100 ng/ml lipopolysaccharide and 100 units/ml interferon-{gamma}.

Flow Cytometry—B220-CD11b+ cells (macrophages) were purified by depletion of B220+ cells (B lymphocytes), followed by positive selection for CD11b+ cells (supplemental Fig. 1) using a magnetic cell sorting system (Miltenyi Biotec, Bergisch Gladbach, Germany). Note that B1 cells are the major subpopulation of B lymphocytes in the peritoneal cavity and weakly express CD11b (22). Aliquots before and after purification were stained with phycoerythrin-conjugated anti-B220 (clone RA3-6B2; Pharmingen), allophycocyanin-conjugated anti-CD11b (clone M1/70; Pharmingen), and a fluorescein isothiocyanate-conjugated antibody (clone CI:A3-1; Serotec, Düsseldorf, Germany) against the macrophage marker F4/80. Phenotypical analysis was performed by flow cytometry using a FACS Canto flow cytometer (BD Biosciences).

RT-PCR Analyses—Total cellular RNA was prepared using a HighPure RNA extraction kit (Roche Applied Science), which included DNase I digestion. After reverse transcription (RT) with a cDNA synthesis kit (MBI Fermentas, St. Leon-Rot, Germany) using oligo(dT)18, PCR analysis was performed using primers (shown in supplemental Table 1) specific for mouse P2Y1, P2Y2, P2Y4, and P2Y6; P2X1, P2X2, P2X3, P2X4, P2X5, and P2X7; GRK2, GRK3, GRK4, GRK5, and GRK6. PCR products were confirmed by sequencing, and where necessary, cDNA prepared from total mouse brain or heart RNA (Ambion, Huntingdon, UK) was used as positive control.

Fluorescence Microscopy—Differential interference contrast and fluorescence images of live peritoneal macrophages, typically 12-14 µm diameter, were acquired using an Olympus IX71 microscope equipped with a software-controlled Sensi-CamQE CCD camera (Chromaphor, Duisburg, Germany). Cells were labeled with the fluorescent nucleic acid stain Hoechst 33342 (Invitrogen) and imaged using a x100 (1.4 numerical aperture) objective lens and immersion oil.

Cytosolic [Ca2+] Measurements—A glass coverslip seeded with macrophages was sealed onto the bottom of a Perspex bath (volume, 100 µl) mounted on the stage of an inverted microscope (Nikon Diaphot 300). Cells were imaged via a x40 (1.4 numerical aperture) oil-immersion objective and superfused at 1 ml/min with modified Hanks' buffered salt solution containing 5% bovine serum albumin, 136 mM NaCl, 5.4 mM KCl, 0.9 mM MgCl2, 4.2 mM NaHCO3, 0.3 mM NaH2PO4, 0.4 mM KH2PO4, 5 mM HEPES, 1.3 mM CaCl2, 0.8 mM probenecid, and5.5 mM D-glucose (pH 7.4). Ca2+-free buffer solution was prepared by omitting CaCl2 and adding 0.5 mM EGTA. Solutions were rapidly switched by means of miniature three-way valves (The Lee Co., Westbrook, CT). To monitor cytosolic [Ca2+], cells were incubated for 15 min with 10 µM fluo-3/AM (Molecular Probes).

Endoplasmic Reticulum [Ca2+] Measurements—To measure ER [Ca2+] ([Ca2+]ER), the low affinity fluorescent Ca2+ indicator mag-fluo-4 was selectively loaded into the ER. Cells were incubated at 37 °C with 5 µM mag-fluo-4/AM (Molecular Probes), washed several times, and incubated overnight at 4 °C before use. Probenecid was omitted from the Hanks' buffered solution to promote leak of cytosolic mag-fluo-4. Fluo-3 or mag-fluo-4 loaded macrophages were excited at 488 nm via a monochromator, whereas fluorescence was detected at 530 ± 15 nm. Only one cell per coverslip was used for experiments, and moreover, only the first response to a given nucleotide concentration was used to establish concentration-response relations. The fluorescence signals were normalized with respect to the resting fluorescence intensity (F0) and expressed as F/F0.

Materials—Chemicals were obtained from Sigma unless stated otherwise. Solutions of UDP or ADP were purified from contaminating nucleotide triphosphates by incubation with 50 units/ml hexokinase and 20 mM glucose for at least 2 h at room temperature. Recombinant mouse interferon-{gamma} was obtained from PeproTech (London, UK), and recombinant mouse complement component C5a was purchased from R & D Systems (Wiesbaden-Nordenstadt, Germany).


Figure 1
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  		FIGURE 1.
Lack of UTP responsiveness in P2Y2/P2Y4 double knock-out macrophages. A and B, plots of [UTP] versus peak [Ca2+]i for macrophages isolated from WT (A) and DKO (B) mice. Each data point (first response to a given [UTP]) is the mean ± S.E. of 4-11 cells.

 

   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
P2Y Receptor Signaling—To determine the effects of extracellular nucleotides on ER Ca2+ release in macrophages, Ca2+-free buffer was used to obviate the potentially confounding effects of store-operated Ca2+ entry and Ca2+ influx via P2X receptors. When single WT macrophages were superfused with UTP in Ca2+-free buffer, a concentration-dependent increase in peak cytosolic [Ca2+] ([Ca2+]i) was evoked (Fig. 1A); UTP increased peak [Ca2+]i with a K0.5 value of 0.7 µM. Activation of either P2Y2 or P2Y4 receptors could be responsible for the transient Ca2+ response to UTP in Ca2+-free buffer. Both of these receptor subtypes have recently been reported to be expressed in mouse macrophages (23). To explore the relative roles of P2Y2 and P2Y4 receptors, we first challenged macrophages isolated from P2Y2 and P2Y4 knock-out mice with UTP. Concentrations of UTP between 0.1 and 100 µM, or as high as 250 µM (n = 7; not shown), had no effect on [Ca2+]i in macrophages isolated from P2Y2/P2Y4 double knock-out mice (Fig. 1B), indicating that P2Y2 or P2Y4 are the only possible Gq-coupled receptors mediating UTP-induced Ca2+ signaling.


Figure 2
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  		FIGURE 2.
Macrophages isolated from P2Y2/P2Y4 double knock-out mice. A, fluorescence image of the nuclei (stained with Hoechst 33342) of typical peritoneal macrophages isolated from a double knock-out (DKO) mouse. Note the bean-shaped morphology of the nuclei, typical for macrophages. B, overlay of fluorescence and differential interference contrast images of the macrophages shown in A. The scale bar is 10 µm. C and D, RT-PCR analyses of RNA obtained from purified macrophages isolated from P2Y2/P2Y4 double knock-out and WT mice. P2Y1 and P2Y6 receptors were detected in both DKO and WT macrophages, and P2Y2, but not P2Y4, could be detected in WT cells (C). In both cases, P2X1, P2X4, and P2X7 receptors were detected (D). All PCR products were confirmed by sequencing. The upper band shown in the P2X4 lanes was not identified.

 
P2Y2/P2Y4 double knock-out mice exhibited no obvious phenotype (20), and macrophages isolated from these animals were indistinguishable from WT cells (Fig. 2, A and B). Moreover, similar to WT cells, macrophages isolated from double knock-out mice expressed mRNA specific for P2Y1 and P2Y6 (Fig. 2C), and P2Y2, but not P2Y4, could be detected in WT cells. Mouse whole brain RNA was used as positive control for the P2Y4-specific primer (not shown). Both WT and double knockout macrophages expressed several ATP-gated, nonselective cation channels as follows: P2X1, P2X4, and P2X7 receptors (Fig. 2D). The upper band in the P2X4 lanes was not identified.

To assess the relative contributions of P2Y2 and P2Y4 receptors, we compared the effects of UTP on Ca2+ signaling in macrophages isolated from Formula or Formula mice. Similar to WT cells, application of 10 µM UTP (in Ca2+-free buffer) elicited a transient and oscillatory increase in cytosolic Ca2+ in macrophages isolated from Formula mice, as shown in Fig. 3A. However, as illustrated by the example in Fig. 3B, macrophages isolated from P2Y2-deficient mice did not respond to UTP, but these cells responded to ATP, provided that Ca2+ was present in the buffer (Fig. 3B). The concentration-response relations for macrophages isolated from Formula and Formula mice are presented in Fig. 3, C and D. Thus, consistent with the RT-PCR data, the concentration-response data reveal that P2Y2 is the sole Gq-coupled receptor for UTP in resident macrophages.

UTP is sequentially degraded to UDP and UMP extracellularly, a reaction catalyzed by the surface membrane enzyme CD39. Because we detected mRNA for P2Y6 in both WT and double knock-out macrophages, we expected to observe ER Ca2+ release induced by UDP. In preliminary experiments, 100 µM UDP (97% pure by high performance liquid chromatography) consistently induced ER Ca2+ release. However, after contaminating UTP (or ATP) was scavenged with hexokinase (24), 10 of 11 WT macrophages did not respond at all to 100 µM UDP, whereas all cells responded to ATP, used as positive control (Fig. 4A). Note that when extracellular Ca2+ was reintroduced after stimulating cells with high ATP (or UTP; not shown) concentrations in Ca2+-free buffer, an increase in intracellular Ca2+, consistent with store-operated Ca2+ entry (25), was always observed (Fig. 4A). Moreover, similar to WT, 12 of 17 P2Y2/P2Y4-deficient macrophages did not respond to 100 µM UDP, and the response in the remaining five cells was characterized by a small, single Ca2+ transient (Fig. 4B). Taken together, the [UDP]-response relations for macrophages from both WT and P2Y2/P2Y4 double knock-out mice were essentially flat in the 1-100 µM range (Fig. 4C). These data reveal that, functionally, P2Y6 is weakly expressed in resident macrophages. However, when P2Y2/P2Y4-deficient macrophages were activated for 48-72 h with lipopolysaccharide and interferon-{gamma}, responsiveness increased, such that most cells (9 of 11) produced a small oscillatory Ca2+ response to 100 µM UDP (peak F/F0 3.0 ± 0.2; not shown).


Figure 3
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  		FIGURE 3.
P2Y40/- but not P2Y2-/- macrophages respond to UTP. A, typical UTP-induced Ca2+ response of a macrophage isolated from a Formula mouse. B, UTP fails to increase Ca2+ in a macrophage isolated from a Formula mouse. However, in the presence of extracellular Ca2+, 100 µM ATP induces a small, transient Ca2+ signal. C and D, plots of [UTP] versus peak [Ca2+]i for macrophages isolated from Formula (C) and Formula the mean ± S.E. of 4-6 cells. (D) mice. Each data point is

 


Figure 4
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  		FIGURE 4.
UDP-selective P2Y6 receptors are weakly expressed in resident macrophages. A, under Ca2+-free conditions, application of 100 µM UDP (purified with hexokinase) had no effect on [Ca2+]i, whereas 100 µM ATP (positive control) induced a large Ca2+ transient. The interruption of the 0 Ca2+ solid line indicates superfusion with buffer containing 1.3 mM Ca2+. B, a subset of macrophages isolated from double knock-out (DKO) mice responded to 100 µM UDP (5 of 17 cells), and the response was typically a single spike. C, overlay of [UDP] versus peak [Ca2+]i relations for macrophages isolated from WT (solid symbols) and double knock-out (open symbols) mice. Each data point is the mean ± S.E. of 4-17 cells. For comparison, the concentration-response relation for UTP is shown (dotted line).

 
To explore the potential role of P2Y1 receptors, we challenged macrophages with ADP under Ca2+-free conditions. Application of 100 µM ADP (purified with hexokinase) had no effect on [Ca2+]i in 9 of 11 WT cells, whereas ATP produced a strong positive control response (Fig. 5A). Concentration-response relations for ADP and ATP in WT macrophages are overlaid in Fig. 5B. Similar to WT, ADP also had little or no effect in macrophages isolated from P2Y2/P2Y4 double knock-out mice, and no response to ATP was observed (Fig. 5C).

P2X Receptor Signaling—Although we could not elicit a Ca2+ response to UTP in macrophages isolated from P2Y2-deficient (Formula or double knock-out) mice, we could observe a small transient response to ATP provided that Ca2+ was present in the buffer (Fig. 6). Thus, targeted disruption of the P2Y2 gene reveals the Ca2+ response to pure P2X receptor activation. The Ca2+ response elicited by ATP was small and diminished upon repeated application of agonist (Fig. 6, A and B). The concentration-response relation in the 0.1-250 µM ATP range is shown in Fig. 6C (data from Formula and double knock-out mice were combined). Note that in RT-PCR analyses we could detect P2X1, P2X4, and P2X7 in macrophages isolated from both WT and P2Y2/P2Y4 double knock-out mice (see Fig. 2D). For comparison, the pure P2Y receptor response to ATP is overlaid in Fig. 6. It can be seen that the P2Y receptor-induced Ca2+ response greatly dominates over the P2X receptor response in the 0.1-10 µM ATP range.

GRK6 Is Not Essential for Rapid Desensitization of P2Y2 Receptors—When extracellular Ca2+ was available, application of 100 µM UTP typically produced a rapid increase in [Ca2+]i followed by fast and slow components of decline (Fig. 7A), the latter of which was absent in Ca2+-free buffer (for example, see Fig. 4A) and can be attributed to store-operated Ca2+ entry. Furthermore, the Ca2+ response to repeated UTP challenge was greatly diminished (Fig. 7A), suggesting that the initial challenge may have desensitized the receptor. GRKs have been implicated in the desensitization mechanism of various G protein-coupled receptors, and to test whether GRK6 is involved in the rapid desensitization of P2Y2 receptors, we isolated macrophages from GRK6-/- mice. Compared with WT cells, there was no enhancement of the second UTP-induced Ca2+ response in GRK6-deficient macrophages, even when cells were pretreated with inhibitors of the candidate regulatory kinases PKC (staurosporine) and CaMKII (KN93) (Fig. 7B). RT-PCR analyses of RNA extracted from purified macrophages indicated that GRK2 and GRK5 were also expressed, but only weak signals for GRK3 and GRK4 were detected (Fig. 7C). We also found that apparent desensitization, assayed by repeated challenges with 100 µM UTP, was not reduced in macrophages isolated from GRK2+/- mice, which express 50% protein compared with WT (26). Thus, the experiments with WT, GRK2+/-, and GRK6-/- macrophages summarized in Fig. 7D (see also supplemental Fig. 2) suggest that the kinases GRK2, GRK6, PKC, and CaMKII are not necessary for the rapid dampening of P2Y2 receptor signaling in macrophages.


Figure 5
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  		FIGURE 5.
Lack of P2Y1 receptor responsiveness in macrophages from WT and P2Y2/P2Y4 double knock-out mice. A, application of 100 µM ADP (purified with hexokinase) under Ca2+-free conditions had no effect on [Ca2+]i. ATP was used as positive control. B and C, overlay of concentration-response relations for ATP (open symbols) and ADP (solid symbols) in macrophages isolated from WT (B) and P2Y2/P2Y4 double knock-out (DKO) (C) mice. Each data point is the mean ± S.E. of 6-10 cells.

 


Figure 6
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  		FIGURE 6.
P2Y2-deficient cells unmask the Ca2+ response to P2X receptor activation. A and B, in the presence of extracellular Ca2+, application of 100 µM ATP (A)or250 µM ATP (B)toP2Y2-deficient macrophages elicits a small and transient Ca2+ response, which is decreased upon repeated agonist challenge. C, plot of [ATP] versus peak [Ca2+]i in P2Y2-deficient macrophages. This plot essentially shows the peak Ca2+ response to P2X receptor activation. For comparison, the P2Y2 receptor response, obtained using WT macrophages under Ca2+-free conditions, is overlaid (dashed line). DKO, double knock-out mice.

 
Stimulation of P2Y2 Receptors Decreases the Subsequent Response to a Non-P2Y Gq-coupled Receptor—If the decreased Ca2+ response to repeated UTP application is because of homologous P2Y2 receptor desensitization, then the response to a non-P2Y Gq-coupled receptor should be unaffected following initial UTP challenge. To test this possibility, we used complement factor C5a as a second agonist, the receptor of which has been shown using WT versus Formula mice to be coupled toGq/15 (27), whereas P2Y receptors are coupled to Gq/11. When a macrophage was first challenged with 100 µM UTP followed by 100 nM C5a, the second Ca2+ response was decreased (Fig. 8A) and vice versa (Fig. 8B), indicating that heterologous mechanisms, such as phosphatidylinositol 4,5-bisphosphate depletion, are contributing to the decayed Ca2+ responses to repeated UTP challenges. On average, the peak of the second Ca2+ response was ~40% of the initial response when UTP was applied twice, whereas the second response was ~60% (relative to the first response) when different agonists were used (Fig. 8C). Another potentially important factor determining the magnitude of the second agonist-induced Ca2+ response is the availability of ER Ca2+. Indeed, there was no response at all to repeated UTP challenge when experiments were performed in Ca2+-free buffer (Fig. 8C), an unphysiological condition that promotes Ca2+ extrusion via the plasma membrane Ca2+-ATPase (Ca2+ pump).

Contribution of ER Ca2+ Depletion to Apparent P2Y2 Receptor Desensitization—Mag-fluo-4 was selectively loaded into the ER lumen to measure [Ca2+]ER. When 100 µM UTP was applied to macrophages, a decrease in [Ca2+]ER was observed (Fig. 9A), which on average recovered 77.3 ± 4.5% (n = 6). Compared with fluo-3, mag-fluo-4 was much more susceptible to photobleaching, and thus the extent of recovery was probably underestimated in these experiments. In Ca2+-free buffer, the recovery of [Ca2+]ER after transient application of UTP was less than 15% (n = 5), as shown in Fig. 9B. However, the ER lumen was rapidly refilled with Ca2+ after switching to buffer containing1.3 mM Ca2+ (Fig. 9B).


Figure 7
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  		FIGURE 7.
Apparent P2Y2 receptor desensitization persists in GRK2+/- macrophages and in GRK6-/- macrophages subjected to PKC and CaMKII inhibition. A, typical response of a WT macrophage to repeated UTP challenges in the continued presence of extracellular Ca2+. Note that the second Ca2+ response to UTP is considerably reduced. B, apparent receptor desensitization (decay of the Ca2+ response with repeated application of UTP) is not obviated in macrophages isolated from GRK6-/- mice and pretreated with inhibitors of PKC (staurosporine (Staurosp.)) and CaMKII (KN93). C, RT-PCR analyses of RNA obtained from purified WT macrophages. The GRKs GRK2, GRK5, and GRK6 were strongly expressed, whereas only weak signals for GRK3 and GRK4 could be detected. D, summary of experiments showing that, compared with control conditions, the second Ca2+ response to repeated agonist challenge (UTP to UTP) was not augmented in macrophages obtained from GRK2+/- or GRK6-/- mice, and no difference was observed with inhibitors of the candidate regulatory kinases PKC and CaMKII. The number of single cells tested in each group is indicated in parentheses.

 


Figure 8
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  		FIGURE 8.
Heterologous mechanisms contribute to apparent P2Y2 receptor desensitization. A and B, the Ca2+ response to complement factor C5a is reduced when a macrophage is first challenged with UTP (A) and vice versa (B). C, summary of data. Note that there is no response to repeated UTP challenge when experiments are performed in Ca2+-free buffer, and there is less decrease of the second response when different Gq-coupled receptor agonists are used. The number of single cells tested in each group is indicated in parentheses.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
Calcium is an important regulatory ion inside cells, and in immune cells it has been implicated in diverse functions, including cytoskeleton reorganization, gene expression, and the gating of various K+ channels (28-31). Here we have identified the subtypes of P2Y and P2X receptors involved in transducing changes in extracellular nucleotide levels to transient Ca2+ signaling in macrophages. We found that UTP- and ATP-induced Ca2+ transients were abolished in macrophages isolated from P2Y2/P2Y4 double knock-out or Formula mice but were unaffected by genetic ablation of P2Y4. This conclusion was supported by RT-PCR analyses that showed that P2Y2, but not P2Y4, receptors were expressed in purified macrophages. Thus, in resident peritoneal macrophages, P2Y2 is the only G protein-coupled receptor linking extracellular UTP and ATP to phospholipase C-beta, which catalyzes the hydrolysis of phosphatidylinositol 4,5-bisphosphate, releasing the second messengers IP3 and diacylglycerol, an activator of PKC. Transient elevation of IP3 promotes the release of Ca2+ from the endoplasmic reticulum by increasing the open probability of IP3 receptors(32). In addition, cytosolic [Ca2+] regulates the various IP3 receptor subtypes in a bell-shaped fashion (inhibition at higher concentrations), a gating property that may facilitate the oscillatory Ca2+ release pattern we observed at submaximal P2Y receptor agonist concentrations.


Figure 9
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  		FIGURE 9.
Contribution of endoplasmic reticulum Ca2+ stores to apparent receptor desensitization. A, in the presence of extracellular Ca2+, the application of UTP to a single macrophage induces a reversible decrease in ER [Ca2+], detected using the low affinity Ca2+ indicator mag-fluo-4. The recovery of ER Ca2+ is underestimated because of photobleaching of the indicator. B, in contrast, under Ca2+-free conditions, there is little recovery of ER Ca2+ stores following UTP-induced Ca2+ release. Recovery of ER Ca2+ stores is seen upon introduction of extracellular Ca2+.

 
In P2Y2-deficient macrophages, ATP-induced Ca2+ mobilization was abolished in strictly Ca2+-free conditions. This lack of responsiveness to ATP underscores that there is no equivalent to the human P2Y11 receptor in mouse macrophages. In the presence of extracellular Ca2+, a small Ca2+ transient induced by ATP is observed and most likely represents the pure P2X receptor response to this agonist. We found that mouse macrophages express P2X1, P2X4, and P2X7 mRNA, which seems to be a common pattern of expression for macrophages from various species (23, 29, 33). P2X7 receptors do not form heteromeric receptors with other P2X subunits, but heteromeric P2X1/P2X4 receptor assembly has been described recently in an oocyte expression system (34). Whether heteromeric P2X1/P2X4 receptors, or even P2Y receptor heteromerization (35), play a functional role in native cells remains to be established. At this stage, we assume that homomeric P2X4 receptors are the major contributors to Ca2+ influx in the concentration range we tested (0.1-250 µM) because the P2X1 receptor response has been reported to terminate within 1 s, and the P2X7 receptor is much less sensitive to ATP (15). Under physiological conditions, P2X receptor signaling is not only switched off by receptor desensitization, which we could observe in superfused cells, but also by rapid degradation of ATP by ecto-nucleotidases.

ADP is the preferential agonist for the Gq-coupled P2Y1 receptor, and RT-PCR analyses showed that this P2Y receptor subtype is expressed in mouse macrophages along with P2Y2.In peritoneal macrophages isolated from BALB/c mice, this agonist has been reported to have no effect on [Ca2+]i at 10 µM but to have a modest effect (possibly overestimated because of contaminating ATP) at 100 µM (23). We also observed that ADP (purified with hexokinase) had negligible effect in macrophages isolated from WT or P2Y2-deficient mice. We additionally detected P2Y6 receptors in RT-PCR analyses; however, UDP-induced Ca2+ release was scant in resident macrophages isolated from either P2Y2/P2Y4 double knock-out or WT mice. A modest response to UDP, though, was observed after macrophages had been activated with lipopolysaccharide and interferon-{gamma}, suggesting that, functionally, P2Y6 receptors are weakly expressed in resting macrophages but, following activation by stimuli such as Toll-like receptor ligands, P2Y6 gene expression is switched on, increasing the scope of uridine nucleotide-induced signaling.

Signal transduction by G protein-coupled receptors, including P2Y receptors, is tightly controlled by mechanisms that dampen signal transmission. The rapid termination of P2Y2 receptor-induced Ca2+ signaling and refractoriness to repeated agonist challenge we observed could, in principle, be due to several mechanisms. The activated Gq/11 subunit may be inhibited by GTPase-activating proteins, such as members of the regulators of G protein signaling family (17, 36), and direct receptor phosphorylation by PKC or GRKs (promoting the recruitment of beta-arrestin) may uncouple the receptor. Our data obtained using GRK2+/- and GRK6-/- mice, as well as pharmacological inhibition of PKC, suggest that GRK6, PKC, or normal levels of GRK2 are not essential for switching off Ca2+ signaling induced by P2Y2 receptor activation. Consistent with our observations, González and co-workers (37, 38) deduced that PKC was not responsible for agonist-induced P2Y2 receptor desensitization, but they provided evidence that receptor phosphorylation by an unidentified kinase was involved. We also found that inhibition of CaMKII did not prevent apparent P2Y2 receptor desensitization. Similarly, Tulapurkar et al. (39) reported that inhibition of CaMKII did not affect desensitization of P2Y1 receptors, but it blocked internalization of activated receptors.


Figure 10
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  		FIGURE 10.
Schematic diagram of nucleotide-induced Ca2+ signaling in a resident macrophage. Abbreviations: PLCbeta, phospholipase C-beta; PMCA, plasma membrane Ca2+-ATPase; CRAC,Ca2+ release-activated Ca2+ channel; SERCA, sarco(endo)plasmic reticulum Ca2+-ATPase.

 
Local depletion of phosphatidylinositol 4,5-bisphosphate (the source of IP3), which has been nicely shown in myocytes to have low mobility in the surface membrane (40), could contribute to switching off signal transmission by activated Gq-coupled receptors. This could explain why the Ca2+ response is greater when the second agonist is C5a instead of UTP after initial challenge with UTP, i.e. the C5a receptor has access to a spatially distinct lipid pool. Downstream of IP3 generation, ER Ca2+ stores may become depleted, intraluminal Ca2+ cycling may be rate-limiting, or the IP3 receptor may be rendered rectractory. ER [Ca2+] measurements suggest that Ca2+ stores are rapidly replenished following P2Y2 receptor stimulation, but under certain conditions favoring plasma membrane Ca2+ transport systems (Ca2+-free buffer) ER stores are depleted, and there is no response to repeated agonist challenge. Thus, the interplay of Ca2+ transport systems is an important determinant of Gq-coupled receptor-mediated Ca2+ signaling.

In conclusion, we can summarize the main findings of this study in the schematic diagram shown in Fig. 10. Various stimuli such as mechanical stress, cell injury, or inflammation release UTP and ATP into the extracellular space. Macrophages sense the increased nucleotide levels through the dominant Gq-coupled P2Y2 receptor that is activated by UTP = ATP but insensitive to UDP and ADP. ATP additionally induces Ca2+ influx via P2X receptors, and UDP activates the P2Y6 receptor, functionally expressed in activated macrophages. Downstream of the activated Gq-coupled receptors, IP3 is generated and releases Ca2+ from the endoplasmic reticulum. At the same time, the recently identified Ca2+-release sensor STIM1 probably translocates to the surface membrane (Fig. 10, dashed arrow) and activates Ca2+ release-activated Ca2+ channels to promote Ca2+ entry and refilling of stores (25). Two Ca2+-ATPases compete to clear Ca2+ from the cytosol as follows: the sarco(endo)plasmic reticulum Ca2+-ATPase and the plasma membrane Ca2+-ATPase. Conditions favoring plasma membrane Ca2+-ATPase activity will lead to diminished ER refilling.


   FOOTNOTES
 
* This work was supported by a grant from the Kempkes-Stiftung (to P. J. H.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

Formula The on-line version of this article (available at http://www.jbc.org) contains supplemental Figs. 1 and 2 and supplemental Table 1. Back

1 To whom correspondence should be addressed. Tel.: 49-6421-286-6546; Fax: 49-6421-286-8960; E-mail: hanley{at}mailer.uni-marburg.de.

2 The abbreviations used are: IP3, inositol 1,4,5-trisphosphate; RT, reverse transcription; ER, endoplasmic reticulum; WT, wild type; KO, knock-out; PKC, protein kinase C; GRK, G protein-coupled receptor kinase; CaMKII, calmodulin-dependent kinase II. Back



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 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
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