Knock-out Mice Reveal the Contributions of P2Y and P2X Receptors to Nucleotide-induced Ca2+ Signaling in Macrophages

Adriana del Rey,Joana E. Matos,Peter J. Hanley,Marylou Zuzarte,Annemieke Kavelaars,Vijay Renigunta,Alexander H. Dalpke,Jens Leipziger,Bernard Robaye

doi:10.1074/jbc.m607713200

Abstract

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 microm) 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, P2Y4(0/-), but not P2Y2-/-, macrophages responded to UTP. In P2Y2-/- 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 approximately 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.

Highlights

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]
By using P2Y2- and/or P2Y4-deficient mice, as well as reverse transcription (RT)-PCR analyses, we show that P2Y2 is the dominant receptor in resident peritoneal macrophages
We have identified the subtypes of P2Y and P2X receptors involved in transducing changes in extracellular nucleotide levels to transient Ca2ϩ signaling in macrophages

Summary

EXPERIMENTAL PROCEDURES

Knock-out (KO) Mice—P2Y2Ϫ/Ϫ, P2Y40/Ϫ, and P2Y2/P2Y4 double KO mice were generated as described recently [20]. Caron (Duke University Medical Center, Durham, NC), and GRK6-deficient mice were generously provided by Dr Richard T. Macrophages were activated by incubation for Ͼ24 h in RPMI medium (with 10% calf serum) containing 100 ng/ml lipopolysaccharide and 100 units/ml interferon-␥. Cells were imaged via a ϫ40 (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, and 5.5 mM D-glucose (pH 7.4). To monitor cytosolic [Ca2ϩ], cells were incubated for 15 min with 10 ␮M fluo-3/AM (Molecular Probes). Probenecid was omitted from the Hanks’ buffered solution to promote leak of cytosolic mag-fluo-4. Each data point (first response to a given [UTP]) is the mean Ϯ S.E. of 4 –11 cells

RESULTS

WT versus

DISCUSSION