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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^*

Adriana del Rey, Vijay Renigunta, Alexander H. Dalpke, Jens Leipziger^¶, Joana E. Matos^¶, Bernard Robaye^||, Marylou Zuzarte, Annemieke Kavelaars^**, and Peter J. Hanley¹

From the 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

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 (K_0.5 <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, , but not , macrophages responded to UTP. In 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-. 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 G_q-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y₆ receptor expression) increases following macrophage activation.

Received for publication, August 11, 2006

^* 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.

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

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.

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