Knock-out mice reveal the contributions of P2Y and P2X receptors to nucleotide-induced Ca2+ signaling in macrophages

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 mu m) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y(2)/P2Y(4) double knock-out mice. Moreover, P2Y(4)(0/-), but not P2Y(2)(-/-), macrophages responded to UTP. In P2Y(2)(-/-) 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 similar to 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 P2Y(2), the sole G(q)-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y(6) receptor expression) increases following macrophage activation.