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 Ca ²⁺ 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, P2Y₄^0/-, 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-γ. Notably, in Ca²⁺-free buffer, UTP-induced Ca ²⁺ 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.