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 (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.
del Rey
Adriana
A
Institute of Physiology, Marburg University, Deutschhausstrasse 2, 35037 Marburg, Germany.
Renigunta
Vijay
V
Dalpke
Alexander H
AH
Leipziger
Jens
J
Matos
Joana E
JE
Robaye
Bernard
B
Zuzarte
Marylou
M
Kavelaars
Annemieke
A
Hanley
Peter J
PJ
D000244:Adenosine Diphosphate / Q000378:metabolism; D000818:Animals; D015220:Calcium Channels; D020013:Calcium Signaling; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D054769:G-Protein-Coupled Receptor Kinase 2; D054768:G-Protein-Coupled Receptor Kinases; D005786:Gene Expression Regulation; D017737:Macrophages, Peritoneal / Q000378:metabolism*; D051379:Mice; D018345:Mice, Knockout; D009711:Nucleotides; D017346:Protein Serine-Threonine Kinases / Q000235:genetics / Q000378:metabolism; D018048:Receptors, Purinergic P2 / Q000235:genetics / Q000378:metabolism*; D058473:Receptors, Purinergic P2Y2; D014530:Uridine Diphosphate / Q000378:metabolism; D051552:beta-Adrenergic Receptor Kinases / Q000235:genetics / Q000378:metabolism
D015220:Calcium Channels; D009711:Nucleotides; C546386:P2ry2 protein, mouse; D018048:Receptors, Purinergic P2; D058473:Receptors, Purinergic P2Y2; C104749:purinoceptor P2Y4; D014530:Uridine Diphosphate; D000244:Adenosine Diphosphate; D017346:Protein Serine-Threonine Kinases; C496568:GRK2 protein, mouse; D051552:beta-Adrenergic Receptor Kinases; D054769:G-Protein-Coupled Receptor Kinase 2; D054768:G-Protein-Coupled Receptor Kinases; C089176:G-protein-coupled receptor kinase 6; D000252:Calcium-Transporting ATPases