Ca2+ signalling by recombinant human CXCR2 chemokine receptors is potentiated by P2Y nucleotide receptors in HEK cells

1 Human embryonic kidney (HEK)-293 cells expressing recombinant Galpha(i)-coupled, human CXC chemokine receptor 2 (CXCR2) were used to study the elevation of the intracellular [Ca2+] ([Ca2+](i)) in response to interleukin-8 (IL-8) following pre-stimulation of endogenously expressed P2Y1 or P2Y2 nucleotide receptors. 2 Pre-stimulation of cells with adenosine 5'-triphosphate (ATP) revealed a substantial Ca2+ signalling component mediated by IL-8 (E-max = 83+/-8% of maximal ATP response, pEC(50) of IL-8 response = 9.7+/-0.1). 3 1 muM 2-methylthioadenosine 5'-diphosphate (2MeSADP; P2Y1 selective) and 100 muM uridine 5'-triphosphate (UTP; P2Y2 selective) stimulated equivalent maximal increases in [Ca2+], elevation. However, UTP caused a sustained elevation, whilst following 2MeSADP [Ca2+](i) rapidly returned to basal levels. 4 Both UTP and 2MeSADP increased the potency and magnitude of IL-8-mediated [Ca2+](i) elevation but the effects of UTP (E-max of IL-8 response increased to 50+/-1% of the maximal response to ATP, pEC50 increased to 9.8+/-0.1) were greater than those of 2MeSADP (E.,,, increased to 36+/-2%, pEC(50) increased to 8.7+/-0.2). 5 The potentiation of IL-8-mediated Ca2+ signalling by UTP was not dependent upon the time of IL-8 addition following UTP but was dependent on the continued presence of UTP. Potentiated IL-8 Ca2+ signalling was apparent in the absence of extracellular Ca2+, demonstrating the release of Ca2+ from intracellular stores. 6 Activation of P2Y1 and P2Y2 receptors also revealed Ca2+ signalling by an endogenously expressed, Galpha(S)-coupled beta-adrenoceptor. 7 In conclusion, pre-stimulation of P2Y nucleotide receptors, particularly P2Y2, facilitates Ca2+ signalling by either recombinant CXCR2 or endogenous beta-adrenoceptors.