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

Abstract

2+ ] i elevation but the e ects of UTP (E max of IL-8 response increased to 50+1% of the maximal response to ATP, pEC 50 increased to 9.8+0.1) were greater than those of 2MeSADP (E max increased to 36+2%, pEC 50 increased to 8.7+0.2). 5 The potentiation of IL-8-mediated Ca 2+ 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 Ca 2+ signalling was apparent in the absence of extracellular Ca 2+ , demonstrating the release of Ca 2+ from intracellular stores. 6 Activation of P2Y1 and P2Y2 receptors also revealed Ca 2+ signalling by an endogenously expressed, Ga s -coupled b-adrenoceptor. 7 In conclusion, pre-stimulation of P2Y nucleotide receptors, particularly P2Y2, facilitates Ca 2+ signalling by either recombinant CXCR2 or endogenous b-adrenoceptors.

Cross Talk between P2Y2Nucleotide Receptors and CXC Chemokine Receptor 2 Resulting in Enhanced Ca2+Signaling Involves Enhancement of Phospholipase C Activity and Is Enabled by Incremental Ca2+Release in Human Embryonic Kidney Cells

“…1a). The magnitude of this Ca 2ϩ response relative to that of the addition of UTP to naive cells is comparable with that seen in our previous studies using a fluorescent light imaging plate reader in which IL-8 was added after, but in the continued presence of UTP (Werry et al, 2002). These data demonstrate that a store of Ca 2ϩ is accessed after coaddition of UTP and IL-8 that is inaccessible to UTP or IL-8 alone.…”

“…Repeated short exposure of HEK-CXCR2 cells to 100 M UTP (which activates P2Y 2 nucleotide receptors in these cells; Werry et al, 2002) in the absence of extracellular Ca 2ϩ resulted in the gradual loss of UTP-mediated [Ca 2ϩ ] i elevation (Fig. 1a).…”

“…The HEK cell line expressing recombinant human CXCR2 at approximately 50,000 sites/ cell was generated and selected as described previously (Werry et al, 2002). This cell line (HEK-CXCR2) was maintained in Dulbecco's modified Eagle's medium (containing 25 mM D-glucose, 4 mM Lalanyl-l-glutamine, and 1 mM sodium pyruvate) supplemented with 10% fetal calf serum, 1% nonessential amino acids, 50 g/ml gentamicin, and 400 g/ml G-418 at 37°C in a 5% CO 2 humidified atmosphere.…”

“…We have also recently demonstrated in human embryonic kidney (HEK) cells that activation of endogenously expressed P2Y 2 nucleotide receptors reveals a Ca 2ϩ signal to stimulation of recombinantly expressed G␣ i -coupled human CXC chemokine receptor 2 (CXCR2) (Werry et al, 2002). The ability of CXCR2 to mediate a Ca 2ϩ response is absolutely dependent upon the presence of P2Y 2 nucleotide receptor agonists.…”

Signaling and Cross-talk by C5a and UDP in Macrophages Selectively Use PLCβ3 to Regulate Intracellular Free Calcium

“…Synergy between G␣ i -and G␣ q -coupled receptors has previously been observed in other cell types. In several systems, including smooth muscles, astrocytes, and kidney epithelial cells, G␣ i -coupled GPCRs may alone not trigger a Ca 2ϩ response, but responses may be facilitated in combination with, or after priming by, G␣ q -coupled receptors (41)(42)(43). This synergy is reflected in the generation of IP 3 , as in our current studies of macrophages, implicating PLC in the pathway of synergy.…”