Ca(2+) 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 G alpha(i)-coupled, human CXC chemokine receptor 2 (CXCR2) were used to study the elevation of the intracellular [Ca(2+)] ([Ca(2+)](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 Ca(2+) 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 microM 2-methylthioadenosine 5'-diphosphate (2MeSADP; P2Y1 selective) and 100 microM uridine 5'-triphosphate (UTP; P2Y2 selective) stimulated equivalent maximal increases in [Ca(2+)](i) elevation. However, UTP caused a sustained elevation, whilst following 2MeSADP [Ca(2+)](i) rapidly returned to basal levels. 4. Both UTP and 2MeSADP increased the potency and magnitude of IL-8-mediated [Ca(2+)](i) elevation but the effects 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. 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, G alpha(s)-coupled beta-adrenoceptor. 7. In conclusion, pre-stimulation of P2Y nucleotide receptors, particularly P2Y2, facilitates Ca(2+) signalling by either recombinant CXCR2 or endogenous beta-adrenoceptors.
Werry
Tim D
TD
Department of Cell Physiology and Pharmacology, Maurice Shock Medical Sciences Building, University Road, Leicester, LE1 9HN, UK. tdw2@le.ac.uk
Christie
Mark I
MI
Dainty
Ian A
IA
Wilkinson
Graeme F
GF
Willars
Gary B
GB
D000255:Adenosine Triphosphate / Q000494:pharmacology; D020013:Calcium Signaling / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D006801:Humans; D058910:Purinergic P2 Receptor Agonists; D020239:Receptor Cross-Talk; D023063:Receptors, Interleukin-8B / Q000378:metabolism / Q000502:physiology*; D018048:Receptors, Purinergic P2 / Q000378:metabolism / Q000502:physiology*; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D011994:Recombinant Proteins / Q000378:metabolism
C546382:P2RY1 protein, human; C546385:P2RY2 protein, human; D058910:Purinergic P2 Receptor Agonists; D023063:Receptors, Interleukin-8B; D018048:Receptors, Purinergic P2; D058472:Receptors, Purinergic P2Y1; D058473:Receptors, Purinergic P2Y2; D011994:Recombinant Proteins; D000255:Adenosine Triphosphate