Werry Tim D, Wilkinson Graeme F, Willars Gary B
Department of Cell Physiology and Pharmacology, Maurice Shock Medical Sciences Building, University of Leicester, University Road, Leicester LE1 9HN, UK.
J Pharmacol Exp Ther. 2003 Nov;307(2):661-9. doi: 10.1124/jpet.103.055632. Epub 2003 Sep 15.
We have shown previously that activation of endogenously expressed, Galphaq/11-coupled P2Y2 nucleotide receptors with UTP reveals an intracellular Ca2+ response to activation of recombinant, Galphai-coupled CXC chemokine receptor 2 (CXCR2) in human embryonic kidney cells. Here, we characterize further this cross talk and demonstrate that phospholipase C (PLC) and inositol 1,4,5-trisphosphate [Ins(1,4,5)P3]-dependent Ca2+ release underlies this potentiation. The putative Ins(1,4,5)P3 receptor antagonist 2-aminoethoxydiphenyl borane reduced the response to CXCR2 activation by interleukin-8, as did sustained inhibition of phosphatidylinositol 4-kinase with wortmannin, suggesting the involvement of phosphoinositides in the potentiation. Against a Li+ block of inositol monophosphatase activity, costimulation of P2Y2 nucleotide receptors and CXCR2 caused phosphoinositide accumulation that was significantly greater than that after activation of P2Y2 nucleotide receptors or CXCR2 alone, and was more than additive. Thus, PLC activity, as well as Ca2+ release, was enhanced. In these cells, agonist-mediated Ca2+ release was incremental in nature, suggesting that a potentiation of Ins(1,4,5)P3 generation in the presence of coactivation of P2Y2 nucleotide receptors and CXCR2 would be sufficient for additional Ca2+ release. Potentiated Ca2+ signaling by CXCR2 was markedly attenuated by expression of either regulator of G protein signaling 2 or the Gbetagamma-scavenger Galphat1 (transducin alpha subunit), indicating the involvement of Galphaq and Gbetagamma subunits, respectively.
我们之前已经表明，用UTP激活内源性表达的、与Gαq/11偶联的P2Y2核苷酸受体，可揭示人胚肾细胞中重组的、与Gαi偶联的CXC趋化因子受体2（CXCR2）激活后的细胞内Ca2+反应。在此，我们进一步表征这种串扰，并证明磷脂酶C（PLC）和依赖肌醇1,4,5-三磷酸[Ins(1,4,5)P3]的Ca2+释放是这种增强作用的基础。推定的Ins(1,4,5)P3受体拮抗剂2-氨基乙氧基二苯硼烷可降低白细胞介素-8对CXCR2激活的反应，用渥曼青霉素持续抑制磷脂酰肌醇4-激酶也有同样效果，这表明磷酸肌醇参与了这种增强作用。针对Li+对肌醇单磷酸酶活性的阻断，P2Y2核苷酸受体和CXCR2的共刺激导致磷酸肌醇积累，其显著大于单独激活P2Y2核苷酸受体或CXCR2后的积累，且超过相加效应。因此，PLC活性以及Ca2+释放均增强。在这些细胞中，激动剂介导的Ca2+释放本质上是递增的，这表明在P2Y2核苷酸受体和CXCR2共激活的情况下，Ins(1,4,5)P3生成的增强足以导致额外的Ca2+释放。CXCR2增强的Ca2+信号传导被G蛋白信号调节剂2或Gβγ清除剂Gαt1（转导素α亚基）的表达显著减弱，分别表明Gαq和Gβγ亚基的参与。
