Endotoxin activation of macrophages does not induce ATP release and autocrine stimulation of P2 nucleotide receptors
Endotoxin activation of macrophages does not induce ATP release and autocrine stimulation of P2 nucleotide receptors
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DOI：
10.4049/jimmunol.165.12.7189
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发表时间：
2000-12-15
影响因子：
4.4
通讯作者：
Dubyak, GR
中科院分区：
文献类型：
--
作者：
Beigi, RD;Dubyak, GR
细胞外核苷酸受体（P2受体，即嘌呤能受体）先前已被认为与巨噬细胞内毒素信号转导有关。最有力的证据是观察到离子型核苷酸（P2X）受体抑制剂，包括高碘酸氧化的ATP（oATP），可减弱内毒素诱导的部分效应，如核因子-κB的激活和诱导型一氧化氮合酶的上调。我们利用对细胞外ATP敏感的在线检测方法，研究内毒素是否会诱导小鼠巨噬细胞系（BAC1.2F5）释放ATP。这些细胞组成性地释放ATP，当以10⁶个贴壁细胞单层置于1 ml培养基中进行检测时，产生的稳态细胞外浓度约为1 nM。然而，在急性或长时间的内毒素刺激过程中，巨噬细胞均未释放额外的ATP。此外，在长时间的内毒素激活后测量细胞外ATP酶活性，发现其没有显著改变。尽管oATP处理显著减弱了内毒素诱导的一氧化氮产生，但当细胞与一种高效的ATP清除剂——三磷酸腺苷双磷酸酶共同孵育时，这种抑制作用并未重现。这些结果表明，内毒素对巨噬细胞的激活不会通过释放到细胞外间隙的内源性ATP诱导P2核苷酸受体的自分泌刺激。此外，数据表明oATP干扰内毒素信号转导的能力是由于其与ATP结合型P2受体以外的分子种类相互作用。
Receptors for extracellular nucleotides (P2, or purinergic receptors) have previously been implicated in the transduction of endotoxin signaling in macrophages, The most compelling evidence has been the observation that inhibitors of ionotropic nucleotide (P2X) receptors, including periodate-oxidized ATP (oATP), attenuate a subset of endotoxin-induced effects such as activation of NF-kappaB and up-regulation of inducible NO synthase, We investigated whether endotoxin induces ATP release from a murine macrophage cell line (BAC1.2F5) using sensitive on-line assays for extracellular ATP. These cells constitutively released ATP, producing steady-state extracellular concentrations of similar to1 nM when assayed as monolayers of 10(6) adherent cells bathed in 1 ml of medium. However, the macrophages did not release additional ATP during either acute or prolonged endotoxin stimulation. In addition, cellular ecto-ATPase activities were measured following prolonged endotoxin activation and were found not to be significantly altered. Although oATP treatment significantly attenuated the endotoxin-induced production of NO, this inhibitory effect was not reproduced when the cells were coincubated with apyrase, a highly effective ATP scavenger. These results indicate that activation of macrophages by endotoxin does not induce autocrine stimulation of P2 nucleotide receptors by endogenous ATP released to extracellular compartments. Moreover, the data suggest that the ability of oATP to interfere with endotoxin signaling is due to its interaction with molecular species other than ATP-binding P2 receptors.