Knock-out mice reveal the contributions of P2Y and P2X receptors to nucleotide-induced Ca2+ signaling in macrophages
Knock-out mice reveal the contributions of P2Y and P2X receptors to nucleotide-induced Ca2+ signaling in macrophages
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DOI：
10.1074/jbc.m607713200
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发表时间：
2006-11-17
影响因子：
4.8
通讯作者：
Hanley, Peter J.
中科院分区：
文献类型：
--
作者：
del Rey, Adriana;Renigunta, Vijay;Hanley, Peter J.
免疫细胞功能受细胞外核苷酸水平变化的调节。在这里，我们使用逆转录-PCR分析，单细胞Ca 2+成像，和基因敲除小鼠，以确定受体介导的核苷酸诱导的Ca 2+信号在居民腹腔巨噬细胞。在无Ca ~（2+）缓冲液中，从P_2Y（2）/P_2Y（4）双基因敲除小鼠分离的细胞中，UTP（或ATP）对内质网（ER）Ca ~（2+）释放的强（K ~（-0.5）< 1 μ m）刺激作用被消除。此外，P2 Y（4）（0/-），而不是P2 Y（2）（-/-），巨噬细胞响应UTP。在P2 Y（2）（-/-）巨噬细胞中，我们可以通过在含有Ca 2+的缓冲液中施加ATP来引发对“纯”P2 X受体激活的Ca 2+反应。纯化的UDP和ADP是无效的激动剂，虽然适度的UDP诱导的Ca 2+反应，可以引起巨噬细胞“激活”后，脂多糖和干扰素-γ。值得注意的是，在无Ca 2+缓冲液中，UTP诱导的Ca 2+瞬变在1分钟内衰减，并且对重复的激动剂激发没有反应。用mag-fluo-4测定ER [Ca ~（2+）]表明，在这些条件下，ER Ca ~（2+）库被耗尽。当细胞外Ca 2+可用时，ER Ca 2+商店重新填充，但Ca 2+在重复UTP挑战后仅增加到初始值的40%。这种明显的受体脱敏在GRK 2（+/-）和GRK 6（-/-）巨噬细胞中持续存在，并且在抑制候选激酶蛋白激酶C和钙调蛋白依赖性激酶II后持续存在。用UTP的初始激发也减少了补体成分C5 a的Ca 2+动员（反之亦然）。总之，同源受体脱敏不是快速抑制P2 Y（2）介导的Ca 2+信号传导的主要机制，P2 Y（2）是巨噬细胞中UTP或ATP的唯一G（q）偶联受体。UDP反应性（P2 Y（6）受体表达）在巨噬细胞活化后增加。
Immune cell function is modulated by changes in extracellular nucleotide levels. Here we used reverse transcription-PCR analyses, single cell Ca2+ imaging, and knock-out mice to define the receptors mediating nucleotide-induced Ca2+ signaling in resident peritoneal macrophages. In Ca2+-free buffer, the potent (K-0.5 < 1 mu m) stimulatory effect of UTP (or ATP) on endoplasmic reticulum (ER) Ca2+ release was abolished in cells isolated from P2Y(2)/P2Y(4) double knock-out mice. Moreover, P2Y(4)(0/-), but not P2Y(2)(-/-), macrophages responded to UTP. In P2Y(2)(-/-) macrophages, we could elicit Ca2+ responses to "pure" P2X receptor activation by applying ATP in buffer containing Ca2+. Purified UDP and ADP were ineffective agonists, although modest UDP-induced Ca2+ responses could be elicited in macrophages after "activation" with lipopolysaccharide and interferon-gamma. Notably, in Ca2+-free buffer, UTP-induced Ca2+ transients decayed within 1 min, and there was no response to repeated agonist challenge. Measurements of ER [Ca2+] with mag-fluo-4 showed that ER Ca2+ stores were depleted under these conditions. When extracellular Ca2+ was available, ER Ca2+ stores refilled, but Ca2+ increased to only similar to 40% of the initial value upon repeated UTP challenge. This apparent receptor desensitization persisted in GRK2(+/-) and GRK6(-/-) macrophages and after inhibition of candidate kinases protein kinase C and calmodulin-dependent kinase II. Initial challenge with UTP also reduced Ca2+ mobilization by complement component C5a (and vice versa). In conclusion, homologous receptor desensitization is not the major mechanism that rapidly dampens Ca2+ signaling mediated by P2Y(2), the sole G(q)-coupled receptor for UTP or ATP in macrophages. UDP responsiveness (P2Y(6) receptor expression) increases following macrophage activation.