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Coordination between ecto-nucleotidases and purinergic receptor-mediated signaling

核酸外切酶和嘌呤能受体介导的信号传导之间的协调

基本信息

批准号：
14570082
负责人：
MATSUOKA Isao
金额：
$ 2.69万
依托单位：
Fukushima Medical University School of Medicine
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (C)
财政年份：
2002
资助国家：
日本
起止时间：
2002 至 2003
项目状态：
已结题

项目摘要

Extracellular ATP produces diverse physiological effects by activating multiple P2 receptors. Although P2 receptor activation is terminated by hydrolysis of ATP by ecto-nucleotidases, this metabolic step results in adenosine (Ade) production, thereby initiating the P1 receptor activation. We demonstrated that extracellular ATP stimulates A_<2A> and A_<2B> receptor through a rapid and localized Ade production on membrane surface. Furthermore, ecto-alkaline phosphatase (eALP) was found to act as ecto-nucleotidases. Heterologous expression of A_<2A> and A_<2B> receptors in Xenopus oocyte revealed that ATP can stimulate each P1 receptor as rapidly as direct stimulation with Ade in an ecto-nucleotidase-dependent manner. The ecto-nucleotidase-dependent P1 receptor activation by ATP was resistant to elimination of Ade by Ade deaminase (ADA). Analysis of extracellular [^3H]ATP metabolism in NG108-15 cells and Xenopus oocyte showed that the metabolically generated [^3H]Ade was retained on the membrane surface even in the presence of ADA. When NG108-15 cells were transfected with eALP-GFP fusion protein, eALP-GFP was localized on membrane surface and co-internalized with A_<2A> receptors upon prolonged A_<2A> receptor activation. These findings suggest that the close association of P1 receptors to ecto-nucleotidases may comprise a functional receptor for ATP, and some physiological responses to ATP would occur through this mechanism.

细胞外ATP通过激活多个P2受体产生多种生理效应。尽管P2受体的激活是由外核苷酸酶水解ATP终止的，但这一代谢步骤导致腺苷（Ade）的产生，从而启动P1受体的激活。我们证明了细胞外ATP通过在膜表面快速和局部地生成Ade来刺激A_<2A>和A_<2B>受体。此外，还发现外碱性磷酸酶（eALP）具有外核苷酸酶的作用。A_<2A>和A_<2B>受体在非洲爪蟾卵母细胞中的异源表达表明，ATP可以像Ade直接刺激P1受体一样以外核苷酸酶依赖的方式快速刺激P1受体。ATP激活的外核苷酸酶依赖的P1受体对Ade脱氨酶（ADA）消除Ade具有抗性。NG108-15细胞和爪蟾卵母细胞胞外[^3H]ATP代谢分析表明，即使存在ADA，代谢产生的[^3H]Ade仍保留在膜表面。将eALP-GFP融合蛋白转染NG108-15细胞后，随着A_<2A>受体激活时间的延长，eALP-GFP定位于膜表面并与A_<2A>受体共内化。这些发现表明P1受体与外核苷酸酶的密切联系可能包含ATP的功能受体，并且通过这种机制可能发生对ATP的一些生理反应。