Coordination between ecto-nucleotidases and purinergic receptor-mediated signaling

细胞外ATP通过激活多种P2受体产生多种生理效应。尽管P2受体激活通过外核苷酸酶水解ATP而终止，但该代谢步骤导致腺苷（Ade）产生，从而启动P1受体激活。我们证明细胞外ATP<2A><2B>通过在膜表面快速和局部产生Ade刺激A_和A_受体。此外，外碱性磷酸酶（eALP）被发现作为外核苷酸酶。在<2A><2B>非洲爪蟾卵母细胞中，A_1和A_2受体的异源表达表明，ATP以依赖于外核苷酸酶的方式刺激每个P1受体，其速度与Ade直接刺激一样快。ATP对P1受体的激活对腺苷脱氨酶（ADA）对腺苷的清除有抵抗作用。对NG 108 -15细胞和非洲爪蟾卵母细胞的细胞外[^3H]ATP代谢的分析表明，即使在ADA存在的情况下，代谢产生的[^3H]Ade仍保留在膜表面。将eALP-GFP融合蛋白转染NG 108 -15细胞后，eALP-GFP定位于细胞膜表面，并与A_受体共内化<2A><2A>。这些结果表明，P1受体与外核苷酸酶的紧密结合可能构成ATP的功能性受体，并且通过这种机制对ATP产生某些生理反应。

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.