Department of Pharmacology and Chemical Biology, University of Pittsburgh School of Medicine, 100 Technology Drive, Room 514, Pittsburgh, PA, 15219, USA.
Purinergic Signal. 2020 Jun;16(2):187-211. doi: 10.1007/s11302-020-09699-x. Epub 2020 May 4.
The goal of this study was to determine the validity of using N-etheno-bridged adenine nucleotides to evaluate ecto-nucleotidase activity. We observed that the metabolism of N-etheno-ATP versus ATP was quantitatively similar when incubated with recombinant CD39, ENTPD2, ENTPD3, or ENPP-1, and the quantitative metabolism of N-etheno-AMP versus AMP was similar when incubated with recombinant CD73. This suggests that ecto-nucleotidases process N-etheno-bridged adenine nucleotides similarly to endogenous adenine nucleotides. Four cell types rapidly (t, 0.21 to 0.66 h) metabolized N-etheno-ATP. Applied N-etheno-ATP was recovered in the medium as N-etheno-ADP, N-etheno-AMP, N-etheno-adenosine, and surprisingly N-etheno-adenine; intracellular N-etheno compounds were undetectable. This suggests minimal cellular uptake, intracellular metabolism, or deamination of these compounds. N-etheno-ATP, N-etheno-ADP, N-etheno-AMP, N-etheno-adenosine, and N-etheno-adenine had little affinity for recombinant A, A, or A receptors, for a subset of P2X receptors (H-α,β-methylene-ATP binding to rat bladder membranes), or for a subset of P2Y receptors (S-ATP-αS binding to rat brain membranes), suggesting minimal pharmacological activity. N-etheno-adenosine was partially converted to N-etheno-adenine in four different cell types; this was blocked by purine nucleoside phosphorylase (PNPase) inhibition. Intravenous N-etheno-ATP was quickly metabolized, with N-etheno-adenine being the main product in naïve rats, but not in rats pretreated with a PNPase inhibitor. PNPase inhibition reduced the urinary excretion of endogenous adenine and attenuated the conversion of exogenous adenosine to adenine in the renal cortex. The N-etheno-bridge method is a valid technique to assess extracellular metabolism of adenine nucleotides by ecto-nucleotidases. Also, rats express an enzyme with PNPase-like activity that metabolizes N-etheno-adenosine to N-etheno-adenine.
本研究旨在确定使用 N-亚乙基桥连腺嘌呤核苷酸评估外核苷酸酶活性的有效性。我们观察到，当与重组 CD39、ENTPD2、ENTPD3 或 ENPP-1 孵育时，N-亚乙基-ATP 与 ATP 的代谢在定量上相似，而当与重组 CD73 孵育时，N-亚乙基-AMP 与 AMP 的代谢在定量上相似。这表明外核苷酸酶对 N-亚乙基桥连腺嘌呤核苷酸的处理类似于内源性腺嘌呤核苷酸。四种细胞类型快速（t，0.21 至 0.66 小时）代谢 N-亚乙基-ATP。应用的 N-亚乙基-ATP 在培养基中作为 N-亚乙基-ADP、N-亚乙基-AMP、N-亚乙基-腺苷回收，令人惊讶的是还有 N-亚乙基-腺嘌呤；细胞内的 N-亚乙基化合物无法检测到。这表明这些化合物的细胞摄取、细胞内代谢或脱氨作用最小。N-亚乙基-ATP、N-亚乙基-ADP、N-亚乙基-AMP、N-亚乙基-腺苷和 N-亚乙基-腺嘌呤对重组 A、A 或 A 受体、一部分 P2X 受体（H-α，β-亚甲基-ATP 与大鼠膀胱膜结合）或一部分 P2Y 受体（S-ATP-αS 与大鼠脑膜结合）的亲和力很小，提示其药理活性很小。N-亚乙基-腺苷在四种不同的细胞类型中部分转化为 N-亚乙基-腺嘌呤；这被嘌呤核苷磷酸化酶 (PNPase) 抑制所阻断。静脉内注射 N-亚乙基-ATP 很快被代谢，N-亚乙基-腺嘌呤是新生大鼠的主要产物，但在预先用 PNPase 抑制剂处理的大鼠中不是。PNPase 抑制减少了内源性腺嘌呤的尿排泄，并减弱了外源性腺苷在肾皮质中转化为腺嘌呤。N-亚乙基桥接方法是一种评估外核苷酸酶对腺嘌呤核苷酸细胞外代谢的有效技术。此外，大鼠表达一种具有 PNPase 样活性的酶，可将 N-亚乙基-腺苷代谢为 N-亚乙基-腺嘌呤。
