Aleo M F, Giudici M L, Sestini S, Danesi P, Pompucci G, Preti A
Departament of Biomedical Science and Biotechnology, University of Brescia, via Valsabbina, 19, 25123 Brescia, Italy.
J Cell Biochem. 2001;80(3):360-6. doi: 10.1002/1097-4644(20010301)80:3<360::aid-jcb90>3.0.co;2-x.
Extracellular NAD is degraded to pyridine and purine metabolites by different types of surface-located enzymes which are expressed differently on the plasmamembrane of various human cells and tissues. In a previous report, we demonstrated that NAD-glycohydrolase, nucleotide pyrophosphatase and 5'-nucleotidase are located on the outer surface of human skin fibroblasts. Nucleotide pyrophosphatase cleaves NAD to nicotinamide mononucleotide and AMP, and 5'-nucleotidase hydrolyses AMP to adenosine. Cells incubated with NAD, produce nicotinamide, nicotinamide mononucleotide, hypoxanthine and adenine. The absence of ADPribose and adenosine in the extracellular compartment could be due to further catabolism and/or uptake of these products. To clarify the fate of the purine moiety of exogenous NAD, we investigated uptake of the products of NAD hydrolysis using U-[(14)C]-adenine-NAD. ATP was found to be the main labeled intracellular product of exogenous NAD catabolism; ADP, AMP, inosine and adenosine were also detected but in small quantities. Addition of ADPribose or adenosine to the incubation medium decreased uptake of radioactive purine, which, on the contrary, was unaffected by addition of inosine. ADPribose strongly inhibited the activity of ecto-NAD-hydrolyzing enzymes, whereas adenosine did not. Radioactive uptake by purine drastically dropped in fibroblasts incubated with (14)C-NAD and dipyridamole, an inhibitor of adenosine transport. Partial inhibition of [(14)C]-NAD uptake observed in fibroblasts depleted of ATP showed that the transport system requires ATP to some extent. All these findings suggest that adenosine is the purine form taken up by cells, and this hypothesis was confirmed incubating cultured fibroblasts with (14)C-adenosine and analyzing nucleoside uptake and intracellular metabolism under different experimental conditions. Fibroblasts incubated with [(14)C]-adenosine yield the same radioactive products as with [(14)C]-NAD; the absence of inhibition of [(14)C]-adenosine uptake by ADPribose in the presence of alpha-beta methyleneADP, an inhibitor of 5' nucleotidase, demonstrates that ADPribose coming from NAD via NAD-glycohydrolase is finally catabolised to adenosine. These results confirm that adenosine is the NAD hydrolysis product incorporated by cells and further metabolized to ATP, and that adenosine transport is partially ATP dependent.
细胞外的烟酰胺腺嘌呤二核苷酸（NAD）通过不同类型的位于表面的酶降解为吡啶和嘌呤代谢物，这些酶在各种人类细胞和组织的质膜上表达不同。在之前的一份报告中，我们证明了烟酰胺腺嘌呤二核苷酸糖水解酶、核苷酸焦磷酸酶和5'-核苷酸酶位于人类皮肤成纤维细胞的外表面。核苷酸焦磷酸酶将NAD裂解为烟酰胺单核苷酸和AMP，5'-核苷酸酶将AMP水解为腺苷。用NAD孵育的细胞会产生烟酰胺、烟酰胺单核苷酸、次黄嘌呤和腺嘌呤。细胞外区室中不存在二磷酸核糖腺苷和腺苷可能是由于这些产物的进一步分解代谢和/或摄取。为了阐明外源性NAD嘌呤部分的去向，我们使用U-[(14)C]-腺嘌呤-NAD研究了NAD水解产物的摄取。发现ATP是外源性NAD分解代谢的主要标记细胞内产物；还检测到了少量的ADP、AMP、肌苷和腺苷。向孵育培养基中添加二磷酸核糖腺苷或腺苷会降低放射性嘌呤的摄取，相反，添加肌苷则不会影响其摄取。二磷酸核糖腺苷强烈抑制胞外NAD水解酶的活性，而腺苷则没有。在用(14)C-NAD和双嘧达莫（一种腺苷转运抑制剂）孵育的成纤维细胞中，嘌呤的放射性摄取急剧下降。在ATP耗尽的成纤维细胞中观察到[(14)C]-NAD摄取的部分抑制，表明转运系统在一定程度上需要ATP。所有这些发现表明腺苷是细胞摄取的嘌呤形式，并且在用(14)C-腺苷孵育培养的成纤维细胞并在不同实验条件下分析核苷摄取和细胞内代谢后，这一假设得到了证实。用[(14)C]-腺苷孵育的成纤维细胞产生的放射性产物与用[(14)C]-NAD产生的相同；在存在5'核苷酸酶抑制剂α-β亚甲基ADP的情况下，二磷酸核糖腺苷对[(14)C]-腺苷摄取没有抑制作用，这表明通过烟酰胺腺嘌呤二核苷酸糖水解酶来自NAD的二磷酸核糖腺苷最终分解代谢为腺苷。这些结果证实腺苷是细胞摄取并进一步代谢为ATP的NAD水解产物，并且腺苷转运部分依赖于ATP。
