Center for Translational Neuromedicine, University of Rochester Medical School, Rochester, New York, USA.
J Cereb Blood Flow Metab. 2012 Jan;32(1):e1-7. doi: 10.1038/jcbfm.2011.142. Epub 2011 Oct 12.
Recent reports based on a chemiluminescent enzymatic assay for detection of adenosine conclude that cultured astrocytes release adenosine during mildly hypoxic conditions. If so, astrocytes may suppress neural activity in early stages of hypoxia. The aim of this study was to reevaluate the observation using high-performance liquid chromatography (HPLC). The HPLC analysis showed that exposure to 20 or 120 minutes of mild hypoxia failed to increase release of adenosine triphosphate (ATP), adenosine diphosphate (ADP), adenosine monophosphate (AMP), and adenosine from cultured astrocytes. Similar results were obtained using a chemiluminescent enzymatic assay. Moreover, since the chemiluminescent enzymatic assay relies on hydrogen peroxide generation, release of free-radical scavengers from hypoxic cells can interfere with the assay. Accordingly, adenosine added to samples collected from hypoxic cultures could not be detected using the chemiluminescent enzymatic assay. Furthermore, addition of free-radical scavengers sharply reduced the sensitivity of adenosine detection. Conversely, use of a single-step assay inflated measured values due to the inability of the assay to distinguish adenosine and its metabolite inosine. These results show that cultured astrocytes do not release adenosine during mild hypoxia, an observation consistent with their high resistance to hypoxia.
最近的一些报告基于用于检测腺苷的化学发光酶检测法得出结论，即在轻度低氧条件下培养的星形胶质细胞会释放腺苷。如果是这样，星形胶质细胞可能会在低氧的早期阶段抑制神经活动。本研究的目的是使用高效液相色谱法（HPLC）重新评估该观察结果。HPLC 分析表明，暴露于 20 或 120 分钟的轻度低氧条件下，未能增加培养的星形胶质细胞中三磷酸腺苷（ATP）、二磷酸腺苷（ADP）、一磷酸腺苷（AMP）和腺苷的释放。使用化学发光酶检测法也得到了类似的结果。此外，由于化学发光酶检测法依赖于过氧化氢的产生，因此来自低氧细胞的自由基清除剂的释放可能会干扰该检测法。因此，无法使用化学发光酶检测法检测从低氧培养物中收集的样品中的添加的腺苷。此外，自由基清除剂的添加大大降低了腺苷检测的灵敏度。相反，由于该检测法无法区分腺苷及其代谢物肌苷，因此使用一步法检测会夸大测量值。这些结果表明，在轻度低氧条件下培养的星形胶质细胞不会释放腺苷，这一观察结果与它们对低氧的高抵抗力一致。
