Hayashi Seiji, Hazama Akihiro, Dutta Amal K, Sabirov Ravshan Z, Okada Yasunobu
Department of Cell Physiology, National Institute for Physiological Sciences, Okazaki 444-8585, Japan.
Sci STKE. 2004 Nov 9;2004(258):pl14. doi: 10.1126/stke.2582004pl14.
Cells release adenosine 5'-triphosphate (ATP) into the extracellular space in response to various stimuli. This released ATP plays an important physiological role in cell-to-cell signal transduction. The bulk ATP concentration can be detected using a conventional luciferin-luciferase assay. However, the ATP concentration in the vicinity of the cell surface is often different from the bulk concentration because of its rapid degradation by ecto-ATPases and because of delayed diffusion due to unstirred layer effects. Here, we describe a simple biosensor method to measure the local ATP concentration on the cell surface in real time. The method is based on the ATP-dependent opening of ligand-gated cation channels of purinergic P2X receptors expressed in undifferentiated pheochromocytoma (PC12) cells or in human embryonic kidney 293 (HEK293) cells stably transfected with recombinant P2X2 purinergic receptors. Under the whole-cell configuration of patch-clamp, a sensor PC12 cell or HEK293 is positioned within the proximity of a target cell, and the P2X-mediated currents induced by ATP released from a given site on the target cell surface is measured. The ATP release is quantified by a calibration procedure utilizing local puff applications of ATP at preset concentrations.
细胞会响应各种刺激将三磷酸腺苷（ATP）释放到细胞外空间。这种释放的ATP在细胞间信号转导中发挥重要的生理作用。可以使用传统的荧光素 - 荧光素酶测定法检测总体ATP浓度。然而，由于细胞外ATP酶对其快速降解以及未搅拌层效应导致的扩散延迟，细胞表面附近的ATP浓度通常与总体浓度不同。在此，我们描述了一种简单的生物传感器方法，用于实时测量细胞表面的局部ATP浓度。该方法基于未分化的嗜铬细胞瘤（PC12）细胞或稳定转染重组P2X2嘌呤能受体的人胚肾293（HEK293）细胞中表达的嘌呤能P2X受体的配体门控阳离子通道的ATP依赖性开放。在膜片钳的全细胞配置下，将一个作为传感器的PC12细胞或HEK293细胞放置在靶细胞附近，并测量由靶细胞表面给定部位释放的ATP诱导的P2X介导的电流。通过利用预设浓度的ATP局部喷射应用的校准程序对ATP释放进行定量。
