Lazarowski E R, Homolya L, Boucher R C, Harden T K
Department of Pharmacology, School of Medicine, University of North Carolina, Chapel Hill, North Carolina 27599, USA.
J Biol Chem. 1997 Sep 26;272(39):24348-54. doi: 10.1074/jbc.272.39.24348.
ATP is released from most cell types and functions as an extracellular signaling molecule through activation of members of the two large families of P2X and P2Y receptors. Although three mammalian P2Y receptors have been cloned that are selectively activated by uridine nucleotides, direct demonstration of the release of cellular UTP has not been reported. Pharmacological studies of the P2Y4 receptor expressed in 1321N1 human astrocytoma cells indicated that this receptor is activated by UTP but not by ATP. Mechanical stimulation of 1321N1 cells also resulted in release of a molecule that markedly activated the expressed P2Y4 receptor. This nucleotide was shown to be UTP by two means. First, high performance liquid chromatography analysis of the medium from [33P]H3PO4-loaded 1321N1 cells illustrated that mechanical stimulation resulted in a large increase in a radioactive species that co-eluted with authentic UTP. This species was degraded by incubation with the nonspecific pyrophosphohydrolase apyrase or with hexokinase and was specifically lost by incubation with the UTP-specific enzyme UDP-glucose pyrophosphorylase. Second, a sensitive assay that quantitates UTP mass at low nanomolar concentrations was devised based on the nucleotide specificity of UDP-glucose pyrophosphorylase. Using this assay, mechanical stimulation of 1321N1 cells was shown to result in an increase of medium UTP levels from 2.6 to 36.4 pmol/10(6) cells within 2 min. This increase was paralleled by a similar augmentation of extracellular ATP levels. A calcein-based fluorescence quenching method was utilized to confirm that none of the increases in medium nucleotide levels could be accounted for by cell lysis. Taken together, these results directly demonstrate the mechanically induced release of UTP and illustrate the efficient coupling of this release to activation of P2Y4 receptors.
三磷酸腺苷（ATP）可从大多数细胞类型中释放出来，并通过激活两个大家族的P2X和P2Y受体成员，作为一种细胞外信号分子发挥作用。尽管已经克隆出三种可被尿苷核苷酸选择性激活的哺乳动物P2Y受体，但尚未有关于细胞内三磷酸尿苷（UTP）释放的直接证据报道。对1321N1人星形细胞瘤细胞中表达的P2Y4受体进行的药理学研究表明，该受体可被UTP激活，而不能被ATP激活。对1321N1细胞进行机械刺激，也会导致一种能显著激活所表达的P2Y4受体的分子释放。通过两种方法证明该核苷酸为UTP。首先，对用[33P]H3PO4标记的1321N1细胞培养基进行高效液相色谱分析表明，机械刺激导致一种与纯UTP共洗脱的放射性物质大幅增加。该物质经与非特异性焦磷酸水解酶——腺苷三磷酸双磷酸酶或己糖激酶孵育后会降解，并经与UTP特异性酶——尿苷二磷酸葡萄糖焦磷酸化酶孵育后特异性消失。其次，基于尿苷二磷酸葡萄糖焦磷酸化酶的核苷酸特异性，设计了一种可在低纳摩尔浓度下定量UTP质量的灵敏检测方法。使用该检测方法，结果显示对1321N1细胞进行机械刺激可导致培养基中UTP水平在2分钟内从2.6皮摩尔/10(6)个细胞增加到36.4皮摩尔/10(6)个细胞。细胞外ATP水平也有类似的升高。采用基于钙黄绿素的荧光猝灭法来确认培养基中核苷酸水平的升高并非由细胞裂解所致。综上所述，这些结果直接证明了UTP的机械诱导释放，并说明了这种释放与P2Y4受体激活的有效偶联。
