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 Aug 15;272(33):20402-7. doi: 10.1074/jbc.272.33.20402.
The P2Y4 receptor is selectively activated by UTP. Although addition of neither ATP nor UDP alone increased intracellular Ca2+ in 1321N1 human astrocytoma cells stably expressing the P2Y4 receptor, combined addition of these nucleotides resulted in a slowly occurring elevation of Ca2+. The possibility that the stimulatory effect of the combined nucleotides reflected formation of UTP by an extracellular transphosphorylating activity was investigated. Incubation of cells with [3H]UDP or [3H]ADP under conditions in which cellular release of ATP occurred or in the presence of added ATP resulted in rapid formation of the corresponding triphosphates. Transfer of the gamma-phosphate from [gamma-33P]ATP to nucleoside diphosphates confirmed that the extracellular enzymatic activity was contributed by a nucleoside diphosphokinase. The majority of this activity was associated with the cell surface of 1321N1 cells, suggesting involvement of an ectoenzyme. Both ADP and UDP were effective substrates for transphosphorylation. Since ecto-nucleotidase(s) has been considered previously to be the primary enzyme(s) responsible for metabolism of extracellular nucleotides, the relative rates of hydrolysis of ATP, ADP, UTP, and UDP also were determined for 1321N1 cells. All four nucleotides were hydrolyzed with similar Km and Vmax values. Kinetic analyses of the ecto-nucleoside diphosphokinase and ecto-nucleotidase activities indicated that the rate of extracellular transphosphorylation exceeds that of nucleotide hydrolysis by up to 20-fold. Demonstration of the existence of a very active ecto-nucleoside diphosphokinase together with previous observations that stress-induced release of ATP occurs from most cell types indicates that transphosphorylation is physiologically important in the extracellular metabolism of adenine and uridine nucleotides. Since the P2Y receptor class of signaling proteins differs remarkably in their respective specificity for adenine and uridine nucleotides and di- and triphosphates, these results suggest that extracellular interconversion of adenine and uridine nucleotides plays a key role in defining activities in nucleotide-mediated signaling.
P2Y4受体可被尿苷三磷酸（UTP）选择性激活。虽然单独添加三磷酸腺苷（ATP）或二磷酸尿苷（UDP）均不会使稳定表达P2Y4受体的1321N1人星形细胞瘤细胞内的钙离子浓度升高，但同时添加这两种核苷酸会导致钙离子浓度缓慢升高。研究了联合添加核苷酸的刺激作用是否反映了细胞外转磷酸化活性形成UTP的可能性。在ATP发生细胞释放的条件下或添加ATP的情况下，用[3H]UDP或[3H]ADP孵育细胞，会导致相应三磷酸酯的快速形成。[γ-33P]ATP的γ-磷酸转移至核苷二磷酸，证实细胞外酶活性由核苷二磷酸激酶产生。这种活性的大部分与1321N1细胞的细胞表面相关，提示有胞外酶参与。ADP和UDP都是转磷酸化的有效底物。由于胞外核苷酸酶以前被认为是负责细胞外核苷酸代谢的主要酶，因此还测定了1321N1细胞对ATP、ADP、UTP和UDP的相对水解速率。所有四种核苷酸的水解具有相似的米氏常数（Km）和最大反应速度（Vmax）值。对胞外核苷二磷酸激酶和胞外核苷酸酶活性的动力学分析表明，细胞外转磷酸化速率比核苷酸水解速率高出20倍。存在一种活性很高的胞外核苷二磷酸激酶，以及先前观察到大多数细胞类型都会发生应激诱导的ATP释放，这表明转磷酸化在腺嘌呤和尿苷核苷酸的细胞外代谢中具有重要生理意义。由于信号蛋白的P2Y受体类别对腺嘌呤和尿苷核苷酸以及二磷酸酯和三磷酸酯的各自特异性有显著差异，这些结果表明腺嘌呤和尿苷核苷酸的细胞外相互转化在确定核苷酸介导的信号传导活性中起关键作用。
