Ectonucleoside triphosphate diphosphohydrolases (NTPDases) control the concentration of released extracellular nucleotides, but the precise physiological roles played by these isozymes in modulation of P2 receptor signaling remain unclear. Activation of the human P2Y 1 receptor was studied in the presence of NTPDase1 or NTPDase2 expressed either in the same cell as the receptor or in P2Y 1 receptor-expressing cells cocultured with NTPDaseexpressing cells. Coexpression of NTPDase1 with the P2Y 1 receptor resulted in increases in the EC 50 for 2Ј-methylthioadenosine 5Ј-diphosphate (2MeSADP; 12-fold), ADP (50-fold), and ATP (10-fold) for activation of phospholipase C. Similar effects were observed when the P2Y 1 receptor and NTPDase1 were expressed on different cells. These results are explained by the capacity of NTPDase1 to hydrolyze both nucleoside triphosphates and diphosphates. NTPDase2 preferentially hydrolyzes nucleoside triphosphates, and the presence of NTPDase2 under either coexpression or coculture conditions did not change the EC 50 of 2MeSADP, ADP, or adenosine 5Ј-O-(2-thiodiphosphate) for activation of the P2Y 1 receptor. However, the EC 50 for ATP was 15-fold lower in the presence of NTPDase2 than in cells expressing the P2Y 1 receptor alone. Whereas expression of NTPDase1 decreased basal activity of the P2Y 1 receptor, the presence of the NTPDase2 resulted in P2Y 1 receptor-dependent increases in basal activity. These results suggest that basal activity of the P2Y 1 receptor is maintained by paracrine or autocrine release of receptor agonists and that the biological and/or pharmacological response mediated by P2Y receptors in target tissues is highly dependent on the types of ectonucleotidases expressed in the vicinity of the receptor.

BackgroundThe leaves of Annona purpurea have yielded several alkaloids with anti-aggregation activities against rabbit platelets. This is promising in the search for agents that might act against platelets and reduce the incidence of cardiovascular diseases. Since significant differences in platelet function have been reported between human and animal platelets, a study focusing on the effect of A. purpurea extracts against human platelet activation is necessary.MethodsThe compounds in an A. purpurea ethanolic extract underwent bio-guided fractionation and were used for in vitro human platelet aggregation assays to isolate the compounds with anti-platelet activity. The bioactive compounds were identified by spectroscopic analysis. Additional platelet studies were performed to characterize their action as inhibitors of human platelet activation.ResultsThe benzylisoquinoline alkaloid norpurpureine was identified as the major anti-platelet compound. The IC50 for norpurpureine was 80 μM against platelets when stimulated with adenosine 5′-diphosphate (ADP), collagen and thrombin. It was pharmacologically effective from 20 to 220 μM. Norpurpureine (220 μM) exhibited its in vitro effectiveness in samples from 30 healthy human donors who did not take any drugs during the 2 weeks prior to the collection. Norpurpureine also gradually inhibited granule secretion and adhesion of activated platelets to immobilized fibrinogen. At the intra-platelet level, norpurpureine prevented agonist-stimulated calcium mobilization and cAMP reduction. Structure–activity relationship analysis indicates that the lack of a methyl group at the nitrogen seems to be key in the ability of the compound to interact with its molecular target.ConclusionNorpurpureine displays a promising in vitro pharmacological profile as an inhibitor of human platelet activation. Its molecular target could be a common effector between Ca2+ and cAMP signaling, such as the PLC-PKC-Ca2+ pathway and PDEs. This needs further evaluation at the protein isoform level.Electronic supplementary materialThe online version of this article (10.1186/s11658-018-0082-4) contains supplementary material, which is available to authorized users.

No abstract

No abstract

To begin to address the functional interactions between constitutively released nucleotides, ectonucleotidase activity, and P2Y receptor-promoted signaling responses, we engineered the human P2Y 1 receptor in a fusion protein with a member of the ectonucleoside triphosphate diphosphohydrolase family, NTPDase1. Membranes prepared from Chinese hamster ovary (CHO)-K1 cells stably expressing either wild-type NTPDase1 or the P2Y 1 receptor-NTPDase1 fusion protein exhibited nucleotide-hydrolytic activities that were over 300-fold greater than activity measured in membranes from empty vector-transfected cells. The molecular ratio for nucleoside triphosphate versus diphosphate hydrolysis was approximately 1:0.4 for both the wild-type NTPDase1 and P2Y 1 -NTPDase1 fusion protein. Stable expression of the P2Y 1 -NTPDase1 fusion protein conferred an ADP and 2MeSADP-promoted Ca 2ϩ response to CHO-K1 cells. Moreover, the maximal capacity of the nonhydrolyzable agonist ADP␤S to stimulate inositol phosphate accumulation was similar, and the EC 50 of ADP␤S was lower in the fusion protein than the wild-type receptor. In contrast, the substantial nucleotide-hydrolyzing activity of the fusion protein resulted in a greater than 50-fold shift to the right of the concentration-effect curve of ADP for activation of phospholipase C compared with the wild-type receptor. Heterologous expression of the P2Y 1 and other P2Y receptors results in marked increases in basal inositol phosphate levels. Given the high nucleotidase activity and apparently normal receptor signaling activity of the P2Y 1 receptor-NTPDase1 fusion protein, we quantitated basal inositol phosphate accumulation in cells stably expressing either the wild-type P2Y 1 receptor or the fusion protein. Although marked elevation of inositol phosphate levels occurred with wild-type P2Y 1 receptor expression, levels in cells expressing the fusion protein were not different from those in wild-type CHO-K1 cells.