Abstract
P2Y2 and P2Y4 receptors are physiologically activated by uridine 5'-triphosphate (UTP) and are widely expressed in many cell types in humans. P2Y2 plays an important role in inflammation and proliferation of tumor cells, which could be attenuated with the use of antagonists. However, little is known about the physiological functions related to P2Y4, due to the lack of selective ligands for these receptors. This can be solved through the search for novel compounds with antagonistic activity. The aim of this study was to discover new potential antagonist candidates for P2Y2 and P2Y4 receptors from natural products. We applied a calcium measurement methodology to identify new antagonist candidates for these receptors. First, we established optimal conditions for the calcium assay using J774.G8, a murine macrophage cell line, which expresses functional P2Y2 and P2Y4 receptors and then, we performed the screening of plant extracts at a cutoff concentration of 50 μg/mL. ATP and ionomycin, known intracellular calcium inductors, were used to stimulate cells. The calculated EC50 were 11 μM and 103 nM, respectively. These cells also responded to the UTP stimulation with an EC50 of 1.021 μM. Screening assays were performed and a total of 100 extracts from Brazilian plants were tested. Joannesia princeps Vell. (stem) and Peixotoa A. Juss (flower and leaf) extracts stood out due to their ability to inhibit UTP-induced responses without causing cytotoxicity, and presented an IC50 of 32.32, 14.99, and 12.98 μg/mL, respectively. Collectively, our results point to the discovery of potential antagonist candidates from Brazilian flora for UTP-activated receptors.
Highlights
Purinergic receptors are plasma membrane receptors activated by extracellular purines and pyrimidines
P2Y2 and P2Y4 are purinergic receptors physiologically activated by uridine triphosphate (UTP), which promote an increase of intracellular calcium via phospholipase Cβ (PLCβ)/ IP3 when activated [2]
Pharmaceutical companies have been developing drugs that act on these P2Y receptors, especially P2Y2, in order to treat these related diseases [5,6,38]
Summary
Purinergic receptors are plasma membrane receptors activated by extracellular purines and pyrimidines. P2Y2 and P2Y4 receptors are physiologically activated by uridine triphosphate (UTP) and promote an increase in intracellular calcium via PLC/IP3 [2] They are expressed in many cell types such as epithelial cells, monocytes, macrophages, neutrophils, cardiomyocytes and organs, including brain, heart, kidneys, liver, spleen, and muscle [3]. Due to a large number of samples for testing, implementation of high-throughput screening methodologies is necessary to meet this demand In this scenario, techniques of intracellular calcium measurement emerge as a tool for screening of new antagonists, since P2Y2 and P2Y4 receptors are coupled to calcium signaling pathway [26]. We found that nine extracts partly inhibited calcium mobilization in J774.G8 cell line, but only one significantly blocked this activity in peritoneal macrophages and did not present cytotoxicity This natural extract may constitute a potential lead in the development of new antagonists for UTP-activated receptors
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