WCP2018 PO3-4-20 Poster session Identification of P2Y receptors with oleamide suppressing microglial inflammatory response

Masahiro Kita1, Yasuhisa Ano1, Asuka Inoue2, Junken Aoki2

1Research Laboratories for Health Science & Food Technologies, Research & Development Division, Kirin Company, Limited, Japan, 2Department of Molecular and Cellular Biochemistry, Graduate School of Pharmaceutical Sciences, Tohoku University, Japan

Microglia is specialized macrophage population in the brain, and excessive activated microglia by chronic stress, aging, and brain injury, produces inflammatory molecules and massive reactive oxygen spieces (ROS), which lead neurological disorder including depression and dementia. Previously, we identified oleamide from fermented dairy products as novel neuroprotective compound suppressing microglial inflammation (Ano Y, et al., PLoS ONE, 2015). Whereas oleamide is known to act as endocannabinoids, and displays anti-inflammatory activity via cannabinoid-2 receptor (CB2), we found the possibility that oleamide suppresses microglial inflammation via other G protein-coupled receptors (GPCRs). In this study, we explored novel GPCRs of oleamide involving in the suppression of microglial inflammation. Primary CD11b-positive microglia from newborn C57BL/6J mice isolated with magnetic cell sorts was treated with oleamide, oleamide analogues (trans-oleamide, oleic acid, oleoylethylamide, oleoylethanolamide, palmitoylethanolamide) or agonists (CP-55940, lisophosphatidylserine, P2Y10 selective agonist, MRS2365) for 24 hours, following pretreatment with antagonists (JTE907, H-89, UBO-QIC, perussis toxin, Y-27632, suramin). After stimulation with lipopolysaccharide (LPS) and interferon-γ, TNF-α in supernatant was quantified by ELISA. For the exploration of novel oleamide's GPCR, 534 human GPCRs were evaluated using transforming growth factor (TGF)-α shedding assay system (Inoue A, et al., Nature Methods, 2012). As a result of serious TGF-α shedding assay, purinergic receptors (P2Y1, P2Y4, P2Y6, P2Y10, and P2Y11) were identified as novel receptors of oleamide. Oleamide significantly increased the detection of TGF-α at concentration dependent manner. CB2, P2Y1 and P2Y10 but not P2Y6 agonists suppressed murine microglial TNF-α production, and P2 receptor antagonist, suramin, attenuated the suppression of microglial TNF-α production treated with oleamide. These results suggest that oleamide partially suppresses microglial inflammatory response via P2Y receptors, especially P2Y1 or P2Y10. The evaluation using oleamide and its structural analogues showed that the agonistic activities of oleamide to human and murine P2Y1 was correlated with that the activities suppressing microglial TNF-α production. These data suggest that agonistic affinities to P2Y1 of fatty acid amid might contribute the suppression of microglial inflammatory response. The present study suggests that P2Y receptors are novel receptors of oleamide and P2Y1 is involved in the suppression of microglial inflammatory response by oleamide.