Functional characterization of purinergic P2Y and P2Y receptors involved in Japanese flounder (Paralichthys olivaceus) innate immune responses.

G-protein-coupled P2Y receptors activated by extracellular nucleotides play important roles under different physiological and pathophysiological conditions in mammals. To investigate the immunological relevance of P2Y receptors in fish, we identified and characterized the P2Y and P2Y receptors in Japanese flounder Paralichthys olivaceus. The P. olivaceus P2Y and P2Y receptors harbor seven transmembrane domains but share only 24% sequence identity. Real-time PCR analysis revealed the constitutive but unequal mRNA expression pattern of P2YR and P2YR in normal Japanese flounder tissues with the dominant expression of P2YR in head kidney and blood and P2YR in hepatopancreas. In addition, the expression of P2Y and P2Y receptors was markedly modulated by PAMPs stimulation and Edwardsiella tarda infection. Furthermore, blockage of P2YR potently increased ADP-activated pro-inflammatory cytokine IL-1beta gene expression in the head kidney macrophages (HKMs). Moreover, inhibition of P2Y and P2Y receptor activity with their respective potent antagonists significantly altered some of the LPS-induced pro-inflammatory cytokine gene expression in the HKMs. However, blockade of P2YR did not affect the poly(I:C)-induced pro-inflammatory cytokine gene expression examined in the HKMs. Collectively, we have for the first time reported the role of purinergic P2Y and P2Y receptors in fish innate immunity. Our findings have also addressed the importance of extracellular ATP and its metabolites in fish innate immune responses.