Cloning and Characterization of the Porcine P2Y6 Receptor: Evidence for Gi Protein‐mediated Signaling in Coronary Smooth Muscle

Abstract

The P2Y6 receptor (P2Y6R) has been thought exclusively coupled with Gq proteins. Our previous work has shown that P2Y6R mRNA is highly expressed in porcine coronary smooth muscles. However, the molecular and pharmacological properties and the functional role of this receptor remain undefined. Here we report the cloning of the cDNA for this receptor that encodes an open reading frame for a protein of 328 amino acids with highest homology to the human P2Y6R. Heterologous expression of this receptor in 1321N1 cells reveals that UDP was more potent and efficacious than UTP for increasing intracellular [Ca2+]i, and ATP/ADP were inactive. However, homologous over-expression of the P2Y6R in sub-cultured coronary smooth muscle cells did not increase intracellular [Ca2+]i in response to UDP, but it enabled UDP to suppress forskolin-induced cAMP elevation, which was eliminated by PTX pretreatment, indicating Gi protein-mediated signaling in coronary smooth muscles. Consistent with this interpretation, UDP stimulation of intact coronary arteries denuded of endothelium decreased isoproterenol-induced vessel relaxation and cAMP elevation. In addition, UDP increased total DNA and protein synthesis in sub-cultured coronary smooth muscle cells over-expressing P2Y6R and in intact coronary arteries, both of which were prevented by PTX. In conclusion, we have cloned and characterized the porcine P2Y6R and demonstrated that this receptor is primarily coupled with Gi protein in coronary smooth muscles to mediate UDP-induced cell proliferation and inhibition of vasorelaxation. (Support: HL29582 to PED and HL062552 to MS)