Cloning, Up-Regulation, and Mitogenic Role of Porcine P2Y2 Receptor in Coronary Artery Smooth Muscle Cells

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Abstract
Previous work has shown up-regulation of a UTP-sensitive P2Y receptor in porcine coronary smooth muscle cells (CSMC) of organ-cultured arteries. However, the molecular identity and functional role of this putative receptor remained undefined. Here we report the cloning of the cDNA for this receptor that encodes an open reading frame for a protein of 373 amino acids with the highest homology to the human P2Y2 receptor (84%). Heterologous expression of this receptor in 1321N1 cells revealed a novel pharmacology in that UTP and ITP were full agonists and UTP was more potent and efficacious than ATP for increasing intracellular [Ca2+] and extracellular signal-regulated kinase phosphorylation. Stimulation of subcultured CSMC with UTP, ITP, or ATP induced a concentration-dependent increase in cellular DNA content, protein synthesis, cell number, and proliferating cell nuclear antigen expression, indicating a mitogenic role for P2Y2 receptors. This was supported by the finding that the treatment of CSMC with antisense oligonucleotides to the cloned cDNA sequence significantly inhibited UTP- and ATP-induced DNA and protein synthesis. In addition, reverse transcription-polymerase chain reaction analysis showed that P2Y2 receptor mRNA was dramatically increased in cells of organ-cultured arteries compared with freshly harvested arteries, whereas the P2Y6 receptor mRNA level was unchanged, and the P2Y4 receptor mRNA was undetectable. This P2Y2 subtype-specific up-regulation was confirmed in cells of coronary arteries stented in vivo. In conclusion, we have cloned the porcine P2Y2 receptor with novel pharmacology and demonstrated that this receptor is up-regulated in CSMC of in vitro organ cultures or in vivo stented coronary arteries to mediate the mitogenic effects of nucleotides.

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