EVIDENCE THAT P2Y(4) NUCLEOTIDE RECEPTORS ARE INVOLVED IN THE REGULATION OF RAT AORTIC SMOOTH-MUSCLE CELLS BY UTP AND ATP

1 Previous studies have shown that ATP and UTP are able to stimulate p hospholipase C (PLC) and proliferation in cultured aortic smooth muscl e cells. Here we set out to characterize the receptor responsible, and investigate a possible role for p42 and p44 mitogen activated protein kinase (MAPK) in the proliferative response. 2 The phospholipase C re sponse of spontaneously hypertensive rat (SHR) derived aortic smooth m uscle cells in culture showed that the response to ATP was partial com pared to the response to UTP. 3 Further studies characterized the resp onses of the SHR derived cells. UTP was the only full agonist with the SHR cells; UDP gave a partial response while ADP, 2-methythio-ATP and alpha,beta-methylene ATP were essentially ineffective. The response t o UDP was almost lost in the presence of hexokinase, consistent with t his being due to extracellular conversion to UTP. These observations a re inconsistent with the response being mediated by either P2Y(1) or P 2Y(6) receptors. 4 When increasing concentrations of ATP were present with a maximally effective concentration of UTP, the size of the respo nse diminished, consistent with UTP and ATP acting at a single populat ion of receptors for which ATP was a partial agonist. This is inconsis tent with a response mainly at P2Y(2) receptors. 5 1321N1 cells transf ected with human P2Y(4) receptors gave a similar agonist response prof ile, with ATP being partial compared to UTP, loss of response to UDP w ith hexokinase treatment, and with the response to UTP diminishing in the presence of increasing concentrations of ATP. 6 Use of the reverse transcriptase-polymerase chain reaction confirmed the presence of mRN A. encoding P2Y(4) receptors in SHR derived vascular smooth muscle cel ls. Transcripts for P2Y(2), P2Y(4 )and P2Y6 receptors, but not P2Y1 re ceptors, were detected. 7 Stimulation of SHR derived cells with UTP en hanced the tyrosine phosphorylation of both p42 and p44 MAPK, and the incorporation of [H-3]-thymidine into DNA. Both these responses were d iminished in the presence of an inhibitor of activation of MAPK. g The se results lead to the conclusion that in SHR derived cultured aortic smooth muscle cells, PLC responses to extracellular UTP and ATP are pr edominantly at P2Y(4) receptors, and suggest that these receptors are coupled to mitogenesis via p42/p44 MAPK.