P2X1 receptor-mediated inhibition of the proliferation of human coronary smooth muscle cells involving the transcription factor NR4A1

Adenine nucleotides acting at P2X(1) receptors are potent vasoconstrictors. Recently, we demonstrated that activation of adenosine A(2B) receptors on human coronary smooth muscle cells inhibits cell proliferation by the induction of the nuclear receptor subfamily 4, group A, member 1 (NR4A1; alternative notation Nur77). In the present study, we searched for long-term effects mediated by P2X(1) receptors by analyzing receptor-mediated changes in cell proliferation and in the expression of NR4A1. Cultured human coronary smooth muscle cells were treated with selective receptor ligands. Effects on proliferation were determined by counting cells and measuring changes in impedance. The induction of transcription factors was assessed by qPCR. The P2X receptor agonist alpha,beta-methylene-ATP and its analog beta,gamma-methylene-ATP inhibited cell proliferation by about 50 % after 5 days in culture with half-maximal concentrations of 0.3 and 0.08 mu M, respectively. The effects were abolished or markedly attenuated by the P2X(1) receptor antagonist NF449 (carbonylbis-imino-benzene-triylbis-(carbonylimino)tetrakis-benzene-1,3-disulfonic acid; 100 nM and 1 mu M). alpha,beta-methylene-ATP and alpha,beta-methylene-ATP applied for 30 min to 4 h increased the expression of NR4A1; NF449 blocked or attenuated this effect. Small interfering RNA directed against NR4A1 diminished the antiproliferative effects of alpha,beta-methylene-ATP and alpha,gamma-methylene-ATP. alpha,beta-methylene-ATP (0.1 to 30 mu M) decreased migration of cultured human coronary smooth muscle cells in a chamber measuring changes in impedance; NF449 blocked the effect. In conclusion, our results demonstrate for the first time that adenine nucleotides acting at P2X(1) receptors inhibit the proliferation of human coronary smooth muscle cells via the induction of the early gene NR4A1.