Nucleotide receptor P2u partially mediates ATP-induced cell cycle progression of aortic smooth muscle cells.

mRNA of the P2u purinoceptor (or nucleotide receptor) is detected both by polymerase chain reaction or Northern blot analyses in cultured aortic smooth muscle cells. When added to the culture medium of these cells, UTP, a specific ligand of the P2u receptor, induces an increased expression of both immediate-early and delayed-early cell cycle-dependent genes. This induction demonstrates similar features (kinetics, concentration dependence) to those obtained after stimulation of aortic smooth cells by exogenous ATP, a common ligand for most P2 purinoceptors. In contrast, 2-methylthioATP, a preferential ligand for P2y purinoceptors, induces only a significant increase of immediate-early genes but not of delayed-early genes. Moreover, the 2-methylthioATP-induced responses (c-fos mRNA increase, free intracellular calcium transient) are lower than those induced by ATP or UTP and are complementary to those of UTP. These results demonstrate that functional P2u receptors are present on cultured aortic smooth muscle cells and suggest that the bulk of responses induced by extracellular ATP on cell cycle progression are mediated via P2u purinoceptors, a hypothesis confirmed by cytofluorometric studies. Since some ATP- or UTP-induced genes code for chemotactic proteins (monocyte chemoattractant protein-1 and osteopontin), this study suggests that these nucleotides may contribute to vascular or blood cell migration and proliferation and consequently to the genesis of arterial diseases.

ATP raises [Ca2+]i via different P2-receptor subtypes in freshly isolated and cultured aortic myocytes.

In vascular smooth muscle, extracellular ATP induces an increase in intracellular [Ca2+] ([Ca2+]i). Various agonists have been used to characterize P2-purinoceptor subtypes involved in the ATP-induced [Ca2+]i rise, measured by indo 1 fluorescence, in both freshly isolated and cultured rat aortic smooth muscle cells. alpha, beta-Methylene-ATP increased [Ca2+]i via Ca2+ entry through P2x-receptor channels in freshly isolated but not in cultured cells. 2-Methylthio-ATP and ADP failed to release Ca2+ via P2y-receptor activation in freshly isolated cells, whereas such a response was obtained in cultured cells. UTP, by stimulating P2u receptors, released Ca2+ from intracellular stores in both freshly isolated and cultured cells. These results suggest that, in the course of the culture process, P2x-receptor activation-induced responses were lost, whereas P2y-receptor activation-induced [Ca2+]i rise appeared, these two phenomena being independent. Responses to P2x-receptor agonist were lost in all culture conditions, whereas functional P2y receptors appeared only in cells that were stimulated with serum to induce cell cycle progression. The phenotypic modulation of vascular myocytes was therefore associated with a change in the functional P2-purinoceptor subtypes.

Exogenous ATP induces a limited cell cycle progression of arterial smooth muscle cells.

Because exogenous ATP is suspected to influence the proliferative process, its effects on the cell cycle progression of arterial smooth muscle cells were studied by investigating changes in the mRNA steady-state level of cell cycle-dependent genes. Stimulation of cultured quiescent smooth muscle cells by exogenous ATP induced chronological activation not only of immediate-early but also of delayed-early cell cycle-dependent genes, which were usually expressed after a mitogenic stimulation. In contrast, ATP did not increase late G1 gene mRNA level, demonstrating that this nucleotide induces a limited cell cycle progression of arterial smooth muscle cells through the G1 phase but is not able by itself to induce crossing over the G1-S boundary and consequently DNA synthesis. An increase in c-fos mRNA level was also induced by ADP but not by AMP or adenosine. Moreover, 2-methylthioadenosine 5'-triphosphate but not alpha, beta-methyleneadenosine 5'-triphosphate mediated this kind of response. Taken together, these results demonstrate that extracellular ATP induces the limited progression of arterial smooth muscle cells through the G1 phase via its fixation on P2 gamma receptors.