CHARACTERIZATION OF THE THROMBOXANE (TP-) RECEPTOR SUBTYPE INVOLVED IN PROLIFERATION IN CULTURED VASCULAR SMOOTH-MUSCLE CELLS OF RAT

1 The effects of the thromboxane A(2) (TxA(2))-mimetic, U-46619, on the proliferation of vascular smooth muscle cells (VSMCs) were examined in a clonal smooth muscle cell line, A10, which was derived from foetal rat aorta. 2 [H-3]-U-46619 bound to A10 cells of passages 18-20 (p18-20) with two classes of sites. The high affinity site showed a B-max of 3.0 +/- 1.8 fmol mg(-1) protein with a K-D value 1.0 +/- 0.1 nM, while the low affinity site showed a B-max of 43.0 +/- 6.0 fmol mg(-1) protein and K-D value of 129.O +/- 7.9 nM. However, [H-3]-U-46619 bound to A10 cells from passages 28-30 (p28-30) at a single class of site with a B-max 111.0 +/- 9.0 fmol mg(-1) protein and a K-D value of 175.4 +/- 22.0 nM. 3 Cinnamophilin and SQ29548 inhibited specific [H-3]-U-46619 binding to p18-20 A10 cells in a concentration-dependent manner with Ki values of 390.0 +/- 3.2 and 4.6 +/- 1.0 nM, respectively at a high affinity site, and 2.6 +/- 0.2 mu M and 310.0 +/- 6.4 nM, respectively at the low affinity site. 4 U-46619 produced isometric contractions of rat aorta in a concentration-dependent manner with an EC(50) 7.0 +/- 1.2 nM. Cinnamophilin and SQ29548 antagonized U-46619-induced aortic contractions with pA(2) values 6.3 +/- 0.1 and 8.2 +/- 0.2, respectively. 5 U-46619 increased [H-3]-thymidine incorporation into DNA of p18-20 and p28-30 A10 cells in a concentration-dependent manner with EC(50) values 362.7 +/- 27.0 and 302.5 +/- 20.1 nM, respectively. The U-46619-induced increase of [H-3]-thymidine incorporation into DNA of p28-30 A10 cells was potentiated by PDGF (1 ng ml(-1)) and FCS (1%) and was inhibited by cinnamophilin (10 mu M) and SQ29548 (1 mu M) with estimated pK(B) values 5.4 +/- 1.2 and 6.3 +/- 0.9, respectively. 6 Cell cycle analysis revealed that U-46619-increased cell cycle progression was primarily due to a rapid transition from the DNA synthetic (S) to the G(2)/mitotic (M) phase. Moreover, U-46619 also increased protein synthesis and cell numbers in VSMC. All these effects of U-46619 were inhibited by cinnamophilin and SQ29548. 7 U-46619 caused phosphoinositide breakdown and increased the intracellular Ca2+ concentration in VSMC, effects which were blocked by cinnamophilin and SQ29548. 8 These data indicate there are two U-46619 binding sites in A10 VSMC. The high affinity site is correlated to U-46619-induced vasoconstriction while the low affinity site is correlated to U-46619-mediated VSMC proliferation. These data also reveal that U-46619 stimulates the cell cycle progression in VSMC primarily through a rapid transition from S to G(2)/M. Since cinnamophilin inhibits TP-receptor-mediated VSMC proliferation, it may thus hold promising potential for the prevention of atherosclerosis or vascular diseases.