Characterization of the thromboxane (TP-) receptor subtype involved in proliferation in cultured vascular smooth muscle cells of rat

Abstract

1 , Jen-Ai Rd., 1st Section, Taipei, Taiwan 1 The effects of the thromboxane A₂ (TxA₂)-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 [³H]-U-46619 bound to A10 cells of passages 18–20 (pl8-20) 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_maxx of 43.0 ±6.0 fmol mg protein and K_D value of 129.0 ±7.9 nM. However, [³H]-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_u value of 175.4 ±22.0 nM 3 Cinnamophilin and SQ29548 inhibited specific [³H]-U-46619 binding to pl8-20 A10 cells in a concentration-dependent manner with K_i values of 390.0 ±3.2 and 4.6 ±1.0 nM, respectively at a high affinity site, and 2.6 ±0.2 μm 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 EC₅₀ 7.0 ±1.2 nM. Cinnamophilin and SQ29548 antagonized U-46619-induced aortic contractions with pA₂ values 6.3 ±0.1 and 8.2 ±0.2, respectively 5 U-46619 increased [³H]-thymidine incorporation into DNA of pi8-20 and p28-30 A10 cells in a concentration-dependent manner with EC₅₀ values 362.7 ±27.0 and 302.5 ±20.1 nM, respectively. The U-46619-induced increase of [³H]-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 μm) and SQ29548 (1 μm) 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 G₂/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 G₂/M. Since cinnamophilin inhibits TP-receptor-mediated VSMC proliferation, it may thus hold promising potential for the prevention of atherosclerosis or vascular diseases.