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

Abstract

, Jen‐Ai Rd., 1st Section, Taipei, Taiwan 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 [3H]‐U‐46619 bound to A10 cells of passages 18–20 (pl8‐20) with two classes of sites. The high affinity site showed a Bmax of 3.0 ±1.8 fmol mg−1 protein with a KD value 1.0 ±0.1 nM, while the low affinity site showed a Bmaxx of 43.0 ±6.0 fmol mg protein and KD 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 Bmax 111.0 ±9.0 fmol mg−1 protein and a Ku value of 175.4 ±22.0 nM Cinnamophilin and SQ29548 inhibited specific [3H]‐U‐46619 binding to pl8‐20 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 μm and 310.0 ±6.4 nM, respectively at the low affinity site 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 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 pKB values 5.4 ±1.2 and 6.3 ±0.9, respectively 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 U‐46619 caused phosphoinositide breakdown and increased the intracellular Ca2+ concentration in VSMC, effects which were blocked by cinnamophilin and SQ29548 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. 1995 British Pharmacological Society