Characterization of the thromboxane (TP-) receptor subtype involved in proliferation in cultured vascular smooth muscle cells of rat.
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 (p18-20) 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 Bmax of 43.0 +/- 6.0 fmol mg-1 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 KD value of 175.4 +/- 22.0 nM. 3. Cinnamophilin and SQ29548 inhibited specific [3H]-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 microM 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 p18-20 and p28-30 A10 cells in aconcentration-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 AO0 cells was potentiatedby PDGF (1 ng ml-1) and FCS (1%) and was inhibited by cinnamophilin (10 microM) and SQ29548 (1 microM)with estimated pKB 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 rapidtransition from the DNA synthetic (S) to the G2/mitotic (M) phase. Moreover, U-46619 also increasedprotein synthesis and cell numbers in VSMC. All these effects of U-46619 were inhibited bycinnamophilin and SQ29548.7. U-46619 caused phosphoinositide breakdown and increased the intracellular Ca2+ concentration inVSMC, effects which were blocked by cinnamophilin and SQ29548.8 These data indicate there are two U-46619 binding sites in AlO 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 TPreceptor-mediated VSMC proliferation, it may thus hold promising potential for the prevention of atherosclerosis or vascular diseases.
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei.
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Teng
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000818:Animals; D001011:Aorta / Q000187:drug effects; D001667:Binding, Competitive; D002478:Cells, Cultured; D004305:Dose-Response Relationship, Drug; D009131:Muscle, Smooth, Vascular / Q000187:drug effects* / Q000502:physiology*; D011450:Prostaglandin Endoperoxides, Synthetic / Q000494:pharmacology*; D051381:Rats; D017482:Receptors, Thromboxane / Q000145:classification* / Q000187:drug effects / Q000502:physiology*; D013928:Thromboxane A2 / Q000031:analogs & derivatives* / Q000494:pharmacology; D013997:Time Factors; D014662:Vasoconstrictor Agents / Q000494:pharmacology*
D011450:Prostaglandin Endoperoxides, Synthetic; D017482:Receptors, Thromboxane; D014662:Vasoconstrictor Agents; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid