15-hydroxy-11-alpha-9-alpha-(epoxymethano)prosta-5-13-dienoic-acid and Hypertrophy

Thromboxane A2 receptor (TPr) stimulation induces cellular hypertrophy in vascular smooth muscle cells (VSMCs); however, regulation of VSMC hypertrophy remains poorly understood. Here we show that TPr stimulation activates AMP-activated kinase (AMPK), which in turn limits TPr-induced protein synthesis in VSMCs. Exposure of cultured VSMCs to either TPr agonists, IBOP and U46619, or exogenous hydrogen peroxide (H2O2) caused time- and dose-dependent AMPK activation, as evidenced by increased phosphorylation of both AMPK-Thr172 and acetyl-coenzyme A carboxylase-Ser79, a downstream enzyme of AMPK, whereas SQ29548, a selective TPr antagonist, significantly attenuated TPr-enhanced AMPK activation. In parallel, both IBOP and U46619 significantly increased the production of reactive oxygen species such as H2O2. Furthermore, adenoviral overexpression of catalase (an H2O2 scavenger) abolished, whereas superoxide dismutase (which catalyzes H2O2 formation) enhanced, IBOP-induced AMPK activation, suggesting that TPr-activated AMPK was mediated by H2O2. Consistently, exposure of VSMCs to either TPr agonists or exogenous H2O2 dose-dependently increased the phosphorylation of LKB1 (at serines 428 and 307), an AMPK kinase, as well as coimmunoprecipitation of AMPK with LKB1. In addition, direct mutagenesis of either Ser428 or Ser307 of LKB1 into alanine, like the kinase-dead LKB1 mutant, abolished both TPr-stimulated AMPK activation and coimmunoprecipitation. Finally, genetic inhibition of AMPK significantly accentuated IBOP-enhanced protein synthesis, whereas adenoviral overexpression of constitutively active AMPK abolished IBOP-enhance protein synthesis in VSMCs. We conclude that TPr stimulation triggers reactive oxygen species-mediated LKB1-dependent AMPK activation, which in return inhibits cellular protein synthesis in VSMCs.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Adenoviridae; AMP-Activated Protein Kinase Kinases; AMP-Activated Protein Kinases; Animals; Bridged Bicyclo Compounds, Heterocyclic; Catalase; Cells, Cultured; Dose-Response Relationship, Drug; Enzyme Activation; Fatty Acids, Unsaturated; Hydrogen Peroxide; Hypertrophy; Multienzyme Complexes; Muscle, Smooth, Vascular; Mutagenesis, Site-Directed; Mutation, Missense; Myocytes, Smooth Muscle; Oxidants; Phosphorylation; Protein Biosynthesis; Protein Serine-Threonine Kinases; Rats; Receptors, Thromboxane A2, Prostaglandin H2; Superoxide Dismutase; Transduction, Genetic; Vasoconstrictor Agents

In the present study, we examined the effect of the thromboxane/prostaglandin endoperoxide analogue U46619 on proliferation and hypertrophy in cultured rat vascular smooth muscle cells and the roles of protein kinase C and transforming growth factor-beta (TGF-beta) in the mediation of the hypertrophic response to U46619. Since an increase in basic fibroblast growth factor (bFGF) was previously shown to mediate the hypertrophic response to U46619, we also assessed the relationship between bFGF and TGF-beta in the expression of U46619 actions. U46619 increased [35S]methionine incorporation into protein and protein content of vascular smooth muscle cells but had no effect on cell number. A role for TGF-beta was supported by the following observations: (1) exogenous human TGF-beta 1 increased protein synthesis; (2) antibody to TGF-beta blocked both TGF-beta- and U46619-induced increases in protein content; (3) U46619 increased active and total TGF-beta bioactivities; and (4) the actions of U46619 on protein content and TGF-beta bioactivity were blocked by the thromboxane/prostaglandin endoperoxide receptor antagonist SQ 29,548. Previous observations had demonstrated a role for bFGF in the expression of U46619 actions on protein synthesis. Results of the present study suggest that TGF-beta and bFGF interact in mediating the protein synthetic response to U46619. First, the concentration of exogenous TGF-beta (10 pmol/L) alone required to produce a protein synthetic response equivalent to that induced by U46619 was much higher than the concentration of endogenous active TGF-beta that accumulated in the media in response to U46619 (0.7 pmol/L). Second, bFGF (20 ng/mL) increased total TGF-beta bioactivity and stimulated protein synthesis. The hyper-trophic response to bFGF was blocked by anti-TGF-beta. The ability of U46619 and bFGF to increase protein synthesis and protein content in vascular smooth muscle cells was associated with TGF-beta-induced suppression of proliferation, as evidenced by the ability of antibody to TGF-beta to enhance U46619- and bFGF-induced increases in [3H]thymidine incorporation into DNA. Results of the present study also supported a role for protein kinase C in the expression of U46619 and bFGF actions. U46619 increased protein kinase C activity in the particulate fraction of vascular smooth muscle cells. Moreover, the protein kinase C inhibitors GF109203X and staurosporine blocked U46619- and bFGF-induced increases in protein synthesis

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Cell Division; Cells, Cultured; Dose-Response Relationship, Drug; Fibroblast Growth Factor 2; Humans; Hypertrophy; Male; Muscle, Smooth, Vascular; Prostaglandin Endoperoxides, Synthetic; Protein Biosynthesis; Protein Kinase C; Rats; Rats, Inbred WKY; Thromboxane A2; Transforming Growth Factor beta; Vasoconstrictor Agents

Vascular smooth muscle cells (VSMC) undergo hypertrophy when exposed to thromboxane A2 and hyperplasia when exposed to phorbol 12-myristate 13-acetate (PMA) or platelet-derived growth factor (PDGF). Each of these three agonists stimulate rapid tyrosine phosphorylation of numerous VSMC proteins. The current studies were undertaken to identify proteins that are specifically tyrosine phosphorylated in one or the other growth response. All three agonists increased the phosphotyrosine content of multiple proteins. In Western analysis of phosphotyrosine immunoprecipitates, the hyperplastic agents PDGF and PMA increased tyrosine phosphorylation of phospholipase C-gamma 1 (PLC-gamma 1), GTPase-activating protein (GAP), and phosphatidylinositol-3-kinase (PI-3-kinase), while the hypertrophic agonist thromboxane failed to tyrosine-phosphorylate either of these three substrates. Tyrosine kinase inhibition with herbimycin A (5 microM) prevented agonist-stimulated tyrosine phosphorylation of PLC-gamma 1, GAP, and PI-3-kinase. In growth studies, herbimycin A inhibited PMA- and PDGF-induced hyperplasia but not thromboxane-stimulated hypertrophy. These results indicate that tyrosine phosphorylation of PLC-gamma 1, GAP, and PI-3-kinase are specific responses for VSMC hyperplasia but not thromboxane-stimulated hypertrophy.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Cells, Cultured; Hyperplasia; Hypertrophy; Muscle, Smooth, Vascular; Phosphatidylinositol 3-Kinases; Phosphorylation; Phosphotransferases (Alcohol Group Acceptor); Platelet-Derived Growth Factor; Prostaglandin Endoperoxides, Synthetic; Protein-Tyrosine Kinases; Rats; Tetradecanoylphorbol Acetate; Thromboxane A2; Type C Phospholipases; Tyrosine

To more clearly define the physiologic roles of thromboxane (TX)A2 and primary prostaglandins (PG) in vascular tissue we examined vascular contractility, cell signaling, and growth responses. The growth-promoting effects of (15S)-hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5Z,13E-dienoic acid (U46619; TXA2 agonist), PGF2 alpha, and PGE2 consisted of protein synthesis and proto-oncogene expression, but not DNA synthesis or cell proliferation. U46619 contracted rat aortas and increased cultured rat aortic vascular smooth muscle cell intracellular free calcium concentration [Ca2+]i, [3H]inositol monophosphate (IP) accumulation, myosin light chain phosphorylation, and protein synthesis ([3H]leucine incorporation) with EC50 values ranging from 10 to 50 nM. Each of these responses was inhibitable with the TXA2 receptor antagonist [1S]1 alpha,2 beta(5Z),3 beta,4 alpha-7-(3-[2- [(phenylamino)carbonyl]hydrazino]methyl)-7-oxabicyclo[2.2.1]hept-2- yl-5-heptenoic acid (SQ29548). In contrast, PGF2 alpha increased [Ca2+]i, [3H]IP, and protein synthesis with EC50 values of 30-230 nM but contracted rat aortas with an EC50 of 4800 nM. PGE2 increased [Ca2+]i, [3H]IP accumulation, protein synthesis, and contracted rat aortas with EC50 values of 2.5-3.5 microM. TXA2 receptor blockade prevented PGF2 alpha- and PGE2-induced aortic contraction and cell myosin light chain phosphorylation, but not cell signaling or protein synthesis. Binding studies to vascular smooth muscle TXA2 receptors using 1S-[1 alpha,2 beta(5Z),3 alpha(1E,3S),4 alpha]-7-(3-[3-hydroxy-4-(p- [125I]iodophenoxy)-1-butenyl]7-oxabicyclo[2.2.1]hept-2-yl)-5-hepte noic acid ([125I]BOP) showed U46619, SQ29548, PGF2 alpha, and PGE2 competition for TXA2 receptor binding at concentrations similar to their EC50 values for aortic contraction, while binding competition with [3H]PGF2 alpha and [3H]PGE2 demonstrated the specificity of [125I]BOP and SQ29548 for TXA2 receptors. The results suggest that 1) PGF2 alpha- and E2-stimulated vessel contraction is due to cross-agonism at vascular TXA2 receptors; 2) PGF2 alpha stimulates TXA2 receptor-independent vascular smooth muscle protein synthesis at nanomolar concentrations, consistent with an interaction at its primary receptor; and 3) TXA2 is a potent stimulus for vascular smooth muscle contraction and protein synthesis. We suggest that the main physiologic effect of PGF2 alpha may be as a stimulus for vascular smooth muscle cell hypertrophy, not as a contractile agonist.

Topics: 15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; Animals; Aorta; Binding, Competitive; Calcium; Cells, Cultured; Cytosol; Dinoprost; Dinoprostone; DNA; Hypertrophy; Inositol Phosphates; Isometric Contraction; Kinetics; Leucine; Male; Muscle, Smooth, Vascular; Prostaglandin Endoperoxides, Synthetic; Prostaglandins; Protein Biosynthesis; Rats; Rats, Sprague-Dawley; Receptors, Prostaglandin; Receptors, Thromboxane; Thymidine; Vasoconstrictor Agents