Low-affinity thromboxane receptor mediates proliferation in cultured vascular smooth muscle cells of rats.
We examined the binding properties and mitogenic effects of U46619, using cultured vascular smooth muscle cells (VSMCs), by ligand-binding assay, measuring [3H]thymidine and [3H]leucine incorporation, checking with flow cytometry, and counting the cell number. The U46619-activated mitogenic signal-transduction pathway was assessed by measuring formation of inositol monophosphate (IP); [Ca2+]i; mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK), and p74raf-1 activities; and GTP-bound Ras. [3H]U46619 bound to cultured VSMCs from Wistar-Kyoto (WKY) rats at a single class of site (Kd: 15.5 +/- 2.6 nmol/L). However, it bound to VSMCs from spontaneously hypertensive rats (SHRs) at two classes of sites (Kd: 2.3 +/- 0.6 nmol/L and 1.4 +/- 0.5 mumol/L). U46619 increased DNA and protein synthesis, cell number, IP formation, [Ca2+]i, and MAPK and MAPKK activities, with EC50 values close to its Kd value for the low-affinity binding site in VSMCs from SHR. Prostaglandin (PG) E2 and PGF2 alpha showed little of such mitogenic effects. All these effects of U46619 were inhibited by SQ29548, staurosporine, or pretreatment of VSMCs with phorbol 12-myristate 13-acetate for 24 hours. However, U46619 stimulation did not lead to a significant increase in the Ras-GTP complex or p74raf-1 activity. In conclusion, the mitogenic effect of U46619 appears to be mediated via the activation of low-affinity thromboxane binding sites that trigger phosphoinositide hydrolysis and activate the MAPK pathway, leading to DNA synthesis and cell proliferation.
Ko
F N
FN
Pharmacological Institute, College of Medicine, National Taiwan University, Taipei.
D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D000595:Amino Acid Sequence; D000818:Animals; D019086:Bridged Bicyclo Compounds, Heterocyclic; D002118:Calcium / Q000378:metabolism; D017871:Calcium-Calmodulin-Dependent Protein Kinases / Q000378:metabolism*; D002453:Cell Cycle; D002455:Cell Division; D002478:Cells, Cultured; D004247:DNA / Q000096:biosynthesis; D004789:Enzyme Activation / Q000187:drug effects; D005231:Fatty Acids, Unsaturated; D006160:Guanosine Triphosphate / Q000378:metabolism; D006834:Hydrazines / Q000494:pharmacology; D007295:Inositol Phosphates / Q000378:metabolism; D008024:Ligands; D020929:Mitogen-Activated Protein Kinase Kinases; D008969:Molecular Sequence Data; D009131:Muscle, Smooth, Vascular / Q000166:cytology*; D010455:Peptides / Q000378:metabolism; D019000:Phosphotyrosine / Q000378:metabolism; D011450:Prostaglandin Endoperoxides, Synthetic / Q000378:metabolism; D011494:Protein Kinases / Q000378:metabolism; D016283:Proto-Oncogene Proteins p21(ras) / Q000378:metabolism; D051381:Rats; D011918:Rats, Inbred SHR; D011921:Rats, Inbred WKY; D017482:Receptors, Thromboxane / Q000378:metabolism*; D013928:Thromboxane A2 / Q000031:analogs & derivatives / Q000378:metabolism
D019086:Bridged Bicyclo Compounds, Heterocyclic; D005231:Fatty Acids, Unsaturated; D006834:Hydrazines; D007295:Inositol Phosphates; D008024:Ligands; D010455:Peptides; D011450:Prostaglandin Endoperoxides, Synthetic; D017482:Receptors, Thromboxane; D019000:Phosphotyrosine; D013928:Thromboxane A2; D019796:15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5,13-dienoic Acid; D006160:Guanosine Triphosphate; C045749:SQ 29548; D004247:DNA; D011494:Protein Kinases; D017871:Calcium-Calmodulin-Dependent Protein Kinases; D020929:Mitogen-Activated Protein Kinase Kinases; D016283:Proto-Oncogene Proteins p21(ras); D002118:Calcium