Low-affinity thromboxane receptor mediates proliferation in cultured vascular smooth muscle cells of rats.

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Abstract
Abstract We examined the binding properties and mitogenic effects of U46619, using cultured vascular smooth muscle cells (VSMCs), by ligand-binding assay, measuring [ 3 H]thymidine and [ 3 H]leucine incorporation, checking with flow cytometry, and counting the cell number. The U46619-activated mitogenic signal-transduction pathway was assessed by mea-suring formation of inositol monophosphate (IP); [Ca 2+ ] i ; mitogen-activated protein kinase (MAPK), MAPK kinase (MAPKK), and p74raf-1 activities; and GTP-bound Ras. [ 3 H]U46619 bound to cultured VSMCs from Wistar-Kyoto (WKY) rats at a single class of site (K d : 15.5±2.6 nmol/L). However, it bound to VSMCs from spontaneously hypertensive rats (SHRs) at two classes of sites ( K d : 2.3±0.6 nmol/L and 1.4±0.5 μmol/L). U46619 increased DNA and protein synthesis, cell number, IP formation, [Ca 2+ ] i , and MAPK and MAPKK activities, with EC 50 values close to its K d value for the low-affinity binding site in VSMCs from SHR. Prostaglandin (PG) E 2 and PGF 2α 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.