Thromboxane prostanoid receptor activation impairs endothelial nitric oxide-dependent vasorelaxations: The role of Rho kinase

Abstract

Activation of thromboxane prostanoid (TP) receptors causes potent vasoconstriction, which contributes to increased vascular tone and blood pressure. The present study examined the hypothesis that stimulation of TP receptor impaired endothelial nitric oxide-mediated vasorelaxation via a Rho kinase-dependent mechanism. The common carotid arteries of Sprague–Dawley rats were isolated and suspended in myograph for measurement of changes in isometric tension. The production of nitric oxide in primary cultured aortic endothelial cells was assayed with an imaging technique and phosphorylated levels of endothelial NOS were determined by Western blot analysis. 9,11-dideoxy-11α,9α-epoxy-methanoprostaglandin F 2α (U46619) inhibited isoprenaline-induced relaxations in rings with or without endothelium. Treatment with Rho kinase inhibitors, Y27632 (2 μM) or HA 1077 (10 μM) prevented the effect of U46619 only in rings with endothelium while protein kinase C inhibitors were without effect. Rho kinase inhibitors did not affect isoprenaline-induced relaxations in endothelium-intact rings treated with L-NAME or 1H-[1,2,4]oxadizolo[4,3-a]quinoxalin-1-one (ODQ). Isoprenaline stimulated rises in nitric oxide (NO) production in cultured rat endothelial cells. The increased NO production was inhibited by U46619 (100 nM) and this effect was prevented by treatment with Y27632 but unaffected by the absence of extracellular calcium ions. U46619 attenuated isoprenaline-stimulated phosphorylation of eNOS, which was sensitive to inhibition by Y27632 and HA 1077. U46619-mediated effects were abolished by TP receptor antagonist, S18886 and the TP receptor was present in endothelial cells. The present results demonstrate that Rho kinase activation is likely to be the primary mechanism that underlies the U46619-stimulated TP-receptor-mediated inhibition of endothelial NO production and subsequent endothelium-dependent relaxations to isoprenaline.