Changes in IP3 receptor expression and function in aortic smooth muscle of atherosclerotic mice

Peroxynitrite is an endothelium - independent vasodilator which induces relaxation via membrane hyperpolarisation. A ctivation of IP3 receptors triggers opening of potassium channels and hyperpolarisation. Previously we found that relaxation to peroxynitrite was maintained during development of atherosclerosis due to changes in expression of calcium regulatory proteins. In this study we investigated 1) the mechanism of peroxynitrite - induced relaxation in mouse aorta 2) the effect of atherosclerosis on relaxation to peroxynitrite and other vasodilators 3) the effect of atherosclerosis on expression and function of the IP3 receptor. Aortic function was studied using wire myography and atherosclerosis was induced by fat - feeding ApoE - / - mice . Expression of IP3 receptors was studied using Western blotting and immunohistochemistry . Relaxation to peroxynitrite was attenuated by the IP3 antagonists 2 - APB and xestospongin C and also the Kv channel blocker 4 - AP. Atherosclerosis attenuated vasodilation to cromakalim and the AMPK activator A769662 but not peroxynitrite. Relaxation was attenuated to a greater extent by 2 - APB in atherosclerotic aortae despite reduced expression of IP3 receptors. 4 - AP was less effective in 4 month fat fed ApoE - / - mice. Peroxynitrite relaxation involves IP3 - induced calcium release and K V channel activation. This mechanism becomes less important as atherosclerosis develops and relaxation to peroxynitrite may be maintained by increased calcium extrusion

Short periods of hypoxia upregulate Sphingosine Kinase 1 and increase vasodilation of arteries to Sphingosine 1-Phosphate via S1P3

Sphingosine kinase [(SK), isoforms SK1 and SK2] catalyzes the formation of the bioactive lipid, sphingosine 1-phosphate (S1P). This can be exported from cells and bind to S1P receptors to modulate vascular function. We investigated the effect of short-term hypoxia on SK1 expression and the response of arteries to S1P. SK1 expression in rat aortic and coronary artery endothelial cells was studied using immunofluorescence and confocal microscopy. Responses of rat aortic rings were studied using wire myography and reversible hypoxia induced by bubbling myography chambers with 95% N2:5% CO2 Inhibitors were added 30 minutes before induction of hypoxia. S1P induced endothelium-dependent vasodilation via activation of S1P3 receptors and generation of nitric oxide. Hypoxia significantly increased relaxation to S1P and this was attenuated by (2R)-1-[[(4-[[3-methyl-5-[(phenylsulfonyl)methyl] phenoxy]methyl]phenyl]methyl]-2-pyrrolidinemethanol [(PF-543), SK1 inhibitor] but not (R)-FTY720 methyl ether [(ROMe), SK2 inhibitor]. Hypoxia also increased vessel contractility to the thromboxane mimetic, 9,11-dideoxy-11α,9α-epoxymethanoprostaglandin F2α, which was further increased by PF-543 and ROMe. Hypoxia upregulated SK1 expression in aortic and coronary artery endothelial cells and this was blocked by PF-543 and 2-(p-hydroxyanilino)-4-(p-chlorophenyl)thiazole [(SKi), SK1/2 inhibitor]. The effects of PF-543 and SKi were associated with increased proteasomal/lysosomal degradation of SK1. A short period of hypoxia increases the expression of SK1, which may generate S1P to oppose vessel contraction. Under hypoxic conditions, upregulation of SK1 is likely to lead to increased export of S1P from the cell and vasodilation via activation of endothelial S1P3 receptors. These data have significance for perfusion of tissue during episodes of ischemia