nitroarginine and 1-6-bis(cyclohexyloximinocarbonyl)hexane

ACh stimulates arachidonic acid (AA) release from membrane phospholipids of vascular endothelial cells (ECs). In rabbit aorta, AA is metabolized through the 15-lipoxygenase pathway to form vasodilatory eicosanoids 15-hydroxy-11,12-epoxyeicosatrienoic acid (HEETA) and 11,12,15-trihydroxyeicosatrienoic acid (THETA). AA is released from phosphatidylcholine (PC) and phosphatidylethanolamine (PE) by phospholipase A2 (PLA2), or from phosphatidylinositol (PI) by phospholipase C (PLC) pathway. The diacylglycerol (DAG) lipase can convert DAG into 2-arachidonoylglycerol from which free AA can be released by monoacylglycerol (MAG) lipase or fatty acid amidohydrolase (FAAH). We used specific inhibitors to determine the involvement of the PLC pathway in ACh-induced AA release. In rabbit aortic rings precontracted by phenylephrine, ACh induced relaxation in the presence of indomethacin and N(omega)-nitro-L-arginine (L-NNA). These relaxations were blocked by the PLC inhibitor U-73122, DAG lipase inhibitor RHC-80267, and MAG lipase/FAAH inhibitor URB-532. Cultured rabbit aortic ECs were labeled with [14C]AA and stimulated with methacholine (10(-5) M). Free [14C]AA was released by methacholine. Methacholine decreased the [14C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [14C]AA was blocked by U-73122, RHC-80267, and URB-532 but not by U-73343, an inactive analog of U-73122. The data suggested that ACh activates PLC, DAG lipase, and MAG lipase pathway to release AA from membrane lipids. This pathway is important in regulating vasodilatory eicosanoid synthesis and vascular relaxation in rabbit aorta.

Topics: Acetylcholine; Animals; Aorta, Thoracic; Arachidonic Acid; Arachidonic Acids; Carbamates; Cells, Cultured; Cyclohexanones; Diazomethane; Endothelium, Vascular; Estrenes; In Vitro Techniques; Indomethacin; Isoenzymes; Lipoprotein Lipase; Methacholine Chloride; Nitroarginine; Phospholipases A; Phospholipases A2; Pyrrolidinones; Rabbits; Type C Phospholipases; Vasodilation

The diacylglycerol lipase inhibitor 1,6-bis(cyclohexyloximinocarbonylamino) hexane (RHC-80267) was tested for its effect on acetylcholine-evoked relaxation in rat mesenteric artery. In artery contracted with either noradrenaline or KCl, RHC-80267 (0.1-10 muM) potentiated the relaxation evoked by acetylcholine. The effect of RHC-80267 was not affected by nitric oxide synthase inhibition or by inhibitors of protein kinase C or of phospholipase A(2). The diacylglycerol analogue 1-oleoyl-2-acetyl-sn-glycerol did not change the relaxation to acetylcholine. RHC-80267 did not affect the relaxation evoked by carbachol, by the nitric oxide donor SNAP (S-nitroso-N-acetylpenicillamine) or by the K(+) channel opener cromakalim. Neostigmine, a cholinesterase inhibitor, produced the same effect as RHC-80267 on acetylcholine-evoked relaxation. When tested on cholinesterase in brain homogenate, RHC-80267 concentration-dependently inhibited cholinesterase activity with an IC(50) of 4 muM. These results indicate that the potentiation of acetylcholine-evoked responses by RHC-80267 in rat mesenteric artery is caused by the inhibition of the cholinesterase activity in the vascular wall.

Topics: Acetylcholine; Animals; Biological Factors; Cholinesterase Inhibitors; Cholinesterases; Cromakalim; Cyclohexanones; Dose-Response Relationship, Drug; Drug Synergism; Endothelium, Vascular; Enzyme Inhibitors; In Vitro Techniques; Lipoprotein Lipase; Male; Mesenteric Arteries; Naphthalenes; Neostigmine; Nitric Oxide; Nitric Oxide Donors; Nitric Oxide Synthase; Nitroarginine; Norepinephrine; Penicillamine; Phospholipases A; Protein Kinase C; Rats; Rats, Inbred WKY; Vasoconstrictor Agents; Vasodilation; Vasodilator Agents