Role of phospholipase C and diacylglyceride lipase pathway in arachidonic acid release and acetylcholine-induced vascular relaxation in rabbit aorta

Abstract

(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 A 2 (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 -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 [ 14 C]AA and stimulated with methacholine (10 Ϫ5 M). Free [ 14 C]AA was released by methacholine. Methacholine decreased the [ 14 C]AA content of PI, DAG, and MAG fractions but not PC or PE fractions. Methacholine-induced release of [ 14 C]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. endothelium-dependent hyperpolarizing factor; phospholipid; monoacylglycerol lipase; trihydroxyeicosatrienoic acid; hydroxyepoxyeicosatrienoic acid VASCULAR ENDOTHELIAL CELLS (ECs) regulate vascular tone by synthesis and release of many bioactive compounds. Ligands such as hormones, ACh, bradykinin, and cytokines stimulate release of arachidonic acid (AA) from cellular membranes (6,15,26,43,55), and AA induces endothelium-dependent vasodilation in isolated rabbit aorta preconstricted with phenylephrine, which is not blocked by nitric oxide (NO) synthase (NOS) and cyclooxygenase (COX) inhibitors (19,39,50). The phospholipase inhibitors mepacrine and dimethyl-eicosadienoic acid (DEDA) decrease relaxation to ACh but not AA, suggesting AA or a metabolite may be involved in the relaxations (11). AA is metabolized by COX, lipoxygenase (LO), and cytochrome P-450 pathways into bioactive eicosanoids (10,30,40,48). Using specific inhibitors in rabbit aorta, Singer and Peach (51) first concluded that a LO metabolite mediates the NO-and PG-resistant relaxation to ACh (51). The LO inhibitors cinnamyl-3,4-dihydroxy-␣-cyanocinnamate, nordihydroguaiaretic acid, and ebselen block ACh and AA-induced vasodilation in rabbit aorta (11,39,42). Previous studies in our lab identified the 15-LO metabolites of AA in rabbit aorta. AA is oxidized by 15-LO to 15-hydroperoxyeicosatetraenoic acid (15-HPETE), which is converted to hydroxyepoxyeicosatrienoic acid (HEETA) and trihydroxyeicosatrienoic acid (THET...

Identification of 15-hydroxy-11,12-epoxyeicosatrienoic acid as a vasoactive 15-lipoxygenase metabolite in rabbit aorta

“…Singer and Peach (45,46) showed that AA relaxed the rabbit aorta, and these relaxations were inhibited by LO inhibitors. Our laboratory (6,48) also demonstrated that ACh-and AA-induced relaxations of rabbit aorta were blocked by LO inhibitors or antisense oligonucleotides against 15-LO-I.…”

“…We have previously shown that AA and ACh cause endotheliumdependent, concentration-related relaxations in Indo-and L-NA-treated aortas (6,36). These relaxations are inhibited by LO inhibitors and antisense oligonucleotides against rabbit 15-LO (6,36,48,49). Thus these relaxations are mediated by a 15-LO metabolite of AA.…”

Membrane fluidity matters: Hyperthermia from the aspects of lipids and membranes

“…Moreover, the heat-induced accumulation of PS and the BA-induced enhancement of DAG may play a positive regulatory role in PKC activation and consequently in HS induction, since both PS and DAG are essential cofactors of PKC [64,65], but can be differently affected by the different stressors. 20:4-DAG can be subsequently metabolised by DAG lipase to 20:4-monoacylglycerol (20:4-MAG) [66,67] after which AA can be released through the action of monoacylglycerol lipase or fatty acid amide hydrolase action [68]. AA released by both PLA 2 and PLC-mediated pathways can mediate signal transduction and be recycled via the Lands pathway, whereas a portion can be lost to b-oxidation.…”

Roles of brain phosphatidylinositol-specific phospholipase C and diacylglycerol lipase in centrally administered histamine-induced adrenomedullary outflow in rats

“…Arachidonic acid may also be released through PLC-and diacylglycerol lipase-mediated pathway. This pathway was reported to be a source of arachidonic acid in human platelets (Bell et al, 1979), murine macrophages (Moscat et al, 1986), rat pancreatic acini (Hou et al 1996) and rabbit aortae (Tang et al, 2006). U-73122 has been shown to be a selective inhibitor of PLC in human platelets and polymorphonuclear neutrophils (Bleasdale et al, 1990;Smith et al, 1990) There are two types of PLCs: phosphatidylinositolspecific PLC and phosphatidylcholine-specific PLC.…”

“…Arachidonic acid has been shown to be released mainly by two different pathways: (1) phospholipase A 2 (PLA 2 ) hydrolyzes the sn-2 ester bond of membrane phospholipids, thereby releasing arachidonic acid (Irvine, 1982;Balsinde et al, 2002); (2) phospholipase C (PLC) cleaves the phosphodiester bond, resulting in the formation of diacylglycerol, which can be hydrolyzed by diacylglycerol lipase to yield arachidonic acid (Bell et al, 1979;Irvine, 1982;Moscat et al, 1986;Hou et al, 1996;Tang et al, 2006). Previously, we reported that the brain PLA 2 is involved in the centrally administered melittin (PLA 2 activator)-induced elevation of plasma noradrenaline and adrenaline (Yokotani et al, 2000), and that the brain PLCdiacylglycerol lipase pathway is involved in the centrally administered corticotropin-releasing factor-, argininevasopressin-and bombesin-induced elevation of plasma catecholamines in rats (Okada et al, 2003b;Shimizu et al, 2004Shimizu et al, , 2005.…”