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Vitamin E enhances the acylation of 1-O-alkyl-sn-glycero-3-phosphocholine in human endothelial cells.

ABSTRACT: 1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) is the precursor of platelet-activating factor. It is formed via the CoA-independent transacylase reaction, which transfers the polyenoyl acyl group from the sn-2 position of a diacyl phospholipid to the sn-2 position of 1-O-alkyl-sn-glycero-3-phosphocholine (alkyl-GPC). We have reported previously that vitamin E alters phospholipid turnover in the endothelial cells by increasing arachidonic acid release and prostacyclin synthesis. In the present study, the role of vitamin E in the formation of alkylacyl-GPC was investigated. Incubation of endothelial cells with vitamin E resulted in an increase in the formation of [3H]alkylacyl-GPC from [3H]alkyl-GPC. The effect of vitamin E was dose-dependent at concentrations below 23 microM. However, vitamin E did not have a direct effect on the transacylase activity. When endothelial cells were incubated with vitamin E, the CoA-independent transacylase activity in the cell homogenate was found to be enhanced. Kinetic analysis of the transacylase activity in the pre-incubated cells showed that the enhancement of enzyme activity was at the enzyme-substrate level. When endothelial cells were incubated with vitamin E analogues (Trolox, tocol and tocopherol acetate), only limited enhancement of the transacylation process was detected. It is clear that vitamin E enhanced the synthesis of alkylacyl-GPC from alkyl-GPC in a very specific manner by an indirect stimulation of the CoA-independent transacylase activity. The regulation by vitamin E of the formation of alkylacyl-GPC may mediate the transfer of arachidonate from the diacyl phospholipid pool into the ether-linked phospholipid pool.

SUBMITTER: Tran K

PROVIDER: S-EPMC1137990 | biostudies-other | 1994 Feb

REPOSITORIES: biostudies-other

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Project description:The generation of phospholipid oxidation products in atherosclerosis, sepsis, and lung pathologies affects endothelial barrier function, which exerts significant consequences on disease outcomes in general. Our group previously showed that oxidized 1-palmitoyl-2-arachidonyl-sn-glycero-3-phosphocholine (OxPAPC) at low concentrations increases endothelial cell (EC) barrier function, but decreases it at higher concentrations. In this study, we determined the mechanisms responsible for the pulmonary endothelial cell barrier dysfunction induced by high OxPAPC concentrations. OxPAPC at a range of 5-20 μg/ml enhanced EC barriers, as indicated by increased transendothelial electrical resistance. In contrast, higher OxPAPC concentrations (50-100 μg/ml) rapidly increased EC permeability, which was accompanied by increased total cell protein tyrosine (Tyr) phosphorylation, phosphorylation at Tyr-418, the activation of Src kinase, and the phosphorylation of adherens junction (AJ) protein vascular endothelial cadherin (VE-cadherin) at Tyr-731 and Tyr-658, which was not observed in ECs stimulated with low OxPAPC doses. The early tyrosine phosphorylation of VE-cadherin was linked to the dissociation of VE-cadherin-p120-catenin/β-catenin complexes and VE-cadherin internalization, whereas low OxPAPC doses promoted the formation of VE-cadherin-p120-catenin/β-catenin complexes. High but not low doses of OxPAPC increased the production of reactive oxygen species (ROS) and protein oxidation. The inhibition of Src by PP2 and ROS production by N-acetyl cysteine inhibited the disassembly of VE-cadherin-p120-catenin complexes, and attenuated high OxPAPC-induced EC barrier disruption. These results show the differential effects of OxPAPC doses on VE-cadherin-p120-catenin complex assembly and EC barrier function. These data suggest that the rapid tyrosine phosphorylation of VE-cadherin and other potential targets mediated by Src and ROS-dependent mechanisms plays a key role in the dissociation of AJ complexes and EC barrier dysfunction induced by high OxPAPC doses.

Project description:In the present study we have investigated the effect of changes in the concentration of cytosolic free Ca2+ ([Ca2+]i) on the deacetylation-reacylation of PAF-acether (alkylacetylglycerophosphocholine, alkylacetyl-GPC) by rabbit platelets. Washed platelets were incubated with alkyl[3H]acetyl-GPC ([3H]acetyl-PAF) or [3H]alkylacetyl-GPC ([3H]alkyl-PAF) and [Ca2+]i was subsequently elevated by the addition of the ionophore A23187 or thrombin. The catabolism of PAF-acether was studied by measuring the release of [3H]acetate or the formation of [3H]alkylacyl-GPC. The ionophore inhibited the release of [3H]acetate and the formation of [3H]alkylacyl-GPC with no accumulation of lyso-[3H]PAF, indicating that the deacetylation of PAF-acether was blocked. The effect of ionophore on the deacetylation of PAF-acether was parallel with the increase of [Ca2+]i and could be reversed by the addition of EGTA. In contrast with the prolonged inhibition evoked by ionophore, thrombin, which induced a transient elevation of [Ca2+]i, merely delayed the deacetylation of PAF-acether. Since intact platelets failed to convert exogenous lyso-PAF, the effect of Ca2+ on its acylation was investigated by using platelet homogenates. These experiments showed that the acylation of lyso-PAF was inhibited by the exogenously added Ca2+, with a maximum effect at 1 mM. When the formation of endogenous lyso-PAF from the labelled pool of alkylacyl-GPC was examined, a prolonged increase in the concentration of lyso-PAF with a parallel and equally prolonged decrease in the cellular level of alkylacyl-GPC were observed after the addition of ionophore to intact platelets. The addition of EGTA reversed the effect of ionophore, thus permitting reacylation of lyso-PAF. In contrast, only a transient change in the level of lyso-PAF and alkylacyl-GPC was evoked by the addition of thrombin. Therefore we conclude that the inhibitory effect of Ca2+ on the deacetylation-reacylation of PAF-acether may have an important role in the regulation of its biosynthesis.

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Vitamin E enhances the acylation of 1-O-alkyl-sn-glycero-3-phosphocholine in human endothelial cells.

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