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Metabolism of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) by cultured rat Kupffer cells.

ABSTRACT: The metabolism of platelet-activating factor (PAF; identified as AGEPC: 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (lyso-GEPC: 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) was investigated in cultured rat Kupffer cells. The rat Kupffer cells accumulated [3H]AGEPC and deacetylated this compound to the corresponding [3H]lyso-GEPC, which was the major metabolic product of [3H]AGEPC. [3H]Lyso-GEPC was distributed primarily in the supernatant fraction of incubated cells throughout the experimental interval. Only a very small portion of the [3H]lyso-GEPC was further converted to 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), indicating that this acylation process was not particularly active in these cells. When [3H]lyso-GEPC was incubated with Kupffer cells, the conversion of lyso-GEPC to AGEPC via the acetyltransferase reaction increased up to 30 min and declined thereafter. Bovine serum albumin (BSA) had a substantial influence on both the cellular uptake and the metabolism of [3H]AGEPC. An increase in the BSA concentration in the incubation media reduced the cellular uptake of [3H]AGEPC and the subsequent formation of lyso-GEPC. The results of this study suggest that the hepatic Kupffer cells play an important role in the metabolism of PAF. Moreover, these results infer that the regulation of the PAF level in certain hepatic pathophysiological situations may be a consequence of the production and subsequent metabolism of this potent lipid autacoid in the Kupffer cells of the liver.

SUBMITTER: Chao W

PROVIDER: S-EPMC1138783 | biostudies-other | 1989 Jul

REPOSITORIES: biostudies-other

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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.

Project description:Recent studies have demonstrated that inflammatory cells can be divided into two groups depending on the type of 2-acetylated phospholipids [1-radyl-2-acetyl-sn-glycero-3-phosphocholine (GPC)] they produce: those that produce predominantly platelet-activating factor (PAF), and those that produce predominantly its 1-acyl analogue (1-acyl-2-acetyl-GPC; AAGPC) [Triggiani, Schleimer, Warner & Chilton (1991) J. Immunol. 147, 660-666]. The present study has examined the factors that regulate the production of these two molecules in mouse bone marrow-derived mast cells (BMMC). Initial experiments indicated that PAF and AAGPC were catabolized by BMMC in a differential manner via two pathways: the first, exclusive for AAGPC, involved a 1-acyl hydrolase that removed the long chain at the sn-1 position of the molecule, and the second, common to AAGPC and PAF, involved acetylhydrolase that removed the acetate at the sn-2 position of the two molecules. Experiments were next designed to identify conditions where the differential catabolism of AAGPC and PAF could be eliminated in order to uncover other factors that regulate the proportions of AAGPC and PAF produced. Phenylmethanesulphonyl fluoride (PMSF) completely blocked the 1-acylhydrolase activity while having little or no effect on the acetyl hydrolase activity, thereby eliminating the influence of the catabolic pathway unique to AAGPC. Moreover, PMSF did not alter the release of arachidonic acid from phospholipid subclasses. PMSF-treated BMMC produced larger quantities of AAGPC than of PAF. The AAGPC/PAF ratio detected in PMSF-treated BMMC was very similar to the ratio of arachidonate contained in and released from 1-acyl-/1-alkyl-linked phosphatidylcholine (PC). BMMC supplemented with arachidonic acid in culture for 3 days increased their total arachidonic acid content in PC as well as the ratio of 1-acyl-2-arachidonoyl-GPC to 1-alkyl-2-arachidonoyl-GPC. These changes resulted in parallel and significant increases in both the total amount of 1-radyl-2-acetyl-GPC and the AAGPC/PAF ration in BMMC. These data indicate that the AAGPC/PAF ratio produced by inflammatory cells is regulated by at least two factors: (1) differential catabolism of these two molecules, and (2) the distribution of arachidonate in 1-acyl- and 1-alkyl-2-arachidonyl-GPC. These observations support the concept of a common pathway for AAGPC and PAF biosynthesis in which the two precursor molecules are 1-acyl-2-arachidonoyl-GPC and 1-alkyl-2-arachidonoyl-GPC, respectively.

Project description:Theophylline and 1-methyl-3-isobutylxanthine (MIX), compounds that block eicosanoid formation and modulate phospholipase A2 activity, inhibited in a dose-dependent manner the formation of both leukotriene B4 (LTB4) and platelet-activating factor (PAF) by human polymorphonuclear leucocytes (PMN) in response to ionophore A23187. Theophylline and MIX lacked any inhibitory effect on acetyl-CoA: lyso-PAF acetyltransferase activity, which is the rate-limiting step for PAF biosynthesis in PMN. The effect of theophylline and MIX on PAF formation could be reversed by incubating the cells in the presence of 1-10 microM exogenous lyso-PAF. Incubation of PMN homogenates in the presence of unsaturated non-esterified fatty acids resulted in dose-dependent inhibition of the acetyltransferase. This effect was linked to the presence of a free carboxyl group, since both arachidonic acid methyl ester and palmitoyl-arachidonoyl phosphatidylcholine lacked inhibitory activity. This inhibitory effect was also dependent on the number of double bonds, since arachidonic acid (C20:4) and eicosapentaenoic acid (C20:5) displayed maximal effect. Kinetic analysis showed that the effect of arachidonic acid was consistent with competitive inhibition, with a Ki value of about 19 microM. Oxidative metabolites of arachidonic acid showed a lesser inhibitory effect with the following order of potency: arachidonic acid greater than 15-HETE (15-hydroxy-6,8,11,14-eicosatetraenoic acid) greater than LTB4 greater than 5-HETE (5-hydroxy-6,8,11,14-eicosatetraenoic acid) greater than lipoxin A4. Examination of enzymes involved in CoA-dependent acylation revealed a low activity of both arachidonoyl-CoA synthetase and arachidonoyl-CoA: lyso-PAF arachidonoyltransferase. These data indicate a strong influence on PAF biosynthesis of the products of the phospholipase A2 reaction, with lyso-PAF disposal being a critical event for PAF formation, and unsaturated fatty acids acting as feed-back inhibitors. The conversion of arachidonic acid via oxidative metabolism into less active inhibitors of acetyl-CoA:lyso-PAF acetyltransferase seems to be an additional mechanism of modulation of this enzyme activity, linked to the function of lipoxygenases. Finally, the enzyme activities involved in arachidonoyl-CoA-dependent acylation of lyso-PAF show a low efficiency in capturing arachidonic acid.

Dataset Information

Metabolism of platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and lyso-PAF (1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine) by cultured rat Kupffer cells.

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