A novel pathway for lipid biosynthesis: the direct acylation of glycerol.

The acylation of glycerol-3-phosphate by acyl-CoA is regarded as the first committed step for the synthesis of the lipoidal moiety in glycerolipids. The direct acylation of glycerol in mammalian tissues has not been demonstrated. In this study, lipid biosynthesis in myoblasts and hepatocytes was reassessed by conducting pulse-chase experiments with [1,3-(3)H]glycerol. The results demonstrated that a portion of labeled glycerol was directly acylated to form monoacylglycerol and, subsequently, diacylglycerol and triacylglycerol. The direct acylation of glycerol became more prominent when the glycerol-3-phosphate pathway was attenuated or when exogenous glycerol levels became elevated. Glycerol:acyl-CoA acyltransferase activity, which is responsible for the direct acylation of glycerol, was detected in the microsomal fraction of heart, liver, kidney, skeletal muscle, and brain tissues. The enzyme from pig heart microsomes displayed optimal activity at pH 6.0 and the preference for arachidonyl-CoA as the acyl donor. The apparent K(m) values for glycerol and arachidonyl-CoA were 1.1 mM and 0.17 mM, respectively. The present study demonstrates the existence of a novel lipid biosynthetic pathway that may be important during hyperglycerolemia produced in diabetes or other pathological conditions.

Modulation of cytosolic phospholipase A(2) by PPAR activators in human preadipocytes.

Cytosolic phospholipase A(2) (cPLA(2)) is responsible for the release of arachidonic acid, a precursor for eicosanoid biosynthesis, from cellular phospholipids. The objective of this study is to examine the regulation of cPLA(2) by peroxisome proliferator-activated receptor (PPAR) activators in preadipocyte SW872 (SW) cells. PPAR belong to the superfamily of nuclear hormone receptors that heterodimerize with the retinoid X receptor. In this study, the presence of both PPARalpha and PPARgamma was confirmed in SW cells by positive identification of their mRNA in the cellular homogenate. Clofibrate, a PPARalpha activator, caused an enhancement of ionophore A-23187-induced arachidonate release in SW cells. This increase resulted from an enhancement of cPLA(2) activity, which was caused by an increase in enzyme protein. Clofibrate at lower concentrations (10-200 microM) produced increases in the mRNA levels of cPLA(2) in a dose-response manner. At higher concentrations (>400 microM), clofibrate treatment resulted in the attenuation of the cPLA(2) mRNA level and protein expression. We postulate that clofibrate, acting through the PPARalpha, caused an induction in the transcription of cPLA(2) gene, which led to an increase in the cPLA(2) protein. The observed increase in arachidonate release in SW cells appeared to be a direct result of the enhanced cPLA(2) activity.

Effects of atorvastatin treatment on the oxidatively modified low density lipoprotein in hyperlipidemic patients.

Atorvastatin is an established HMG-CoA reductase inhibitor which effectively reduces the plasma low density lipoprotein (LDL)-cholesterol level in hyperlipidemic patients. The present study was designed to investigate whether atorvastatin treatment can modify the biochemical content of oxidized LDL in hyperlipidemic patients and the ability of oxidized LDL to impair the endothelium-dependent relaxation of blood vessels. With atorvastatin (10 mg/day) treatment for 4 weeks in 19 type IIa hyperlipidemic patients, total cholesterol level was lowered by 23%, LDL-cholesterol was lowered by 32% and triacylglycerol was lowered by 19% as compared with dietary therapy alone. High density lipoprotein levels increased by approximately 9%. The ability of oxidized LDL from hyperlipidemic patients after atorvastatin treatment to impair the endothelium-dependent relaxation was significantly reduced as compared with dietary intervention alone. Analysis of the biochemical contents of oxidized LDL from this group revealed that there was an 11% reduction in lysophosphatidylcholine (LPC) as compared with the group that received only dietary counseling. A decrease in the C16:0 moiety with a corresponding increase in the C18:0 moiety of LPC in the oxidized LDL was also observed in the atorvastatin treated group. We propose that the observed reduction and the change in composition of acyl groups in LPC in the oxidized LDL of the atorvastatin-treated group results from a combination of the continued dietary treatment as well as drug therapy. In view of an observation that both C16:0 and C18:0 LPC species are equally potent in the impairment of endothelium-dependent relaxation of the aortic rings, we feel that the reduced level of LPC in the oxidized LDL produced by atorvastatin treatment is partially responsible for the improvement in endothelium control of vascular tone.

Effect of Flos carthami on stress-activated protein kinase activity in the isolated reperfused rat heart.

The apoptotic death of cardiomyocytes due to ischemia/reperfusion is one of the major complications of heart disease. Ischemia/reperfusion has been shown to lead to the activation of the stress-activated protein (SAP) kinases and the p38/reactivating kinase (p38/RK). In this study, the direct effect of an aqueous Flos carthami (FC) extract on SAP kinases was investigated. When isolated rat hearts were perfused by Langendorff mode with media containing FC extract prior to the induction of global ischemia and the subsequent reperfusion, SAP kinase activity was inhibited 95%. Untreated ischemic/reperfused hearts showed a 57% elevation in the activity of SAP kinase. The in vitro effect of these FC extracts on SAP kinase was also tested. At a concentration of 10 microg/ml, the aqueous FC extract resulted in 50% inhibition of SAP kinase activity in ischemic heart tissue. Our results showed that FC affected both the interaction of SAP kinase with c-jun as well as the phosphotransferase reaction. These results clearly demonstrate that extracts from Flos carthami exerted inhibitory effects on SAP kinase. The administration of the FC extract may lead to a modulation of the apoptotic effect of SAP kinase activation induced during ischemia/reperfusion.

Phosphatidylcholine metabolism in human endothelial cells: modulation by phosphocholine.

Phosphatidylcholine is the principal phospholipid in mammalian tissues, and a major source for the production of arachidonic acid. In this study, the effect of exogenous phosphocholine, a precursor of phosphatidylcholine biosynthesis, on the metabolism of phosphatidylcholine in human umbilical vein endothelial cells was investigated. Incubation of endothelial cells with exogenous phosphocholine at concentrations of 1 to 5 mM was found to inhibit choline uptake and its subsequent incorporation into phosphatidylcholine. Phosphocholine appeared to inhibit choline uptake in a competitive manner. Since phosphatidylcholine is metabolized mainly by the action of phospholipase A2, with the release of arachidonic acid and other fatty acids, the effect of phosphocholine on arachidonic acid release in endothelial cells was also examined. The induction of arachidonic acid release by ATP was enhanced in cells treated with 1 mM phosphocholine. In vitro assays of phospholipase A2 activity in cells incubated with phosphocholine, however, did not produced any significant change in the activity of this enzyme. The results of this study show that phosphocholine modulates the biosynthesis and catabolism of phosphatidylcholine in an indirect manner.

The effect of fenofibrate treatment on endothelium-dependent relaxation induced by oxidative modified low density lipoprotein from hyperlipidemic patients.

The objective of the research project was to investigate whether fenofibrate treatment may alter the biochemical content of the oxidized LDL and consequently its ability to impair the endothelium-dependent relaxation in hyperlipidemic patients. We hypothesized that fenofibrate treatment of hyperlipidemic patients may attenuate the ability of their oxidized LDL to impair the endothelium-dependent relaxation of the blood vessels as a consequence of fenofibrate-induced changes to the content and composition of lysoPC in the LDL molecule. Hyperlipidemic patients (Type IIb and Type IV) were recruited from the Lipid Clinic, HSC, Winnipeg, Canada, for this study. A blood sample was taken immediately after the recruitment, a second sample was taken after 6 weeks of dietary treatment, and a third sample was taken after 8 weeks of fenofibrate treatment. LDL was isolated from the plasma and oxidized by copper sulfate. Fenofibrate was shown to be highly effect in the reduction of total cholesterol, LDL cholesterol and triglycerides in these patients. Fenofibrate treatment also caused the attenuation of impairment of endothelium-dependent relaxation by the oxidized LDL from these patients. A slight reduction of lysophosphatidylcholine level was also found in the oxidized LDL of the fenofibrate treated patients, relative to LDL isolated after dietary treatment. In addition there were no changes in the fatty acid levels of the lysophosphatidylcholine isolated from LDL. Taken together, our results suggest that while the reduced lysophosphatidylcholine levels may contribute to the attenuated impairment of the endothelium-dependent relaxation of the aortic ring, other unidentified factors impacted by fenofibrate are likely to contribute to the attenuated effects.

Atherosclerosis risk factors: the possible role of homocysteine.

Atherosclerosis is the leading cause of death in North America. It is characterized by thickening of the coronary artery wall by the formation of plaques, resulting in reduced blood flow. Plaque rupture and the consequent thrombosis may lead to sudden blockage of arteries and causing stroke and heart attack. In the last several decades, more than 250 factors associated with the development of coronary artery disease have been identified. Recently, a relationship between atherosclerosis and elevated homocysteine level in the blood has been established. The mechanism for the production of atherosclerosis by homocysteine has been investigated. When human hepatoma cells (HepG2) were incubated with 4 mM homocysteine, enhancements in the production of cholesterol and secretion of apolipoprotein B-100 were observed. The stimulatory effect on cholesterol synthesis was mediated via the enhancement of HMG-CoA reductase, which catalyzes the rate-limiting step in cholesterol biosynthesis. Cholesterol appears to play an important role in the regulation of apoB-100 secretion by hepatocytes. It is plausible that the increase in apoB secretion was caused by the elevated cholesterol level induced by homocysteine. The ability of homocysteine to produce a higher amount of cholesterol and promote the secretion of apoB would provide a plausible mechanism for the observed relationship between hyperhomocysteinemia and the development of atherogenesis and coronary artery disease.

Evaluation of a new apolipoprotein(a) isoform-independent assay for serum Lipoprotein(a).

The risk factor, Lipoprotein(a), [(Lp(a)], has been measured in numerous clinical studies by a variety of immunochemical assay methods. It is becoming apparent that for many of these assays antibody specificity towards the apolipoprotein(a) [apo(a)] repetitive component [the kringle 4-type 2 repeats] and apo(a) size heterogeneity can significantly affect the accuracy of serum Lp(a) measurements. To address this issue, we investigated whether our current in house Lp(a) [Mercodia] assay showed such bias compared to a recently available assay [Apo-Tek], claiming to possess superior capability for isoform-independent measurement of Lp(a). Levels of Lipoprotein(a) by both Apo-Tek and Mercodia assays correlated inversely with apo(a) isoform sizes. No significant differences were observed between assays in ranges of Lp(a) concentration within each isoform group. The Mercodia assay exhibited similar isoform-independent behaviour to that of Apo-Tek for the quantitation of serum Lipoprotein(a). Essentially identical results were obtained by the two methods, suggesting that Mercodia assay's capture monoclonal antibody also (as is the case for Apo-Tek) does not recognize the kringle 4-type 2 repetitive domain of apo(a). Correlation of Lp(a) concentrations in patient specimens between Apo-Tek and Mercodia assays showed good agreement, although an overall higher degree of imprecision and non-linearity was noted for the Apo-Tek procedure. A change-over to the Apo-Tek assay would therefore not improve on our current assessment of risk contribution from Lp(a) for atherosclerotic vascular disease in individuals with measurable levels of circulating Lipoprotein(a).

Lysophosphatidylethanolamine acyltransferase activity is elevated during cardiac cell differentiation.

We examined if elevation in lysophosphatidylethanolamine acyltransferase activity was associated with elevation in phosphatidylethanolamine content during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of 1% dimethylsulfoxide to the medium. Immunofluorescence microscopy revealed the presence of striated myosin at 8 days post-dimethylsulfoxide addition confirming differentiation into cardiac cells. The content of phosphatidylethanolamine was increased 2.1-fold (P<0.05) in differentiated cells compared to undifferentiated cells, whereas the content of phosphatidylcholine was reduced 29% (P<0.05). There were no alterations in the pool sizes of other phospholipids, including cardiolipin. The relative abundance of fatty acids in phospholipids of P19 cells was 18:1 > 18:0 > 16:1 = 18:2 > 16:0 = 14:0 > 20:4 and differentiation did not affect the relative amounts of these fatty acids within individual phospholipids. When cells were incubated with [1,3-(3)H]glycerol, radioactivity incorporated into phosphatidylethanolamine was elevated 5.8-fold, whereas radioactivity incorporated into phosphatidylcholine was unaltered. Ethanolaminephosphotransferase, cholinephosphotransferase and membrane CTP:phosphocholine cytidylyltransferase activities were elevated in differentiated cells compared to undifferentiated cells, whereas membrane and cytosolic phospholipase A2 activities were unaltered. Lysophosphatidylethanolamine acyltransferase activities were elevated 2.4-fold (P<0.05). Lysophosphatidylcholine acyltransferase, monolysocardiolipin acyltransferase, acyl-Coenzyme A synthetase and acyl-Coenzyme A hydrolase activities were unaltered in differentiated cells compared to undifferentiated cells. We postulate that during cardiac cell differentiation, the observed elevation in lysophosphatidylethanolamine acyltransferase activity accompanies the elevation in phosphatidylethanolamine mass, possibly to maintain the fatty acyl composition of this phospholipid within the membrane.

Elevation in phosphatidylethanolamine is an early but not essential event for cardiac cell differentiation.

The biosynthesis of phosphatidylethanolamine was examined during differentiation of P19 teratocarcinoma cells into cardiac myocytes. P19 cells were induced to undergo differentiation into cardiac myocytes by the addition of dimethyl sulfoxide to the medium. Immunofluorescence labeling confirmed the expression of striated myosin 10 days postinduction of differentiation. The content of phosphatidylethanolamine increased significantly within the first 2 days of differentiation. [1,3-(3)H]Glycerol incorporation into phosphatidylethanolamine was increased 7.2-fold during differentiation, indicating an elevation in de novo synthesis from 1, 2-diacyl-sn-glycerol. The mechanism for the increase in phosphatidylethanolamine levels during cardiac cell differentiation was a 2.8-fold increase in the activity of ethanolaminephosphotransferase, the 1,2-diacyl-sn-glycerol utilizing reaction of the cytidine 5'-diphosphate-ethanolamine pathway of phosphatidylethanolamine biosynthesis. Incubation of P19 cells with the phosphatidylethanolamine biosynthesis inhibitor 8-(4-chlorophenylthio)-cAMP inhibited the differentiation-induced elevation in phosphatidylethanolamine levels but did not affect the expression of striated myosin. The results suggest that elevation in phosphatidylethanolamine is an early event of P19 cell differentiation into cardiac myocytes, but is not essential for differentiation to proceed.

Lysophosphatidylcholine induces arachidonic acid release and calcium overload in cardiac myoblastic H9c2 cells.

Lysophosphatidylcholine (lyso-PC) and arachidonate are products of phosphatidylcholine hydrolysis by phospholipase A(2). In this study, the modulation of arachidonate release by exogenous lyso-PC in rat heart myoblastic H9c2 cells was examined. Incubation of H9c2 cells with lyso-PC resulted in an enhanced release of arachidonate in both a time- and dose-dependent fashion. Lyso-PC species containing palmitoyl (C(16:0)) or stearoyl (C(18:0)) groups evoked the highest amount of arachidonate release, while other lysophospholipid species were relatively ineffective. Cells treated with phospholipase A(2) inhibitors resulted in the attenuation of the enhanced arachidonate release in the presence of lyso-PC. Lyso-PC caused the translocation of phospholipase A(2) from the cytosol to the membrane fraction and induced an increase in Ca2+ flux from the medium into the cells. Nimodipine, a specific Ca(2+)-channel blocker, partially attenuated the lyso-PC-induced rise in intracellular Ca2+. Concurrent with Ca2+ influx, lyso-PC caused an enhancement of protein kinase C activity. The lyso-PC-induced arachidonate release was attenuated when cells were pre-incubated with specific protein kinase C and mitogen activated protein kinase kinase inhibitors. Taken together, these results strongly indicate that the lyso-PC-induced increases in levels of intracellular calcium and stimulation of protein kinase C lead to the activation of cytosolic phospholipase A(2) which results in the enhancement of arachidonate release in H9c2 cells.

Inhibition of endothelium-dependent vascular relaxation by lysophosphatidylcholine: impact of lysophosphatidylcholine on mechanisms involving endothelium-derived nitric oxide and endothelium derived hyperpolarizing factor.

Hyperlipidemia has been associated with an increase in the incidence of atherosclerosis. The oxidation of low density lipoprotein (LDL) plays an important role in the initiation and progression of atherosclerosis, one of its effects being the inhibition of endothelium dependent relaxation (EDR). The elevated level of lysophosphatidylcholine (LPC) in oxidatively modified LDL has been shown to be a biochemical factor responsible for the impairment of EDR in vascular ring preparations. Several endothelium-derived modulators are thought to control vascular responsiveness. The present work examined whether acetylcholine (ACh)-induced EDR in rat aorta (pre-contracted with phenylephrine, PE) involved both endothelium-derived nitric oxide (EDNO) and endothelium-dependent hyperpolarizing factor (EDHF) and whether LPC inhibited either of these selectively. Indomethacin (10(-5) M), had no significant effect on EDR, indicating that products of cyclooxygenase, including prostacyclin, are not involved. Treatment with either N(W)-nitro-L-arginine methyl ester (L-NAME, 6.8 microM) to inhibit the production of EDNO or with elevated K+ (15 mM), to block the hyperpolarizing effect of EDHF impaired EDR considerably (each of these shifting the inhibitory dose-response relationship to ACh by almost one log unit); in muscles treated with both of these agents EDR was completely inhibited. In each of L-NAME- and K-treated muscles, the addition of LPC (20 microM) further impaired EDR. LPC did not independently raise the tone of resting- or PE-contracted aorta. We conclude that the inhibition of EDR of rat aorta by LPC involves the actions of both EDNO and EDHF.

Homocysteine stimulates the production and secretion of cholesterol in hepatic cells.

Homocysteinemia and hypercholesterolemia are important risk factors associated with the occurrence of arteriosclerotic vascular diseases. A positive correlation between plasma levels of homocysteine and cholesterol was found in homocysteinemic patients as well as in experimental animals. In the present study, the effect of homocysteine on the production and secretion of cholesterol in human hepatoma cell line HepG2 cells was investigated. When cells were incubated with 4 mM homocysteine, the amounts of total cholesterol produced as well as the cholesterol secreted by these cells were significantly increased (from 32 +/- 5 to 74 +/- 5 nmol/mg cellular protein). Further biochemical analyses revealed that the increase in cholesterol was resulted from an enhancement in the production and secretion of the unesterified cholesterol with no concomitant change in the level of cholesteryl esters. The activity of intracellular 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase was markedly elevated by 131% and 190% after cells were incubated with homocysteine for 24 and 48 h. Homocysteine also stimulated the secretion of apo B100 by HepG2 cells (from 0.84 +/- 0.11 to 1.37 +/- 0.12 micrograms apolipoprotein B/mg cellular protein). Our results demonstrate that homocysteine stimulates the production and secretion of cholesterol and apolipoprotein B100 in HepG2 cells. The increase in the production of cholesterol induced by homocysteine may contribute to the pathogenesis of arteriosclerosis.

Lysophosphatidylcholine stimulates the release of arachidonic acid in human endothelial cells.

Lysophosphatidylcholine (lyso-PC) is a product of phosphatidylcholine hydrolysis by phospholipase A2 (PLA2) and is present in cell membranes, oxidized lipoproteins, and atherosclerotic tissues. It has the ability to alter endothelial functions and is regarded as a causal agent in atherogenesis. In this study, the modulation of arachidonate release by lyso-PC in human umbilical vein endothelial cells was examined. Incubation of endothelial cells with lyso-PC resulted in an enhanced release of arachidonate in a time- and concentration-dependent manner. Maximum arachidonate release was observed at 10 min of incubation with 50 microM lyso-PC. Lyso-PC species containing palmitoyl (C16:0) or stearoyl (C18:0) groups elicited the enhancement of arachidonate release, while other lysolipids such as lysophosphatidylethanolamine, lysophosphatidylserine, lysophosphatidylinositol, or lysophosphatidate were relatively ineffective. Lyso-PC-induced arachidonate release was decreased by treatment of cells with PLA2 inhibitors such as para-bromophenacyl bromide and arachidonoyl trifluoromethyl ketone. Furthermore, arachidonate release was attenuated in cells grown in the presence of antisense oligodeoxynucleotides that specifically bind cytosolic PLA2 mRNA. Treatment of cells with lyso-PC resulted in a translocation of PLA2 activity from the cytosolic to the membrane fractions of cells. Lyso-PC induced a rapid influx of Ca2+ from the medium into the cells, with a simultaneous enhancement of protein kinase C (PKC) activity in the membrane fractions. The lyso-PC-induced arachidonate release was attenuated when cells were preincubated with specific inhibitors of PKC (staurosporine and Ro31-8220) or a specific inhibitor of mitogen-activated protein kinase/extracellular regulated kinase kinase (PD098059). Taken together, the results of this study show that lyso-PC caused the elevation of cellular Ca2+ and the activation of PKC, which stimulated cytosolic PLA2 in an indirect manner and resulted in an enhanced release of arachidonate.

Vitamin E up-regulates arachidonic acid release and phospholipase A2 in megakaryocytes.

The release of arachidonic acid is the rate limiting step in eicosanoid synthesis. In mammalian cells, the release of arachidonic acid is catalyzed by several enzymes. The 85 kDa cytosolic phospholipase A2 (cPLA2) is the key enzyme for the release reaction because of its specific acyl selectivity in phospholipid substrates. We have previously reported that vitamin E enrichment potentiates the arachidonic acid release as well as the spontaneous prostacyclin release in human endothelial cells. In contrast, similar enrichment of diets caused a dose-dependent suppression of platelet thromboxane synthesis. Therefore, the present study was undertaken to determine the effect of vitamin E on arachidonate release and phospholipaseA2 activity in a platelet precursor cell, the MEG-01 megakaryocyte cell line. When these cells were incubated with different concentrations of vitamin E, cellular incorporation was linear with the dosages of this vitamin. Determination of arachidonate release after labeling cells with [3H]-arachidonate showed that vitamin E enrichment caused a dose-dependent increase in ionophore A23187-induced [3H]-arachidonic acid release. Analysis of PLA2 activity showed that activity was detected in the cytosol and this activity was completely abolished by the addition of anti-cPLA2, antibody. Determination of cPLA2 activity demonstrated that vitamin E enrichment caused an increase in enzyme activity. Analysis of cPLA2 protein by Western blot revealed that vitamin E caused an increase in enzyme protein. These data showed that the potentiation of arachidonic acid release and cPLA2, activity by vitamin E was mediated by the enhanced expression of cPLA2 protein.

Uptake and metabolism of lipoprotein-X in mesangial cells.

Progressive glomerulosclerosis is a major complication in patients with familial lecithin:cholesterol acyltransferase (LCAT) deficiency. The lack of LCAT activity results in the accumulation of an abnormal lipoprotein, lipoprotein-X (Lp-X), in the plasma of these patients. Lipoprotein-X contains high levels of unesterified cholesterol and phosphatidylcholine. Lp-X may play a role in the accumulation of lipids in the kidney, which in turn may lead to glomerulosclerosis. The objective of this study is to examine the uptake and metabolism of Lp-X by rat mesangial cells. Our results suggest that Lp-X is taken up by mesangial cells and that the lipids in Lp-X are metabolized. Lysosomes containing unesterified cholesterol and phosphatidylcholine, in a molar ratio similar to Lp-X, were synthesized to investigate the roles individual apolipoproteins (apo CI, II, III and E) play in the uptake of Lp-X. Both apo CI and CIII inhibited its uptake while apo CII (1.5 fold) and E (4 fold) stimulated the uptake of Lp-X. Very low density lipoprotein (VLDL) and low density lipoprotein (LDL) inhibited Lp-X uptake by mesangial cells. However, at higher concentrations of high density lipoprotein (HDL), the uptake of Lp-X was stimulated. Proteoglycans have an important role in regulating the uptake of Lp-X, while cytoskeleton-dependent phagocytosis and the scavenger receptor do not appear to be involved.

Acyl-GPC and alkenyl/alkyl-GPC:acyl-CoA acyltransferases.

In mammalian tissues, phosphatidylcholine, or 1,2-diacyl-glycerophosphocholine (GPC), is the most abundant form of choline-containing phospholipids. In some electrically active tissues, a significant portion of the choline-containing phospholipids is 1-alkenyl-2-acyl-GPC (plasmenylcholine). The 1-alkyl-2-acyl-GPC is found in significant amounts in circulating cells such as neutrophils and macrophages but in low amounts in other tissues. Structural studies of phosphatidylcholine indicate that there is an asymmetric distribution of acyl groups on the molecule. Saturated fatty acids are usually esterified at the sn-1 position of the glycerol backbone, whereas unsaturated fatty acids are esterified at the sn-2 position. Similarly, unsaturated acyl groups are usually found in the sn-2 position of plasmenylcholine. The remodelling of the sn-2 acyl group in phosphatidylcholine by the deacylation-reacylation process has been demonstrated in a number of tissues. Phospholipase A2 is responsible for the hydrolysis of the acyl group at the sn-2 position, whereas 1-acyl-GPC:acyl-CoA acyltransferase is responsible for the reacylation reaction. The acyltransferase is located in the microsomal fraction and displays specificity towards the polyunsaturated acyl groups. The enzyme can be solubilized by detergent, but the enzyme activity in soluble form is difficult to maintain. The acyltransferase for the reacylation of 1-alkenyl-GPC is also located in the microsomal fraction and is somewhat specific towards polyunsaturated acyl groups. In guinea pig heart mitochondria, however, a new form of 1-alkenyl-GPC acyltransferase was identified which appeared to be different from the microsomal form. The acyltransferase for the acylation of 1-alkyl-GPC into platelet-activating factor has been studied in several tissues including human neutrophils. At present, the contribution of the acyltransferase in attaining the observed molecular composition of the choline-containing phospholipids in the tissue has not been defined. We postulate that the intrinsic acyl-CoA specificity of the acyltransferase, the flux of 1-acyl-GPC, 1-alkenyl-GPC and 1-alkyl-GPC, as well as the pool size of acyl-CoA are major factors in producing the final composition of the molecular species of the choline-containing phospholipids.

On the mechanism of the losartan-mediated inhibition of phosphatidylcholine biosynthesis in H9c2 cells.

Phosphatidylcholine is the major phospholipid in mammalian tissues and the biosynthesis of phosphatidylcholine in H9c2 cells was previously shown to be stimulated by angiotensin II. In this study, we used the potent AT1 receptor antagonist, losartan, to determine if the angiotensin II-mediated stimulation of phosphatidylcholine biosynthesis was mediated by AT1 receptors. H9c2 cells were incubated with angiotensin II in the absence or presence of various concentrations of losartan. The cells were then incubated with [methyl-3H]choline for an additional 60 min and the radioactivity incorporated into phosphatidylcholine and its choline-containing metabolites determined. Losartan at concentrations which block AT1 receptors did not effect phosphatidylcholine biosynthesis mediated by angiotensin II. In contrast, higher concentrations of losartan inhibited radioactivity incorporated into phosphatidylcholine and its metabolites and this was due to a losartan-mediated reduction in choline uptake. Kinetic studies revealed that the losartan-mediated inhibition of choline uptake was competitive. High concentrations of losartan caused a translocation of CTP:phosphocholine cytidylyltransferase from the cytosolic (inactive) to the membrane (active) fraction likely as a compensatory mechanism for the losartan-mediated reduction in new phosphatidylcholine biosynthesis. Incubation of cells with PD123319, a potent AT2-receptor antagonist, did not block the angiotensin II-mediated stimulation of phosphatidylcholine biosynthesis. The results suggest that angiotensin II stimulates phosphatidylcholine biosynthesis independent of AT1- and AT2-receptor activation and losartan inhibits phosphatidylcholine biosynthesis by reducing choline uptake in H9c2 cells.

Effect of lipoprotein-X on lipid metabolism in rat kidney.

Lipoprotein-X (Lp-X) is found in the plasma of patients with familial lecithin: cholesterol acyltransferase (LCAT) deficiency syndromes. The majority of the patients with this disorder develop progressive glomerulosclerosis. In this study, the effect of Lp-X on lipid metabolism in perfused rat kidney was investigated. Lp-X was isolated from plasma of patients with familial LCAT deficiency by sequential ultracentrifugation and gel filtration column chromatography. Rat kidneys were perfused for 1-2 h with Krebs-Henseleit buffer containing 20 microM [1-(14)C]acetate or 20 microM [Me-3H]choline. In the presence of Lp-X, no significant difference in the incorporation of radioactivity into triglycerides, cholesterol, phosphocholine, CDP-choline and sphingomyelin was observed. However, incorporation of radioactivity into cholesteryl esters and phosphatidylcholine was significantly elevated in Lp-X perfused kidneys. The contents of cholesterol, cholesteryl esters and phosphatidylcholine were also significantly increased in Lp-X perfused kidneys. The increase in lipid content in the Lp-X perfused kidney is attributed to the direct deposition of Lp-X lipids into the organ. The increase in the labelling of cholesteryl esters was attributed to the increase of available substrate (cholesterol) for the acyl-CoA:cholesterol acyltransferase (ACAT) reaction. The increase in phosphatidylcholine labelling was caused by a reduced turnover of the newly synthesized labelled phosphatidylcholine during Lp-X perfusion.

Biosynthesis of plasmenylethanolamine (1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine) in the guinea pig heart.

In this study, the isolated guinea pig heart was pulse-labeled with a precursor of ethanolamine glycerophospholipid, and then chased with the non-radioactive compound for 0-8 h. Labeling with hexadecanol revealed that plasmanylethanolamine was the immediate precursor of plasmenylethanolamine, but a substantial portion of the label was also found in phosphatidylethanolamine. When ethanolamine was used as the precursor, the labeling of plasmenylethanolamine was between 50-65% of the labeling of phosphatidylethanolamine, and this ratio was maintained throughout the perfusion. The ratio of labeling is similar to the ratio of pool sizes of these ethanolamine glycerophospholipid in the heart, which implies that the CDP-ethanolamine pathway is also important for plasmenylethanolamine biosynthesis. The role of diradylglycerol in the synthesis of each ethanolamine glycerophospholipid was also investigated. The ratio of 1-alkenyl-2-acyl glycerol to total diradylglycerol content was 7% in the homogenate and 32% in the microsomes. However, ethanolamine phosphotransferase displayed a distinct selectivity towards 1-alkenyl-2-acyl glycerol. Kinetic studies revealed that the synthesis of phosphatidylethanolamine was inhibited by 1-alkenyl-2-acyl glycerol, but the formation of plasmenylethanolamine was not affected by 1,2-diacylglycerol. In addition, the inhibition of ethanolamine phosphotransferase by 1-alkyl-2-acyl glycerol appears to be an important mechanism for the coordination of plasmenylethanolamine biosynthesis via the desaturase reaction and the CDP-ethanolamine pathway.