Coenzyme A-dependent and -independent acyl transfer between dog heart microsomal phospholipids

We have recently shown that dog heart microsomes catalyze the transfer of acyl groups from the sn-2 position of exogenous phosphatidylcholine to lysophosphatidylethanolamine with strong preference for arachidonate over linoleate (Biochem. Biophys. Res. Commun. 129, 381-388 (1985)). We now report that the addition of 0.5 mM CoA enhances the acyl transfer activity 3-4-fold but reduces the selectivity for arachidonate. Acyl transfer in the absence of CoA exhibits a pH optimum of 7.5-8.5, whereas two pH optima (7.5 and 4.5) are observed in the presence of CoA with transfer activity at pH 4.5 exceeding that of pH 7.5 by 4-5-fold. The plasmalogen (alkenyl) analog of lysophosphatidylethanolamine is an equally effective acyl acceptor in the absence of CoA but less effective in its presence. The microsomal acyl-CoA/lysophosphatidylethanolamine acyltransferase does not favor arachidonate over linoleate. Therefore, transacylation from phosphatidylcholine may account for the high arachidonate content of dog heart microsomal phosphatidylethanolamine and its plasmalogen analog. In fact, acyl transfer from endogenous lipids to 1-[1'-14C]palmitoyl-2-lyso-sn-glycerophosphoethanolamine results in the generation of mostly (over 80%) tetraunsaturated phosphatidylethanolamine. This proportion is reduced by the addition of CoA and, even more, by CoA plus acyl-CoA-generating cofactors. We conclude that in dog heart microsomes, lysophosphatidylethanolamine can be acylated by different mechanisms, of which the CoA-independent transacylase exhibits the greatest acyl selectivity.     

Acylation of dog heart lysophosphatidylserine by transacylase activity

Dog heart microsomes catalyze the transfer of acyl groups from the sn-2 position of phosphatidylcholine (PC) to lysophosphatidylserine (lysoPS) in the presence of coenzyme A (CoA) at pH optima of 4.5-5.0 and 7.5. Acyl transfer activity at acidic pH is about three times higher than at neutral pH. Transacylation of lysoPS by acyl transfer from PC with dog heart microsomes at neutral pH favors arachidonate over linoleate by a factor of 2.1, whereas free linoleic acid is favored by a factor of 3.7 over arachidonic acid for lysoPS acylation in the presence of acyl-CoA-generating cofactors. Considering the location and acyl composition of myocardial PS, it appears that both acyl transfer from PC and utilization of unesterified fatty acids may be involved in the acylation of lysoPS at its sn-2 position.     

Selective acylation of lyso platelet activating factor by arachidonate in human neutrophils

Human polymorphonuclear leukocytes (PMN) incubated with 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-[3H]alkyl-2-acetyl-GPC; platelet activating factor) inactivated the compound by removing the acetyl group and replacing it with a long chain acyl residue. The nature of the acyl group added at the 2-position of the 1-O-[3H]alkyl-2-acyl-GPC formed was examined by argentation chromatography and by reverse phase high performance liquid chromatography. A striking selectivity for arachidonate was observed in the acylation reaction. The major labeled component of the starting material was the 1-O-hexadecyl-linked species; high performance liquid chromatography analysis revealed that 75 to 80% of this component was acylated by arachidonate. Similarly, based on argentation thin layer chromatography, approximately 80% of the total starting material was acylated by tetraenoic acyl residues. The incorporation of 1-O-[3H]alkyl-2-lyso-GPC into 1-O-alkyl-2-acyl-GPC by the PMN was compared; no difference in the acylation pattern was observed with the 2-acetyl and 2-lyso precursors. Thus, activation of the PMN does not appear to be required to elicit the selectivity for arachidonate. When labeled 1-palmitoyl-2-lyso-GPC was compared in the system under the same conditions, it was also preferentially acylated by arachidonate; thus, it is not clear at this time whether or not the selectivity for arachidonate is physiologically limited to platelet activating factor. Our findings suggest a close relationship exists between the metabolism of platelet activating factor and arachidonate in human PMN.     

C20 polyunsaturated fatty acids and phorbol myristate acetate enhance agonist-stimulated synthesis of 1-radyl-2-acetyl-sn-glycero-3-phosphocholine in vascular endothelial cells

This study has investigated the effect of supplementation of vascular endothelial cells with arachidonate and other polyunsaturated fatty acids on the agonist-stimulated synthesis of platelet activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; 1-alkyl-2-acetyl-GPC). Incubation of calf pulmonary artery endothelial cells for 48 h in medium containing 40 microM arachidonate resulted in a 2-3-fold enhancement of [3H]acetate incorporation into 1-radyl-2[3H]acetyl-GPC in response to either bradykinin or calcium ionophore A23187. The effects of arachidonate supplementation were both dose- and time-dependent, requiring a minimum exogenous arachidonate concentration of 2.5 microM and an incubation time of 4-6 h. Eicosapentaenoate and docosahexaenoate also enhanced the synthesis of 1-radyl-2-[3H]acetyl-GPC, but were less potent than arachidonate; alpha-linolenate, linoleate and oleate were without effect. Although not effective as an agonist, phorbol myristate acetate potentiated A23187- and bradykinin-stimulated synthesis of 1-radyl-2-[3H]acetyl-GPC. The effects of arachidonate supplementation were synergistic with potentiation by phorbol myristate acetate. Sphingosine inhibited agonist-stimulated incorporation of [3H]acetate into 1-radyl-2-[3H]acetyl-GPC both in the presence and absence of PMA. Characterization of the radiolabeled material indicated that the primary product was the acyl analogue of PAF (1-acyl-2-acetyl-GPC) rather than PAF. The results from this study suggest that agonist-stimulated synthesis of 1-radyl-2-acetyl-GPC in vascular endothelial cells is modulated both by cellular fatty acyl composition and activation of protein kinase C. Enrichment of vascular endothelial cells with fatty acids, which are mobilized by agonist-stimulated phospholipase A2, may enhance subsequent deacylation of choline phospholipids and, thus, increase synthesis of both 1-acyl-2-acetyl-GPC and PAF.     

Evidence for the reversibility of the acyl-CoA:lysophosphatidylcholine acyltransferase in microsomal preparations from developing safflower (Carthamus tinctorius L.) cotyledons and rat liver.

Acyl exchange between acyl-CoA and position 2 of sn-phosphatidylcholine occurs in the microsomal preparations of developing safflower cotyledons. Evidence is presented to show that the acyl exchange is catalysed by the combined back and forward reactions of an acyl-CoA:lysophosphatidylcholine acyltransferase (EC 2.3.1.23). The back reaction of the enzyme was demonstrated by the stimulation of the acyl exchange with free CoA and by the observation that the added CoA was acylated with acyl groups from position 2 of sn-phosphatidylcholine. Re-acylation of the, endogenously produced, lysophosphatidylcholine with added acyl-CoA occurred with the same specificity as that observed with added palmitoyl lysophosphatidylcholine. A similar acyl exchange, catalysed by an acyl-CoA:lysophosphatidylcholine acyltransferase, occurred in microsomal preparations of rat liver. The enzyme from safflower had a high specificity for oleate and linoleate, whereas arachidonate was the preferred acyl group in the rat liver microsomal preparations. The rate of the back reaction was 3-5% and 0.2-0.4% of the forward reaction in the microsomal preparations of safflower and rat liver respectively. Previous observations, that the acyl exchange in safflower microsomal preparations was stimulated by bovine serum albumin and sn-glycerol 3-phosphate, can now be explained by the lowered acyl-CoA concentrations in the incubation mixture with albumin and in the increase in free CoA in the presence of sn-glycerol 3-phosphate (by rapid acylation of sn-glycerol 3-phosphate with acyl groups from acyl-CoA to yield phosphatidic acid). Bovine serum albumin and sn-glycerol 3-phosphate, therefore, shift the equilibrium in acyl-CoA:lysophosphatidylcholine acyltransferase-catalysed reactions towards the rate-limiting step in the acyl exchange process, namely the removal of acyl groups from phosphatidylcholine. The possible role of the acyl exchange in the transfer of acyl groups between complex lipids is discussed.     

Phospholipid biosynthesis in human platelets. The acylation of lyso-platelet-activating factor.

Acyl-CoA:1-alkyl-sn-glycero-3-phosphocholine acyltransferase of human platelets is membrane-bound, has a pH optimum of 7.5, is insensitive to 1 mM-Mg2+, is inhibited by 1 mM-Ca2+, and is stimulated slightly by 1 mM-EDTA. Maximal formation of 1-alkyl-2-acyl-sn-glycero-3-phosphocholine is observed at 150 microM-1-alkyl-sn-glycero-3-phosphocholine and 20 microM unsaturated fatty acyl-CoA. The transfer of unsaturated fatty acyl groups to 1-alkyl-sn-glycero-3-phosphocholine is 3-14 times slower than to 1-acyl-sn-glycero-3-phosphocholine. The CoA esters of linoleate and arachidonate, two unsaturated fatty acyl groups commonly found in platelet phospholipids, are the preferred fatty acyl group donors.     

ACBP and cholesterol differentially alter fatty acyl CoA utilization by microsomal ACAT

Microsomal acyl CoA:cholesterol acyltransferase (ACAT) is stimulated in vitro and/or in intact cells by proteins that bind and transfer both substrates, cholesterol, and fatty acyl CoA. To resolve the role of fatty acyl CoA binding independent of cholesterol binding/transfer, a protein that exclusively binds fatty acyl CoA (acyl CoA binding protein, ACBP) was compared. ACBP contains an endoplasmic reticulum retention motif and significantly colocalized with acyl-CoA cholesteryl acyltransferase 2 (ACAT2) and endoplasmic reticulum markers in L-cell fibroblasts and hepatoma cells, respectively. In the presence of exogenous cholesterol, ACAT was stimulated in the order: ACBP > sterol carrier protein-2 (SCP-2) > liver fatty acid binding protein (L-FABP). Stimulation was in the same order as the relative affinities of the proteins for fatty acyl CoA. In contrast, in the absence of exogenous cholesterol, these proteins inhibited microsomal ACAT, but in the same order: ACBP > SCP-2 > L-FABP. The extracellular protein BSA stimulated microsomal ACAT regardless of the presence or absence of exogenous cholesterol. Thus, ACBP was the most potent intracellular fatty acyl CoA binding protein in differentially modulating the activity of microsomal ACAT to form cholesteryl esters independent of cholesterol binding/transfer ability.     

Coenzyme A-mediated transacylation of sn-2 fatty acids from phosphatidylcholine in rat lung microsomes

Evidence was obtained for a CoA-dependent transfer of linoleate from rat lung microsomal phosphatidylcholine to lysophosphatidylethanolamine without the intervention of a Ca2+-requiring phospholipase A2 activity and ATP. To study this CoA-mediated transacylation process, microsomes were prepared in which the endogenous phosphatidylcholine was labeled by protein-catalyzed exchange with phosphatidylcholines containing labeled fatty acids in the sn-2-position. The apparent Km for CoA in the transfer of arachidonate from phosphatidylcholine to 1-acyllysophosphatidylethanolamine was 1.5 microM. At saturating lysophosphatidylethanolamine concentrations, the transacylation was linear with the amount of microsomal protein, i.e., a fixed percentage of the labeled fatty acid was transferred independent of the amount of microsomal protein. A maximal transfer of 12.2% for arachidonate and 2.0% for linoleate from the respective phosphatidylcholines to lysophosphatidylethanolamine was observed in 30 min. With 1-acyl-2-[1-14C]arachidonoylphosphatidylcholine as acyl donor, lysophosphatidylethanolamine was the best acceptor followed by lysophosphatidylglycerol and lysophosphatidylserine. Lysophosphatidate barely functioned as acceptor. These data provide further evidence for the widespread occurrence of CoA-mediated transacylation reactions. The arachidonate transacylation from phosphatidylcholine to other phospholipids in lung tissue may contribute to the low level of arachidonate in pulmonary phosphatidylcholine.     

Differential metabolism of exogenous and endogenous arachidonic acid in human neutrophils.

Leukotrienes can be produced by cooperative interactions between cells in which, for example, arachidonate derived from one cell is oxidized to leukotriene A(4) (LTA(4)) by another and this can then be exported for conversion to LTB(4) or cysteinyl leukotrienes (cys-LTs) by yet another. Neutrophils do not contain LTC(4) synthase but are known to cooperate with endothelial cells or platelets (which do have this enzyme) to generate cys-LTs. Stimulation of human neutrophils perfusing isolated rabbit hearts resulted in production of cys-LTs, whereas these were not seen with perfused hearts alone or isolated neutrophils. In addition, the stimulated, neutrophil-perfused hearts generated much greater amounts of total LTA(4) products, suggesting that the hearts were supplying arachidonate to the neutrophils and, in addition, that this externally derived arachidonate was preferentially used for exported LTA(4) that could be metabolized to cys-LTs by the coronary endothelium. Stable isotope-labeled arachidonate and electrospray tandem mass spectrometry were used to differentially follow metabolism of exogenous and endogenous arachidonate. Isolated, adherent neutrophils at low concentrations (to minimize transcellular metabolism between them) were shown to generate higher proportions of nonenzymatic LTA(4) products from exogenous arachidonate (deuterium-labeled) than from endogenous (unlabeled) sources. The endogenous arachidonate, on the other hand, was preferentially used for conversion to LTB(4) by the LTA(4) hydrolase. This result was not because of saturation of the LTA(4) hydrolase, because it occurred at widely differing concentrations of exogenous arachidonate. Finally, in the presence of platelets (which contain LTC(4) synthase), the LTA(4) synthesized from exogenous deuterium-labeled arachidonate was converted to cys-LTs to a greater degree than that from endogenous sources. These experiments suggest that exogenous arachidonate is preferentially converted to LTA(4) for export (not intracellular conversion) and raises the likelihood that there are different intracellular pathways for arachidonate metabolism.     

Role of exogenous arachidonate in the biosynthesis of 5-lipoxygenase metabolites of arachidonic acid by human polymorphonuclear leukocytes induced by the calcium ionophore A23187

By using high performance liquid chromatography with simultaneous detection of unlabeled and radiolabeled product of lipoxygenase oxidation of arachidonic acid, the mechanism of exogenous arachidonate involvement in leukotriene synthesis in human neutrophils induced by the Ca2+ ionophore A23187 was studied. It was found that after addition of labeled arachidonate the specific radioactivity of the reaction product (leukotriene B4) does not change on a time scale, i.e., the free arachidonic acid exchange between the cell and extracellular space is a very rapid process. Exogenous arachidonic acid was found to be the substrate of the lipoxygenase reaction which acts in parallel with the endogenous one. The dependence of specific radioactivity of leukotriene B4 in added arachidonic acid concentration is described by a hyperbolic curve with saturation. When exogenous arachidonate is used at a concentration of 10.8 +/- 3.9 microM, that of intracellular arachidonic acid increases twofold at the expense of the exogenously added acid.     

Arachidonate cannot be released directly from diacyl-sn-glycero-3-phosphocholine in thrombin-stimulated platelets.

The origin of the arachidonate released from platelets on stimulation with thrombin was investigated by comparing the specific activities of released arachidonate and of arachidonoyl-containing phospholipids using rat platelets prelabelled with arachidonate. Quantification of the released arachidonate was determined in the presence of BW 755 C, a dual cyclo-oxygenase/lipoxygenase inhibitor, which was found not to modify the arachidonate mobilization between the platelet phospholipids. The phospholipid molecular species were analysed by h.p.l.c. of diradylglycerol benzoate derivatives of diacyl, alkylacyl and alkenylacyl classes. The labelled/unlabelled arachidonate ratio varied greatly in the phospholipids depending on whether an ether or acyl bond was present in sn-1 position of the glycerol, on the length and degree of unsaturation of this fatty chain and on the polar head group. Between 15 s and 5 min of stimulation by thrombin, the released arachidonate kept a constant specific activity which was considerably lower than the specific activity of diacyl-GPC. The specific activity of the released arachidonate was intermediate between the specific activities of the 16:0-20:4 and 18:0-20:4 species of diacyl-GPI and diacyl-GPE, and corresponded to the mean specific activity of alkylacyl-GPC. The data indicate that the released arachidonate cannot come directly from diacyl-GPC, and that two phospholipids in particular can act as direct precursors of the released arachidonate. These are (1) the alkylacyl-GPC and (2) the diacyl-GPE whose hydrolysis would induce an arachidonate transfer from diacyl-GPC.     

Properties of canine myocardial phosphatidylethanolamine N-acyltransferase

Dog heart contains a membrane bound N-acyltransferase (transacylase) which transfers acyl groups from the sn-1 position of membrane phospholipids to the amino group of ethanolamine phospholipids in the presence of millimolar Ca2+ concentrations. Using crude membrane preparations, we found this N-acyltransferase activity to be heat sensitive and inhibited by sulfhydryl reagents. Pretreatment of a membrane fraction with trypsin reduced N-acyltransferase activity to 60% while pretreatment with trypsin and Triton X-100 together reduced it to 30% of the control value. At pH 8.0 both Sr2+ and Mn2+ could fully substitute for Ca2+ with respect to optimum ion concentration and molecular species of the product formed in dog heart membranes from endogenous substrates. Ba2+ was equally effective in achieving N-acylation of ethanolamine phospholipids while other divalent cations were less effective or ineffective. The reaction exhibited a pH optimum of 8.5 to 9.0 with both Ca2+ and Sr2+ while Mn2+ precipitated above pH 8.0 resulting in decreased N-acylation activity. Both phosphatidylcholine and 1-acyl lysophosphatidylcholine could serve as acyl donors. Triton X-100 at a concentration of 0.1% stimulated acyl transfer from exogenous phosphatidylcholine but inhibited acyl transfer from lysophosphatidylcholine.     

Some unique features of the metabolic conversion of platelet-activating factor (AGEPC) to alkyl acyl PC by washed rabbit platelets

Recently several synthetic analogs of 1-0-alkyl-2-acetyl-sn-glycero-3-phosphocholine (AGEPC; platelet-activating factor) were characterized as selective inhibitors of this agonist's effects on rabbit platelets (Tokumura, A., Homma, H., and Hanahan, D. J. (1985) J. Biol. Chem. 260, 12710-12714). In this current investigation, these studies have been extended to include a further inquiry into the biochemical nature of the metabolic inactivation of AGEPC in rabbit platelets, and the effect of these analogs on this process. Two of the latter components (U66985 and CV3988), which blocked AGEPC biological activity on rabbit platelets, also blocked the metabolism of this agonist. The metabolic conversion of AGEPC to alkyl acyl PC was inhibited nearly sevenfold by the most potent analog, U66985. Those analogs with low (U68043) or no biological inhibitory activity (lysoGEPC) had marginal effects on the metabolism of AGEPC. The effects of these compounds on the metabolism of AGEPC was not simply due to competitive inhibition. In platelets which had been pretreated with AGEPC in absence of extracellular Ca2+ (desensitized) and washed, the metabolic conversion of AGEPC to alkyl acyl PC was actually enhanced. This enhanced metabolic inactivation of AGEPC was also observed upon the treatment of the cells with thrombin, collagen, or ionophore A23187, indicating that the metabolism of AGEPC in platelets was enhanced not only by AGEPC itself but by other agonists as well. Nearly 85% of the fatty acyl residues was arachidonate in the alkyl acyl PC derived from AGEPC. This specific acylation with arachidonate was observed in the presence and absence of the inhibitor and in desensitized cells, indicating that selectivity for arachidonate is not dependent on the enhancement of the metabolism of AGEPC. The alkyl acyl PC found in the cells treated with thrombin, collagen, or A23187 was also predominantly alkyl arachidonoyl PC. Thus it has been shown that the inactivation of AGEPC by its conversion to alkyl acyl PC by rabbit platelets is enhanced by this agonist itself and that excess amounts of AGEPC could be further inactivated by the enhanced capacity of the metabolism process.     

Reacylation of platelet activating factor with eicosapentaenoic acid in fish-oil-enriched monkey neutrophils

Platelet activating factor (PAF) is rapidly metabolized via a deacetylation: reacylation pathway which shows striking specificity for arachidonate at the sn-2 position of the 1-O-alkyl-2-acyl-GPC thus formed. We have now examined the effects of a diet enriched in fish oils on the metabolism of PAF and specificity for arachidonate in the reacylation reaction. [3H]PAF was incubated for various lengths of time with neutrophils from monkeys fed a control diet or one enriched in fish oils. The [3H]PAF added to the cell suspension was rapidly converted to 1-O-alkyl-2-acyl-GPC. Reverse-phase HPLC analysis of the acyl chains added at the sn-2 position revealed that arachidonate was the major fatty acid incorporated into the 1-O-alkyl-2-acyl-GPC formed by neutrophils from monkeys on the control diet. In contrast, both 1-O-alkyl-2-arachidonoyl-GPC and 1-O-alkyl-2-eicosapentaenoyl-GPC were formed by the fish-oil-enriched neutrophils. We also report on the fatty acid composition of neutrophil phospholipids during such a diet.     

Effects of dietary vitamin E on the biosynthesis of 5-lipoxygenase products by rat polymorphonuclear leukocytes (PMNL)

Activation of polymorphonuclear neutrophils (PMNL) leads to the release of arachidonate from cellular phospholipids via a phospholipase A2, and conversion of products of the 5-lipoxygenase pathway. Evidence to date indicates the dietary vitamin E ((R,R,R)-alpha-tocopherol) can influence both cyclooxygenase and phospholipase A2 activities and that the effect of this vitamin is cell/tissue specific. The present study was undertaken in order to examine the effects of varying dietary tocopherol on PMNL tocopherol content and 5-lipoxygenase product profile using the ionophore A23187 as stimulant in the presence and absence of exogenous arachidonate. Feeding semi-purified diets containing 0, 30 or 3000 ppm of (R,R,R)-alpha-tocopherol acetate to weanling rats for 17 weeks resulted in a dose-related enrichment of PMNL tocopherol. Stimulation of PMNL elicited a significant and rapid loss of tocopherol. When PMNL were stimulated with A23187 alone, the synthesis of 5-HETE, LTB4 and 19-hydroxy-LTB4 was decreased in proportion to increasing dietary tocopherol concentrations. However, when exogenous arachidonate was provided with A23187, intermediate amounts of dietary tocopherol (30 ppm) still suppressed the formation of 5-lipoxygenase products, but high doses (3000 ppm) did not have any additional inhibitory effect. This differential response to high concentrations of vitamin E in the presence and absence of exogenous arachidonate highly suggest that at these concentrations, tocopherol may act principally at the level of substrate release whereas at lower concentrations, 5-lipoxygenase is inhibited. Data from this study demonstrated that attenuation of the formation of 5-lipoxygenase products in PMNL can be achieved by dietary vitamin E enrichment.     

Inhibition of arachidonic acid incorporation into erythrocyte phospholipids by peracetic acid and other peroxides. Role of arachidonoyl-CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase

To explore possible mechanisms of the arachidonic acid deficiency of the red blood cell membrane in alcoholics, we compared the effect of ethanol and its oxidized products, acetaldehyde and peracetic acid, with other peroxides on the accumulation of [14C]arachidonate into RBC membrane lipids in vitro. Incubation of erythrocytes with 50 mM ethanol or 3 mM acetaldehyde had no effect on arachidonate incorporation. Pretreatment of erythrocytes with 10 mM hydrogen peroxide, 0.1 mM cumene hydroperoxide or 0.1 mM t-butyl hydroperoxide had little effect on [14C]arachidonate incorporation in the absence of azide. However, pretreatment of cells with N-ethylmaleimide, 0.1 mM peracetic acid or performic acid, with or without azide, inhibited arachidonate incorporation into phospholipids but not neutral lipids. In chase experiments, peracetate also inhibited transfer of arachidonate from neutral lipids to phospholipids. To investigate a possible site of this inhibition of arachidonate transfer into phospholipids by percarboxylic acids, we assayed a repair enzyme, arachidonoyl CoA: 1-palmitoyl-sn-glycero-3-phosphocholine acyl transferase (EC 2.3.1.23). As in intact cells, phospholipid biosynthesis was inhibited more by N-ethylmalemide and peracetic acid than by hydrogen peroxide, cumene hydroperoxide, and t-butyl hydroperoxide. Peracetic acid was the only active inhibitor among ethanol and its oxidized products studied and may deserve further examination in ethanol toxicity.     

Prostaglandin release from in vitro guinea-pig gallbladder

In order to study prostaglandin release from guinea pig gallbladder, full thickness tissue sections were incubated for one hour in Krebs solution. Extraction and two dimensional chromatography of incubation media obtained in the presence of radio-labelled arachidonic acid demonstrated the presence of PGE2, PGF2 alpha, 6-keto-PGF1 alpha and thromboxane B2. These results were supported by radioimmunoassay of incubations conducted in the absence of exogenous arachidonate and in the presence of varying concentrations of unlabelled exogenous arachidonate. The previously reported predominance of PGE2 was only seen at high concentrations of exogenous arachidonate.     

Mass quantitation of agonist-induced arachidonate release and icosanoid production in a fibrosarcoma cell line. Effect of time of agonist stimulation, amount of cellular arachidonate, and type of agonist

The mass of total arachidonate released from phospholipids upon agonist stimulation of the cell and the fraction of released arachidonate which is converted to icosanoids are two parameters of arachidonate metabolism which have been difficult to quantitate because the mass of arachidonate released upon cell stimulation is very low. We have been able to quantitate both of these parameters under a variety of experimental conditions using a unique essential fatty acid-deficient mouse fibrosarcoma cell line (EFD-1), which when repleted with arachidonate, produces prostaglandin E2 (PGE2). Because there is no endogenous pool of arachidonate in these cells, the specific activity of exogenous arachidonate does not change upon incorporation into cells, an advantage which permits mass determination of very small quantities of arachidonate directly from radioactive counts. EFD-1 cells were incubated with various concentrations of [14C]arachidonate (for release studies) or unlabeled arachidonate (for PGE2 radioimmunoassays) for 24 h and then stimulated with bradykinin. The time courses for arachidonate release and PGE2 production demonstrated that free arachidonate was rapidly converted to PGE2 with plateau levels attained for both parameters within 240 s of agonist exposure for 2 microM and for 10 microM arachidonate-repleted cultures. There was a linear relationship (r = 0.94) between the mass of arachidonate in the cell and the mass of arachidonate released upon stimulation, up to a cellular concentration of 11 nmol of arachidonate/10(6) cells, a concentration 10-20% above normal for the parent mouse fibrosarcoma cell line (HSDM1C1) which is not essential fatty acid-deficient. Importantly, the percent of released arachidonate which was converted to PGE2 decreased from 90 to 15% with increasing concentrations of cellular arachidonate, because PGE2 production plateaued at greater than or equal to 6 nmol of arachidonate/10(6) cells, but total arachidonate release continued to rise. Finally, we demonstrated that agonist stimulation with thrombin, A23187, and bradykinin all showed the same percent conversion of released arachidonate to PGE2, implying that the determination of this fraction is not a function of the mechanism of release. These studies with our unique cell line indicate that, when the concentration of arachidonate in the cell is not elevated above amounts normally found in our HSDM1C1 cell line, released arachidonate is rapidly and almost quantitatively converted to PGE2, independent of the agonist used to stimulate the cells.(ABSTRACT TRUNCATED AT 400 WORDS)     

Serum Albumin Washing Specifically Enhances Arachidonate Incorporation into Synaptosomal Phosphatidylinositols

Arachidonate incorporation into synaptosomal phospholipids was shown to be affected by factors including the procedure for preparation of the membrane fractions and preincubation of synaptosomes prior to assay of incorporation of arachidonate into both phosphatidylcholine (PC) and phosphatidylinositol (PI). However, the inhibition toward incorporation into PIs, but not PCs, was fully reversed when the membranes were washed with bovine serum albumin. A twofold increase in arachidonate incorporation into PIs was also observed when freshly prepared synaptosomes were washed with serum albumin immediately before assay of incorporation activity. The inhibitory action is thought to be due to an increase in polyunsaturated fatty acids and/or their oxidation products which may then elicit a special effect on the acyltransferase responsible for transferring arachidonate into phosphatidylinositols. The differences in fatty acid uptake and response to serum albumin also suggest the presence of different acyltransferase for acyl transfer to PIs and PCs.     

Biochemical actions of vasoactive peptide hormones. Time-synchronized activation of lipolysis and decreased fatty-acid release by bradykinin and angiotensin in the perfused rabbit kidney.

Bradykinin and angiotensin administered to the isolated perfused rabbit kidney activate two sequential processes: (1) a selective release of the prostaglandin precursor arachidonate with concomitant partial conversion of the arachidonate into prostaglandin E2; (2) activation of a process that leads to decreased release of all fatty acids in the perfusate. There is a time lag of approx. 1 min between the initial activation of the arachidonate-specific deacylation reaction that is coupled to prostaglandin generation, and the subsequent decrease in the release of all fatty acids. This synchronized cycle provides for instant generation of required amounts of prostaglandins and at the same time serves to conserve cellular arachidonate.