1-O-alkyl-2-arachidonoyl-sn-glycero-3-phosphocholine. A common source of platelet-activating factor and arachidonate in human polymorphonuclear leukocytes

1-O-[3H]Alkyl-2-lyso-sn-glycero-3-phosphocholine (1-O-[3H]alkyl-2-lyso-GPC) incubated with human polymorphonuclear leukocytes (PMN) for 30 min is metabolized to 1-O-alkyl-2-acyl-GPC containing greater than 80% arachidonate at the 2 position (Chilton, F. H., O'Flaherty, J. T., Ellis, J. M., Swendsen, C. L., and Wykle, R. L. (1983) J. Biol. Chem. 258, 7268-7271). PMN containing 1-O-[3H]alkyl-2-arachidonoyl-GPC incorporated into their cellular phospholipids in this manner were stimulated with Ca2+ ionophore (A23187). Within 5 min after stimulation, 14%, 7%, and 7% of the total 1-O-[3H]alkyl-2-arachidonoyl-GPC in the cells had been converted to 1-O-[3H]alkyl-2-acetyl-GPC (platelet-activating factor), 1-O-[3H]alkyl-2-lyso-GPC, and 3H-labeled neutral lipid, respectively. Stimulation by opsonized zymosan yielded similar results. In related studies, cells were labeled with 1-O-hexadecyl-2-arachidonoyl-GPC containing a [methyl-14C] choline moiety. The nature of the long-chain acyl residues in the sn-2 position of the labeled 1-O-hexadecyl-2-acyl-GPC remaining after stimulation with A23187 was examined. Analysis by high-performance liquid chromatography using synthetic 1-O-hexadecyl-2-acyl-GPC standards indicated there is a time-dependent loss of arachidonate from the 2 position of the labeled 1-O-hexadecyl-2-arachidonoyl-GPC followed by reacylation by other fatty acids (primarily linoleic and oleic). This shift in the acylation pattern exhibited after Ca2+ ionophore stimulation was further examined in PMN preincubated with A23187 and subsequently incubated with labeled 1-O-alkyl-2-lyso-GPC; the stimulated cells produced 1-O-[3H]alkyl-2-acetyl-GPC (greater than 15% of total label) and 1-O-[3H]alkyl-2-acyl-GPC containing linoleic acid and oleic acid, rather than arachidonic acid in the sn-2 position. The findings demonstrate that upon stimulation of PMN, 1-O-alkyl-2-arachidonoyl-GPC can yield arachidonate and 1-O-alkyl-2-lyso-GPC; the 1-O-alkyl-2-lyso-GPC formed may be acetylated producing platelet-activating factor or reacylated with fatty acyl residues other than arachidonate.     

Biosynthesis of platelet activating factor in rabbit polymorphonuclear neutrophils

The synthesis of platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) was studied in rabbit peritoneal polymorphonuclear neutrophils. Upon stimulation with ionophore A23187 and Ca2+, these cells are able to incorporate [3H]acetate or 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine into platelet activating factor. Under the same incubation conditions, however, the cells do not synthesize platelet activating factor from [14C]hexadecanol, which is an immediate precursor of O-alkyl chains in the de novo pathway. In the absence of ionophore, [14C] hexadecanol is incorporated into 1-O-alkyl-2-acyl-sn-glycerol-3-phosphate and subsequently into the 1-O-alkyl-linked choline and ethanolamine phosphoglyceride pools. However, in the presence of ionophore, [14C] hexadecanol incorporation is limited to phosphatidic acid, perhaps due to the inhibition of choline phosphotransferase. These findings provide strong evidence that platelet activating factor is synthesized by a deacylation-reacylation mechanism. Upon stimulation, these cells can utilize both plausible substrates of this pathway to make the final product, while under the same conditions it appears that a key step of the de novo pathway is inhibited.     

Biosynthesis and metabolism of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine in rat glomerular mesangial cells

The ability of rat mesangial cells to synthesize 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (1-O-alkyl-2-acetyl-GPC), also known as platelet activating factor (PAF), was studied in mesangial cell cultures originating from isolated rat glomeruli. In response to the phospholipase A2 agonist A23187 mesangial cells synthesized PAF primarily via an acetyltransferase utilizing either [3H]lyso-PAF or [3H]acetate/[3H]acetyl-CoA substrates. The major PAF species synthesized was 1-O-hexadecyl-2-acetyl-GPC. PAF was also synthesized from 1-O-[3H]alkyl-2-acetyl-sn-3-glycerol, indicating the presence of a CDP-cholinephosphotransferase. Mesangial cells incorporated [3H]lyso-PAF to 1-O-[3H]alkyl-2-acyl-GPC. Subsequent stimulation with A23187 (2 microM) resulted in formation and release of [3H]PAF following 3 h, and this was associated with concomitant decrements in intracellular 1-O-[3H]alkyl-2-acyl-GPC and [3H]lyso-PAF levels, indicating a precursor-product relationship among these alkyl ether lipids. Mesangial cells rapidly converted exogenous [3H]PAF to [3H]lyso-PAF and 1-O-[3H]alkyl-2-acyl-GPC, and this process was inhibited by diisopropyl fluorophosphate (10 microM). The demonstration of PAF activation-inactivation pathways in mesangial cells may be of importance in regulating their function and in glomerular injury.     

Metabolism of platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-alkyl-2-acetyl-sn-glycerol by human endothelial cells

The metabolism of platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine) and 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol was studied in cultures of human umbilical vein endothelial cells. Human endothelial cells deacetylated 1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine to the corresponding lyso compound (1-[1,2-3H]alkyl-2-lyso-sn-glycerol-3-phosphocholine) and a portion was converted to 1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine. Lyso platelet activating factor (lyso-PAF) (1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine) was detected in the media very early during the incubation and the amount remained higher than the level of the lyso product observed in the cells. Cellular levels of 1-[1,2-3H]alkyl-2-lyso-sn-glycero-3-phosphocholine were significantly higher than the acylated product (1-[1,2-3H]alkyl-2-acyl(long-chain)-sn-glycero-3-phosphocholine) at all times during the 60-min incubation period, which suggests that the ratio of acetylhydrolase to acyltransferase activities is greater in endothelial cells than in most other cells. When endothelial cells were incubated with 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol, a known precursor of PAF, 1-[1,2-3H]alkyl-sn-glycerol was the major metabolite formed (greater than 95% of the 3H-labeled metabolites during 20- and 40-min incubations). At least a portion of the acetate was removed from 1-[1,2-3H]alkyl-2-acetyl-sn-glycerol by a hydrolytic factor released from the endothelial cells into the medium during the incubations. Only negligible amounts of the total cellular radioactivity (0.2%) was incorporated into platelet activating factor (1-[1,2-3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine); therefore, it is unlikely that the previously observed hypotensive activity of 1-alkyl-2-acetyl-sn-glycerols can be explained on the basis of the conversion to platelet activating factor (1-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by endothelial cells. Results of this investigation indicate that endothelial cells play an important role in PAF catabolism. Undoubtedly, the endothelium is important in the regulation of PAF levels in the vascular system.     

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.     

Biosynthesis of platelet-activating factor by two-cell mouse embryos.

Incubation of two-cell mouse embryos with a range of radiolabelled compounds resulted in the incorporation of label into platelet-activating factor (PAF; 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) in the culture media. The demonstration that known precursors ([1-14C]hexadecanol, [1-3H]hexadecanol, 1-O-[alkyl-1'2'-3H]lyso-PAF, 1-O-[alkyl-1'2'-3H]acetyl-glycerol and [methyl-3H]choline chloride) were incorporated into PAF showed that embryo-derived PAF biosynthesis occurred via pathways present in other PAF-producing cells. The enzyme responsible for the formation of the ether linkage of the PAF molecule, alkyl-dihydroxyacetone-phosphate synthase, was present in the preimplantation embryo as [1-3H]hexadecanol was incorporated into PAF. Incorporation of label from alkylacetyl-glycerol and choline chloride into lyso-PAF was also observed, suggesting a role for lyso-PAF in the metabolism of embryo-derived PAF. Incubation of embryos with each of three [14C]carbohydrate energy substrates resulted in the incorporation of label into PAF in culture media, indicating that the composition of embryo culture media is important in the synthesis of PAF precursors. Incorporation of label from [2-14C]pyruvate was greatest and is consistent with the suggestion that pyruvate is the major energy source at the two-cell stage of development. L-[U-14C]Lactate was also incorporated into embryo-derived PAF, but the mean amount incorporated relative to the concentration of labelled substrate in the medium was 40 times less. The incorporation of D-[U-14C]glucose into PAF was 2405 times less than that from pyruvate, relative to the concentration in the medium.     

Metabolism of platelet-activating factor in primary cultured adult rat hepatocytes by a new pathway involving phospholipase C and alkyl monooxygenase

The metabolic fate of 1-O-[3H]alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF-acether) upon interaction with primary cultured adult rat hepatocytes was investigated. [3H]PAF-acether was transformed time-dependently into [3H]lyso-PAF-acether, 1-O-[3H]alkylglycerol and finally converted to 3H-labeled fatty aldehyde. 1-O-[3H]Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC) was formed after a long incubation time and with a smaller amount compared with that formed in platelets and neutrophils. When lipids from cells, cell surfaces and incubation medium were analyzed separately, most of the transformed products of [3H]PAF-acether remained in the cells. When 1-O-[3H]alkyl-2-lyso-sn-glycero-3-phosphocholine was incubated with hepatocytes, it was mainly converted into 1-O-[3H]alkylglycerol. 3H-labeled fatty aldehyde and [3H]alkylacyl-GPC were also found. Hepatocytes metabolized slowly from 1-O-[1-14C]hexadecylglycerol to 3H-labeled fatty aldehyde and 3H-labeled phospholipid. These findings suggest that cultured hepatocytes mainly catabolize exogeneous PAF-acether by removing the acetyl residue and the polar head group and, finally, by cleaving an ether bond. The deacetylation-reacylation step, which is important in platelets and neutrophils, was not shown to be a main metabolic pathway of PAF-acether in cultured hepatocytes.     

Metabolism of long-chain alcohols in cell suspension cultures of soya and rape

Heterotrophic cell suspension cultures of soya (Glycine max) and photomixotrophic cell suspension cultures of rape (Brassica napus) were incubated with cis-9-[1-¹⁴C]octadecenol for 3–48 h. It was found that under aerobic conditions large proportions of the alcohol are oxidized to oleic acid, which is incorporated predominantly into phospholipids, whereas up to 30% of the substrate is esterified to wax esters. This is true for both the heterotrophic and the photomixotrophic cell suspension cultures, but the metabolic rates are much higher in the latter. Under anaerobic conditions only small proportions of the radioactively labeled alcohol are oxidized to oleic acid, whereas a major portion of the alcohol is esterified to wax esters both in heterotrophic and photomixotrophic cultures. Incubations of homogenates of photomixotrophic rape cells with labeled cis-9-octadecenol showed that pH 6 is optimum for the formation of wax esters. This monounsaturated alcohol is preferred as a substrate over saturated longchain alcohols, whereas short-chain alcohols, cholesterol, and glycerol are not acylated. Incubations of an enzyme concentrate from a homogenate of rape cells with unlabeled cis-9-octadecenol and [1-¹⁴C]oleic acid, or [1-¹⁴C]stearoyl-CoA, or di[1-¹⁴C]palmitoyl-sn-glycero-3-phosphocholine showed that acylation of the longchain alcohol proceeds predominantly through acyl-CoA. Direct esterification of the alcohol with fatty acid as well as acyl transfer from diacylglycerophosphocholine could be demonstrated to occur to a much smaller extent.     

A novel mechanism of diglyceride formation. 12-O-tetradecanoylphorbol-13-acetate stimulates the cyclic breakdown and resynthesis of phosphatidylcholine

12-O-Tetradecanoylphorbol-13-acetate (TPA) treatment of Madin-Darby canine kidney cells resulted in an increased incorporation of 32Pi and [methyl-3H]choline into choline-containing phosphoglycerides (PC). In pulse-chase experiments, TPA treatment caused an increased release of [methyl-3H]choline from the PC fraction of prelabeled cells. When cells were prelabeled with [3H]arachidonic acid and [14C]palmitic acid, TPA treatment resulted in an increased synthesis of 14C, 3H-diglycerides. Further studies were done to determine the relationship between PC breakdown and diglyceride synthesis. Cells were preincubated with ether-linked 1-O-[3H]hexadecyl-2-lyso-sn-glycero-3-phosphocholine which was acylated to form 1-O-[3H]hexadecyl-2-acyl-sn-glycero-3-phosphocholine. Subsequent treatment of these cells with TPA resulted in an increased synthesis of 1-O-[3H]hexadecyl-2-acyl-sn-glycerol compared to cells not stimulated with TPA. These findings demonstrate that TPA stimulates PC turnover in Madin-Darby canine kidney cells and provide evidence for a novel mechanism of diglyceride formation.     

Biosynthetic preparation of radioactively labelled ethanolamine plasmalogen (1-O-[1'-14C]octadec-1'-enyl-2-acyl-sn-glycero-3-phosphoethanola mine) using a protozoan cell culture

Ethanolamine plasmalogen radiolabelled mainly in the O-alkenyl moiety was prepared from cell suspension cultures of the flagellate Leishmania donovani previously incubated with [1-14C]octadecanol over one growth period. The optimal concentration of [1-14C]octadecanol for labelling was shown to be 1 microM, when 60% of total lipid radioactivity appeared in the 1,2-diradyl-sn-glycero-3-phosphoethanolamine fraction, with an overall yield of approx. 35%. Analysis of this fraction revealed that 93% of the label was present in O-octa-dec-1-enyl, 3% in O-alkyl and 4% in acyl moieties. A specific radioactivity of approx. 14 mCi/mmol was determined. Raising the culture medium concentration of [1-14C]octadecanol to 2 microM yielded a product with a specific radioactivity of 25 mCi/mmol.     

Metabolism of ether glycolipids with potentially antineoplastic activity by Ehrlich ascites tumor cells

Ehrlich ascites tumor cells were incubated in vitro with rac-1-O-[1'-14C]octadecyl-2-O-methylglycero-3-beta-D-glucopyranosi de for 24 h. The potentially antineoplastic ether glycolipid was rapidly metabolized by the cells to radioactive 1-O-octadecyl-2-O-methylglycerol (70 pmol/10(6) cells per h) and further acylated to 1-O-octadecyl-2-O-methyl-3-acylglycerols. Incubation of Ehrlich ascites cells with synthetic rac-1-O-[1'-14C]octadecyl-2-O-methyl-3-palmitoylglycerol showed that this metabolite is reconverted by deacylation to 14C-labeled 1-O-octadecyl-2-O-methylglycerol. The latter compound or a metabolite derived therefrom may be the 'toxic principle' of both the ether glyceroglycolipids and ether glycerophospholipids having a 1-O-alkyl-2-O-methylglyceryl moiety, as suggested by Unger et al. (J. Natl. Cancer Inst. 78 (1987) 219-222).     

Metabolism of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) by human promyelocytic leukemic HL60 cells. Stimulated expression of phospholipase A2 and acetyltransferase requires differentiation

Human promyelocytic leukemia (HL60) cells can be induced to differentiate into mature granulocytes by exposure to dimethyl sulfoxide. The addition of N-formylMet-Leu-Phe or the Ca2+ ionophore A23187 to these differentiated cells generated 15-30 pmol of platelet-activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (alkylacetyl-GPC)/10(6) cells as quantified by platelet aggregation assays. Under identical conditions, uninduced cells produced little alkylacetyl-GPC. Upon the addition of ionophore A23187, differentiated cells, and not uninduced ones, released [14C]arachidonate from prelabeled phospholipids including ether-linked phosphatidylcholines, formed both 3H-labeled 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine (alkyllyso-GPC) and [3H]alkylacetyl-GPC from endogenous 3H-labeled 1-O-alkyl-2-(long chain) acyl-sn-glycero-3-phosphocholine (alkylacyl-GPC), and incorporated exogenously added [3H]acetate or [3H]alkyllyso-GPC into alkylacetyl-GPC. These results are suggestive that both phospholipase A2 and acetyltransferase activities are involved in alkylacetyl-GPC biosynthesis by HL60 cells and that these activities appear during differentiation. However, when measured in cell extracts, the activities of phospholipase A2 and acetyltransferase of uninduced cells were virtually indistinguishable from those of differentiated cells. Uninduced cells exhibited enhanced incorporation of [3H]alkyllyso-GPC or [3H]alkylacetyl-GPC into alkylacyl-GPC and of [14C]arachidonate and [14C]oleate into various phospholipids including phosphatidylcholine. However, such enhanced expression of acylation reactions could not account for the lack of accumulation of arachidonate or of alkylacetyl-GPC by uninduced cells. Furthermore, analyses of phospholipid classes by phosphorus determination showed no significant alterations in phospholipid composition of HL60 cells during differentiation. Together these data are suggestive that mechanisms regulating the activation of phospholipase A2 and acetyltransferase activities are defective in uninduced cells and that an increased concentration of cytosolic free Ca2+ alone is not a sufficient requirement for these mechanisms.     

Partial purification and characterization of 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine:acetyl-CoA acetyltransferase from rat spleen.

The enzyme 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine: acetyl-CoA acetyltransferase (EC 2.3.1.67) was purified from rat spleen approx. 1500-fold in 1.6% yield. The specific activity of the purified enzyme was 0.317 +/- 0.089 mumol/min per mg of protein (mean +/- S.D., n = 6). The Km for the substrate acetyl-CoA was 137 +/- 13 microM and the pH optimum was about 8. Incubation of the purified enzyme was 1-O-[3H]octadecyl-2-lyso-sn-glycero-3-phosphocholine followed by electrophoresis resulted in the incorporation of radioactivity into a protein of Mr 29,000. The enzyme was most active towards 1-O-alkyl-2-lyso-sn-glycero-3-phosphocholine as substrate, 1-palmitoyl-2-lyso-glycero-3-phosphocholine being a poor substrate. In addition, the enzyme preferred acetyl-CoA to palmitoyl-CoA or oleoyl-CoA as substrate.     

Incorporation of arachidonic acid into 1-acyl-2-lyso-sn-glycero-3-phosphocholine of the human neutrophil

In this study, the initial incorporation of arachidonic acid into human neutrophils has been examined. Neutrophils pulse labeled for 5 min with [3H]arachidonic acid rapidly incorporated this fatty acid into 1,2-diacylglycerophosphocholine. However, when neutrophils were pulse labeled with [3H]arachidonic acid for 5 min, washed, and allowed to incubate for an additional 120 min, the relative amount of [3H]arachidonic acid increased in alkylacylglycerophosphocholine molecular species. Similar, when neutrophils were pulse labeled, washed, and allowed to incubate in the presence of 30 microM unlabeled arachidonic acid for 120 min, [3H]arachidonic acid was also remodeled into alkylacylglycerophosphocholine. These results implied that the initial incorporation of [3H]arachidonic acid proceeded via a free fatty acid intermediate into 1,2-diacyl-GPC, while the subsequent remodeling of arachidonate-containing glycerophospholipids did not. This initial incorporation was further investigated in a number of cell-free systems. Disrupted neutrophils incubated with [14C]arachidonoyl-CoA incorporated [14C]arachidonic acid into 1,2-diacyl-GPC containing 16:0, 18:0, and 18:1 at their sn-1 position in a pattern similar to that seen when whole neutrophils were incubated with arachidonic acid for 5 min. A small percentage of [14C]arachidonate from [14C]arachidonoyl-CoA was incorporated into 1-alkyl-2-acyl-GPC. The enzymatic activity responsible was found predominately in the membrane fraction of the broken cell preparation. This selectivity of the CoA-dependent acyltransferase for 1-acyl-linked glycerophosphocholine was further examined by adding [14C]arachidonoyl-CoA and various 1-radyl-2-lyso-GPC to neutrophil membrane preparations. These studies provide evidence that the initial incorporation of arachidonic acid into sn-glycero-3-phosphocholine takes place by an arachidonoyl-CoA: lysophosphatidylcholine acyltransferase(s) which is selective for the 1-acyl-2-lyso-GPC.     

Clearance and tissue distribution of functionalized polymeric liposomes from the blood stream of rats

Polymeric liposomes containing a synthetic porphinato-iron-imidazole complex (hemoglobin or red blood cell model) were labeled by introducing 1,2-di[1-14C]palmitoyl-sn-glycero-3-phosphocholine into their polymerized bilayers. After intravenous injection into rats, their clearance from a blood stream was measured. The apparent half-life time (50% disappearance time) was about 14 +/- 2 h. Their tissue distribution was determined with time by whole autoradiographic measurement.     

1-O-Alkyl-2-acyl-sn-glycero-3-phosphocholine: A novel source of arachidonic acid in neutrophils stimulated by the calcium ionophore A23187

Rabbit peritoneal neutrophils incorporated [¹⁴C]arachidonic acid into seven molecular species of choline-containing phosphoglycerides. These 2-[¹⁴C]arachidonoyl species differed with respect to the alkyl ether or acyl residue bound at the $\text{sn}$-1 position; four of the seven were ether-linked. Stimulation with calcium ionophore A23187 induced a proportionate release of arachidonate from all seven molecular species: 40% of the released arachidonate came from alkyl ether species. Thus, 1-$\text{O}$-alkyl-2-arachidonoyl-$\text{sn}$-glycero-3-phosphocholine (GPC) is a significant source of metabolizable arachidonic acid. Since 1-$\text{O}$-alkyl-2-lyso-GPC is the metabolic precussor of platelet activating factor, these results further interrelate pathways forming arachidonate metabolites and platelet activating factor; they also supply a rationale for the observation that both classes of stimuli form concomitantly during cell activation.     

Structures of minor ether lipids isolated from the aceticlastic methanogen, Methanothrix concilii GP6

Structures were determined for two phospholipids and three glycolipids purified from chloroform-methanol extracts of Methanothrix concilii GP6. Together they accounted for 14% of the total lipid and were based on a C20,20-diether core structure consisting of either 2,3-di-O-phytanyl-sn-glycerol or its 3'-hydroxy analog, namely, 2-O-[3,7,11,15-tetramethylhexadecyl]-3-O-[3'- hydroxy-3',7',11',15'-tetramethylhexadecyl]-sn-glycerol. These two core lipids formed phosphodiester bonds to ethanolamine and glycosidic bonds to beta-D-galactopyranose. A third glycolipid consisted of the triglycosyl head group beta-D-galactopyranosyl-(1----6)-[beta-D-glucopyranosyl-(1----3)]-beta-D - galactopyranose in glycosidic linkage to the 3'-hydroxydiether core lipid.     

Conversion of 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholine to 1-O-alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine. A novel pathway for the metabolism of ether-linked phosphoglycerides.

Madin Darby canine kidney (MDCK) cells convert 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphocholine [( 3H]alkylacylGPC) to a product tentatively identified as an ethanolamine-containing phosphoglyceride (PE) (Daniel, L. W., Waite, B. M., and Wykle, R. L. (1986) J. Biol. Chem. 261, 9128-9132). In the present study, analysis of the radiolabeled phosphoglycerides as diradylglycerobenzoate derivatives indicated that [3H] alkylacylGPC was initially converted to 1-O-[3H]alkyl-2-acyl-sn-glycero-3-phosphoethanolamine [( 3H]alkylacylGPE) which was subsequently desaturated to 1-O-[3H]alk-1'-enyl-2-acyl-sn-glycero-3-phosphoethanolamine [( 3H]alkenylacylGPE). The conversion of [3H]/[32P]alkyl-lysoGPC to [3H]alkenylacylGPE indicated that base exchange enzymes were not involved in this pathway. A phosphono analog of alkyl-lysoGPC, resistant to phospholipase D hydrolysis and radiolabeled in the 1-O-alkyl chain was readily incorporated, acylated, and subsequently metabolized to [3H]alkylacylGPC and [3H]alkenylacylGPE. Therefore, the involvement of phospholipase D in the conversion pathway was ruled out. The conversion of [3H]alkylacylGPC or its phosphono analog to [3H]alkenylacylGPE was significantly enhanced by the addition of 100 microM ethanolamine to the culture media, suggesting that [3H]alkylacylglycerol is an intermediate in the cytidine-dependent pathway of PE synthesis. MDCK cell cytosol and microsomes contained no detectable phospholipase C activity. However, incubation of microsomes with CMP resulted in the degradation of [3H]alkylacylGPC and accumulation of [3H]alkylacylglycerol. Furthermore, the addition of CDP-ethanolamine to microsomes following preincubation with CMP, resulted in a decrease in [3H]alkylacylglycerol with a concomitant increase in [3H]alkenylacylGPE. Overall, these results suggest that the reverse reaction of choline phosphotransferase may be responsible for the conversion of alkylacylGPC to alkylacylGPE.     

Stimulation of platelet-activating factor synthesis by a nonmetabolizable bioactive analog of platelet-activating factor and influence of arachidonic acid metabolites

Platelet-activating factor (PAF) is a potent neutrophil agonist operating through specific receptors located on the cell surface. Binding of PAF to its receptor may also stimulate further PAF synthesis, thus providing a means of amplifying the PAF signal for the cell of origin and/or other responsive cells. In this report we demonstrate that 1-O-alkyl-2-N-methylcarbamyl-sn-glycero-3-phosphocholine (C-PAF), a nonmetabolizable bioactive analog of PAF, stimulates human neutrophils to synthesize PAF, as detected by [3H]acetate incorporation into PAF. This approach allowed us to conclude that [3H]acetate-labeled PAF was formed from endogenous precursor rather than mere turnover of the stimulatory dose of PAF. PAF's ability to initiate further PAF synthesis was confirmed by measuring the PAF-stimulated conversion of 1-O-[3H]alkyl-2-acylglycerophosphocholine to 1-O-[3H]alkyl-2-acetylglycerophosphocholine by prelabeled human neutrophils and by determining the molecular species of 1-O-alkyl-2-[3H]acetylglycerophosphocholine produced by cells stimulated with a single molecular species of PAF (C15:0). Degradation of exogenously added [3H]PAF was not inhibited by C-PAF/5-hydroxyeicosatetraenoic acid treatment. Thus, inhibition of PAF degradation was ruled out as the mechanism accounting for the appearance of labeled PAF in the stimulated cells. Synthesis of PAF in response to C-PAF was not dependent on cytochalasin B pretreatment but was dramatically potentiated by 5-hydroxyeicosatetraenoic acid, which alone was without effect. Additionally, we have demonstrated that another major arachidonate metabolite of neutrophils, leukotriene B4, stimulates PAF production. Thus, at least three products of activated neutrophils, including PAF itself, can promote PAF synthesis by these cells. This positive feedback effect may amplify autacoid production and the final cellular response.     

A simplified procedure for synthesis of di-[14C]acyl-labeled lecithins.

A simplified procedure for synthesis of 1,2-di-[1'-14C]oleoyl-, 1,2-di-[1'-14C]linoleoyl-, and 1,2-di-[1'-14C]eicosatrienoyl-sn-glycero-3-phosphorylcholine is described. The method involves acylation of the CdCl2 complex of glycerophosphorylcholine with a 14C-labeled fatty acid in the presence of trifluoracetic anhydride and pyridine. The 14C-labeled lecithin is isolated in pure form by preparative thin-layer chromatography and alumina column chromatography in an overall yield of 12-24%. No isomerization or peroxidation of the unsaturated acids was detected.