Production of complex ether glycerolipids from exogenous alkylglycerols by cell suspension cultures of rape

Summary Neutral and ionic ether glycerolipids, especially alkylacylglycerophosphocholines and 1-alkenylacylglycerophosphocholines, are formed from exogenous 1-O-alkylglycerols, 1-O-(1-alkenyl)glycerols or 2-O-alkylglycerols by photomixotrophic cell suspension cultures of rape (Brassica napus). Best yields of ether glycerolipids were obtained by incubating rape cells with optically active 1-O-alkyl-sn-glycerols. Racemic or symmetric alkylglycerols are also utilized by rape cell suspension cultures for the biosynthesis of optically active ionic ether glycerolipids. In contrast, 3-O-hexadecyl-sn-glycerol is not incorporated into ether glycerophospholipids of rape cells. Incorporation of the substrates into ionic ether lipids is dependent on chain length (C₁₄>C₁₆>C₁₈) and degree of unsaturation (C_18:1C_18:0) of alkyl chains.Stereochemically uniform 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines and 2-O-alkyl-1-acyl-sn-glycero-3-phosphocholines with defined alkyl moieties can be prepared from exogenous alkylglycerols. This method recommends itself especially for the preparation of 1-O-(1-alkenyl)-2-acyl-sn-glycero-3-phosphocholines (choline plasmalogens) from 1-O-(1-alkenyl)-sn-glycerols. Ether glycerophospholipids with physiological activity, such as 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholines (platelet activating factor, PAF) and 1-O-alkyl-sn-glycero-3-phosphocholines (lyso PAF), were synthesized from 1-O-alkyl-2-acyl-sn-glycero-3-phosphocholines formed by cell suspension cultures of rape.     

Formation of complex ether lipids from 1-O-alkylglycerols in cell suspension cultures of rape

Photomixotrophic cell suspension cultures of rape, Brassica napus, were incubated with rac-1-O-[1′-¹⁴C]hexadecylglycerol. Radioactivity was incorporated predominantly into choline glycerophospholipids. Prolonged incubation led also to considerable proportions of labeled ethanolamine glycerophospholipids. In addition to these ionic lipids,isomeric hexadecylacylglycerols as well as hexadecyldiacylglycerols were formed. About a third of the hexadecylglycerol supplied as substrate was cleaved within 48 h incubation. The palmitic acid formed by oxidative cleavage of the substrate was incorporated predominantly into choline glycerophospholipids, ethanolamine glycerophospholipids, and triacylglycerols. Incubation of an equimolar mixture of homologous saturated rac-1-O-[1′¹⁴C]alkylglycerols (C₁₄, C₁₆, C₁₈, C₂₀) with rape cells showed that alkylglycerols with alkyl moieties having 16 and 18 carbon atoms were incorporated preferentially. Incubation of labeled hexadecyglycerol with a homogenate of rape cells led also predominantly to choline glycerophospholipids; highest yields were obtained at pH 7. Neither the 1-O-alkyl moieties in choline glycerophospholipis nor those in ethanolamine glycerophospholipids were desaturated to 1-O-alk-1′-enylmoieties. The results of these experiments led to the following conclusions: (1) The acylation of 1-O-alkylglycerols to isomeric alkylacylglycerols is catalyzed by two acyltransferases differing in their specificity with regard to the chain length of the alkyl moiety in the substrate. (2) CDP-Choline: diacylglycerol cholinephosphotransferase and CDP-ethanolamine: diacylglycerol ethanolaminephosphotransferase are two enzymes differing in various respects. Cholinephosphotransferase exhibits a much higher affinity for 1-O-alkyl-2-O-acylglycerols than ethanolaminephosphotransferase. The two enzymes show marked differences with regard to their specificity for 1-O-alkyl-2-O-acylglycerols differing in the chain lengths of their alkyl moieties.     

Formation of optically active ether lipids from race-mic-1-O-tetradecylglycerol in plant cell cultures

Photomixotrophic rape cells in culture specifically incorporate 1-O-tetradecyl-sn-glycerol from a racemic mixture into complex alkyl glycerolipids. Thus, both neutral and ionic 1-O- alkyl-2-O-acyl-sn-glycerolipids with defined alkyl moieties can be prepared from racemic mixtures of alkylglycerols.     

Biologically active lipids. Semi-synthesis of 3H-labeled ether glycerophospholipids and ether glyceroglycolipids from ratfish liver oil

1-O-Alkyl-2,3-diacyl-sn-glycerols, the major constituents of ratfish (Chimaera monstrosa) liver oil, serve as starting material for the preparation of 1-O-alkyl-sn-glycero-3-phosphocholines, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholines, 1-O-alkyl-2-O-methyl-sn-glycero-3-phosphocholines, and 1-O-alkyl-2-O-methyl-sn-glycero-3-beta-D-glucopyranosides. Catalytic tritiation of the unsaturated alkyl moieties in these biologically active ether lipids affords the corresponding 3H-labeled substances.     

Biosynthesis of wax esters in tissues of Sinapis alba L. seeds

The biosynthesis of wax esters has been investigated in maturing seeds of Sinapis alba. Exogenous long-chain alcohols are incorporated exclusively into alkyl moieties of wax esters. Oxidation of the long-chain alcohols is not detected. Exogenous fatty acids are incorporated into acyl moieties of wax esters to a low extent. A reduction of fatty acids to alcohols is not observed. Synthesis of wax esters is localized exclusively in the testa; both outer and inner integument are equally active in wax ester biosynthesis. The biosynthesis of wax esters is specific with regard to both chain length and degree of unsaturation of long-chain alcohols. Exogenous and endogenous sterols are not esterified.     

Identification of 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols, a new type of lipid class, in harderian gland tumors of mice

A new class of alkyl glycerolipids, 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols, was identified in lipid extracts prepared from harderian gland tumors of mice. After saponification, this lipid class yielded 1-alkyl-3-(1'-glycerol)glycerols. Identification was based on mass spectrometry, proton nuclear magnetic resonance spectroscopy, infrared spectroscopy, and chromatography of various derivatives and appropriate standards that were synthesized. The alkyl moieties of this unique lipid class consisted of saturated aliphatic chains with chain lengths of 14 to 20 carbon atoms. The acyl moieties were mostly saturated and monounsaturated aliphatic chains ranging from 14 to 24 carbon atoms. The alkyl and acyl moieties of 1-alkyl-2-acyl-3-(2',3'-diacylglycerol)glycerols were similar to those of alkyldiacylglycerols present in the same tissue, except for the presence of monounsaturated alkyl moieties in the latter. 1-Alkyl-2-acyl-3-(2', 3'-diacylglycerol)glycerols were only found in trace amounts in the normal harderian glands of mice. The total quantity of the alkyl and acyl moieties with a chain length greater than 20 carbon atoms in the alkyldiacylglycerols from tumors were considerably lower than those found in normal harderian glands of mice. This is the first report of the presence of bisglyceryl ether lipids in mammalian tissue; its unique chemical structure is consistent with the type of ether-linked lipid products that could be synthesized in the reaction catalyzed by alkyldihydroxyacetone-P synthase.     

Formation of ether lipids from isomeric cis-octadecen-1-ols in normal and neoplastic cells: substrate specificity of enzymes with regard to position of double bonds

The pattern of incorporation of isomeric cis-[1-14C]octadecen-1-ols into alkyl moieties of glycerophospholipids of rat brain as well as of L 1210 and S 180 murine ascites cells revealed the following: 1) The enzymes involved in the biosynthesis of acylalkylglycerophospholamines have no substrate specificity with regard to position of the double bond of either cis-oectadecen-1-ols or of intermediate ether lipids derived therefrom. 2) CDPcholine: diradylglycerol cholinephosphotransferases exhibit a strong preference for acylalkylglycerols with cis-8, cis-9 and cis-10-octadecenyl moieties as substrates.     

Occurrence of glyceryl ethers in the phosphatidylcholine fraction of surfactant from dog lungs

The molecular species of ether-linked lipids in the phosphatidylcholine (PC) fraction of the pulmonary surfactant obtained from the lavage fluid of dog were characterized. A combination of base-catalyzed methanolysis, phospholipase C treatment, gas-liquid chromatography, and mass spectrometry procedures were applied. The phospholipid composition of the surfactant, obtained by phosphorus assay of lipids separated by silica gel G thin-layer chromatography (TLC), was: PC (75%), phosphatidylglycerol (10%), phosphatidylethanolamine (7%), plus small amounts of sphingomyelin, phosphatidylinositol, and phosphatidylserine. The major components of the PC were 1,2-diacylPC (95%), and 1-O-alkyl-2-acylPC (5%). No detectable amounts of 1-O-alkyl-1'-enyl-2-acylPC or di-alkyl-1-enylPC were observed. The acyl groups present in the diacylPC were 14:0 (5%), 16:0 (68%), 16:1 (12%), 18:0 (6%), 18:1 (7%) and 18:2 (2%). The predominant alkyl ether chains located at the carbon 1 position of the 1-O-alkyl-2-acylPC were 16:0 (84%), 18:0 (5%) and 18:1 (14%). At the carbon 2 position only a 16:0 fatty acyl residue was detected. In three out of seven animals platelet-activating factor-like activity, as determined by a platelet aggregation assay, was isolated by TLC. This aggregating activity was lost upon base-catalyzed methanolysis, but was restored by functional levels after acetylation.     

Effects of platelet activating factor and related lipids on phase transition of dipalmitoylphosphatidylcholine

Recent evidence localizing the inflammatory mediator, platelet activating factor, (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine) to the membranes of stimulated neutrophils raises the possibility that PAF may, in addition to its activities as a mediator, alter the physical properties of membranes. Accordingly, the effects of PAF and related alkyl ether and acyl analogs on phase transition thermodynamics of dipalmitoylphosphatidylcholine (DPPC) were studied using fluorescence polarization of the fluorescent probe, 1,6-diphenyl-1,3,5-hexatriene (DPH). PAF, its ester analog (1-palmitoyl-2-acetylphosphatidylcholine) and both the corresponding alkyl and acyl lysophospholipid analogs (each at a concentration of 10 mol%) significantly decreased the phase transition temperature and broadened the phase transition of DPPC (P less than 0.05). The relative potency of the lipids in causing this effect was ester-PAF greater than or equal to PAF greater than or equal to lyso-PAF greater than lyso-PC suggesting that the fluidization of the synthetic membranes was attributable to both the 2-position acetyl group and the 1-position alkyl linkage. Furthermore, using various related compounds, increases in chain length and degree of unsaturation in the 2-position were shown to enhance the depression in transition temperature and broadening of the phase transition. Phase transition thermodynamics were also assessed using differential scanning calorimetry. Similar depression in the phase transition temperature was measured for PAF and both the alkyl and acyl lysophospholipids. Broadening of the phase transition for DPPC by the various analogs was assessed by calculation of transition peak width and cooperative unit. Data from fluorescence polarization and differential scanning calorimetry provide similar though not identical results and support the hypothesis that the unique features of PAF may alter membrane physical properties and could ultimately explain some of its biologic actions.     

On the Role of Long-Chain Aldehydes in Mammalian Plasmalogen Biosynthesis

Abstract: [1-³H, 1-¹⁴C]Palmitaldehyde(³H:¹⁴C= 15) was injected intracerebrally to 18-day-old rats and incorporation of radioactivity into brain lipids was followed over a 24-h period. The substrate was metabolized primarily by oxidation to palmitic acid with loss of tritium and, to a lesser extent, by reduction to hexadecanol. The alkyl moieties of the ethanolamine phospholipids showed considerably lower ³H:¹⁴C ratios than the substrate, indicating a substantial participation in ether lipid synthesis by tritium-free alcohols derived from ¹⁴C-labeled fatty acids. Virtually no ³H radioactivity was found in alkenyl moieties, indicating stereospecificity of both reduction of aldehyde and dehydrogenation of alkyl to alkenyl glycerolipid. The data are consistent with the general concept that plasmalogen biosynthesis proceeds exclusively through fatty alcohols and alkyl glycerolipids and that fatty aldehydes cannot be utilized directly.     

Alkyl acyl ethanolamine glycerophosphatides in rat intestinal mucosa

The ethanolamine glycerophosphatides (EGP) were isolated from rat mucosal lipids by column and thin-layer chromatography and were found to contain alkyl acyl EGP. After hydrolysis and acetylation, the resulting alkyl diacetyl glycerols were separated by gas-liquid chromatography and the paraffinic moieties were shown to consist of C18- and C20-saturated and C18-monounsaturated residues. The fatty acid compositions of the 1,2-diacyl glycerols and 1-alkyl-2 acyl glycerols obtained by treatment of the phosphatides with phospholipase C were determined. The 1-alkyl 2-acyl glycerols were rich in polyunsaturated acyl residues.     

A method for the quantitative determination of glycerolipids containing O-alkyl and O-alk-1-enyl moieties.

We have developed a spectrophotometric procedure, based on a combination of established methods, for the quantitative determination of aklyl and alk-1-enyl (plasmalogens) ether-linked glycerolipids. It depends upon the release of alkylglycerols and alk-1-enylglycerols from phospholipids by phosphlipase C (Bacillus cereus) followed by saponification or by Vitride reduction the phospholipids; aldehydes are subsequently formed and measured colorimetrically after reacting them with a fuchsin reagent. The total alkyl and alk-1-enyl content of glycerolipids is determined oxidation of the sample withperiodate to form aldehydes and alkylglycolic aldehydes. The O-alk-1-enyl lipid content is determined on a separate sample by measuring the aldehydes produced after acid hydrolysis. The quantity of O-alkyl lipids is calculated from the difference between the values obtained for the total ether-lipid content and that of the O-alk-1enyl lipid content. Alternately, direct determination of alk-1-enylglycerols and alkylglycerols can be made if these hydrolytic products are first separated by thin-layer chromatography.     

Metabolism of orally administered rac-1-O-[1'-14C]dodecylglycerol and nutritional effects of dietary rac-1-O-dodecylglycerol in mice

The metabolism of orally administered rac-1-O-[1'-14C]dodecylglycerol was investigated in mice. The substrate was rapidly absorbed in the intestine and then incorporated into ether glycerolipids of various organs and tissues in high proportions. In intestine and liver, however, large amounts of rac-1-O-[1'-14C]dodecylglycerol were catabolized by oxidative cleavage of the ether bond followed by degradation of the radioactive cleavage product, i.e., lauric acid, to water-soluble metabolites that were excreted in the urine at a fast rate. The feeding of a rac-1-O-dodecylglycerol-containing diet (1 g rac-1-O-dodecylglycerol/kg body weight X day) given over a period of 4 weeks did not significantly alter body weights or organ weights of mice. Analysis of total lipids revealed that high proportions of the substrate were incorporated into ether lipids of all organs and tissues during the feeding period, generally accompanied by a remarkable increase in saturated acyl moieties and a concomitant decrease of linoleoyl moieties of total lipids. Yet, 4 weeks after removal of the rac-1-O-dodecylglycerol-containing diet, the lipids of murine organs and tissues showed a close resemblance to those of the control group.     

Acyloxyacyl hydrolase, a leukocyte enzyme that deacylates bacterial lipopolysaccharides, has phospholipase, lysophospholipase, diacylglycerollipase, and acyltransferase activities in vitro.

Human acyloxyacyl hydrolase (AOAH) is a leukocyte enzyme that hydrolyzes acyloxyacyl bonds in the lipid A region of bacterial lipopolysaccharide (LPS), thereby detoxifying the LPS. We report here that the enzyme also acts in vitro on glycerophospholipids, lysophospholipids, and diacylglycerol. While AOAH preferentially removes palmitate or stearate from the sn-1 position of phospholipid and diacylglycerol substrates that have unsaturated acyl chains in the sn-2 position, it is able to cleave both palmitates from sn-1,2-dipalmitoylphosphatidylcholine and sn-1,2-dipalmitoylglycerol. This apparent preference for removing saturated (or shorter) acyl chains from glycerolipids is consistent with its ability to cleave laurate more rapidly than palmitoleate from lipopolysaccharide (Erwin, A. L., and Munford, R. S. (1990) J. Biol. Chem. 265, 16444-16449). AOAH also catalyzes acyl transfer from LPS and phosphatidylethanolamine to acceptor lipids; approximately equal amounts of laurate and myristate are transferred from LPS to monooleoylglyceryl ether, forming acyloleoylglyceryl ether. The demonstration that AOAH has phospholipase, lysophospholipase, diacylglycerol lipase, and acyltransferase activities in vitro suggests that the enzyme may have roles in addition to LPS deacylation (detoxification) in phagocytic cells.     

The acyl dihydroxyacetone phosphate pathway enzymes for glycerolipid biosynthesis are present in the yeast Saccharomyces cerevisiae.

The presence of the acyl dihydroxyacetone phosphate (acyl DHAP) pathway in yeasts was investigated by examining three key enzyme activities of this pathway in Saccharomyces cerevisiae. In the total membrane fraction of S. cerevisiae, we confirmed the presence of both DHAP acyltransferase (DHAPAT; Km = 1.27 mM; Vmax = 5.9 nmol/min/mg of protein) and sn-glycerol 3-phosphate acyltransferase (GPAT; Km = 0.28 mM; Vmax = 12.6 nmol/min/mg of protein). The properties of these two acyltransferases are similar with respect to thermal stability and optimum temperature of activity but differ with respect to pH optimum (6.5 for GPAT and 7.4 for DHAPAT) and sensitivity toward the sulfhydryl blocking agent N-ethylmaleimide. Total membrane fraction of S. cerevisiae also exhibited acyl/alkyl DHAP reductase (EC 1.1.1.101) activity, which has not been reported previously. The reductase has a Vmax of 3.8 nmol/min/mg of protein for the reduction of hexadecyl DHAP (Km = 15 microM) by NADPH (Km = 20 microM). Both acyl DHAP and alkyl DHAP acted as substrates. NADPH was the specific cofactor. Divalent cations and N-ethylmaleimide inhibited the enzymatic reaction. Reductase activity in the total membrane fraction from aerobically grown yeast cells was twice that from anaerobically grown cells. Similarly, DHAPAT and GPAT activities were also greater in aerobically grown yeast cells. The presence of these enzymes, together with the absence of both ether glycerolipids and the ether lipid-synthesizing enzyme (alkyl DHAP synthase) in S. cerevisiae, indicates that non-ether glycerolipids are synthesized in this organism via the acyl DHAP pathway.     

Mono- and Dialkyl Glycerol Ether Lipids in Anaerobic Bacteria: Biosynthetic Insights from the Mesophilic Sulfate Reducer Desulfatibacillum alkenivorans PF2803T

Bacterial glycerol ether lipids (alkylglycerols) have received increasing attention during the last decades, notably due to their potential role in cell resistance or adaptation to adverse environmental conditions. Major uncertainties remain, however, regarding the origin, biosynthesis, and modes of formation of these uncommon bacterial lipids. We report here the preponderance of monoalkyl- and dialkylglycerols (1-O-alkyl-, 2-O-alkyl-, and 1,2-O-dialkylglycerols) among the hydrolyzed lipids of the marine mesophilic sulfate-reducing proteobacterium Desulfatibacillum alkenivorans PF2803^T grown on n-alkenes (pentadec-1-ene or hexadec-1-ene) as the sole carbon and energy source. Alkylglycerols account for one-third to two-thirds of the total cellular lipids (alkylglycerols plus acylglycerols), depending on the growth substrate, with dialkylglycerols contributing to one-fifth to two-fifths of the total ether lipids. The carbon chain distribution of the lipids of D. alkenivorans also depends on that of the substrate, but the chain length and methyl-branching patterns of fatty acids and monoalkyl- and dialkylglycerols are systematically congruent, supporting the idea of a biosynthetic link between the three classes of compounds. Vinyl ethers (1-alken-1′-yl-glycerols, known as plasmalogens) are not detected among the lipids of strain PF2803^T. Cultures grown on different (per)deuterated n-alkene, n-alkanol, and n-fatty acid substrates further demonstrate that saturated alkylglycerols are not formed via the reduction of hypothetic alken-1′-yl intermediates. Our results support an unprecedented biosynthetic pathway to monoalkyl/monoacyl- and dialkylglycerols in anaerobic bacteria and suggest that n-alkyl compounds present in the environment can serve as the substrates for supplying the building blocks of ether phospholipids of heterotrophic bacteria.     

Synthesis and biological properties of the fluorescent ether lipid precursor 1-O-[9'-(1'-pyrenyl)]nonyl-sn-glycerol

The synthesis of an omega-pyrene-labeled 1-O-alkyl-sn-glycerol was performed using a chirospecific method starting from R-(-)-2,3-O-isopropylidene-sn-glycerol. The product, 1-O-[9'-(1'-pyrenyl)]nonyl-sn-glycerol (pAG), is a fluorescent ether lipid that has a pyrene moiety covalently attached at the alkyl chain terminus. pAG was taken into CHO-K1 cells and a plasmalogen-deficient variant of CHO-K1, NRel-4. This variant is defective in dihydroxyacetonephosphate acyltransferase, which catalyzes the first step in plasmenylethanolamine (PlsEtn) biosynthesis. pAG was incorporated primarily into ethanolamine and choline phospholipids as well as a neutral lipid fraction tentatively identified as alkyldiacylglycerol. NRel-4 accumulated more fluorescence in the phospholipid fraction than CHO-K1, specifically in the ethanolamine phospholipids. Analysis of the fluorescent lipids showed that 93% of the pAG was incorporated into glycerolipids with the ether bond intact. Although the addition of 20 microM 1-O-hexadecyl-sn-glycerol to the medium fully restored PlsEtn biosynthesis in NRel-4 cells, pAG only partially restored PlsEtn synthesis. Incubation of cells with pAG followed by irradiation with long-wavelength (>300 nm) ultraviolet light resulted in cytotoxicity. NRel-4 cells displayed an increased sensitivity to this treatment compared with CHO-K1 cells. This photodynamic cytotoxicity approach could be used to select for mutants that are defective in downstream steps in ether lipid biosynthesis.     

Incorporation of D-[3-3H, U-14C] glucose into glycerolipid via acyl dihydroxyacetone phosphate untransformed and viral-transformed BHK-21-c13 fibroblasts.

Untransformed BHK-21-c13 fibroblasts as well as 4 polyoma-transformed strains were incubated with D-[U-14C,3-3H]glucose. This substrate generates intracellular labeled glycerol, and also [4-3H]NADPH via the phosphogluconate oxidative pathway. The latter selectively transfers hydrogen to C-2 of glycerol in glycerolipid via the acyl dihydroxyacetone phosphate pathway. After incubation, the distribution of radioactivity and the ratios of 3H/14C at the three positions of recovered glycerol were determined in sn-glycerol 3-phosphate, saponifiable glycerolipids, alkyl ether glycerolipids, and plasmalogens. In each of the cell types examined, 3H in the sn-1 position of glycerol in the recovered ether-containing glycerolipids was negligible, yet this position contained most of the recovered 3H in sn-glycerol 3-phosphate and saponifiable glycerolipids. The 3H/14C ratio in position 2 of glycerol, measured at various incubation times, was from 5- to 200-fold greater in the saponifiable glycerolipids than in free sn-glycerol 3-phosphate. The ratio in position 2 of ether-containing glycerolipids was the same or greater than that in the saponifiable glycerolipids in all of the cell types employed. A similar pattern in the 3H/14C ratio was observed when BHK-21-c13 cells were incubated with D-[U-14C,1-3H]glucose. These observations demonstrate significant participation of the acyl dihydroxyacetone phosphate pathway in glycerolipid synthesis in BHK cells.     

Behaviour of a glycosphingolipid with unsaturated fatty acid in phosphatidylcholine bilayers

N-(Oleoyl)galactosylceramide with perdeuterated acyl chain was prepared by partial synthesis, and studied by wide line ²H-NMR in phospholipid liposomes. Spectra were obtained for low glycolipid concentrations in bilayers of dimyristoyl-, distearoyl-, and 1-palmitoyl-2-oleoylphosphatidylcholines. In an attempt to isolate the effects of glycosphingolipid fatty acid cis unsaturation on glycolipid behaviour in membranes, spectral findings related to the above species were compared to literature NMR data for pure 1-palmitoyl-2-oleoylphosphatidylcholine bilayers in which the oleoyl chain of the phospholipid had been deuterated, and to analogously deuterated glycerol based lipids in Acholeplasma laidlawii membranes. The results for N-(oleoyl-d₃₃)galactosylceramide proved to be qualitatively and quantitatively very similar to published data dealing with glycerol based lipids at comparable temperatures. In addition, the results were strikingly similar for glycolipids dispersed in saturated and unsaturated phospholipid host matrices. It would appear that the primary effects of cis 9,10 fatty acid unsaturation in glycosphingolipids (at low concentration in fluid phospholipid membranes) are the same as those of fatty acid cis unsaturation in glycerolipids. It further appears that the overall dynamic behaviour of N-(oleoyl)galactosylceramide in fluid phospholipid membranes is very similar to that of glycerolipids with comparable acyl chains.     

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.