A comparative study of the effect of the antineoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine and some homologous compounds on PKC alpha and PKC epsilon

The effects of the anti-neoplastic ether lipid ET-18-OCH3 and some structural homologues on the activity of protein kinase C alpha (PKC alpha) were studied and compared with the effects the same had on the activity of PKC epsilon. ET-18-OCH3 progressively inhibited the activity of PKC alpha as the concentration was increased up to 30 mol% of the total lipid, above which the effect was one of activation. The experiments carried out with the homologues showed that the methoxy group bound at the sn-2 position of the glycerol of ET-18-OCH3 is essential for both the initial inhibitory effect and the subsequent activation effect. On the other hand, variations in the type of bond linking substitutions in the sn-1 position, ether or ester, do not seem to play an important role in determining the activity of the enzyme. The effects were different on PKC epsilon since ET-18-OCH3 had a triphasic effect, activating the enzyme at low concentrations, inhibiting it at slightly higher concentrations and then activating it again at higher concentrations. In this case, when the homologues were used, it was observed that the presence of the methoxy group linked to the sn-2 position of glycerol and the type of bond linking substitutions to the sn-1 position were important for activating the enzyme, so that only homologues with ester bonds as LPC and PAPC were able to induce the initial activation step in a way similar to ET-18-OCH3. Substitution of the phosphocholine group of ET-18-OCH3 by phosphoserine led to a greater activation of PKC alpha, an effect that comes from the Ca(2+)-phospholipid binding site probably because of the specific interaction of this site with the phosphoserine group. The action of ET-18-OCH3 and its homologues, as demonstrated in this paper, may permit the selective inhibition or activation of PKC alpha and PKC epsilon by using the most suitable range of concentrations.     

Surface-active properties of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine)

The surface activity and interaction with lipid monolayers and bilayers of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine) have been studied. Edelfosine is a surface-active soluble amphiphile, with critical micellar concentrations at 3.5 microM and 19 microM in water. When the air-water interface is occupied by a phospholipid, edelfosine becomes inserted in the phospholipid monolayer, increasing surface pressure. This increase is dose-dependent, and reaches a plateau at ca. 2 microM edelfosine bulk concentration. The ether lipid can become inserted in phospholipid monolayers with initial surface pressures of up to 33 mN/m, which ensures its capacity to become inserted into cell membranes. Upon interaction with phospholipid vesicles, edelfosine exhibits a weak detergent activity, causing release of vesicle contents to a low extent (<5%), and a small proportion of lipid solubilization. The weak detergent properties of edelfosine can be related to its very low critical micellar concentrations. Its high affinity for lipid monolayers combined with low lytic properties support the use of edelfosine as a clinical drug. The surface-active properties of edelfosine are similar to those of other "single-chain" lipids, e.g. lysophosphatidylcholine, palmitoylcarnitine, or N-acetylsphingosine.     

Correlation between the effect of the anti-neoplastic ether lipid 1-O-octadecyl-2-O-methyl-glycero-3-phosphocholine on the membrane and the activity of protein kinase Calpha.

The antineoplastic ether phospholipid 1-O-octadecyl-2-O-methyl-sn-glycero-3-phophocholine (ET-18-OCH3) was incorporated into dimyristoylglycerophosphocholine (Myr2Gro-PCho)/dimyristoylglycerophosphoserine (Myr2Gro-PSer) (4 : 1 molar ratio) mixtures. Electron microscopy showed that the addition of ET-18-OCH3 reduced the size of the vesicles. Small vesicles could be detected even at 60 mol% ET-18-OCH3. Sedimentation studies showed the increasing presence of phospholipids in the supernatant, while turbidity measurements indicated a decrease in absorbance as the ET-18-OCH3 concentration was increased. These findings may be explained by the formation of small vesicles and/or mixed micelles. Infrared spectroscopy showed that at 60 mol% the fluidity of the membrane was considerably increased at temperatures below the phase transition, with only a small increase in the proportion of gauche isomers after the gel-to-fluid phase transition of this sample. On the other hand, protein kinase Calpha (PKCalpha) activity progressively decreased when ET-18-OCH3 was incorporated into multilamellar vesicles, reaching a minimum value at 20 mol%, this inhibition being attributed to the modification of the membrane produced by a cone-shaped molecule. At higher concentrations, however, ET-18-OCH3 activated the enzyme with a maximum being attained at 50 mol%. This activation being attributed to the formation of small vesicles and/or micelles. At still higher concentrations of ET-18-OCH3 the enzyme was once again inhibited, inhibition being almost complete at 80 mol%. When PKC was assayed using large unilamellar vesicles a slight activation was observed at very low ET-18-OCH3 concentrations.     

Increased cell-surface receptor expression on U-937 cells induced by 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine

Association of the ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH₃) with liposomes (ELL-12) reduces acute toxicity while maintaining or enhancing anticancer activity in experimental tumor models. ELL-12 has been shown to induce apoptosis by a cytochrome-c-dependent caspase-mediated pathway, which results in proteolytic cleavage of poly(ADP-ribose) polymerase and lamins, but the antitumor effects of ET-18-OCH₃ or ELL-12 could result from tumor cell differentiation or activation. Here we compared the effects of ET-18-OCH₃ and ELL-12 on the expression of cell-surface proteins associated with cell differentiation and/or activation in U-937 cells. Phorbol 12-myristate 13-acetate and all-trans-retinoic acid, which induce differentiation in U-937 cells, up-regulated CD11b (MAC1 α-integrin) and CD82 and down-regulated CD71 (transferrin receptor) in a time- and dose-dependent manner. In contrast, ET-18-OCH₃ and ELL-12 up-regulated both CD71 and CD11b and did not have any effect on expression of CD82 in U-937 cells, suggesting that the ELL-12 may activate these cells rather than induce differentiation. Further evidence of activation was that ET-18-OCH₃ and ELL-12 strongly induced tumor necrosis factor α production by U-937 cells. Received: 25 February 1999 / Accepted: 4 August 1999     

Stability of association of 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine with liposomes is composition dependent

The ether lipid, 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-OCH₃), has anticancer activity, but it has serious side-effects, including hemolysis, which prevent its optimal use. We surmised if ET-18-OCH₃ could be stably associated with liposomes, less free ET-18-OCH₃ would be available for lytic interaction with red cells. Liposome composition variables investigated included acyl chain saturation, phospholipid head group and mole ratio of Chol and ET-18-OCH₃. It was found that attenuation of hemolysis was strongly liposome composition dependent. Some ET-18-OCH₃ liposome compositions were minimally hemolytic. For example, whereas the HI₅ (drug concentration required to cause 5% human red cell lysis) was 5–6 μM for free ET-18-OCH₃, it was approximately 250 μM for DOPC (dioleoylphosphatidylcholine):Chol (cholesterol):DOPE-GA (glutaric acid derivatized DOPE):ET-18-OCH₃, (4:3:1:2) and 640 μM for DOPE (dioleyolphosphatidylethanolamine):Chol:DOPE-GA:ET-18-OCH₃ (4:3:1:2) liposomes. Efflux of carboxyfluorescein (CF) from liposomes and Langmuir trough determinations of mean molecular area of lipids in monolayers (MMAM) were used as indicators of membrane packing and stability. Incorporation of ET-18-OCH₃ in liposomes reduced the MMAM. Reduction in CF permeation was correlated with reduction in hemolysis. The most stable liposomes included components, such as cholesterol, DOPC and DOPE, which have complementary shapes to ET-18-OCH₃.     

Surface-active properties of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine)

The surface activity and interaction with lipid monolayers and bilayers of the antitumour ether lipid 1-O-octadecyl-2-O-methyl-rac-glycero-3-phosphocholine (edelfosine) have been studied. Edelfosine is a surface-active soluble amphiphile, with critical micellar concentrations at 3.5 μM and 19 μM in water. When the air-water interface is occupied by a phospholipid, edelfosine becomes inserted in the phospholipid monolayer, increasing surface pressure. This increase is dose-dependent, and reaches a plateau at ca. 2 μM edelfosine bulk concentration. The ether lipid can become inserted in phospholipid monolayers with initial surface pressures of up to 33 mN/m, which ensures its capacity to become inserted into cell membranes. Upon interaction with phospholipid vesicles, edelfosine exhibits a weak detergent activity, causing release of vesicle contents to a low extent (< 5%), and a small proportion of lipid solubilization. The weak detergent properties of edelfosine can be related to its very low critical micellar concentrations. Its high affinity for lipid monolayers combined with low lytic properties support the use of edelfosine as a clinical drug. The surface-active properties of edelfosine are similar to those of other “single-chain” lipids, e.g. lysophosphatidylcholine, palmitoylcarnitine, or N-acetylsphingosine.     

Structure and thermotropic properties of hydrated 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine bilayer membranes

The ether-linked phosphatidylcholines 1-eicosyl-2-dodecyl-rac-glycero-3-phosphocholine (EDPC) and 1-dodecyl-2-eicosyl-rac-glycero-3-phosphocholine (DEPC) have been investigated by differential scanning calorimetry (DSC) and X-ray diffraction. DSC of hydrated EDPC shows a single endothermic transition at 34.8 degrees C (delta H = 11.2 kcal/mol) after storage at -4 degrees C while DEPC shows three endothermic transitions at 7.7 and approximately 9.0 degrees C (combined delta H approximately 0.4 kcal/mol) and at 25.2 degrees C (delta H = 4.7 kcal/mol). Both the single transition of EDPC and the two higher temperature transitions of DEPC are reversible, while the approximately 7.7 degrees C transition of DEPC increases in enthalpy on low-temperature incubation. At 23 degrees C, X-ray diffraction of hydrated EDPC shows a sharp reflection at 4.2 A together with lamellar reflections corresponding to a bilayer periodicity, d = 56.2 A. Electron density profiles derived from swelling experiments show a phosphate-phosphate intrabilayer distance, dp-p, of 36 A at all hydrations. This, together with calculated lipid thickness and molecular area considerations, suggests an interdigitated, three chains per head group, bilayer gel phase, L beta*, with no hydrocarbon chain tilt. This is structurally analogous to the bilayer gel phase of hydrated 18:0/10:0 ester PC [McIntosh, T. J., Simon, S. A., Ellington, J. C., Jr., & Porter, N. A. (1984) Biochemistry 23, 4038]. In contrast, DEPC at -4 degrees C shows an L beta' bilayer gel phase with tilted hydrocarbon chains (d = 61.1 A). However, this transforms above 9 degrees C to an interdigitated, triple-chain, L beta* bilayer gel phase (identical with that of EDPC) with d = 56.6 A and a phosphate-phosphate distance of 36 A. Above their respective chain melting transitions, Tm, EDPC and DEPC exhibit liquid-crystalline L alpha bilayer phases with d = 64.5 and 65.0 A at 55 and 45 degrees C, respectively. The ability of both EDPC and DEPC to form triple-chain interdigitated gel-state bilayers suggests that the conformational inequivalence at the sn-1 and sn-2 positions is less pronounced in the ether-linked PCs compared to the ester-linked PCs, where only one of the positional isomers, e.g., 18:0/10:0 PC but not 10:0/18:0 PC, forms the triple-chain structure (J. Mattai, unpublished results). Thus, a different conformation around the glycerol is predicted for ether-linked PC compared to ester-linked PC.     

Modulation of the physical properties of dielaidoylphosphatidylethanolamine membranes by a dirhamnolipid biosurfactant produced by Pseudomonas aeruginosa

Rhamnolipids are bacterial biosurfactants produced by Pseudomonas spp. These compounds have been shown to present several interesting biological activities, restricting the growth of Bacillus subtilis and showing zoosporicidal activity on zoosporic phytopathogens. It has been suggested that the interaction with the membrane could be the ultimate responsible for these actions. Therefore, it is of great interest to get insight into the molecular mechanism of the interaction of purified rhamnolipids with the various phospholipid components of biological membranes. In this paper we report on the phase behaviour of mixtures of dielaidoylphosphatidylethanolamine (DEPE) with a purified dirhamnolipid (DiRL) fraction from Pseudomonas aeruginosa, as studied by a number of physical techniques such as differential scanning calorimetry, FTIR, small angle X-ray (SAX) diffraction and dynamic light scattering. Our data indicate that the presence of DiRL counteracts the tendency of DEPE to form vesicular aggregates of large size, forming vesicles of smaller diameter which most probably have a lower lamellarity index. The partial phase diagram obtained from calorimetric data shows a complex behaviour with a solid-phase immiscibility. X-ray diffraction shows that DiRL has a bilayer stabilizing effect, impeding formation of the inverted hexagonal-HII phase of DEPE. The presented data are discussed focussing into how DiRL/DEPE interactions could help to explain the membrane perturbing activities of this biosurfactant.     

Altered mitochondrial function and overgeneration of reactive oxygen species precede the induction of apoptosis by 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine in p53-defective hepatocytes.

The mechanism of induction of apoptosis by the novel anti-cancer drug 1-O-octadecyl-2-methyl-rac-glycero-3-phosphocholine (ET-18-OCH3) was investigated in p53-defective SV40 immortalized rat hepatocytes (CWSV1). Exposure to 12 microM ET-18-OCH3 for 36 h induced apoptosis as determined using classical morphological features and agarose gel electrophoresis of genomic DNA. Increased levels of reactive oxygen species (ROS) were detected spectrophotometrically using a nitroblue tetrazolium (NBT) assay in cells treated with ET-18-OCH3. Both the increased generation of ROS and the induction of apoptosis were inhibited when cells were treated concurrently with ET-18-OCH3 in the presence of the antioxidant alpha-tocopherol. Similar results were achieved when cells were switched acutely to choline-deficient (CD) medium in the presence of the antioxidant. The possible role of mitochondria in the generation of ROS was investigated. Both ET-18-OCH3 and CD decreased the phosphatidylcholine (PC) content of mitochondrial and associated membranes, which correlated with depolarization of the mitochondrial membrane as analyzed using 5,5',6,6'-tetramethylbenzimidazolcarbocyanine iodide (JC-1), a sensitive probe of mitochondrial membrane potential. Rotenone, an inhibitor of the mitochondrial electron transport chain, significantly reduced the intracellular level of ROS and prevented mitochondrial membrane depolarization, correlating with a reduction of apoptosis in response to either ET-18-OCH3 or CD. Taken together, these results suggest that the form of p53-independent apoptosis induced by ET-18-OCH3 is mediated by alterations in mitochondrial membrane PC, a loss of mitochondrial membrane potential, and the release of ROS, resulting in completion of apoptosis.     

Modulation of endothelial cell proliferation and capillary network formation by the ox-LDL component: 1-palmitoyl-2-archidonoyl-sn-glycero-3-phosphocholine (ox-PAPC)

Atherosclerotic plaque formation is often associated with pathological angiogenesis. Modified phospholipids, including oxidized lipoproteins such as LDL, are found to induce adhesion of the monocytes to the endothelial cells and to stimulate their chemotaxis. Effects of oxidized 1-palmitoyl-2-archidonoyl-sn-glycero-3-phosphocholine (ox-PAPC) mimic actions of minimally modified LDL in vivo. Interleukin-8 (IL-8) and interleukin-15 (IL-15) are known to induce both inflammation and angiogenesis. The goal of our study was to analyze a potential synergism between ox-PAPC and IL-15 in the in vitro model of angiogenesis carried out in the human endothelial cells (HUVECs). Increasing IL-15 concentrations led to formation of the tube-like structures in the matrigel 3D-model of angiogenesis (P < 0.05), in contrast to ox-PAPC that inhibited this process. HUVECs incubation with ox-PAPC led to reduced IL-15 gene basal expression (P = 0.033) along with parallel increase, however statistically insignificant, of basal gene expression of IL-8 (P = 0.086). Our findings point to the ox-PAPC opposite effects on the IL-8- and IL-15-mediated angiogenic responses that contribute to pathological angiogenesis induced by ox-LDL.     

Modulation of the Cs2CO3-promoted catalytic amination by a crown ether

The catalytic addition of 18-Crown-6 in some Cs2CO3-promoted amination of triflates and bromides was beneficial to improve sluggish reaction with suppression of the unwanted side products. The protocol was useful for the preparation of chlorinated aryl piperazines from phenol derivatives.     

Metabolism of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine in the human neutrophil

The biosynthesis of 1-acyl-2-acetyl-sn-glycero-3-phosphocholine (1-acyl-2-acetyl-GPC) together with that of 1-alkyl-2-acetyl-GPC (platelet-activating factor) has been demonstrated in a variety of inflammatory cells and tissues. It has been hypothesized that the relative proportion of these phospholipids produced upon cell activation may be influenced by their rates of catabolism. We studied the catabolism of 1-acyl-2-acetyl-GPC in resting and activated human neutrophils and compared it to that of 1-alkyl-2-acetyl-GPC. Neutrophils rapidly catabolize both 1-alkyl-2-acetyl-GPC and 1-acyl-2-acetyl-GPC; however, the rate of catabolism of 1-acyl-2-acetyl-GPC is approximately 2-fold higher than that of 1-alkyl-2-acetyl-GPC. In addition, most of 1-acyl-2-acetyl-GPC is catabolized through a pathway different from that of 1-alkyl-2-acetyl-GPC. The main step in the catabolism of 1-acyl-2-acetyl-GPC is the removal of the long chain at the sn-1 position; the long chain residue is subsequently incorporated either into triglycerides or into phosphatidylcholine. The 1-lyso-2-acetyl-GPC formed in this reaction is then further degraded to glycerophosphocholine, choline, or phosphocholine. 1-Acyl-2-acetyl-GPC is also catabolized, to a lesser extent, through deacetylation at the sn-2 position and reacylation with a long chain fatty acid. Stimulation of neutrophils by A23187 results in a higher rate of catabolism of 1-acyl-2-acetyl-GPC by increasing both the removal of the long chain at the sn-1 position and the deacetylation-reacylation at the sn-2 position. In a broken cell preparation, the cytosolic fraction of the neutrophil was shown to contain an enzyme activity which cleaved the sn-1 position of 1-acyl-2-acetyl-GPC and 1-acyl-2-lyso-GPC but not of 1,2-diacyl-GPC. Taken together, these data demonstrate that the human neutrophil is able to catabolize 1-acyl-2-acetyl-GPC in a manner both quantitatively and qualitatively different from that of platelet-activating factor. The differential catabolism may regulate the relative proportion of these two bioactive phospholipids in the neutrophil.     

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

In platelets, and in several other cell systems, pre-treatment with protein kinase C activators such as phorbol 12-myristate 13-acetate (PMA) results in the inhibition of receptor-mediated responses, suggesting that protein kinase C may play an important role in the termination of signal transduction. In the present study, we have attempted to locate the site of action of phorbol ester by comparing thrombin-induced (i.e. receptor-mediated) platelet activation with that induced by guanosine 5'-[gamma-thio]triphosphate (GTP[S]) and NaF, two agents which by-pass the receptor and initiate platelet responses by directly modulating G-protein function. After a 10 s pre-treatment with PMA (16 nM), dense-granule secretion induced by thrombin (0.2 unit/ml), GTP[S] (40 microM) and NaF (30 mM) was potentiated, resulting in a greater than additive response to agent plus PMA. However, after a 5 min pre-treatment, thrombin-induced secretion alone was inhibited, whereas PMA plus GTP[S]/NaF-induced release remained greater than additive. [32P]Phosphatidate formation in response to all three agents, in contrast, was inhibited by 50-70% in PMA (5 min)-treated platelets. That secretion induced by these agents is a protein kinase C-dependent event was demonstrable by using staurosporine, a protein kinase C inhibitor which at concentrations of 1-10 nM inhibited (70-90%) PMA-induced as well as thrombin- and NaF-induced secretion and protein phosphorylation. In membranes from PMA-treated platelets, thrombin-stimulated GTPase activity was significantly enhanced compared with that in untreated membranes (59% versus 82% increase over basal activity). The results suggest that inhibition of receptor-mediated responses by PMA may be directed towards two sites relating to G-protein activation: (i) receptor-stimulated GTPase activity and (ii) G-protein-phospholipase C coupling. Furthermore, the lack of inhibition of NaF- and GTP[S]-induced secretion by PMA suggests that different mechanisms may be involved in thrombin-induced and G-protein-activator-induced secretion.     

Biosynthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor and a hypotensive lipid) by cholinephosphotransferase in various rat tissues

The unique alkyl phospholipid, 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine, has been reported to exhibit powerful antihypertensive activity (Blank, M.L., Snyder, F., Byers, L.W., Brooks, B. and Muirhead, E.E. (1979) Biochem. Biophys. Res. Commun. 90, 1194-1200) and appears to be an extremely potent platelet-activating factor (Demopoulos, C.A., Pinckard, R.N. and Hanahan, D.J. (1979) J. Biol. Chem. 254, 9355-9358). In the present study, microsomal preparations from several rat tissues were found to catalyze the synthesis of 1-alkyl-1-acetyl-sn-glycero-3-phosphocholine by 1-alkyl-2-acetyl-sn-glycerol:CDPcholine cholinephosphotransferase reaction. Optimal conditions to measure enzyme activity were established. A subcellular survey of this cholinephosphotransferase activity showed that the enzyme was of microsomal origin. Enzyme activity was found in microsomes from several tissues; however, spleen has the highest activity of the tissues examined. Three different species of 1-alkyl-2-acetyl-sn-glycerol were all found to be substrates. The 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine synthesized in the microsomes could be hydrolyzed by adding the 100,000 x g supernatant fraction to the incubation medium. The optimum pH for formation of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine was 8.0, which was different from the pH optimum of 8.5 observed for the long-chain diacylglycerol cholinephosphotransferases. Activity of cholinephosphotransferase towards 1-alkyl-2-acetyl-sn-glycerol was slightly enhanced and stabilized by dithiothreitol, whereas the activity towards a diacylglycerol was inhibited by dithiothreitol. The possible involvement of two different enzymes in the conversion of 1-alkyl-2-acetyl-sn-glycerol and diacylglycerol to their respective phospholipid products is discussed.     

1-O-hexadec-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine and its biological activity

1-O-Alk-1'-enyl analog of platelet-activating factor (PAF, 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine, alkylacetyl-GPC) was prepared semi-synthetically from choline plasmalogens of beef heart muscle. The main compound was identified mass spectrometrically as 1-hexadec-1'-enyl-2-acetyl-sn-glycero-3-phosphocholine (16:O alk-1'-enylacetyl-GPC, 16:O vinyl form of PAF) and its platelet aggregation activity was about one-fifth of that of the corresponding 16:O alkylacetyl-GPC. The irreversible platelet aggregation activity induced by 5X10(-10) M 16:O alk-1'-enylacetyl-GPC was completely inhibited by 5X10(-7) M CV-3988 and 1X10(-7) M L-652, 731, specific PAF antagonists, and more than 99% of the activity was also lost by acid treatment. The hydrogenated product, alkylacetyl analog, showed quite same activity as that of authentic 16:O alkylacetyl-GPC. The platelets desensitized with 16:O alkylacetyl-GPC and with 16:O alk-1'-enylacetyl-GPC were not aggregated with 5X10(-10) M 16:O alk-1'-enylacetyl-GPC, suggesting that alk-1'-enylacetyl-GPC occupied the same receptor site of alkylacetyl-GPC.     

Synthesis of optically active 1-acyl-2-O-alkyl-sn-glycero-3-phosphocholine via 1-O-benzyl-sn-glycerol 3-arenesulfonate

A stereocontrolled route to 1-palmitoyl-2-O-hexadecyl-sn-glycero-3-phosphocholine from (R)-glycidyl tosylate is described. This method gives very high enantioselectivity (93-96% enantiomeric excess) and can be used to prepare 3-acyl-2-O-alkyl-sn-glycero-1-phosphocholines from (S)-glycidyl tosylate. The key step is the preparation of 1-O-benzyl-sn-glycerol 3-tosylate by the boron trifluoride etherate catalyzed regio- and stereo-specific opening of the epoxide ring with excess benzyl alcohol. The alkyl group is introduced using alkyl trifluoromethanesulfonate in the presence of excess 2,6-di-tert-butyl-4-methylpyridine. Debenzylation gives 2-O-alkyl-sn-glycerol 3-arenesulfonate, which is acylated and then converted into the phosphocholine. The use of chiral glycidyl derivatives as starting materials for the synthesis of glycerophospholipids is discussed.     

Hydrolysis of 1-triacontanoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine by human pancreatic phospholipase A2

A novel fluorescent phospholipid analogue, 1-triacontanoyl-2-(pyren-1-yl)hexanoyl-sn-glycero-3-phosphocholine (C30PHPC) was employed as a substrate for human pancreatic phospholipase A2. C30PHPC has a main endothermic phase transition with Tm at 46 degrees C as determined by differential scanning calorimetry (DSC). For an aqueous dispersion of C30PHPC the ratio of the intensities of pyrene excimer and monomer fluorescence emission, (IE/IM) has a maximum between 32 and 36 degrees C. The excimer emission intensity (at 480 nm) exceeds the monomer emission intensity (at 400 nm) 6.5-fold thus indicating a close packing of the phospholipid pyrene moieties in the lipid phase. C30PHPC has a limiting mean molecular area of 37 A2 at surface pressure 35 dyn cm-1 as judged by the compression isotherm at an air-water interphase. The hydrolysis of C30PHPC by human pancreatic phospholipase A2 was followed by monitoring the increase in the pyrene monomer fluorescence emission intensity occurring as a consequence of transfer of the reaction product, pyren-1-yl hexanoic acid into the aqueous phase. The enzyme reaction exhibited an apparent Km of 2.0 microM substrate. Calcium at a concentration of 0.2 mM activated the enzyme 4-fold. Maximal hydrolytic rates were obtained at 45 degrees C and at pH between 5.5 and 6.5. The enzyme reaction could be inhibited by 5 mM EDTA, confirming the absolute requirement for Ca2+ of this enzyme. The present fluorimetric assay easily detects hydrolysis of C30PHPC in the pmol min-1 range. Accordingly, less than nanogram levels of human pancreatic phospholipase A2 can be detected.     

Metabolic fate of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine by human spermatozoa: biosynthesis and biodegradation

Human spermatozoa can synthesise 1-alkyl-2-acetyl-glycerophosphocholine (AAGPC) in small amount by acetyltransferase (AT) in absence of any stimulus, but can actively catabolise it by acetylhydrolase (HY). Seminal plasma, on the other hand, was devoid of anabolic enzyme albeit enrich in catabolic enzyme, suggesting as an active site for biodegradation of AAGPC secreted by spermatozoa. Both, AT and HY exhibited pH-optima in range of 7.0-7.6 at which spermatozoa are maximum viable and motile. Ionophore A23187 and EGTA inhibited AT, reversibly, whereas HY was inhibited by BSA, calcium-channel blockers, and phospholipase A2-inhibitors. Effect of aging-time on ejaculates exhibited decreased AT activity with increased HY activity along with unchanged calcium content of spermatozoa. Serotonin in vitro studies showed a pro-aggregator role on agglutination of spermatozoa. Viscid/long liquefaction time ejaculates exhibited raised AT activity and calcium contents with decreased HY activity in spermatozoa and high degree of agglutination. Studies with dithiothreitol-treatment indeed helped in liquefaction but levels of both enzymes remained status quo, suggesting existence of both pathways: remodelling of membrane phospholipids and de novo synthesis of AAGPC in spermatozoa, earlier being pre-dominant. We have proposed a role of AAGPC-Serotonin-Calcium in agglutination and liquefaction of spermatozoa, a vital aspect in normal fertility.     

Biosynthesis of 1-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet-activating factor) from 1-alkyl-2-acyl-sn-glycero-3-phosphocholine by rat alveolar macrophages. Phospholipase A2 and acetyltransferase activities during phagocytosis and ionophore stimulation

1-Alkyl-2-acyl-sn-glycero-3-phosphocholine (alkyl-acyl-GPC) comprises 11% of the total phospholipids of rat alveolar macrophages. This endogenous pool of alkylacyl-GPC was prelabeled by incubating the macrophages with [1,2-3H]alkyllyso-GPC (54 Ci/mmol), which enters the cells and is acylated. The effect of various stimuli on the synthesis and release into the media of labeled alkylacetyl-GPC (platelet-activating factor) from the cells was used to establish the role of inactive alkylacyl-GPC as a precursor of the biologically active derivative. A phagocytic agent (zymosan, 100 micrograms/ml) and an ionophore (A23187, 2 microM) stimulated the release of both alkylacetyl-GPC and alkyllyso-GPC into the media at the expense of cellular alkylacyl-GPC. Phospholipase A2 activity (at pH 4.5 and in 1 mM EDTA) was also increased in the media. The stimulatory effect of zymosan and the ionophore on alkylacetyl-GPC release was prevented by mepacrine (0.1 mM), an agent that inhibits the release of fatty acids from phospholipids. These data indicate that phospholipase activity is required for the biosynthesis of alkylacetyl-GPC. However, since the inhibitory effect of mepacrine was not apparent when acetate was present, it appears that the acetylation step is rate limiting. Exposure of alveolar macrophages in culture to zymosan or A23187 stimulated acetyltransferase activity 250-300%. In contrast, phorbol myristate acetate (1.6 microM), which stimulated the accumulation of lysophospholipids but not the level of alkylacetyl-GPC in the media, did not substantially increase acetyltransferase activity. We conclude that alkylacyl-GPC serves as a precursor of alkylacetyl-GPC and that the production of this potent mediator by rat alveolar macrophages can be stimulated by agents that affect phospholipase A2 and acetyltransferase activities. The latter enzyme appears to have a regulatory function in the biosynthesis of alkylacetyl-GPC.