Saturated and unsaturated 1-O-alkyl-2-O-acetoyl-sn-glycero-3-phosphocholines derived from ratfish liver oil: effect on human leukocyte migration

A mixture of 1-O-alkyl-2-O-acetoyl-sn-glycero-3-phosphocholines containing saturated alkyl moieties and a mixture of such compounds with saturated and unsaturated alkyl moieties, prepared from ratfish (Hydrolagus colliei) liver oil, were studied for their in vitro effect on human neutrophil migration. The mixture containing unsaturated compounds (II) was more active compared to the saturated (I) ones at a range from 10^-6 M to 10^-10 M concentrations. At 10^-4 M, II was cytotoxic. Both I and II were more potent than synthetic ‘PAF-acether’ (III) and the material prepared from beef heart plasmalogens (IV). Preincubation and checker-board titration experiments showed that the ether phospholipids derived from ratfish liver oil have primarily chemokinetic, but also chemotactic effects on neutrophils, as has been reported for compound III. These compounds are therefore highly potent stimulants of human neutrophils with potentially unique membrane-activating properties.     

Phosphono-platelet activating factor I. synthesis of 1-O-hexadecyl-2-O-acetyl-glyceryl-3-(2-trimethyl ammonium-methyl) phosphonate and its platelet activating potency

The total synthesis of 1-O-alkyl-2-acetyl-3-glyceryl-(2-trimethyl ammoniummethyl) phosphonate, the phosphono analogue of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine, is described. The phosphonolipid shows much lower activity than the phospholipid stimulating serotonin release from rabbit platelets.     

Synthesis of 1-(1-alkenyl)-2-acetyl-sn-glycero-3-phosphocholines, the aldehyded analogs of platelet activating factor

1-(1-Hexadecenyl)- and 1-(1-octadecenyl)-2-acetyl-sn-glycero-3-phosphocholines were synthesized. To this goal, synthetically prepared racemic phosphatidal-cholines were hydrolyzed with phospholipase A2, and lysophosphatidal-cholines obtained were acetylated with acetic anhydride.     

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.     

Effect of 1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF-acether) on leukocytes I. Analysis of the in vitro migration of human neutrophils

The effect of synthetic 1-O-octadecyl-2-O-acetyl-sn-glycero-3-phosphocholine (PAF-acether) and of 1-O-octadecyl-sn-glycero-3-phosphocholine (lyso-PAF-acether) on human neutrophil migration was studied in modified Boyden chambers, with the following results: (1) By checker-board analysis and deactivation experiments, the factors are chemokinetic at low (10^?8 M) and chemotactic at higher concentrations (10^?6 M), with lyso-PAF-acether being less potent at all concentrations. (2) Cross-deactivation occurs between the two PAF compounds, but not with two other chemotactic factors, suggesting a specific, common receptor for the PAFs on the neutrophil membrane. (3) Other chemotactic substances may act as potentiating or additive factors to the PAF compounds. (4) Inhibition of arachidonic acid turnover during chemotaxis by compound BW 755 C enhances leukocyte chemotaxis towards the PAF compounds and towards other chemotactic factors. The data suggest that PAF and its lyso-derivate may contribute in a unique and potent fashion to leukocyte accumulation at inflammatory sites.     

A facile synthesis of 1-O-alkyl-2-(R)-hydroxypropane-3-phosphonocholine (lyso-phosphono-platelet activating factor).

The synthesis of 1-O-alkyl-2-(R)-hydroxypropane-3-phosphonocholine is described. An efficient alkylation procedure using (NaH/DMSO) catalysis is also described and applied to the synthetic scheme. The key intermediate 1-O-alkyl-2-(R)-O-benzyl-3-bromopropane was phosphonylated using tris(methylsilyl)phosphite; the resulting phosphonic acid was coupled to choline using trichloroacetonitrile/pyridine or triisopropylbenzenesulfonyl chloride/pyridine followed by catalytic hydrogenation to yield 1-O-alkyl-2(R)-hydroxypropane-3-phosphonocholine.     

Antibody to platelet activating factor (1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine; PAF)

Specific antibodies to platelet activating factor (PAF) were prepared by immunizing rabbits with a hapten-bovine serum albumin (BSA) conjugate. As the hapten we used the synthetic PAF derivative which is resistant against enzymatic inactivation by plasma or tissues and which can bind to BSA through covalent bonding. Antibody activity was determined by an enzyme-linked immunosorbent assay (ELISA). Anti-PAF IgG reacted strongly with PAF. By means of the ELISA inhibition assay, we found that the antibody did not cross-react with phosphocholine, glycerophosphocholine, dilaurylglycerophosphocholine or PAF analogues which have ethanolamine-type polar head groups instead of choline group.     

Activation of rabbit platelet phospholipase and thromboxane synthesis by 1-o-hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (platelet activating factor)

1-O-Hexadecyl/octadecyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AGEPC), structurally identical with platelet activating factor, is a potent stimulus for rabbit platelet aggregation and serotonin secretion. AGEPC at concentrations between 10⁻¹⁰ and 10⁻⁸ M induced stimulation of rabbit platelet synthesis of thromboxane B₂. The dose vs. response curve for platelet thromboxane B₂ synthesis was displaced slightly towards higher stimulus concentrations compared to [³H]serotonin secretion, with half-maximal concentrations of 2.5 · 10⁻⁹ and 8 · 10^t-10 M, respectively. Rates of thromboxane B₂ synthesis and secretion were similar with a $\text{t}{}_{\mathrm{<mtext>1</mtext><mtext>2</mtext><mtext>max</mtext>}}$ of 4.0–4.5 s for both processes. AGEPC induced a decrease in platelet [¹⁴C]arachidonic acid in both phosphatidylinositol and phosphatidylcholine, although [¹⁴C]arachidonic acid turnover in phosphatidylcholine was not observed below 1 · 10⁻⁸ M AGEPC. Concomitantly, this decrease in phospholipid [¹⁴C]arachidonic acid was associated with a marked increase of radiolabel in platelet diacylglycerol and phosphatidic acid 15s after AGEPC addition, suggesting the possibility of a phospholipase C-diacylglycerol lipase mechanism of fatty acid cleavage. As observed previously with secretion and aggregation, removal of the 2-acetyl group from AGEPC abrogated all capacity of this molecule to stimulate platelet phospholipase. This study indicates that AGEPC (or platelet activating factor) activation of rabbit platelet phospholipase occurs in a time-course and concentration range similar to that required for [³H] serotonin secretion.     

Semi-synthetic preparation of 1-O-[1'-14C]hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) using plant cell cultures

Incubation of photomixotrophic cell suspension cultures of rape (Brassica napus) and heterotrophic cell suspension cultures of soya (Glycine max) with 1-O-[1'-14C]hexadecyl-sn-glycerol or rac-1-O-[1'-14C]hexadecylglycerol leads in high yield (up to 78%) to labeled 1-O-hexadecyl-2-acyl-sn-glycero-3-phosphocholines. Alkaline hydrolysis of the choline glycerophospholipids yields pure 1-O-[1'-14C]hexadecyl-sn-glycero-3-phosphocholine. 1-O-[1'-14C]Hexadecyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) is obtained by acetylating the lyso compound. The semi-synthetic preparation described leads to labeled platelet activating factor in an overall yield of 50-60% without loss of specific activity.     

The role of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AcGEPC) and palmitoyl-lysophosphatidate in the responses of human blood platelets to collagen and thrombin

Desensitisation of human blood platelets to the effects of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (1-O-alkylAcGEPC) and palmityl-lysophosphatidate by pre-incubation with these agonists has no effect on the aggregatory or secretory responses to collagen but causes 30–40% inhibition of these responses to thrombin in aspirin-treated platelets. The effects of 1-O-alkylAcGEPC and palmitoyl-lysophosphatidate are not additive. The results are not consistent with the proposal that 1-O-alkylAcGEPC or lysophosphatidate are the mediators for the responses to collagen observed when prostaglandinendo-peroxide synthesis is prevented, although they may play some role in the responses to thrombin under these conditions.     

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.     

Specific receptor sites for 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) on rabbit platelet and guinea pig smooth muscle membranes

By using tritiated 1-O-alkyl-2-O-acetyl-sn-glycero-3-phosphocholine (3H-PAF), we have directly identified its specific binding sites on rabbit platelet plasma membranes. The equilibrium dissociation constant for 3H-PAF is 1.36 (+/- 0.05) X 10(-9) M at 0 degrees C. The number of binding sites is 1.61 (+/- 0.34) X 10(12)/mg of membrane, which corresponds to approximately 150-300 receptors/platelet (depending on membrane vesicle orientation). Binding of 3H-PAF to rabbit platelet plasma membrane is rapid (t1/2 less than 5 min at 0 degrees C) and reversible. For a series of PAF analogues, their affinity for the receptor sites parallels with their relative potency to induce platelet aggregation. PAF can cause contraction of smooth muscle of heart, parenchymal strip, trachea, and ileum. Specific PAF receptor binding was demonstrated with purified plasma membrane from several smooth muscles and from polymorphonuclear leukocytes but not from presumably PAF nonresponsive cells such as erythrocytes and alveolar macrophages. It is likely that the interaction of PAF with these binding sites initiates the specific responses of platelets, polymorphonuclear leukocytes, and smooth muscles.     

Activation of phospholipase A2 by 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine in vitro

Oxidative stress leads to drastic modifications of both the biophysical properties of biomembranes and their associated chemistry imparted upon the formation of oxidatively modified lipids. To this end, oxidized phospholipid derivatives bearing an aldehyde function, such as 1-palmitoyl-2-(9'-oxo-nonanoyl)-sn-glycero-3-phosphocholine (PoxnoPC) can covalently react with proteins that come into direct contact. Intriguingly, we observed PoxnoPC in a 1,2-dipalmitoyl-sn-glycero-3-phosphocholine (DPPC) matrix to shorten and abolish the lag time in the action of phospholipase A2 (PLA2) on this composite substrate, with concomitant augmented decrement in pH, indicating more extensive hydrolysis, which was in keeping with enhanced 90° light scattering. The latter was abolished by the aldehyde scavenger methoxyamine, thus suggesting the involvement of Schiff base. Enhanced hydrolysis of a fluorescent phospholipid analogue was seen for PLA2 preincubated with PoxnoPC. Mixing PLA2 with submicellar (22 µM) PoxnoPC caused a pronounced increase in Thioflavin T fluorescence, in keeping with the formation of amyloid-type fibers, which were seen also by electron microscopy.     

Synthesis and antibacterial activity of novel 3-O-arylalkylcarbamoyl-3-O-descladinosyl-9-O-(2-chlorobenzyl)oxime clarithromycin derivatives

A novel series of 3-O-arylalkylcarbamoyl-3-O-descladinosyl-9-O-(2-chlorobenzyl)oxime clarithromycin derivatives, were designed, synthesized and evaluated for their in vitro antibacterial activity. These derivatives were found to have strong activity against susceptible and resistant bacteria strains. Among them, compounds 7a and 7q showed the most potent activity (0.125?µg/mL) against erythromycin-resistant S. pneumoniae expressing the mefA gene. Moreover, compounds 7f, 7i, 7p and 7z displayed remarkably improved activity (4?µg/mL) against penicillin-resistant S. aureus ATCC31007, and compounds 7a, 7b, 7f, 7p and 7z showed improved activity (8?µg/mL) against erythromycin-resistant S. pyogenes. In particular, compound 7z exhibited potent and balanced activity against the tested drug-susceptible and -resistant bacterial strains.     

Stimulation of high-affinity hormone-sensitive GTPase of human platelets by 1-O-alkyl-2-O-acetyl-sn-glyceryl-3-phosphocholine (platelet activating factor)

1-O-Alkyl-2-O-acetyl-sn-glyceryl-3-phosphocholine (platelet activating factor) inhibits human platelet adenylate cyclase via the GTP-dependent mechanism. Inhibition of adenylate cyclase correlates with the stimulation of high affinity hormone-sensitive GTPase. The half-maximal effects of PAF on both enzymes are observed at concentrations about 10(-8) M. Phentolamine, an alpha-adrenergic antagonist, does not abolish the PAF-induced inhibition of adenylate cyclase. The obtained data suggest that PAF receptors are coupled with the GTP-binding inhibitory protein.     

Effects of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (platelet activating factor) on cardiac function in perfused guinea-pig heart

The direct cardiac action of 1-O-alkyl-2-acetyl-sn-glycero-3-phosphocholine (PAF) was studied in isolated perfused guinea-pig heart preparations. PAF produced a fall in left ventricular pressure, decreases in the rate of rise of the left ventricular pressure (dp/dt) and coronary flow, but had no effect on heart rate. These results indicate that PAF is a cardiodepressant with inotropic selectivity and this effect on heart is blocked by CV-3988, a specific PAF antagonist.     

A facile method to determine the CO2/O2 specificity factor for ribulose bisphosphate carboxylase/oxygenase

A rapid method to determine the CO₂/O₂ specificity factor of ribulose 1,5-bisphosphate carboxylase/oxygenase is presented. The assay measures the amount of CO₂ and O₂ fixation at varying CO₂/O₂ ratios to determine the relative rates of each reaction. CO₂ fixation is measured by the incorporation of the moles of¹⁴CO₂ into 3-phosphoglycerate, while O₂ fixation is determined by subtraction of the moles of CO₂ fixed from the moles of RuBP consumed in each reaction. By analyzing the inorganic phosphate specifically hydrolyzed from RuBP under alkaline conditions, the amount of RuBP present before and after catalysis by rubisco can be determined.     

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.