Secreted and intracellular phospholipases A2 inhibition by 1-decyl 2-octyl-glycerophosphocholine in rat peritoneal macrophages.

Compounds able to inhibit phospholipases A2 can be considered as potential anti-inflammatory drugs. In this respect, the inhibitory effect of the phospholipid analogue 1-decyl 2-octyl-rac-glycero-3-phosphocholine (decyloctyl-GPC) added to the culture medium of rat peritoneal macrophages stimulated with ionophore A23187 was determined. (a) The substrate of phospholipase A2 1-octadecanoyl 2-[14C]eicosatetraenoyl-sn-glycero-3-phosphocholine ([14C]20:4-GPC) was added to the culture medium.

High rate of intestinal absorption of the phospholipid anlaogue 1-dodecyl 2-[1-14C] octanamido-sn-2-deoxy-glycero-3-phosphocholine in the rat.

The phospholipid analogue with two short fatty chains, 1-dodecyl-2-[1-14C] octanamido-sn-2-deoxy-glycero-3-phosphocholine ([14C] phospholipid analogue), with a non-hydrolyzable bond at position 2 of the glycerol, is an inhibitor of phospholipase A2. It was obtained after chemical synthesis and 0.5 micromol was solubilized in Na+ taurocholate with an equimolar amount of 1-octadecanoyl 2-[3H]eicosatetraenoyl-sn- glycero-3-phosphocholine which is the current substrate of phospholipases A2.

Uptake of the phospholipase A2 inhibitor 1-dodecyl 2-[1-14C] octanamido-sn-2-deoxy glycero-3-phosphocholine by peritoneal macrophages.

The [14C] phospholipid analogue 1-dodecyl-2-[1-14C] octanamido-sn-2-deoxy glycero-3-phosphocholine was synthetized. With 2 short fatty chains linked by alkyl and amido bonds to positions 1 and 2 of the glycerophosphate backbone, it was an inhibitor of phospholipase A2 in ionophore A23187-stimulated macrophages. Its uptake by rat peritoneal macrophages and its resistance towards phospholipases A2 were determined at nanomolar or micromolar concentrations in the culture medium.

Structure-activity of three phospholipid analogues towards inhibition of phospholipase A2 in macrophages.

At concentrations 1-20 microns in culture medium of rat peritoneal macrophages which were stimulated with ionophore A23187, the phospholipid analogues 1-decyl-2-octyl-glycerophosphocholine and 1-dodecyl-2-octanamido-2-deoxy glycerophosphocholine were found more potent inhibitors than 1-octyl-2-deoxy glycerophosphocholine to lower the phospholipase A2 activities. The inhibitory effect was measured by [3H] eicosatetraenoic acid ([3H]20:4) release in macrophages and extracellular fluids and synthesis of [3H] eicosanoids after incubation of macrophages with traces of the molecular species of lecithin 1-octadecanoyl-2-[3H] eicosatetraenoyl glycerophosphocholine. The three phospholipid analogues developed higher inhibitory effects than mepacrine, dexamethasone or bromophenacyl bromide, at corresponding concentrations in medium.

Dissociate arachidonic acid release from eicosanoid synthesis in rat peritoneal macrophages is possible using concomitantly eicosapentanoic acid as a phospholipid analogue.

Activated macrophages exposed to the association of eicosapentanoic acid 20:5 n-3 and a synthetic non hydrolysable phospholipid analogue maintained a discrete synthesis of active eicosanoids. 20:4 n-6 split from the internalized 20:4-GPC was accumulated in cells and extracellular fluids. This combination thus represents a novel approach to reduce the 20:4 n-6 cascade.

New antiinflammatory compounds that inhibit tumor necrosis factor production: probable interaction with protein kinase C activation.

We have previously described a family of benzamide derivatives that showed antiinflammatory activity in vivo on carragenin-induced paw edema and experimental cerebral edema. Those compounds inhibited eicosanoids production from activated macrophages (M phi) without inhibiting cyclooxygenase. To further investigate their antiinflammatory activity and compare it to that of classical cyclooxygenase inhibitors, we analyzed their effect on the production of a major proinflammatory cytokine, tumor necrosis factor (TNF-alpha), by in vitro-activated peritoneal macrophages.

Severe modifications of ether- ester- or diester-linked glycerolipid and non glycerolipid synthesis in human neuroblastoma LAN-1 cells cultured with octadecylmethylglycerophosphocholine.

The growth rate of the human neuroblastoma LAN-1 cells was decreased half after 48 h of incubation with dialkylglycerophosphocholine 1-O-octadecyl 2-O-methyl-sn-glycero-3-phosphocholine (ET-18-O-CH3), at 4 microM. Four radiolabelled precursors, [3H]hexadecanol, [3H]hexadecanoic, [3H]arachidonic acids, or N-acetyl[14C] neuraminic acid were added in the culture medium to follow their cell incorporation among various glycerolipids, gangliosides and eicosanoids. Several modifications of the glycerophospholipid synthesis induced by ET-18-O-CH3 were observed.

Influence of alkyl chain lengths in dialkylglycerophosphocholines towards phospholipase A2 inhibition in macrophages.

Rat peritoneal macrophages were cultured with a specific and potent phospholipase A2 activator A 23187, with 1-stearoyl-2-[3H]arachidonoyl-sn-GPC as source of [3H] arachidonic acid, and with a dialkyl-GPC, at 2, 10 or 20 microM. Four dialkyl-GPCs were prepared by chemical synthesis. Position 2 of rac-glycerol was alkylated with an alkane chain of 8 carbons and position 1 was alkylated with various alkane chains (8, 10, 12, or 16 carbons).

Comparison of macrophage phospholipid radiolabelling methods for measuring phospholipase A2 inhibition.

Rat peritoneal macrophages activated by ionophore A 23187, were labelled after introduction in the culture medium of 1-0-stearoyl 2-0-[3H] arachidonylglycero-3-phosphocholine (as unique source of tritiated arachidonic acid), or [3H] arachidonic acid which was esterified by cells in phospholipids and triglycerides or remained non esterified. With either cell-labelling method, stimulated macrophages produced tritiated nonesterified fatty acids and eicosanoids which were isolated from cell and medium lipids. When introduced into the culture medium at 1, 5 or 10 microM, the membrane phospholipid analogue 1,2 di-O-hexadecylglycerophosphocholine (dihexadecyl-GPC), but not the lysolecithin analogue 1-0-octadecyl 2-0-methylglycero phosphocholine, lowered phospholipase A2 activity with either labelling method.

Effect of octadecylmethylglycerophosphocholine and hexadecylmethylglycerol on arachidonic acylation or release in macrophages.

1-O-Octadecyl-2-O-methyl-sn-glycero-3-phosphocholine (ET-18-O-CH3) and 1-O-hexadecyl-2-O-methyl-sn-glycerol (hexadecylmethylglycerol) are dialkylglycerolipids poorly split by the lipolytic enzymes which can activate macrophages in culture. This study concerned the incorporation of [3H]arachidonate into glycerolipids of rat peritoneal macrophages and its subsequent release. Cells cultured with ET-18-O-CH3 or hexadecylmethylglycerol were labelled according to two procedures: (1) Cells were incubated first with dialkylglycerolipid and then with the tritiated arachidonate: hexadecylmethylglycerol was practically inactive.

[Inhibition of phospholipase A2 of peritoneal macrophages in rats by 1,2-di-O-hexadecyl-rac-glycero-3-phosphocholine].

The 1-O-stearoyl-2-O-[3H] arachidonyl-sn-glycero-3-phosphocholine, introduced in the culture medium, was taken up by the peritoneal macrophages activated by the ionophore A 23187. After intracellular phospholipase A2 activity, the [3H] arachidonic acid was found in cells and in extracellular fluids. It also reached the eicosanoid synthesis.

The 1-O-octadecyl-2-O-methyl-sn-glycero-3-phosphocholine causes a differential incorporation of hexadecanol into neutral ether ester glycerolipids of 2 variant cell lines of rat colon carcinoma.

The 1-O-octadecyl 2-O-methyl-sn-glycerophosphocholine (ET-18-O-CH3), when incubated for 24-48h with cells in culture, exerts a highly selective cytotonic activity against a variety of tumor cells that is not seen in normal ones. In this study, we present data which indicate that this exogenous molecule altered the endogenous synthesis of the neutral ether, ester-sn-glycerols, in 2 variant cell lines of a rat colon carcinoma. ET-18-O-CH3, at 20 microM in the medium and for an incubation of 48h, inhibited the growth rates of the PRO cells which have the ability to metastasize and of the REG cells (the regressive cell line), by, respectively, 54 and 67%, as measured after [3H] thymidine uptakes.

Hexadecanoic and neuraminic acid incorporations in two rat colon carcinoma cell lipids: selective influence of 1-O-octadecyl 2-O-methyl-3-phosphocholine on glycerolipid and ganglioside biosynthesis.

[3H] hexadecanoic and N-acetyl [14C] neuraminic acids were incorporated in glycerolipids or gangliosides of 2 rat colon carcinoma cell lines, having (PRO cells), or not (REG cells) invasive capacities when inoculated in syngeneic BD IX rats. The cells were cultured (48 h) in presence of 1-0-octadecyl-2-0-methyl-3-phosphocholine (ET 18-0-CH3) 20 or 40 microM, which, on transformed cells, inhibits the cell growth, modifies the glycerolipid biosynthesis, and activates the sialyltransferases. ET 18-0-CH3 20 microM activated, in PRO and in REG cells the incorporation of [3H] hexadecanoate in monosialogangliosides (1.

[Comparative study on the digestion of radiolabelled vicilin, legumin and lectin of Pisum sativum in the rat].

Storage proteins, vicilin, legumin as well as the lectin, purified from pea seed proteins, were radiolabelled with [14C]- or [3H]-formaldehyde, then mixed with the pea flour which was incorporated in a liquid meal and given to rats through a gastric tubing. Radiolabelled casein (75% of the total proteins in the meal) and triolein were added to the meal and the animals were killed after 2 and 7 h. Samples were taken from the stomach and intestinal contents and from the intestinal mucosa and the liver.

Effect of micellar lipids on rabbit intestinal brush-border membrane phospholipid bilayer integrity studied by 31P NMR.

The effect of biliary salts and fatty acids on the bilayer structure of rabbit intestinal brush-border membranes was studied using the nonperturbing probe 31P NMR. The broad, asymmetric lineshape of the 31P NMR spectrum of isolated brush-border vesicles demonstrates that their component phospholipids are organized in extended bilayers. These membranes are not significantly perturbed by incubation with physiological concentrations of biliary salts (3, 9, 18 mM), demonstrating that the vesicles are highly stable, corresponding to their biological function.

[High phosphatidylcholine weights compared to lysophosphatidylcholine weights, in micellar intestinal contents, in the rat (author's transl)].

Casein hydrolysat, lactose and lipids (100 mg of fatty acids) were introduced in the stomach of rats by a gastric tube: either pure tri-oleoylglycerol, or phospholipids, or phosphatidylcholines, or the mixture 9/1 to fatty acid weight of tri-oleoylglycerol-phospholipids or phosphatidylcholines. The rats were killed 2 h later. The intraluminal intestinal lipids of the oil and micellar phases were separated after microfiltration (Millipore filters) in preference to the filtration by gel chromatography on polyacrylamide agarose, as an hydrolysis of intraluminal phospholipid occurred after the column elution.

In vitro and in vivo effects of exogenous lipids on the enzymatic hydrolysis of rat bile phospholipids.

The addition of total phospholipids, phosphatidylcholines, triglycerides, cholesterol or glycerol to incubation media containing rat pancreatic juice and bile labeled with [9, 10 3H2] oleic acid (90% of the radioactivity present as phospholipids) had no effect on the hydrolysis of bile endogenous phospholipids. The introduction of 2 or 10 mg of phosphatidylcholines and 0.5 ml of bile (approximately 1.

The behavior of rat bile phospholipids in the intestine and in incubation media containing pancreatic juice.

Samples of radioactive bile were collected from rats after intravenous injection of potassium soaps ([9-10 3H2] or [1 14C] oleate, [1 14C] linoleate or [9-10 3H2] palmitate). These radioactive acids were chosen because it is well established that, in natural phosphatidyl cholines, palmitic acid is located chiefly at the 1 position and linoleic and oleic acids at the 2 position. After incubation of bile with pancreatic juice, the labeling of unchanged biliary phospholipids was higher when native bile was labeled with oleic acid than with palmitic or linoleic acids.

The effects of administering N-(2-benzoyloxyethyl) norfenfluramine to rats on the hepatic synthesis of glycerolipids.

N-(2-Benzoyloxyethyl) norfenfluramine (S-780) was administered to rats by stomach tube at a dose of 50 mg kg-1 of body weight. Livers of the rats which were given an acute dose of the drug synthesized more triacylglycerol, phosphatidylcholine and phosphatidylethanolamine from [1,3-3H]glycerol and [14C]palmitate than did those of control rats. The measurements were made by injecting a mixture of the radioactive precursors into the portal veins of anaesthetized rats and freeze clamping a portion of the liver 1 min later.

[Variations in the lipid and fatty acid composition of cardiac tissue during perfusion of hearts isolated from rats on a high colza oil diet].

After perfusion, during one hour, of isolated working hearts extracted from Rats fed (60 days) with diet containing 15% of rapeseed oil, we have found a decrease of triglycerides and phosphatidic-cardiolipin fractions, particularly of fractions containing erucic acid. In contrast with this finding, lipids and phospholipids of hearts from Rat feed with peanut oil is not decreased and fatty acid composition slightly altered.