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Biomedical subjects

M Lagarde

Publications and source records attributed to M Lagarde.

At least 181 records · Page 10Linked to original sources
PubMed1990

Modulation of prostanoid formation by various polyunsaturated fatty acids during platelet-endothelial cell interactions.

Previous studies have reported that polyunsaturated fatty acids (PUFAs) of nutritional interest may influence arachidonic acid (20:4n-6) metabolism in both platelets and endothelium, when tested separately. In the present study, platelets (PL) and cultured endothelial cells (EC) were first pre-enriched with eight different PUFAs for a two hour incubation in the presence of free fatty acid albumin pre-coated with each acid. EC, PL or both cell populations in combination, were then stimulated by thrombin (0.1 U/ml) for five minutes. Prostanoids were extracted, purified by thin-layer chromatography, and TxB2, 6-keto-PGF1 alpha and PGE2 were quantitated by radioimmunoassays. Prostanoids or dihomoprostanoids formed from cyclooxygenase substrates other than 20:4n-6 were measured by gas chromatography-negative chemical ionisation mass-spectrometry (GC-MS). When co-incubated with EC, PL produced less TxB2 (-15 and -85% in the absence and presence of thrombin, respectively). In contrast, 6-keto-PGF1 alpha increased by 189 (basal conditions) and 358% (thrombin stimulation) when PL were added to EC, in agreement with PGH2 transfers from PL to EC. PGE2, produced by both cell populations, reached amounts which roughly represent the sum of those measured in PL and EC alone, except when cells were pre-enriched with linoleic (18:2n-6) and the n-3 family fatty acids (18:3-, 20:5- and 22:6n-3). 6-keto-PGF1 alpha was markedly inhibited by adrenic acid (22:4n-6), while this acid was converted into dihomo-6-keto-PGF1 alpha, the stable metabolite of dihomoprostacyclin. 22:4n-6 also inhibited TxB2 formation and was converted into dihomo-TxA2.(ABSTRACT TRUNCATED AT 250 WORDS)

6-Ketoprostaglandin F1 alpha↗
PubMed1990

Decreased cyclic nucleotide phosphodiesterase activity in human peripheral blood mononuclear cells from elderly women.

1. Both adenosine 3':5'-cyclic monophosphate and guanosine 3':5'-cyclic monophosphate phosphodiesterase activities of peripheral blood mononuclear cells were markedly decreased in elderly women as compared with young control women. 2. In contrast, the ability of these cells to bind guanosine 5'-[beta, gamma-imido]triphosphate, a non-hydrolysable analogue of guanosine 5'-triphosphate, was the same in both groups. 3. These findings are discussed in the context of the decline in immune function which occurs with increasing age.

2',3'-Cyclic-Nucleotide Phosphodiesterases↗
PubMed1990

Characterization of A-2 receptors in postmortem human pineal gland.

We have examined the binding of the adenosine agonist radioligands [3N]N6-cyclohexyladenosine ([3H]CHA) and [3H]5'-N-ethylcarboxamidoadenosine ([3H]NECA) to membranes prepared from postmortem human pineal glands. The results showed that the A-1-specific ligand CHA did not bind to membranes. By contrast, [3H]NECA, a nonselective A-1/A-2 ligand, gave 68% specific binding of the total binding. This specific binding was nearly insensitive to the N-ethyl-maleimide pretreatment method. To characterize this binding, we used cyclopentyladenosine (50 nM). Under those conditions [3H]NECA binding at 30 degrees C was rapid and reversible; the KD determined from the kinetic studies was 141 nM. In postmortem human pineal gland, the rank order of potency of adenosine analogues and drugs competing with [3H]NECA showed the specificity for an A-2 receptor: NECA greater than 2-chloroadenosine greater than L-N6(2-phenylisopropyl)adenosine greater than 8-phenyltheophylline greater than 3-isobutyl-1-methylxanthine greater than caffeine. Guanylylimidodiphosphate (100 microM) induced a decrease in the affinity of [3H]NECA, a result suggesting the involvement of a G protein mechanism in the coupling of the adenosine receptor to other components of the receptor complex. Scatchard analysis revealed one class of binding sites for [3H]NECA with KD and Bmax ranging from 175 to 268 nM and 11.0 to 14.1 pmol/mg protein, respectively. The binding of [3H]NECA was not affected by age, sex, or postmortem delay. [3H]NECA should be a useful tool to assess brain A-2 receptor density in a variety of neuropsychiatric disorders.

Adenosine↗
PubMed1990

Benzodiazepine analogues inhibit arachidonate-induced aggregation and thromboxane synthesis in human platelets.

1. Benzodiazepine analogues inhibit human platelet aggregation induced by arachidonate with an EC50 value of 0.68 microM for PK 11195, the most potent analogue used. 2. There was a highly significant correlation between the inhibition of arachidonate-induced aggregation and the affinity for the peripheral-type of benzodiazepine binding sites. 3. There was no significant correlation between the inhibition of the platelet activating factor (PAF)-induced aggregation and the binding to the peripheral-type of benzodiazepine binding sites. 4. The inhibition of platelet aggregation seems to result from the inhibition of arachidonic acid cyclo-oxygenation, since the synthesis of thromboxane and 12-hydroxy-heptadecatrienoic acid, both cyclo-oxygenase products, was reduced. 5. Our results suggest that peripheral-type of benzodiazepine binding sites on human platelets could be linked to cyclo-oxygenase.

Arachidonic Acid↗
PubMed1990

Trans n-3 eicosapentaenoic and docosahexaenoic acid isomers exhibit different inhibitory effects on arachidonic acid metabolism in human platelets compared to the respective cis fatty acids.

N-3 trans geometrical isomers of 20:5 n-3 and 22:6 n-3 were isolated from rats fed heated linseed oil. The ability of these acids to inhibit 20:4 n-6 metabolism by human platelets was examined. The concentrations required to inhibit 50% of platelet aggregation (IC50) induced by 2.5 microM 20:4 n-6 were higher for the 20:5 delta 17t isomer compared to all cis 20:5 n-3; means 29.2 and 7.6 microM, respectively (P less than 0.05). There were no significant differences in IC50 between 22:6 delta 19t and all cis 22:6 n-3; means 4.3 and 5.6 microM, respectively (P greater than 0.05). Inhibition of action of cyclooxygenase on 20:4 n-6 was similar for 20:5 delta 17t and 20:5 n-3 when examined at their IC50s, but comparison at equal concentrations indicated that 20:5 n-3 was a significantly better inhibitor (P less than 0.05). The ability to inhibit platelet aggregation was paralleled by cyclooxygenase inhibition as determined by thromboxane B2 and 12-hydroxyheptadecatrienoic acid formation. 22:6 delta 19t appeared to inhibit cyclooxygenase more completely than 22:6 n-3, examined at their IC50s or at similar concentrations (P less than 0.05). Isomers of 20:5 n-3 and 22:6 n-3 having an n-3 cis or trans bond appear to have similar modes of action, although levels required for effectiveness are different for the C20 acids.

Arachidonic Acids↗
PubMed1989

Requirement and role of arachidonic acid in the differentiation of pre-adipose cells.

The terminal adipose differentiation of Ob1771 cells, characterized by glycerol-3-phosphate dehydrogenase activity and triacylglycerol accumulation, was studied in serum-free hormone-supplemented medium containing growth hormone, tri-iodothyronine, insulin, transferrin and fetuin. Arachidonic acid was able to substitute for a crude adipogenic fraction isolated from fetal bovine serum but not for growth hormone or tri-iodothyronine. Arachidonic acid was also able to increase in a rapid and dramatic manner cyclic AMP production; moreover it was able to amplify the adipose conversion promoted by other agents elevating cyclic AMP concentrations and to induce inositol phospholipid breakdown. Both phorbol 12-myristate 13-acetate, a protein kinase C activator and ionomycin, a Ca2+-mobilizing agent, showed potent synergy with agents elevating cyclic AMP concentrations for the promotion of adipose conversion, whereas 8-bromo cyclic GMP and 4 alpha-phorbol 12,13-dibutyrate were ineffective. The triggering of both the cyclic AMP and inositol phospholipid pathways was accompanied by a single round of cell division, and within a few days all the cells became differentiated. Similar results were obtained, after exposure to arachidonic acid, with preadipose 3T3-F442A cells and with rat adipose precursor cells in primary culture. The availability of arachidonic acid from intracellular stores and/or of exogenous origin should play a major role for the onset of critical mitoses leading to terminal differentiation in pre-adipose cells.

Adipose Tissue↗
PubMed1989

Inhibitory effect of stearidonic acid (18:4 n-3) on platelet aggregation and arachidonate oxygenation.

The effect of stearidonic acid (18:4 n-3) present in fish and some plant oils, such as black currant seed oil, was studied on human platelets. When added to platelets simultaneously with collagen, arachidonic acid or endoperoxide mimetic U46619, 18:4 n-3 appeared as a weak inhibitor of platelet aggregation. In addition, 18:4 n-3 did not alter the metabolism of exogenous arachidonic acid. In contrast, when preincubated with platelets after precoating onto albumin, 18:4 n-3 inhibited platelet aggregation induced by thrombin, collagen, arachidonic acid or U46619, and was as potent as eicosapentaenoic acid (20:5 n-3) tested under similar conditions. Stearidonic acid also altered the endogenous arachidonate oxygenation stimulated by low doses of thrombin, but to a significantly lesser extent than did 20:5 n-3. It seems therefore that, in addition to competing with endogenous arachidonate metabolism, 18:4n-3 may affect platelet aggregation by another mechanism.

15-Hydroxy-11 alpha,9 alpha-(epoxymethano)prosta-5↗
PubMed1989

Increased in vivo biosynthesis of prostacyclin and thromboxane A2 in chronic idiopathic thrombocytopenic purpura.

The production of thromboxane A2 (TxA2) and prostacyclin (PGI2) was studied in patients with chronic idiopathic thrombocytopenic purpura (10 patients) compared to central thrombocytopenia (five patients) and healthy subjects (10 subjects). This production was monitored by the assay of urinary 2,3-dinor-TxB2 and 2,3-dinor-6-keto-PGF1 alpha as respective breakdown products of TxA2 and PGI2 by stable isotope dilution assays employing negative ion-chemical gas-chromatography-mass-spectrometry. Evidence is presented for the existence of an enhanced PGI2 and TxA2 urinary excretion in the group of idiopathic thrombocytopenic purpura (ITP) patients. Moreover, production of serum TxB2 per platelet was decreased in ITP group. These results provide arguments for an in vivo platelet cyclooxygenase hyperactivity during chronic ITP.

6-Ketoprostaglandin F1 alpha↗
PubMed1989

Effects of small concentrations of eicosapentaenoic acid on platelets.

Increased platelet functions have been associated with enhanced arachidonic acid (AA) metabolism in diabetics and elderly people. We investigated these parameters in both groups after feeding low doses of pure eicosapentaenoic acid (EPA). After such an intake, platelet aggregation induced by various agonists was decreased whereas the oxygenated metabolism of endogenous AA was not substantially altered, and the fatty acid composition of plasma and platelet lipid pools did not change. In the most recent study, the measurement of vitamin E revealed that although its level in plasma was not modified, it was increased in platelets after EPA intake. It is concluded that small amounts of EPA intake do not reduce platelet aggregation in competing with AA metabolism but rather in depressing the peroxide tone of the cell.

Aged↗
PubMed1989

Differential effect of SIN-1 on thromboxane and prostacyclin formation in platelets and endothelial cells.

The effect of SIN-1, the active metabolite of molsidomine that inhibits platelet aggregation, was tested upon the oxidation of arachidonic acid in platelets and endothelial cells. The metabolism of arachidonic acid from both exogenous and endogenous sources was investigated by determining the formation of thromboxane and prostacyclin. These prostanoids were measured in platelets and endothelial cells alone or during their interaction, in the absence or presence of SIN-1. The presence of endothelial cells decreased the generation of thromboxane by the platelets, especially from endogenous arachidonate, whereas the platelets tended to increase that of prostacyclin under basal conditions. SIN-1 significantly reduced the production by the platelets of metabolites from endogenous arachidonic acid but did not affect those from exogenous sources. The reduction in metabolism of endogenous arachidonate was more pronounced in the presence of endothelial cells. In contrast, SIN-1 did not alter the production of cyclo-oxygenase metabolites of arachidonic acid in endothelial cells. Thus, the liberation of arachidonic acid, leading to prostanoid synthesis, may be regulated differentially in platelets and endothelial cells: molsidomine might be a potential antithrombogenic drug because it alters specifically the phospholipase activity in the platelets.

Arachidonic Acids↗
PubMed1989

Metabolism of endogenous arachidonic acid in weakly activated platelets. Absence of leukocyte cooperative products in whole blood.

Human platelets were incubated alone or in whole blood with specific agonists such as thrombin or collagen, and 12-hydroxy-heptadecatrienoic (HHT) and 12-hydroxy-eicosatetraenoic (12-HETE) acids were measured by HPLC as indices of platelet cyclooxygenase and lipoxygenase activities, respectively. We found that both arachidonic acid metabolites are significantly formed at concentrations of thrombin insufficient to provoke platelet aggregation. The ratio HHT/12-HETE varied with increasing concentrations of thrombin, with an increase in the absence and a decrease in the presence of albumin in the incubation. When platelets were stimulated in whole blood, this ratio favoured HHT and the addition of albumin to isolated platelets had the same effect. The formation of oxygenated products of 12-HETE by leukocyte LTB w-hydroxylase and 5-lipoxygenase in unstimulated and stimulated leukocytes, respectively, was also investigated. We failed to detect any significant amounts of these products in whole blood incubated with relatively high concentrations of collagen in the presence or absence of the chemotactic peptide FMLP. We conclude that, although 12-HETE is a good substrate for leukocyte oxygenases when incubated at high concentration with the cells alone, its oxygenation is unlikely to occur in whole blood, making 12-HETE and/or HHT potential markers of platelet activation in vivo, provided they are not substantially degraded during passage of the blood through various organs.

12-Hydroxy-5,8,10,14-eicosatetraenoic Acid↗
PubMed1989

Differential susceptibility to biological detergents of the particulate cGMP-stimulated phosphodiesterase from rat heart: preservation of the allosteric properties of the solubilized enzyme.

The effects of various biological detergents on the particulate cGMP-stimulated cAMP phosphodiesterase activity from rat heart were investigated. When added to particulate fractions, anionic and non-ionic detergents diversely increased both cAMP and cGMP phosphodiesterase activities and slightly decreased the stimulatory effect of cGMP on cAMP hydrolysis whereas cationic detergents were rather inhibitory and drastically lowered the stimulatory effect of cGMP. Among the most efficient detergents, only sodium cholate was able to solubilize phosphodiesterase activity and preserve the stimulatory effect of cGMP on cAMP hydrolysis. Furthermore, the addition of glycerol significantly improved the conservation of the allosteric properties of the enzyme. Kinetic properties of the cholate-solubilized phosphodiesterase were quite identical to those of the membrane-bound enzyme.

3',5'-Cyclic-AMP Phosphodiesterases↗
PubMed1988

Monohydroxylated fatty acid substrate specificity of human leukocyte 5-lipoxygenase and omega-hydroxylase.

Various monohydroxylated fatty acids were synthesized from eicosapolyenoic acids, namely arachidonic (20:4 omega-6), timnodonic (20:5 omega-3), dihomogammalinolenic (20:3 omega-6) and mead (20:3 omega-9) acids. 12-Hydroxy derivatives, as well as 12-hydroxy-5,8,10-heptadecatrienoic acid (HHT), were produced with platelets as the enzyme source, and 15-hydroxy derivatives were produced by soya bean lipoxygenase treatment. Each monohydroxylated fatty acid was incubated with human leukocytes in the presence or absence of the calcium ionophore A23187, and dihydroxylated products were analysed by h.p.l.c. 12-Hydroxy derivatives of 20:4 omega-6, 20:5 omega-3 and 20:3 omega-9 were similarly oxygenated by both the 5-lipoxygenase and the omega-hydroxylase. As expected, the 12-hydroxy derivative of 20:3 omega-6 was not a substrate for 5-lipoxygenase, but surprisingly, omega-6 oxygenated products, like 15-OH-20:4 or HHT, were not converted by the enzyme, although being potential substrates because of the presence of two double bonds at C-5 and C-8. omega-6 oxygenated derivatives were also poorly converted by leukotriene B4 omega-hydroxylase, a cytochrome P-450-dependent enzyme. It is concluded that both leukocyte 5-lipoxygenase and omega-hydroxylase exhibit a substrate specificity towards monohydroxylated fatty acids with respect to their double bonds and/or the carbon position of the alcohol function.

Arachidonate 5-Lipoxygenase↗
PubMed1988

Modulation of prostacyclin/thromboxane formation by molsidomine during platelet-endothelial cell interactions.

Platelet and endothelial cell metabolism of both exogenous and endogenous arachidonic acid, via the cyclooxygenase pathway, was evaluated according to thromboxane and prostacyclin formations. This was investigated in platelets, endothelial cells alone or during their interactions, in the presence or absence of SIN-1, the active anti-anginal metabolite of molsidomine. This revealed that, in contrast to the generation of thromboxane, which was decreased in the presence of endothelial cells, especially from endogenous arachidonate, that of prostacyclin increased under basal conditions as well as from endogenous arachidonate, that of prostacyclin increased under basal conditions as well as from endogenous arachidonate to a lesser extent. SIN-1 reduced thromboxane formation solely from endogenous arachidonate, and this was more pronounced when both cell populations interacted. In contrast, SIN-1 failed to decrease prostacyclin formation, which would emphasize its anti-aggregating potential. We conclude that the liberation of arachidonic acid leading to prostanoid synthesis may be differently regulated in platelets and endothelial cells, and that molsidomine might be a potential anti-aggregating drug in altering specifically thromboxane formation.

Arachidonic Acid↗