Patrick Molière

Preferential Transfer of 2-Docosahexaenoyl-1-Lysophosphatidylcholine Through an In Vitro Blood-Brain Barrier Over Unesterified Docosahexaenoic Acid

Abstract : The passage of either unesterified docosahexaenoic acid (DHA) or lysophosphatidylcholine‐containing DHA (lysoPC‐DHA) through an in vitro model of the blood‐brain barrier was investigated. The model was constituted by a brain capillary endothelial cell monolayer set over the medium of an astrocyte culture. Cells were incubated for 4 h with a medium devoid of serum, then the endothelial cell medium was replaced by the same medium containing labeled DHA or lysoPC‐DHA and incubations were performed for 2 h. DHA uptake by cells and its transfer to the lower medium (astrocyte medium when they were present) were measured. When the lower medium from preincubation and astrocytes were maintained during incubation, the passage of lysoPC‐DHA was higher than that of unesterified DHA. The passage of both forms decreased when astrocytes were removed. The preference for lysoPC‐DHA was not seen when the lower medium from preincubation was replaced by fresh medium, and was reversed when albumin was added to the lower medium. A preferential lysoPC‐DHA passage also occurred after 2 h with brain endothelial cells cultured without astrocytes but not with aortic endothelial cells cultured and incubated under the same conditions. Altogether, these results suggest that the blood‐brain barrier cells released components favoring the DHA transfer and exhibit a preference for lysoPC‐DHA.

Fatty acid composition of the rat pineal gland. Dietary modifications

When compared to brain, the fatty acid composition of the rat pineal gland revealed that the total proportion of n-6 polyunsaturated fatty acids (PUFA) was 2.3-fold higher, whereas the proportion of n-3 fatty acids was similar. Specifically, 20:4(n-6) and 18:2(n-6) were respectively 1.56- and 11.80-fold higher in the pineal than in the brain, while the proportions of 22:6(n-3) were similar in both tissues. In addition, 18:1(n-9) was found 2.15-fold lower in the pineal. Feeding adult rats with fish oil concentrates induced a significant alteration of the polyunsaturated fatty acid composition of the pineal. There was a reciprocal replacement of the n-6 by the n-3 fatty acids. Conversely, in rats fed a n-3 fatty acid-deficient diet (sunflower oil or coconut oil diet), the pineal gland contained reduced proportions of n-3 fatty acids. We conclude that the pineal gland (i) differs from the brain in containing much higher proportions of 18:2(n-6) and from the other tissues for its high proportions of 22:6(n-3) and (ii) is highly sensitive to the n-3 fatty acid diet in contrast to what is known for the brain. These findings are discussed in the context of melatonin biosynthesis, the major hormone of the pineal gland.

Leukotriene B4 level in stimulated blood neutrophils and alveolar macrophages from healthy and asthmatic subjects. Effect of beta-2 agonist therapy.

Abstract. Leukotriene B4 levels were measured after stimulation by calcium ionophore A23187: (i) in peripheral neutrophils (PMN) from allergic asthmatics, rhinitis and healthy subjects; (ii) in macrophages collected by bronchoalveolar lavage. LTB4 levels in PMNs were significantly higher in non‐treated allergic asthmatics and non‐treated subjects with rhinitis compared to controls. Beta‐2 agonist‐treated asthmatics showed a significantly decreased LTB4 production which was not different from those of controls. In vitro, LTB4 production decreased significantly after PMN incubation with Salbutamol (10‐6 mo11‐1). LTB4 produced by AM collected by BAL was measured in non‐treated (n = 5) and treated (n= 11) asthmatics with inhaled beta‐2 agonist. AM collected from all controls and non‐treated asthmatics produced LTB4. By contrast, no production of LTB4 was observed in the treated group. LTB4 production decreased when normal AM were incubated in vitro with Salbutamol (10‐8 mol 1‐1). These results suggest that biochemical differences occur in PMN and macrophages from subjects treated with beta‐2 agonist, presumably in changing the 5‐lipoxygenase pathway.

Synthesis of Amino-Dideoxy-Dl-Pentopyranoses and Their Ureido Derivatives

Abstract Various 4-deoxy-α or β-erythro or threo-pentopyranoses aminated on position 1 [6 and 19]. 2 [38] or 3 [9, 22, 23, 24, 32 and 35] were synthesised from 2-methoxy-5,6-dihydro-2H-pyran [1] and methyl 2,3-anhydro-4-deoxy-α and β-DL-erythro-pentopyranoside [11]. Different reactions were investigated including azidation. cis-oxyamination, epimine formation and oxirane aminolysis. All these amino sugars were converted into their chloroethylureido derivatives.

Platelets may inhibit leucotriene biosynthesis by human neutrophils at the integrin level

Summary. Polymorphonuclear leucocytes and blood platelets co‐operate in several pathophysiological processes, and arachidonic acid (AA) metabolites produced in response to the activation of these cells are potent mediators of their functions. We studied the role of platelets in the formation of 5‐lipoxygenase products from AA by autologous neutrophils, especially the chemotactic agent leucotriene (LT) B4. The formation of all products, namely 5‐hydroxy‐eicosatetraenoic acid (5‐HETE), LTB4 and the other LTA4‐derived metabolites, in response to the calcium ionophore A23187 was evaluated by high‐performance liquid chromatography. All the 5‐lipoxygenase products were significantly diminished by physiological concentrations of platelets. This inhibitory effect was lost when platelets were previously degranulated by thrombin in non‐aggregating conditions. Peptides containing the Arg‐Gly‐Asp‐Ser or His‐His‐Leu‐Gly‐Gly‐Ala‐Lys‐Gln‐Ala‐Gly‐Asp‐Val sequence, which prevent the adhesion of platelets to neutrophils via the fibrinogen released from platelet granules and the integrin glycoprotein IIb/IIIa, markedly decreased the inhibitory effect of non‐degranulated platelets. The production of transcellular metabolites of AA such as LTC4, the dual 5‐ and 12‐lipoxygenase product 5,12‐diHETE and lipoxins could not account for the decreased formation of 5‐HETE and LTA4‐derived metabolites. It is concluded that platelets may inhibit the neutrophil 5‐lipoxygenase activity at the integrin level and in turn may play a role in slowing down the production of LTB4 in the course of inflammation.

Modulation of norepinephrine-stimulated cyclic AMP accumulation in rat pinealocytes by n-3 fatty acids.

This work showed that docosahexaenoic (22:6n-3) and eicosapentaenoic (20:5n-3) acid supplementation for 48 h have opposite effects on the norepinephrine-stimulated cyclic AMP accumulation in rat pinealocytes. We found that 22:6n-3 supplementation of pineal cells, done by increasing specifically 22:6n-3 in phospholipid and triacylglycerol pools, led to inhibition of norepinephrine-stimulated cyclic AMP production whereas 20:5n-3 supplementation, by increasing 20:5n-3, and 22:5n-3 and 22:6n-3 in the same pools, stimulated it. In contrast, direct treatment of pinealocytes with each fatty acid (50 microM) did not affect cyclic AMP production in the presence of (0.1-10 microM) norepinephrine. The results indicate that, using pharmacological agents such as forskolin or prazosin: (a) neither basal nor forskolin-stimulated cyclic AMP levels were modified in fatty acid-supplemented cells compared to control cells; (b) in the presence of 1 microM prazosin, the activation by 20:5n-3 was still effective whereas no additional inhibition of norepinephrine stimulation was observed in 22:6n-3-supplemented cells. Taken together our results suggest that 22:6n-3 or 20:5n-3 supplementation modulates specifically the alpha 1- or beta-adrenoceptors in the rat pineal gland.

Distribution and metabolism of arachidonic and docosahexaenoic acids in rat pineal cells. Effect of norepinephrine

The time-course incorporation of 10 microM [14C]arachidonic (AA) and docosahexaenoic (DHA) acids into glycerolipids was studied in rat pineal cells. The incorporation of both labeled fatty acids into total lipids was approximately equal, but their distribution profiles among the various cell lipids showed marked differences. The esterification of [14C]DHA in the neutral lipids, triacylglycerols (TAG) and cholesterol esters (CE), was 2-fold higher than that of [14C]AA whereas the opposite could be observed in total phospholipids (PL). The order of incorporation into PL was phosphatidylcholine (PC) > phosphatidylinositol (PI) = phosphatidylethanolamine (PE) for [14C]AA and PC = PE for [14C]DHA, the incorporation of both fatty acids being not detected in phosphatidylserine (PS) and that of DHA not in PI. When using 0.5 microM [3H] fatty acids, the respective distribution patterns resembled that of fatty acids at 10 microM, except for a lower proportion in TAG. The stimulation of 3H-labeled cells by 100 microM norepinephrine induced a 170% increase of basal release of [3H]AA into the medium, while [3H]DHA was virtually not released. However, the analysis of cell labeling revealed that both [3H] fatty acid levels were decreased in PL and increased in TAG. These findings suggest different involvement for AA and DHA in the pineal function. The preferential incorporation of DHA in TAG suggests that TAG might play an important role in the pineal enrichment with DHA. The absence of DHA release after NE stimulation, which however cannot be ascertained, may raise the question of the role of DHA in NE transduction.

Uptake of 12-HETE by human bronchial epithelial cells (HBEC): effects on HBEC cytokine production.

12-HETE, the major lipoxygenase end-product of platelets and macrophages, may be released in contact of bronchial epithelium in inflammatory diseases of the lung. We have studied the outcome of 12-HETE in presence of human bronchial epithelial cells (HBEC). When HBEC were incubated with [3H]12-HETE for 30 minutes, 27.5% of total radioactivity was found in HBEC and 72.5% in supernatants. Unesterified 12-HETE accounted for 22.4% of total radioactivity, 4.5% being recovered in phospholipids, preferentially in phosphatidylcholine and phosphatidylethanolamine. No incorporation in neutral lipids was detected. 72.9% of the incubated radioactivity was recovered in un identified metabolites. As 12-HETE has been shown to modulate the expression and production of various proteins, the consequence of the 12-HETE uptake on the release of GM-CSF and IL8 by HBEC was assessed. HBEC from control subjects were cultured for 24 hours with 12-HETE (10(-9) to 10(-7)M) in the presence or absence of TNF alpha. Detectable amounts of both cytokines were released in the supernatant in basal conditions at 24hr, and TNF alpha increased significantly the release of GM-CSF. 12-HETE at 10(-7)M weakly but significantly decreased the TNF-induced release of GM-CSF from HBEC. Thus the uptake of 12-HETE could affect the epithelial cell function in some situations.

Platelets abrogate leukotriene B(4) generation by human blood neutrophils stimulated with monosodium urate monohydrate or f-Met-Leu-Phe in vitro.

Neutrophils are physiologically associated with platelets in whole blood. Inflammatory reactions can be modulated by the presence of platelets. To investigate the influence of platelets on neutrophil activity, we studied the 5-lipoxygenase (5-LOX) metabolic pathway in normal human blood neutrophils stimulated with f-Met-Leu-Phe (fMLP) or monosodium urate monohydrate (MSUM) in the presence of autologous platelets. Platelets inhibited by more than 90% the synthesis of leukotriene B4 and 5-HETE in neutrophils activated with fMLP or MSUM. The addition of exogenous arachidonic acid did not reverse the inhibitory effect of platelets on 5-LOX–generated metabolites in fMLP- or MSUM-activated neutrophils. Preincubation of neutrophils with adenosine deaminase reversed the inhibitory effect of platelets in fMLP-treated neutrophils, indicating that adenosine was responsible for the platelet inhibition of leukotriene B4 and 5-HETE formation. In contrast, adenosine deaminase had no influence on the inhibitory effects of platelets in MSUM-stimulated cells. These results suggest that platelets can inhibit the synthesis of 5-LOX products (a) by acting mainly downstream to phospholipase A2 in cells stimulated by fMLP or MSUM, (b) through adenosine when neutrophils are activated with fMLP, and (c) by an adenosine-independent mechanism in MSUM-activated neutrophils by an as-yet-unidentified mediator.