Biomedical subjects

M Lagarde

Publications and source records attributed to M Lagarde.

At least 73 records · Page 4Linked to original sources

PubMed1998

Hyperglycaemia modifies the reaction of microvessels to insulin in rat skeletal muscle.

The role played by glucose and/or insulin in local vascular regulation of tissue glucose uptake is largely unknown. Thus, the aim of this study was to examine microvascular changes induced either by hyperinsulinaemia alone or in combination with hyperglycaemia. The effects of insulin or glucose on the diameter and periodic vasomotion of precapillary arterioles (diameter < 20 microm) were determined by using the spinotrapezius muscle preparation in fasted, anaesthetized rats. Ten minutes after s.c. insulin administration, the blood insulin level was greatly increased whereas plasma glucose remained unchanged. This was associated with a marked and durable vasodilation of terminal arterioles without significant changes in vasomotion. When similar plasma insulin levels were attained by glucose infusion, tissue glucose uptake was increased in spite of a partial constriction and increased vasomotion of precapillary arterioles. Importantly, local tissue blood flow was not reduced despite the diminution in microvascular diameters. These results indicate that hyperinsulinaemia alone produces an increase in the diameter of terminal arterioles. This effect seems to be offset when the same level of hyperinsulinaemia is associated with hyperglycaemia (such as occurs postprandially), as illustrated by vasoconstriction of the muscle terminal arterioles. Our data suggest that the vasoconstriction of precapillary arterioles may be part of an active regulation for optimal glucose supply to the tissue in acute hyperglycaemic episodes. These data provide the first direct evidence that insulin and glucose can act as regulators of microflow in the skeletal muscle, as illustrated by changes in precapillary haemodynamics.

Animals↗

PubMed1998

Covalent modifications of aminophospholipids by 4-hydroxynonenal.

Lipid oxidation is implicated in a wide range of pathophysiological disorders, which leads to reactive compounds such as aldehydes. Among them 4-hydroxynonenal (4-HNE) reacts strongly with the NH2 groups of amino acids and forms mainly Michael adducts and minor Schiff-base adducts. Such reactions occur also with compounds containing thiol groups. No data are available describing 4-HNE interactions with amino-phospholipids. To investigate such a possibility, 4-HNE was incubated with either phosphatidylethanolamine (PE) or phosphatidylserine (PS) in an aqueous-organic biphasic system and the resulting products were identified by liquid chromatography-mass spectrometry (LC-MS). Our study points out the potential capacity of 4-HNE to react with phospholipids containing amino groups and particularly PE. The main resulting compounds found were a Michael adduct plus a minor Schiff base adduct, which was partly cyclized as a pyrrole derivative via a loss of water. Its stabilization as a pyrrole derivative allows to differentiate 4-HNE from the other aldehydes generated via lipid oxidation (e.g., malondialdehyde, 2-nonenal) that lack the 4-hydroxyl group. Their formation seems not to be affected when the pH varies from 6.5 to 8.5. Surprisingly, PS reacted poorly producing only a small amount of Michael adduct, the Schiff-base adduct being nondetectable. We conclude that such adducts, if they are formed in cell membranes, could alter the phospholipase-dependent cell signaling.

Aldehydes↗

PubMed1998

In vivo effect of 8-epi-PGF2alpha on retinal circulation in diabetic and non-diabetic rats.

Retinal hemodynamic responses to a F2-isoprostane, 8-epi-PGF2alpha, were quantitated in vivo in non-diabetic and diabetic rats using a video fluorescein angiography system. Vascular diameters and retinal mean circulation time were determined before and after 5 microl intra-vitreous injection of 8-epi-PGF2alpha (10(-5) to 10(-3) M), 10(-4) M 8-epi-PGF2alpha, + 10(-3) M SQ29,548 or 10(-3) M LCB2853 (two inhibitors of TXA2 receptor), 10(4) M 9beta-PGF2alpha, or the carrier in non-diabetic animals. Diabetic rats received either 8-epi-PGF2alpha 10(-4) M, or the carrier. Compared to control animals, diabetic rats presented in the basal state a venous vasodilation (P<0.01), without modification of retinal mean circulation time or blood flow. After intravitreous injection of 8-epi-PGF2alpha, a significant arterial vasoconstriction was observed in control but not in diabetic animals. This vasoconstriction was concomitant with increased retinal mean circulation time in control but not in diabetic rats, inducing an impaired reduction of blood flow. No vasoconstriction was observed after injection of either the carrier, 9beta-PGF2alpha or the isoprostane associated to the inhibitors of TXA2 receptors. This is the first direct observation that the isoprostane 8-iso-PGF2alpha is a potent vasoconstricting agent in the retina. It occurs at the arterial but not venous level, and is likely mediated through a TXA2-like receptor. Differences observed between control and diabetic animals suggest altered adaptative mechanisms toward vasoconstrictor substances (such as isoprostanes) in diabetic rats.

Angiography↗

PubMed1998

Nuclear location of PLA2-I in proliferative cells.

We have previously demonstrated that pancreatic PLA2 (PLA2-I) stimulates the proliferation of UIII cells, a stromal cell line derived from normal rat uterus. In order to gain further insight into the mechanism of action of PLA2-I, we have investigated the intracellular processing of PLA2-I. Either highly proliferative or growth arrested UIII cells were analyzed. Growth arrested cells were obtained from a contact inhibited monolayer or from aristolochic acid-treated cultures. Using cellular fractionation, western blotting, immunocytochemistry and confocal microscopy, we demonstrate that endogenous PLA2-I was mainly located in the nucleus in highly proliferative cells whereas its location was cytoplasmic in non proliferative cells. When non confluent UIII cells were incubated with nanomolar amounts of exogenous PLA2-I, the enzyme was internalized and, in the majority of cells, appeared within the nucleus. Both internalization and nuclear location of exogenous PLA2-I were suppressed by the addition of aristolochic acid to the culture medium. Binding experiments performed on purified nuclear preparations showed the presence of specific cooperative binding sites for PLA2-I. Collectively our data suggest that the proliferative effect exerted by pancreatic PLA2 in UIII cells is mediated by a direct interaction of the enzyme at the nuclear level. Putative mechanisms and targets are discussed.

Animals↗

PubMed1998

Docosahexaenoic acid lowers phosphatidate level in human activated lymphocytes despite phospholipase D activation.

N-3 polyunsaturated fatty acids from marine oil have been shown to decrease T cell-mediated immune function both in animals and humans, and to inhibit the mitogen-induced lymphoproliferative response when added to lymphocyte culture medium. As phosphatidic acid (PA) is a key mediator of the mitogenic process, the present study aims to investigate whether docosahexaenoic (DHA) and eicosapentaenoic (EPA) acids, the main n-3 fatty acids from fish oil, are able to alter the mitogen-induced synthesis of PA, when added to the culture medium of human peripheral blood mononuclear cells (PBMC). Incubation of PBMC in a medium containing 5 microM DHA bound to 5 microM human delipidated serum albumin induced a 2-fold increase in the basal PA mass whereas incubation with EPA, in the same conditions, had no effect. In contrast, both fatty acids markedly reduced the concanavalin A (ConA)-induced production of PA as compared with untreated cells. Paradoxically, phospholipase D (PLD) activity, evidenced by the synthesis of phosphatidylbutanol, was only detected in DHA-treated cells further stimulated by ConA, indicating that both DHA and ConA are required for PLD activation. Similarly, an increased diacylglycerol (DAG) mass was only observed in DHA-treated cells stimulated by ConA, whereas no modification occurred in control or EPA-treated cells stimulated or not by ConA. Furthermore, 1-butanol suppressed the ConA-induced increase of DAG mass observed in DHA-treated cells, indicating that phosphatidate was the source of the newly synthesized diacylglycerol. Altogether, these results show that, in concanavalin A-activated human peripheral blood mononuclear cells, docosahexaenoate stimulates both phospholipase D and phosphatidate phosphohydrolase activities, which ultimately results in an increased diacylglycerol production at the expense of phosphatidate.

Concanavalin A↗

PubMed1998

Astrocytes are mainly responsible for the polyunsaturated fatty acid enrichment in blood-brain barrier endothelial cells in vitro.

To determine the respective roles of endothelial cells from brain capillaries and astrocytes in the conversion of circulating 18:2n-6 and 18:3n-3 into 20:4n-6 and 22:6n-3, respectively, a coculture of the two cell types mimicking the in vivo blood-brain barrier was used. During the culture period, endothelial cells cultured on an insert were set above the medium of a Petri dish containing or not a stabilized culture of astrocytes. Five days after confluence, labeled 18:2n-6 and 18:3n-3 (10 microM each) were added to the endothelial cells and incubated for 48 h. Analogous experiments were also performed by using each cell type cultured alone in the culture device. The distribution of radioactivity in lipids and fatty acids was studied in all the compartments of the culture device. Endothelial cells cultured alone weakly converted the precursor fatty acids into 20:4n-6 and 22:6n-3. When endothelial cells were cocultured with astrocytes, their content of polyunsaturated fatty acids increased dramatically. This effect was associated with the uptake of polyunsaturated fatty acids from the lower medium (astrocyte medium). These fatty acids were released by astrocytes after they were synthesized from the precursor fatty acids that passed through the endothelial cell monolayer into the lower medium. Polyunsaturated fatty acids were released by astrocytes as unesterified fatty acids and as phospholipids (mainly phosphatidylcholine and lysophosphatidylcholine) even when the medium was devoid of serum. These results suggest that astrocytes could play a major role in the delivery of essential polyunsaturated fatty acids to the barrier itself and to the brain.

Animals↗

PubMed1997

Eicosapentaenoic and docosahexaenoic acids reduce PGH synthase 1 expression in bovine aortic endothelial cells.

To enlighten the mechanism of inhibition of prostacyclin (PGI2) production by n-3 fatty acids, eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids, cultured endothelial cells were incubated with albumin bound-EPA or -DHA for 22 h. Under these conditions, PGI2 formation in response to bradykinin, calcium ionophore or exogenous arachidonic acid was equally inhibited by 50%, suggesting that the inhibition might occur downstream the phospholipase step, likely at the level of PGH synthase and/or PGI2 synthase activities. Western blot analysis indicated that the mass of the constitutive isoform of PGH synthase (PGH synthase 1), but not PGI2 synthase, was significantly reduced in n-3 fatty acid-enriched cells. In subsequent experiments, PGH synthase 1 mRNA level, measured by northern blotting, was also decreased in n-3 supplemented cells. This reduction was not due to mRNA destabilization. None of these parameters were altered by similar enrichment with oleic acid (OA). These results suggest that EPA and DHA may affect PGH synthase 1 expression, presumably at the transcriptional level.

Animals↗

PubMed1997

Selective stimulation of a cAMP-specific phosphodiesterase (PDE4A5) isoform by phosphatidic acid molecular species endogenously formed in rat thymocytes.

We have previously reported that concanavalin A (ConA) stimulation of rat thymocytes induces an increase in the cellular phosphatidic acid mass as well as a change in its fatty acid composition. An increase in phosphodiesterase (PDE) activity, mostly due to cAMP-specific (PDE4) isoforms, has also been observed in thymocytes stimulated by ConA. Furthermore, phosphatidic acid was able to stimulate PDE4 activity in vitro. In the present study, cAMP levels have been shown to decrease upon ConA stimulation of thymocytes. Decreasing phosphatidic acid level using diacylglycerol kinase inhibitors induced a parallel decrease of the ConA-stimulated cAMP-specific PDE activity in these cells. Analyses of phosphatidic acid molecular species in cells stimulated for 5 min by ConA revealed a significant increase in 1-stearoyl-2-arachidonoyl-sn-glycerol-3-phosphate and a relative decrease in the other molecular species of phosphatidic acid, mainly species containing palmitate. On the other hand, phosphatidic acid extracted from ConA-stimulated cells activated more efficiently the recombinant PDE4A5 isoform in vitro, as compared to phosphatidic acid extracted from unstimulated cells. In addition, phosphatidic acid species containing unsaturated fatty acids were stimulatory, while those containing two saturated fatty acids had only a marginal effect on the enzyme activity. Taken together, these data suggest that the mitogenic stimulation of thymocytes is accompanied by the synthesis of peculiar phosphatidic acid molecular species able to activate a PDE4 isoform. This activation might be of physiological relevance since cAMP is a major negative effector of the mitogenic response.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed1997

Low concentrations of lipid hydroperoxides prime human platelet aggregation specifically via cyclo-oxygenase activation.

There is mounting evidence that lipid peroxides contribute to pathophysiological processes and can modulate cellular functions. The aim of the present study was to investigate the effects of lipid hydroperoxides on platelet aggregation and arachidonic acid (AA) metabolism. Human platelets, isolated from plasma, were incubated with subthreshold (i.e. non-aggregating) concentrations of AA in the absence or presence of hydroperoxyeicosatetraenoic acids (HPETEs). Although HPETEs alone had no effect on platelet function, HPETEs induced the aggregation of platelets co-incubated with non-aggregating concentrations of AA, HPETEs being more potent than non-eicosanoid peroxides. The priming effect of HPETEs on platelet aggregation was associated with an increased formation of cyclo-oxygenase metabolites, in particular thromboxane A2, and was abolished by aspirin, suggesting an activation of cyclo-oxygenase by HPETEs. It was not receptor-mediated because the 12-HPETE-induced enhancement of AA metabolism was sustained in the presence of SQ29, 548 or RGDS, which blocked the aggregation. These results indicate that physiologically relevant concentrations of HPETEs potentiate platelet aggregation, which appears to be mediated via a stimulation of cyclo-oxygenase activity.

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

PubMed1997

Advanced glycation end products induce specific glycoprotein alterations in retinal microvascular cells.

In order to investigate the mechanisms involved in diabetic retinopathy, we studied the effects of advanced glycosylation end products (AGE) on retinal microvascular cell glycoproteins. Bovine retinal pericytes (BRP) and endothelial cells (BREC) were incubated in the presence of AGE-modified albumin and cell glycoproteins analyzed by lectin affinoblotting and metabolic radiolabeling with sugar precursors. Selective modifications in the glycoprotein sugar chains were observed mainly in BREC and for a 210 kDa membrane glycoprotein. Indeed, a 40% decrease of alpha(2,3) sialic acid, beta(1,3) galactose or alpha(1,6) fucose content was observed without significant protein amount changes. These glycoprotein alterations were related to the concentration of AGE. Neither BRP nor BREC glycoproteins were modified when cells were incubated with high glucose or fructose concentrations. These results suggest a new diabetic pathogenic mechanism in which a protein post-translational modification, in this case glycation, could modify another post-translational process such as the enzymatic glycosylation.

Animals↗

PubMed1997

Correlation between arachidonic acid oxygenation and luminol-induced chemiluminescence in neutrophils: inhibition by diethyldithiocarbamate.

Neutrophils from allergic subjects were hypersensitive to stimulation by low calcium ionophore concentration (0.15 microM), resulting in an increased formation of leukotriene B4 (LTB4), 5S-hydroxy-6,8,11,14-(E,Z,Z,Z)-eicosatetraenoic acid (5-HETE), and other arachidonic acid metabolites through the 5-lipoxygenase pathway. In parallel, luminol-dependent chemiluminescence was also higher in neutrophils from allergic patients at the basal state and after stimulation by calcium ionophore, revealing an enhancement of radical oxygen species and peroxide production. The activity of glutathione peroxidase, the main enzyme responsible for hydroperoxide reduction, was lowered in these cells. Diethyl-dithiocarbamate (DTC) induced a concentration-dependent decrease in chemiluminescence and arachidonic acid metabolism after neutrophil stimulation. These data show that the elevation of arachidonic acid metabolism in neutrophils from allergic patients is strongly correlated with oxidative status. This elevation may be the consequence of an increased cellular hydroperoxide known to activate 5-lipoxygenase (5-LOX) activity and/or an increased arachidonic acid availability, due either to phospholipase A2 (PLA2) activation or inhibition of arachidonate reesterification into phospholipids. Lowering this oxidative status was associated with a concomitant decrease of this metabolism. Our results suggest that the effect of DTC may be the consequence of an inhibition of peroxyl radical and cellular lipid hydroperoxide production. Thus, DTC may modulate arachidonic acid metabolism in neutrophils by modulating the cellular hydroperoxide level.

Adult↗

PubMed1997

Effects of fatty acids on human platelet glutathione peroxidase: possible role of oxidative stress.

Highly polyunsaturated fatty acids of the n-3 family are known to be inhibitors of platelet functions, but these fatty acids (FA) may alter the platelet antioxidant status, depending on their concentrations. The present study was aimed to investigate the effect of various FA on glutathione-dependent peroxidase (GPx), the required antioxidant enzyme for degrading FA hydroperoxides. Human platelets were enriched in vitro with either n-3 (18:3, 20.5, or 22.6), n-6 (18:2 or 18:3) FA, 18:1 n-9 or 16:0, and the GPx activity was then measured. It was found that n-3 FA enhanced the GPx activity whereas the others did not affect the enzyme activity. The increased GPx activity was associated with an increased amount of the enzyme measured by Western blotting. The enhanced activity and amount of GPx induced by 22:6n-3, the most potent activator among the n-3 FA, was completely abolished in the presence of cycloheximide at a concentration known to inhibit platelet protein synthesis. Because platelets are devoid of nucleus, which rules out the involvement of transcriptional factors, this suggests that 22:6n-3 might act at a translational level. On the other hand, 22:6n-3 treatment increased the malondialdehyde formation and decreased the vitamin E level in platelets, both events that could be prevented by the antioxidant epicatechin. Because epicatechin also suppressed the enhancement of both the activity and amount of GPx induced by 22:6n-3, we conclude that the increased GPx activity (possibly via protein synthesis) might be associated with an oxidative stress induced by 22:6n-3 and/or 20:4n-6 released from the platelet endogenous pool in the course of the 22:6n-3 enrichment.

Blood Platelets↗

PubMed1997

Growth modulation of retinal microvascular cells by early and advanced glycation products.

To investigate the possible implication of non-enzymatic glycosylation in the etiopathogenesis of the diabetic retinopathy, we studied the effect of early and advanced glycation products on the growth of retinal microvascular cells. Glucose modified products were obtained by incubating bovine serum albumin or fetal bovine serum with 0.5 M glucose for 10 (early glycation products: EG-BSA and EG-FBS, respectively) or 60 days (advanced glycation end products: AGE-BSA and AGE-FBS, respectively). Cell growth was assessed by cell counting and DNA content determination. EG-BSA or AGE-BSA significantly decreased pericyte proliferation after 8 days of culture (33 and 13% inhibition, respectively). Concerning endothelial cells, EG-BSA reduced proliferation to 40% whereas AGE-BSA increased it to 156% after 4 days of culture. The glucose-treated sera didn't exhibit the same growth effects, neither the EG-FBS nor the AGE-FBS significantly affected endothelial cell proliferation. Only the AGE-FBS showed a significant inhibitory effect on pericyte proliferation (40% inhibition). We conclude that retinal microvascular cell growth in vitro could be differently modulated by early and advanced glycation products. The inhibitory effect of AGEs observed on pericyte growth, suggests that glycoxidation could be implicated in the pericyte loss observed in diabetic retinopathy.

Animals↗

PubMed1997

Involvement of lipid peroxidation in platelet signalling.

A well-known signalling pathway in blood platelets consists in the release of arachidonic acid (AA) from membrane phospholipids and its specific oxygenation into bioactive derivatives. In particular, cyclic prostaglandin endoperoxides and thromboxane A2 are potent inducers of platelet functions and are produced in greater amounts when the level of lipid hydroperoxides is higher than normal, as 'physiological concentrations' of such peroxides activate the cyclooxygenation of AA. In this context, a lower activity of platelet glutathione peroxidase (GPx), the key-enzyme for the degradation of lipid hydroperoxides, has been reported in aging, which will ensure a longer life span to those peroxides. Accordingly, the biosynthesis of pro-aggregatory prostanoids is elevated in platelets from the elderly. On the other hand, fatty acids from marine origin have been recognized as inhibitors of platelet functions, and they may alter the redox status of cells. They may for instance increase the platelet GPx activity, an effect that can be prevented by antioxidants. Overall, these data point out the relevance of the redox status in platelet functions.

Aged↗

PubMed1997

Involvement of cell-cell interactions in the pathogenesis of diabetic retinopathy.

Retinopathy is a severely disabling complication of diabetes mellitus whose underlying mechanisms are still obscure. The key question is why retinal microvessels are so reactive to the diabetic environment, whereas other microvessels show no evidence of alteration. The answer could lie in the particular structure and location of retinal microvessels since they are composed of, and surrounded by, various types of cells, thereby favouring cell-cell interactions which occur between cells of the capillary wall itself but also with circulating blood cells and retinal neural cells. In the retinal capillary wall, pericytes are in close relation with underlying endothelial cells, and both cell types have close contacts with the capillary basement membrane. Adhesion molecules and cell surface glycoconjugates appear to be the main mediators of interactions between circulating blood cells and capillary endothelial cells, whereas growth factors seem to play a major role in interactions between glial and capillary wall cells in the retina. Biochemical dysfunctions observed in diabetes, such as glycation of proteins and enhanced oxidative stress, could modify these cell-cell and cell-matrix interactions, thereby disturbing the complex cellular organization in which retinal microvessels are embedded. The aim of this review was to provide an overall, nonexhaustive description of some types of cellular interactions that may underlie the pathogenic mechanisms involved in flow and growth changes leading to diabetic retinopathy.

Animals↗

PubMed1997

Human plasma albumin transports [13C]docosahexaenoic acid in two lipid forms to blood cells.

Docosahexaenoic acid (22:6) decreases blood platelet function and is highly concentrated in the brain where its depletion leads to functional impairments. Because the platelets and blood brain barrier capillary endothelium cannot hydrolyze the complex lipids for fatty acid (FA) uptake, nonesterified FA (NEFA) bound to albumin are assumed to be the delivery route of FA to these cells. The supply of 13C-labeled 22:6 to blood cells by plasma albumin was studied in humans after a single ingestion of this FA esterified in a triglyceride (TG). The 22:6 13C/12C ratio, measured by gas chromatography combustion-isotope ratio mass spectrometry was measured in lipid classes from albumin, platelets, leukocytes, and erythrocytes (taken as a tentative index of the brain uptake). Nonesterified [13C]22:6 bound to albumin was rapidly produced after ingestion, as a result of the hydrolysis of very low density lipoprotein (VLDL) plus chylomicron TG. We found that albumin carried another source of 22:6, lyso-phosphatidylcholines (lyso-PC), in which [13C]22:6 accumulated while the nonesterified [13C]22:6 reached its minimal plasma concentrations. Computation of the relative contribution of NEFA and lyso-PC for the [13C]22:6 delivery to platelets and erythrocytes showed that the [13C]22:6 supply to platelets occurred uniquely through NEFA, whereas this pool was weakly involved in the delivery to erythrocytes. In contrast, lyso-PC was uniquely concerned with the 22:6 delivery to erythrocytes and represented the major part of this supply. We conclude that plasma albumin carries 22:6 in two lipid forms that are involved differently in the delivery of this FA to target cells.

Biological Transport, Active↗

PubMed1997

Basal concentrations of free and esterified monohydroxylated fatty acids in human blood platelets.

Monohydroxylated fatty acids (HO-FA), namely 12-hydroxyeicosatetraenoic and 12-hydroxyheptadecatrienoic acids, are enzymatically formed in response to platelet activation. Different techniques, including gas chromatography (GC) and liquid chromatography-mass spectrometry (LC-MS), have been described to measure HO-FA in activated cells, but they are not well-adapted to resting cells. Measurements of free and esterified HO-FA at basal concentration require the prevention of platelet activation. For this purpose, such an activation was minimized by adding various inhibitors to the anticoagulant. Platelet recovery was greater in the protected group than in controls (473 x 10(9) +/- 4.0 x 10(9) platelets/L vs 410 x 10(9) +/- 4.53 x 10(9) platelets/L, respectively) (mean +/- SEM, n = 9, P < 0.05). Lipids were extracted and immediately hydrogenated to avoid fatty acid autoxidation occurring during the workup. Unesterified and esterified HO-FA were analyzed by GC-MS, and the former were lower in the protected group (1.52 +/- 0.84 pmol/10(9) platelets) than in the unprotected one (12.63 +/- 10.52 pmol/10(9) platelets) (mean +/- SEM, n = 9, P < 0.05). Interestingly, only traces of HO-FA were detected in both the triglyceride and sterol ester fractions, and they were also weakly esterified in phospholipids.

Alprostadil↗

PubMed1996

Arachidonic acid up-regulates and prostaglandin E2 down-regulates the expression of pancreatic-type phospholipase A2 and prostaglandin-endoperoxide synthase 2 in uterine stromal cells.

It is well known that arachidonic acid, as a substrate of prostaglandin G/H synthase (PGHS), is converted into prostaglandins of the two-series. In this work, we attempted to determine whether arachidonic acid and prostaglandin E2 might regulate the expression of PGHS and the pancreatic-type phospholipase A2 (PLA2I), which may be involved in the liberation of arachidonic acid from membrane phospholipids. For this purpose, we used the uterine stromal cell line UIII, which produces prostaglandin E2 and expresses both the constitutive and inducible PGHS enzymes (PGHS1 and PGHS2) and PLA2 I. The results show that PGHS1, which is expressed at a high level in UIII cells, was not modified by arachidonic acid. The expression of PGHS2 and PLA2 I was up-regulated by increasing arachidonate concentrations (1-10 microM). The maximal response was obtained at 24 h, reaching a 2.3-fold and 2.6-fold increase for PGHS2 and PLA2 I expression, respectively, compared to the control level. To discriminate between the effect of arachidonic acid and that of prostaglandins, which are highly increased in the presence of exogenous arachidonic acid, we treated the cells with two inhibitors of PGHS activity, aspirin and meclofenamic acid. Both inhibitors failed to suppress the arachidonate-induced increase of PLA2 I and PGHS2 expression and even enhanced it either in the presence or absence of arachidonic acid. In contrast, the addition of prostaglandin E2 to the culture medium decreased the expression of both enzymes in a dose-dependent manner, the maximal response being reached at 1 microM. We conclude that arachidonic acid up-regulates the expression of PLA2 I and PGHS2 in the uterine stromal cells, independently of prostanoids, and that prostaglandin E2 is capable of down-regulating enzyme expression.

Animals↗