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Biomedical subjects

M F Simon

Publications and source records attributed to M F Simon.

At least 19 recordsLinked to original sources
PubMed2000

Identification of pancreatic type I secreted phospholipase A2 in human epidermis and its determination by tape stripping.

Phospholipases A2 (PLA2) catalyse the release of fatty acids from the sn-2 position of phospholipids and have been suggested to play a key part in permeability barrier homeostasis. Using a sensitive and versatile fluorometric method, significant PLA2 activity has been detected in both human skin homogenates and tape strippings of stratum corneum. Based on various properties (resistance to heat and sulphuric acid treatment, neutral optimal pH, absolute requirement for millimolar calcium concentrations, inhibition by dithiothreitol and p-bromophenacyl bromide, and resistance to a trifluoromethyl ketone derivative of arachidonic acid, AACOCF3, a specific inhibitor of cytosolic PLA2), this enzyme was characterized as a secretory PLA2 (sPLA2). Immunohistochemistry revealed strong labelling of type I pancreatic sPLA2 at the stratum corneum-stratum granulosum junction, type II sPLA2 being undetectable. An increase in PLA2 activity in tape-stripped material from the deepest level of the stratum corneum was correlated with partial morphological disappearance of type I sPLA2 immunolabelling. Our data thus provide the first convincing evidence that pancreatic sPLA2 is significantly expressed in human epidermis, where it might participate in the accumulation of free fatty acids contributing to the permeability barrier. In addition, our method for determining PLA2 activity in easily available tape strippings should allow further clinical studies aimed to explore possible PLA2 abnormalities in various dermatoses.

Adolescent↗
PubMed2000

Identification of two secreted phospholipases A2 in human epidermis.

Phospholipases A2 are enzymes that catalyze the release of fatty acids from the sn-2 position of phospholipids. Fatty acids have been suggested to play a key role in the barrier function of the epidermis. The aim of this study was to identify and characterize the type of secretory phospholipase A2 expressed in human epidermis. We report the molecular cloning of two secretory phospholipase A2 in the human epidermis. The first enzyme is identical to human pancreatic type IB phospholipase A2. Western blots revealed a 14 kDa protein localized in the soluble fraction. The second phospholipase A2 is identical to human synovial type IIA enzyme and is localized in the membrane fraction. By semiquantitative reverse transcription-polymerase chain reaction performed on horizontal sections of the epidermis, we found that the mRNAs of both phospholipases A2 were expressed mainly in the basal layers of the epidermis. Our data thus provide evidence for the expression of two secretory phospholipases A2 in human epidermis. The different localization of these two secretory proteins strongly suggests that each enzyme might have a specific role in skin physiology and probably in the barrier function. Taken together, these data validate the reverse transcription-polymerase chain reaction technique performed on thin sections as a first approach to detect gene expression in different layers of the epidermis.

Amino Acid Sequence↗
PubMed2000

A simple and highly sensitive radioenzymatic assay for lysophosphatidic acid quantification.

The objective of the present work was to develop a simple and sensitive radioenzymatic assay to quantify lysophosphatidic acid (LPA). For that, a recombinant rat LPA acid acyltransferase (LPAAT) produced in Escherichia coli was used. In the presence of [(14)C]oleoyl-CoA, LPAAT selectively catalyzes the transformation of LPA and alkyl-LPA into [(14)C]phosphatidic acid. Acylation of LPA was complete and linear from 0 to 200 pmol with a minimal detection of 0.2 pmol. This method was used to quantify LPA in butanol-extracted lipids from bovine sera, as well as from human and mouse plasma. This radioenzymatic assay represents a new, simple, and highly sensitive method to quantify LPA in various biological fluids.

Acyltransferases↗
PubMed1999

Structure-activity analysis of the effects of lysophosphatidic acid on platelet aggregation.

Lysophosphatidic acid (1-acyl-sn-glycero-3-phosphate or LPA) is a phospholipid mediator displaying numerous and widespread biological activities and thought to act via G-protein-coupled receptors. Here we have studied the effects on human platelets of a number of LPA analogues, including two enantiomers of both N-palmitoyl-(L)-serine-3-phosphate ((L) and (D)NAPS for N-acyl-phosphoserine) and 2-(R)-N-palmitoyl-norleucinol-1-phosphate ((R) and (S)PNPA), cyclic analogues of 1-acyl-sn-glycero-3-phosphate (cPA) and of 1-O-hexadecyl-sn-glycero-3-phosphate (cAGP), sphingosine-1-phosphate (SPP), as well as two palmitoyl derivatives of dioxazaphosphocanes bearing either a P-H or a P-OH bond (DOXP-H and DOXP-OH, respectively). Nine of these compounds induced platelet aggregation with the following order of potency: SPP < cAGP < DOXP-OH < (L)NAPS = (D)NAPS < (R)PNPA = (S)PNPA < LPA < AGP, EC50 varying between 9.8 nM and 8.3 microM. Two of these compounds (SPP and cAGP) appeared as weak agonists inducing platelet aggregation to only 33% and 41%, respectively, of the maximal response attained with LPA and other analogues. In cross-desensitization experiments, all of these compounds specifically inhibited LPA-induced aggregation, suggesting that they were all acting on the same receptor(s). In contrast, cPA and DOXP-H did not trigger platelet aggregation but instead specifically inhibited the effects of LPA in a concentration-dependent manner. The inhibitory action of cPA did not vary with the acyl chain length or the presence of a double bond and did not involve an increase in cAMP. These data thus confirm the lack of stereospecificity of platelet LPA receptor(s). In addition, since the order of potency of some analogues is different from that described in other cells, our results suggest that platelets contain (a) pharmacologically distinct receptor(s) whose molecular identity still remains to be established. Finally, this unique series of compounds might be used for further characterization of other endogenous or recombinant LPA receptors.

Humans↗
PubMed1999

Effects of lysophosphatidic acid on proliferation and cytosolic Ca++ of human adult vascular smooth muscle cells in culture.

Lysophosphatidic acid (LPA) is a lipid mediator generated by activated platelets and having various effects on numerous cell types. We investigated some effects of 1-oleyl LPA on vascular smooth muscle cells cultured from adult human normal arteries. At micromolar concentrations, LPA induced a mitogenic effect ([3H]-thymidine incorporation and cell proliferation) on quiescent cells, without an additional growth factor being required. This effect was equipotent to that of 10% fetal calf serum, and it was accompanied by early (5 minutes) and late (1-3 hours) phosphorylation of mitogenactivated protein kinase. LPA inhibited cell migration through collagen coated membranes, with or without platelet-derived growth factor BB as chemoattractant. LPA induced a typical biphasic Ca2+ signal response made up of a rapid first phase due to Ca2+ release from intracellular stores followed by a second wave due to external Ca2+ influx. These findings support the proposal that LPA released from activated platelets is a mediator for smooth muscle cell response at the site of vessel injury in humans.

Adult↗
PubMed1999

New developments in phospholipase A2.

Some of the most recent data concerning various phospholipases A2, with special emphasis on secretory, cytosolic, and calcium-independent phospholipases A2 are summarized. Besides their contribution to the production of proinflammatory lipid mediators, the involvement of these enzymes in key cell responses such as apoptosis or tumor cell metastatic potential is also discussed, taking advantage of transgenic models based on gene invalidation by homologous recombination. The possible role of secretory and cytosolic platelet-activating factor acetyl hydrolases is also briefly mentioned. Finally, the ectopic expression in epididymis of an intestinal phospholipase B opens some novel issues as to the possible function of phospholipases in reproduction.

Animals↗
PubMed1999

Membrane sidedness of biosynthetic pathways involved in the production of lysophosphatidic acid.

Lysophosphatidic acid (LPA) is a novel phospholipid mediator with diverse biological activities such as smooth muscle contraction, and proliferative effects or modifications of cytoskeleton. Activated blood platelets are the best identified source, explaining accumulation of LPA in serum upon blood coagulation. However, the metabolic pathways responsible for LPA synthesis are still poorly known. Using a model of human erythrocytes treated with the calcium ionophore A23187, we have shown that type II secretory phospholipase A2 (sPLA2) is able to produce LPA by hydrolyzing phosphatidic acid exposed on the cell surface after phospholipid scrambling. A similar mechanism does not appear to occur in platelets, where inhibitors of sPLA2 or genetic lack of the enzyme do not modify LPA production. However, this does not definitely eliminate the possibility that LPA is also produced in platelets in the external leaflet of the membrane by other phospholipases, which have to be better characterized.

Animals↗
PubMed1998

Regulation of secretory type-II phospholipase A2 and of lysophosphatidic acid synthesis.

Secretory non-pancreatic phospholipase A2 (sPLA2), also called type II-PLA2, is produced in large amounts under inflammatory conditions, thus accumulating in inflammatory fluids. Since the enzyme is virtually inactive on phospholipids from intact cells, we have searched for conditions allowing the action of sPLA2 on membrane phospholipids. Based on an in vitro model, our studies suggest that only those membranes where the transverse distribution of phospholipids has been disturbed offer a convenient surface able to interact with the enzyme, which then achieves significant degradation of all glycerophospholipids. This results in the accumulation of various lysophospholipids such as lysophosphatidylcholine, lysophosphatidylethanolamine and lysophosphatidylserine. However, lysophosphatidic acid (LPA) can also be generated under these conditions involving accumulation of phosphatidic acid in the cytoplasmic leaflet of the membrane, followed by its transfer to the outer monolayer. Since LPA is now considered as a novel phospholipid mediator, this pathway deserves further studies concerning mainly platelets, the main source of LPA identified so far.

Animals↗
PubMed1997

Lysophosphatidic acid as a phospholipid mediator: pathways of synthesis.

From very recent studies, including molecular cloning of cDNA coding for membrane receptors, lysophosphatidic acid (LPA) reached the status of a novel phospholipid mediator with various biological activities. Another strong argument supporting this view was the discovery that LPA is secreted from activated platelets, resulting in its appearance in serum upon blood coagulation. The metabolic pathways as well as the enzymes responsible for LPA production are poorly characterized. However, a survey of literature data indicates some interesting issues which might be used as the basis for further molecular characterization of phospholipases A able to degrade phosphatidic acid.

Animals↗
PubMed1995

Lysophosphatidic acid-induced Ca2+ mobilization in human A431 cells: structure-activity analysis.

Lysophosphatidic acid (LPA; 1-acyl-sn-glycero-3-phosphate) is a platelet-derived lipid mediator that activates its own G-protein-coupled receptor to trigger phospholipase C-mediated Ca2+ mobilization and other effector pathways in numerous cell types. In this study we have examined the structural features of LPA that are important for activation of the Ca(2+)-mobilizing receptor in human A431 carcinoma cells, which show an EC50 for oleoyl-LPA as low as 0.2 nM. When the acyl chain at the sn-1 position is altered, the rank order of potency is oleoyl-LPA > arachidonoyl-LPA > linolenoyl-LPA > linoleoyl-LPA > stearoyl-LPA = palmitoyl-LPA > myristoyl-LPA. The shorter-chain species, lauroyl- and decanoyl-LPA, show little or no activity. Ether-linked LPA (1-O-hexadecyl-sn-glycero-3-phosphate) is somewhat less potent than the corresponding ester-linked LPA; its stereoisomer is about equally active. Deletion of the glycerol backbone causes a 1000-fold decrease in potency. Replacement of the phosphate group in palmitoyl-LPA by a hydrogen- or methyl-phosphonate moiety results in complete loss of activity. A phosphonate analogue with a methylene group replacing the oxygen at sn-3 has strongly decreased activity. All three phosphonate analogues induce cell lysis at doses > 15 microM. Similarly, the methyl and ethyl esters of palmitoyl-LPA are virtually inactive and become cytotoxic at micromolar doses. None of the LPA analogues tested has antagonist activity. Sphingosine 1-phosphate, a putative messenger with some structural similarities to LPA, elicits a transient rise in intracellular [Ca2+] only at micromolar doses; however, cross-desensitization experiments indicate that sphingosine 1-phosphate does not act through the LPA receptor. The results indicate that, although many features of the LPA structure are important for optimal activity, the phosphate group is most critical, suggesting that this moiety is directly involved in receptor activation.

Animals↗
PubMed1995

Secretory phospholipase A2 generates the novel lipid mediator lysophosphatidic acid in membrane microvesicles shed from activated cells.

Nonpancreatic secretory phospholipase A2 (sPLA2) displays proinflammatory properties; however, its physiological substrate is not identified. Although inactive toward intact cells, sPLA2 hydrolyzed phospholipids in membrane microvesicles shed from Ca(2+)-loaded erythrocytes as well as from platelets and from whole blood cells challenged with inflammatory stimuli. sPLA2 was stimulated upon degradation of sphingomyelin (SPH) and produced lysophosphatidic acid (LPA), which induced platelet aggregation. Finally, lysophospholipid-containing vesicles and sPLA2 were detected in inflammatory fluids in relative proportions identical to those used in vitro. We conclude that upon loss of phospholipid asymmetry, cell-derived microvesicles provide a preferential substrate for sPLA2. SPH hydrolysis, which is provoked by various cytokines, regulates sPLA2 activity, and the novel lipid mediator LPA can be generated by this pathway.

Animals↗
PubMed1995

Inhibition of platelet type II phospholipase A2 by an acylamino phospholipid does not alter arachidonate liberation.

An acylamino phospholipid analogue (2-(R)-N-palmitoylnorleucinol-1-phosphoglycol or (R)-PNPG) was examined for its inhibitory effects against type II phospholipase A2 (PLA2) acting on membranes from Escherichia coli. Using two enzyme sources (rat platelet membranes or recombinant human type II PLA2), (R)-PNPG inhibited phospholipid hydrolysis to a maximal value of 80-85%, half-maximal effect being attained at a substrate/inhibitor molar ratio of 80-250. In contrast, (S)-PNPG was 12-fold less potent and thus provided a control for possible non-specific effects of these polar lipids. However, both analogues exerted only marginal effects on the liberation of [3H]arachidonic acid from rat platelets challenged with calcium ionophore A23187. Since, among various animal species, rat platelets contain by far the highest amounts of this enzyme, our data rule out any possible involvement of secretory PLA2 in arachidonic acid liberation from platelet phospholipids, cytosolic PLA2 appearing in this case as the best candidate able to regulate eicosanoid biosynthesis.

Animals↗
PubMed1994

Virtual lack of annexins in human platelets argues against a role in phospholipase A2 regulation and platelet secretion.

Annexins are calcium-phospholipid binding proteins which share structural similarities and common biochemical properties. These proteins seem to be involved in different pathways of cell activation such as regulation of phospholipase A2 activity, membrane-cytoskeleton interaction, and exocytosis. The aim of this study was to attempt to characterize annexins in human platelets. Our results were based on specific EGTA extraction of proteins from platelet homogenates, immunodetection with specific antibodies raised against annexins I, II, V and VI, and measurement of phospholipase A2 inhibition. Antibodies raised against annexins I, V and VI revealed only trace amounts of these proteins in platelet EGTA extracts which did not promote phospholipase A2 inhibition in an in vitro assay. In addition, upon precipitation of membranes in the presence of calcium followed by EGTA extraction, the main substrate of protein kinase C (40-47-kDa protein) displayed a behaviour strictly different from that of annexins. Although one cannot exclude that a small amount of annexin(s) becomes phosphorylated in activated human platelets, these data argue against a role of annexins in the regulation of intracellular phospholipase A2 or in the processes of exocytosis in activated human platelets.

Annexin A1↗
PubMed1992

Inhibition of platelet arachidonic acid liberation by endothelium-derived relaxing factor (EDRF) as studied with sin-1, a nitric oxide generating drug. Evidence for calcium-dependent and calcium-independent mechanisms.

In order to investigate possible effects of endothelium-derived relaxing factor (EDRF or NO.) on platelet phospholipase A2 activity, human platelets labelled with [3H]arachidonic acid ([3H]AA) were stimulated with thrombin (0.5 IU/ml) in the absence or in the presence of sin-1, a vasodilator and platelet inhibitor releasing NO. by spontaneous decomposition at physiological pH. Sin-1 promoted a dose-dependent inhibition of [3H]AA liberation, which was identical in the presence or in the absence of 1 mM Ca2+ in the external medium, suggesting that a reduction of Ca2+ influx was not responsible for this metabolic effect. Using fura-2 as a fluorescent Ca2+ indicator, sin-1 was found to inhibit similarly both Ca2+ influx and Ca2+ mobilization, the latter effect being directly related to a reduction of inositol 1,4,5-tris phosphate production by phospholipase C. However, comparison of cytoplasmic free calcium concentrations ([Ca2+]i) and of [3H]AA liberation attained by platelets treated under various experimental conditions indicated the lack of a direct relationship between [Ca2+]i and platelet phospholipase A2 activity. The effects of sin-1 on [3H]AA liberation could be reproduced by a membrane-permeant analogue of cGMP (8-bromo cyclic GMP), with no evidence of additional effects of sin-1 under these conditions. These data bring further support to the view that Ca2+, although being a necessary cofactor of intracellular phospholipase A2, is not the only regulator of the enzyme. Owing to the multiple effects of this drug on various events involved in membrane-signal transduction (Ca2+ influx, phospholipase C and phospholipase A2 activation), it is suggested that sin-1 inhibits platelet function at an early step of signal transduction, probably by elevating cGMP through a direct effect of NO. on cytosolic guanylate cyclase.

Arachidonic Acid↗
PubMed1989

Inhibition of calcium influx in thrombin-stimulated platelets by SIN-1, an activator of soluble guanylate cyclase.

A previous study revealed that elevation of platelet cyclic GMP induced by a pharmacological activator of soluble guanylate cyclase, 3-morpholinosydnonimine (SIN-1), induced a major inhibition of Ca2+ influx caused by thrombin, as detected by monitoring the fluorescence of the Ca2+ indicator quin-2. In contrast, activation of phospholipase C as well as Ca2+ mobilization presumably promoted by inositol-1,4,5-trisphosphate was less affected by SIN-1 treatment. In the present study, the effects of SIN-1 on Ca2+ influx have been investigated in more detail using platelets loaded with millimolar concentrations of quin-2. Under these conditions, Ca2+ entry from the medium into the platelet cytoplasm could be followed either by detecting fluorescence quenching by Mn2+ or by determination of 45Ca2+ uptake. Both events were inhibited by SIN-1 in a dose-dependent manner. Furthermore, the inhibition of 45Ca2+ uptake and of fluorescence increase observed in the presence of extracellular Ca2+ displayed remarkably parallel dose-response curves, suggesting that elevation of cyclic GMP brought about by SIN-1 inhibits the opening of "receptor-operated channels" whose precise nature remains to be determined.

Biological Transport↗
PubMed1988

Effect of PCR 4099 on ADP-induced calcium movements and phosphatidic acid production in rat platelets.

Antiplatelet activity of PCR 4099, an analogue of ticlopidine, resides in its specific effect against exogenous as well as released ADP. This study investigated in rat platelets the effects of the drug on ADP-induced shape change, elevation of cytosolic free Ca2+ concentration ([Ca2+]i) and hydrolysis of inositol phospholipids, monitored as [32P]phosphatidic acid formation. Shape change and influx of Ca2+ ions across the plasma membrane were not modified after PCR 4099 administration using aspirin-treated platelets. On the other hand, phosphatidic acid formation and calcium mobilization from internal stores were strongly inhibited. These results suggest that PCR 4099 leaves intact the machinery involved in ADP-induced platelet shape change and influx of calcium ions, but inhibits an early step in the ADP-response coupling leading to inositol phospholipid hydrolysis and aggregation.

Adenosine Diphosphate↗
PubMed1988

Effect of a stimulant of guanylate cyclase, sin 1, on calcium movements and phospholipase C activation in thrombin-stimulated human platelets.

The effects of sin 1, a metabolite of an antianginal agent, molsidomine, were investigated on human platelet activation induced by thrombin. This drug promoted a slight inhibition of serotonin release in a medium containing 1 mM Ca2+ or 1 mM EGTA (from 63% to 46% and from 57% to 41% of total serotonin secretion, respectively, with the highest dose used). Under these conditions, Ca2+ movements, monitored by quin 2 fluorescence, were markedly impaired. The most pronounced effect was towards Ca2+ influx, which presented a rapid inhibition with low doses. In the presence of external calcium, thrombin raised cytoplasmic free Ca2+ concentration from 100 nM to 1277 nM. This was reduced to 466 nM and 175 nM with 10(-7) M and 10(-4) M sin 1, respectively. Ca2+ mobilization from internal stores was less inhibited, since cytoplasmic free Ca2+ movements, sin 1 was tested on [32P] phosphatidic acid synthesis resulting from phospholipase C activation induced by thrombin. Phosphatidic acid labelling displayed a maximal inhibition of 43-50% with the highest doses of sin 1 (10(-4) M-10(-3) M) with or without Ca2+ in the incubation medium. However, this effect appeared much more sensitive to sin 1 in the presence of external Ca2+ (25% at 10(-7) M sin 1 with external Ca2+ against 12% at the same sin 1 concentration with EGTA). This discrepancy might be explained by the difference of cGMP level obtained when platelets were treated by sin 1 in the presence or in the absence of Ca2+ in the medium. This study shows that the major target of sin 1 via cGMP is not platelet phospholipase C as previously described, but inhibition of Ca2+ influx through plasma membrane.

Blood Platelets↗