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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.

Thrombin-induced accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) but not of PtdIns(3,4,5,)P3 is strongly correlated with the relocation to the cytoskeleton of 29% of the p85 alpha regulatory subunit of phosphoinositide 3-kinase (PtdIns 3-kinase) and is accompanied by a significant increase in PtdIns 3-kinase activity in this subcellular fraction. Actually, PtdIns(3,4)P2 accumulation and PtdIns 3-kinase, pp60c-src, and p125FAK translocations as well as aggregation were concomitant events occurring with a distinct lag after actin polymerization. The accumulation of PtdIns(3,4)P2 and the relocalization of PtdIns 3-kinase to the cytoskeleton were both dependent on tyrosine phosphorylation, integrin signaling, and aggregation. Furthermore, although p85 alpha was detected in anti-phosphotyrosine immunoprecipitates obtained from the cytoskeleton of thrombin-activated platelets, we failed to demonstrate tyrosine phosphorylation of cytoskeletal p85 alpha. Tyrphostin treatment clearly reduced its presence in this subcellular fraction, suggesting a physical interaction of p85 alpha with a phosphotyrosyl protein. These data led us to investigate the proteins that are able to interact with PtdIns 3-kinase in the cytoskeleton. We found an association of this enzyme with actin filaments: this interaction was spontaneously restored after one cycle of actin depolymerization-repolymerization in vitro. This association with F-actin appeared to be at least partly indirect, since we demonstrated a thrombin-dependent interaction of p85 alpha with a proline-rich sequence of the tyrosine-phosphorylated cytoskeletal focal adhesion kinase, p125FAK. In addition, we show that PtdIns 3-kinase is significantly activated by the p125FAK proline-rich sequence binding to the src homology 3 domain of p85 alpha subunit. This interaction may represent a new mechanism for PtdIns 3-kinase activation at very specific areas of the cell and indicates that the focal contact-like areas linked to the actin filaments play a critical role in signaling events that occur upon ligand engagement of alpha IIb/beta 3 integrin and platelet aggregation evoked by thrombin.

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.

Phospholipase A2 (PLA2) activity was tested in the seminal receptacle of the firebrat female during an ovarian cycle and in relation to insemination. The PLA2 activity was weak up to the time of insemination, while a strong increase of the enzyme activity occurred at the end of the ovarian cycle in both inseminated and non-inseminated females, indicating that PLA2 is an endogenous enzyme not transferred by the spermatophore. Females received various drugs whose anti-PLA2 effects were checked on the seminal receptacle tissues. The effects of the drugs were investigated on ovarian maturation and on the number of eggs layed by inseminated females. Dose-response relationships provide crucial indications that PLA2 inhibition interferes with the firebrat fecundity. The emergent picture is that oocyte maturation is insensitive to PLA2 activity, but that the treatments which reduce the release of arachidonic acid from cellular phospholipids interrupt some physiological link leading to egg deposition. The arachidonate metabolites are designated as the primer of the ovulation mechanisms; the possible role of development hormones in PLA2 regulation is discussed.

Nonpancreatic secretory phospholipase A 2 (sPLA 2 ) displays proinflammatory properties; however, its physiological substrate is not identified. Although inactive toward intact cells, sPLA 2 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. sPLA 2 was stimulated upon degradation of sphingomyelin (SPH) and produced lysophosphatidic acid (LPA), which induced platelet aggregation. Finally, lysophospholipid-containing vesicles and sPLA 2 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 sPLA 2 . SPH hydrolysis, which is provoked by various cytokines, regulates sPLA 2 activity, and the novel lipid mediator LPA can be generated by this pathway.

The lipoprotein profile, the HDL subfractions and the apolipoprotein (apo) A-I conformation of HDL were determined in 119 patients with myocardial infarction and 119 controls, paired for age. Apo A-I conformation was assessed by its immunoreactivity towards two monoclonal antibodies, 5F6 and 3G10. HDL phospholipid levels and fatty acid composition were also determined. HDL and HDL3 cholesterol, apo A-I, LpA-I and LpA-I:A-II levels were significantly lower in patients than in controls, whereas the levels of total and LDL-cholesterol and of apo B were not different. Apo A-I immunoreactivity against MAb 5F6 was significantly better in patients than in controls, while the immunoreactivity against 3G10 was similar. Paired stepwise logistic regression showed only apo A-I, HDL3 cholesterol and 5F6 immunoreactivity to be significantly related to myocardial infarction. After adjustment for HDL cholesterol, lower levels of HDL phospholipids and polyunsaturated fatty acids (PUFA) were found in patients compared with controls. Hence, of all HDL markers, apo A-I and HDL3 cholesterol appear to be the most informative. Also, lower HDL phospholipid and PUFA content may explain a different apo A-I conformation in patients with myocardial infarction.

Insulin receptor (IR) purified from human placenta by wheat germ agglutinin affinity chromatography was incubated in the presence of insulin, [gamma-32P]ATP and annexin I. In parallel to its own tyrosine phosphorylation, annexin I promoted a dose-dependent inhibition of IR autophosphorylation (IC50 0.5 microM). This effect was specific for insulin-stimulated tyrosine kinase activity and required the N-terminal end of the protein containing the phosphorylatable Tyr21 residue. A pentadecapeptide encompassing residues 16-30 of human annexin I displayed a similar activity, but at higher concentrations. These data underscore a specific interaction of IR with annexin I, which should be considered as a potential physiological regulator of the effects of insulin on its target tissues.

A significant protein tyrosine phosphatase (PTP) activity was found to be associated with the cytoskeleton of thrombin-stimulated platelets. Translocation of the enzyme became maximal within 1-2 min of thrombin stimulation and was suppressed by cytochalasin D or upon inhibition of aggregation. Immunoblotting as well as immunoprecipitation revealed that a PTP with two SH2 domains (SH-PTP1) displayed the same behaviour, translocation to the cytoskeleton showing the same time course as that observed for pp60c-src. We conclude that SH-PTP1 might represent a critical enzyme in the complex interplay between the various proteins regulating protein tyrosine phosphorylation in the cytoskeletal matrix.

Beside 4- and 5-phosphatases playing a role in the interconversion between the D-3 phosphorylated polyphosphoinositides, the only enzyme described so far to be responsible for a phosphomonoesterasic activity on the D-3 position of inositol lipids is a specific 3-phosphatase that hydrolyzes PtdIns(3)P in NIH 3T3 cells. We report here the presence of a potent 3-phosphatase activity in different cell types. This activity is detected both in cytosol and membranes of A431 cells and is inhibited by VO43− and Zn2+. Interestingly, the cytosolic activity from A431 cells selectively hydrolyzes in vitro PtdIns(3)P and PtdIns(3,4)P2, whereas PtdIns(3,4,5)P3 remains a very poor substrate under the same conditions. Finally, assays of phosphatidylinositol 3-kinase and 3-phosphatase activities in the pool of phosphotyrosine-containing proteins isolated from EGF-stimulated A431 cells suggest a compartmentation of these two antagonistic activities during cell activation.

In this paper, we present the first evidence that free apoA-I, without association with lipids, binds only to a high-affinity binding site (Kd = 1.8 microgram/mL, Bmax = 63.12 ng/mL). This is a new binding site of higher affinity (80-100 times) but of lower capacity than the binding sites already described for HDL. This is also the first evidence on HepG2 cells both of a high-affinity site (Kd = 0.685 microgram/mL, Bmax = 39.86 ng/mL) and of a low-affinity site (Kd = 55.65 micrograms/mL, Bmax = 665.45 ng/mL) for HDL. ApoA-I-DMPC complexes also present two binding components comparable to the HDL3 binding sites. This free apoA-I binding is specific, as shown by competition experiments, and allowed us to specifically study this high-affinity site, without interference of the low-affinity one. Kinetic rates of association/dissociation for the high-affinity site were faster than for the low-affinity site (10 and 20 min versus 40 and 30 min, respectively). The kinetic Kd values, derived from association and dissociation rate constants (Kd = 55.14 and 2.91 micrograms/mL), were of similar magnitude as the Kd values calculated by Scatchard analysis. These data confirm that HDL3 binding sites characterized by saturation experiments follow the law of mass action, indicative of ligand-receptor interaction. In summary, HepG2 cells present high HDL3 binding sites which are able to bind free apoA-I in contrast with the low-affinity HDL3 binding sites.

The role of hepatic triacylglycerol lipase (H-TGL) in promoting the liver uptake of high density lipoprotein (HDL) free and esterified cholesterol was studied in a recirculating rat liver perfusion, a situation where the enzyme is physiologically expressed and is active at the vascular bed. For this purpose, reconstituted HDL of defined phospholipid composition were prepared, containing either 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, a substrate for H-TGL, or 1-O-hexadecyl-2-oleoyl-sn-glycero-3-phosphocholine, a non-hydrolyzable analog. Reconstituted HDL were then used in the perfused rat liver system. The main results are the following. 1) Reconstituted HDL were obtained by sonication of lipids and apolipoproteins and isolated by ultracentrifugation in the 1.07-1.21 g/ml density interval. Reconstituted HDL containing either diacylphosphatidylcholine or alkyl-acyl-phosphatidylcholine were similar in terms of chemical composition, apparent size, and apolipoprotein A-I immunoreactivity, and were comparable to native HDL3. 2) Reconstituted HDL were labeled with free [14C]cholesterol and [3H]cholesteryl ether, a non-hydrolyzable tracer of esterified cholesterol, and were perfused through the rat liver. Liver uptake of [3H]cholesteryl ether was 2.5-fold higher from reconstituted HDL containing diacylphospholipid than from HDL reconstituted with alkyl-acyl-phospholipids. Liver uptake of free [14C]cholesterol was identical in both cases. 3) H-TGL-depleted rat livers were obtained by a 12-min preperfusion in the presence of heparin, displacing 90% of the enzymatic activity. The residual activity in the perfusate was inhibited by a specific antibody directed against rat H-TGL. Liver uptake of [3H]cholesteryl ether from reconstituted HDL containing diacylphospholipid was reduced by 35% in hepatic lipase-depleted livers compared to controls. On the other hand, hepatic lipase depletion had no effect on the liver uptake of esterified cholesterol from HDL reconstituted with alkyl-acyl-phospholipids. The above findings support a role for the phospholipase A1 activity of H-TGL in stimulating the delivery of HDL esterified cholesterol to liver cells.

The effect of phorbol 12-myristate 13-acetate (PMA) on [3H]choline incorporation into phosphatidylcholine (PtdCho) and on the ‘de novo’ pathway of PtdCho synthesis has been investigated, compared with that of oleic acid, in ascitic-strain Krebs-II cells. Both compounds stimulated [3H]choline incorporation into PtdCho, but the PMA-induced incorporation was saturable at concentrations of the agonist around 100 nM, whereas no saturation was noticed with oleic acid up to 1 mM. Chase experiments showed no effect of PMA on the conversion of phosphocholine into CDP-choline. The phorbol ester did not stimulate any of the enzyme activities of the ‘de novo’ pathway, whereas oleic acid increased specifically by 2.5-fold the CTP:phosphocholine cytidylyltransferase (CT, EC 2.7.7.15) activity. In addition, no change in the subcellular distribution of CT was observed upon incubation with PMA, in contrast with oleic acid treatment. Cells challenged with oleic acid showed a 25-fold increase in diradylglycerol (DG) content, which was not modified upon incubation with 200 nM PMA, the most effective concentration of phorbol ester promoting choline incorporation. Subcellular fractionation of Krebs-II cells on Percoll gradients revealed that [3H]PMA and 1-radyl-2-[3H]oleoyl-glycerol, derived from exogenously supplied [3H]oleic acid, both exhibited the same enrichment in the endoplasmic reticulum. We have previously shown that the labelled fatty acid also accumulated in the endoplasmic reticulum [Tercé, Record, Tronchère, Ribbes and Chap (1992) Biochem. J. 282, 333-338]. However, PMA induced a stimulation of choline uptake, which was not provoked by PMA 4-O-methyl ether, which interacts poorly with protein kinase C. Our data provide evidence that the enhancement of [3H]choline incorporation into PtdCho triggered by PMA and oleic acid proceeds via completely distinct mechanism(s).

In vitro phosphorylation of annexin 1 by purified rat brain protein kinase C (PKC) has been studied in the presence of annexin 5, which is not a substrate for PKC. Annexin 5 promoted a dose-dependent inhibition of annexin 1 phosphorylation, which could be overcome by increasing the concentration of phosphatidylserine (PtdSer). In addition, a close relationship was found between the amount of PtdSer uncovered by annexin 5 and the residual phosphorylation of annexin 1. These data fit with the ‘surface depletion model’ explaining the antiphospholipase activity of annexins. In order to check the possibility that the in vitro effect of annexin 5 could be of some physiological relevance, annexins 1, 2, and 5, as well as the light chain of calpactin 1 (p11), have been quantified in human endothelial cells by measuring the radioactivity bound to the proteins after Western blotting with specific antibodies and 125I-labelled secondary antibody. Our data indicate that annexins 1 and 5, PKC and PtdSer are present in human endothelial cells in relative amounts very similar to those used in vitro under conditions permitting the detection of the inhibitory effect of annexin 5. Since annexin 1 remained refractory to PKC-dependent phosphorylation in intact cells, we suggest that annexin 5 might exert its inhibitory effect towards PKC in vivo, provided that its binding to phospholipids can occur at physiological (micromolar) concentrations of Ca2+. This was previously shown to occur in vitro using phosphatidylethanolamine/phosphatidic acid vesicles [Blackwood and Ernst (1990) Biochem. J. 266, 195-200]. Using identical assay conditions, which also allowed expression of PKC activity, annexin 5 again inhibited annexin 1 phosphorylation without interfering with PKC autophosphorylation. These data suggest that annexins 1 and 5 might interact with each other on the lipid surface, resulting in a specific inhibition of annexin 1 phosphorylation by PKC. Whether a similar mechanism also occurs in vivo remains to be determined.

By means of selective extraction in a Ca(2+)-chelating medium and immunoblotting, four annexins (I, II, V, and VI) were identified in both isolated rat renal glomeruli and rat glomerular mesangial cells. Upon 32P labeling of these cells in culture, annexin I was immunoprecipitated using a specific polyclonal antibody and was found to incorporate radioactivity in a constitutive manner. However, as with epidermal growth factor (200 ng/ml), addition of angiotensin II (10(-7) M), arginine-vasopressin (10(-7) M), or endothelin I (10(-7) M) resulted in a 2-3-fold stimulation of annexin I phosphorylation. The basal phosphorylation as well as the stimulating effect of angiotensin II were also detected by immunoblotting annexin extracts using an antiphosphotyrosine antibody. In addition, among various phosphotyrosyl proteins isolated from EGTA extracts by adsorption onto an anti-phosphotyrosine antibody, annexin I was specifically recognized by Western blotting using a monoclonal anti-annexin I antibody, and displayed the same increase upon cell stimulation with angiotensin II. Moreover, thin layer chromatographic analysis of phosphoamino acids present in immunoprecipitated [32P]annexin I showed an exclusive labeling of phosphotyrosine residue(s). Finally, the effect of angiotensin II was detectable after 10 min, maximal at 6 h, and present until 12 h of incubation. Using 12-h stimulation, tyrosine phosphorylation of annexin I displayed a maximum at 10(-7) to 10(-6) M angiotensin II. These data report for the first time the stimulation of annexin I tyrosine phosphorylation by biologically active peptides acting via receptors belonging to the superfamily of seven hydrophobic domain, G-protein-linked receptors, which lack an intrinsic protein tyrosine kinase. This suggests a possible role of annexin I in the mitogenic effect of angiotensin II, arginine-vasopressin, and endothelin I, which was previously observed on rat glomerular mesangial cells as well as on other cells.

Cyclophosphamide injection into the fasted rabbit induces a hypertriglyceridemia (4.6 mM vs. 0.8 mM in controls) and a defect of lipoprotein lipase (LPL), as measured in post- heparin plasma (PHP). In contrast, administration of the drug into fed animals tends to increase PHP-LPL. The effects of cyclophosphamide on LPL activity and synthesis, depending on the nutritional state, were thus studied in two sites: peri- epididymal adipose tissue and heart. In adipose tissue, fasting decreased LPL activity to 45.2 mIU/g (P < 0,001) compared to 667.9 mIU/g in fed animals. PHP-LPL activity was also decreased by 45% upon starvation. These modulations ap- peared to be related to plasma insulin levels. The relative rate of synthesis of fat tissue LPL was decreased from 0.32% total protein synthesis in fed animals to 0.10% in fasted rabbits, con- cordant with a reduction in the expression of LPL specific mRNA. Cyclophosphamide administration to the fed rabbit led to decreases of LPL activity and synthesis in the adipose tissue, similar to those observed upon starvation. However, when in- jected into fasted animals, the drug did not further depress fat tissue LPL. Fasting did not change heart LPL activity (288.3 mIU/g vs. 239.3 in fed animals) nor its relative rate of synthesis (0.21% of total protein synthesis). However, cyclophosphamide induced opposite effects, depending on the nutritional state: after injection into fed animals, heart LPL activity increased up to 477.2 mIU/g (P < 0.01) with a concomitant increase in the LPL synthesis rate. Conversely, drug administration into fasted rabbits led to a decrease of heart LPL activity to 133.9 mIU/g. Similar qualitative variations were recorded in postheparin plasma. Hence, although insensitive to nutritional modulations, heart LPL responded differently to cyclophosphamide, depend- ing on the nutritional state. In spite of those different modula- tions of heart and adipose tissue LPL, the enzyme isolated from these two sources displayed similar molecular mass, immuno- reactivity, and catalytic properties. The effects of cyclophospha- mide injection on very low density lipoprotein (VLDL)-triacyl- glycerol (TG) synthesis were also investigated, as a possible determinant of hypertriglyceridemia. The drug stimulated TG synthesis in both nutritional states, and maximally by 45% in fed animals. Hence, a defect of heart and postheparin plasma LPL appears as a major determinant of hypertriglyceridemia in cyclophosphamide-treated fasted rabbits. The opposite varia- tions of heart LPL towards cyclophosphamide suggest that the drug may unveil some effects of insulin on heart LPL. -Lespine, A., C. Azema, M. Gafvels, J. Manent, N. Dousset, H. Chap, and B. Perret. Lipoprotein lipase regulation in the cyclophosphamide-treated rabbit: dependence on nutritional status. J Lipid Res. 1993. 34: 23-36.

Upon in vitro incubation with [ 14 ]arachidonic acid, reproductive tissues of Thermobia domestica incorporated radioactivity, mostly into phospholipids. Stimulation with calcium ionophore A 23187 modified the distribution of the labeling between the various phospholipids, and promoted a release of arachidonic acid as well as metabolites comigrating with authentic HETEs. The lipolytic activity was tested against three exogenous phosphatidylcholines. It is concluded that arachidonic acid availability for eicosanoid biosynthesis is mostly regulated by a calcium-dependent phospholipase A 2 . The specific activity of PLA 2 was assayed in reproductive tissues. After insemination, the phospholipase activity strongly increased in the female seminal receptacle, but the rise was independent of the number of transfered spermatophores.

Phosphatidylinositol (PtdIns)-4- and -3-kinases, PtdIns(4)P-5-kinase, diacylglycerol (DAG) kinase, and PtdIns-phospholipase C were all detected in cytoskeletons of resting human platelets. The total cytoskeletal enzyme activities were greatly increased upon thrombin stimulation of the intact cells. Those reached a maximum after a 60-s stimulation for PtdIns(4)P-5-kinase and phospholipase C, while the other kinases appeared to be slightly delayed. Specific activities were stimulated from about 4-fold (PtdIns-3-kinase) to about 6-fold (PtdIns-4-kinase). Thrombin treatment also promoted a co-extraction of pp60c-src with the cytoskeletons and its disappearance from the Triton X-100 soluble fraction. These results suggest that stimulation of platelets by thrombin causes the association of enzymes responsible for lipid phosphorylation and hydrolysis with the cytoskeletons. This could occur at cytoskeleton anchoring points to the membranes.

Protein-tyrosine phosphatases (PTPases) are becoming an important family of enzymes that might regulate key events in cell growth and transformation. While isolating a new member of this family via amplification of human lung cDNA by the polymerase chain reaction, we found a clone identical to but truncated at the 3'-end of the coding region of human PTPase beta (HPTP beta) mRNA. This difference in sequence is situated in the most conserved part of the catalytic domain of the enzyme. The expression level of the truncated form of HPTP beta mRNA in human lung was lower than its normal form.

Short time effect of oleate and 1-O-alkyl-2-O-methyl-rac-glycero-3-phosphocholine (AMGPC) on choline incorporation into phosphatidylcholines were studied in HL-60 cells. The non lytic concentration of 50 microM oleate induced a three-fold increase in [3H]choline incorporation into phosphatidylcholine. This stimulation was accompanied by a translocation of the CTP:phosphocholine cytidylyltransferase (EC 2.7.7.15) from cytosol to membranes. By contrast, the ether-lipid AMGPC inhibited [3H]choline incorporation into phosphatidylcholine by 60% at 10 microM. AMGPC had no effect on choline kinase or choline phosphotransferase activities. When AMGPC was added separately to an homogenate, a particulate or a cytosolic fraction, cytidylyltransferase inhibition was observed only in the homogenate. However on particulates recovered from homogenates treated with increasing concentrations of AMGPC, membranous cytidylyltransferase activity decreased dose-dependently. Thus AMGPC had no effect on cytidylyltransferase activity itself but inhibited its translocation from cytosol to membrane. At variance with the well-established positive effect on cytidylyltransferase translocation induced by fatty acids, this is the first demonstration that AMGPC can inhibit cytidylyltransferase translocation in cell-free system.

Plasma membranes were isolated from A431 cells previously labelled with myo-[3H]inositol during exponential growth, using a rapid procedure on Percoll gradients. They displayed a significant phospholipase (PLC) activity against phosphoinositides, which was stimulated by guanosine 5'-O-(3-thiotriphosphate) (GTP gamma S), epidermal growth factor (EGF) and fetal calf serum (FCS) (24%, 11% and 97% over controls, respectively). The effect of EGF was not significantly increased by GTP gamma S. Upon addition of cytosol, EGF promoted an almost 100% stimulation of inositol 1,4,5-trisphosphate and inositol bisphosphate generation, which displayed an absolute requirement for GTP gamma S. This dose-dependent effect of cytosol was linear until 60 micrograms/ml of cytosolic protein and decreased afterwards; it was abolished by heat treatment and trypsin hydrolysis, and it was not reproduced by an identical amount of bovine serum albumin. The same biphasic stimulation was observed with phosphotyrosyl proteins immunopurified from cytosol of A431 cells previously stimulated by EGF. Since phosphotyrosyl proteins displayed PLC activity, our data suggest that soluble protein substrates of EGF receptor tyrosine kinase, including PLC, could be involved in the regulation of phosphoinositide hydrolysis in response to EGF. Using phosphatidyl[3H]inositol 4,5-bisphosphate (PIP2) dispersed with unlabelled phosphatidylethanolamine and phosphatidylserine as an exogenous substrate, no stimulation of PLC activity by EGF could be detected, either with membranes or with membranes plus cytosol. It is concluded that EGF might stimulate hydrolysis of phosphoinositides by PLC through complex interactions between plasma membrane and cytosolic factors which still remain to be identified.

The reproductive tissues of the primitive insect Thermobia domestica synthesize several eicosanoids when incubated with exogenous arachidonic acid. The enzymatic system was characterized with respect to kinetic parameters, Ca 2+ requirement and pH- and cofactor-dependence. Five monohydroxylated eicosatetraenoic acids (HETEs) were identified by mass spectrometry procedures, demonstrating the existence of the lipoxygenase pathway. Quantitative studies were performed by high-pressure liquid chromatography. It is confirmed that the 8-lipoxygenase activity remains the major pathway in tissues from males and from inseminated females. In female tissues, the amounts of metabolite depend on the number of spermatophores contained in the seminal receptacles. These data are in agreement with the hypothesis of a transfer of the enzyme from male to female during mating. The mechanisms involved are discussed in comparison with those of other insect species in which the synthesis of prostaglandins has been reported.

The presence of a calcium- and phospholipid-dependent kinase activity in the soluble extracts of Neurospora crassa has been reported earlier. This activity phosphorylated in vitro two endogenous substrates, one of apparent molecular weight ( M r ) 85 000, the other much lower than 14 000. This paper reports that the smaller substrate is a lipid. The lower M r phosphorylated compound could be extracted from proteins by organic solvents. Various thin-layer chromatographic analyses revealed that it did not comigrate with any of the phospholipids tested. Chemical and enzymatic structural analyses indicated that the phosphorylated compound was sensitive to mild alkaline methanolysis, alkaline phosphate and phospholipase A 1 but resistant to phospholipases A 2 , C and D or nitrous acid treatment. A similar compound was detected in the organic phase after extraction of N. crassa mycelia labelled in vivo with either 32 P i or [ 3 H]glycerol. It could not be detected when [ 3 H]inositol was used as label. The addition of in vivo 32 P-labelled lipids in phosphorylation reactions revealed that the low M r substrate of the calcium- and phospholipid-dependent kinase already contained some phosphate which was itself sensitive to alkaline phosphatase. An identical apolar component was generated by alkaline phosphatase treatment of both the substrate of the kinase and the hosphorylated substrate. From these data it is concluded that the low M r phosphorylated compound is a lipid, presumably consisting of a diacylglycerol backbone with an as yet undetermined structure linked to the glycerol moiety. This structure contains at least two phosphate groups.

Platelet-activating factor is a proinflammatory lipid active at subnanomolar concentrations. The intermembrane transfer of a biologically active PAF analog has been previously demonstrated in macrophages. Here we demonstrate that the specific activity of this transfer activity increases when HL-60 cells are induced to differentiate by treatment with dimethyl sulfoxide, dibutyryl cAMP or phorbol diester. In undifferentiated HL-60 cells, methylcarbamyl-PAF transfer activity was only 0.56 U·min −1 ·mg −1 . This basal value was increased 2.6 and 6.7 times upon granulocytic and macrophagic differentiation, respectively. On the other hand, the transfer of 2-O-methyl-PAF, a cytotoxic analog with no PAF biological activity, remained very low and did not vary during differentiation.

Radioactive PtdIns(3)P was detected in human platelets incubated with [32P]Pi, but remained unaffected by thrombin treatment. In contrast, [32P]PtdIns(3,4)P2 was absent from resting platelets, but was produced by thrombin-activated platelets in a dose- and time-dependent manner. [32P]PtdInsP3 was never found under these conditions. These changes are similar to those elicited in other cells by platelet-derived growth factor or the oncogene product pp60c-src.

Lysophosphatidic acid (LPA), platelet-activating factor (PAF), and thromboxane A(2) are proinflammatory lipid mediators that activate surface receptors on platelets, producing increased intracellular calcium, which is necessary for aggregation. We investigated propofol's effect on platelet aggregation and intracellular calcium mobilization caused by these three agonists. Platelets from human volunteers were incubated in buffers containing LPA (1 microM), U46619 (thromboxane A(2) analog; 1 microM), or PAF (10 nM). Propofol emulsion or 2,6-diisopropylphenol (propofol without fat emulsion) dissolved in ethanol was added to achieve concentrations of propofol used clinically: 5 or 10 microg/mL. After 2 min, aggregation or intracellular calcium concentrations were measured with optical techniques. Propofol emulsion and propofol in ethanol produced similar inhibition of platelet aggregation induced by LPA, PAF, and U46619 in a dose-dependent fashion. LPA, PAF, and U46619 each caused significant increases in intracellular calcium that were not modified by propofol. Because propofol does not significantly alter intracellular calcium increases caused by receptor activation, inhibition appears to act distal to platelet receptors, inositol phosphate 3, and phospholipase C. Because the three lipid mediators play a key role in inflammation, their inhibition by propofol might be clinically important.

The involvement of phosphoinositide 3-kinase C2alpha in vascular smooth muscle cell migration was investigated. Products of phosphoinositide 3-kinase, phosphatidylinositol-3-phosphate, and phosphatidylinositol-3,4-bis-phosphate were increased upon smooth muscle cell migration but their synthesis was affected only partially by phosphoinositide 3-kinase inhibitors, wortmannin and LY-294002. Using specific antibody, we showed that the wortmannin/LY-294002 poorly sensitive phosphoinositide 3-kinase C2alpha is expressed in smooth muscle cells. Measurement of phosphoinositide 3-kinase C2alpha activity in vitro, after immunoprecipitation, clearly demonstrated its activation upon smooth muscle cell migration. Moreover, for the first time, phosphoinositide 3-kinase C2alpha was found to be differentially regulated by alpha(v)beta(3) and alpha(v)beta(5) integrin engagement. Finally, we have identified two new potential phosphoinositide 3-kinase C2alpha-binding proteins, p70 and p110, which both may be tyrosine phosphorylated. Thus, phosphoinositide 3-kinase C2alpha might represent a new regulatory pathway of cell migration downstream of integrin engagement.

Guinea pig phospholipase B (GPPLB) is a glycosylated ectoenzyme of intestinal brush border membrane. It displays a broad substrate specificity and is activated by trypsin cleavage. The primary sequence contains four tandem repeat domains (I to IV) and several serines in lipase consensus sequences. We used site-directed mutagenesis to demonstrate that only the serine 399 present in repeat II is responsible for the various enzymatic activities of GPPLB. Furthermore, we characterized for the first time the retinyl esterase activity of the enzyme. We also constructed and expressed in COS-7 cells, an NH2-terminal repeat I deletion mutant which was detected at a very low level by immunoblot. However, confocal microscopy study showed a strong intracellular accumulation with a weak membrane expression of the mutated protein, indicating a role of the NH2-terminal repeat I in the processing of GPPLB. Nevertheless, the Western blot-detected protein presented a glycosylation and trypsin sensitivity patterns similar to wild type PLB. The mutant is also fully active without trypsin treatment, in contrast to native enzyme. Thus, we propose a structural model for GPPLB, in which the repeat I constitutes a lid covering the active site and impairing enzymatic activity, its removal by trypsin leading to an active protein.

Phospholipase B (PLB) is an enzyme that displays both phospholipase A(2) and lysophospholipase activities. Analysis of human epidermis homogenates indicated the presence of a 97 kDa PLB protein, as well as a phospholipase A(2) activity, both being enriched in the soluble fraction. Immunolabelling and in situ hybridization experiments showed that this enzyme is expressed in the different layers of epidermis with an accumulation at the dermo-epidermis junction. RT-PCR data indicated that PLB is specifically expressed in natural and reconstructed epidermis. By 3'-RACE-PCR and screening of human genome databases, we obtained a 3600 bp cDNA coding for human PLB highly homologous to already described intestinal brush border PLBs. These data led us to conclude that the soluble PLB corresponds to a proteolytic cleavage of the membrane anchored protein. Altogether, our results provide the first characterization of human PLB which should play an important role in epidermal barrier function.

Although Gbetagamma is thought to mediate mitogen-activated protein kinase (MAPK) activation in response to G protein-coupled receptor stimulation, the mechanisms involved in this pathway have not been clearly defined. Phosphoinositide 3-kinase (PI3K) has been proposed as an early intermediate in this process, but its role has remained elusive. We have observed that dominant negative mutants of p110beta, but not of p110gamma, inhibited MAPK stimulation in response to lysophosphatidic acid (LPA). The role of p110beta was located upstream from Ras. To determine which of the lipid or protein kinase activities of p110beta were important for Ras activation, we produced a mutant p110beta lacking the lipid but not the protein kinase activity. This protein displayed a dominant negative activity similar to a kinase-dead mutant, indicating that p110beta lipid kinase activity was essentially involved in Ras activation. In agreement, overexpression of the lipid phosphatase PTEN was found to specifically inhibit Ras stimulation induced by LPA. In addition, we have observed that the PH domain-containing adapter protein Gab1, which is involved in p110beta activation during LPA stimulation, is also implicated in this pathway downstream of p110beta. Indeed, both membrane redistribution and phosphorylation of Gab1 were reduced in the presence of PI3K inhibitors or dominant negative p110beta. Downstream of Gab1, the tyrosine phosphatase SHP2 was found to mediate Ras activation in response to LPA and to be recruited through PI3K and Gab1, because transfection of Gab1 mutant deficient for SHP2 binding inhibited Ras activation without interfering with PI3K activation. We conclude that LPA-induced Ras activation is mediated by a p110beta/Gab1/SHP2 pathway. Moreover, we present data indicating that p110beta is effectively the target of betagamma in this pathway, suggesting that the p110beta/Gab1/SHP2 pathway provides a novel link between betagamma and Ras by integrating two early events of LPA signaling, i.e. Gbetagamma release and tyrosine kinase receptor transactivation.

Recent reports have shown that phosphoinositide 3-kinases (PI3Ks) mediate various biological activities of lysophosphatidic acid (LPA), including cell proliferation or survival. In addition, these enzymes have been proposed to be early intermediates of mitogen-activated protein kinase (MAPK) activation. Here we summarize our current knowledge of the mechanisms underlying these observations. p110gamma is an isoform of PI3K that can be activated in vitro by Gbetagamma subunits and was therefore considered as the logical candidate to mediate responses induced by G protein-coupled receptor (GPCR) agonists. In agreement with this, p110gamma has been involved in different biochemical models linking Gbetagamma to MAPK activation. Nevertheless, its apparent tissue-specific distribution has raised questions regarding the physiological relevance of these models. In addition, LPA can activate p110beta, a member of the phosphotyrosine-dependent PI3K subfamily that participates in the mitogenic effect of LPA. Its activation is thought to involve a synergistic effect of Gbetagamma and phosphotyrosine motifs provided by a transactivated EGF receptor/Gab1 pathway. We are currently studying a possible role of p110beta upstream from Ras, suggesting that this protein could provide a novel connection between betagamma and the MAPK pathway.

Glycosphingolipid- and cholesterol-enriched membrane microdomains, called rafts, can be isolated from several mammalian cells, including platelets. These microdomains appear to play a critical role in signal transduction in several hematopoietic cells, but their function in blood platelets remains unknown. Herein, we first characterized the lipid composition, including the fatty acid composition of phospholipids, of human platelet rafts. Then their role in platelet activation process was investigated. Interestingly, thrombin stimulation led to morphological changes of rafts correlating with the production of lipid second messengers in these microdomains. Indeed, we could demonstrate for the first time that a large part of the stimulation-dependent production of phosphatidic acid and phosphoinositide 3-kinase products was concentrated in rafts. Moreover, cholesterol depletion with methyl-beta-cyclodextrin disrupted platelet rafts, dramatically decreased the agonist-dependent production of these lipid signaling molecules, and impaired platelet secretion and aggregation. Cholesterol repletion restored the physiological platelet responses. Altogether our data indicate that rafts are highly dynamic platelet membrane structures involved in critical signaling mechanisms linked to the production of lipid second messengers. The demonstration of phosphatidylinositol 3,4,5-trisphosphate production in rafts may have general implications for the understanding of the role of this key second messenger found ubiquitously in higher eucaryotic cells.

Fibrinogen deposition and smooth muscle cell migration are important causes of atherosclerosis and angiogenesis. Involvement of calpains in vascular smooth muscle cell adhesion onto fibrinogen was investigated. Using calpain inhibitors, we showed that activation of calpains was required for smooth muscle cell spreading. An increase of 32 P-labeled phosphatidic acid and phosphatidylinositol-3,4- bis phosphate, respective products of phospholipase C and phosphoinositide 3-kinase activities, was measured in adherent cells. Addition of the calpain inhibitor calpeptin strongly decreased phosphatidic acid and phosphatidylinositol-3,4- bis phosphate. However, smooth muscle cell spreading was prevented by the phospholipase C inhibitor U-73122, but poorly modified by phosphoinositide 3-kinase inhibitors wortmannin and LY-294002. Moreover, PLC was found to act upstream of the PI 3-kinase IA isoform. Thus, our data provide the first evidence that calpains are required for smooth muscle cell spreading. Further, phospholipase C activation is pointed as a key step of cell-spreading regulation by calpains.

Recent studies highlight the existence of an autonomous nuclear polyphosphoinositide metabolism related to cellular proliferation and differentiation. However, only few data document the nuclear production of the putative second messengers, the 3-phosphorylated phosphoinositides, by the phosphoinositide 3-kinase (PI3K). In the present paper, we examine whether GTP-binding proteins can directly modulate 3-phosphorylated phosphoinositide metabolism in membrane-free nuclei isolated from pig aorta smooth muscle cells (VSMCs). In vitro PI3K assays performed without the addition of any exogenous substrates revealed that guanosine 5'-(gamma-thio)triphosphate (GTPgammaS) specifically stimulated the nuclear synthesis of phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P(3)), whereas guanosine 5'-(beta-thio)diphosphate was ineffective. PI3K inhibitors wortmannin and LY294002 prevented GTPgammaS-induced PtdIns(3,4,5)P(3) synthesis. Moreover, pertussis toxin inhibited partially PtdIns(3,4,5)P(3) accumulation, suggesting that nuclear G(i)/G(0) proteins are involved in the activation of PI3K. Immunoblot experiments showed the presence of Galpha(0) proteins in VSMC nuclei. In contrast with previous reports, immunoblots and indirect immunofluorescence failed to detect the p85alpha subunit of the heterodimeric PI3K within VSMC nuclei. By contrast, we have detected the presence of a 117-kDa protein immunologically related to the PI3Kgamma. These results indicate the existence of a G protein-activated PI3K inside VSMC nucleus that might be involved in the control of VSMC proliferation and in the pathogenesis of vascular proliferative disorders.

Although the mechanisms involved in the activation of mitogen-activated protein kinases (MAPK) by receptor tyrosine kinases do not display an obvious role for phosphoinositide 3-kinases (PI3Ks), we have observed in the nontransformed cell line Vero stimulated with epidermal growth factor (EGF) that wortmannin and LY294002 nearly abolished MAPK activation. The effect was observed under strong stimulation and was independent of EGF concentration. In addition, three mutants of class Ia PI3Ks were found to inhibit MAPK activation to an extent similar to their effect on Akt/protein kinase B activation. To determine the importance of PI3K lipid kinase activity in MAPK activation, we have used the phosphatase PTEN and the pleckstrin homology domain of Tec kinase. Overexpression of these proteins, but not control mutants, was found to inhibit MAPK activation, suggesting that the lipid products of class Ia PI3K are necessary for MAPK signaling. We next investigated the location of PI3K in the MAPK cascade. Pharmacological inhibitors and dominant negative forms of PI3K were found to block the activation of Ras induced by EGF. Upstream from Ras, although association of Grb2 with its conventional effectors was independent of PI3K, we have observed that the recruitment of the tyrosine phosphatase SHP2 required PI3K. Because SHP2 was also essential for Ras activation, this suggested the existence of a PI3K/SHP2 pathway leading to the activation of Ras. In addition, we have observed that the docking protein Gab1, which is involved in PI3K activation during EGF stimulation, is also implicated in this pathway downstream of PI3K. Indeed, the association of Gab1 with SHP2 was blocked by PI3K inhibitors, and expression of Gab1 mutant deficient for binding to SHP2 was found to inhibit Ras stimulation without interfering with PI3K activation. These results show that, in addition to Shc and Grb2, a PI3K-dependent pathway involving Gab1 and SHP2 is essential for Ras activation under EGF stimulation.

Phospholipases A 2 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 A 2 expressed in human epidermis. We report the molecular cloning of two secretory phospholipase A 2 in the human epidermis. The first enzyme is identical to human pancreatic type IB phospholipase A 2 . Western blots revealed a 14 kDa protein localized in the soluble fraction. The second phospholipase A 2 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 A 2 were expressed mainly in the basal layers of the epidermis. Our data thus provide evidence for the expression of two secretory phospholipases A 2 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.

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.

Pretreatment of intact platelets with cytochalasin D prevented actin polymerization and cytoskeleton reorganization induced by thrombin, but did not affect platelet aggregation. Under these conditions, synthesis of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2) stimulated by thrombin was strongly inhibited, while production of phosphatidic acid was unaffected. The inhibitory effect of cytochalasin D was not observed when platelet aggregation was prevented by the RGDS peptide. We also found that cytochalasin D did not affect PtdIns(3,4)P2 synthesis induced by concanavalin A (ConA), which is known to occur through an aggregation-independent mechanism. Moreover, thrombin, but not ConA, induced the translocation of phosphatidylinositol 3-kinase to the cytoskeleton. This process was equally inhibited by both the RGDS peptide and cytochalasin D. These results demonstrate that the cytoskeleton represents a functional link between thrombin-induced aggregation and synthesis of PtdIns(3,4)P2.

Phosphoinositide 3-kinase (PI3K) has been shown to play an essential role in G protein-induced signaling even in non-myeloid cells where few agonists of G protein-coupled receptors are known to activate PI3K. We have identified adherent cell lines where lysophosphatidic acid (LPA) strongly and rapidly activates the accumulation of PI3K lipid products. The process is not modified by expression of a kinase-dead mutant of the Gbetagamma-responsive PI3K p110gamma. In contrast, it is inhibited by genistein or expression of a dominant negative mutant of p85 and potentiated by overexpressing wild-type p110alpha or -beta but not -gamma. By using a specific chemical inhibitor of the epidermal growth factor receptor (EGFR) and expression of a dominant negative mutant, we have observed that recruitment of p85/p110 PI3Ks occurs through transactivation of the EGFR by LPA and downstream mobilization of the docking protein Gab1 that associates with p85 upon LPA stimulation. Finally, we show that LPA cannot activate PI3K in cell lines lacking the EGFR/Gab1 pathway, including cells that transactivate the PDGF receptor. Altogether, these results demonstrate that activation of PI3K by LPA is conditioned by the ability of LPA to transactivate an EGFR/Gab1 signaling pathway.

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.

Flavonoids provide a large number of interesting natural compounds that are consumed daily and exhibit more or less potent and selective effects on some signaling enzymes as well as on the growth and proliferation of certain malignant cells in vitro. Among the identified signal transducers, phosphoinositide 3-kinase (PI 3-kinase) and protein kinase C (PKC) are now considered key players in many cellular responses including cell multiplication, apoptosis, and transformation. Despite their lack of strict specificity, some flavonoids provide valuable bases for the design of analogues that could be used to specifically block particular isoforms of PI 3-kinase or PKC and their downstream-dependent cellular responses.

The hepatic lipase acting on triglyceride-rich high-density lipoprotein2 (HDL2) induces the formation of pre beta 1-HDL, leaving a residual alpha-migrating HDL particle that was named "remnant-HDL2" (Barrans, A., Collet, X., Barbaras, R., Jaspard, B., Manent, J., Vieu, C., Chap, H., and Perret, B. (1994) J. Biol. Chem. 269, 11572-11577.]. In this study, these two product particles generated by hepatic lipase were isolated by density gradient ultracentrifugation. Particles were first characterized in terms of chemical composition, density, and mass. The pre beta 1-HDL obtained in vitro contain one to two molecules of apoA-I, associated with phospholipids, and free and esterified cholesterol. When compared to triglyceride-rich HDL2, remnant-HDL2 have lost on average one molecule of apoA-I, 60% of triacylglycerols, and 15% of phospholipids. The estimated composition is concordant with the hypothesis of the splitting of a substrate particle into one pre beta 1-HDL and one remnant-HDL2. Spectroscopic studies were carried out to monitor changes in lipid fluidity upon lipolysis. The fluorescence anisotropy was measured using (1,6)-diphenyl-hexa-(1,3, 5)-triene as a probe, and the degree of order was calculated from electron spin resonance spectra using the 5-nitroxy-derivative of stearic acid. Both approaches showed a decreased lipid fluidity in remnant-HDL2, as compared to triglyceride-rich HDL2. The immunoreactivity of apoA-I toward several monoclonal antibodies was assayed as a reflection of changes of apoA-I conformation. In remnant-HDL2, as compared to triglyceride-rich HDL2, a lower reactivity was noted with the 2G11 antibody, which interacts in the NH2 terminal part of apoA-I. Finally, remnant-HDL2 was clearly different from HDL3 with respect to all of the parameters studied, demonstrating that hepatic lipase does not promote the direct conversion of HDL2 to HDL3. Thus, hepatic lipase produces remnant-HDL2 particles, which display modifications of apoA-I conformation and of fluidity of the lipid environment. This newly described HDL2 subfraction may play a major role in the reverse cholesterol transport.

An insertion/deletion (I/D) polymorphism of the angiotensin I-converting enzyme (ACE) has been associated with an increased risk of coronary artery disease (CAD) and myocardial infarction (MI). However, this finding has not been fully investigated in European populations with very low CAD risk. In a case-control study on a population from Southern Europe (Toulouse, France), we evaluated the ACE I/D polymorphism in 405 men, aged 35-65 years, who underwent coronary angiography and in 357 representative control men within the same age range. We also explored associations in the patients between this polymorphism and CAD severity. The ACE genotype was not associated with the presence of either CAD or MI. The ACE genotype was not a marker for angiographically assessed CAD severity. In a sample in one of the European populations with the lowest CAD risk, ACE I/D polymorphism was not associated with an increased risk for CAD or MI and did not influence the extent of CAD.

Bruton tyrosine kinase (Btk) plays a crucial role in the differentiation of B lymphocytes and belongs to the group of Tec kinases, which are characterised by the presence of a pleckstrin homology domain. Here we show that Btk is activated and undergoes tyrosine phosphorylation upon challenge of platelet thrombin receptor, these responses requiring engagement of αIIb/β3 integrin and phosphoinositide 3-kinase activity. These data unravel a novel signalling pathway involving Btk downstream of an adhesive receptor via a complex regulation implicating the products of phosphoinositide 3-kinase, which might act to anchor Btk at the membrane.

A number of reports suggest that under different conditions leading to cytoskeleton reorganization the GTPase Rac1 and possibly RhoA are downstream targets of phosphoinositide 3-kinase (PI 3-kinase). In order to gain more insight into this particular signaling pathway, we have addressed the question of a possible direct interaction of PI 3-kinase products with the Rho family GTPases RhoA, Rac1, and Cdc42. Using recombinant proteins, we found that Rac1 and, to a lesser extent, RhoA but not Cdc42 were capable to selectively bind to phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) in a mixture of crude brain phosphoinositides. Nucleotide-depleted Rac1 was the most efficient, but the GDP- and GTP-bound forms retained significant PtdIns(3,4,5)P3 binding activity. This protein-lipid association involved electrostatic as well as hydrophobic interactions, since both phosphate groups located at specific positions of the inositol ring and fatty-acyl chains were absolutely required. Based on the sequence of Rac1, two potential binding sites were identified, one at the C terminus and one in the extra alpha-helical domain. Deletion of these two domains resulted in a complete loss of binding to PI 3-kinase products. Finally, PtdIns(3, 4,5)P3 strongly stimulated GDP dissociation from Rac1 in a dose-dependent manner. In agreement, data obtained in intact cells suggest that PtdIns(3,4,5)P3 might target Rac1 to peculiar membrane domains, allowing formation of specific clusters containing not only small GTPases but other partners bearing pleckstrin homology domains such as specific exchange factors required for Rac1 and RhoA activation.

International audience; We have shown previously that ADP released upon platelet adhesion mediated by IIb 3 integrin triggers accumulation of phosphatidylinositol 3,4-bisphosphate (PtdIns-3,4-P 2) (Gironcel, D., Racaud-Sultan, C., Payras-tre, B., Haricot, M., Borchert, G., Kieffer, N., Breton, M., and Chap, H. (1996) FEBS Lett. 389, 253–256). ADP has also been involved in platelet spreading. Therefore, in order to study a possible role of phosphoinositide 3-ki-nase in platelet morphological changes following adhesion , human platelets were pretreated with specific phosphoinositide 3-kinase inhibitors LY294002 and wortmannin. Under conditions where PtdIns-3,4-P 2 synthesis was totally inhibited (25 M LY294002 or 100 nM wortmannin), platelets adhered to the fibrinogen matrix , extended pseudopodia, but did not spread. Moreover , addition of ADP to the medium did not reverse the inhibitory effects of phosphoinositide 3-kinase inhibi-tors on platelet spreading. Although synthetic dipalmi-toyl PtdIns-3,4-P 2 and dipalmitoyl phosphatidylinositol 3,4,5-trisphosphate restored only partially platelet spreading, phosphatidylinositol 4,5-bisphosphate (Pt-dIns-4,5-P 2) was able to trigger full spreading of wort-mannin-treated adherent platelets. Following 32 P labeling of intact platelets, the recovery of [ 32 P]PtdIns-4,5-P 2 in anti-talin immunoprecipitates from adherent plate-lets was found to be decreased upon treatment by wort-mannin. These results suggest that the lipid products of phosphoinositide 3-kinase are required but not sufficient for ADP-induced spreading of adherent platelets and that PtdIns-4,5-P 2 could be a downstream messenger of this signaling pathway.

Cyclophosphamide administration into fasted rabbits induces a hypertriglyceridaemia and a defect in vascular lipoprotein lipase. Heart LPL activity was more than 50% decreased after antimitotic treatment in fasted animals. The tissue distribution of lipoprotein lipase activity was followed in heart using recycling perfusion. Cyclophosphamide administration resulted in a profound decline in the heparin-releasable lipoprotein lipase activity, concordant with a higher recovery in the residual heart tissue. The effects were more pronounced in fasted than in fed animals. In agreement, the proportion of neosynthesized [35S]methionine-labelled lipoprotein lipase released by heparin was decreased by 50% following antimitotic treatment. The lipolysis of very low density lipoprotein-labelled triacylglycerols was found 2.5-fold reduced in hearts from cyclophosphamide-treated rabbits as compared to controls. These results suggest that a defective secretion of lipoprotein lipase may contribute to the poor expression of lipolytic activity in the vascular bed and to the occurrence of hypertriglyceridaemia during cyclophosphamide treatment.

In thrombin-stimulated platelets αIIbβ3 integrin engagement triggers both phosphatidylinositol 3′,4′-bisphosphate synthesis and calpain activation. We checked the possible involvement of calpains in phosphatidylinositol 3-kinase signalling pathway using a cell permeant specific inhibitor of calpains, calpeptin. In conditions where thrombin-induced platelet aggregation and secretion were not impaired, we found a dose-dependent inhibition of phosphatidylinositol 3,4-bisphosphate synthesis by calpeptin from 50 μg/ml. Moreover, pretreatment of platelets by both calpeptin and the peptide RGDS, an inhibitor of fibrinogen binding to activated αIIbβ3 integrin, did not induce additive effects on phosphatidylinositol 3,4-bisphosphate inhibition. Finally, the p85 regulatory subunit of phosphatidylinositol 3-kinase was still translocated to the cytoskeleton in calpeptin-treated platelets. These data indicate that calpains are involved in the regulation of αIIbβ3 integrin-dependent phosphatidylinositol 3-kinase signalling pathway.©1997 Federation of European Biochemical Societies.

The aim of our study was to evaluate the effect of ADP and the role of cytoskeleton reorganization during reversible and irreversible platelet aggregation induced by ADP and thrombin, respectively, on the heterodimeric (p85alpha-p110) phosphoinositide 3-kinase translocation to the cytoskeleton and its activation. Reversible ADP-induced aggregation was accompanied by a reversible reorganization of the cytoskeleton and an increase in levels of the regulatory subunit p85alpha in this cytoskeleton similar to the increase observed in thrombin-activated platelets. This translocation followed a course parallel to the amplitude of aggregation. No increase in levels of both phosphatidylinositol (3, 4)-bisphosphate (PtdIns(3,4)P2) and phosphatidylinositol-(3,4,5)P3 could, however, be detected even at the maximum aggregation and PI 3-kinase alpha translocation. Moreover, in contrast to the situation for thrombin stimulation, the GTP-binding protein RhoA was hardly translocated to the cytoskeleton when platelets were stimulated with ADP, whereas translocation of pp60(c-)src and focal adhesion kinase did occur. These results suggest (i) translocation of signaling enzymes does not necessarily imply their activation, (ii) the reversibility of ADP-induced platelet aggregation may be the cause or the result of a lack of PI 3-kinase activation and hence of PtdIns(3,4)P2 production, and (iii) RhoA does not seem to be involved in the ADP activation pathway of platelets. Whether PtdIns(3,4)P2 or RhoA may contribute to the stabilization of platelet aggregates remains to be established.