Biomedical subjects

H Chap

Publications and source records attributed to H Chap.

At least 37 records · Page 2Linked to original sources

PubMed1998

Phosphatidylinositol 3,4,5-trisphosphate-dependent stimulation of phospholipase C-gamma2 is an early key event in FcgammaRIIA-mediated activation of human platelets.

Platelets express a single class of Fcgamma receptor (FcgammaRIIA), which is involved in heparin-associated thrombocytopenia and possibly in inflammation. FcgammaRIIA cross-linking induces platelet secretion and aggregation, together with a number of cellular events such as tyrosine phosphorylation, activation of phospholipase C-gamma2 (PLC-gamma2), and calcium signaling. Here, we show that in response to FcgammaRIIA cross-linking, phosphatidylinositol (3,4, 5)-trisphosphate (PtdIns(3,4,5)P3) is rapidly produced, whereas phosphatidylinositol (3,4)-bisphosphate accumulates more slowly, demonstrating a marked activation of phosphoinositide 3-kinase (PI 3-kinase). Inhibition of PI 3-kinase by wortmannin or LY294002 abolished platelet secretion and aggregation, as well as phospholipase C (PLC) activation, indicating a role of this lipid kinase in the early phase of platelet activation. Inhibition of PLCgamma2 was not related to its tyrosine phosphorylation state, since wortmannin actually suppressed its dephosphorylation, which requires platelet aggregation and integrin alphaIIb/beta3 engagement. In contrast, the stable association of PLCgamma2 to the membrane/cytoskeleton interface observed at early stage of platelet activation was fully abolished upon inhibition of PI 3-kinase. In addition, PLCgamma2 was able to preferentially interact in vitro with PtdIns(3,4,5)P3. Finally, exogenous PtdIns(3,4,5)P3 restored PLC activation in permeabilized platelets treated with wortmannin. We propose that PI 3-kinase and its product PtdIns(3,4,5)P3 play a key role in the activation and adequate location of PLCgamma2 induced by FcgammaRIIA cross-linking.

Androstadienes↗

PubMed1998

Lipid products of phosphoinositide 3-kinase and phosphatidylinositol 4',5'-bisphosphate are both required for ADP-dependent platelet spreading.

We have shown previously that ADP released upon platelet adhesion mediated by alphaIIb beta3 integrin triggers accumulation of phosphatidylinositol 3',4'-bisphosphate (PtdIns-3,4-P2) (Gironcel, D. , Racaud-Sultan, C., Payrastre, 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-kinase 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-P2 synthesis was totally inhibited (25 microM 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 inhibitors on platelet spreading. Although synthetic dipalmitoyl PtdIns-3,4-P2 and dipalmitoyl phosphatidylinositol 3',4', 5'-trisphosphate restored only partially platelet spreading, phosphatidylinositol 4',5'-bisphosphate (PtdIns-4,5-P2) was able to trigger full spreading of wortmannin-treated adherent platelets. Following 32P labeling of intact platelets, the recovery of [32P]PtdIns-4,5-P2 in anti-talin immunoprecipitates from adherent platelets was found to be decreased upon treatment by wortmannin. 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-P2 could be a downstream messenger of this signaling pathway.

Adenosine Diphosphate↗

PubMed1998

Ectopic epididymal expression of guinea pig intestinal phospholipase B. Possible role in sperm maturation and activation by limited proteolytic digestion.

Guinea pig intestinal phospholipase B is a calcium-independent phospholipase hydrolyzing sequentially the acyl ester bonds at sn-2 and sn-1 positions of glycerophospholipids, promoting the formation of sn-glycero-3-phosphocholine from phosphatidylcholine. This 140-kDa glycoprotein from the brush border membrane of differentiated enterocytes contributes to lipid digestion as an ectoenzyme. The cDNA coding for guinea pig phospholipase B was revealed to be the homologue of AdRab-B, an mRNA appearing in rabbit upon intestine development. The sequence predicts a polypeptide of 1463 amino acids displaying four homologous repeats, two of them containing the lipase consensus sequence GXSXG. A 5-kilobase transcript was particularly abundant in mature ileal and jejunal enterocytes but was also detected in epididymis, where phospholipase B displayed a higher molecular mass (170 kDa versus 140 kDa in intestine), with no obvious evidence for enzyme activity. Trypsin treatment of phospholipase B immunoprecipitated from epididymal membranes reduced its size to 140 kDa, coinciding with the appearance of a significant phospholipase A2 activity. The same results were obtained in COS cells transfected with phospholipase B cDNA. Since sn-glycero-3-phosphocholine present at high concentrations in seminal plasma mainly stems from epididymis, this suggests a possible role of phospholipase B in male reproduction. This novel localization also unravels a mechanism of phospholipase B activation by limited proteolysis involving either trypsin in the intestinal lumen or a trypsin-like endopeptidase in the male reproductive tract.

Amino Acid Sequence↗

PubMed1998

Overexpression of the myristoylated alanine-rich C kinase substrate in human choroidal melanoma cells affects cell proliferation.

Reduced expression of the myristoylated alanine-rich C kinase substrate (MARCKS) has been described in various cell lines after oncogenic or chemical transformation, leading to the question of whether this protein may be involved in cell proliferation. Here we compare the expression of MARCKS in human tumor-derived choroidal melanoma cells (OCM-1) and in primary cultures of normal choroidal melanocytes. We found an important down-regulation of the protein in the melanoma cell line. Stable transfection of these cells with the cDNA coding for MARCKS led to the selection of several clones expressing variable levels of the protein. Proliferation experiments performed with four of these clones revealed that cell growth was reduced by 35-40% when compared with control cells. Upon serum starvation, cell proliferation was almost abolished when the expression level of MARCKS was high, whereas it was only partially reduced in the controls. MARCKS overexpression induced a higher percentage of cells in the G0-G1 phase of the cell cycle upon serum starvation, as well as the inhibition of colony formation in soft agar. Finally, the expression of the CDK inhibitor p27 was increased in the cells presenting a high level of MARCKS protein. Altogether, these data suggest that the expression of this protein kinase C substrate affects the proliferation and partially reverts the transformed phenotype of the OCM-1 cells.

Cell Cycle Proteins↗

PubMed1998

Phosphatidylinositol 3-kinase inhibitors block aortic smooth muscle cell proliferation in mid-late G1 phase: effect on cyclin-dependent kinase 2 and the inhibitory protein p27KIP1.

In the present study, we investigated the involvement of phosphatidylinositol 3-kinase (PI 3-kinase) activity in the progression of vascular smooth muscle cells (VSMCs) throughout the G1 phase of cell cycle. Addition of two selective inhibitors of PI 3-kinase, LY 294002 or wortmannin, to quiescent VSMCs prevented serum-induced DNA synthesis in a dose-dependent manner with IC50 of 8.7 +/- 2.0 microM and 53.9 +/- 8.5 nM, respectively. Time course studies revealed that the two PI 3-kinase inhibitors blocked VSMC proliferation in mid-late G1 phase, about 6 h before the G1/S transition. This G1 growth arrest was due, at least in part, to the reduction of the CDK2 associated kinase activity resulting mainly from the upregulation of the inhibitory protein p27KIP1.

Androstadienes↗

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↗

PubMed1998

[Prevalence and management of major risk factors in 500 men with stable coronary disease].

Management of major risk factors (smoking, hypercholesterolaemia, hypertension), in the context of secondary prevention, has an impact on recurrence and life expectancy. However, there is a delay between the publication of therapeutic trials and their diffusion within the medical community. The objective of this study was to evaluate the prevalence and management of the main risk factors in a sample of 500 men, with a mean age of 55.1 +/- 7.5 years, presenting with stable coronary artery disease. 11% of subjects had a family history of premature myocardial infarction. Smoking was frequent: 21% of smokers, 60% of ex-smokers. Hypercholesterolaemia (LDL-C > 3.4 mmole/l or treatment) was present in 82% of subjects. Only 45% of treated subjects had an LDL-C < 3.4 mmole/l. Hypertension (systolic blood pressure > or = 140 mmHg or diastolic blood pressure > or = 90 mmHg or treatment) was present in 61% of subjects. Only 33% of treated subjects were controlled. Non-insulin-dependent diabetes mellitus (blood glucose > or = 7.7 mmole/l or treatment) was present in 21% of subjects. Only 43% of treated subjects were controlled. Calculation of the distribution of major risk factors (smoking, pathological obesity, hypercholesterolaemia, hypertension, diabetes) showed that 90% of coronary patients presented at least two risk factors. Overall, the prevalence and management of risk factors in patients with stable coronary artery disease is far from optimal.

Adult↗

PubMed1997

Tyrosine phosphorylation and relocation of SHIP are integrin-mediated in thrombin-stimulated human blood platelets.

The SH2 domain-containing inositol 5-phosphatase, SHIP, known to dephosphorylate inositol 1,3,4,5-tetrakisphosphate and phosphatidylinositol 3,4,5-trisphosphate has recently been shown to be expressed in a variety of hemopoietic cells. This 145-kDa protein is induced to associate with Shc by multiple cytokines and may play an important role in the negative regulation of immunocompetent cells mediated by FcgammaRIIB receptor. We report here that SHIP is present in human blood platelets and may be involved in platelet activation evoked by thrombin. Platelet SHIP was identified by Western blotting as a single 145-kDa protein. Both phosphatidylinositol 3,4,5-trisphosphate and inositol 1,3,4, 5-tetrakisphosphate 5-phosphatase activities could be demonstrated in anti-SHIP immunoprecipitates of platelet lysate. Thrombin stimulation induced a tyrosine phosphorylation of SHIP, this effect being prevented if platelets were not shaken or if RGD-containing peptides were present, indicating an aggregation-dependent, integrin-mediated event. Moreover, although the intrinsic phosphatase activity of SHIP did not appear to be significantly increased, tyrosine-phosphorylated SHIP was relocated to the actin cytoskeleton upon activation in an aggregation- and integrin engagement-dependent manner. Finally, the striking correlation observed between phosphatidylinositol 3,4-bisphosphate production and the tyrosine phosphorylation of SHIP, as well as its relocation to the cytoskeleton upon thrombin stimulation, suggest a role for SHIP in the aggregation-dependent and GpIIb-IIIa-mediated accumulation of this important phosphoinositide.

Blood Platelets↗

PubMed1997

Integrin-dependent tyrosine phoshorylation and cytoskeletal translocation of Tec in thrombin-activated platelets.

Using a specific polyclonal anti-Tec antibody, we have shown that Tec is expressed in human platelets. In addition, Tec was found to undergo tyrosine phosphorylation during platelet activation. The phosphorylation increased after 1 min and remained stable after 3 min of thrombin treatment. The tetrapeptide RGDS inhibited more than 90% of thrombin-induced tyrosine phosphorylation of Tec and blocked its translocation to the cytoskeleton. These results suggest that Tec participates in platelet signaling downstream of integrin activation.

Biological Transport↗

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↗

PubMed1997

Relationship between flavonoid structure and inhibition of phosphatidylinositol 3-kinase: a comparison with tyrosine kinase and protein kinase C inhibition.

Depending on their structure, flavonoids display more or less potent inhibitory effects on the growth and proliferation of certain malignant cells in vitro, and these effects are thought to be due to inhibition of various enzymes. We investigated the inhibitory action of fourteen flavonoids of different chemical classes on phosphatidylinositol 3-kinase alpha (PI 3-kinase alpha) activity, an enzyme recently shown to play an important role in signal transduction and cell transformation. Of the fourteen flavonoids tested, myricetin was the most potent PI 3-kinase inhibitor (IC50 = 1.8 microM), while luteolin and apigenin were also effective inhibitors, with IC50 values of 8 and 12 microM, respectively. Fisetin and quercetin, as previously reported, were also found to significantly inhibit PI 3-kinase activity. The same flavonoids were also analyzed for inhibition of epidermal growth factor receptor (EGF-R), intrinsic tyrosine kinase and bovine brain protein kinase C (PKC). At elevated doses, some of these flavonoids were found to also cause significant inhibition of PKC and tyrosine kinase activity of EGF-R. A structure-activity study indicated that the position, number and substitution of the hydroxyl group of the B ring, and saturation of the C2-C3 bond are important factors affecting flavonoid inhibition of PI 3-kinase. They may also play a significant role in specificity of inhibition and could help to provide a basis for the further design of specific inhibitors of this lipid kinase. Finally, possible relationships between the antitumoral properties of these flavonoids and their biological activities are discussed.

Dose-Response Relationship, Drug↗

PubMed1997

Impaired secretion of heart lipoprotein lipase in cyclophosphamide-treated rabbit.

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.

Animals↗

PubMed1997

Calpains are involved in phosphatidylinositol 3',4'-bisphosphate synthesis dependent on the alpha IIb beta 3 integrin engagement in thrombin-stimulated platelets.

In thrombin-stimulated platelets alpha IIb beta 3 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 micrograms/ml. Moreover, pretreatment of platelets by both calpeptin and the peptide RGDS, an inhibitor of fibrinogen binding to activated alpha IIb beta 3 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 alpha IIb beta 3 integrin-dependent phosphatidylinositol 3-kinase signalling pathway.

Biological Transport↗

PubMed1997

Reversible translocation of phosphoinositide 3-kinase to the cytoskeleton of ADP-aggregated human platelets occurs independently of Rho A and without synthesis of phosphatidylinositol (3,4)-bisphosphate.

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.

Adenosine Diphosphate↗

PubMed1997

Studies on ether-phospholipids of vascular smooth muscle cells. Identification of a rapid Ca(2+)-dependent hydrolysis of alkyl-phosphatidylethanolamine promoted by endothelin-l.

We have investigated the metabolism of 1-O-[3H]octadecyl-sn-glycero-3-phosphocholine ([3H]lyso PAF) and [3H] myristic acid in secondary cultures of aortic smooth muscle cells (SMC) to characterize the origin of second messengers generated upon stimulation with endothelin-l (ET-l). When cells were labelled with [3H]lyso PAF, we observed a transfer of the label from phosphatidylcholine (PC) to phosphatidylethanolamine (PE) In contrast, incubation with [3H]lyso PAF labelled mainly alkyl-subclasses while [3H]myristate was associated with diacyl-subclasses. Using these specific labelling procedures, we have shown that ET-l induced a strong hydrolysis of PE. This hydrolysis was specific for alkyl-PE with a maximum after 5 s of stimulation. We have also observed an extracellular Ca(2+)-dependent increase in diglyceride (DG), phosphatidic acid (PA) and mainly triglyceride (TG) concomitant to alkyl-PE hydrolysis. Thus, alkyl-DG generated from alkyl-PE appears to be a major product in ET-l stimulation of SMC. These results suggest a new level of complexity in the signal transduction cascade involving a specificity for phospholipid subclasses.

Animals↗

PubMed1997

Dual effect of lysophosphatidic acid on proliferation of glomerular mesangial cells.

Among the variety of factors able to contribute to mesangial hypertrophy by altering mesangial cell growth, lysophosphatidic acid (LPA) is the focus of increasing attention. It is produced in plasma following platelet activation, as well as by mesangial cells stimulated by secretory phospholipase A2. As mitogenic/antimitogenic properties of LPA are already described in a variety of cells, knowledge of its specific actions on mesangial cells is of potential interest regarding the pathophysiology of glomerulus damage in situ. We tested the effect of LPA on cultured rat mesangial cell growth. At 10 to 20 microM, LPA stimulated thymidine incorporation as well as phosphorylation of mitogen-activated protein kinases (MAP-kinases) p42-p44 in dose- and time-dependent manner, which demonstrated a positive effect on cell proliferation. However, higher concentrations of LPA (100 microM) were unable to stimulate thymidine incorporation and partly inhibited the proliferative effect as well as p42-p44 phosphorylation evoked by serum. Finally, whereas lysophosphatidylcholine (10 to 20 microM) was lytic for mesangial cells, no cell lysis could be detected at the highest concentrations of LPA. Taken together, these results suggest that LPA exerts a dual effect on mesangial cell proliferation, which could be due to activation of distinct specific signaling pathways, in dose-dependent fashion. Specific actions of LPA able to modify mesangial cell proliferation in a positive or negative manner are also likely to influence the pathophysiological processes involved in the progression of glomerulosclerosis in the kidney.

Animals↗

PubMed1997

Structural and functional comparison of HDL from homologous human plasma and follicular fluid. A model for extravascular fluid.

In the preovulatory period, follicular fluid contains only HDL. Biochemical characterization of such lipoproteins showed that follicular fluid HDLs were cholesterol-poor particles compared with serum HDLs, whereas the amount of phospholipids, expressed as percent weight, was significantly higher in follicular fluid HDLs (28.5%) than in serum HDLs (25.0%, P < .05). The amount of apolipoprotein (apo) A-IV per apo A-I was significantly higher in follicular fluid than in serum (0.77 versus 0.58 mg/g apo A-I, P < .02). To explore the role of HDLs as cholesterol acceptors in physiological media, we compared the ability of either whole human follicular fluids or homologous sera to promote cellular cholesterol efflux using Fu5AH rat hepatoma cells. At equivalent concentrations of HDL cholesterol in follicular fluid and in serum, t1/2 values for cholesterol efflux were in the same range. In addition, estimated maximal efflux values were not significantly different in follicular fluid and serum (45.9% and 49.6%, respectively), as were K(m) values (0.064 and 0.071 mmol/L HDL cholesterol respectively). In addition, isolated HDLs displayed the same capacity to promote cellular cholesterol efflux in both media. Thus, the kinetics and dose-response data between these two physiological media showed that HDLs play the major role in cellular cholesterol efflux. The rate of cholesterol esterification, as measured in the presence of cells, was significantly higher in follicular fluid than in serum at constant HDL cholesterol concentrations, whereas the rate of esterified cholesterol transfer toward added LDL was lower. In contrast, in a cell-free system, lecithin:cholesterol acyltransferase activity represented only 26% of that in serum HDL, whereas cholesterol ester transfer protein activities were comparable. In summary, in this particular model, we confirmed the essential role of HDLs as physiological acceptors in the removal of cellular cholesterol.

Animals↗

PubMed1997

Protein tyrosine phosphatase SHP-1 fails to associate with cytoskeleton but is normally phosphorylated upon thrombin stimulation of thrombasthenic platelets.

SHP-1 is a cytoplasmic protein tyrosine phosphatase predominantly expressed in hematopoietic cells. Upon thrombin stimulation of human platelets, SHP-1 is rapidly phosphorylated on both serine and tyrosine residues, and becomes associated with the cytoskeleton, where it could participate in the formation of multiprotein signalling complexes. In order to discriminate between signalling events occurring downstream of G-protein-coupled thrombin receptor and those subsequent to integrin alpha IIb beta 3 engagement, SHP-1 behaviour was examined in platelets from two patients lacking integrin alpha IIb beta 3 (Glanzmann's thrombasthenia). Upon thrombin stimulation, phosphorylation of SHP-1 occurred normally in thrombasthenic platelets, whereas association with the cytoskeleton was abolished. Moreover, inhibition of normal platelet aggregation with the tetrapeptide arg-gly-asp-ser (RGDS) which impairs fibrinogen binding to integrin alpha IIb beta 3, did not alter significantly SHP-1 phosphorylation. It is concluded that SHP-1 phosphorylation is not a consequence of integrin signalling but might rather occur downstream of thrombin receptor and heterotrimeric G-proteins.

Blood Platelets↗