Biomedical subjects

Michel Lagarde

Publications and source records attributed to Michel Lagarde.

42 records · Page 3Linked to original sources

PubMed2003

IGF-I-induced differentiation of L6 myogenic cells requires the activity of cAMP-phosphodiesterase.

Inhibition of type 4 cAMP-specific phosphodiesterase (PDE4) activity in L6-C5 and L6-E9 abolished myogenic differentiation induced by low-serum medium and IGF-I. L6-C5 cells cultured in low-serum medium displayed a PDE4 activity higher than cells cultured in serum-free medium, a condition not sufficient to induce differentiation. In the presence of serum, PDE4D3, the major isoform natively expressed in L6-C5 cells, translocated to a Triton-insoluble fraction, which increased the PDE specific activity of the fraction, and exhibited a Mr shift typical of phosphorylation of this isoform. Furthermore, serum promoted the localization of PDE4D3 to a vesicular subcellular compartment. In L6-C5 cells, IGF-I is a stronger inducer of myogenic differentiation in the presence than in absence of serum. Its ability to trigger differentiation in the absence of serum was restored by overexpressing wild-type PDE4D3, but not a phosphorylation-insensitive mutant. This finding was confirmed in single cells overexpressing a GFP-PDE4D3 fusion protein by assessing nuclear accumulation of myogenin in both L6-C5 and L6-E9. Overexpression of other PDE isoforms was less efficient, confirming that PDE4D3 is the physiologically relevant phosphodiesterase isoform in the control of myogenesis. These results show that downregulation of cAMP signaling through cAMP-phosphodiesterase stimulation is a prerequisite for induction of myogenesis.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMed2003

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

Neutrophils are physiologically associated with platelets in whole blood. Inflammatory reactions can be modulated by the presence of platelets. To investigate the influence of platelets on neutrophil activity, we studied the 5-lipoxygenase (5-LOX) metabolic pathway in normal human blood neutrophils stimulated with f-Met-Leu-Phe (fMLP) or monosodium urate monohydrate (MSUM) in the presence of autologous platelets. Platelets inhibited by more than 90% the synthesis of leukotriene B(4) and 5-HETE in neutrophils activated with fMLP or MSUM. The addition of exogenous arachidonic acid did not reverse the inhibitory effect of platelets on 5-LOX-generated metabolites in fMLP- or MSUM-activated neutrophils. Preincubation of neutrophils with adenosine deaminase reversed the inhibitory effect of platelets in fMLP-treated neutrophils, indicating that adenosine was responsible for the platelet inhibition of leukotriene B(4) and 5-HETE formation. In contrast, adenosine deaminase had no influence on the inhibitory effects of platelets in MSUM-stimulated cells. These results suggest that platelets can inhibit the synthesis of 5-LOX products (a). by acting mainly downstream to phospholipase A(2) in cells stimulated by fMLP or MSUM, (b). through adenosine when neutrophils are activated with fMLP, and (c). by an adenosine-independent mechanism in MSUM-activated neutrophils by an as-yet-unidentified mediator.

Adenosine Deaminase↗

PubMed2002

The mechanism of docosahexaenoic acid-induced phospholipase D activation in human lymphocytes involves exclusion of the enzyme from lipid rafts.

Docosahexaenoic acid (DHA), an n-3 polyunsaturated fatty acid that inhibits T lymphocyte activation, has been shown to stimulate phospholipase D (PLD) activity in stimulated human peripheral blood mononuclear cells (PBMC). To elucidate the mechanisms underlying the DHA-induced PLD activation, we first characterized the PLD expression pattern of PBMC. We show that these cells express PLD1 and PLD2 at the protein and mRNA level and are devoid of oleate-dependent PLD activity. DHA enrichment of PBMC increased the DHA content of cell phospholipids, which was directly correlated with the extent of PLD activation. The DHA-induced PLD activation was independent of conventional protein kinase C but inhibited by brefeldin A, which suggests ADP-ribosylation factor (ARF)-dependent mechanism. Furthermore, DHA enrichment dose-dependently stimulated ARF translocation to cell membranes. Whereas 50% of the guanosine 5'-3-O-(thio)triphosphate plus ARF-dependent PLD activity and a substantial part of PLD1 protein were located to the detergent-insoluble membranes, so-called rafts, of non-enriched PBMC, DHA treatment strongly displaced them toward detergent-soluble membranes where ARF is present. Collectively, these results suggest that the exclusion of PLD1 from lipid rafts, due to their partial disorganization by DHA, and its relocalization in the vicinity of ARF, is responsible for its activation. This PLD activation might be responsible for the immunosuppressive effect of DHA because it is known to transmit antiproliferative signals in lymphoid cells.

ADP-Ribosylation Factors↗

PubMed2002

Advanced glycation end-products induce apoptosis of bovine retinal pericytes in culture: involvement of diacylglycerol/ceramide production and oxidative stress induction.

One of the earliest changes observed in retinal microvessels in diabetic retinopathy is the selective loss of intramural pericytes. We tested the hypothesis that AGE might be involved in the disappearance of retinal pericytes by apoptosis and further investigated the signaling pathway leading to cell death. Chronic exposure of pericytes to methylglyoxal-modified bovine serum albumin (AGE-BSA) (3 microM) leads to a 3-fold increase of apoptosis (8.9 +/- 1.1%), associated with an increase in cellular ceramide (185 +/- 12%) and diacylglycerol (194 +/- 9%) levels. Ceramide formation was almost inhibited (95%) by an acidic sphingomyelinase inhibitor, desipramine (0.3 microM). Dual inhibition of ceramide (95%) and diacylglycerol (80%) production was observed with a phosphatidylcholine-phospholipase C inhibitor, D609 (9.4 microM). Taken together, these results suggest activation of phosphatidylcholine-phospholipase C coupled to acidic sphingomyelinase. However, both inhibitors only partially protected pericytes against apoptosis, suggesting another apoptotic pathway independent of diacylglycerol/ceramide production. Treatments with various antioxidants completely inhibited pericyte apoptosis, suggesting oxidative stress induction during this apoptotic process. Inhibition of diacylglycerol/ceramide production by N-acetyl-L-cysteine suggests that oxidative stress acts upstream of the two metabolic pathways. AGE treated with metal chelators were also able to induce pericyte apoptosis, suggesting a specific effect of AGE on intracellular oxidative stress independent of redox-active metal ions bound to AGE. In conclusion, these results identify new biochemical targets involved in pericyte loss, which can provide new therapeutic perspectives in diabetic retinopathy.

Adrenergic Uptake Inhibitors↗

PubMed2002

Synthesis of photoreactive phosphatidic acid analogues displaying activatory properties on cyclic AMP-phosphodiesterases. Photoaffinity labeling of an isoform of phosphodiesterase.

We have previously shown that phosphatidic acid (PA) is a specific activator of some isoforms of type 4 cyclic nucleotide phosphodiesterases (PDE 4) and that accumulation of endogenous PA can, in this way, influence the cAMP signaling pathway in different cell types. Enzyme activation depends on direct binding of the effector to specific sites carried by the enzyme. To identify the binding domain, photoactivatable phosphatidic acid analogues 1-azidoPA (12) and 2-azidoPA (7 and 15), potentially suitable for covalent labeling of PDE4, have been synthesized. The ability of phospholipases A(2) and D to hydrolyze unnatural phospholipids has been considered in this paper. The effect of 1-azidoPA (12) and 2-azidoPA (7 and 15) on the activity of a recombinant PA-sensitive isoform PDE4D3 was evaluated. The three compounds were able to activate the enzyme with different efficiencies. A tritiated analogue of 15 was synthesized and used in PDE4D3 labeling experiments, which showed that this PA analogue was specifically and covalently linked to the enzyme after UV irradiation. Photoactivatable analogues thus appear as suitable tools for the characterization of PA binding sites.

3',5'-Cyclic-AMP Phosphodiesterases↗

PubMedDate not supplied

Hydrolysis of nuclear phospholipids in relation with proliferative state in uterine stromal cells.

The current study examined the metabolism of phospholipid (PL) in the whole cell homogenate and in the nuclear fraction in proliferative and non-proliferative uterine stromal cells (U(III) cells). Growth arrested cells were obtained either from contact-inhibited confluent cells or from proliferative cells treated with aristolochic acid (AR) for 2 days. Fatty acid composition and fatty acid amount of both total and nuclear PL were not significantly different between proliferative, confluent and AR-treated cells. In contrast, marked differences were observed in the incorporation of [(3)H]AA, with greater incorporation in proliferative cells than in confluent or AR-treated cells, particularly in nuclear PL. Considering endogenous level of arachidonic acid (AA) in total and nuclear PL, we found that AA turnover in nuclear PL was especially high compared to that in total PL and that this difference was accentuated in proliferative cells compared to non-proliferative cells. Interestingly, [(3)H]AA incorporation and AA turnover in proliferative, confluent and AR-treated cells vary accordingly to the expression, activity and/or content of pancreatic phospholipase A(2) (PLA(2)-I) in the nuclear compartment of these cells that we reported in previous studies. The changes in metabolism of nuclear PL during cell proliferation are consistent with an enhanced PL hydrolysis that could involve PLA(2)-I.

Arachidonic Acid↗