Maryse Lenfant

Interaction of benzodiazepines with mouse macrophages.

Mouse peritoneal inflammatory macrophages have a saturable binding site for [3H]flunitrazepam with a KD of 43 +/- 7 nM and Bmax of 391 +/- 58 fmol per 10(6) cells, which corresponds to 250 000 sites per cell. The IC50 values for PK 11195, Ro 5-4864, diazepam, flunitrazepam, clonazepam, ethyl-beta carboline 3-carboxylate and Ro 15-1788 were 5.6, 6.5, 21, 40, 3 000, 12 000 and 80 000 nM respectively; gamma-aminobutyric acid did not displace [3H]flunitrazepam. These results indicate that the benzodiazepine binding site on the macrophage is of the peripheral type. In vivo experiments show that, at 1 mg/kg, peripheral and mixed type molecules stimulate the humoral response to sheep red blood cells while central type benzodiazepines are inactive. The hypothesis that this effect is mediated through the binding on the macrophage may be considered.

Nitric oxide formation during microsomal hepatic denitration of glyceryl trinitrate: Involvement of cytochrome P-450

Glyceryl trinitrate was denitrated by rat liver microsomes in the presence of NADPH with formation of a mixture of glyceryl dinitrates and glyceryl mononitrates. The highest activity was obtained under anaerobic conditions and the reaction was inhibited by O2 indicating that it is a reductive denitration. It was also inhibited by CO, metyrapone and miconazole showing that it was catalyzed by cytochrome P-450. Finally the formation of the cytochrome P-450-Fe(II)-NO complex during this reaction was shown by visible spectroscopy. These data demonstrate that microsomal reductive denitration of glyceryl trinitrate is catalyzed by cytochrome P-450 and can be involved in the formation of the endothelium-derived relaxing factor (EDRF = nitric oxide).

Involvement of thymosin β4 and endoproteinase Asp-N in the biosynthesis of the tetrapeptide AcSerAspLysPro a regulator of the hematopoietic system

It is shown that AcSDKP a new regulator of the hematopoietic system can he generated from thymosin β4 by a one‐step enzymatic cleavage in vitro and in vivo. AcSDKP and Tβ4 were both detected in bone marrow cells (BMC). Incubation of [3H]Tβ4 with either intact or lysed BMC led to the formation of [3H]AcSDKP whereas the labelled tetrapeptide was not degraded under these conditions. Model enzymatic degradation of Tβ4 carried out with bacterial enzymes suggests that a mammalian endoproteinase Asp‐N might be involved in the formation of AcSDKP through the specific cleavage of the 4Pro‐5 Asp peptide bond of T β4.

Effect of heparin on the stimulation of non-vascular cells by human acidic and basic FGF

We have purified acidic and basic fibroblast growth factors from human brain (h-aFGF, h-bFGF) and studied the effect of heparin on the growth stimulation by these factors of hamster fibroblast CC139 cells and bovine epithelial lens (BEL) cells. In both the presence and the absence of foetal calf serum (FCS) heparin cooperates with h-aFGF in a dose dependent manner to stimulate both types of cells. The cooperation with h-bFGF is much less. An unpurified human brain fraction containing both factors behaves differently: in the absence of FCS, heparin enhances the activity of the crude fraction on BEL cells, while in the presence of FCS, it decreases this activity. These results indicate that heparin cooperates strongly with h-aFGF to stimulate non-vascular cell proliferation while in a partially purified extract and in the presence of serum it can induce the opposite effect.

Nitration of catecholamines with nitrogen oxides in mild conditions: a hypothesis for the reactivity of NO in physiological systems

Abstract Dopamine, norepinephrine and epinephrine react at room temperature, in acetate buffer (36) with sodium nitrite or in non-deaerated phosphate buffer (pH 7.4) with NO. The corresponding 6-nitro derivatives are formed.

Nitroglycerin metabolism by Phanerochaete chrysosporium: evidence for nitric oxide and nitrite formation

We have demonstrated that a filamentous fungus Phanerochaete chrysosporium converts glyceryl trinitrate (GTN) into its di- and mononitrate derivatives concurrently with the formation of nitric oxide detected by electron paramagnetic resonance (EPR), and the formation of nitrite. The metabolisms of nitrite and nitrate by the fungus are evaluated and taken into account when considering GTN degradation. Lack of evidence for nitrate formation from GTN suggests that an esterase-type activity is not involved. Furthermore, the kinetics of appearance of the hemoprotein-NO and non-heme protein-NO (FeS-NO) complexes indicate that an enzymatic process producing NO directly from GTN may be involved concurrently with a glutathione transferase-like system.

In vivo effect of the tetrapeptide, N-Acetyl-Ser-Asp-Lys-Pro, on the G1-S transition of rat hepatocytes

Abstract. The synthetic molecule N‐Acetyl‐Ser‐Asp‐Lys‐Pro (Ac‐SDKP), corresponding to the low molecular weight inhibitory factor preventing in vivo haematopoietic stem cell (CFU‐S) entry into DNA synthesis, was tested in two heterologous systems in vivo: adult regenerating rat liver and 10‐day‐old rat hepatocytes synchronized by an irritating trigger. In both systems, it was shown that doses of 2–8 μg kg‐1 of tetrapeptide inhibited 50–70% of the hepatocyte G1‐S transitions.

Interaction of G-actin with thymosin ?4 and its variants thymosin ?9 and thymosin ? 9 met

SummaryThymosin β4 is a major actin sequestering peptide in vertebrate cells and plays a role in the regulation of actin monomer/polymer ratio. Thymosin β9 and thymosin β9met are minor variants of thymosin β4. The possible function of these peptides has been investigated by comparing the actin binding properties of these β-thymosins. Thymosin β9 and thymosin β9met were found to inhibit polymerization of ATP-actin with identical Kds of 0.7–0.8 μM (as compared to 2±0.3 μM for thymosin β4); like thymosin β4, they bound to ADP-G-actin with a 100-fold lower affinity than to ATP-G-actin. The interaction of thymosin β4 and thymosin β9met with G-actin was weakened 20-fold upon oxidation of methionine-6 into methionine sulfoxide. Binding of thymosin β4 to G-actin was accompanied by a 15% increase in the fluorescence intensity of actin tryptophans, and a 10 nm emission blue shift. Methionine-6 played an important role in this effect. The fluorescence change was used to monitor the kinetics of thymosin β4 binding to G-actin in the stopped-flow. The reaction was bimolecular, with association and dissociation rate constants of ∼1.5 μM-1 s-1 and 2s-1 respectively, under physiological conditions. The possible physiological significances of methionine-6 oxidation and of the relatively slow binding kinetics in regulating thymosin β4 function in vivo is discussed.

Particular ability of cytochrome P-450 CYP3A to reduce glyceryl trinitrate in rat liver microsomes: Subsequent formation of nitric oxide

Glyceryl trinitrate was denitrated in rat hepatic subcellular fractions, with formation of glyceryl dinitrates and glyceryl mononitrates. Among differently treated-rat liver microsomes, the highest microsomal activity was obtained under anaerobic conditions with microsomal preparations from dexamethasone-treated rats and NADPH. The reaction was inhibited by O2, CO, miconazole, dihydroergotamine and troleandomycin showing that it was catalyzed by cytochrome P-450 CYP3A isoforms. The formation of a transient cytochrome P-450 Fe(II)-NO complex during this reaction was shown by visible spectroscopy. The cytosolic activity was shown to be dependent on glutathione and glutathione transferase and was not inhibited by dioxygen. In the hepatic 9000 x g supernatant containing both NADPH and cytochrome P-450 and glutathione and glutathione transferase, the cytochrome P-450-dependent reaction accounts for 30-40% of the total denitration activity observed under anaerobic conditions, using 100 microM GTN.

In vivo immunomodulating activity of PK 1195, a structurally unrelated ligand for “peripheral” benzodiazepine binding sites — I. Potentiation in mice of the humoral response to sheep red blood cells

PK 11195, an isoquinoline carboxamide compound with the highest affinity for the peripheral type benzodiazepine binding site has been shown to enhance the humoral response of mice to sheep red blood cells, a T cell dependent antigen. The compound was active when administered i.p. 1 day following immunization. The active doses ranged from 10 ng to 1 mg/kg. Such an immunomodulating activity might be related to the presence of peripheral type benzodiazepine binding sites, previously detected on the macrophage.