Evaluation de la toxicité de cinq plantes antiasthmatiques de la médecine traditionnelle ivoirienne

Les feuilles de Boerhavia diffusa Linn. Sp. (Nyctaginaceae), Baphia nitida Lodd., Cassia occidentalis Linn. Sp., Desmodium adscendens (Sw.) DC. et l’écorce de racines de Dichrostachys cinerea (L.) Wight etArn. (Fabaceae) sont traditionnellement utilisées pour traiter l’asthme en Côte d’Ivoire. Les propriétés antispasmodiques de quatre extraits totaux (décocté, hydro-alcoolique, méthanolique et chlorométhylénique) deces substances végétales sur la musculature lisse du tractus respiratoire ont précédemment été montrées sur la trachée isolée de souris, justifiant certainement leur usage antiasthmatique en médecine traditionnelle. Laprésente investigation a consisté en l’étude de la toxicité de ces extraits totaux de plantes. Les tests de cytotoxicité réalisés in vitro sur cellules KB en culture ainsi que les tests in vivo chez la souris avec des doses similaires aux concentrations testées sur la trachée isolée n’ont pas mis d’effets toxiques en évidence à ces doses, suggérant une certaine sécurité d’emploi de ces substances végétales dans le traitement traditionnel de l’asthme

Alteration of vascular reactivity in heart failure: role of phosphodiesterases 3 and 4

International audienc

The negative inotropic effect of beta3-adrenoceptor stimulation is mediated by activation of a nitric oxide synthase pathway in human ventricle.

Beta1- and beta2-adrenoceptors in heart muscle cells mediate the catecholamine-induced increase in the force and frequency of cardiac contraction. Recently, in addition, we demonstrated the functional expression of beta3-adrenoceptors in the human heart. Their stimulation, in marked contrast with that of beta1- and beta2-adrenoceptors, induces a decrease in contractility through presently unknown mechanisms. In the present study, we examined the role of a nitric oxide (NO) synthase pathway in mediating the beta3-adrenoceptor effect on the contractility of human endomyocardial biopsies. The negative inotropic effects of a beta3-adrenoceptor agonist, BRL 37344, and also of norepinephrine in the presence of alpha- and beta1-2-blockade were inhibited both by a nonspecific blocker of NO, methylene blue, and two NO synthase (NOS) inhibitors, L-N-monomethyl-arginine and L-nitroarginine-methyl ester. The effect of the NOS inhibitors was reversed by an excess of L-arginine, the natural substrate of NOS, but not by D-arginine. Moreover, the effects of the beta3-adrenoceptor agonist on contractility were associated with parallel increases in the production of NO and intracellular cGMP, which were also inhibited by NOS inhibitors. Immunohistochemical staining of human ventricular biopsies showed the expression of the endothelial constitutive (eNOS), but not the inducible (iNOS) isoform of NOS in both ventricular myocytes and endothelial cells. These results demonstrate that beta3-adrenoceptor stimulation decreases cardiac contractility through activation of an NOS pathway. Changes in the expression of this pathway may alter the balance between positive and negative inotropic effects of catecholamines on the heart potentially leading to myocardial dysfunction

C006 Nadph-oxidases and uncoupled endothelial NO-synthase in pulmonary arterial hypertension induced by chronic hypoxia

Nitric oxide (NO) production by endothelial NO-synthase (eNOS) is critically dependent on the cofactor, tetrahydrobiopterin (BH4). Depletion in BH4 consecutive to an increase of reactive oxygen species (ROS) production by NADPH-oxidases and/or eNOS over-expression, favour eNOS uncoupling. This study investigates the potential role of NADPH-oxidases and uncoupled eNOS in pulmonary arterial hypertension induced by chronic hypoxia.Male C57BL/6 and eNOS knockout (eNOS-/-) mice were exposed or not to hypobaric hypoxia (0.5atm) for 21 days. Fulton index (right ventricular / left ventricular + septum weight ratio) was determined. Lungs were used for measurement of BH4 (by HPLC), for expression of eNOS (by western-blotting) and of the NADPH-oxidases subunits Nox1, Nox2 and Nox4 (by RT-PCR). Pulmonary arteries were also mounted in a wire myograph for evaluation of vasomotor responses.Chronic hypoxia induced a marked up-regulation of Nox1, Nox2 and Nox4 mRNAs in lungs, and an increase of ROS levels in pulmonary arteries. BH4 levels, as well as eNOS expression, were enhanced in lungs from hypoxic WT mice (1.25 and 4 fold increase compared to normoxic WT mice, respectively). In pulmonary arteries from hypoxic WT mice, the contractile response to phenylephrine was about 1.8 greater than in those from normoxic WT mice. The use of ROS scavengers (PEG-SOD or catalase) and NOS inhibitor (L-NAME) revealed the involvement of ROS in hypoxia-induced hyper-reactivity to phenylephrine, and a loss of NO-dependent relaxation. Chronic treatment of hypoxic WT mice with the BH4 precursor sepiapterin preserved the vasorelaxant effect of NO. This treatment and the deletion of eNOS gene abolished the inhibitory effect of catalase on phenylephrine-induced contraction, and also attenuated hypoxia-induced right ventricular hypertrophy.These data show that chronic hypoxia induced an up-regulation of Nox isoforms and eNOS in lungs. They suggest that uncoupled eNOS participates to right ventricular hypertrophy and to alterations of vasomotor responses in pulmonary arteries in hypoxia-induced pulmonary hypertension. The weak increase in BH4 and the large over-expression of eNOS suggest the existence of compensatory mechanisms on BH4 synthesis, which may moderate eNOS dysfunction.Grants: Fondation de France, ANR, GRRC (E.D PhD grant

Influence of cell confluence on the cAMP signalling pathway in vascular smooth muscle cells

International audienceThe influence of cell confluence on the β-adrenoceptor (β-AR)/cAMP/phosphodiesterase (PDE) pathway was investigated in cultured rat aortic smooth muscle cells (RASMCs).Cells were plated either at low density (LD: 3.103 cells/cm2) or high density (HD: 3.104 cells/cm2) corresponding to non-confluent or confluent cells, respectively, on the day of experiment.β-AR-stimulated cAMP was monitored in real-time using the fluorescence resonance energy transfer (FRET)-based cAMP sensor, Epac2-camps. A brief application (15 s) of the β-AR agonist isoprenaline (Iso) induced a typical transient FRET signal, reflecting cAMP production followed by its rapid degradation. The amplitude of this response, which increased with the concentration of Iso (10 or 100 nM), was higher in HD than in LD cells, whatever the Iso concentration used. However, activation of adenylyl cyclase by L-858051 (100 µM) induced a similar saturating response in both LD and HD cells. A β1-AR antagonist (CGP 20712A, 100 nM) reduced the Iso (100 nM) response in HD but not LD cells, whereas a β2-AR antagonist (ICI 118,551, 5 nM) reduced this response in HD cells and almost abolished it in LD cells. Competitive [125I]-ICYP binding experiments with betaxolol, a β-AR ligand, identified two binding sites in HD cells, corresponding to β1- and β2-ARs with a proportion of 11% and 89%, respectively, but only one binding site in LD cells, corresponding to β2-ARs. Total cAMP-PDE activity (assessed by a radioenzymatic assay) was increased in HD cells compared to LD cells. This increase was associated with a rise in mRNA expression of five cAMP-PDEs subtypes (PDE1A, 3A, 4A, 4B and 7B) in HD cells, and a decrease in basal [cAMP]i (assessed by an EIA assay). PDE4 inhibition with Ro-20-1724 (10 µM) strongly prolonged the Iso response in LD and HD cells, whereas PDE3 inhibition with cilostamide (1 µM) slightly prolonged Iso response only in LD cells. Interestingly, inhibition of PDE4 unmasked an effect of PDE3 in HD cells. Our results show that in cultured RASMCs, the β-AR/cAMP/PDE signalling pathway is substantially modulated by the cell density. In HD cells, Iso response involves both β1- and β2-AR stimulation and is mainly controlled by PDE4, PDE3 being recruited only after PDE4 inhibition. In LD cells, Iso response involves only β2-AR stimulation and is controlled by PDE4 and to a lower degree by PDE3. This low density state is associated with an absence of membrane expression of the β1-AR, a lower cAMP-PDE activity and a higher basal [cAMP]i. This study highlights the critical role of the cellular environment in controlling the vascular β-AR signalling