Véronique Leblais

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.

Beta 3‐adrenoceptor stimulation induces vasorelaxation mediated essentially by endothelium‐derived nitric oxide in rat thoracic aorta

The relaxant effects of isoprenaline may result from activation of another β‐adrenoceptor subtype in addition to β1 and β2. This study evaluated the role of a third β‐adrenoceptor subtype, β3, in β‐adrenoceptor‐induced relaxation of rat thoracic aorta by isoprenaline. Isoprenaline produced a concentration‐dependent relaxation of phenylephrine pre‐contracted rings of the thoracic aorta (pD2=7.46±0.15; Emax=85.9±3.4%), which was partially attenuated by endothelium removal (Emax=66.5±6.3%) and administration of the nitric oxide (NO) synthase inhibitor, L‐NG‐monomethyl arginine (L‐NMMA) (Emax=61.3±7.9%). In the presence of nadolol, a β1‐ and β2‐adrenoceptor antagonist, isoprenaline‐induced relaxation persisted (Emax=55.6±5.3%), but occurred at higher concentrations (pD2=6.71±0.10) than in the absence of nadolol and lasted longer. Similar relaxant effects were obtained with two β3‐adrenoceptor agonists: SR 58611 (a preferential β3‐adrenoceptor agonist), and CGP 12177 (a partial β3‐adrenoceptor with β1‐ and β2‐adrenoceptor antagonistic properties). SR 58611 caused concentration‐dependent relaxation (pD2=5.24±0.07; Emax=59.5±3.7%), which was not modified by pre‐treatment with nadolol but antagonized by SR 59230A, a β3‐adrenoceptor antagonist. The relaxation induced by SR 58611 was associated with a 1.7 fold increase in tissue cyclic GMP content. Both relaxation and the cyclic GMP increase induced by SR 58611 were greatly reduced by endothelium removal and in the presence of L‐NMMA. We conclude that in the rat thoracic aorta, β3‐adrenoceptors are mainly located on endothelial cells, and act in conjuction with β1‐ and β2‐adrenoceptors to mediate relaxation through activation of an NO synthase pathway and subsequent increase in cyclic GMP levels.

Role of reactive oxygen species and gp91phox in endothelial dysfunction of pulmonary arteries induced by chronic hypoxia.

1 This study investigates the role of nitric oxide (NO) and reactive oxygen species (ROS) on endothelial function of pulmonary arteries in a mice model of hypoxia‐induced pulmonary hypertension. 2 In pulmonary arteries from control mice, the NO‐synthase inhibitor Nω‐nitro‐L‐arginine methyl ester (L‐NAME) potentiated contraction to prostaglandin F2α (PGF2α) and completely abolished relaxation to acetylcholine. In extrapulmonary but not intrapulmonary arteries, acetylcholine‐induced relaxation was slightly inhibited by polyethyleneglycol‐superoxide dismutase (PEG‐SOD) or catalase. 3 In pulmonary arteries from hypoxic mice, ROS levels (evaluated using dihydroethidium staining) were higher than in controls. In these arteries, relaxation to acetylcholine (but not to sodium nitroprusside) was markedly diminished. L‐NAME abolished relaxation to acetylcholine, but failed to potentiate PGF2α‐induced contraction. PEG‐SOD or catalase blunted residual relaxation to acetylcholine in extrapulmonary arteries, but did not modify it in intrapulmonary arteries. Hydrogen peroxide elicited comparable (L‐NAME‐insensitive) relaxations in extra‐ and intrapulmonary arteries from hypoxic mice. 4 Exposure of gp91phox–/– mice to chronic hypoxia also decreased the relaxant effect of acetylcholine in extrapulmonary arteries. However, in intrapulmonary arteries from hypoxic gp91phox–/– mice, the effect of acetylcholine was similar to that obtained in mice not exposed to hypoxia. 5 Chronic hypoxia increases ROS levels and impairs endothelial NO‐dependent relaxation in mice pulmonary arteries. Mechanisms underlying hypoxia‐induced endothelial dysfunction differ along pulmonary arterial bed. In extrapulmonary arteries from hypoxic mice, endothelium‐dependent relaxation appears to be mediated by ROS, in a gp91phox‐independent manner. In intrapulmonary arteries, endothelial dysfunction depends on gp91phox, the latter being rather the trigger than the mediator of impaired endothelial NO‐dependent relaxation.

Impairment of NO-dependent relaxation in intralobar pulmonary arteries: comparison of urban particulate matter and manufactured nanoparticles.

Background and Objectives Because pulmonary circulation is the primary vascular target of inhaled particulate matter (PM), and nitric oxide is a major vasculoprotective agent, in this study we investigated the effect of various particles on the NO–cyclic guanosine monophosphate (cGMP) pathway in pulmonary arteries. Methods We used intrapulmonary arteries and/or endothelial cells, either exposed in vitro to particles or removed from PM-instilled animals for assessment of vasomotricity, cGMP and reactive oxygen species (ROS) levels, and cytokine/chemokine release. Results Endothelial NO-dependent relaxation and cGMP accumulation induced by acetylcholine (ACh) were both decreased after 24 hr exposure of rat intrapulmonary arteries to standard reference material 1648 (SRM1648; urban PM). Relaxation due to NO donors was also decreased by SRM1648, whereas responsiveness to cGMP analogue remained unaffected. Unlike SRM1648, ultrafine carbon black and ultrafine and fine titanium dioxide (TiO2) manufactured particles did not impair NO-mediated relaxation. SRM1648-induced decrease in relaxation response to ACh was prevented by dexamethasone (an anti-inflammatory agent) but not by antioxidants. Accordingly, SRM1648 increased the release of proinflammatory mediators (tumor necrosis factor-α, interleukin-8) from intrapulmonary arteries or pulmonary artery endothelial cells, but did not elevate ROS levels within intrapulmonary arteries. Decreased relaxation in response to ACh was also evidenced in intrapulmonary arteries removed from rats intratracheally instilled with SRM1648, but not with fine TiO2. Conclusion In contrast to manufactured particles (including nanoparticles), urban PM impairs NO but not cGMP responsiveness in intrapulmonary arteries. We attribute this effect to oxidative-stress–independent inflammatory response, resulting in decreased guanylyl cyclase activation by NO. Such impairment of the NO pathway may contribute to urban-PM–induced cardiovascular dysfunction.

Role of Gi/o-Src kinase-PI3K/Akt pathway and caveolin-1 in β2-adrenoceptor coupling to endothelial NO synthase in mouse pulmonary artery

Activation of the β₂-adrenoceptor (β₂-AR) elicits an endothelial nitric oxide synthase (eNOS)-dependent relaxation in mouse pulmonary artery, which, contrary to the muscarinic receptor-dependent relaxation, is preserved in hypoxic pulmonary arterial hypertension. We therefore characterized the signaling pathways underlying the β₂-AR-mediated eNOS activation, with special focus on G(i/o) proteins, protein kinases and caveolae. Functional studies (for evaluation of vasorelaxant response), Western blotting (for assessment of eNOS and caveolin-1 phosphorylation) and transmission electron microscopy (for visualization of caveolae) were conducted in pulmonary arteries from wild-type or caveolin-1 knockout mice. In wild-type isolated arteries, relaxation to the selective β₂-AR agonist procaterol was reduced by inhibitors of G(i/o) proteins (pertussis toxin, PTX), phosphatidylinositol 3-kinase (PI3K; wortmannin or LY 294002), Akt (Akt inhibitor X) and Src-kinase (PP2) and by cholesterol depletion (using methyl-β-cyclodextrin). Procaterol induced eNOS phosphorylation at Ser(1177), which was prevented by PTX, PP2 or Akt inhibitor. Procaterol also promoted caveolin-1 phosphorylation at Tyr(14), which was decreased by PTX or PP2. Caveolin-1 gene deletion resulted in endothelial caveolae disruption in mouse pulmonary artery and in potentiation of procaterol-induced relaxation. Unlike procaterol, acetylcholine-induced relaxation was unaffected by PTX, methyl-β-cyclodextrin or caveolin-1 gene deletion. To conclude, the mouse pulmonary endothelial β₂-AR is coupled to a G(i/o)-Src kinase-PI3K/Akt pathway to promote eNOS phosphorylation at Ser(1177) leading to a NO-dependent vasorelaxation. Caveolin-1 exerts a negative control on this response that is abrogated by its phosphorylation at Tyr(14), through a G(i/o)-Src kinase pathway. Since pulmonary β₂-AR- and muscarinic receptor-mediated relaxations differentiate in their respective signaling pathways leading to eNOS activation and sensitivities during hypoxia-induced pulmonary arterial hypertension, mechanisms underlying eNOS activation might be key determinants of pulmonary endothelial dysfunction.

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

This study examined the role of the main vascular cAMP‐hydrolysing phosphodiesterases (cAMP‐PDE) in the regulation of basal vascular tone and relaxation of rat aorta mediated by β‐adrenoceptors, following heart failure (HF).

β-Adrenergic cAMP signals are predominantly regulated by phosphodiesterase type 4 in cultured adult rat aortic smooth muscle cells.

Background We investigated the role of cyclic nucleotide phosphodiesterases (PDEs) in the spatiotemporal control of intracellular cAMP concentrations in rat aortic smooth muscle cells (RASMCs). Methodology/Principal Findings The rank order of PDE families contributing to global cAMP-PDE activity was PDE4> PDE3  =  PDE1. PDE7 mRNA expression but not activity was confirmed. The Fluorescence Resonance Energy Transfer (FRET)-based cAMP sensor, Epac1-camps, was used to monitor the time course of cytosolic cAMP changes. A pulse application of the β-adrenoceptor (β-AR) agonist isoproterenol (Iso) induced a transient FRET signal. Both β1- and β2-AR antagonists decreased the signal amplitude without affecting its kinetics. The non-selective PDE inhibitor (IBMX) dramatically increased the amplitude and delayed the recovery phase of Iso response, in agreement with a role of PDEs in degrading cAMP produced by Iso. Whereas PDE1, PDE3 and PDE7 blockades [with MIMX, cilostamide (Cil) and BRL 50481 (BRL), respectively] had no or minor effect on Iso response, PDE4 inhibition [with Ro-20-1724 (Ro)] strongly increased its amplitude and delayed its recovery. When Ro was applied concomitantly with MIMX or Cil (but not with BRL), the Iso response was drastically further prolonged. PDE4 inhibition similarly prolonged both β1- and β2-AR-mediated responses. When a membrane-targeted FRET sensor was used, PDE3 and PDE4 acted in a synergistic manner to hydrolyze the submembrane cAMP produced either at baseline or after β-AR stimulation. Conclusion/Significance Our study underlines the importance of cAMP-PDEs in the dynamic control of intracellular cAMP signals in RASMCs, and demonstrates the prominent role of PDE4 in limiting β-AR responses. PDE4 inhibition unmasks an effect of PDE1 and PDE3 on cytosolic cAMP hydrolyzis, and acts synergistically with PDE3 inhibition at the submembrane compartment. This suggests that mixed PDE4/PDE1 or PDE4/PDE3 inhibitors would be attractive to potentiate cAMP-related functions in vascular cells.

Tracheal relaxation of five Ivorian anti-asthmatic plants: Role of epithelium and K + channels in the effect of the aqueous-alcoholic extract of Dichrostachys cinerea root bark

ETHNOPHARMACOLOGICAL RELEVANCE Leaves of Boerhavia diffusa (Nyctaginaceae), Baphia nitida, Cassia occidentalis, Desmodium adscendens (Fabaceae), and root bark of Dichrostachys cinerea (Fabaceae) are used in Ivory Coast for the treatment of asthma. The aim of this study was to evaluate the potential airway relaxant activity of different extracts of these plants. MATERIALS AND METHODS Extracts of different polarities (H(2)O, EtOH/H(2)O, MeOH and CH(2)Cl(2)) were obtained from these five plants. Their ex vivo relaxant activity was tested in mice isolated trachea precontracted with carbachol (1 μM). RESULTS Cumulative concentrations of most extracts induced moderate to strong relaxation, the methanolic extracts being the most potent and the polar extracts the most active at the concentrations used, supporting the traditional use of these five plants as anti-asthmatic remedies. We further investigated the molecular and cellular mechanisms of the mouse trachea relaxant effect of the aqueous-alcoholic extract of Dichrostachys cinerea root bark, the most potent extract. Its effect was not modified in the presence of β-adrenoceptor antagonists (propranolol or ICI 118,551) or a PKA inhibitor (H89). By contrast, it was decreased after depolarization-induced precontraction (with 80 mM KCl), in the presence of some K(+) channels blockers [4-aminopyridine as voltage-dependent K(+) (K(v)) channel blocker and tetraethylammonium chloride as large conductance Ca(2+)-activated K(+) (BK(Ca)) channel blocker, but not with glibenclamide, an ATP-sensitive K(+) (K(ATP)) channel blocker] or after epithelium removal. CONCLUSIONS The mouse tracheal relaxant effect of Dichrostachys cinerea EtOH/H(2)O extract was independent of β(2)-adrenoceptors activation and cAMP/PKA pathway, but dependent on epithelium and K(+) channels, namely K(v) and BK(Ca) channels. Further investigation will be required to identify the component(s) responsible for this airways relaxant activity.

Phosphodiesterase types 3 and 4 regulate the phasic contraction of neonatal rat bladder smooth myocytes via distinct mechanisms.

Activation of the cyclic AMP (cAMP) pathway reduces bladder contractility. However, the role of phosphodiesterase (PDE) families in regulating this function is poorly understood. Here, we compared the contractile function of the cAMP hydrolyzing PDEs in neonatal rat bladder smooth myocytes. RT-PCR and Western blotting analysis revealed that several isoforms of PDE1-4 were expressed in neonatal rat bladder. While 8-methoxymethyl-3-isobutyl-1-methylxanthine (a PDE1 inhibitor) and BAY-60-7550 (a PDE2 inhibitor) had no effect on the carbachol-enhanced phasic contractions of bladder strips, cilostamide (Cil, a PDE3 inhibitor) and Ro-20-1724 (Ro, a PDE4 inhibitor) significantly reduced these contractions. This inhibitory effect of Ro was blunted by the PKA inhibitor H-89, while the inhibitory effect of Cil was strongly attenuated by the PKG inhibitor KT 5823. Application of Ro in single bladder smooth myocytes resulted in an increase in Ca(2+) spark frequency but a decrease both in Ca(2+) transients and in sarcoplasmic reticulum (SR) Ca(2+) content. In contrast, Cil had no effect on these events. Furthermore, Ro-induced inhibition of the phasic contractions was significantly blocked by ryanodine and iberiotoxin. Taken together, PDE3 and PDE4 are the main PDE isoforms in maintaining the phasic contractions of bladder smooth myocytes, with PDE4 being functionally more active than PDE3. However, their roles are mediated through different mechanisms.

Antihypertensive and vasodilator effects of methanolic extract of Inula viscosa: Biological evaluation and POM analysis of cynarin, chlorogenic acid as potential hypertensive

BACKGROUND Inula viscosa L. (Asteraceae) is a medicinal plant widely used as a folk medicine in oriental Morocco, to treat hypertension. The antihypertensive effect of the methanolic extract obtained from I. viscosa leaves was evaluated in hypertensive L-NAME rats. Systolic blood pressure (SBP) was measured using a non-invasive indirect tail-cuff plethysmographic method. Four groups of rats were used: a control group; a hypertensive group treated with L-NAME (32mg/kg/day); a positive control group treated with L-NAME plus enalapril (15mg/kg/day) as a reference antihypertensive agent; and a group treated with L-NAME plus MeOH-extract (40mg/kg/day). METHODS Treatment with L-NAME alone caused a progressive increase in SBP. After 4 weeks, the value of SBP reached 160±2mmHg which shows the installation of hypertension. Enalapril prevented the increase in SBP, which remained normal at 123±1mmHg after 4 weeks of treatment. The administration of MeOH-extract along with L-NAME prevented the increase in SBP as well, which remained constant at 115±1mmHg after 4 weeks of treatment. In ex-vivo models, MeOH-extract produced a relaxation of pre-contracted ring aorta (54 ± 2% of relaxation at 3g/L). But, when the rings were denuded, MeOH-extract failed to relax pre-contracted rings of aorta. Phytochemical study of I. viscosa MeOH-extract revealed the presence of phenolic compounds, such as cynarin and chlorogenic acid. RESULTS The present results suggest that I. viscosa MeOH-extract has an antihypertensive, predominantly mediated by an endothelium-dependent vasodilatory effect. Cynarin and chlorogenic acid, which have a strong vasorelaxant effect may be involved in the antihypertensive effect of the plant extract. The bioinformatic POM analysis confirms the therapeutic potential of cynarin and chlorogenic acids as inhibitors of various biotargets. Based on the results, the coordination of these phytochemicals with calcium and transition metals should be studied, for better scope at antihypertensive drug development.