Thierry Chataigneau

Acetylcholine‐induced relaxation in blood vessels from endothelial nitric oxide synthase knockout mice

Isometric tension was recorded in isolated rings of aorta, carotid, coronary and mesenteric arteries taken from endothelial nitric oxide synthase knockout mice (eNOS(−/−) mice) and the corresponding wild‐type strain (eNOS(+/+) mice). The membrane potential of smooth muscle cells was measured in coronary arteries with intracellular microelectrodes. In the isolated aorta, carotid and coronary arteries from the eNOS(+/+) mice, acetylcholine induced an endothelium‐dependent relaxation which was inhibited by Nω‐L‐nitro‐arginine. In contrast, in the mesenteric arteries, the inhibition of the cholinergic relaxation required the combination of Nω‐L‐nitro‐arginine and indomethacin. The isolated aorta, carotid and coronary arteries from the eNOS(−/−) mice did not relax in response to acetylcholine. However, acetylcholine produced an indomethacin‐sensitive relaxation in the mesenteric artery from eNOS(−/−) mice. The resting membrane potential of smooth muscle cells from isolated coronary arteries was significantly less negative in the eNOS(−/−) mice (−64.8±1.8u2003mV, n=20 and −58.4±1.9u2003mV, n=17, for eNOS(+/+) and eNOS(−/−) mice, respectively). In both strains, acetylcholine, bradykinin and substance P did not induce endothelium‐dependent hyperpolarizations whereas cromakalim consistently produced hyperpolarizations (−7.9±1.1u2003mV, n=8 and −13.8±2.6u2003mV, n=4, for eNOS(+/+) and eNOS(−/−) mice, respectively). These findings demonstrate that in the blood vessels studied: (1) in the eNOS(+/+) mice, the endothelium‐dependent relaxations to acetylcholine involve either NO or the combination of NO plus a product of cyclo‐oxygenase but not EDHF; (2) in the eNOS(−/−) mice, NO‐dependent responses and EDHF‐like responses were not observed. In the mesenteric arteries acetylcholine releases a cyclo‐oxygenase derivative.

Epoxyeicosatrienoic acids, potassium channel blockers and endothelium‐dependent hyperpolarization in the guinea‐pig carotid artery

Using intracellular microelectrodes, we investigated the effects of 17‐octadecynoic acid (17‐ODYA) on the endothelium‐dependent hyperpolarization induced by acetylcholine in the guinea‐pig isolated internal carotid artery with endothelium. In the presence of Nω‐nitro‐L‐arginine (L‐NOARG, 100u2003μM) and indomethacin (5u2003μM) to inhibit nitric oxide synthase and cyclo‐oxygenase, acetylcholine (1u2003μM) evoked an endothelium‐dependent hyperpolarization which averaged −16.4u2003mV starting from a resting membrane potential of −56.8u2003mV. There was a negative correlation between the amplitude of the hyperpolarization and the absolute values of the resting membrane potential. The acetylcholine‐induced endothelium‐dependent hyperpolarization was not altered by charybdotoxin (0.1u2003μM) or iberiotoxin (30u2003nM). It was partially but significantly reduced by apamin (0.5u2003μM) to −12.8±1.2u2003mV (n=10) or the combination of apamin plus iberiotoxin (−14.3±3.4u2003mV, n=4). However, the combination of charybdotoxin and apamin abolished the hyperpolarization and under these conditions, acetylcholine evoked a depolarization (+7.1±3.7u2003mV, n=8). 17‐ODYA (10u2003μM) produced a significant hyperpolarization of the resting membrane potential which averaged −59.6u2003mV and a partial but significant inhibition of the acetylcholine‐induced endothelium‐dependent hyperpolarization (−10.9u2003mV). Apamin did not modify the effects of 17‐ODYA but in the presence of charybdotoxin or iberiotoxin, 17‐ODYA no longer influenced the resting membrane potential or the acetylcholine‐induced hyperpolarization. When compared to solvent (ethanol, 1% v/v), epoxyeicosatrienoic acids (EpETrEs) (5,6‐, 8,9‐, 11,12‐ and 14,15‐EpETrE, 3u2003μM) did not affect the cell membrane potential and did not relax the guinea‐pig isolated internal carotid artery. These results indicate that, in the guinea‐pig internal carotid artery, the involvement of metabolites of arachidonic acid through the cytochrome P450 pathway in endothelium‐dependent hyperpolarization is unlikely. Furthermore, the hyperpolarization mediated by the endothelium‐derived hyperpolarizing factor (EDHF) is probably not due to the opening of BKCa channels.

Cannabinoid CB1 receptor and endothelium-dependent hyperpolarization in guinea-pig carotid, rat mesenteric and porcine coronary arteries.

1 The purpose of these experiments was to determine whether or not the endothelium‐dependent hyperpolarizations of the vascular smooth muscle cells (observed in the presence of inhibitors of nitric oxide synthase and cyclo‐oxygenase) can be attributed to the production of an endogenous cannabinoid. 2 Membrane potential was recorded in the guinea‐pig carotid, rat mesenteric and porcine coronary arteries by intracellular microelectrodes. 3 In the rat mesenteric artery, the cannabinoid receptor antagonist, SRu2003141716 (1u2003μM), did not modify either the resting membrane potential of smooth muscle cells or the endothelium‐dependent hyperpolarization induced by acetylcholine (1u2003μM) (17.3±1.8u2003mV, n=4 and 17.8±2.6u2003mV, n=4, in control and presence of SRu2003141716, respectively). Anandamide (30u2003μM) induced a hyperpolarization of the smooth muscle cells (12.6±1.4u2003mV, n=13 and 2.0±3.0u2003mV, n=6 in vessels with and without endothelium, respectively) which could not be repeated in the same tissue, whereas acetylcholine was still able to hyperpolarize the preparation. The hyperpolarization induced by anandamide was not significantly influenced by SRu2003141716 (1u2003μM). HU‐210 (30u2003μM), a synthetic CB1 receptor agonist, and palmitoylethanolamide (30u2003μM), a CB2 receptor agonist, did not influence the membrane potential of the vascular smooth muscle cells. 4 In the rat mesenteric artery, the endothelium‐dependent hyperpolarization induced by acetylcholine (1u2003μM) (19.0±1.7u2003mV, n=6) was not altered by glibenclamide (1u2003μM; 17.7±2.3u2003mV, n=3). However, the combination of charybdotoxin (0.1u2003μM) plus apamin (0.5u2003μM) abolished the acetylcholine‐induced hyperpolarization and under these conditions, acetylcholine evoked a depolarization (7.7±2.7u2003mV, n=3). The hyperpolarization induced by anandamide (30u2003μM) (12.6±1.4u2003mV, n=13) was significantly inhibited by glibenclamide (4.0±0.4u2003mV, n=4) but not significantly affected by the combination of charybdotoxin plus apamin (17.3±2.3u2003mV, n=4). 5 In the guinea‐pig carotid artery, acetylcholine (1u2003μM) evoked endothelium‐dependent hyperpolarization (18.8±0.7u2003mV, n=15). SRu2003141716 (10u2003nM to 10u2003μM), caused a direct, concentration‐dependent hyperpolarization (up to 10u2003mV at 10u2003μM) and a significant inhibition of the acetylcholine‐induced hyperpolarization. Anandamide (0.1 to 3u2003μM) did not influence the membrane potential. At a concentration of 30u2003μM, the cannabinoid agonist induced a non‐reproducible hyperpolarization (5.6±1.3u2003mV, n=10) with a slow onset. SRu2003141716 (1u2003μM) did not affect the hyperpolarization induced by 30u2003μM anandamide (5.3±1.5u2003mV, n=3). 6 In the porcine coronary artery, anandamide up to 30u2003μM did not hyperpolarize or relax the smooth muscle cells. The endothelium‐dependent hyperpolarization and relaxation induced by bradykinin were not influenced by SRu2003141716 (1u2003μM). 7 These results indicate that the endothelium‐dependent hyperpolarizations, observed in the guinea‐pig carotid, rat mesenteric and porcine coronary arteries, are not related to the activation of cannabinoid CB1 receptors.

Red Wine Polyphenolic Compounds Inhibit Vascular Endothelial Growth Factor Expression in Vascular Smooth Muscle Cells by Preventing the Activation of the p38 Mitogen-Activated Protein Kinase Pathway

Objective—Moderate consumption of red wine has a beneficial effect on the cardiovascular system. This study examines whether red wine polyphenolic compounds (RWPCs) affect vascular endothelial growth factor (VEGF) expression, a major angiogenic and proatherosclerotic factor in vascular smooth muscle cells (VSMCs). Methods and Results—VEGF mRNA expression was assessed by Northern blot analysis and the release of VEGF by immunoassay in cultured VSMCs. Short-term and long-term exposure of VSMCs to RWPCs inhibited VEGF mRNA expression and release of VEGF in response to platelet-derived growth factor AB (PDGFAB), transforming growth factor-&bgr;1, or thrombin. The PDGFAB-induced expression of VEGF was markedly reduced by SB203580 (inhibitor of p38 mitogen-activated protein kinase [MAPK]), antioxidants, and diphenylene iodonium (inhibitor of flavin-dependent enzymes), slightly reduced by PD98059 (inhibitor of MEK), and not significantly affected by wortmannin (inhibitor of PI-3-kinase) and L-JNKI (inhibitor of JNK). Short-term and long-term treatment of VSMCs with RWPCs markedly reduced PDGFAB-induced production of reactive oxygen species and phosphorylation of p38 MAPK. Conclusions—These data indicate that RWPCs strongly inhibit growth factor–induced VEGF expression in VSMCs by preventing the redox-sensitive activation of the p38 MAPK pathway. The potential antiangiogenic and antiatherosclerotic properties of RWPCs are likely to contribute to cardiovascular protection by preventing the development of atherosclerotic lesions.

Induction of apoptosis by thymoquinone in lymphoblastic leukemia Jurkat cells is mediated by a p73-dependent pathway which targets the epigenetic integrator UHRF1

The salvage anti-tumoral pathway which implicates the p53-related p73 gene is not yet fully characterized. We therefore attempted to identify the up- and down-stream events involved in the activation of the p73-dependent pro-apoptotic pathway, by focusing on the anti-apoptotic and epigenetic integrator UHRF1 which is essential for cell cycle progression. For this purpose, we analyzed the effects of a known anti-neoplastic drug, thymoquinone (TQ), on the p53-deficient acute lymphoblastic leukemia (ALL) Jurkat cell line. Our results showed that TQ inhibits the proliferation of Jurkat cells and induces G1 cell cycle arrest in a dose-dependent manner. Moreover, TQ treatment triggers programmed cell death, production of reactive oxygen species (ROS) and alteration of the mitochondrial membrane potential (DeltaPsim). TQ-induced apoptosis, confirmed by the presence of hypodiploid G0/G1 cells, is associated with a rapid and sharp re-expression of p73 and dose-dependent changes of the levels of caspase-3 cleaved subunits. These modifications are accompanied by a dramatic down-regulation of UHRF1 and two of its main partners, namely DNMT1 and HDAC1, which are all involved in the epigenetic code regulation. Knockdown of p73 expression restores UHRF1 expression, reactivates cell cycle progression and inhibits TQ-induced apoptosis. Altogether our results showed that TQ mediates its growth inhibitory effects on ALL p53-mutated cells via the activation of a p73-dependent mitochondrial and cell cycle checkpoint signaling pathway which subsequently targets UHRF1.

Red wine polyphenols induce EDHF-mediated relaxations in porcine coronary arteries through the redox-sensitive activation of the PI3-kinase/Akt pathway.

Red wine polyphenolic compounds (RWPCs) are potent inducers of endothelium‐dependent relaxations of coronary arteries, which involve both nitric oxide and endothelium‐derived hyperpolarizing factor (EDHF). The EDHF‐mediated relaxation to RWPCs is critically dependent on the formation of reactive oxygen species by a flavin‐dependent enzyme. The aim of the present study was to determine the role of redox‐sensitive protein kinases including p38 MAPK, ERK1/2 and PI3‐kinase/Akt in RWPCs‐induced EDHF‐mediated relaxation. Porcine coronary artery rings were suspended in organ chambers for measurement of changes in isometric tension. Confluent cultures of porcine coronary artery endothelial cells were used to determine the phosphorylation level of p38 MAPK, ERK1/2 and Akt by Western blot analysis. All experiments were performed in the presence of indomethacin and Nω‐nitro‐L‐arginine. RWPCs caused pronounced endothelium‐dependent relaxations, which were significantly reduced by wortmannin and LY294002, two inhibitors of PI3‐kinase, and not affected by PD98059 (an inhibitor of ERK1/2 kinase kinase) and SB203580 (an inhibitor of p38 MAPK). In contrast, wortmannin did not affect relaxations to bradykinin or levcromakalim. RWPCs elicited within minutes a sustained and concentration‐dependent phosphorylation of p38 MAPK, ERK1/2 and Akt in endothelial cells. The phosphorylation of Akt in response to RWPCs was abolished by wortmannin and LY294002, and by the membrane‐permeant analogue of superoxide dismutase Mn(III)tetrakis(1‐methyl‐4‐pyridyl)porphyrin. The present findings demonstrate that RWPCs cause EDHF‐mediated relaxations of coronary arteries; these responses are critically dependent on the redox‐sensitive activation of the PI3‐kinase/Akt pathway in endothelial cells.

eNOS Activation Induced by a Polyphenol-Rich Grape Skin Extract in Porcine Coronary Arteries

Background/Aims: Drinking red wine is associated with a decreased mortality from coronary heart diseases. This study examined whether polyphenols contained in a grape skin extract (GSE) triggered the endothelial formation of nitric oxide (NO) and investigated the underlying mechanism. Methods: Vascular reactivity was assessed in organ chambers using porcine coronary artery rings in the presence of indomethacin (a cyclooxygenase inhibitor) and charybdotoxin plus apamin (inhibitors of endothelium-derived hyperpolarizing factor-mediated responses). The phosphorylation level of Src, Akt and endothelial NO synthase (eNOS) were assessed by Western blot analysis, and the formation of reactive oxygen species (ROS) was investigated using dihydroethidine and dichlorodihydrofluorescein. Results: GSE-induced endothelium-dependent relaxations were abolished by NG-nitro-L-arginine (an eNOS inhibitor) and ODQ (a soluble guanylyl cyclase inhibitor), and they were reduced by MnTMPyP, polyethyleneglycol catalase, PP2 (an inhibitor of Src kinase) and wortmannin (an inhibitor of phosphoinositide 3-kinase). GSE caused phosphorylation of Src, which was prevented by MnTMPyP. It also caused phosphorylation of Akt and eNOS, which were prevented by MnTMPyP, polyethyleneglycol catalase, PP2, wortmannin and LY294002. GSE elicited the formation of ROS in native and cultured endothelial cells, which was prevented by MnTMPyP. Conclusions: GSE causes endothelium-dependent NO-mediated relaxations of coronary arteries. This effect involves the intracellular formation of ROS in endothelial cells leading to the Src kinase/phosphoinositide 3-kinase/Akt-dependent phosphorylation of eNOS.

Molecular structure and function of P2X receptors.

ATP-gated P2X receptors are trimeric ion channels selective to cations. Recent progress in the molecular biophysics of these channels enables a better understanding of their function. In particular, data obtained from biochemical, electrophysiogical and molecular engineering in the light of recent X-ray structures now allow delineation of the principles of ligand binding, channel opening and allosteric modulation. However, although a picture emerges as to how ATP triggers channel opening, there are a number of intriguing questions that remain to be answered, in particular how the pore itself opens in response to ATP and how the intracellular domain, for which structural information is limited, moves during activation. In this review, we provide a summary of functional studies in the context of the post-structure era, aiming to clarify our understanding of the way in which P2X receptors function in response to ATP binding, as well as the mechanism by which allosteric modulators are able to regulate receptor function. This article is part of the Special Issue entitled Purines in Neurodegeneration and Neuroregeneration.

3-Morpholinosydnonimine (SIN-1) and K+ channels in smooth muscle cells of the rabbit and guinea pig carotid arteries

Experiments were designed to determine the subtype of K(+) channels activated by the nitrovasodilator 3-morpholinosydnonimine (SIN-1) in smooth muscle cells of the rabbit and guinea pig carotid arteries. Membrane potential was recorded in isolated segments with intracellular microelectrode and K(+) currents in freshly dissociated smooth muscle cells, with the patch-clamp technique. In the guinea pig carotid artery, SIN-1 caused a glibenclamide-sensitive hyperpolarization. The nitrovasodilator did not affect the whole-cell K(+) current, but activated a glibenclamide-sensitive K(+) current. In the rabbit carotid artery, SIN-1 induced only an iberiotoxin-sensitive repolarization in phenylephrine-depolarized tissue and in isolated cells, enhanced the activity of an iberiotoxin-sensitive K(+) current. These findings demonstrate that the population of K(+) channels activated by nitric oxide (NO) is species-dependent and support the conclusion that, in the guinea pig carotid artery, in contrast to the rabbit carotid artery, the release of NO cannot account for the responses attributed to endothelium-derived hyperpolarizing factor (EDHF).

Vascular effects of ovariectomy and chronic oestrogen treatment in rats: controversy or experimental protocol diversity?

Several clinical studies have indicated that oestrogens have protective properties on the cardiovascular system. Although the beneficial effect has been attributable, at least in part, to their ability to stimulate the endothelial fomation of nitric oxide (NO) and endothelium‐derived hyperpolarizing factor (EDHF), the underlying mechanism still remains unclear. In a study from this issue of British Journal of Pharmacology, Nawate et al. have examined the effects of rat ovariectomy and chronic treatment with 17β‐oestradiol on the endothelial function as assessed ex vivo. The data indicate that acetylcholine‐induced endothelium‐dependent relaxations of the isolated mesenteric artery are affected by neither ovariectomy nor chronic hormonal treatment. Despite the maintained endothelium‐dependent relaxation, the contribution of the two major endothelial factors NO and EDHF was changed. Indeed, ovariectomy increased the NO‐mediated component of the relaxation, most likely as a consequence of the downregulation of the physiological allosteric inhibitor of endothelial NO synthase, caveolin‐1. In addition, ovariectomy decreased the EDHF‐mediated component of the relaxation and membrane hyperpolarization of the smooth muscle cells, an effect which might be explained by a concomitant decrease of the expression of the gap junction connexin‐40 and connexin‐43. Furthermore, chronic administration of 17β‐estradiol to ovariectomized rats normalized all these effects. This study provides further experimental evidence indicating that the hormonal status plays a determinant role in the control of the endothelial formation of both NO and EDHF.