Endothelium-independent and endothelium-dependent vasorelaxation by a dichloromethane fraction from Anogeissus leiocarpus (dc) guill. Et Perr. (combretaceae): possible involvement of cyclic nucleotide phosphodiesterase inhibition

Many traditional medicinal herbs from Burkina Faso are used to treat arterial hypertension (HTA). Among them, Anogeissus leiocarpus (A. Leiocarpus) which is well known and widely used in Burkina traditional medicine. Herein we assess the effects of dichloromethane fraction from A. leiocarpus stem bark (ALF), selected as the most active on cyclic nucleotide phosphodiesterases (PDEs) and characterized its specificity towards purified vascular PDE1 to PDE5 isoenzymes and study its effects on a vascular model. ALF potently and preferentially inhibits (IC50=1.6 ± 0.6 ìg/mL) the calmodulindependent phosphodiesterase PDE1, being mainly present in vascular smooth muscle and preferentially hydrolyses cGMP. In the same range (IC50 =2.8 ±0.2 ìg/ml) ALF inhibits PDE2, a cGMP-activated enzyme that is only present in endothelial cells and hydrolyses both cAMP and cGMP. PDE5, which specifically hydrolyses cGMP and which mainly contributes to cGMP hydrolysis is also potently inhibited by ALF (IC50=7.6±3.5 ìg/ml). The potencies of ALF on cAMP hydrolyzing isoenzymes was lesser, being more effective on PDE4 (IC50= 17.6±3.5 ìg/ml) than on PDE3 (60.9 ± 1.8 ìg/ml). Since the major effect of ALF were against cGMP hydrolysis and since cGMP is implicated in endothelium-dependent relaxation, the endotheliumdependent vasorelaxation was studied on isolated porcine coronary arteries rings pre-contracted with U46619. The endothelium-dependent vasorelaxation is significantly inhibited by Nù-nitro-L-arginine (LNA 300 ìmol/L, an inhibitor of endothelial NO synthase), but not affected by charybdotoxin (CTX, 100nM) plus apamin (APA, 100nM) (two inhibitors of EDHF-mediated responses). The combination of 4-aminopyridine (4-AP, 1 mmol/L, inhibitor of voltage-dependent potassium channels, Kv) plus baryum (Ba2+, 30 ìmol/L, inhibitor of the potassium channels with entering correction, Kir) plus ouabain (3 ìmol/L, inhibitor of ATPase Na+/K+ channels) partially inhibits endothelium-independent vasorelaxant effect. This endothelium-independent relaxant effect was also sensitive to combination of 1H-[1,2,4]-oxadiazole-[4,3-á]-quinoxalin- 1-one (ODQ, 10 ìM, soluble guanylyl cyclase inhibitor) and N-[2-(p-Bromocinnamylamino)ethyl]-5-isoquinoline sulfonamide dihydrochloride (H89, 100 nM, Protein Kinase A inhibitor). Taken together, these results indicate that ALF is a powerful vasodilator modulated by the formation of NO from endothelium, but also act by directly relaxing the vascular smooth muscle cells, by inhibiting cGMP hydrolyzing PDEs (PDE1, PDE2 and PDE5) and to a lesser extend on cAMP degradation (PDE3 and PDE4), cAMP and cGMP being second messengers involved in vascular relaxation

Nitric Oxide-Induced Activation of the AMP-Activated Protein Kinase α2 Subunit Attenuates IκB Kinase Activity and Inflammatory Responses in Endothelial Cells

BACKGROUND: In endothelial cells, activation of the AMP-activated protein kinase (AMPK) has been linked with anti-inflammatory actions but the events downstream of kinase activation are not well understood. Here, we addressed the effects of AMPK activation/deletion on the activation of NFκB and determined whether the AMPK could contribute to the anti-inflammatory actions of nitric oxide (NO). METHODOLOGY/PRINCIPAL FINDINGS: Overexpression of a dominant negative AMPKα2 mutant in tumor necrosis factor-α-stimulated human endothelial cells resulted in increased NFκB activity, E-selectin expression and monocyte adhesion. In endothelial cells from AMPKα2(-/-) mice the interleukin (IL)-1β induced expression of E-selectin was significantly increased. DETA-NO activated the AMPK and attenuated NFκB activation/E-selectin expression, effects not observed in human endothelial cells in the presence of the dominant negative AMPK, or in endothelial cells from AMPKα2(-/-) mice. Mechanistically, overexpression of constitutively active AMPK decreased the phosphorylation of IκB and p65, indicating a link between AMPK and the IκB kinase (IKK). Indeed, IKK (more specifically residues Ser177 and Ser181) was found to be a direct substrate of AMPKα2 in vitro. The hyper-phosphorylation of the IKK, which is known to result in its inhibition, was also apparent in endothelial cells from AMPKα2(+/+) versus AMPKα2(-/-) mice. CONCLUSIONS: These results demonstrate that the IKK is a direct substrate of AMPKα2 and that its phosphorylation on Ser177 and Ser181 results in the inhibition of the kinase and decreased NFκB activation. Moreover, as NO potently activates AMPK in endothelial cells, a portion of the anti-inflammatory effects of NO are mediated by AMPK

Impact of short-term dietary modification on postprandial oxidative stress

<p>Abstract</p> <p>Background</p> <p>We have recently reported that short-term (21-day) dietary modification in accordance with a stringent vegan diet (i.e., a Daniel Fast) lowers blood lipids as well as biomarkers of oxidative stress. However, this work only involved measurements obtained in a fasted state. In the present study, we determined the postprandial response to a high-fat milkshake with regards to blood triglycerides (TAG), biomarkers of oxidative stress, and hemodynamic variables before and following a 21-day Daniel Fast.</p> <p>Methods</p> <p>Twenty-two subjects (10 men and 12 women; aged 35 ± 3 years) completed a 21-day Daniel Fast. To induce oxidative stress, a milkshake (fat = 0.8 g·kg^-1; carbohydrate = 1.0 g·kg^-1; protein = 0.25 g·kg^-1) was consumed by subjects on day one and day 22 in a rested and 12-hour fasted state. Before and at 2 and 4 h after consumption of the milkshake, heart rate (HR) and blood pressure were measured. Blood samples were also collected at these times and analyzed for TAG, malondialdehyde (MDA), hydrogen peroxide (H₂O₂), advanced oxidation protein products (AOPP), nitrate/nitrite (NOx), and Trolox Equivalent Antioxidant Capacity (TEAC).</p> <p>Results</p> <p>A time effect was noted for HR (<it>p </it>= 0.006), with values higher at 2 hr post intake of the milkshake as compared to pre intake (<it>p </it>< 0.05). Diastolic blood pressure was lower post fast as compared to pre fast (<it>p </it>= 0.02), and a trend for lower systolic blood pressure was noted (<it>p </it>= 0.07). Time effects were noted for TAG (<it>p </it>= 0.001), MDA (<it>p </it>< 0.0001), H₂O₂(<it>p </it>< 0.0001), AOPP (<it>p </it>< 0.0001), and TEAC (<it>p </it>< 0.0001); all concentrations were higher at 2 h and 4 h post intake compared to pre intake, except for TEAC, which was lower at these times (<it>p </it>< 0.05). A condition effect was noted for NOx (<it>p </it>= 0.02), which was higher post fast as compared to pre fast. No pre/post fast × time interactions were noted (<it>p </it>> 0.05), with the area under the curve from pre to post fast reduced only slightly for TAG (11%), MDA (11%), H₂O₂(8%), and AOPP (12%), with a 37% increase noted for NOx.</p> <p>Conclusion</p> <p>Partaking in a 21-day Daniel Fast does not result in a statistically significant reduction in postprandial oxidative stress. It is possible that a longer time course of adherence to the Daniel Fast eating plan may be needed to observe significant findings.</p

Weekly Intra-Amniotic IGF-1 Treatment Increases Growth of Growth-Restricted Ovine Fetuses and Up-Regulates Placental Amino Acid Transporters

Frequent treatment of the growth-restricted (IUGR) ovine fetus with intra-amniotic IGF-1 increases fetal growth. We aimed to determine whether increased growth was maintained with an extended dosing interval and to examine possible mechanisms. Pregnant ewes were allocated to three groups: Control, and two IUGR groups (induced by placental embolization) treated with weekly intra-amniotic injections of either saline (IUGR) or 360 µg IGF-1 (IGF1). IUGR fetuses were hypoxic, hyperuremic, hypoglycemic, and grew more slowly than controls. Placental glucose uptake and SLC2A1 (GLUT2) mRNA levels decreased in IUGR fetuses, but SLC2A3 (GLUT3) and SLC2A4 (GLUT4) levels were unaffected. IGF-1 treatment increased fetal growth rate, did not alter uterine blood flow or placental glucose uptake, and increased placental SLC2A1 and SLC2A4 (but not SLC2A3) mRNA levels compared with saline-treated IUGR animals. Following IGF-1 treatment, placental mRNA levels of isoforms of the system A, y+, and L amino acid transporters increased 1.3 to 5.0 fold, while the ratio of phosphorylated-mTOR to total mTOR also tended to increase. Weekly intra-amniotic IGF-1 treatment provides a promising avenue for intra-uterine treatment of IUGR babies, and may act via increased fetal substrate supply, up-regulating placental transporters for neutral, cationic, and branched-chain amino acids, possibly via increased activation of the mTOR pathway

Nitric oxide and vascular smooth muscle homeostasis | LE MONOXYDE D'AZOTE ET L'HOMEOSTASIE DU MUSCLE LISSE VASCULAIRE

Nitric oxide is synthesised from an amino acid, L-arginine, by a family of enzymes called nitric oxide (NO) synthase, by most cells in the vessel wall. In healthy vessels, the production of NO is due to the constitutive and calcium dependent NO synthase present in the endothelial cells. On the other hand, when the vascular system is diseased and defense mechanisms are activated, the mediators of inflammatory and immunitary reactions induce and NO synthase non-responsive to calcium which produces large quantities of NO in most of the cells of the vessel wall. Nitric oxide is a liposoluble radical with a short half-life. It plays a central role in the regulation of the motricity and proliferation of blood vessels and in the interaction of the blood cells with the vessel wall. An inadequate production of nitric oxide could play a role in many vascular diseases such as hypertension, atherosclerosis, restenosis or vascular hyporeactivity associated with septicaemic shock.link_to_subscribed_fulltex

SIN-1 stimulates the production of cyclic GMP but not cyclic AMP in porcine aortic endothelial cells

The purpose of the present investigations was to determine whether or not SIN-1, a metabolite of molsidomine that spontaneously releases nitric oxide, stimulates the production of adenosine-3',5'-cyclic monophosphate (cyclic AMP) and of guanosine-3',5'-cyclic monophosphate (cyclic GMP) in endothelial cells. All experiments were performed on first or second passage cultured porcine aortic endothelial cells. SIN-1 induced a time- and concentration-dependent accumulation of cyclic GMP but not of cyclic AMP. The production of cyclic GMP evoked by SIN-1 but not evoked by human α-natriuretic polypeptide was inhibited by treatment of the cells with either methylene blue (an inhibitor of soluble guanylate cyclase) and hemoglobin (a scavenger of nitric oxide). These data suggest that SIN-1 enhances the activity of soluble guanylate cyclase, which in turn induces the accumulation of cyclic GMP in endothelial cells. This response is probably due to the spontaneous release of nitric oxide, which is a potent activator of soluble guanylate cyclase.link_to_subscribed_fulltex

Endothelin-1: A potent vasoactive peptide

link_to_subscribed_fulltex

L-arginine evokes relaxations of the rat aorta in both the presence and absence of endothelial cells

The effect of L-arginine [the substrate for the formation of nitric oxide (NO) in endothelial cells] on the reactivity of isolated vascular preparations was studied. Rings of rat aorta, with or without endothelium, were suspended in organ chambers for the measurement of isometric force. After various incubation periods in physiological salt solution (37°C, 95% O2 and 5% CO2), they were contracted with phenylephrine (10-6 M) before the addition of cumulative concentrations of L-arginine. L-Arginine evoked only minor changes in tension in preparations incubated for 0.5 h. However, when the incubation period was longer than 2 h, the amino acid evoked concentration- and time-dependent relaxations in preparations both with and without endothelium. The relaxations evoked by L-arginine were impaired by nitro-L-arginine and methylene blue. Other basic cationic amino acids (D-arginine, L-homoarginine, L-citrulline, L-lysine, and L-ornithine) evoked only small or no relaxations in both types of preparations. These observations demonstrate that L-arginine stereoselectively and specifically relaxes the rat aorta whether or not it contains the endothelium; this response depends on the duration of the prior incubation of the rings in physiological salt solution. The relaxations are impaired by inhibitors of both the formation and the action of NO, demonstrating that the endothelial cells and the vascular smooth muscle of the rat aorta possess an L-arginine - NO pathway(s) associated with relaxation.link_to_subscribed_fulltex