BACKGROUND & AIMS: Advanced stages of portal hypertension are characterized by generalized vasodilatation and a hyperdynamic syndrome that leads to complications such as hepatopulmonary syndrome. We assessed the endothelial function-particularly the formation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF)-in rats following common bile duct ligation (CBDL) to determine the underlying mechanisms of these processes. METHODS: Reactivity of mesenteric artery rings from male Wistar rats was determined in organ chambers. The expression levels of connexins (Cx) (Cx37, Cx40, Cx43), intermediate and small conductance Ca2+-activated K+ channels (IKCa, SKCa), endothelial NO synthase (eNOS), NADPH oxidase subunits, and nitrotyrosines were assessed by immunohistochemistry in mesenteric and pulmonary arteries. The vascular formation of reactive oxygen species (ROS) was evaluated using dihydroethidine. Control rats or those that had undergone CBDL were given either the NADPH oxidase inhibitor apocynin or the angiotensin II receptor type 1 antagonist losartan. RESULTS: Decreased EDHF-mediated relaxations to acetylcholine and red wine polyphenols were observed in CBDL rats, compared with controls, whereas the level of NO-mediated relaxation was similar. Impaired EDHF-mediated relaxations were associated with reduced vascular expression of Cx37, Cx40, Cx43, IKCa and SKCa; increased expression of eNOS and NADPH oxidase subunits; and increased vascular formation of ROS and peroxynitrites. These effects were prevented by exposure to apocynin or losartan. CONCLUSIONS: CBDL is associated with reduced EDHF-mediated relaxations in the mesenteric artery, whereas NO-mediated relaxations persisted. These findings indicate that impaired EDHF-mediated relaxation involves an excessive vascular oxidative stress, most likely following activation of angiotensin II type 1 receptors.

Purpose: This study determined whether the Crataegus (Hawthorn species) special extract WS (R) 1442 stimulates the endothelial formation of nitric oxide (NO), a vasoprotective factor, and characterized the Underlying, mechanism.Methods and Results: Vascular reactivity was assessed in porcine coronary artery rings, reactive oxygen species (ROS) formation in artery sections by microscopy, and phosphorylation of Akt and endothelial NO synthase (eNOS) in endothelial cells by Western blot analysis. WS (R) 1442 caused endothelium-dependent relaxations in coronary, artery rings, which were reduced by N-omega-nitro-L-arginine (it competitive inhibitor of NO synthase) and by charybdotoxin plus apamin (two inhibitors of endothelium-derived hyperpolarizing factor-mediated responses). Relaxations to WS (R) 1442 were inhibited by intracellular ROS scavengers and inhibitors of Src and P13-kinase, but not by an estrogen receptor antagonist. WS (R) 1442 stimulated the endothelial formation of ROS in artery sections, and a redox-sensitive phosphorylation of Akt and eNOS in endothelial cells.Conclusions: WS (R) 1442 induced endothelium-dependent NO-mediated relaxations of coronary artery rings through the redoxsensitive Src/P13-kinase/Akt-dependent phosphorylation of eNOS.

Hypertension has been shown to be associated with impaired endothelium-derived hyperpolarizing factor (EDHF)-mediated arterial relaxation and hyperpolarization. Treatments of hypertensive rats with inhibitors of the renin-angiotensin system have been shown to restore both EDHF-mediated responses and the expression of connexins involved in the intercellular transfer of the hyperpolarization in mesenteric arteries. The present study was designed to determine whether chronic treatment of rats with angiotensin II impairs EDHF-mediated responses and the expression of connexins in the mesenteric arterial wall. Male Wistar rats were treated with angiotensin II (0.4 mg/kg/day) for 21 days using osmotic minipumps. Arterial pressure was measured by tail-cuff plethysmography. Contractile responses and membrane potential were measured in isolated mesenteric arteries. The expression of the three connexins (Cxs), Cx37, Cx40, and Cx43, was quantified in segments of mesenteric arteries by immunohistochemistry and quantitative real-time reverse transcriptase-polymerase chain reaction. Angiotensin II administration increased the mean systolic blood pressure. EDHF-mediated relaxation and hyperpolarization to acetylcholine and red wine polyphenols were significantly impaired in mesenteric arteries from angiotensin II-treated rats in comparison with control animals, whereas nitric oxide-mediated relaxation was unaltered. The expression of connexins Cx37, Cx40, and Cx43 was significantly decreased in the mesenteric artery from angiotensin II-treated rats. These findings indicate that angiotensin II-induced hypertension is associated with a selective impairment of EDHF-mediated relaxation and hyperpolarization in the rat mesenteric artery. The inhibition of EDHF-mediated responses is due, at least in part, to a decreased expression of connexins Cx37, Cx40, and Cx43 in the arterial wall.

RhoA-activated kinase (ROK) is involved in the disorders of smooth muscle contraction found in hypertension model animals and patients. We examined whether the alpha 1-adrenergic receptor agonist-induced ROK signal is perturbed in resistance small mesentery artery (SMA) of Lyon genetically hypertensive (LH) rats, using a ROK antagonist, Y27632. Smooth muscle strips of SMA and aorta were isolated from LH and Lyon normotensive (LN) rats. After Ca2+-depletion and pre-treatment with phenylephrine (PE), smooth muscle contraction was induced by serial additions of CaCl2. In LH SMA Ca2+ permeated cells to a lesser extent as compared with LN SMA, while CaCl2-induced contraction of LH SMA was greater than that of LN SMA, indicating a higher ratio of force to Ca2+ in LH SMA contraction (Ca2+ sensitization). No hyper-contraction was observed in LH aorta tissues. Treatment of LH SMA with Y27632 restored both Ca2+ permeability and Ca2+-force relationship to levels seen for LN SMA. In response to PE stimulation, phosphorylation of CPI-17, a phosphorylation-dependent myosin phosphatase inhibitor protein, and MYPT1 at Thr853, the inhibitory phosphorylation site of the myosin phosphatase regulatory subunit, was increased in LN SMA, but remained unchanged in LH SMA. These results suggest that the disorder in ROK-dependent Ca2+ permeability and Ca2+-force relationship is responsible for LH SMA hyper-contraction. Unlike other hypertensive models, the ROK-induced hyper-contractility of LH SMA is independent of MYPT1 and CPI-17 phosphorylation, which suggests that ROK-mediated inhibition of myosin phosphatase does not affect SMA hyper-contractility in LH SMA cells.

BACKGROUND Several epidemiological studies have shown that regular consumption of moderate amounts of wine, in particular red wine, is associated with a decreased total mortality due, in part, to a reduced risk of cardiovascular diseases. The protective effect has been attributable to polyphenols, which are potent vasodilators and have anti-thrombotic properties. Polyphenols have been shown to induce pronounced endothelium-dependent relaxations of arteries by causing the redox-sensitive PI3-kinase-dependent formation of nitric oxide (NO) and endothelium-derived hyperpolarizing factor (EDHF). The aim of the present study was to determine the role of the AMP-activated protein kinase (AMPK) in the red wine polyphenols (RWPs)-induced endothelial formation of NO and EDHF. METHODS AND RESULTS Vascular reactivity was assessed in organ chambers. Cultured porcine coronary artery endothelial cells porcine coronary artery segements were used to study the phosphorylation level of endothelial NO synthase (eNOS) at serine 1177, and AMPK at the Threonine 172 by Western blot analysis and immunohistochemical staining. RWPs caused endothelium-dependent relaxations in rings from rat aorta and mesenteric artery, and in those from porcine coronary artery. NO-mediated relaxations to RWPs as assessed in the presence of indomethacin and charybdotoxin plus apamin, were inhibited by compound C (an inhibitor of AMPK). Compound C also reduced EDHF-mediated relaxations as assessed in the presence of indomethacin and N(G)-nitro L-arginine. In contrast, compound C did not affect endothelium-dependent relaxations to acetylcholine and those to sodium nitroprusside. Moreover, RWPs induced the phosphorylation of AMPK at threonine 172 and eNOS at serine 1177 in endothelial cells; these responses were inhibited by compound C. CONCLUSION The present findings indicate that RWPs cause both NO and EDHF-mediated relaxations in several types of isolated arteries and that these effects are dependent on the activation of the AMP-activated protein kinase pathway.

We have previously demonstrated that in endothelium-denuded arteries, S-nitrosation of cysteine residues is a mechanism of formation of releasable nitric oxide (NO) stores, accounting for the long-lasting relaxation induced by S-nitrosating agents like S-nitrosoglutathione (GSNO). Here, we have investigated whether such effects could also be obtained in arteries exhibiting oxidative stress-associated endothelial dysfunction. Rats were implanted or not with a minipump delivering saline or angiotensin II for 14 days. As expected, aorta from angiotensin II-infused rats exhibited increased level of superoxide anions (as evaluated with dihydroethidine as fluorescent probe) and a reduced relaxation to acetylcholine in comparison to saline group. Unlike aortic rings with endothelium from controls, those from angiotensin II-infused rats exhibited persistent hyporesponsiveness to phenylephrine after pre-exposure to GSNO, as well as relaxation upon addition of N-acetylcysteine (NAC, which can displace NO from cysteine–NO residues) or HgCl2 (which cleaves S–NO bonds). In aorta from angiotensin II-infused rats, GSNO also induced a persistent increase in cysteine–NO residues (as determined using anti-cysteine–NO antiserum), which was blunted by NAC and HgCl2. These data indicate that (i) the vasorelaxant influence of releasable NO stores is unmasked by endothelial dysfunction (ii) S-nitrosation of cysteine residues remains an effective mechanism of formation of releasable NO stores in arteries exhibited oxidative stress-associated endothelial dysfunction. Thus, formation of releasable NO stores by S-nitrosating agents allows targeted vasculoprotective effects of NO at sites of endothelial dysfunction.

OBJECTIVE:Chronic administration of moderate amounts of red wine has been associated with a protective effect on the cardiovascular system. This study examined whether red wine polyphenols prevent the angiotensin II (Ang II)-induced hypertension and endothelial dysfunction in rats, and, if so, to elucidate the underlying mechanism.METHODS:Hypertensive rats were obtained by a 14-day infusion of Ang II. Red wine polyphenols were administered in the drinking water one week before and during the Ang II infusion. Arterial pressure was measured in conscious rats. Ex vivo vascular relaxation was assessed in organ chambers, vascular superoxide anion production by dihydroethidine and vascular NADPH oxidase expression by immunohistochemistry.RESULTS:Ang II-induced hypertension was associated with decreased relaxation to acetylcholine but not to red wine polyphenols. The Ang II treatment also increased vascular superoxide anion production and expression of nox1 and p22phox NADPH oxidase subunits. Intake of red wine polyphenols prevented the Ang II-induced hypertension and endothelial dysfunction and normalized vascular superoxide anion production and NADPH oxidase subunit expression. Red wine polyphenol treatment alone did not affect blood pressure.CONCLUSION:Intake of red wine polyphenols prevents Ang II-induced hypertension and endothelial dysfunction. Prevention of vascular NADPH oxidase induction and preservation of arterial nitric oxide availability during Ang II administration likely contribute to this effect.

Angiotensin II (Ang II)-induced hypertension is associated with vascular oxidative stress and an endothelial dysfunction. This study examined the role of reactive oxygen species (ROS) and endothelium-derived contracting factors in Ang II-induced endothelial dysfunction and whether these effects are prevented by red wine polyphenols (RWPs), a rich source of natural antioxidants. Rats were infused with Ang II for 14 days. RWPs were administered in the drinking water 1 week before and during the Ang II infusion. Arterial pressure was measured in conscious rats. Vascular reactivity was assessed in organ chambers and cyclooxygenase-1 (COX-1) and COX-2 expression by Western blot and immunofluorescence analyses. Ang II-induced hypertension was associated with blunted endothelium-dependent relaxations and induction of endothelium-dependent contractions in the presence of nitro-L-arginine in response to acetylcholine (Ach). These effects were not affected by the combination of membrane permeant analogs of superoxide dismutase and catalase but were abolished by the thromboxane A(2) (TP) receptor antagonist GR32191B and the COX-2 inhibitor NS-398. The COX-1 inhibitor SC-560 also prevented contractile responses to Ach. Ang II increased the expression of COX-1 and COX-2 in the aortic wall. RWPs prevented Ang II-induced hypertension, endothelial dysfunction, and upregulation of COX-1 and COX-2. Thus, Ang II-induced endothelial dysfunction cannot be explained by an acute formation of ROS reducing the bioavailability of nitric oxide but rather by COX-dependent formation of contracting factors acting on TP receptors. RWPs are able to prevent the Ang II-induced endothelial dysfunction mostly due to their antioxidant properties.

Nω-hydroxy-L-arginine (L-NOHA), the stable intermediate of the nitric oxide synthase (NOS)-catalyzed reaction, can induce NO/cyclic GMP-dependent relaxation in the rat aorta, in an endothelium- and NOS-independent manner. In this study, the role of the adventitia in the endothelium-independent effect of L-NOHA was investigated. Despite a decrease in norepinephrine (NE)-induced precontraction, adventitia removal in the rat aorta did not markedly alter the relaxant effect of forskolin, S-nitroso-N-acetylpenicillamine or glyceryl trinitrate. In contrast, both inhibition of NE-induced contraction and relaxation of NE-precontracted rings produced by L-NOHA were diminished in the absence of adventitia. Moreover, exposure to L-NOHA significantly enhanced the cyclic GMP level in the media of the aorta with, but not without adventitia. These findings demonstrate the role of the adventitia in the L-NOHA-induced decrease in tone and increase in cyclic GMP in the endothelium-denuded rat aorta. They suggest that NO or an NO-related compound formed from L-NOHA in the adventitia may produce paracrine effects.

Endothelium-independent relaxant activities of Nω-hydroxy-l-arginine (l-NOHA) homologues and hydroxylamine, a possible intermediate in nitric oxide (NO) formation, were examined in rat aortic rings. Addition of one –CH2– group to the –(CH2)x– chain between the α-amino acid and the hydroxyguanidine group (x=4) almost abolished–while deletion of one or two –CH2– (x=1 or 2) enhanced–the relaxant activity of l-NOHA homologues. Nω-hydroxy-nor-l-arginine- (x=2) and hydroxylamine-induced relaxations were blunted by a NO scavenger and by inhibitors of the guanylyl cyclase pathway, but not by NO synthase or cytochrome P450 inhibitors (except 7-ethoxyresorufin). However, aortic NO formation was detected (using electron paramagnetic resonance) in the presence of concentrations of these compounds higher than those producing relaxation. These findings support the view that endothelium-independent vasorelaxations induced by both l-NOHA homologues with a required chain length x≤3 and hydroxylamine are mediated by NO-dependent activation of guanylyl cyclase, through a 7-ethoxyresorufin-inhibited mechanism.

This study was designed to investigate the cardiovascular consequences of oral administration of Cedrelopsis grevei (CG) in normotensive rats. Experiments were designed to investigate hemodynamic parameters in vivo as well as the consequences of CG treatment on the vasoconstriction response to norepinephrine and the vasorelaxant response to ACh ex vivo in isolated aortas and small mesenteric arteries (SMA). Treatment of male Wistar rats with 80 mg/kg CG for 4 wk induced a progressive decrease in systolic blood pressure. In the aorta, CG did not significantly alter the response to norepinephrine despite the participation of extraendothelial nitric oxide (NO)-induced hyporeactivity. In the SMA, contraction to norepinephrine was not modified by CG treatment even though it enhanced the participation of endothelial NO. Endothelium-dependent relaxation to ACh was increased in both the aorta and SMA from CG-treated rats. In the aorta from CG-treated rats, the mechanism involved superoxide dismutase (SOD)- and catalase-sensitive free radical production. The latter was associated with enhanced expression of Cu/Zn SOD and endothelial NO synthase. These results suggest that oral administration of CG produces a decrease in blood pressure in normotensive rats. This hemodynamic effect was associated with enhanced endothelium-dependent relaxation and an induction of Cu/Zn SOD and endothelial NO synthase expressions in the vessel wall. They also show subtle mechanisms that compensate for the increased participation of NO to maintain unchanged agonist-induced contractility. These data provide a pharmacological basis for the empirical use of CG against cardiovascular diseases.

Aqueous extracts of Tapinanthus dodoneifolius DC Danser. (Loranthaceae) (AETD) were investigated for cardiovascular activities on isolated rat aorta and heart. AETD did not affect heart rate but significantly enhanced heart contraction force and relaxation capacity. AETD (0.001-1mg/ml) elicited a dose-dependent relaxation on arteries which was previously contracted with phenylephrine [10-6M] (rat aorta). AETD induces relaxation in endothelium dependent manner. When Indomethacin failed to inhibit AETD vasodilatory activity, in the presence of L-NAME, the vasodilatory activity of AETD was completely abolished. These results suggest a cardiotropic activity without any tachycardia as a side effect for AETD. However, AETD elicited vasodilatory activity which involved NO from endothelium. Key words : Tapinanthus dodoneifolius, endothelium , vasodilator , rat. Afr. J. Trad. Comp. Alt. Med. , 2005, 2 (1): 25-30

The present study was aimed to characterize the effects of ageing on vascular contraction by noradrenaline in rat isolated arteries. The existence of vascular bed heterogeneity was investigated in endothelium‐denuded conductance (aorta) and resistance (small mesenteric artery, SMA) arteries, with respect to Ca2+ handling, Ca2+ sensitization or Ca2+‐independent mechanisms. In both arteries, contractions to noradrenaline were not different between adult and aged rats. In Ca2+‐ free medium, noradrenaline elicited a transient increase in tension that was reduced by the Ca2+ mobilizing agents, ryanodine and thapsigargin, in arteries from adult rats. A loss of the thapsigargin‐ but not the ryanodine‐sensitive component of noradrenaline‐induced contraction was observed in the two arteries from aged rats. After depletion of Ca2+ stores with noradrenaline, addition of exogenous CaCl2 produced a sustained contraction that was decreased to the same extent by the protein kinase C inhibitor, GF 109203X and the tyrosine kinase inhibitor, tyrphostin A‐23, in arteries from adult and aged rats. The Rho‐associated protein kinase inhibitor, Y‐27632, caused identical relaxation of noradrenaline pre‐contracted arteries from both age groups. Basal intracellular calcium ([Ca2+]i) was higher in SMA from aged than from adult rats. In addition, the noradrenaline [Ca2+]i‐force relationship was significantly shifted to the right in the SMA from aged rats. Altogether, these data indicate that responsiveness to noradrenaline is preserved both in conductance and resistance arteries with ageing. The latter results from the association of increased basal [Ca2+]i, changes in Ca2+ handling at the level of thapsigargin‐sensitive sarcoplasmic reticulum Ca2+‐ATPases and decreased myofilament sensitivity to Ca2+. British Journal of Pharmacology (2003) 138, 745–750. doi:10.1038/sj.bjp.0705104