EFFECT OF TOLERANCE TO GLYCERYL TRINITRATE ON VASCULAR RESPONSES IN CONSCIOUS RABBITS

1. The effect of tolerance to glyceryl trinitrate (GTN) on vasodilator and vasoconstrictor responses was examined in conscious rabbits and isolated rabbit aortic rings. 2. In conscious rabbits, depressor responses to 5 min infusions of GTN (10-40 micrograms/kg per min intravenously (i.v.)), sodium nitroprusside (SNP, 5-20 micrograms/kg per min i.v.) and acetylcholine (ACh, 3-12 micrograms/kg per min i.v.) were examined before and after transdermal treatment with GTN (20 mg/48 h). GTN pretreatment significantly attenuated GTN-induced depressor responses, indicating the development of tolerance, but did not affect the reductions in arterial pressure induced by SNP or ACh. 3. Similarly, aortic rings taken from GTN pretreated rabbits exhibited tolerance to GTN but the relaxant responses to SNP or the calcium ionophore A23187 were not affected. In the aortic rings from GTN-tolerant rabbits contractile responses to serotonin or the thromboxane-mimetic U46619 were significantly attenuated, in contrast to the responses to the alpha 1-adrenoceptor agonist phenylephrine (PE) which were significantly enhanced. 4. Similarly, in conscious rabbits, PE-induced increases in arterial pressure and hindlimb vascular resistance were significantly enhanced by GTN pretreatment but the responses to the alpha 2-adrenoceptor agonist BHT 920 were unaffected. 5. In conclusion, tolerance to GTN does not affect endothelium-dependent vasodilatation but does cause a selective enhancement of alpha 1- but not alpha 2-adrenoceptor-mediated vasoconstriction.     

Effect of resveratrol on nitrate tolerance in isolated human internal mammary artery

The present study aims to examine whether resveratrol, a natural antioxidant present in red wine, restores the tolerance to nitroglycerin (GTN) on isolated human internal mammary artery (IMA), using an in vitro model of nitrate tolerance. IMA rings were obtained from 53 male patients undergoing coronary bypass operation. Nitrate tolerance was induced by incubating the artery ring with 100 microM GTN for 90 minutes. Concentration-response curves to GTN (10(-9) to 10(-4) M) were obtained on IMA rings precontracted with noradrenaline. A low concentration (5 microM) of lucigenin was used as a tool to measure superoxide production in IMA segments. GTN produced concentration-dependent relaxation in isolated human IMA rings. Preexposure of artery rings to GTN reduced the relaxations to GTN [E(max) values: 105 +/- 2% and 76 +/- 3%, n = 10 to 12, P < 0.05; EC(50) values (-log M): 6.72 +/- 0.05 and 4.95 +/- 0.06, P < 0.05, respectively]. Relaxation to sodium nitroprusside remained unchanged. Diminished relaxation to GTN is partially restored after removing endothelium or L(G)-nitro-L-arginine (L-NOARG, 10 M) or superoxide dismutase (20 and 200 U/mL) or catalase (200 U/mL) pretreatments. Pretreatments with resveratrol (1, 10, and 20 microM) for 20 minutes relatively improved the reduced relaxation to GTN in tolerant IMA rings. Coadministration of L-NOARG with resveratrol did not abolish the beneficial effect of resveratrol on nitrate tolerance. The inhibitory effect of resveratrol on GTN-induced tolerance was not abolished in arterial rings without endothelium. Exposure to GTN increased superoxide production in IMA segments with endothelium. Endothelium denudation, L-NOARG, or superoxide dismutase pretreatments markedly inhibited the increased superoxide production in tolerant arteries. Resveratrol (1 and 10 microM) almost completely abolished basal or NAD(P)H-stimulated superoxide production in tolerant and nontolerant arteries. Vascular tolerance to GTN, in in vitro tolerant human IMA rings, can be induced by endothelial superoxide anions. Resveratrol partially restored the reduced relaxation to GTN by inhibiting NAD(P)-derived superoxide production in endothelium.     

Role of mitochondrial aldehyde dehydrogenase in nitrate tolerance

Glyceryl trinitrate (GTN) is used in the treatment of angina pectoris and cardiac failure, but the rapid onset of GTN tolerance limits its clinical utility. Research suggests that a principal cause of tolerance is inhibition of an enzyme responsible for the production of physiologically active concentrations of NO from GTN. This enzyme has not conclusively been identified. However, the mitochondrial aldehyde dehydrogenase (ALDH2) is inhibited in GTN-tolerant tissues and produces NO2- from GTN, which is proposed to be converted to NO within mitochondria. To investigate the role of this enzyme in GTN tolerance, cumulative GTN concentration-response curves were obtained for both GTN-tolerant and -nontolerant rat aortic rings treated with the ALDH inhibitor cyanamide or the ALDH substrate propionaldehyde. Tolerance to GTN was induced using both in vivo and in vitro protocols. The in vivo protocol resulted in almost complete inhibition of ALDH2 activity and GTN biotransformation in hepatic mitochondria, indicating that long-term GTN exposure results in inactivation of the enzyme. Treatment with cyanamide or propionaldehyde caused a dose-dependent increase in the EC50 value for GTN-induced relaxation of similar magnitude in both tolerant and nontolerant aorta, suggesting that although cyanamide and propionaldehyde inhibit GTN-induced vasodilation, these inhibitors do not affect the enzyme or system involved in tolerance development to GTN. Treatment with cyanamide or propionaldehyde did not significantly inhibit 1,1-diethyl-2-hydroxy-2-nitrosohydrazine-mediated vasodilation in tolerant or nontolerant aorta, indicating that these ALDH inhibitors do not affect the downstream effectors of NO-induced vasodilation. Immunoblot analysis indicated that the majority of vascular ALDH2 is present in the cytoplasm, suggesting that mitochondrial biotransformation of GTN by ALDH2 plays a minor role in the overall vascular biotransformation of GTN by this enzyme.     

3', 4'-dihydroxyflavonol enhances nitric oxide bioavailability and improves vascular function after ischemia and reperfusion injury in the rat

We hypothesized that 3',4'-dihydroxyflavonol (DiOHF) by scavenging superoxide anions (O2-*) would increase the bioavailability of NO and potentiate NO-mediated relaxation in the rat aorta. Furthermore we hypothesized that DiOHF, by its antioxidant activity, would preserve responses to acetylcholine (ACh) in the presence of O2-* generators in the aorta in vitro and after ischemia and reperfusion of the rat hindquarters vasculature in situ. Using lucigenin-enhanced chemiluminescence we demonstrated that DiOHF caused a concentration-dependent reduction in O2-* accumulation whether generated by xanthine/xanthine oxidase in a cell-free system or by rat isolated aorta in the presence of NADPH. DiOHF also prevented the inhibitory effects of xanthine/xanthine oxidase and pyrogallol on vasorelaxation to ACh and sodium nitroprusside (SNP) in the rat aorta in vitro, and attenuated the vascular dysfunction caused by 2 h ischemia and 2 h reperfusion (I/R) in the rat hindquarters. I/R significantly reduced the dilator responses to both ACh and SNP; however, this effect was attenuated when DiOHF was given before the onset of ischemia or reperfusion. In conclusion, DiOHF, by scavenging O2-*, increases the relaxant activity of ACh and SNP and reduces the degree of inhibition of xanthine/xanthine oxidase or pyrogallol on the response to ACh. DiOHF reduces the adverse effects of I/R on vascular function by increasing NO bioavailability suggesting that it may be useful in preventing reperfusion injury.     

Involvement of H2O2 in superoxide-dismutase-induced enhancement of endothelium-dependent relaxation in rabbit mesenteric resistance artery

1 The mechanism underlying the enhancement by superoxide dismutase (SOD) of endothelium-dependent relaxation was investigated in rabbit mesenteric resistance arteries. 2 SOD (200 U ml(-1)) increased the production of H(2)O(2) in smooth muscle cells (as indicated by the use of an H(2)O(2)-sensitive fluorescent dye). 3 Neither SOD nor catalase (400 U ml(-1)) modified either the resting membrane potential or the hyperpolarization induced by acetylcholine (ACh, 1 micro M) in smooth muscle cells. 4 In arteries constricted with noradrenaline, the endothelium-dependent relaxation induced by ACh (0.01-1 micro M) was enhanced by SOD (200 U ml(-1)) (P<0.01). This action of SOD was inhibited by L-N(G)-nitroarginine (nitric oxide (NO)-synthase inhibitor) but not by either charybdotoxin+apamin (Ca(2+)-activated-K(+)-channel blockers) or diclofenac (cyclooxygenase inhibitor). 5 Neither ascorbate (50 micro M) nor tiron (0.3 mM), superoxide scavengers, had any effect on the ACh-induced relaxation, but each attenuated the enhancing effect of SOD on the ACh-induced relaxation. Similarly, catalase (400 U ml(-1)) inhibited the effect of SOD without changing the ACh-induced relaxation. 6 In endothelium-denuded strips constricted with noradrenaline, SOD enhanced the relaxation induced by the NO donor 1-hydroxy-2-oxo-3-(N-methyl-3-aminopropyl)-3-methyl-1-triazene (NOC-7) (P<0.05). Ascorbate and catalase each attenuated this effect of SOD. 7 H(2)O(2) (1 micro M) enhanced the relaxation on the noradrenaline contraction induced by NOC-7 and that induced by 8-bromo-cGMP, a membrane-permeable analogue of guanosine 3',5' cyclic monophosphate (cGMP). 8 SOD had no effect on cGMP production, whether measured in endothelium-intact strips following an application of ACh (0.1 micro M) or in endothelium-denuded strips following an application of NOC-7 (0.1 micro M). 9 It is suggested that in rabbit mesenteric resistance arteries, SOD increases the ACh-induced, endothelium-dependent relaxation by enhancing the action of NO in the smooth muscle via its H(2)O(2)-producing action (rather than via a superoxide-scavenging action).     

Vasorelaxations induced by calcitonin gene-related peptide,vasoactive intestinal peptide,and acetylcholine in aortic rings of endothelial and inducible nitric oxide synthase-knockout mice

The objective of this study was to determine if vasorelaxant responses to calcitonin gene-related peptide (CGRP), vasoactive intestinal peptide (VIP), and acetylcholine are altered in aortic rings of mice lacking genetic expression of endothelial nitric oxide synthase (eNOS) or inducible nitric oxide synthase (iNOS) genes (i.e., eNOS- and iNOS-knockout mice) as compared with control (wild-type) mice. Aortic rings from eNOS-knockout (eNOS (-/-)) mice did not relax in response to acetylcholine, thereby confirming previous reports. Aortic rings from iNOS-knockout (iNOS (-/-)) mice relaxed in response to acetylcholine in an endothelium-dependent manner. However, maximum relaxations in endothelium-intact rings were significantly (p < 0.05) larger than in control mice (85.3 +/- 3.1% in iNOS (-/-) mice vs. 67.9 +/- 5.6% in controls). CGRP caused concentration-dependent relaxations in aortas of all three types of mice: control mice, iNOS (-/-) mice, and eNOS (-/-) mice. Vasorelaxant responses to CGRP in control and iNOS (-/-) mice had identical relationships; both were partially dependent on endothelium. In eNOS (-/-) mice, dose-response curves of CGRP in endothelium-intact and endothelium-denuded rings were not significantly different, indicating loss of the partial dependence on endothelium. The vasorelaxant responses to VIP were completely dependent on endothelium in control and iNOS (-/-) mice. Maximum relaxations to VIP in iNOS (-/-) mice (77.4 +/- 2.7%) were significantly greater than in control mice (64.0 +/- 5.5%). Vasorelaxant responses to VIP in eNOS (-/-) aortic rings were also endothelium-dependent, but responses were greatly attenuated compared with wild-type mice. Relaxations induced by VIP (1 x 10 ) in endothelium-intact aortic rings of eNOS (-/-) mice and control mice were 18.3 +/- 5.4% and 64.0 +/- 5.5%, respectively. These findings demonstrated that, in eNOS (-/-) mice, aortic vasorelaxant responses to CGRP were fully present but no longer dependent on the endothelium, and responses to VIP were greatly attenuated compared with control and responses to acetylcholine were abolished. In iNOS (-/-) mice, aortic vasorelaxant responses to VIP and acetylcholine were significantly greater than wild-type control, suggesting that induction of iNOS may have attenuated vascular responses to VIP and acetylcholine in wild-type controls.     

Divergent effects of vitamin C on relaxations of rabbit aortic rings to acetylcholine and NO-donors

1. Vitamin C may influence NO-dependent relaxation independently of effects on oxidant stress. 2. We investigated effects of vitamin C (0.1 -- 10 mmol l(-1)) on relaxation of pre-constricted rabbit aortic rings to acetylcholine (ACh), authentic NO and the NO-donors glyceryl trinitrate (GTN), nitroprusside (NP) and S-nitroso-N-acetyl-penicillamine (SNAP). DETCA (2 -- 6 mmol l(-1)), a cell permeable inhibitor of endogenous Cu-Zn superoxide dismutase (SOD) was used to increase intracellular superoxide anion (O(2)(-)). 3. Vitamin C reduced the response to ACh (71 +/- 7% inhibition of maximum relaxation at 10 mmol l(-1)) and inhibited relaxation to authentic NO. Vitamin C inhibited relaxation to GTN but potentiated relaxations to NP and SNAP, causing a parallel shift to a lower concentration range of the log dose-response curve by approximately one log unit at the highest dose. 4. Vitamin C increased the concentration of NO in bath solution (plus EDTA, 1.0 mmol l(-1)) following the addition of SNAP from 53 +/- 14 to 771 +/- 101 nmol l(-1) over the range 0.1-3.0 mmol l(-1). 5. DETCA inhibited relaxation to ACh (71 +/- 9% inhibition of maximum relaxation). This inhibition was abolished by a cell permeable SOD mimetic, but not by vitamin C. DETCA inhibited relaxation to SNAP but not that to NP nor to GTN. 6. Vitamin C inhibits endothelium-dependent relaxations of rabbit aortic rings to ACh and authentic NO and does not reverse impaired relaxation resulting from increased intracellular oxidant stress. Vitamin C potentiates relaxation to the NO-donors NP and SNAP by a mechanism that could involve release of NO from nitrosothiols.     

Role of oxidative stress and nitric oxide in regulation of spontaneous tone in aorta of DOCA-salt hypertensive rats

1. The roles of nitric oxide (NO), superoxide anion (O(2)(-)), and hydrogen peroxide (H(2)O(2)) in the modulation of spontaneous tone were investigated in isolated aorta from deoxycorticosterone acetate (DOCA)-salt hypertensive rats. 2. Increases in preload from 1 to 5 g were accompanied by increases in spontaneous tone in aortic rings from DOCA-salt hypertensive rats but not from SHAM-normotensive rats. 3. Tone was higher in endothelium-denuded aortic rings than in endothelium-intact vessels. Inhibition of nitric oxide synthase (NOS) with 300 microM N(G)-nitro-L-arginine methyl ester (l-NAME) increased spontaneous tone. 4. Basal O(2)(-) generation was higher in aortic rings from DOCA-salt hypertensive rats than in those from SHAM-normotensive rats. Stretch increased O(2)(-) levels even further in the DOCA-salt group. In rings isolated from DOCA-salt hypertensive rats, administration of the O(2)(-) scavenger, superoxide dismutase (SOD, 150 U ml(-1)), or the nicotinamide adenine dinucleotide phosphate (NADPH)-oxidase inhibitor, apocynin (100 microM), completely abolished the development of spontaneous tone in endothelium-intact aortic rings but not in endothelium-denuded or in L-NAME-treated rings. SOD and apocynin decreased the generation of O(2)(-) in endothelium-intact, endothelium-denuded, and L-NAME-treated aortic rings. 5. Oral treatment of DOCA-salt hypertensive rats with the O(2)(-) scavengers, tempol or tiron, or with apocynin for 3 weeks prevented the development of hypertension and abolished the increases in O(2)(-) generation and spontaneous tone. 6. Administration of catalase (1000 U ml(-1)) to aortic rings increased spontaneous tone in vessels from DOCA-salt hypertensive rats. 7. Administration of the cyclooxygenase (COX) inhibitor, valeroyl salicylate, or the thromboxane/prostaglandin antagonist, SQ 29548, to aortic rings abolished tone. 8. The results suggest that NO plays a major role in preventing the generation of spontaneous tone in isolated aortic rings from DOCA-salt hypertensive rats. NADPH-oxidase-derived O(2)(-) enhanced spontaneous tone by inactivating NO. Endogenous H(2)O(2) appears to mitigate the increase in tone. In addition, a COX component may also contribute to spontaneous tone.     

Captopril-induced reversal of nitroglycerin tolerance: role of sulfhydryl group vs. ACE-inhibitory activity.

The angiotensin-converting enzyme (ACE) inhibitor captopril has been shown to reverse vascular tolerance to nitroglycerin (NTG). Whether captopril reverses NTG tolerance by providing sulfhydryl (SH) groups or by inhibiting ACE is not clear. To examine this issue, we treated rat aortic rings with buffer, captopril (SH +, ACE inhibitory activity +), enalaprilat (SH-, ACE inhibitory activity +), or N-acetylcysteine (NAC, SH+, ACE inhibitory activity-) prior to their contraction with epinephrine and subsequent relaxation with NTG. Previous exposure of NTG-treated rings resulted in marked resistance to the vasorelaxant effect of a subsequent exposure to NTG in buffer-treated rings. Both NAC and captopril, but not enalaprilat, potentiated the vasorelaxant effects of NTG during the first exposure of vascular rings to NTG and also prevented the development of tolerance to NTG during a second exposure. Buffer-treated rings showed an inability to accumulate cyclic guanosine monophosphate (GMP) in response to a second exposure to NTG. In contrast, both NAC and captopril-pretreated rings demonstrated a persistence of cyclic GMP accumulation during the second NTG exposure. The endothelium-dependent vasodilator acetylcholine (ACh) caused relaxation of the NTG-tolerant rings and also induced cyclic GMP accumulation in these rings. In other experiments, we found that prior exposure of vascular rings to ACh did not cause resistance to the subsequent vasorelaxant effects of ACh. NAC, captopril, and enalaprilat did not modulate the effects of ACh during either the first or subsequent exposures to ACh. In addition, indomethacin did not influence the "protective" effects of NAC or captopril against NTG tolerance.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasorelaxant effect of harman

The in vivo cardiovascular effect and in vitro vasorelaxant effect of harman, one of harmala alkaloids isolated from Peganum harmala, were examined in this study. Harman (1-10 mg/kg, i.v.) dose-dependently produced transient hypotension and long-lasting bradycardia in pentobarbital-anesthetized rats, which were attenuated by N(G)-nitro-L-arginine pretreatment. In isolated rat endothelium-intact thoracic aortic rings, harman concentration dependently relaxed phenylepherine-induced contractions with an IC(50) value around 9 microM. This vasorelaxant effect was attenuated by endothelium removal or N(omega)-nitro-L-arginine methyl ester pretreatment, but not by tetraethylammonium or indomethacin pretreatment. In cultured rat aortic endothelial cells, harman (1-100 microM) concentration dependently increased nitric oxide (NO) release, which was dependent on the presence of external Ca(2+). Harman pretreatment (3-30 microM) also concentration dependently inhibited the contractions induced by phenylephrine, 5-hydroxytryptamine (5-HT), and KCl in endothelium-denuded aortic rings in a non-competitive manner. In addition, harman pretreatment reduced both phasic and tonic phases of phenylephrine-induced contractions. Receptor binding assays further indicated that harman (K(i) values around 5-141 microM) interacted with the cardiac alpha(1)-adrenoceptors, brain 5-HT(2) receptors, and cardiac 1, 4-dihydropyridine binding site of L-type Ca(2+) channels. Therefore, the present results suggested that the vasorelaxant effect of harman was attributed to its actions on the endothelial cells to release NO and on the vascular smooth muscles to inhibit the contractions induced by the activation of receptor-linked and voltage-dependent Ca(2+) channels. The vasorelaxant effect may be involved in the hypotensive effect of harman.     

Heterozygous deficiency of manganese superoxide dismutase in mice (Mn-SOD+/-): a novel approach to assess the role of oxidative stress for the development of nitrate tolerance

Nitroglycerin (GTN)-induced tolerance was reported to be associated with increased levels of reactive oxygen species (ROS) in mitochondria. In the present study, we further investigated the role of ROS for the development of nitrate tolerance by using heterozygous manganese superoxide dismutase knock-out mice (Mn-SOD+/-). Mn-SOD is acknowledged as a major sink for mitochondrial superoxide. Vasodilator potency of mouse aorta in response to acetylcholine and GTN was assessed by isometric tension studies. Mitochondrial ROS formation was detected by 8-amino-5-chloro-7-phenylpyrido[3,4-d]pyridazine-1,4-(2H,3H)dione sodium salt (L-012)-enhanced chemiluminescence and mitochondrial aldehyde dehydrogenase (ALDH-2) activity was determined by a high-performance liquid chromatography-based assay. Aortic rings from Mn-SOD+/- mice showed normal endothelial function and vasodilator responses to GTN. In contrast, preincubation of aorta with GTN or long-term GTN infusion caused a marked higher degree of tolerance as well as endothelial dysfunction in Mn-SOD+/- compared with wild type. Basal as well as GTN-stimulated ROS formation was significantly increased in isolated heart mitochondria from Mn-SOD+/- mice, correlating well with a marked decrease in ALDH-2 activity in response to in vitro and in vivo GTN treatment. The data presented indicate that deficiency in Mn-SOD leads to a higher degree of tolerance and endothelial dysfunction associated with increased mitochondrial ROS production in response to in vitro and in vivo GTN challenges. These data further point to a crucial role of ALDH-2 in mediating GTN bioactivation as well as development of GTN tolerance and underline the important contribution of ROS to these processes.     

Endothelial function in spontaneously hypertensive rats: influence of quinapril treatment.

1. Angiotensin converting enzyme (ACE) inhibition has been shown to restore the impaired endothelial function in hypertension, but the mediators underlying the promoted endothelium-dependent dilatation have not been fully characterized. Therefore, we investigated the effects of 10-week-long quinapril therapy (10 mg kg-1 day-1) on responses of mesenteric arterial rings in vitro from spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto (WKY) rats. 2. Endothelium-dependent relaxations of noradrenaline (NA)-precontracted rings to acetylcholine (ACh) and adenosine 5'-diphosphate (ADP) were similar in WKY rats and quinapril-treated SHR and more pronounced than in untreated SHR. The nitric oxide (NO) synthase inhibitor NG-nitro-L-arginine methyl ester (L-NAME) attenuated the relaxations in both WKY groups and quinapril-treated SHR, and completely inhibited them in untreated SHR. When endothelium-dependent hyperpolarization was prevented by precontraction of the preparations with potassium chloride (KCl), no differences were found in relaxations to ACh and ADP between the study groups. In addition, in NA-precontracted rings the L-NAME- and indomethacin-resistant relaxations to ACh were partially prevented by apamin, an inhibitor of calcium-activated potassium channels. 3. Interestingly, in quinapril-treated SHR but not in the other groups, exogenous bradykinin potentiated the relaxations to ACh in both NA- and KCl-precontracted arterial rings. 4. Contractile sensitivity of endothelium-intact rings to NA was reduced in SHR by quinapril, and was more effectively increased by L-NAME in quinapril-treated than untreated SHR.(ABSTRACT TRUNCATED AT 250 WORDS)     

The possible implication of trans-Resveratrol in the cardioprotective effects of long-term moderate wine consumption.

trans-Resveratrol (t-RESV; 1-10 microM), a phenolic component of wines, had no effect on phenylephrine-(PE; 1 microM) and high KCl-(60 mM) induced contractions in endothelium-denuded rat aortic rings. However, it relaxed the contractile response produced by these vasoconstrictor agents in intact rat aorta. The vasorelaxing effects of t-RESV were completely inhibited by N(G)-nitro-L-arginine (L-NOARG; 0.1 mM) and methylene blue (10 microM), but they were unaffected by atropine (10 microM) and yohimbine (1 microM). The reversal effect produced by L-NOARG was antagonized by L-arginine but not by D-arginine (0.1 mM). t-RESV (1-10 microM) did not significantly modify rat aorta constitutive nitric-oxide synthase activity. However, this natural compound decreased NADH/NADPH oxidase activity in rat aortic homogenates. In addition, t-RESV (1-10 microM) was ineffective in scavenging superoxide anions (O(2)*) generated enzymatically by a hypoxanthine/xanthine oxidase (HX/XO) system and/or to inhibit XO. The above data demonstrate that the characteristic endothelium-dependent vasorelaxant effect of t-RESV in rat aorta seems to be caused by the inhibition of vascular NADH/NADPH oxidase and the subsequent decrease of basal cellular O(2)* generation and, therefore, of NO biotransformation. Under the assumption that t-RESV exhibits a similar behavior in human blood vessels and bearing in mind that an overactivity of NADH/NADPH oxidase has been found in a number of cardiovascular pathologies, the results obtained in this work suggest that t-RESV could play an important role in the cardioprotective effects induced by the long-term moderate wine consumption.     

Persistence of the response to SIN1 on isolated coronary arteries rendered tolerant to nitroglycerin in vitro or in vivo

Experiments were performed on isolated canine and human coronary arteries to provide more insight into the mechanisms responsible for the vascular tolerance to nitroglycerin that is induced under in vitro or in vivo conditions. In vitro tolerance was produced after an incubation of coronary ring segments with nitroglycerin (10 microM for 30 min at physiological pH). After elevation of tone with KCl (15 mM), dose-response curves were constructed for nitroglycerin or SIN1 (3-morpholino-syndnonimin) on control and tolerant rings. On canine tolerant rings the dose-response curve for nitroglycerin-induced relaxations was significantly (p less than 0.001) shifted to the right, and 50% of the maximal relaxation (ED50) increased from 55 +/- 9 nM to 1.2 +/- 0.2 microM. Pretreatment of tolerant rings with N-acetylcysteine (NAC, 10 microM 10 min before KCl-induced contraction) partially restored the responsiveness to nitroglycerin, with ED50 reducing to 0.56 +/- 0.03 microM (p less than 0.02). On the other hand, the dose-response curves to SIN1 were not significantly altered. Similar results were obtained on human preparations. On isolated canine coronary rings rendered tolerant in vivo by subcutaneous injections of 15 mg/kg nitroglycerin (two times daily for 4 consecutive days), ED50 for nitroglycerin was 0.67 +/- 0.08 microM (p less than 0.001 versus control rings), and NAC again partially restored the responsiveness to nitroglycerin. As for the in vitro tolerance, the relaxations to SIN1 were not significantly altered on these canine rings rendered nitrate tolerant in vivo.(ABSTRACT TRUNCATED AT 250 WORDS)     

Hydrogen peroxide is an endothelium-dependent contracting factor in rat renal artery

In addition to endothelium-derived relaxing factor and hyperpolarizing factor, vascular endothelium also modulates smooth muscle tone by releasing endothelium-derived contracting factor(s) (EDCF), but the identity of EDCF remains obscure. We studied here the involvement of hydrogen peroxide (H2O2) in endothelium-dependent contraction (EDC) of rat renal artery to acetylcholine (ACh). ACh (10(-6), 10(-5), and 10(-4) M) induced a transient contraction of rat renal artery with intact endothelium in a concentration-related manner, but not in the artery with endothelium removed. In phenylephrine-precontracted renal arteries, ACh induced an endothelium-dependent relaxation response at lower concentrations (10(-8)-10(-6) M), and a relaxation followed by a contraction at higher concentrations (10(-5) M). Inhibition of nitric oxide synthase by N(omega)-nitro-L-arginine (10(-4) M) enhanced the EDC to ACh. Catalase (1000 U ml(-1)) reduced the EDC to ACh. H2O2 (10(-6), 10(-5), and 10(-4) M) induced a similar transient contraction of the renal arteries as ACh, but in an endothelium-independent manner. Inhibition of NAD(P)H oxidase and cyclooxygenase by diphenylliodonium chloride and diclofenac greatly attenuated ACh-induced EDC, while inhibition of xanthine oxidase (allopurinol) and cytochrome P450 monooxygenase (17-octadecynoic acid) did not affect the contraction. Antagonist of thromboxane A2 and prostaglandin H2 receptors (SQ 29548) and thromboxane A2 synthase inhibitor (furegrelate) attenuated the contraction to ACh and to H2O2. In isolated endothelial cells, ACh (10(-5) M) induced a transient H2O2 production detected with a fluorescence dye sensitive to H2O2 (2',7'-dichlorofluorescein diacetate). The peak concentration of H2O2 was 5.1 x 10(-4) M at 3 min and was prevented by catalase. Taken together, these results show that ACh triggers H2O2 production through NAD(P)H oxidase activation in the endothelial cells, and that ACh and H2O2 share the same signaling pathway in causing smooth muscle contraction. Therefore, H2O2 is most likely the EDCF in rat renal artery in response to ACh stimulation.     

Involvement of nitric oxide in the endothelium-dependent relaxation induced by hydrogen peroxide in the rabbit aorta.

1. The effects of hydrogen peroxide (H2O2, 0.1-1 mM) on the tone of the rings of rabbit aorta precontracted with phenylephrine (0.2-0.3 microM) were studied. 2. H2O2 induced a concentration-dependent relaxation of both the intact and endothelium-denuded rings. However, in the presence of intact endothelium, H2O2-induced responses were 2-3 fold larger than in its absence, demonstrating the existence of endothelium-independent and endothelium-dependent components of the vasorelaxant action of H2O2. 3. The endothelium-dependent component of H2O2-induced relaxation was prevented by NG-nitro-L-arginine methyl ester (L-NAME, 30 microM) or NG-monomethyl-L-arginine (300 microM), inhibitors of nitric oxide synthase (NOS), in a manner that was reversible by L-, but not by D-arginine (2mM). The inhibitors of NOS did not affect the responses of denuded rings. 4. Methylene blue (10 microM), an inhibitor of soluble guanylate cyclase, blocked H2O2-induced relaxation of both the intact and denuded rings. 5. H2O2 (1 mM) enhanced the efflux of cyclic GMP from both the endothelium-intact and denuded rings. The effect of H2O2 was 4 fold greater in the presence of intact endothelium and this endothelium-dependent component was abolished after the inhibition of NOS by L-NAME (30 microM). 6. In contrast to the effects of H2O2, the vasorelaxant action of stable organic peroxides, tert-butyl hydroperoxide or cumene hydroperoxide, did not have an endothelium-dependent component. Moreover, they did not potentiate the efflux of cyclic GMP from the rings of rabbit aorta.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vasorelaxation by amentoflavone isolated from Selaginella tamariscina

In the courses of in vitro screening for the vasorelaxant effect of the various extracts from medicinal plants, an ethyl acetate-soluble extract of Selaginella tamariscina was found to exhibit distinctive vasorelaxant activity. Further purifications of the extract as guided by in vitro vasorelaxant assay afforded an active biflavonoid, amentoflavone. Amentoflavone induced concentration-dependent relaxation of the phenylephrine-precontracted aorta, which disappeared by removal of functional endothelium. Pretreatment of the aortic tissues with N(G)-nitro- L-arginine methyl ester (L-NAME), methylene blue, or 1 H- -oxadiazolo[4,3- a]quinoxalin-1-one (ODQ) inhibited the relaxation induced by amentoflavone. Amentoflavone-induced relaxations were also markedly attenuated by addition of tetraethylammonium (TEA) or verapamil. However, the relaxant effect of amentoflavone was not blocked by pretreatment with indomethacin, glibenclamide, atropine, or propranolol. Incubation of endothelium-intact aortic rings with amentoflavone increased the production of cGMP, but this effect was blocked by endothelium-denudation or pretreatment with L-NAME or ODQ. These results suggest that amentoflavone relaxes vascular smooth muscle via endothelium-dependent nitric oxide-cGMP signaling, with possible involvement of non-specific K (+) and Ca (2+) channels. Abbreviations. EDRF:endothelium-derived relaxing factor EDHF:endothelium-derived hyperpolarizing factor NO:nitric oxide cGMP:guanosine 3',5'-cyclic monophosphate DMSO:dimethyl sulfoxide L-NAME: N(G)-nitro- L-arginine methyl ester ODQ:1 H-[1,2,4]-oxadiazole-[4,3- a]-quinoxalin-1-one IBMX:3-isobutyl-1-methylxanthine K (Ca):Ca (2+)-dependent K (+) channel K (ATP):adenosine triphosphate (ATP)-sensitive K (+) channel TEA:tetraethylammonium     

Developmental changes in endothelium-dependent pulmonary vasodilatation in pigs.

1. We compared in vitro endothelium-dependent vasorelaxant responses to acetylcholine (ACh) and the endothelium-independent vasodilator response to sodium nitroprusside (SNP) in prostaglandin F2 alpha (PGF2 alpha)-precontracted muscular pulmonary arteries (PA) from pigs aged 5 min to 2 h (neonatal), 3-10 days, 3-8 weeks and adults. 2. In the pulmonary artery (PA) rings from neonatal animals, the vasodilator response to ACh was negligible. However, responses to ACh were present in all PA rings from older animals, being greatest at 3-10 days and then decreasing with age (P less than 0.001, ANOVA). ACh (30 microM) induced a 1 +/- 1%, 92 +/- 9%, 62 +/- 5% and 51 +/- 6% reduction of the PGF2 alpha-generated tension in neonatal, 3-10 days, 3-8 weeks and adult groups, respectively. 3. The relaxant response to SNP was present in the PA rings from all age groups and increased with age (P less than 0.001, ANOVA). SNP (1 microM)-induced relaxation was 55 +/- 9%, 73 +/- 7%, 97 +/- 5% and 93 +/- 6% in neonatal, 3-10 days, 3-8 week and adult groups, respectively. 4. Removal of the vascular endothelium abolished the relaxant response to ACh but had no effect on the response to SNP in any groups. 5. NG-monomethyl-L-arginine (30 microM), a nitric oxide synthesis inhibitor, inhibited the response to ACh but not to SNP. The lipoxygenase inhibitor, nordihydroguaiaretic acid, had no significant effect on responses to ACh or SNP in any group.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of vascular nitric oxide in vitro and in vivo; a new role for endogenous hydrogen sulphide?

BACKGROUND AND PURPOSE: The aim of these experiments was to evaluate the significance of the chemical reaction between hydrogen sulphide (H2S) and nitric oxide (NO) for the control of vascular tone. EXPERIMENTAL APPROACH: The effect of sodium hydrosulphide (NaHS; H2S donor) and a range of NO donors, such as sodium nitroprusside (SNP), either alone or together, was determined using phenylephrine (PE)-precontracted rat aortic rings and on the blood pressure of anaesthetised rats. KEY RESULTS:Mixing NaHS with NO donors inhibited the vasorelaxant effect of NO both in vitro and in vivo. Low concentrations of NaHS or H2S gas in solution reversed the relaxant effect of acetylcholine (ACh, 400 nM) and histamine (100 microM) but not isoprenaline (400 nM). The effect of NaHS on the ACh response was antagonized by CuSO(4) (200 nM) but was unaffected by glibenclamide (10 microM). In contrast, high concentrations of NaHS (200-1600 microM) relaxed aortic rings directly, an effect reduced by glibenclamide but unaffected by CuSO4. Intravenous infusion of a low concentration of NaHS (10 micromol kg(-1) min(-1)) into the anaesthetized rat significantly increased mean arterial blood pressure. L-NAME (25 mg kg(-1), i.v.) pretreatment reduced this effect. CONCLUSIONS AND IMPLICATIONS: These results suggest that H2S and NO react together to form a molecule (possibly a nitrosothiol) which exhibits little or no vasorelaxant activity either in vitro or in vivo. We propose that a crucial, and hitherto unappreciated, role of H2S in the vascular system is the regulation of the availability of NO.