Effect of trandolapril on vascular responsiveness in cholesterol-fed rabbit-isolated arteries

According to the World Health Organisation, cardiovascular disorders are one of the main causes of morbi/mortality in the western world. The effect of trandolapril (0.3 mg kg(-1) day(-1)), a non-sulphydryl angiotensin-converting enzyme (ACE) inhibitor, on the vascular responsiveness in aorta isolated from hypercholesterolemic rabbits was examined. Three groups of rabbits (n=30) were used: Group 0 (control group); Group 1 (hypercholesterolemic group, 0.5% (wt/wt) cholesterol-enriched diet) and Group 2 (hypercholesterolemic+trandolapril 0.3 mg kg(-1) day(-1)). After 18 weeks of treatment, the rabbits were killed and the thoracic aorta, proximal coronary and mesenteric (5th branch) arteries were isolated, cleaned off and mounted in an organ bath. Trandolapril had no significant effect on plasma cholesterol, high density lipoprotein (HDL) or low density lipoprotein (LDL). Despite the lack of effect of the drug on the above-mentioned parameters, treatment with trandolapril improved endothelium-dependent relaxation induced by acetylcholine in aortic and mesenteric rings from hypercholesterolemic rabbits treated with trandolapril. The relaxation induced by 10(-5) M acetylcholine were 65.0+/-4.0%; 24. 0+/-9.4% (P<0.01, n=10) and 51.3+/-7.0% (P<0.01, n=10) in aortic rings from Groups 0, 1 and 2, respectively, and 50.0+/-12.0%; 10. 1+/-10.0% (P<0.01, n=10); 61.0+/-9.7% (P<0.01, n=10) in small mesenteric rings from Groups 0, 1 and 2, respectively. In addition, trandolapril treatment improved the increase in serotonin-induced contraction in proximal coronary arteries with respect to the hypercholesterolemic group. On the other hand, we did not find any differences among the group in endothelium-independent relaxation induced by sodium nitroprusside. These results provide evidence that trandolapril restores endothelium-dependent relaxation in hypercholesterolemic rabbit-isolated arteries. These data suggest that trandolapril might have beneficial action in the prevention of vascular alteration involved in atherosclerosis.     

Effect of prolonged incubation with copper on endothelium-dependent relaxation in rat isolated aorta

1 We investigated the effects of prolonged exposure to copper (Cu(2+)) on vascular functioning of isolated rat aorta. 2 Aortic rings were exposed to CuSO(4) (3-24 h) in Dulbecco's modified Eagle medium with or without 10% foetal bovine serum (FBS) and then challenged with vasoconstrictors or vasodilators in the absence of Cu(2+). 3 Exposure to 2 micro M Cu(2+) in the absence of FBS did not modify the response to phenylephrine (PE) or acetylcholine (ACh) in aortic rings incubated for 24 h. Identical exposure in the presence of FBS increased the contractile response to 1 micro M PE by 30% (P<0.05) and impaired the relaxant response to 3 micro M ACh or 1 micro M A23187 (ACh, from 65.7+/-7.1 to 6.2+/-1.1%, n=8; A23187, from 74.6+/-8.2 to 12.0+/-0.8%, n=6; P<0.01 for both). Cu(2+) exposure did not affect the relaxant response to NO-donors. 4 Impairment of vasorelaxation appeared 3 h after incubation with 2 micro M Cu(2+) and required 12 h to attain a steady state. Vasorelaxation to ACh was partially restored by 1 mM tiron (intracellular scavenger of superoxide ions; maximum relaxation 34.2+/-6.4%, n=10, P<0.01 vs Cu(2+) alone), whereas catalase, superoxide dismutase or cycloheximide were ineffective. 5 Twenty-four hour-exposure to 2 micro M Cu(2+) did not affect endothelium integrity or eNOS expression, and increased the Cu content in arterial rings from 6.8+/-1.1 to 18.9+/-2.9 ng mg(-1) wet weight, n=8; P<0.01. 6 Our results show that, in the presence of FBS, prolonged exposure to submicromolar concentrations of Cu(2+) impaired endothelium-dependent vasorelaxation in aortic rings, probably through an intracellular generation of superoxide ions. British Journal of Pharmacology (2002) 136, 1185-1193     

Investigation of the inhibitory effects of homocysteine and copper on nitric oxide-mediated relaxation of rat isolated aorta

1. Elevated plasma levels of homocysteine (HC) and copper have both been associated with the development of inflammatory vascular diseases, such as atherosclerosis. In this study, the effects of a combination of HC and copper on nitric oxide (NO)-mediated relaxation of isolated rat aortic rings were investigated. 2. Exposure to HC (10-100 microM; 30 min) had no effect on relaxation to acetylcholine (ACh; 0.01-10 microM, n=4). Pre-incubation of aortic rings with a higher concentration of HC for an extended period (1 mM; 180 min) significantly inhibited endothelium-dependent relaxation (n=4), but this inhibition was prevented by the presence of the copper chelator bathocuprione (10 microM, 180 min, n=6). 3. Exposure to HC (100 microM) and copper (10-100 microM; 30 min) caused a copper concentration-dependent inhibition of endothelium-dependent relaxation (n=4). This inhibitory effect was reduced in the presence of either superoxide dismutase (SOD; 100 u ml(-1); n=4) or catalase (100 u ml(-1); n=4), and further reduced by the presence of both enzymes (n=5). 4. HC and copper (100 microM; 30 min) significantly inhibited endothelium-independent relaxation to glyceryl trinitrate (0.01-10 microM; n=8). In contrast, HC (1 mM), alone or in combination with copper (100 microM), did not inhibit relaxation to the endothelium-independent relaxant sodium nitroprusside (0.01-10 microM; n=4). 5. These data indicate that the presence of copper greatly enhances the inhibitory actions of HC on NO-mediated relaxation of isolated aortic rings. The reduction of inhibition by catalase and SOD indicates a possible role for copper-catalyzed generation of superoxide and hydrogen peroxide leading to an increased inactivation or decreased production of endothelium-derived NO.     

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.     

Co-incubation of native and oxidized low-density lipoproteins: potentiation of relaxation impairment

The influence of native low-density lipoprotein (LDL) on the inhibition of endothelium-dependent relaxation previously induced by oxidized LDL was investigated with intact rabbit aortic rings. We also tried to assess oxysterol involvement in the native lipoprotein effects. Lipoprotein fractions (1 mg protein/ml) were tested for their ability to inhibit the vasorelaxation induced by acetylcholine in aorta rings previously precontracted by noradrenaline vs. that in control strips in Krebs buffer. Co-incubation of oxidized and native LDL reinforced the oxidized LDL-induced inhibition, compared to the impairment evoked by oxidized LDL alone (E(max)=43.3+/-6.7% and 61. 4+/-5.4%, respectively; P<0.05). Finally, smaller amounts of 7-oxy-cholesterols were recovered in organ baths after co-incubation of native and oxidized LDL than after incubation of oxidized LDL alone. Conversely, more oxy-cholesterols were found in the strip vessels under the same conditions (% of oxysterol incorporation: 0. 05158 vs. 0.10199, r=0.703). Together these results suggest that the strengthening of oxidized LDL-induced inhibition by native LDL is dependent on an oxysterol effect on arterial wall cells. Mechanisms involved in this phenomenon remain to be investigated.     

Time course of changes in endothelium-dependent and -independent relaxation of chronically diabetic aorta: role of reactive oxygen species

In the present study, the role of reactive oxygen species and the contribution of antioxidant defence in the time course of changes in acetylcholine-stimulated endothelium-dependent and sodium nitroprusside-stimulated endothelium-independent relaxation were investigated in aortic rings isolated from 6-month streptozotocin-diabetic and age-matched control rats. Although there were no significant differences in the degree of the peak relaxations produced by a single administration of acetylcholine (1 microM) or sodium nitroprusside (0.01 microM) between control and diabetic rings, the endothelium-dependent and -independent relaxant responses were more transient and the time required to reach a peak relaxation after addition of acetylcholine was shorter in diabetic vessels. Pretreatment of diabetic vessels with superoxide dismutase (100 U/ml) normalized the recovery phases of endothelium-dependent and -independent relaxations, but had no effect on the peak responses to acetylcholine and sodium nitroprusside. In the presence of diethyldithiocarbamate (5 mM), an inhibitor of superoxide dismutase, the transient nature of the relaxant response to acetylcholine or sodium nitroprusside was more marked and the peak relaxations were inhibited; these effects of diethyldithiocarbamate were more pronounced in diabetic than in control rings. Catalase, 160 U/ml, decreased the peak relaxant response to acetylcholine and accelerated fading of the relaxation in diabetic aorta. Similar results were obtained for control aorta with a higher concentration of catalase (550 U/ml). Pretreatment with 3-amino-1,2,4 triazole (5 mM), a catalase inhibitor, inhibited the peak relaxant response to acetylcholine in diabetic rings. The combination of superoxide dismutase (100 U/ml) plus 3-amino-1,2,4 triazole (5 mM) produced an increase of the transient nature of endothelium-dependent relaxation of diabetic rings greater than that with 3-amino-1,2,4 triazole alone. Neither catalase nor 3-amino-1,2,4 triazole affected the characteristics of sodium nitroprusside-induced relaxation. Desferrioxamine, an inhibitor of hydroxyl radical (.OH) production, or mannitol, a.OH scavenger, had no effect on the characteristics of either acetylcholine- or sodium nitroprusside-induced relaxation in control and diabetic rings. Biochemical measurements revealed an inhibited superoxide dismutase activity in diabetic aorta together with activated catalase. Our findings suggest that, during the chronic phase of streptozotocin-diabetes, excess superoxide (O(2)(. -)) is responsible for the enhanced transient nature of endothelium-dependent and -independent relaxation of aorta via a reduction in bioavailable concentrations of nitric oxide (NO). However, the involvement of hydrogen peroxide (H(2)O(2)) in the establishment of acetylcholine-stimulated relaxation may be increased, which is likely to account for the maintenance of the relaxant effect of acetylcholine in chronically diabetic vessels.     

Effect of pravastatin on impaired endothelium-dependent relaxation induced by lysophosphatidylcholine in rat aorta

Aim: To investigate the effects of pravastatin, a potent 3-hydroxy-3-methylglutaryl coenzyme A (HMG-CoA) reductase inhibitor, on impaired endothelium-dependent relaxation induced by lysophosphatidylcholine (LPC), the major component of oxidized low-density lipoprotein, in rat thoracic aorta. Methods: Both the endothelium-dependent relaxation response to acetylcholine and the endotheliumindependent relaxation response to sodium nitroprusside of aortic rings were measured by recording isometric tension after the rings were exposed to LPC in the absence or presence of pravastatin to estimate the injury effect of LPC and the protective effect of pravastatin on the aortic endothelium, respectively. Results: Exposure of aortic rings to LPC (1-10μmol/L) for 30 min induced a significant concentration-dependent inhibition of endothelium-dependent relaxation to acetylcholine, but did not affect endothelium-independent relaxation in response to sodium nitroprusside. Pre-incubation of aortic rings with pravastatin (0.3-3mmol/L) for 15 min and then co-incubation of the rings with LPC (3 μmol/L) for another 30 min significantly attenuated the inhibition of endothelium-dependent relaxation induced by LPC. This protective effect of pravastatin (1 mmol/L) was abolished by N^G-nitro-L-arginine methyl ester (30 μmol/L), an inhibitor of nitric oxide synthase, but not by indomethacin (10 μmol/L), an inhibitor of cyclooxygenase. Moreover, protein kinase C inhibitor chelerythrine (1μmol/L) the superoxide anion scavenger superoxide dismutase (200 kU/L), and the nitric oxide precursor L-arginine (3 mmol/L) also improved the impaired endotheliumdependent relaxation induced by LPC, similar to the effects of pravastatin.C onclusion: Pravastatin can protect the endothelium against functional injury induced by LPC in rat aorta, a fact which is related to increasing nitric oxide bioavailability.     

Endothelium dysfunction in LDL receptor knockout mice: a role for H2O2

1. In this study, the role of endogenous H(2)O(2) as an endothelium-dependent relaxant factor was characterised in aortas from C57BL/6J and LDL receptor-deficient mice (LDLR(-/-)). 2. Aortic rings from LDLR(-/-) mice showed impaired endothelium-dependent relaxation to acetylcholine (ACh; 0.001-100 micro M) and to the Ca(2+) ionophore A23187 (0.001-3 micro M) compared with aortic rings from control mice. Endothelium-independent relaxation produced by the NO donor, 3-morpholino-sydnonimine (SIN-1) was not different between strains. 3. Pretreatment of vessels with L-NNA (100 micro M) or L-NNA (100 micro M) plus L-NAME (300 micro M) plus haemoglobin (10 micro M) markedly decreased, but did not abolish the relaxation to ACh in control mice. In the aortas from LDLR(-/-) mice treated with L-NNA (100 micro M), ACh induced a contractile effect. Catalase (800 and 2400 U ml(-1)) shifted to the right the endothelium-dependent relaxation to ACh in aortas from control but not from LDLR(-/-) mice. Aminotriazole (50 mM), which inhibits catalase, abolished its effect on control mice. Treatment of vessels with L-NNA and catalase abolished vasorelaxation induced by ACh. Indomethacin (10 micro M) did not modify the concentration-response curve to ACh. Superoxide dismutase (300 U ml(-1)) did not change ACh-induced relaxation in both strains. 4. Exogenous H(2)O(2) produced a concentration-dependent relaxation in endothelium-denuded aortic rings, which was not different between strains. 5. It is concluded that H(2)O(2) greatly contributes to relaxation to ACh in aorta from control mice. Endothelial-dependent relaxation to ACh is impaired in LDLR(-/-) mice. Reduced biosynthesis or increased inactivation of H(2)O(2) is the possible mechanism responsible for endothelial dysfunction in aortas of atherosclerosis-susceptible LDLR(-/-) mice.     

Na+/Ca2+ exchanger inhibitor ameliorates impaired endothelium-dependent Na+ relaxation induced by high glucose in rat aorta

The present study was designed to investigate the effects of KB-R7943, an inhibitor of the Na+/Ca2+ exchanger, on impaired endothelium-dependent relaxation (EDR) induced by high glucose in rat isolated aorta. Both acetylcholine (ACh)-induced EDR and sodium nitroprusside (SNP)-induced endothelium-independent relaxation (EIR) were measured after aortic rings had been exposed to high glucose in the absence and presence of KB-R7943. Coincubation of aortic rings with high glucose (25 mmol/L) for 24 h resulted in a significant inhibition of EDR, but had no effect on EIR. After incubation of aortic rings in the presence of both KB-R7943 (0.1-10 micromol/L) and high glucose for 24 h, significantly attenuation of impaired EDR was observed. This protective effect of KB-R7943 (10 micromol/L) was abolished by superoxide dismutase (SOD; 200 U/mL) and l-arginine (3 mmol/L), whereas d-arginine (3 mmol/L) had no effect. Similarly, high glucose decreased SOD activity and the release of nitric oxide (NO) and increased superoxide anion (O2(-)) production in aortic tissue. KB-R7943 significantly decreased O2(-) production and increased SOD activity and NO release. These results suggest that KB-R7943 can restore impaired EDR induced by high glucose in rat isolated aorta, which may be related to the scavenging of oxygen free radicals and enhanced NO production.     

An increase in opening of BK(Ca) channels in smooth muscle cells in streptozotocin-induced diabetic mice

INTRODUCTION Diabetes mellitus (DM) is a kind of disease thatmetabolism decompensates with hyperglycemia and re-sults in multi-organ damage[1]. Thus, the risk of coro-nary disease, cerebrovascular disease, and other car-diovascular complications increase. These changes, atleastpartially, due todiabetes functionalchanges inbloodvessels including endothelial cell dysfunction. Simulta-neously, altered ion channelfunction in vascular smoothmuscle are also involved[2,3]. In vascular…     

Effects of sulphite supplementation on vascular responsiveness in sulphite oxidase-deficient rats

1. The aim of the present study was to explore the effect of dietary sulphite supplementation on vascular responsiveness in sulphite oxidase (SO)-deficient rats. 2. Male albino rats were divided into four groups, namely control (n = 8), sulphite-treated (n = 8), SO-deficient (n = 8) and sulphite-treated SO-deficient (n = 8) groups. Sulphite oxidase deficiency was induced by administration of a low-molybdenum diet with concurrent addition of 200 p.p.m. tungsten in the form of sodium tungstate in the drinking water for 9 weeks. Sulphite, in the form of sodium metabisulphite (Na(2)O(5)S(2); 25 mg/kg) was given in the drinking water to sulphite-treated and sulphite-treated SO-deficient groups for the last 6 weeks. The vascular responsiveness of isolated aortic rings to acetylcholine (ACh), sodium nitroprusside (SNP) and histamine was investigated in organ baths. 3. The responsiveness of aortic rings to SNP and histamine did not differ significantly between groups. Conversely, there was a significant decrease in ACh-induced relaxation in aortic rings from the sulphite-treated SO-deficient group compared with the control group (pD(2) 6.2 +/- 0.3 and 7.5 +/- 0.1, respectively; P < 0.05). Incubation of aortic rings in the presence of either l-arginine or superoxide dismutase significantly improved the ACh-induced vasorelaxation in sulphite-treated SO-deficient group (pD(2) 7.2 +/- 0.3 and 7.4 +/- 0.3, respectively). 4. The findings of the present study suggest that the increased production of reactive oxygen species and the resultant increment in l-arginine/nitric oxideconsumption may play a role in the reduced endothelium-dependent vasorelaxation in sulphite-treated SO-deficient rats.     

Effects of superoxide dismutase mimetics on the activity of nitric oxide in rat aorta.

A number of structurally distinct superoxide dismutase (SOD) mimetics were examined to determine if they shared the ability of authentic Cu/Zn SOD to produce endothelium-dependent relaxation of rings of rat aorta by protecting basal nitric oxide from destruction by endogenously produced superoxide anion. MnCl2 (10 nM-100 microM), CuSO4 (100 nM-1 mM) and CuDIPS (Cu [II]-[diisopropylsalicylate]2; 100 nM-30 microM) each mimicked the ability of Cu/Zn SOD (0.1-300 u ml(-1)) to produce relaxation of phenylephrine-precontracted aortic rings in a manner inhibited by endothelial removal or treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM). In contrast, MnTMPyP (Mn [III] tetrakis [1-methyl-4-pyridyl] porphyrin; 10 nM-30 microM) augmented phenylephrine-induced contraction and this was blocked by endothelial removal or treatment with L-NAME (100 microM), consistent with destruction rather than protection of basal nitric oxide activity. Pretreatment with Cu/Zn SOD (250 u ml(-1)) blocked this augmentation suggesting that it arose paradoxically through destruction of nitric oxide by superoxide anion. The spin trap agents tiron (100 nM-1 mM), tempol (100 nM-1 mM) and PTIYO (4-phenyl-2,2,5,5-tetramethyl imidazolin-1-yloxy-5-oxide; 100 nM-300 microM) all failed to promote endothelium-dependent relaxation. In fact, the last two augmented phenylephrine-induced tone and this was blocked by endothelial removal or treatment with L-NAME (100 microM), consistent with destruction of basal nitric oxide activity. This destruction was unaffected by pretreatment with Cu/Zn SOD (250 u ml(-1)) and probably reflected the direct ability of tempol and PTIYO to destroy nitric oxide. Thus, the ideal SOD mimetic for protection of nitric oxide activity in conditions of oxidant stress still awaits development.     

Effects of chronic ethanol consumption on alpha-adrenergic-induced contractions and endothelium-dependent relaxations in rat thoracic aorta.

The effects of chronic oral administration of ethanol (7.2% daily during 24 weeks) on the contractions induced by phenylephrine (Phe) and the endothelium-dependent relaxation responses to acetylcholine (ACh) were studied in rat thoracic aorta. Ethanol pretreatment significantly attenuated the contractile responses to Phe, resulting in parallel shift of the concentration-response curve to the right. EC(50)values of Phe were 64.6+/-11.2 and 95.5+/-8.5 nmol l(-1)in control and ethanol-fed rats, respectively. On the other hand, either calcium-induced contractions or relaxation responses to ACh and sodium nitroprusside were similar in the vessels of the control and ethanol-treated rats. These results suggest that chronic ethanol ingestion significantly attenuates the alpha(1)-adrenergic-induced contractions but does not affect the relaxation responses mediated by nitric oxide in rat aortic rings.     

Impairment of endothelium-dependent relaxation of rat aortas by homocysteine thiolactone and attenuation by captopril

To explore the effects of angiotensin-converting enzyme (ACE) inhibitors on endothelial dysfunction induced by homocysteine thiolactone (HTL). Both endothelium-dependent relaxation and nondependent relaxation of thoracic aortic rings in rats induced by acetylcholine (Ach) or sodium nitroprusside (SNP) and biochemical parameters including malondialdehyde (MDA) and nitric oxide (NO) were measured in rat isolated aorta. Exposure of aortic rings to HTL (3 to 30 mM) for 90 minutes made a significant inhibition of endothelium-dependent relaxation induced by Ach, decreased contents of NO, and increased MDA concentration in aortic tissue. After incubation of aortic rings with captopril (0.003 to 0.03 mM) attenuated the inhibition of endothelium-dependent relaxation (EDR) and significantly resisted the decrease of NO content and elevation of MDA concentration caused by HTL (30 mmol/L) in aortic tissues, a similarly protective effect was observed when the aortic rings were incubated with both N-acetylcysteine (0.05 mM). Treatment with enalaprilat (0.003 to 0.01 mM) made no significant difference with the HTL (30 mM) group regarding EDR, but enalaprilat (0.03 mM) and losartan (0.03 mM) could partly restore the EDR in response to HTL (30 mM). Captopril was more effective than enalaprilat and losartan in attenuation of the inhibition of on acetylcholine-stimulated aortic relaxation by HTL in the same concentration. Moreover, superoxide dismutase (SOD, 200 U/mL), which is a scavenger of superoxide anions, apocynin (0.03 mM), which is an inhibitor of NADPH oxidase, and l-Arginine (3 mmol/L), a precursor of nitric oxide (NO), could reduce HTL (30 mM)-induced inhibition of EDR. After pretreatment with not only the NO synthase inhibitor Nomega-nitro-l-arginine methyl ester (L-NAME, 0.01 mM) but also the free sulfhydryl group blocking agent p-hydroxymercurybenzoate (PHMB, 0.05 mM) could abolish the protection of captopril and N-acetylcysteine, respectively. These results suggest that mechanisms of endothelial dysfunction induced by HTL may include the decrease of NO and the generation of oxygen free radicals and that captopril can restore the inhibition of EDR induced by HTL in isolated rat aorta, which may be related to scavenging oxygen free radicals and may be sulfhydryl-dependent.     

Regulation of nitric oxide synthesis by dimethylarginine dimethylaminohydrolase.

1. Dimethylarginine dimethylaminohydrolase (DDAH), an enzyme that metabolizes the endogenous nitric oxide synthase inhibitors NG-monomethyl-arginine and NG,NG-dimethy-L-arginine to citrulline, was identified by Western blotting in rat and human tissue homogenates. 2. S-2-amino-4(3-methylguanidino)butanoic acid (4124W) inhibited the metabolism of [14C]-NG-monomethyl-L-arginine to [14C]-citrulline by rat liver homogenates (IC50 416 +/- 66 microM; n = 9), human cultured endothelial cells (IC50 250 +/- 34 microM; n = 9) and isolated purified dimethylarginine dimethylaminohydrolase. 3. Addition of 4124W to culture medium increased the accumulation of endogenously-generated NG,NG-dimethy-L-arginine in the supernatant of human cultured endothelial cells from 3.1 +/- 0.3 to 5 +/- 0.7 microM (n = 15; P < 0.005). 4. 4124W (1 microM - 1 mM) had no direct effect on endothelial nitric oxide synthase activity but caused endothelium-dependent contraction of rat aortic rings (1 mM 4124W increased tone by 81.5 +/- 9.6% of that caused by phenylephrine 100 nM). This effect was reversed by L-arginine (100 microM). 4124W reversed endothelium-dependent relaxation of human saphenous vein (19.2 +/- 6.7% reversal of bradykinin-induced relaxation at 1 mM 4124W). 5. These data suggest that inhibition of dimethylarginine dimethylaminohydrolase increases the intracellular contraction of NG,NG-dimethyl-L-arginine sufficiently to inhibit nitric oxide synthesis. Inhibiting the activity of DDAH may provide an alternative mechanism for inhibition of nitric oxide synthases and changes in the activity of DDAH could contribute to pathophysiological alterations in NO generation.     

Poly(ADP-ribose) polymerase inhibition improves endothelial dysfunction induced by reactive oxidant hydrogen peroxide in vitro

Reactive oxygen species, such as hydrogen peroxide (H(2)O(2)) induce oxidative stress and DNA-injury. The subsequent activation of poly(ADP-ribose) polymerase (PARP) has been implicated in the pathogenesis of various cardiovascular diseases including ischaemia-reperfusion injury, circulatory shock, diabetic complications and atherosclerosis. We investigated the effect of PARP-inhibition on endothelial dysfunction induced by H(2)O(2). In vascular reactivity measurements on isolated rat aortic rings we investigated the phenylephrine-induced contraction, and endothelium-dependent and -independent vasorelaxation by using cumulative concentrations of acetylcholine and sodium nitroprusside. Endothelial dysfunction was induced by exposing the rings to H(2)O(2) (200 and 400 muM) for 30 min. In the treatment group, rings were preincubated with the potent PARP-inhibitor INO-1001. DNA strand breaks were assessed by the terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling (TUNEL) method. Immunohistochemical analysis was performed for poly(ADP-ribose) (the enzymatic product of PARP) and for apoptosis inducing factor (a pro-apoptotic factor regulated by PARP). Exposure to H(2)O(2) resulted in reduced contraction forces and a dose-dependent impairment of endothelium-dependent vasorelaxation of aortic rings (maximal relaxation to acetylcholine: 86.21+/-1.574% control vs. 72.55+/-1.984% H(2)O(2) 200 muM vs. 66.86+/-1.961% H(2)O(2) 400 muM; P<0.05). PARP-inhibition significantly improved the acetylcholine-induced vasorelaxation (77.75+/-3.019% vs. 66.86+/-1.961%; P<0.05), while the contractility remained unaffected. The dose-response curves of endothelium-independent vasorelaxation to sodium nitroprusside did not differ in any groups studied. In the H(2)O(2) groups immunohistochemical analysis showed enhanced PARP-activation and nuclear translocation of apoptosis inducing factor, which were prevented by INO-1001. Our results demonstrate that PARP activation contributes to the pathogenesis of H(2)O(2)-induced endothelial dysfunction, which can be prevented by PARP inhibitors.     

Preservation of endothelium-dependent relaxation in cholesterol-fed and streptozotocin-induced diabetic mice by the chronic administration of cholestyramine.

1. Experiments were designed to investigate the effects of the low density lipoprotein (LDL)-lowering drugs cholestyramine on serum LDL levels and endothelium-dependent relaxation to acetylcholine (ACh) in cholesterol-fed or streptozotocin (STZ)-induced diabetic mice. 2. In aortic rings from control mice, ACh or A23187 caused concentration-dependent relaxation. The relaxations caused by ACh or A23187 were significantly attenuated in aortic rings from cholesterol-fed and STZ-diabetic mice. The attenuated vasodilatation in both cholesterol-fed and diabetic mice was returned to normal by chronic administration of cholestyramine. The endothelium-independent relaxations of aortic rings induced by sodium nitroprusside (SNP) were not significantly different between control, cholesterol-fed and STZ-induced diabetic mice. 3. The increased LDL levels in cholesterol-fed and diabetic mice were returned to normal by the chronic administration of cholestyramine. Chronic administration of cholestyramine had no effects on serum glucose levels. 4. These results suggest that attenuated endothelium-dependent vasodilatations in both cholesterol-fed and STZ-diabetic mice are improved by the chronic administration of cholestyramine, and these effects are, at least in part, due to lowering serum LDL levels.     

Indomethacin potentiates acetylcholine-induced vasodilation by increasing free radical production

We studied the effects of indomethacin on endothelium-dependent and -independent vascular relaxation in rat thoracic aortic rings and its role in superoxide anion (O(2)(-)) production. We measured isometric force changes in response to acetylcholine (Ach, 1 nM-0.1 mM), sodium nitroprusside (SNP, 0.1 nM-0.1 microM; a nitric oxide (NO) donor) and cromakalim (1 nM-0.1 mM; a K(ATP)-channel opener) in aorta rings contracted with norepinephrine (NE, 0.1 microM). Indomethacin (10 microM; 20 min) significantly increased Ach-induced vasodilation (EC(50) decreased from 8.99 microM to 16 nM). The free radical scavengers superoxide dismutase and 4-hydroxy-2,2,6,6-tetramethylpiperidine-N-oxyl completely reverted these effects. Indomethacin did not affect SNP- or cromakalim-induced vasodilation. Neither acetylsalicylic acid (ASA, 5-100 microM; 15 min) nor ketoprofen (1-100 microM; 15 min) affected Ach, SNP and cromakalim concentration-response curves. Incubation of the aorta with Ach (1 microM) rapidly and markedly increased intracellular NO fluorescence in the aorta endothelium. Indomethacin did not affect Ach-induced NO production. We measured intracellular O(2)(-) in the aorta endothelium with dihydroethidium (DHE) dye. Indomethacin significantly increased O(2)(-) fluorescence versus controls. Neither ASA nor ketoprofen affected O(2)(-) fluorescence. Nitrotyrosine staining was increased in indomethacin-treated aorta sections exposed to Ach, which indicates endogenous formation of peroxynitrite. It was low in aorta sections exposed to Ach alone or with ASA or ketoprofen. We cannot judge if indomethacin-induced endothelium-dependent vasodilation damages or protects the cardiovascular system. Here, we show that indomethacin acts on the cardiovascular system regardless of cyclooxygenase inhibition.     

Acute effect of sidestream cigarette smoke extract on vascular endothelial function

Acute exposure to passive smoking adversely affects vascular function by promoting oxidative stress and endothelial dysfunction. However, it is not known whether tobacco sidestream (SS) smoke has a greater deleterious effect on the endothelium than non-tobacco SS smoke and whether these effects are related to nicotinic endothelial stimulation. To test these hypotheses, endothelial-dependent relaxation and superoxide anion production were assessed in isolated rat aortas incubated with tobacco SS smoke, non-tobacco SS smoke, or pure nicotine. Tobacco SS smoke decreased the maximal relaxation to acetylcholine (Ach) from 79 +/- 6% to 57 +/- 7.3% (% inhibition of phenylephrine-induced plateau, P < 0.001) and increased superoxide anion production from 31 +/- 9.7 to 116 +/- 24 count/10 sec/mg (P < 0.01, lucigenin-enhanced chemiluminescence technique). The non-tobacco SS smoke extract had no significant effect on the response to Ach but increased superoxide anion production in the aortic wall to 133 +/- 2 count/10 sec/mg (P < 0.001). Furthermore, concentration-response curves to Ach and superoxide production remained unaltered with nicotine (0.001, 0.01, or 0.1 mM). In conclusion, despite similar increases in vascular wall superoxide production with tobacco and non-tobacco SS smoke, only the tobacco SS smoke extracts affected endothelium-dependent vasorelaxation. Nicotine alone does not reproduce the effects seen with tobacco SS smoke, suggesting that the acute endothelial toxicity of passive smoking cannot simply be ascribed to a nicotine-dependent mechanism.     

Captopril restores endothelium-dependent relaxation of rat aortic rings after exposure to homocysteine

This study was designed to investigate the effects of captopril, an angiotensin-converting enzyme inhibitor, on inhibition of endothelium-dependent relaxation induced by homocysteine in isolated rat aorta. Isometric tension recordings were used to assess inhibitory effects of homocysteine and protective effects of captopril on endothelium-dependent relaxation of aortic rings. Exposure of aortic rings to homocysteine (0.3 approximately 3 mmol/L) for 30 min induced a significant concentration-dependent inhibition of endothelium-dependent relaxation response to acetylcholine (ACh), but did not affect endothelium-independent relaxation response to sodium nitroprusside. Pre-incubation of aortic rings with captopril (3 approximately 30 micromol/L) for 15 min and co-incubation of aortic rings with homocysteine (1 mmol/L) for another 30 min attenuated the inhibition of homocysteine in a dose-dependent manner. Moreover, superoxide dismutase (SOD, 200 U/mL), a scavenger of superoxide anions, reduced homocysteine-induced inhibition. L-Arginine (3 mmol/L), a precursor of nitric oxide (NO), also attenuated the impairment of vasorelaxation induced by homocysteine. However, in the combined presence of SOD and L-arginine, the inhibitory effect of homocysteine was reversed, which was very similar to the effect of 30 micromol/L captopril. These results suggest that captopril can prevent the inhibition of endothelium-dependent relaxation induced by homocysteine in isolated rat aorta, which may be related to scavenging oxygen free radicals and enhancing NO production.