Effect of melatonin on vascular reactivity in pancreatectomized rats

The present study was undertaken to assess whether the improvement of contractile performance of aortic rings by melatonin described in streptozotocin diabetic rats also occurs in another model of type I diabetes, the pancreatectomized rats. Adult male Wistar rats submitted to a subtotal pancreatectomy and exhibiting altered levels of fasting glucose and an abnormal tolerance glucose test, were used. Sham-operated laparotomized rats were employed as controls. Dose-response curves for acetylcholine-induced, endothelium-related relaxation of aortic rings (after previous exposure to phenylephrine) and for phenylephrine-induced vasoconstriction were conducted. This protocol was repeated with rings pre-incubated in a high glucose solution (44 mmol/l). Pancreatectomy decreased significantly acetylcholine-induced relaxation of aortic rings, but not phenylephrine-induced vasoconstriction, the effect being amplified by preincubation in high glucose solution. The deleterious effect of a high glucose medium was more pronounced in pancreatectomized rats. Melatonin (10(-5) M) did not modify acetylcholine-induced relaxation in normal glucose concentration but was effective to prevent the impairment of relaxation brought about by exposure to high glucose solution. The contractile response to phenylephrine of aortic rings obtained from pancreatectomized rats was not affected by melatonin. The results further support the improvement by melatonin of endothelial-mediated relaxation in blood vessels of diabetic rats.     

Comparative effects of melatonin and vitamin E in restoring aortic relaxation in pancreatectomized rats

In a previous study we reported the efficacy of melatonin to restore the decreased relaxation response to acetylcholine (ACh) or to sodium nitroprusside (SNP) in aortic rings of rats turned hyperglycemic by subtotal pancreatectomy. The effect was amplified by pre-incubation in a high (44 mmol/l) glucose solution, a situation that resulted in oxidative stress. We hereby compare the effect of another antioxidant, vitamin E, with that of melatonin on ACh response in intact aortic rings or on SNP response in endothelium-denuded aortic rings obtained from pancreatectomized or sham-operated rats. Dose-response curves to ACh or SNP were performed in the presence or absence of melatonin or vitamin E (10-5 mol/1) in 10 or 44 mmol/1 glucose medium. Melatonin was more effective than vitamin E in restoring ACh- or SNP-induced relaxation of aortic rings in a high glucose medium. The differences between the two antioxidants may rely on the ability of melatonin to diffuse readily into intracellular compartments.     

Menadione induces endothelial dysfunction mediated by oxidative stress and arylation

Our previous studies showed that menadione causes endothelial dysfunction which results in decreased relaxation and increased contraction of blood vessels. This investigation examined the role of two possible mechanisms (oxidative stress and arylation) in menadione-induced endothelial dysfunction. Menadione increased superoxide anion generation in aortic rings in a dose-dependent manner. Superoxide dismutase (SOD), reversed the inhibitory effects of menadione on vascular relaxation. The relaxation induced by the NO donor, sodium nitroprusside, was inhibited by menadione pretreatment in a dose-dependent manner. Endothelial nitric oxide synthase activity (eNOS) was suppressed by menadione. Menadione resulted in a dose-dependent reduction of cGMP levels accumulated by acetylcholine. This reduction of cGMP levels was blocked by SOD treatment, suggesting that superoxide anion generated by menadione could play a role in the inhibition of the nitric oxide pathway. Evidence supporting a possible role for arylation in impaired vascular relaxation was suggested by the observation that benzoquinone, which does not induce oxidative stress in aortic rings, inhibited acetylcholine-induced vascular relaxation to the same extent as menadione. Collectively, these results suggest that menadione can cause endothelial dysfunction in blood vessels by the inhibition of the nitric oxide pathway via superoxide anion generation and that arylation activity may also be another important mechanism.     

Oleoyl-Lysophosphatidylcholine Limits Endothelial Nitric Oxide Bioavailability by Induction of Reactive Oxygen Species

Previously we reported modulation of endothelial prostacyclin and interleukin-8 production, cyclooxygenase-2 expression and vasorelaxation by oleoyl- lysophosphatidylcholine (LPC 18:1). In the present study, we examined the impact of this LPC on nitric oxide (NO) bioavailability in vascular endothelial EA.hy926 cells. Basal NO formation in these cells was decreased by LPC 18:1. This was accompanied with a partial disruption of the active endothelial nitric oxide synthase (eNOS)- dimer, leading to eNOS uncoupling and increased formation of reactive oxygen species (ROS). The LPC 18:1-induced ROS formation was attenuated by the superoxide scavenger Tiron, as well as by the pharmacological inhibitors of eNOS, NADPH oxidases, flavin-containing enzymes and superoxide dismutase (SOD). Intracellular ROS-formation was most prominent in mitochondria, less pronounced in cytosol and undetectable in endoplasmic reticulum. Importantly, Tiron completely prevented the LPC 18:1-induced decrease in NO bioavailability in EA.hy926 cells. The importance of the discovered findings for more in vivo like situations was analyzed by organ bath experiments in mouse aortic rings. LPC 18:1 attenuated the acetylcholine-induced, endothelium dependent vasorelaxation and massively decreased NO bioavailability. We conclude that LPC 18:1 induces eNOS uncoupling and unspecific superoxide production. This results in NO scavenging by ROS, a limited endothelial NO bioavailability and impaired vascular function.     

Inhibition of acetylcholine-induced EDHF response by elevated glucose in rat mesenteric artery

The effects of high glucose on endothelium-derived hyperpolarizing factor (EDHF)-mediated relaxations of isolated rat mesenteric artery and the possible involvement of reactive oxygen species in these responses were investigated. After precontraction with phenylephrine (3 x 10(-8)-10(-7) M), acetylcholine (10(-8)-3 x 10(-6) M) and A 23187 (10(-8)-3 x 10(-6) M), a calcium ionophore, induced concentration-dependent relaxations in the presence of N(W)-nitro-l-arginine methyl ester (L-NAME) (10(-4) M) and indomethacin (10(-5) M). These relaxations were abolished in the presence of charybdotoxin (2 x 10(-7) M) plus apamin (10(-7) M) and were assumed to be mediated by EDHF. Effects of elevated glucose were examined by incubating the arterial rings for 6 h in Krebs-Henseleit solution containing 22.2 mM glucose. Under these conditions relaxation to acetylcholine was significantly attenuated but was unchanged when the tissues were incubated for 6 h in solution containing 11.1 mM mannitol used as hyperosmotic control. Addition of superoxide dismutase (SOD) (75 U/ml) and combination of SOD with catalase (200 U/ml) during incubation with high glucose significantly preserved the impairment of EDHF-mediated relaxations to acetylcholine. A 23187-induced endothelium-dependent relaxation was not affected by high glucose. Similarly, relaxations to pinacidil (10(-10)-10(-5) M) and to sodium nitroprusside (SNP) (10(-10)-3 x 10(-7) M) were also unchanged in the rings exposed to high glucose. These results suggest that in rat mesenteric arteries exposed to elevated glucose receptor-dependent EDHF-mediated relaxations (acetylcholine-induced) are impaired whereas receptor-independent ones (A 23187-induced) and responses to smooth muscle relaxants that exert their effects through mechanisms independent of endothelium are unaffected. Our findings lead us to propose that reactive oxygen species like superoxide ((.)O(2)(-)) and hydrogen peroxide (H(2)O(2)) do seem to play a role in the impairment of EDHF-mediated relaxations in the presence of elevated glucose.     

Coronary reperfusion in dogs inhibits endothelium-dependent relaxation: role of superoxide radicals

Previous studies indicate that release of superoxide radicals during coronary reperfusion following occlusion may relate to the loss of endothelium-dependent coronary arterial relaxation. We examined coronary arterial ring relaxation in dogs subjected to temporary circumflex (Cx) coronary artery occlusion and treated with saline or the superoxide radical scavenger superoxide dismutase (SOD). In dogs treated with saline, Cx coronary ring relaxation in response to leukotriene D4 (LTD4) and acetylcholine (ACh) was attenuated (p less than 0.01), but coronary relaxation in response to nitroglycerin was preserved, suggesting loss of endothelium-dependent relaxation following coronary reperfusion. In contrast, Cx coronary relaxation in response to LTD4 and ACh was preserved in the SOD-treated dogs (p less than 0.01 compared to saline-treated dogs). To further examine the role of superoxide radicals in the loss of endothelium-dependent relaxation, normal nonischemic canine coronary artery and rat aortic rings were exposed to a superoxide radical generating system of xanthine and xanthine oxidase in vitro. Xanthine plus xanthine oxidase treatment caused a significant (p less than 0.01) decrease in the relaxant effects of ACh. Pretreatment of rat aortic rings with SOD protected against the loss of ACh-induced relaxation. These observations suggest that release of superoxide radicals during reperfusion is the basis of loss of endothelium-dependent coronary arterial relaxation. Treatment with superoxide radical scavengers prior to coronary reperfusion protects against this loss.     

Increased superoxide dismutase and Na+, K+-ATPase activities in aortic strips from potassium-adapted rats: implication for altered vascular reactivity

The contributions of superoxide dismutase (SOD) and Na(+), K(+)-ATPase to the altered vascular reactivity in potassium-adapted rats were investigated to test the hypothesis that smooth muscle hyperpolarisation may be involved. Isometric contractions to noradrenaline (NA), 5-hydroxytryptamine (5-HT), and relaxations to acetylcholine (ACh), levcromakalim (LEV) and sodium nitroprusside (SNP), were measured in aortic rings from potassium-adapted rats. Pieces of the aortae were also excised from the animals and assayed for SOD and Na(+), K(+)-ATPase. Maximum contractile responses were significantly attenuated (P<0.05) in aortic rings from the potassium-adapted rats to NA and 5-HT, while relaxations were also significantly augmented (P<0.05) in the same rings to LEV and SNP, but not to ACh. Both SOD and Na(+), K(+)-ATPase activities were significantly higher (P<0.05) in the aortae from the potassium-adapted rats compared to controls. It is concluded that the alteration in vascular smooth muscle reactivity may be due to hyperpolarisation caused by the activities of SOD and Na(+), K(+)-ATPase.     

Effects of 17ß-estradiol on endothelium-dependent relaxation induced by acetylcholine in female rat aorta

Estrogens have been postulated to play an important role in modulation of vascular responses to endogenous reactive substances. The effects of chronic in vivo treatment with 17ß-estradiol on relaxant responses to acetylcholine were investigated in the rat aorta isolated from prepubertal female rats. The selectivity of effects of 17ß-estradiol on acetylcholine-induced relaxation was evaluated using histamine, another endothelium-dependent relaxant in the rat aorta. 17ß-Estradiol significantly enhanced endothelium-dependent relaxation induced by acetylcholine, but did not alter the vascular responses to acetylcholine in endothelium-denuded aortic rings isolated from prepubertal female rats. In contrast, 17ß-estradiol did not change endothelium-dependent relaxation induced by histamine in endothelium-intact aortic rings. The results of the present study demostrate that 17ß-estradiol selectively enhanced acetylcholine-induced endothelium-dependent relaxation in the rat aorta.     

Oxidant stress from uncoupled nitric oxide synthase impairs vasodilation in fetal lambs with persistent pulmonary hypertension

Persistent pulmonary hypertension of newborn (PPHN) is associated with decreased NO release and impaired pulmonary vasodilation. We investigated the hypothesis that increased superoxide (O(2)(*-)) release by an uncoupled endothelial nitric oxide synthase (eNOS) contributes to impaired pulmonary vasodilation in PPHN. We investigated the response of isolated pulmonary arteries to the NOS agonist ATP and the NO donor S-nitroso-N-acetylpenicillamine (SNAP) in fetal lambs with PPHN induced by prenatal ligation of ductus arteriosus and in sham-ligated controls in the presence or absence of the NOS antagonist nitro-L-arginine methyl ester (L-NAME) or the O(2)(*-) scavenger 4,5-dihydroxy-1,3-benzenedisulfonate (Tiron). ATP caused dose-dependent relaxation of pulmonary artery rings in control lambs but induced constriction of the rings in PPHN lambs. L-NAME, the NO precursor L-arginine, and Tiron restored the relaxation response of pulmonary artery rings to ATP in PPHN. Relaxation to NO was attenuated in arteries from PPHN lambs, and the response was improved by L-NAME and by Tiron. We also investigated the alteration in heat shock protein (HSP)90-eNOS interactions and release of NO and O(2)(*-) in response to ATP in the pulmonary artery endothelial cells (PAEC) from these lambs. Cultured PAEC and endothelium of freshly isolated pulmonary arteries from PPHN lambs released O(2)(*-) in response to ATP, and this was attenuated by the NOS antagonist L-NAME and superoxide dismutase (SOD). ATP stimulated HSP90-eNOS interactions in PAEC from control but not PPHN lambs. HSP90 immunoprecipitated from PPHN pulmonary arteries had increased nitrotyrosine signal. Oxidant stress from uncoupled eNOS contributes to impaired pulmonary vasodilation in PPHN induced by ductal ligation in fetal lambs.     

Taurine Supplementation Reduces Blood Pressure and Prevents Endothelial Dysfunction and Oxidative Stress in Post-Weaning Protein-Restricted Rats

Introduction

Taurine is a sulfur-containing amino acid that exerts protective effects on vascular function and structure in several models of cardiovascular diseases through its antioxidant and anti-inflammatory properties. Early protein malnutrition reprograms the cardiovascular system and is linked to hypertension in adulthood. This study assessed the effects of taurine supplementation in vascular alterations induced by protein restriction in post-weaning rats.

Methods and Results

Weaned male Wistar rats were fed normal- (12%, NP) or low-protein (6%, LP) diets for 90 days. Half of the NP and LP rats concomitantly received 2.5% taurine supplementation in the drinking water (NPT and LPT, respectively). LP rats showed elevated systolic, diastolic and mean arterial blood pressure versus NP rats; taurine supplementation partially prevented this increase. There was a reduced relaxation response to acetylcholine in isolated thoracic aortic rings from the LP group that was reversed by superoxide dismutase (SOD) or apocynin incubation. Protein expression of p47^phox NADPH oxidase subunit was enhanced, whereas extracellular (EC)-SOD and endothelial nitric oxide synthase phosphorylation at Ser 1177 (p-eNOS) were reduced in aortas from LP rats. Furthermore, ROS production was enhanced while acetylcholine-induced NO release was reduced in aortas from the LP group. Taurine supplementation improved the relaxation response to acetylcholine and eNOS-derived NO production, increased EC-SOD and p-eNOS protein expression, as well as reduced ROS generation and p47^phox expression in the aortas from LPT rats. LP rats showed an increased aortic wall/lumen ratio and taurine prevented this remodeling through a reduction in wall media thickness.

Conclusion

Our data indicate a protective role of taurine supplementation on the high blood pressure, endothelial dysfunction and vascular remodeling induced by post-weaning protein restriction. The beneficial vascular effect of taurine was associated with restoration of vascular redox homeostasis and improvement of NO bioavailability.     

Vascular protective actions of a nitric oxide aspirin analog in both in vitro and in vivo models of diabetes mellitus

BACKGROUND: Defective endothelium-dependent relaxation is observed in experimental and human diabetes mellitus. The nature of this defect is not fully understood but may involve decreased nitric oxide (NO) bioactivity due to enhanced production of reactive oxygen species (ROS). In this paper, we examine the benefits and actions of a novel NO-donating, antioxidant called 2-acetoxybenzoic acid 2-(2-nitrooxymethyl) phenyl ester, and denoted as NCX4016, on NO-mediated endothelium-dependent relaxation in normal arteries exposed to acute elevations in glucose or in arteries derived from chronic diabetic animals. MATERIAL AND METHODS: Intrinsic free radical scavenging by NO-NSAIDs in solution were evaluated using electron paramagnetic resonance (EPR) spectroscopy and spin trapping with 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). In acute studies, normal rat aortas were exposed in tissue culture for 18 h to 5.5 mM or 40 mM in the presence or absence of NCX4016, a NO-donating NSAID unrelated to aspirin (NCX2216) or aspirin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined. For chronic hyperglycemia, diabetes was induced in rats by intravenous injection with streptozotocin. Vascular reactivity of thoracic aortic rings to endothelium-dependent relaxation to acetylcholine in vitro was determined after 8 wks in untreated animals or animals chronically-treated with NCX4016. Antioxidant efficacy in vivo was determined by measurement of plasma isoprostanes and by nuclear binding activity of NF-kappaB in nuclear fractions of aortae. RESULTS: Incubation with NCX4016 and NCX2216 produced a concentration-dependent inhibition of DMPO-OH formation indicating scavenging of hydroxyl radicals (HO(*)). In contrast, little efficacy to scavenge superoxide anion radicals was noted. Acute incubation of normal arteries with elevated glucose concentration caused inhibition of normal relaxation to acetylcholine. This impairment was prevented by co-incubation with NCX4106 but not by mannitol, the parent compound (aspirin) or by NCX2216. In addition, chronic treatment with NCX4016 prevented the development of defective endothelium-dependent relaxation to acetylcholine. This protection did not occur as a result to any changes in blood glucose concentration or hemoglobin glycation. Treatment with NCX4016 did decrease the elevation in plasma isoprostanes and normalized the diabetes-induced increase in NF-kappaB binding activity in nuclear fractions derived from aortic tissue. CONCLUSIONS: Collectively, these studies suggest that antioxidant interventions using NO-donating NSAIDs may provide an important novel therapeutic strategy to protect the diabetic endothelium.     

Dietary soy modulates endothelium-dependent relaxation in aged male rats: Increased agonist-induced endothelium-derived hyperpolarising factor and basal nitric oxide activity

We examined the effects of dietary soy on the contributions of endothelium-derived hyperpolarising factor (EDHF), nitric oxide (NO), and oxidative stress to vascular tone in isolated aortic rings and small mesenteric and pulmonary arteries in vitro. Male Wistar rats were either continuously fed a soy-deficient diet (SD) or switched from a soy-deficient diet to a soy-rich one for 6 months (SW). Contractile responses were generally smaller in arteries from SW rats. In mesenteric arteries, this difference was blunted by L-NAME, but not by charybdotoxin and apamin. Preconstricted SW mesenteric arteries were more sensitive to acetylcholine (ACh) than SD ones. This difference was unaffected by L-NAME but was abolished by charybdotoxin and apamin. Exogenous superoxide dismutase (SOD) and catalase induced powerful relaxations in aortic rings, which were smaller in those from SW rats. In mesenteric and pulmonary arteries, however, they partially inhibited ACh-mediated relaxation, and enhanced PGF(2alpha)-mediated contraction, respectively. Our results suggest that feeding aging male rats a soy-rich diet results in improved agonist-mediated EDHF production and a generalized reduction in contractile force, which is partly due to elevated basal NO. Our data also suggest a prorelaxant role for endogenous H(2)O(2) in small arteries, which is modulated by a soy diet.     

Propolis protects against high glucose-induced vascular endothelial dysfunction in isolated rat aorta

While propolis is known to have abundant bioactive constituents and a variety of biological activities, it is not clear whether propolis has beneficial effects on high glucose-mediated vascular endothelial impairment. The aim of the present study was to investigate the potential protective effect of propolis extract against the acute vascular endothelial dysfunction resulting from exposure to high glucose load and to elucidate its underlying mechanism. Rat aortic rings were incubated with normal glucose (11 mM), high glucose (44 mM), or mannitol (44 mM) for 3 h with or without propolis extract (400 μg/ml). Contraction to phenylephrine (Phe, 10^?9–10^?5 M) and relaxation to acetylcholine (ACh, 10^?9–10^?5 M) and sodium nitroprusside (SNP, 10^?9–10^?5 M) were measured before and after incubation. Changes in malondialdehyde (MDA), reduced glutathione (GSH), and superoxide dismutase (SOD) were also measured. Phe-induced contraction was impaired by high glucose as the E _max decreased from 138.87?±?11.43 to 103.65?±?11.5 %. In addition, ACh-induced relaxation was impaired as the E _max decreased from 99.80?±?7.25 to 39.20?±?6.5 %. SNP-induced relaxation was not affected. Furthermore, high glucose decreased the levels of both SOD (by 6 U/ml) and GSH (by 68 %) and increased levels of MDA (by 85 %). Propolis extract prevented high glucose-induced impairment of Phe and ACh responses and increased both SOD and GSH, leading to decreased MDA levels. In conclusion, propolis can protect against high glucose-induced vascular dysfunction by reducing oxidative stress.     

Increased activity of H2O2 in aorta isolated from chronically streptozotocin-diabetic rats: effects of antioxidant enzymes and enzymes inhibitors.

The effects of hydrogen peroxide (H2O2, 1 nM-5 mM) on the tone of the rings of aorta precontracted with phenylephrine (PE) were studied in 4-5 months streptozotocin (STZ)-diabetic rats and their age-matched controls. H2O2 induced brief contraction before relaxation in endothelium-containing rings that was more pronounced in diabetic rats. Removal of the endothelium or pretreatment of rings with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM) abolished H2O2-induced immediate and transient increase in tone, but preincubation with indomethacin (10 microM) had no effect on contractions induced by H2O2 in both group of animals. Pretreatment with L-NAME or indomethacin as well as absence of endothelium produced an inhibition of H2O2-induced relaxation that was more pronounced in diabetic rings. Chronically STZ-diabetes resulted in a significant increase in H2O2-induced maximum relaxation that was largely endothelium-dependent. Decreased sensitivity (pD2) of diabetic vessels to vasorelaxant action of H2O2 was normalized by superoxide dismutase (SOD, 80 U/ml). Pretreatment with SOD had no effect on H2O2-induced maximum relaxations in both group of animals but led to an increase in H2O2-induced contractions in control rats. When the rings pretreated with diethyldithiocarbamate (DETCA, 5 mM), H2O2 produced only contraction in control rats, and H2O2-induced relaxations were markedly depressed in diabetic rats. H2O2 did not affect the tone of intact or endothelium-denuded rings in the presence of catalase (2000 U/ml). Aminotriazole (AT, 10 mM) failed to affect H2O2-induced contractions or relaxations in all rings. Our observations suggest that increased production of oxygen-derived free radicals (OFRs) in diabetic state leads to a decrease in SOD activity resulting an increase in endogenous superoxide anions (O2*-), that is limited cytotoxic actions, and an increase in catalase activity resulting a decrease in both H2O2 concentrations and the production of harmful hydroxyl radical (*OH) in diabetic aorta in long-term. Present results indicate that increased vascular activity of H2O2 may be an important factor in the development of vascular disorders associated with chronically diabetes mellitus. Enhanced formation of *OH, that is a product of exogenous H2O2 and excess O2*, seems to be contribute to increased relaxations to exogenously added H2O2 in chronically diabetic vessels.     

Quercetin, a flavonoid antioxidant, modulates endothelium-derived nitric oxide bioavailability in diabetic rat aortas

The present work examined the effect of chronic oral administration of quercetin, a flavonoid antioxidant, on blood glucose, vascular function and oxidative stress in STZ-induced diabetic rats. Male Wistar-Kyoto (WKY) rats were randomized into euglycemic, untreated diabetic, vehicle (1% w/v methylcellulose)-treated diabetic, which served as control, or quercetin (10mgkg(-1) body weight)-treated diabetic groups and treated orally for 6 weeks. Quercetin treatment reduced blood glucose level in diabetic rats. Impaired relaxations to endothelium-dependent vasodilator acetylcholine (ACh) and enhanced vasoconstriction responses to alpha(1)-adrenoceptor agonist phenylephrine (PE) in diabetic rat aortic rings were restored to euglycemic levels by quercetin treatment. Pretreatment with N(omega)-nitro-l-arginine methyl ester (l-NAME, 10microM) or methylene blue (10microM) completely blocked but indomethacin (10microM) did not affect relaxations to ACh in aortic rings from vehicle- or quercetin-treated diabetic rats. PE-induced vasoconstriction with an essentially similar magnitude in vehicle- or quercetin-treated diabetic rat aortic rings pretreated with l-NAME (10microM) plus indomethacin (10microM). Quercetin treatment reduced plasma malonaldehyde (MDA) plus 4-hydroxyalkenals (4-HNE) content as well as increased superoxide dismutase activity and total antioxidant capacity in diabetic rats. From the present study, it can be concluded that quercetin administration to diabetic rats restores vascular function, probably through enhancement in the bioavailability of endothelium-derived nitric oxide coupled to reduced blood glucose level and oxidative stress.     

Garlic extract attenuates time-dependent changes in the reactivity of isolated aorta in streptozotocin-diabetic rats

In the present study, the effect of aqueous garlic extract (Allium sativum; 100 mg/Kg/day; i.p.) on the vascular reactivity of streptozotocin (STZ)-diabetic rats was investigated. Vascular reactivity of isolated aortic rings was assessed by measuring phenylephrine-induced contractile and acetylcholine-induced relaxation responses. The results showed that aortas prepared from 8-week, but not 4-week, STZ-diabetic rats exhibited significantly increased contractile responses, which were partially attenuated by garlic extract treatment. The maximum vasorelaxant response of pre-contracted aortic rings exposed to acetylcholine also significantly decreased in 8-week STZ-diabetic animals which was attenuated to some extent by extract treatment. It is concluded that long-term administration of garlic extract (i.p.) can attenuate various functional alterations induced by STZ-diabetes in the vascular system of an insulin-dependent model of uncontrolled diabetes, and this may suggest a potential approach to future therapeutic strategies.     

Gene transfer of extracellular superoxide dismutase improves relaxation of aorta after treatment with endotoxin

Lipopolysaccharide (LPS) impairs vascular function, in part by generation of reactive oxygen species. One goal of this study was to determine whether gene transfer of extracellular SOD (ECSOD) improves vascular responsiveness in LPS-treated rats. A second goal was to determine whether effects of ECSOD are dependent on the heparin-binding domain of the enzyme, which facilitates binding of ECSOD to the outside of cells. Adenoviruses containing ECSOD (AdECSOD), ECSOD with deletion of its heparin-binding domain (AdECSOD-HBD), or a control virus (AdLacZ) were injected intravenously into rats. Three days later, vehicle or LPS (10 mg/kg ip) was injected. After 24 h, vascular reactivity was examined in aortic rings in vitro. Maximum relaxation to acetylcholine was 95 +/- 1% (means +/- SE) after AdlacZ plus vehicle and 77 +/- 3% after AdlacZ plus LPS (P < 0.05). Responses to calcium ionophore A-23187 and submaximal concentrations of nitroprusside also were impaired by LPS. Gene transfer of ECSOD, but not AdECSOD-HBD, improved (P < 0.05) relaxation to acetylcholine and A-23187 after LPS. Maximum relaxation to acetylcholine was 88 +/- 3% after LPS plus AdECSOD. Superoxide was increased in aorta after LPS, and the levels were reduced after AdECSOD but not AdECSOD-HBD. LPS-induced adhesion of leukocytes to aortic endothelium was reduced by AdECSOD but not by AdECSOD-HBD. We conclude that after gene transfer in vivo, binding of ECSOD to arteries effectively decreases the numbers of adherent leukocytes and levels of superoxide and improves impaired endothelium-dependent relaxation produced by LPS.     

Effect of high-salt diet on NO release and superoxide production in rat aorta

Sprague-Dawley rats were fed either a high-salt (HS) diet (4.0% NaCl) or a low-salt (LS) diet (0.4% NaCl) for 3 days. Nitric oxide (NO) and superoxide production were assessed in the thoracic aorta by evaluating the fluorescence signal intensity from 4,5-diaminofluorescein (DAF-2DA) and dihydroethidine, respectively. Methacholine caused increased NO release in the aortas from rats on a LS but not HS diet. The SOD mimetic tempol restored methacholine-induced NO release in aortas from rats on a HS diet. Methacholine also caused superoxide production in the aortas of rats on a HS diet but not in the aortas of rats on a LS diet. Tempol and N(G)-monomethyl-l-arginine eliminated methacholine-induced superoxide production in the aortas of rats on a HS diet. Aortic rings from rats on the HS diet showed impaired methacholine-induced relaxation, which was improved by tempol. Tempol alone caused a NO-dependent relaxation of norepinephrine-precontracted aortas that was significantly greater in the aortas of rats on the HS diet than in vessels from rats on the LS diet. These data suggest that a HS diet impairs endothelium-dependent relaxation via reduced NO levels and increased superoxide production.     

Augmented nitric oxide production and up-regulation of endothelial nitric oxide synthase during cecal ligation and perforation

Nitric oxide (NO) has been pointed out as being the main mediator involved in the hypotension and tissue injury taking place during sepsis. This study aimed to investigate the cellular mechanisms implicated in the acetylcholine (ACh)-induced relaxation detected in aortic rings isolated from rats submitted to cecal ligation and perforation (CLP group), 6h post-CLP. The mean arterial pressure was recorded, and the concentration-effect curves for ACh were constructed for endothelium-intact aortic rings in the absence (control) or after incubation with one of the following NO synthase inhibitors: L-NAME (non-selective), L-NNA (more selective for eNOS), 7-nitroindazole (more selective for nNOS), or 1400W (selective for iNOS). The NO concentration was determined by using confocal microscopy. The protein expression of the NOS isoforms was quantified by Western blot analysis. The prostacyclin concentration was indirectly analyzed on the basis of 6-keto-prostaglandin F(1α) (6-keto-PGF(1α)) levels measured by enzyme immunoassay. There were no differences between Sham- and CLP-operated rats in terms of the relaxation induced by acetylcholine. However, the NOS inhibitors reduced this relaxation in both groups, but this effect remained more pronounced in the CLP group as compared to the Sham group. The acetylcholine-induced NO production was higher in the rat aortic endothelial cells of the CLP group than in those of the Sham group. eNOS protein expression was larger in the CLP group, but the iNOS protein was not verified in any of the groups. The basal 6-keto-PGF(1α) levels were higher in the CLP group, but the acetylcholine-stimulated levels did not increase in CLP as much as they did in the Sham group. Taken together, our results show that the augmented NO production in sepsis syndrome elicited by cecal ligation and perforation is due to eNOS up-regulation and not to iNOS.     

Metformin reduces blood pressure and restores endothelial function in aorta of streptozotocin-induced diabetic rats

Effect of metformin treatment on blood pressure, endothelial function and oxidative stress in streptozotocin (STZ)-induced diabetes in rats was studied. In vitro effect of metformin on vascular reactivity to various agonist in the presence of metformin in untreated nondiabetic and STZ-diabetic rats were also studied. Sprague-Dawley rats were randomized into nondiabetic and STZ-diabetic groups. Rats were further randomized to receive metformin (150 mg/kg) or vehicle for 4 weeks.Metformin treatment reduced blood pressure without having any significant effect on blood glucose level in STZ-diabetic rats. Enhanced phenylephrine (PE)-induced contraction and impaired acetylcholine (Ach)-induced relaxation in STZ-diabetic rats were restored to normal by metformin treatment. Enhanced Ach-induced relaxation in metformin-treated STZ-diabetic rats was blocked due to pretreatment with 100 μM of Nω-nitro-l-arginine-methyl ester (l-NAME) or 10 μM of methylene blue but not 10 μM of indomethacin. Metformin treatment significantly increased antioxidant enzymes and reduced lipid peroxidation in STZ-diabetic rats. In vitro studies in aortic rings of untreated nondiabetic and STZ-diabetic rats showed that the presence of higher concentration of metformin (1 mM and 10 mM) significantly reduced PE-induced contraction and increased Ach-induced relaxation. Metformin per se relaxed precontracted aortic rings of untreated nondiabetic and STZ-diabetic rats in a dose-dependent manner. Pretreatment with l-NAME or removal of endothelium blocked metformin-induced relaxation at lower concentration (up to 30 μM) but not at higher concentration (above 30 μM). Metformin-induced relaxation was blocked in the presence of 1 mM of 4-aminopyridine, or 1 mM of tetraethylammonium but not in the presence of 100 μM of barium ion or 10 μM of glybenclamide. The restored endothelial function along with direct effect of metformin on aortic rings and reduced oxidative stress contributes to reduced blood pressure in STZ-diabetic rats. From the present study, it can be concluded that metformin administration to STZ-diabetic rats lowers blood pressure, and restores endothelial function.