Acidic reoxygenation protects against endothelial dysfunction in rat aortic rings submitted to simulated ischemia

Ischemia-reperfusion causes endothelial dysfunction. Prolongation of acidosis during initial cardiac reperfusion limits infarct size in animal models, but the effects of acidic reperfusion on vascular function are unknown. The present work analyzes the effects of acidic reoxygenation on vascular responses to different agonists in rat aortic rings. Arterial rings obtained from Sprague-Dawley rat aorta were placed in organ baths containing a Krebs solution oxygenated at 37 degrees C (pH 7.4). After equilibration (30 mN, 1 h), the effects of acidosis (pH 6.4) on aortic responses to acetylcholine and norepinephrine were initially assessed under normoxic conditions. Thereafter, the effects of acidosis during hypoxia (1 h) or reoxygenation on aortic responses to acetylcholine, norepinephrine, or sodium nitroprusside were analyzed and compared with those observed in control rings. Acidosis did not modify aortic responses to acetylcholine or adrenaline during normoxia. In contrast, rings submitted to hypoxia and reoxygenated at pH 7.4 showed a reduction in vasodilator responses to acetylcholine and in contractions to norepinephrine with no change in responses to sodium nitroprusside. Reoxygenation at pH 6.4 did not modify the depressed response to norepinephrine but enhanced the recovery of acetylcholine-induced vasorelaxation. Cumulative concentration-response curves to acetylcholine showed an increased responsiveness to this drug in rings reoxygenated at a low pH. This functional improvement was associated with the preservation of aortic cGMP content after stimulation of reoxygenated rings with acetylcholine. In conclusion, acidic reoxygenation preserves endothelial function in arterial rings submitted to simulated ischemia, likely through the preservation of cGMP signaling.     

L-arginine ameliorates kidney function and urinary bladder sensitivity in experimentally-induced renal dysfunction in rats

Effects of L-arginine and NG-nitro-L-arginine methyl ester (L-NAME) on the renal dysfunction that is induced by cisplatin (CDDP) were investigated. A single dose of CDDP (7.5 mg/kg i.p.) induced renotoxicity, which was manifested by increasing the sensitivity of isolated urinary bladder rings to acetylcholine (ACh), together with a significant elevation of serum urea and creatinine, and a severe decrease in serum albumin. Moreover, renal dysfunction was further confirmed by a significant decrease of enzyme activities, such as glutathione peroxidase, GSH-Px (E.C 1.11.1.9), catalase (E.C 1.11.1.6), as well as a significant increase in lipid peroxides that were measured as malondialdhyde (MDA) in kidney tissue homogenates. The administration of L-arginine (70 mg/kg/d p.o in drinking water 5 d before and 5 d after the CDDP injection) significantly ameliorated the renotoxic effects of CDDP, as judged by restoring the normal responses of isolated bladder rings to Ach, and also by an improvement in a range of renal function indices, which included serum urea and creatinine concentrations and kidney weight. In addition, L-arginine prevents the rise of MDA, as well as a reduction of GSH-Px and catalase activities in kidney tissues homogenates. On the other hand, the administration of L-NAME (4 mg/kg/d p.o) resulted in no protection against renal dysfunction that was induced by CDDP treatment. The findings of this study suggest that L-arginine can attenuate kidney injury that is produced by CDDP treatment. In addition, L-arginine may be a beneficial remedy for CDDP-induced renal toxicity, and could be used to improve the therapeutic index of CDDP.     

Diosgenin improves vascular function by increasing aortic eNOS expression, normalize dyslipidemia and ACE activity in chronic renal failure rats

In recent years, the role of endothelial dysfunction (ED) and excessive oxidative stress in the development of cardiovascular diseases has been highlighted. The aim of the present study is to evaluate the effect of diosgenin, an antioxidant on chronic renal failure (CRF) induced vascular dysfunction. CRF was induced by feeding the rats with a diet containing 0.75 % adenine and diosgenin was given orally (everyday at the dose of 40 mg/kg). Isometric force measurement was performed on isolated aortic rings in organ baths. Levels of reduced glutathione (GSH), nitric oxide metabolites, and endothelial nitric oxide synthase mRNA in rat aorta were examined. Further, plasma lipid profile, activity of enzymes of lipid metabolism, and aortic angiotensin converting enzyme (ACE) also studied. The overall results have proved that diosgenin attenuates CRF-induced impairment in acetylcholine induced endothelium-dependent and sodium nitroprusside induced endothelium-independent vascular relaxation. Moreover, it elevates the GSH and restores the eNOS mRNA expression level. CRF-induced dyslipidemia and ACE activity was also inhibited by diosgenin treatment. This study indicates that diosgenin have enough potential to protect vasculature against oxidative stress, dyslipidemia which in turn improves the vascular function in CRF milieu.     

Contributions of prostacyclin and nitric oxide to carbon monoxide-induced cerebrovascular dilation in piglets

Carbon monoxide (CO) is an endogenous dilator in the newborn cerebral microcirculation. Other dilators include prostanoids and nitric oxide (NO), and interactions among the systems are likely. Experiments on anesthetized piglets with cranial windows address the hypothesis that CO-induced dilation of pial arterioles involves interaction with the prostanoid and NO systems. Topical application of CO or the heme oxygenase substrate heme-L-lysinate (HLL) produced dilation. Indomethacin, N(omega)-nitro-L-arginine (L-NNA), and either iberiotoxin or tetraethylammonium chloride (TEA) were used to inhibit prostanoids, NO, and Ca(2+)-activated K(+) (K(Ca)) channels, respectively. Indomethacin, L-NNA, iberiotoxin, or TEA blocked cerebral vasodilation to CO and HLL. Vasodilations to both CO and HLL were returned to indomethacin-treated piglets by topical application of iloprost. Vasodilations to both CO and HLL were returned to L-NNA-treated piglets by sodium nitroprusside but not iloprost. In iberiotoxin- or TEA-treated piglets, dilations to CO and HLL could not be restored by either iloprost or sodium nitroprusside. The dilator actions of CO involve prostacyclin and NO as permissive enablers. The permissive actions of prostacyclin and NO may alter the K(Ca) channel response to CO because neither iloprost nor sodium nitroprusside could restore dilation to CO when these channels were blocked.     

Glutamine-dependent inhibition of pial arteriolar dilation to acetylcholine with and without hyperammonemia in the rat

Glutamine has been shown to influence endothelial-dependent relaxation and nitric oxide production in vitro, possibly by limiting arginine availability, but its effects in vivo have not been well studied. Hyperammonemia is a pathophysiological condition in which glutamine is elevated and contributes to depressed CO(2) reactivity of cerebral arterioles. We tested the hypothesis that acute hyperammonemia decreases pial arteriolar dilation to acetylcholine in vivo and that this decrease could be prevented by inhibiting glutamine synthetase with L-methionine-S-sulfoximine (MSO) or by intravenous infusion of L-arginine. Pial arteriolar diameter responses to topical superfusion of acetylcholine were measured in anesthetized rats before and at 6 h of infusion of either sodium or ammonium acetate. Ammonium acetate infusion increased plasma ammonia concentration from approximately 30 to approximately 600 microM and increased cerebral glutamine concentration fourfold. Arteriolar dilation to acetylcholine was intact after infusion of sodium acetate in groups pretreated with vehicle or with MSO plus methionine, which was coadministered to prevent MSO-induced seizures. In contrast, dilation in response to acetylcholine was completely blocked in hyperammonemic groups pretreated with vehicle or methionine alone. However, MSO plus methionine administration before hyperammonemia, which maintained cerebral glutamine concentration at control values, preserved acetylcholine dilation. Intravenous infusion of L-arginine during the last 2 h of the ammonium acetate infusion partially restored dilation to acetylcholine without reducing cerebral glutamine accumulation. Superfusion of 1 or 2 mM L-glutamine through the cranial window for 1 h in the absence of hyperammonemia attenuated acetylcholine dilation but had no effect on endothelial-independent dilation to nitroprusside. We conclude that 1) hyperammonemia reduces acetylcholine-evoked dilation in cerebral arterioles, 2) this reduction depends on increased glutamine rather than ammonium ions, and 3) increasing arginine partially overcomes the inhibitory effect of glutamine.     

Endothelial dysfunction is induced by proinflammatory oxidant hypochlorous acid

The myeloperoxidase (MPO)-derived oxidant hypochlorous acid (HOCl) plays a role in tissue injury under inflammatory conditions. The present study tests the hypothesis that HOCl decreases nitric oxide (NO) bioavailability in the vasculature of Sprague-Dawley rats. Aortic ring segments were pretreated with HOCl (1-50 microM) followed by extensive washing. Endothelium-dependent relaxation was then assessed by cumulative addition of acetylcholine (ACh) or the calcium ionophore A23187. HOCl treatment significantly impaired both ACh- and A23187-mediated relaxation. In contrast, endothelium-independent relaxation induced by sodium nitroprusside was unaffected. The inhibitory effect of HOCl on ACh-induced relaxation was reversed by exposure of ring segments to L-arginine but not D-arginine. In cellular studies, HOCl did not alter endothelial NO synthase (NOS III) protein or activity, but inhibited formation of the NO metabolites nitrate (NO3(-) and nitrite (NO2(-). The reduction in total NO metabolite production in bovine aortic endothelial cells was also reversed by addition of L-arginine. These data suggest that HOCl induces endothelial dysfunction via modification of L-arginine.     

Bone medullary arterioles from ovariectomized rats have smaller baseline diameters but normal eNOS expression and NO-mediated dilation

This study was designed to test the hypothesis that endogenous estrogens decrease the expression of endothelial nitric oxide synthase (eNOS) in resistance-size bone arterioles, thereby reducing endothelium-dependent vasodilator function. Sexually mature female rats were ovariectomized to reduce endogenous estrogens. Age-matched female rats served as controls. Seven to ten days after ovariectomy, bone marrow tissue was collected from the femoral canal. Immuno-histochemistry was performed to detect expression of estrogen receptors, alpha and beta and eNOS. eNOS protein content in medullary bone arterioles was compared using Western blot analysis. Endothelial cell function was assessed by quantitating the dilation of isolated, pressurized bone arterioles in response to acetylcholine. The results indicate that the endothelium of bone arterioles from ovariectomized and control rats express ER-alpha, ER-beta and eNOS. eNOS protein content in the two groups of arterioles did not differ. However, the baseline diameter of arterioles from ovariectomized rats (63+/-4 microm) was significantly smaller than the diameter of arterioles from control rats (75+/-3 microm, p<0.05). The two groups of arterioles dilated equally in response to acetylcholine. L-NAME, an inhibitor of eNOS, almost completely abolished the dilator responses to acetylcholine, but not to sodium nitroprusside. L-Arginine restored acetylcholine-induced dilation after L-NAME treatment. Thus, arteriole dilation to acetylcholine appears to be mediated almost exclusively by NO. The smaller diameter of arterioles from ovariectomized rats suggests that endogenous estrogens exert a significant dilator influence on bone arterioles. However, the dilator influence does not appear to be mediated by an increase in eNOS expression or enhanced NO-dependent vasodilation. These results indicate that estrogens do not decrease eNOS expression or diminish NO-mediated dilation of bone medullary arterioles.     

Vasoactivity of the ventral aorta of the American eel (Anguilla rostrata), Atlantic hagfish ( Myxine glutinosa), and sea lamprey (Petromyzon marinus)

To determine if vascular smooth muscle from teleost and agnathan fishes expresses receptors for signaling agents that are important in vascular tension in other vertebrates, we exposed rings of aortic vascular smooth muscle from the eel (Anguilla rostrata), the hagfish (Myxine glutinosa), and the lamprey (Petromyzon marinus) to a suite of putative agonists, including: acetylcholine, endothelin, nitric oxide, natriuretic peptides, and prostanoids. Acetylcholine constricted aortic rings from the eel, but had no effect on the rings from lamprey. On the other hand, endothelin constricted rings from all three species. Use of receptor-specific ET agonists demonstrated that only ET(A) receptors are expressed in the eel and lamprey aorta. The nitric oxide donor sodium nitroprusside or nitric oxide itself dilated rings from the eel, but both agonists constricted rings from the hagfish and NO produced a biphasic response (constriction followed by dilation) in the lamprey. Two natriuretic peptides, eel ANP and porcine CNP, produced marginally significant dilation in the eel aorta, human ANP dilated the hagfish rings, and pCNP and eANP dilated the lamprey rings. The prostanoids PGE(1) and PGE(2) both dilated the eel aortic rings, and PGE(1) and carbaprostacyclin (stable PGI(2) agonist) dilated the hagfish and lamprey rings. Our results suggest that receptors for a variety of vasoactive signaling agents are expressed in the aortic smooth muscle of the earliest vertebrates (lamprey and hagfish), as well as the more advanced teleosts (eel).     

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.     

Twenty-four-hour variation of L-arginine/nitric oxide/cyclic guanosine monophosphate pathway demonstrated by the mouse visceral pain model

The L-arginine/nitric oxide (NO)/cyclic guanosine monophosphate (cGMP) pathway is known to be involved in central and peripheral nociceptive processes. This study evaluated the rhythmic pattern of the L-arginine/NO/cGMP pathway using the mouse visceral pain model. Experiments were performed at six different times (1, 5, 9, 13, 17, and 21 h after light on) per day in male mice synchronized to a 12 h:12 h light-dark cycle. Animals were injected s.c. with saline, 2 mg/kg L-arginine (a NO precursor), 75 mg/kg L-N(G)-nitroarginine methyl ester (L-NAME, a NOS inhibitor), 40 mg/kg methylene blue (a soluble guanylyl cyclase and/or NOS inhibitor), or 0.1 mg/kg sodium nitroprusside (a nonenzymatic NO donor) 15 min before counting 2.5 mg/kg (i.p.) p-benzoquinone (PBQ)-induced abdominal constrictions for 15 min. Blood samples were collected after the test, and the nitrite concentration was determined in serum samples. L-arginine or L-NAME caused both antinociception and nociception, depending on the circadian time of their injection. The analgesic effect of methylene blue or sodium nitroprusside exhibited significant biological time-dependent differences in PBQ-induced abdominal constrictions. Serum nitrite levels also displayed a significant 24 h variation in mice injected with PBQ, L-NAME, methylene blue, or sodium nitroprusside, but not saline or L-arginine. These results suggest that components of L-arginine/NO/cGMP pathway exhibit biological time-dependent effects on visceral nociceptive process.     

Leptin resistance extends to the coronary vasculature in prediabetic dogs and provides a protective adaptation against endothelial dysfunction

Hyperleptinemia, associated with prediabetes, is an independent risk factor for coronary artery disease and a mediator of coronary endothelial dysfunction. We previously demonstrated that acutely raising the leptin concentration to levels comparable with those observed in human obesity significantly attenuates coronary dilation/relaxation to acetylcholine (ACh) both in vivo in anesthetized dogs and in vitro in isolated canine coronary rings. Accordingly, the purpose of this investigation was to extend these studies to a model of prediabetes with chronic hyperleptinemia. In the present investigation, experiments were conducted on control and high-fat-fed dogs. High-fat feeding caused a significant increase (131%) in plasma leptin concentration. Furthermore, in high-fat-fed dogs, exogenous leptin did not significantly alter vascular responses to ACh in vivo or in vitro. Coronary vasodilator responses to ACh (0.3-30.0 microg/min) and sodium nitroprusside (1.0-100.0 microg/min) were not significantly different from those observed in control dogs. Also, high-fat feeding did not induce a switch to an endothelium-derived hyperpolarizing factor as a major mediator of muscarinic coronary vasodilation, because dilation to ACh was abolished by combined pretreatment with N(omega)-nitro-l-arginine methyl ester (150 microg/min ic) and indomethacin (10 mg/kg iv). Quantitative, real-time PCR revealed no significant difference in coronary artery leptin receptor gene expression between control and high-fat-fed dogs. In conclusion, high-fat feeding induces resistance to the coronary vascular effects of leptin, and this represents an early protective adaptation against endothelial dysfunction. The resistance is not due to altered endothelium-dependent or -independent coronary dilation, increased endothelium-derived hyperpolarizing factor, or changes in coronary leptin receptor mRNA levels.     

Dietary zinc modifies the characteristics of endothelial dilation in normozincemic rats

Because zinc attenuates endothelial cell dysfunction that proceeds atherosclerosis, depressed zinc status may be involved in the initiation of endothelial dysfunction. However, before recommending a zinc-enriched diet to reduce the risks for atherosclerosis, the effect of excess zinc on endothelial cell functions in normozincemic status should be known. Therefore, in this study, the effect of dietary zinc on normal endothelial cell functions in animals subjected to a diet containing 334 +/- 58 ppm zinc for 30 d was studied to see whether supplemented zinc has an effect on endothelial cells. Despite a slight increase in blood zinc, unaltered aortic and kidney zinc contents were associated with unchanged blood pressure in rats subjected to a zinc-enriched diet. Increased basal nitric oxide and prostacyclin were accompanied by a normal response to phenylephrine. Dietary zinc influenced neither endothelial-dependent nor endothelial-independent relaxations significantly. However, it elevated the share of M1-type cholinoceptor response as well as dilator prostaglandin release, which seems to be nitric oxide dependent. There was a strong correlation (r=0.826, p<0.05) between M1-type cholinoceptor response and prostacyclin release in zinc-treated rings. These results suggested that zinc ions increases M1-mediated prostacyclin release in normal endothelial cells without altering intracellular pathways.     

Endothelum-dependent and-independent relaxations in aortic rings obtained from hypertensive hooded (Aguti) rats.

Experimental hypertension studies are few in the hooded (Aguti) rat. The present study was designed to investigate the usefulness of this rat strain for experimental hypertension studies and to test the hypothesis that the hypertension may be associated with a diminution of endothelium dependent and independent relaxations. Hypertension was induced in inbred hooded rats (n=8 each) by administering 8% salt in the diet and /or 100 mg/kg/day Nomega-nitro-L-arginine-methyl-ester (L-NAME) in the drinking water for six and/or four weeks respectively. The rats were anaesthetized using a 25% urethane and 1% chloralose mixture given intraperitoneally at a dose of 5 mg/kg. Their blood pressure was measured invasively. Thereafter, relaxations of rat aortic preparations to acetylcholine, histamine and sodium nitroprusside (SNP) were assessed using standard organ bath conditions. Probabiliity level of 0.05 was taken as statistically significant. The mean arterial pressure (MAP;mm Hg) rose significantly in all test groups (Salt: 148.3 +/- 4.6; L-NAME: 181.7 +/-8.3; Salt+L-NAME:154.9 +/-8.7) compared with control (94.2 +/-6.8; [P < 0.05]. The MAP was significantly [P < 0.05] higher in the L-NAME group than in all the other groups. The heart rate fell significantly in the salt + L-NAME group compared to control [P <0.05].The IC50 of acetylcholine in aortic rings from L-NAME rats (7.9 x 10(-1) +/- 6.0 x 10(-3)) was significantly higher than in rings from control (9.4 x 10(-8) +/- 2.8 x 10(-8)), salt (7.8 x 10(-7) +/- 4.7 x 10(-7)) and salt + L-NAME (3.3 x 10 (-7) +/- 2.1 x 10(-7)) rats [P < 0.05]. The IC50 of histamine and SNP in the rings from the test groups of rats showed no significant difference from control. Also, endothelium dependent and independent relaxations were preserved in the various forms of hypertension studied except in chronic NOS inhibition where the former was attenuated in response to acetylcholine.     

Increased calcium influx mediates increased cardiac stiffness in hyperthyroid rats

Cardiac remodeling (hypertrophy and fibrosis) and an increased left ventricular diastolic stiffness characterize models of hypertension such as the SHR and DOCA-salt hypertensive rats. By contrast, hyperthyroidism induces hypertrophy and hypertension, yet collagen expression and deposition is unchanged or decreased, whereas diastolic stiffness is increased. We determined the possible role of increased calcium influx in the development of increased diastolic stiffness in hyperthyroidism by administering verapamil (15 mg/[kg x d] orally) to rats given triiodothyronine (T3) (0.5 mg/[kg x d] subcutaneously for 14 d). Administration of T3 significantly increased body temperature (control: 36.7 +/- 0.2 degrees C; T3: 39.6 +/- 0.2 degrees C), left ventricular wet weight (control: 2.09 +/- 0.02 mg/kg; T3 3.07 +/- 0.07 mg/kg), systolic blood pressure (control: 128 +/- 5 mmHg; T3: 156 +/- 4 mmHg), and left ventricular diastolic stiffness (control: 20.6 +/- 2.0; T3: 28.8 +/- 1.4). Collagen content of the left ventricle was unchanged. Contractile response to noradrenaline in thoracic aortic rings was reduced. Relaxation in response to acetylcholine (ACh) was also reduced in T3-treated rats, whereas sodium nitroprusside response was unchanged. Verapamil treatment of hyperthyroid rats completely prevented the increased diastolic stiffness and systolic blood pressure while attenuating the increased body temperature and left ventricular weight; collagen content remained unchanged. ACh response in thoracic aortic rings was restored by verapamil. Thus, in hyperthyroid rats, an increased calcium influx is a potential mediator of the increased diastolic stiffness independent of changes in collagen.     

Compositional changes of the xiphoid process and costal cartilage with aging

Because zinc attenuates endothelial cell dysfunction that proceeds atherosclerosis, depressed zinc status may be involved in the initiation of endothelial dysfunction. However, before recommending a zinc-enriched diet to reduce the risks for atherosclerosis, the effect of excess zinc on endothelial cell functions in normozincemic status should be known. Therefore, in this study, the effect of dietary zinc on normal endothelial cell functions in animals subjected to a diet containing 334±58 ppm zinc for 30 d was studied to see whether supplemented zinc has an effect on endothelial cells. Despite a slight increase in blood zinc, unaltered aortic and kidney zinc contents were associated with unchanged blood pressure in rats subjected to a zinc-enriched diet. Increased basal nitric oxide and prostacyclin were accompanied by a normal response to phenylephrine. Dietary zinc influenced neither endothelial-dependent nor endothelial-independent relaxations significantly. However, it elevated the share of M1-type cholinoceptor response as well as dilator prostaglandin release, which seems to be nitric oxide dependent. There was a strong correlation (r=0.826, p<0.05) between M1-type cholinoceptor response and prostacyclin release in zinc-treated rings. These results suggested that zinc ions increases M1-mediated prostacyclin release in normal endothelial cells without altering intracellular pathways.     

Beneficial effects of L-arginine supplementation in experimental hyperlipemia-hyperglycemia in the hamster

The objective of this study was to evaluate whether administration of L-arginine, the substrate for nitric oxide synthesis, was able to ameliorate the endothelial dysfunction and the morphological changes induced by the combined insult of hyperlipemia and hyperglycemia. To this purpose, golden Syrian hamsters were rendered simultaneously hyperlipemic and diabetic (HD group) for 24 weeks, and then orally treated with 622.14 mg/kg per day L-arginine, for 12 weeks (HD + L-arg group). The following assays were carried out: (1) spectrophotometric: concentrations of circulating glucose, cholesterol, and creatinine, the activity of angiotensin-converting enzyme (ACE), and the osmotic fragility of erythrocyte plasmalemma; (2) myographic: the endothelium-dependent and -independent relaxation of the resistance arteries (i.d. 210-250 microm) to 10(-8) to 10(-4) M acetylcholine (ACh) or sodium nitroprusside (SNP); and (3) electron-microscopic: the ultrastructure of the resistance arteries, myocardium, and kidney glomeruli, which are main targets of hypertensive complications.The results showed that oral supplementation with L-arginine in simultaneous hyperlipemia-hyperglycemia induced in hamsters had favorable effects on: (1) homeostasis, i.e., diminished the concentration of circulating glucose (by ~63%) and cholesterol (by approximately 10%), reduced the ACE activity (by approximately 45%), and lowered the osmotic fragility of erythrocyte plasmalemma (as marker for the oxidative stress in plasma); (2) mesenteric resistance arteries, which showed (in 10(-4) M ACh) an improved endothelium-dependent relaxation (72.40+/-4.6% in the HD + L-arg group vs 61.90+/-1.45% in the HD group) and a reduced thickness (approximately 1.32-fold) of the smooth muscle cells' extracellular matrix; and (3) the heart, which displayed approximately 16% diminishing of the thickness of the left ventricular wall, and an apparently normal structure of the myocardium; the restoration of the thickness of the pericapillary extracellular matrix to almost normal dimensions was also observed. Administration of L-arginine did not modify the high level of plasma creatinine determined for the HD group (approximately 48% increased vs control group) and had no effect on the thickened, nodular basal lamina of the kidney capillaries. The results indicate that endothelial dysfunction established in combined hyperlipemia-diabetes is distinctive for each vascular bed (mesenteric arterioles, heart capillaries, kidney glomerular capillaries), and there is a reversible stage of the dysfunction in which L-arginine oral supplementation induced beneficial effects.     

Reactivity of mesenteric and aortic rings from trained rats fed with high caloric diet

A strong association between the benefits of physical exercise on the cardiovascular disease with an improvement of the endothelium-derived relaxing factor production has been consistently shown. The purpose of this study was to evaluate the effect of exercise training associated with high caloric diet in the reactivity of rat mesenteric and aortic rings. Experimental protocol consisted of 4 weeks of high caloric diet consumption previous to 4 weeks of run training (1.2 km/h, 0% grade, in sessions of 60 min, 5 days/week). Concentrations of triglycerides, glucose, insulin and nitrite/nitrate levels were measured and atherogenic index was calculated. Concentration-response curves to acetylcholine (10 nM-100 microM), sodium nitroprusside (100 pM-100 nM) and phenylephrine (1 nM-3 microM) were obtained. Exercise training reduced body mass (6%) and triglyceride levels (about 54%), without changes in glucose and insulin concentrations. An improvement of endothelium-dependent relaxation responses to acetylcholine in mesenteric and aortic rings was observed in trained group. No changes were seen for sodium nitroprusside and phenylephrine. In conclusion, our study is the first to show clearly that run training promotes an improvement of the endothelium-dependent relaxing response in aorta and mesenteric rings from rats fed with high caloric diet and that is associated with increase of NO production.     

Effect of tumor necrosis factor on hypoxic pulmonary vasoconstriction.

The effects of tumor necrosis factor (TNF) on hypoxic pulmonary vasoconstriction (HPV) and endothelium-dependent relaxation were examined in a blood-perfused rat lung preparation. Lungs from TNF-treated rats (0.26 mg/kg iv 12 h before experimentation) had a significantly greater HPV and a reduced vasorelaxant response to the endothelium-dependent vasodilator acetylcholine (ACh) but a similar vasorelaxant response to the endothelium-independent vasodilator nitroprusside compared with lungs from control rats (pretreated with 0.1 ml saline iv). Pentoxifylline (20 mg/kg iv and ip 20 min before administration of TNF) had no detectable effect on either HPV or ACh-induced relaxation but completely negated the augmentation on HPV and the inhibiting action on ACh-induced relaxation caused by TNF. The TNF effect on ACh relaxation was unaffected by pretreatment with L-arginine. These results indicate that TNF induces endothelial dysfunction and enhances HPV, effects that are inhibited by pentoxifylline.     

Hydrogen peroxide promotes endothelial dysfunction by stimulating multiple sources of superoxide anion radical production and decreasing nitric oxide bioavailability.

Hydrogen peroxide (H(2)O(2)) is an oxidant implicated in cell signalling and various pathologies, yet relatively little is known about its impact on endothelial cell function. Herein we studied the functional and biochemical changes in aortic vessels and cultured porcine aortic endothelial cells (PAEC) exposed to H(2)O(2). Exposure of aortic rings to 25 or 50 microM, but not 10 microM, H(2)O(2) for 60 min prior to constriction significantly decreased subsequent relaxation in response to acetylcholine (ACh), but not the nitric oxide ((.)NO) donor sodium nitroprusside. Treatment of PAEC with 50 microM H(2)O(2) significantly decreased ACh-induced accumulation of (.)NO, as measured with a (.)NO-selective electrode, yet such treatment increased nitric oxide synthase activity approximately 3-fold, as assessed by conversion of L-arginine to L-citrulline. Decreased (.)NO bioavailability was reflected in decreased cellular cGMP content, associated with increased superoxide anion radical (O(2)(-.)), and overcome by addition of polyethylene glycol superoxide dismutase. Increased cellular O(2)(-.) production was inhibited by allopurinol, diphenyliodonium and rotenone in an additive manner. The results show that exposure of endothelial cells to H(2)O(2) decreases the bioavailability of agonist-induced (.)NO as a result of increased production of O(2)(-.) likely derived from xanthine oxidase, NADPH-oxidase and mitochondria. These processes could contribute to H(2)O(2)-induced vascular dysfunction that may be relevant under conditions of oxidative stress such as inflammation.     

Role of nitric oxide in mediating cardiovascular alterations accompanying heart failure in rats

The present study was designed to evaluate the role of endothelial NO in the hemodynamics and vascular changes that occur in heart failure following myocardial infarction in rats. Left ventricular systolic pressure (LVSP), mean blood pressure (MBP), aortic morphology (media thickness) and reactivity were evaluated in rats with coronary artery ligation (heart failure, HF) or sham operation (SO) untreated or treated for four weeks with either a low dose of NG-nitro-L-arginine methyl ester (L-NAME, 6 mg.kg(-1).day(-1)) or L-arginine (1.5 g.kg(-1).day(-1)). In rats with HF LVSP (HF = 111 +/- 8 mmHg; SO = 143 +/- 6 mmHg, p < 0.05), MBP (HF = 98 +/- 8 mmHg; SO = 127 +/- 6 mmHg, p < 0.05) and aortic media thickness (HF = 68 +/- 6 microm; SO = 75 +/- 2 microm, p < 0.05) were significantly reduced. The contractile response to phenylephrine and the endothelium-independent relaxation to sodium nitroprusside were similar in HF and SO aortas, but the sensitivity (pD2) to acetylcholine (HF = 7.5 +/- 0.06; SO = 7.1 +/- 0.08, p < 0.05) was significantly increased in HF aortas, indicating an enhanced basal NO release. Treatment with L-NAME (LN) reversed the effects of HF on LVSP (HF-LN = 143 +/- 9 mmHg, p < 0.05 vs. HF), MBP (HF-LN = 128 +/- 8 mmHg, p < 0.05 vs. HF), sensitivity to acetylcholine (HF-LN = 6.9 +/- 0.10, p < 0.05 vs. HF) and aortic media thickness (HF-LN = 79 +/- 2 microm, p < 0.05 vs. HF), without changing these parameters in SO rats. L-NAME also selectively increased the maximal response to phenylephrine in HF aortas (HF-LN = 2.4 +/- 0.20 g; HF = 1.6 +/- 0.17 g, p < 0.05). L-arginine (LA) did not change the effects of HF on LSVP, MBP or aortic media thickness, but it reduced the sensitivity to phenylephrine in aortas from SO rats (SO-LA = 6.5 +/- 0.12; SO = 7.0 +/- 0.09, p < 0.05). Taken together, these results suggest an important role for endothelial NO in mediating the reduced vascular growth, myocardial dysfunction and hypotension in rats with HF.