Endothelium-dependent Nitric Oxide Release Research Articles

Tracking endothelium-dependent NO release in pressurized arteries.

Background: Endothelial cell (EC) dysfunction is an early hallmark of cardiovascular disease associated with the reduced bioavailability of nitric oxide (NO) resulting in over-constriction of arteries. Despite the clear need to assess NO availability, current techniques do not reliably allow this in intact arteries. Methods: Confocal fluorescence microscopy was used to compare two NO-sensitive fluorescent dyes (NO-dyes), Cu2FL2E and DAR-4M AM, in both cell-free chambers and isolated, intact arteries. Intact rat mesenteric arteries were studied using pressure myography or en face imaging to visualize vascular smooth muscle cells (SMCs) and endothelial cells (ECs) under physiological conditions. Both NO-dyes irreversibly bind NO, so the time course of accumulated fluorescence during basal, EC-agonist (ACh, 1µM), and NO donor (SNAP, 10µM) responses were assessed and compared in all experimental conditions. To avoid motion artefact, we introduced the additional step of labelling the arterial elastin with AF-633 hydrazide (AF) and calculated the fluorescence ratio (FR) of NO-dye/elastin over time to provide data as FR/FR0. Results: In cell-free chambers using either Cu2FL2E or DAR-4M AM, the addition of SNAP caused a time-dependent and significant increase in fluorescence compared to baseline. Next, using pressure myography we demonstrate that both Cu2FL2E and DAR-4M AM could be loaded into arterial cells, but found each also labelled the elastin. However, despite the use of different approaches and the clear observation of NO-dye in SMCs or ECs, we were unable to measure increases in fluorescence in response to either ACh or SNAP when cells were loaded with Cu2FL2E. We then turned our attention to DAR-4M AM and observed increases in FR/FR0 following stimulation with either ACh or SNAP. The addition of each agent evoked an accumulating, time-dependent, and statistically significant increase in fluorescence within 30min compared to time controls. These experiments were repeated in the presence of L-NAME, an NO synthase inhibitor, which blocked the increase in fluorescence on addition of ACh but not to SNAP. Conclusion: These data advance our understanding of vascular function and in the future will potentially allow us to establish whether ECs continuously release NO, even under basal conditions.

Oxidonitrergic and antioxidant effects of a low molecular weight peptide fraction from hardened bean (Phaseolus vulgaris) on endothelium.

About 3000 tons of beans are not used in human food due to hardening. Several studies on bean-derived bioactive peptides have shown potential to treat some diseases, including those relying on oxidative dysfunctions. We assessed the effects of peptides extracted from hardened bean Phaseolus vulgaris (PV) on reactive oxygen species (ROS) and nitric oxide (NO) production, cytotoxic and cytoprotective effects in endothelial cells, and oxidonitrergic-dependent vasodilating effects. Extract was composed by peptide fraction <3 kDa (PV3) from hardened common bean residue. PV3 sequences were obtained and analyzed with bioinformatics. Human umbilical vein endothelial cells were treated with 10, 20, 30, and 250 µg/mL PV3. Oxidative stress was provoked by 3% H2O2. Cytotoxicity and cytoprotective effects were evaluated by MTT assay, whereas, ROS and NO were quantified using DHE and DAF-FM fluorescent probes by confocal microscopy. NO- and endothelium-dependent vasodilating effects of PV3 were assessed in isolated aortic rings. We found 35 peptides with an average mass of 1.14 kDa. There were no cell deaths with 10 and 20 μg/mL PV3. PV3 at 30 μg/mL increased cell viability, while cytotoxicity was observed only with 250 μg/mL PV3. PV3 at 10 μg/mL was able to protect cells from oxidative stress. PV3 also increased NO release without causing cell death. It also reduced relative ROS production induced by H2O2. PV3 vasodilating effects relied on endothelium-dependent NO release. PV3 obtained from low-commercial-value bean displays little cytotoxicity and exerts antioxidant effects, whereas it increases endothelial NO release.

Homology modeling, vasorelaxant and bradykinin-potentiating activities of a novel hypotensin found in the scorpion venom from Tityus stigmurus.

In a recent work by our group involving a transcriptomics approach applied to the venom glands from Tityus stigmurus we identified a new family of peptides called Hypotensins (TSTI0006C) (Almeida etal., 2012). The cluster TSTI0006C was analyzed in the main 25 amino acid residues and named T.stigmurus Hypotensin (TistH), showing a molecular mass of 2.7kDa, an absence of cysteines and the presence of two C-terminal proline residues, which are a bradykinin-potentiating peptide (BPP) signature. Here, we describe the homology modeling of the three-dimensional structure of TistH. In addition, we evaluated the cardiovascular effects elicited by TistH in normotensive rats. Firstly, TistH showed no cytotoxic effect on horse erythrocyte. Furthermore, in normotensive rats TistH was able to potentiate the hypotensive action of bradykinin (BK) and induced a vasorelaxant effect in mesenteric artery rings by endothelium-dependent release of nitric oxide (NO) and demonstrated independent inhibition of angiotensin converting enzyme (ACE). Our data can contribute to a better understanding of the structural and functional characteristics of TistH and suggest its potential use in cardiovascular diseases.

Des-aspartate angiotensin I (DAA-I) reduces endothelial dysfunction in the aorta of the spontaneously hypertensive rat through inhibition of angiotensin II-induced oxidative stress

Des-aspartate angiotensin I (DAA-I), an endogenous nonapeptide, counteracts several effects of angiotensin II on vascular tone. The aim of this study was to investigate the acute protective effect of DAA-I on endothelial function in the spontaneously hypertensive rat (SHR) as well as its effect on angiotensin II-induced contractions and oxidative stress. Aortic rings were incubated with DAA-I (0.1μM) for 30min prior to the assessment of angiotensin II-induced contractions (0.1nM–10μM) in WKY and SHR aortas. Total nitrate and nitrite levels were assessed using a colorimetric method and reactive oxygen species (ROS) were measured by dihydroethidium (DHE) fluorescence and lucigenin-enhanced chemiluminescence. The effect of DAA-I was also assessed against endothelium-dependent and -independent relaxations to acetylcholine and sodium nitroprusside, respectively. Angiotensin II-induced contractions were significantly reduced by DAA-I, losartan and tempol. Incubation with ODQ (soluble guanylyl cyclase inhibitor) and removal of the endothelium prevented the reduction of angiotensin II-induced contractions by DAA-I. Total nitrate and nitrite levels were increased in DAA-I, losartan and tempol treated-SHR tissues while ROS level was reduced by DAA-I and the latter inhibitors. In addition, DAA-I significantly improved the impaired acetylcholine-induced relaxation in SHR aortas whilst sodium nitroprusside-induced endothelium-independent relaxation remained unaffected. The present findings indicate that improvement of endothelial function by DAA-I in the SHR aorta is mediated through endothelium-dependent release of nitric oxide and inhibition of angiotensin II-induced oxidative stress.

Molecular Biomarkers of Vascular Dysfunction in Obstructive Sleep Apnea

Untreated and long-lasting obstructive sleep apnea (OSA) may lead to important vascular abnormalities, including endothelial cell (EC) dysfunction, hypertension, and atherosclerosis. We observed a correlation between microcirculatory reactivity and endothelium-dependent release of nitric oxide in OSA patients. Therefore, we hypothesized that OSA affects (micro)vasculature and we aimed to identify vascular gene targets of OSA that could possibly serve as reliable biomarkers of severity of the disease and possibly of vascular risk. Using quantitative RT-PCR, we evaluated gene expression in skin biopsies of OSA patients, mouse aortas from animals exposed to 4-week intermittent hypoxia (IH; rapid oscillations in oxygen desaturation and reoxygenation), and human dermal microvascular (HMVEC) and coronary artery endothelial cells (HCAEC) cultured under IH. We demonstrate a significant upregulation of endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha-induced protein 3 (TNFAIP3; A20), hypoxia-inducible factor 1 alpha (HIF-1α?? and vascular endothelial growth factor (VEGF) expression in skin biopsies obtained from OSA patients with severe nocturnal hypoxemia (nadir saturated oxygen levels [SaO2]<75%) compared to mildly hypoxemic OSA patients (SaO2 75%–90%) and a significant upregulation of vascular cell adhesion molecule 1 (VCAM-1) expression compared to control subjects. Gene expression profile in aortas of mice exposed to IH demonstrated a significant upregulation of eNOS and VEGF. In an in vitro model of OSA, IH increased expression of A20 and decreased eNOS and HIF-1α expression in HMVEC, while increased A20, VCAM-1 and HIF-1αexpression in HCAEC, indicating that EC in culture originating from distinct vascular beds respond differently to IH stress. We conclude that gene expression profiles in skin of OSA patients may correlate with disease severity and, if validated by further studies, could possibly predict vascular risk in OSA patients.

Hydralazine and Isosorbide Dinitrate in Heart Failure

Until the 1970s, treatment options for heart failure were limited to digitalis and diuretics.1 Although effective for symptoms, there was no evidence of mortality benefit with this combination, and it was an inadequate option for many patients with advanced symptoms. The search for more effective options led to strategies that modulated hemodynamics.1 Numerous physiological studies showed the dependence of ventricular function on vascular resistance, and drugs that reduced systemic vascular resistance improved cardiac performance.2,–,5 A pivotal study by Franciosa et al6 showed that sodium nitroprusside in patients with heart failure in the setting of acute myocardial infarction reduced left ventricular filling pressures from 22.7±2.0 to 11.3±1.6 mm Hg and led to a modest increase in cardiac output. A subsequent study revealed more striking improvements in patients with refractory heart failure, in whom nitroprusside reduced systemic vascular resistance by 50%, increased cardiac output by 56%, and reduced left ventricular filling pressure by 47%.7 These hemodynamic benefits with nitroprusside led to studies with oral agents, including hydralazine, isosorbide dinitrate (ISDN), prazosin, phentolamine, and minoxidil. Although these drugs did affect hemodynamics, none was as effective as nitroprusside individually.8,–,12 In 1977, Massie et al13 studied the combination of 2 oral agents, hydralazine and ISDN (H-ISDN) in class III to IV heart failure patients, proposing that simultaneously reducing preload with ISDN and afterload with hydralazine would result in a better response than with either drug individually. They found that H-ISDN reduced left ventricular filling pressure by 36%, increased cardiac index by 58%, and reduced systemic vascular resistance by 34%. Later, Pierpont et al14 compared H-ISDN with nitroprusside and showed that the 2 therapies had similar effects on wedge pressure reduction and cardiac index increase. Considering these hemodynamic benefits with …

Eicosapentaenoic acid-induced endothelium-dependent and -independent relaxation of sheep pulmonary artery

It is known that long chain polyunsaturated fatty acids, such as eicosapentaenoic acid (EPA), have beneficial effects on cardiovascular function including pulmonary hypertension. The purpose of the present study was to examine the mechanisms involved in EPA-induced relaxation of sheep isolated pulmonary artery by measuring isometric tension. Nitric oxide (NO) derived from constitutive nitric oxide synthase (cNOS) was measured by Greiss method in the presence of the inducible nitric oxide synthase (iNOS) selective inhibitor N-[[3-(aminomethyl) phenyl]methyl]-ethanimidamide, dihydrochloride (1400 W). EPA (10(-)(7)-10(-)(4)M) caused concentration-dependent relaxation of sheep pulmonary artery with a pD(2) of 5.56+/-0.09 and E(max) of 87.40+/-3.10% (n=9). N(G)-nitro-L-arginine methyl ester (L-NAME) 100 microM significantly attenuated (E(max) 41.95+/-6.70%; n=8) EPA-induced relaxation of endothelium intact arterial rings. Similarly, endothelium denudation markedly inhibited (E(max) 17.60+/-1.21%; n=4) EPA-induced relaxation. EPA (30 microM) significantly increased the cNOS-derived NO release (10.17+/-0.96; n=8 versus control 7.43+/-0.78 pmol/mg tissue wet wt./h; n=7) in endothelium intact vessels. However, EPA-stimulated NO release was markedly blunted by either 100 microM L-NAME (7.07+/-0.54 pmol/mg tissue wet wt./h; n=8) or endothelium removal (6.97+/-0.87 pmol/mg tissue wet wt./h; n=17). In endothelium-denuded K(+) (80 mM)-depolarized arterial rings, EPA (30 microM) significantly inhibited CaCl(2)-induced contractions (E(max) 42.77+/-5.90% versus control 94.78+/-9.82%; n=5). The fatty acid also inhibited nifedipine (1 microM)-insensitive 5-HT-induced contractions in this vessel (E(max) 70.57+/-4.88% versus control 161.50+/-17.46%; n=5). In conclusion, EPA relaxes sheep pulmonary artery primarily through endothelium-dependent NO release, and the residual endothelium-independent relaxation may result from inhibition of Ca(2+)-influx through L-type calcium channels, as well as 5-HT-stimulated intracellular Ca(2+) release.

Noninvasive Assessment of Myocardial Perfusion

Noninvasive assessment of myocardial perfusion is important in the diagnosis and risk stratification of patients with known or suspected coronary artery disease (CAD). Although single-photon emission computed tomography (SPECT) is most commonly used, multiple modalities including myocardial contrast echocardiography (MCE), positron emission tomography (PET), cardiac MRI (CMR), and cardiac computed tomography (CT) have emerged as promising techniques. This article will critically evaluate the strengths and weakness of these modalities for evaluating myocardial perfusion. Myocardial perfusion is a highly regulated process that includes epicardial vessels, resistance vessels, and the endothelium. Endothelial dysfunction is an early manifestation of vascular disease and plays a role in the development of CAD.1 In normal coronaries, sympathetic stimulation causes a flow-mediated endothelium-dependent release of nitric oxide resulting in epicardial and arteriolar vasodilation. With endothelial dysfunction, vasoconstriction from acetylcholine predominates, resulting an attenuation or absence of the normal flow-mediated vasodilation.2 When coronary arteries are narrowed by atherosclerotic disease, coronary autoregulation attempts to normalize myocardial blood flow by reducing the resistance of distal perfusion beds to preserve adequate myocardial oxygen supply.3 A stenosis must exceed 85% to 90% of luminal diameter before significant reductions in resting blood flow occur.4 However, under vasodilator stimulus, maximal coronary flow has been shown to decrease with stenosis of >45% (Figure 1).4 This has been demonstrated clinically using quantitative PET myocardial perfusion imaging (MPI).5,6 Because perfusion is an early change in the ischemic cascade,7 stress modalities that assess coronary perfusion reserve have a higher sensitivity in detecting flow-limiting stenoses than analysis of stress-induced wall motion abnormalities or ECG changes alone.8 Abnormal coronary flow reserve with vasodilator stress in the absence of a significant coronary stenosis occurs and has been attributed to microvascular and/or endothelial dysfunction.9 Figure 1. Relationship between percent diameter stenosis and …

Oleanolic acid induces relaxation and calcium‐independent release of endothelium‐derived nitric oxide

The present study investigated the mechanisms by which oleanolic acid, a component of olive oil, increases release of nitric oxide (NO). Measurements of isometric tension, NO concentration, or endothelial cell calcium were made in rat isolated mesenteric arteries. Immunoblotting for endothelial NOS (eNOS) and Akt kinase were performed in primary cultures of human umbilical vein endothelial cells (HUVECs). Oleanolic acid (3-30 microM) evoked endothelium-dependent relaxations in noradrenaline-contracted rat superior and small mesenteric arteries. In rat superior mesenteric arteries, oleanolic acid induced simultaneous increases in NO concentration and relaxation, and these responses were inhibited by an inhibitor of NOS, asymmetric dimethyl-L-arginine (300 microM) and by the NO scavenger, oxyhaemoglobin (10 microM). Oleanolic acid-evoked NO increases were not reduced in Ca(2+)-free solution and in the presence of an inhibitor of endoplasmic reticulum calcium-ATPase, thapsigargin (1 microM). Oleanolic acid evoked relaxation without changes in endothelial cell calcium, but decreased smooth muscle calcium in arterial segments. Oleanolic acid failed to increase calcium in HUVECs, but increased time-dependently phosphorylation of Akt kinase at Serine(473) (Akt-Ser(473)) and eNOS at Serine(1177) (eNOS-Ser(1177)), which was attenuated by inhibitors of phosphoinositide-3-kinase. This study provides direct evidence that a component of olive oil, oleanolic acid, activated endothelium-dependent release of NO and decreased smooth muscle cell calcium followed by relaxation. The oleanolic acid-evoked endothelium-derived NO release was independent of endothelial cell calcium and involved phosphoinositide-3-kinase-dependent phosphorylation of Akt-Ser(473) followed by phosphorylation of eNOS-Ser(1177).

Effects of dietary salt loading on the responses of isolated rat mesenteric arteries to leptin

Leptin induces relaxation of vascular smooth muscle through an endothelium-dependent release of nitric oxide (EDNO) and administration of a high-salt diet reduces the relaxation of vessels to EDNO. We would, therefore, predict that salt loading would reduce the leptin-induced dilatation. However, in salt-loaded animals the relaxation to acetylcholine is maintained through an endothelial-dependent hypopolarizing factor instead of EDNO. These experiments were, therefore, designed to examine whether in salt-loaded animals the response to leptin would be reduced or whether, as for acetylcholine, an alternative mechanism would be substituted. Weanling rats were given diets containing either 0.4% (n = 10) or 8% (n = 9) sodium chloride for 4 weeks. They were then killed and a length of second order mesenteric artery was mounted in a perfusion myograph with diameter changes measured using a microscope-video tracking system. The vessels were preconstricted with norepinephrine and then the effects of graded concentrations of leptin were determined. In vessels from the low salt animals leptin caused a dose-dependent dilatation (maximum change 31.4% +/- 5.8% of the initial norepinephrine-induced constriction) but in the high salt animals the change was only 3.4% +/- 1.1%. The nitric oxide synthase blocker Nomega-nitro-L-arginine methyl ester (L-NAME) abolished the responses, although responses could still be obtained in vessels from both groups to the NO donor, sodium nitroprusside. These results indicate that salt loading to rats almost completely abolishes the vasodilatation to leptin. This implies endothelial disruption and, unlike the response to acetylcholine, no other vasodilator mechanism is implicated. This could provide a link between high salt intake and hypertension because the known increase in sympathetic activity caused by leptin would not be countered by a direct vasorelaxation.

Inferior Canine Hindlimb Ischemia and Reperfusion Impairs Femoral Artery Endothelium-Dependent Wall Relaxation

This experimental investigation was carried out to study the endothelium-dependent ischemia-reperfusion injury upon the release of nitric oxide (NO) in the canine femoral artery. The tested experimental model was the inferior canine hindlimb ischemia induced by infrarenal abdominal aortic clamping followed by reperfusion. The research protocol was standardized in four experimental groups (n=6): (1) Control group; (2) Ischemia (120 minutes) group; (3) Ischemia (90 minutes) and reperfusion (60 minutes) group; and (4) Ischemia (120 minutes) and reperfusion (90 minutes) group. The femoral artery vascular reactivity was studied in vitro with the aid of an eight "organ chambers'' setup, where segments from 4 to 5 mm, with and without endothelium, were suspended and connected to force transducers. The fundamental data of this study were as follows: (1) Ischemia caused by 120 minutes of infrarenal aortic clamping did not cause femoral artery endothelium dysfunction. (2) Ischemia caused by 90 minutes of clamping followed by 60 minutes of reperfusion also did not cause endothelium dysfunction, with an observed tendency to signal transduction impairment (studied by the sodium fluoride dose response curves). (3) Ischemia caused by 120 minutes of clamping followed by 90 minutes of reperfusion caused endothelium dysfunction, observed by the femoral artery impaired capacity of the endothelium-dependent NO release evoked by acetylcholine, adenosine diphosphate, and sodium fluoride. The present pharmacologic in vitro observations are concordant with the evidence that the NO-release impairment, caused by inferior canine hindlimb ischemia followed by reperfusion, is a consequence of endothelium cell membrane receptors, and G-proteins signal transduction dysfunction.

Inferior Canine Hindlimb Ischemia and Reperfusion Impairs Femoral Artery Endothelium-Dependent Wall Relaxation

This experimental investigation was carried out to study the endothelium-dependent ischemiareperfusion injury upon the release of nitric oxide (NO) in the canine femoral artery. The tested experimental model was the inferior canine hindlimb ischemia induced by infrarenal abdominal aortic clamping followed by reperfusion. The research protocol was standardized in four experimental groups (n=6): (1) Control group; (2) Ischemia (120 minutes) group; (3) Ischemia (90 minutes) and reperfusion (60 minutes) group; and (4) Ischemia (120 minutes) and reperfusion (90 minutes) group. The femoral artery vascular reactivity was studied in vitro with the aid of an eight “organ chambers” setup, where segments from 4 to 5 mm, with and without endothelium, were suspended and connected to force transducers. The fundamental data of this study were as follows: (1) Ischemia caused by 120 minutes of infrarenal aortic clamping did not cause femoral artery endothelium dysfunction. (2) Ischemia caused by 90 minutes of clamping followed by 60 minutes of reperfusion also did not cause endothelium dysfunction, with an observed tendency to signal transduction impairment (studied by the sodium fluoride dose response curves). (3) Ischemia caused by 120 minutes of clamping followed by 90 minutes of reperfusion caused endothelium dysfunction, observed by the femoral artery impaired capacity of the endothelium-dependent NO release evoked by acetylcholine, adenosine diphosphate, and sodium fluoride. The present pharmacologic in vitro observations are concordant with the evidence that the NO-release impairment, caused by inferior canine hindlimb ischemia followed by reperfusion, is a consequence of endothelium cell membrane receptors, and G-proteins signal transduction dysfunction.

Balloon angioplasty and induction of non-endothelial nitric oxide synthase in rabbit carotid arteries

The purpose of the study was to evaluate whether balloon angioplasty is associated with changes in nitric oxide synthase (NO synthase) activity. Normal rabbit carotid arteries were examined 10 min or 1, 2, 3 or 10 weeks after angioplasty with 2 or 2.5-mm balloons. Immunohistology was used to evaluate intimal thickening and endothelial cell regeneration. The NO synthase activity was studied functionally using isolated segments in organ chambers. Immunohistochemistry of the endothelial cell markers von Willebrand factor and platelet endothelial cell adhesion molecule-1 indicated that the regeneration of endothelial cells from patchy islands that remained after angioplasty was virtually complete within 2 weeks. However, the endothelium-dependent relaxations elicited by acetylcholine remained impaired up to 10 weeks after dilatation. Contractions elicited by 5-hydroxytryptamine (5-HT) were attenuated, but were significantly augmented by the NO synthase blocker, nitro- l-arginine. Furthermore, in contrast to normal arteries, the balloon-treated arteries developed marked contractions in response to nitro- l-arginine methyl ester ( l-NAME), contractions which could be reversed by l-arginine. The latter contractions and relaxations were not influenced by endothelial removal. These results suggest that although the endothelium quickly regenerates after severe balloon injury, the endothelium-dependent release of nitric oxide remains disturbed. However, the functional data also suggest that angioplasty led to a significant induction of NO synthase in ‘non-endothelial’ cells of the artery.

Differential effects of prolonged septicemia on isolated pulmonary arteries and veins from sheep.

Isolated third-order pulmonary arteries and veins from sheep were examined for the effects of septicemia on norepinephrine-induced contractions, nitric oxide (NO)-mediated dilation, and basal cyclic GMP levels. The groups studied were as follows: control sheep (n = 7); sheep given live Pseudomonas aeruginosa (Ps, n = 6) for 48 h; and sheep given NG-mono-methyl-L-arginine during the last 24 h of Ps infusion (Ps-L-NMMA, n = 4). The norepinephrine-induced contractions were significantly greater (p < .05) in arteries from septic (Ps and Ps-L-NMMA) sheep. Basal cyclic GMP levels were similar in all of the arteries. The norepinephrine-induced contractions were significantly depressed (p < .05) in veins from septic (Ps and Ps-L-NMMA) sheep. Basal cyclic GMP levels in veins from Ps sheep were markedly elevated (p < .01). N omega-nitro-L-arginine methyl ester (L-NAME) ex vivo decreased cyclic GMP in both arteries and veins. Removal of endothelium enhanced contractions and decreased cyclic GMP in arteries and veins only from control sheep. The results show that septicemia differently affects the pulmonary artery and vein. The enhanced vasoconstriction of the artery is due to decreased endothelium-dependent NO release; the attenuated vasoconstriction of the vein is associated with NO-mediated increased cyclic GMP levels.

Angiotensin-(1-7) Dilates Canine Coronary Arteries Through Kinins and Nitric Oxide

Angiotensin-(1-7) [Ang-(1-7)] was recently recognized to have novel biological functions that are distinct from those of Ang II. In these studies, we determined the vasoactive effects of Ang-(1-7) together with the endothelium-dependent mediator(s) of these responses in canine coronary arteries. Isometric tension was measured in intact canine coronary artery rings suspended in organ chambers perfused with 95% O2/5% CO2 at 37 degrees C. Ang-(1-7) caused significant concentration-dependent vascular relaxation (2.73 +/- 0.58 micromol/L, EC50) of rings precontracted with the thromboxane A2 analogue U46,619. Pretreatment with the nitric oxide synthase inhibitor N(omega)-nitro-L-arginine (1 mol/L) abolished the vasodilator response to Ang-(1-7), whereas treatment with the cyclooxygenase inhibitor indomethacin (10 micromol/L) was without effect. The vasodilator response produced by Ang-(1-7) was blocked by 75% with the bradykinin B2 receptor antagonist Hoe 140 (1 micromol/L) or by 80% with the nonselective Ang II antagonist [Sar1,Thr8]-Ang II (1 micromol/L). In contrast, the selective AT1 or AT2 Ang II antagonists CV 11974 (1 micromol/L), and PD 123319 (1 micromol/L), respectively, were ineffective in inhibiting the Ang-(1-7)-elicited vasodilation. Furthermore, pretreatment of the coronary rings with 2 micromol/L Ang-(1-7) markedly potentiated the bradykinin response. These results suggest that Ang-(1-7) elicits coronary vasodilation that is specifically mediated by the endothelium-dependent release of nitric oxide. These responses involve a B2 bradykinin receptor and a non-AT1, non-AT2, angiotensin receptor. These data suggest that increases in circulating levels of Ang-(1-7) accompanying long-term administration of converting enzyme inhibitors or Ang II receptor blockers may contribute to the cardioprotective actions of these drugs.

930-104 Nitrate Tolerance: Impaired Vascular Effects of Stimulation and Inhibition of Nitric Oxide Release in Vivo

Nitroglycerin (NTG) is metabolized to nitric oxide (NO) in vascular smooth muscle cells. It has been suggested that prolonged exposure to NO results in reduced vascular sensitivity to NO and/or down regulation of NO synthesis. Whether such changes contribute to the in vivodevelopment of nitrate tolerance is currently not clear. This study investigates NO-mediated vasodilation in conscious rats before and after development of nitrate tolerance. Tolerance was induced by a 72 hours iv. infusion of NTG and confirmed by a 68% reduction in the response to NTG (from 25 ± 3 to 8 ± 1 mmHg, p &lt; 0.05, n = 7). The hypotensive effects of acetylcholine (ACh, endothelium dependent NO release, 10 μg/kg), and sodium nitroprusside (SNP, releases NO spontaneously, 20 μg/kg), were examined before and after infusion of NTG. Nitrate tolerance was associated with a attenuated hypotensive response to ACh (before 24 ± 3; after 17 ± 2 mmHg, n = 7, P &lt; 0.05). Similarly, the response to SNP was reduced from 32 ± 1 to 26 ± 3 mmHg, n = 7, P &lt; 0.05). Infusion of placebo (NTG solvens, n = 6) for 72 hours did not affect the response to ACh and SNP (p &gt; 0.05). An effect of NTG treatment on endogenous NO regulation was further substantiated by the finding of a 62% reduction in the hypertensive response to the NO synthase inhibitor, L-NAME (n = 6), after induction of nitrate tolerance (from 29 ± 6 to 11 ± 1 mmHg, p &lt; 0.05). The results suggest that prolonged infusion of NTG attenuates the vasodilator effects of NO, whether it is derived from the endothelium (ACh) or from exogenous sources (SNP) and that reduced vascular sensitivity to NO dependent vasodilation may contribute to the development of nitrate tolerance in vivo.

Influence of aortic baroreceptor denervation on adenosine receptor-mediated relaxation of isolated rat aorta

The effect of aortic baroreceptor denervation on the vasorelaxant activity of the adenosine analogue, 5′-N-ethylcarboxamidoadenosine (NECA) was evaluated in the isolated thoracic aortic rings from rats. The responses were evaluated at 3 h after baroreceptor denervation when the blood pressure was significantly higher than that of control (149±3 vs. 112±2 mmHg) and at 48 h after aortic baroreceptor denervation when blood pressure returned to control level. Sham operation had no effect on blood pressure at either time interval. A concentration-dependent relaxation of rat aorta elicited by NECA was observed in all groups. However, the responsiveness to NECA was reduced in aortic baroreceptor-denervated rats. The nitric oxide (NO) synthase inhibitor, L-monomethyl- L-arginine (30 μM) shifted the dose-response curve for NECA to the right in all groups suggesting that the vascular response to NECA is partially mediated through the release of NO. Removal of the endothelium abolished the differences in the response to NECA in sham and aortic baroreceptor-denervated rats suggesting that the decrease in the responsiveness of aortic smooth muscle to NECA is dependent on the release of NO. Vasorelaxant responses to acetylcholine were not altered by aortic baroreceptor denervation. The ability of aortic baroreceptor denervation to attenuate vasorelaxant responses to NECA but not to acetylcholine indicated the possibility of functional changes involving adenosine receptor-mediated relaxation in endothelium rather than structural changes due to aortic baroreceptor denervation. These findings suggest that short-term elevation of blood pressure following aortic baroreceptor denervation modulates the endothelium-dependent release of NO mediated by adenosine receptor activation.

Differences in EDNO contribution to arteriolar diameters at rest and during functional dilation in striated muscle.

This study was designed to determine the physiological role of endothelium-dependent nitric oxide (EDNO) in the control of arteriolar diameter during rest and muscle stimulation. Diameters of first-, second-, and third-order arterioles in the superfused hamster cremaster muscle were measured before and throughout 1 min of field stimulation before and after inhibition of EDNO release. ENDO inhibition by intravenous N omega-nitro-L-arginine methyl ester (L-NAME) significantly attenuated the arteriolar vasodilation in response to 1 microM acetylcholine. First-order arterioles averaged 65 +/- 5 microns at rest and dilated to 86 +/- 6 microns during muscle stimulation (n = 9), second-order arterioles averaged 45 +/- 6 microns and dilated to 72 +/- 3 microns during muscle stimulation (n = 6), with third-order arterioles averaging 29 +/- 2 microns, and dilating to 53 +/- 3 microns during muscle stimulation (n = 7). EDNO inhibition significantly decreased both the resting diameter of first-order arterioles (57 +/- 4 microns) and functional dilation (68 +/- 3 microns; P < 0.05). EDNO inhibition had no effect on the resting diameter of second-order arterioles (45 +/- 5 microns) yet significantly attenuated the functional dilation (64 +/- 4 microns; P < 0.05). EDNO inhibition had no effect on either the resting diameter of third-order arterioles (30 +/- 2 microns) or the functional dilation (49 +/- 2 microns).(ABSTRACT TRUNCATED AT 250 WORDS)