Release Of Endothelium-derived Nitric Oxide Research Articles

Aortic Relaxation in Castrated Male Rats and Responses to Estrogen Treatment

Previous studies suggest that risk factors for cardiovascular diseases (CVD) have increased in the transgender population after they undergo cross‐sex hormone therapy (CSHT). Several studies, including ours in animal models as well as in humans, have shown that estrogen exerts direct beneficial effects on the vessel wall in healthy females. A growing body of evidence also suggests that androgen exhibits protective actions on the cardiovascular system. However, the effects of CSHT in cardiovascular physiology in males or females are less understood.The current study was, therefore, undertaken to evaluate the effects of estrogen treatment on aortic reactivity in castrated (CAS) male rats. Specifically, we investigated the effects of in vivo treatment with 17β‐estradiol (E2) on the aortic responses of CAS male as well as ovariectomized (OVX) female rats.Age‐matched CAS male and OVX female rats were implanted subcutaneously with either 17β‐estradiol (E2,1.5 mg) or a placebo‐containing pellet for 30 days. Endothelium‐dependent vasorelaxation (EDV) in response to acetylcholine (ACh, 10‐8 to 10‐5 M) in aorta precontracted with phenylephrine (PE, 2 µM) was measured before and after pretreatment with indomethacin (10 µM, cyclooxygenase inhibitor), followed by N‐nitro‐ L‐arginine methyl ester [L‐NAME, 200 µM, a nonselective nitric oxide synthase (NOS) inhibitor]. Endothelium‐independent vasorelaxation was assessed by measuring sodium nitroprusside (SNP)‐induced relaxation response in endothelium‐denuded rings in addition to the contractile response curves to PE before and after pretreatment with L‐NAME in aortic rings.EDV to ACh was significantly impaired in aortic rings from E2‐treated CAS rats compared to their placebo‐treated counterparts and E2‐treated OVX rats. The impairment of EDV to ACh in E2‐treated CAS rats was accompanied by a significant decrease in nitric oxide (NO) contribution to ACh‐induced relaxation in this group. The smooth muscle sensitivity to NO as measured by SNP relaxation response curve was not altered, whereas the responsiveness to PE was slightly enhanced in the aortas of E2‐treated CAS group compared to those in other experimental groups. Endothelium‐derived NO release during smooth muscle contraction, as assessed by the potentiation of the response to PE after NOS inhibition, was not different in the aortas of experimental groups.Overall, our data demonstrate that aortic function was altered in E2‐treated CAS male rats. Here, we provide the first evidence of impairment in aortic relaxation in E2‐treated CAS male rats, possibly via a significant decrease in the relative contribution of NO to the regulation of vascular relaxation response. Furthermore, the comparison of current data in the CAS male rats with our recently published data on the aortic reactivity in the intact males appears to rule out any effect of testosterone deprivation on the stimulated release of NO in male rat aortas. Additional studies will be needed to document the direction and magnitude of CSHT interaction with vasculatures.

Moringa oleifera leaf extract enhances endothelial nitric oxide production leading to relaxation of resistance artery and lowering of arterial blood pressure

A mass of evidence has identified a promoting of nitric oxide (NO) production in endothelial cells using natural products as a potential strategy to prevent and treat hypertension. This study investigated whether the aqueous extract of Moringa oleifera leaves (MOE) could lower mean arterial pressure (MAP) and relax mesenteric arterial beds in rats via stimulating endothelium-derived NO production. Intravenous administration of MOE (1–30 mg/kg) caused a dose-dependent reduction in MAP in anesthetized rats. In rats pretreated with the NO-synthase inhibitor, Nω-nitro-L-arginine methyl ester (L-NAME, 30 mg/kg, i.v.), the effect of MOE on MAP was significantly reduced. MOE (0.001–3 mg) induced relaxation in methoxamine (10 μM) pre-contracted mesenteric arterial beds, which was abolished by endothelium denudation. This endothelium-dependent vasorelaxation was reduced by L-NAME (100 μM) or the NO-sensitive guanylyl cyclase inhibitor, 1H- [1,2,4]-oxadiazolo-[4,3-a]-quinoxalin-1-one (10 μM). In primary human pulmonary artery endothelial cells, MOE (3–30 μg/mL) induced NO production, which was inhibited by L-NAME (100 μM) pretreatment. These findings show that MOE stimulates the endothelium-derived NO release for driving its vasorelaxation to lower arterial blood pressure. These suggest the development of MOE as a natural antihypertensive supplement.

Angiotensin-Converting Enzyme 2 Activation Improves Endothelial Function

Diminished release and function of endothelium-derived nitric oxide coupled with increases in reactive oxygen species production is critical in endothelial dysfunction. Recent evidences have shown that activation of the protective axis of the renin-angiotensin system composed by angiotensin-converting enzyme 2, angiotensin-(1-7), and Mas receptor promotes many beneficial vascular effects. This has led us to postulate that activation of intrinsic angiotensin-converting enzyme 2 would improve endothelial function by decreasing the reactive oxygen species production. In the present study, we tested 1-[[2-(dimetilamino)etil]amino]-4-(hidroximetil)-7-[[(4-metilfenil)sulfonil]oxi]-9H-xantona-9 (XNT), a small molecule angiotensin-converting enzyme 2 activator, on endothelial function to validate this hypothesis. In vivo treatment with XNT (1 mg/kg per day for 4 weeks) improved the endothelial function of spontaneously hypertensive rats and of streptozotocin-induced diabetic rats when evaluated through the vasorelaxant responses to acetylcholine/sodium nitroprusside. Acute in vitro incubation with XNT caused endothelial-dependent vasorelaxation in aortic rings of rats. This vasorelaxation effect was attenuated by the Mas antagonist D-pro7-Ang-(1-7), and it was reduced in Mas knockout mice. These effects were associated with reduction in reactive oxygen species production. In addition, Ang II-induced reactive oxygen species production in human aortic endothelial cells was attenuated by preincubation with XNT. These results showed that chronic XNT administration improves the endothelial function of hypertensive and diabetic rat vessels by attenuation of the oxidative stress. Moreover, XNT elicits an endothelial-dependent vasorelaxation response, which was mediated by Mas. Thus, this study indicated that angiotensin-converting enzyme 2 activation promotes beneficial effects on the endothelial function and it is a potential target for treating cardiovascular disease.

Salsolinol participation in mechanism of oxytocin release in nursing ewe

OBJECTIVE: Prostaglandins liberated from the uterus in response to chemical and physical stimuli would be important modulators of uterine arterial tone and blood flow. This study was aimed at analyzing mechanisms of vasodilator action of prostaglandins in uterine arteries.STUDY DESIGN: Canine uterine artery strips were suspended in Ringer-Locke solutions for isometric tension recording.RESULTS: Prostaglandin F2α-induced relaxation was reversed to contraction by cyclooxygenase inhibitors and suppressed by tranylcypromine (prostaglandin I2 synthesis inhibitor) or diphloretin phosphate (an inhibitor of prostaglandin F2α and prostaglandin I2 actions) but was unaffected by endothelium denudation. Prostaglandin E2-induced relaxation was not attenuated by indomethacin but was partially inhibited by a nitric oxide synthase inhibitor and endothelium denudation. Relaxation induced by beraprost (prostaglandin I2 analog) was suppressed by diphloretin phosphate but was not influenced by indomethacin and endothelium denudation.CONCLUSION: It appears that prostaglandin F2α-induced relaxation is mediated by prostaglandin I2 released from subendothelial tissues, whereas prostaglandin E2-induced relaxation is caused by release of endothelium-derived nitric oxide and also by an endothelium-independent mechanism.

P20 Glucocorticoids suppress hydrogen sulfide production in liver and kidneys

Chronic exposure to high glucocorticoid levels is often associated with alterations in the haemostatic system, arterial hypertension and atherosclerosis. Increased frequency of both thromboembolic events and high blood pressure has been reported in patients with Cushing’s syndrome. From the other side, recent research has shown that the endogenous gas hydrogen sulphide (H 2 S) induces relaxation of vessels through a direct effect on KATP-channels causing hyperpolarization and closure of voltage-dependent Ca 2+ -channels followed by a reduction in intracellular calcium as well as through the release of endothelium-derived hyperpolarizing factor and nitric oxide from the endothelium. Therefore, we hypothesized that probably the cardiovascular complications occurring in hypercorticism are determined at least partially by the decrease in H 2 S production.Wistar rats were treated with saline, Dexamethasone (Dex) (4 mg/kg/d intraperitoneally for 2 weeks) or Dex + Pyridoxine. Pyridoxine (10 mg/kg/d intraperitoneally) was administered every day during glucocorticoid treatment. Concentration of H 2 S in blood plasma was measured by the spectrophotometric methylene-blue method. The activities of cystathionine- γ -lyase (CSE), cysteine aminotransferase (CAT) and cystathionine- β -synthase (CBS) were measured in the homogenates of liver and kidneys. Rats were euthanized on the 8th and 15th days after the beginning of Dex administration.In general, in both terms of experiment Dex caused almost a 2-fold decrease in H 2 S blood plasma concentration compared to the control. Analysis of the time-dependent effect of Dex on the functioning of H 2 S producing enzymes showed the significant inhibition of CSE, CAT and CBS activities. CSEis expressed abundantly in mammalian cardiovascular system but it also appears to be the main H 2 S-forming enzyme in the liver and kidneys. After Dex injection for 7 days the liver CSE activity decreased in 1.2 times, whereas administration of hormone for 14 days resulted in the decrease of this index in 1.5 times. Another enzyme that has been reported to generate H 2 S in rat liver is CAT. We observed 1.2- and 1.6-fold decrease of CAT activity in liver after Dex administration for 7 and 14 days correspondingly. The CSE and CAT activities in kidneys of rats with hyperglucocorticoidemia did not change significantly compared with the control rats. CBS expression is significant mainly in the brain and it is not so considerable in other tissues. Despite this fact in our experiments we showed that in rats treated with Dex the significant inhibition of CBS activity occurs in both liver and kidneys on 8th and 15th days of experiment. Pyridoxine partially reversed both inhibition of CSE, CAT and CBS activities and H 2 S concentration in blood of rats treated with Dex.Thus, our results clearly demonstrated that Dex reduces H 2 S production in liver and kidneys and H 2 S level in blood. This allows to assume that reduced endogenous H 2 S production in the diseases associated with chronic increase of blood glucocorticoids concentration may play a role in the development of hypertension, atherosclerosis, heart ischemia and administration of Pyridoxine or H 2 S donors may be beneficial in prevention of such cardiovascular complications.

Increased Vascular Contractility and Oxidative Stress in β2-Adrenoceptor Knockout Mice: The Role of NADPH Oxidase

Background/Aims: β₂-adrenoceptor (β₂-AR) activation induces smooth muscle relaxation and endothelium-derived nitric oxide (NO) release. However, whether endogenous basal β₂-AR activity controls vascular redox status and NO bioavailability is unclear. Thus, we aimed to evaluate vascular reactivity in mice lacking functional β₂-AR (β₂KO), focusing on the role of NO and superoxide anion. Methods and Results: Isolated thoracic aortas from β₂KO and wild-type mice (WT) were studied. β₂KO aortas exhibited an enhanced contractile response to phenylephrine compared to WT. Endothelial removal and L-NAME incubation increased phenylephrine-induced contraction, abolishing the differences between β₂KO and WT mice. Basal NO availability was reduced in aortas from β₂KO mice. Incubation of β₂KO aortas with superoxide dismutase or NADPH inhibitor apocynin restored the enhanced contractile response to phenylephrine to WT levels. β₂KO aortas exhibited oxidative stress detected by enhanced dihydroethidium fluorescence, which was normalized by apocynin. Protein expression of eNOS was reduced, while p47^phox expression was enhanced in β₂KO aortas. Conclusions: The present results demonstrate for the first time that enhanced NADPH-derived superoxide anion production is associated with reduced NO bioavailability in aortas of β₂KO mice. This study extends the knowledge of the relevance of the endogenous activity of β₂-AR to the maintenance of the vascular physiology.

Effects of polycythemia on systemic endothelial function in chronic hypoxic lung disease

Chronic obstructive pulmonary disease (COPD) is a major risk factor for cardiovascular disease. Polycythemia, a common complication of hypoxic COPD, may affect systemic vascular function by altering blood viscosity, vessel wall shear stress (WSS), and endothelium-derived nitric oxide (NO) release. Here, we evaluated the effects of hypoxia-related polycythemia on systemic endothelial function in patients with COPD. We investigated blood viscosity, WSS, and endothelial function in 15 polycythemic and 13 normocythemic patients with COPD of equal severity, by recording brachial artery diameter variations in response to hyperemia and by using venous occlusion plethysmography (VOP) to measure forearm blood flow (FBF) responses to a brachial artery infusion of acetylcholine (ACh), bradykinin (BK), sodium nitroprusside (SNP), substance P (SP), isoptin, and N-monomethyl-L-arginine (L-NMMA). At baseline, polycythemic patients had higher blood viscosity and larger brachial artery diameter than normocythemic patients but similar calculated WSS. Flow-mediated brachial artery vasodilation was increased in the polycythemic patients, in proportion to the hemoglobin levels. ACh-induced vasodilation was markedly impaired in the polycythemic patients and negatively correlated with hemoglobin levels. FBF responses to endothelium- (BK, SP) and non-endothelium-dependent (SNP, isoptin) vasodilators were not significantly different between the two groups. L-NMMA infusion induced a similar vasoconstrictor response in both groups, in accordance with their similar baseline WSS. In conclusion, systemic arteries in polycythemic patients adjust appropriately to chronic or acute WSS elevations by appropriate basal and stimulated NO release. Overall, our results suggest that moderate polycythemia has no adverse effect on vascular function in COPD.

Arterial stiffness, hypertension, and rational use of nebivolol

Arterial stiffness plays a key role in the pathophysiology of the cardiovascular system. Some indices of arterial stiffness (pulse wave velocity, augmentation index, characteristics of central blood pressure waveform) may be presently calculated and evaluated in the clinical setting. Age and blood pressure are the two major clinical determinants of increased arterial stiffness, while molecular determinants of arterial stiffness are related to fibrotic components of the extracellular matrix, mainly elastin, collagen and fibronectin. Increased arterial stiffness has been consistently observed in conditions such as hypertension, dyslipidemia and diabetes. Arterial stiffness evaluated by means of carotid-femoral pulse wave velocity yielded prognostic significance beyond and above traditional risk factors. A more favorable effect of calcium channel blockers, diuretics and ACE inhibitors compared with beta-blockers on indices of arterial stiffness was observed in several studies. It is conceivable that newer beta-blockers with additional vasodilating properties, such as nebivolol, which has favorable effects on carbohydrate and lipid metabolism, as well as on endothelial function and on oxidative stress, may have favorable effects on arterial stiffness, compared with atenolol. In fact, in recent studies, nebivolol was demonstrated to improve artery stiffness to a greater extent than older beta-blockers. Because endothelial dysfunction and increased arterial stiffness play an important role in the early atherosclerotic processes and are associated with poor outcomes and increased mortality, independently of blood pressure, the ability of nebivolol to enhance release of endothelium-derived nitric oxide, and consequently improve endothelial function and arterial stiffness, may have significant clinical implications for the use of this agent in the treatment of hypertension and cardiovascular diseases.

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).

Cardioactive and Vasoactive Effects of Natural Wild Honey Against Cardiac Malperformance Induced by Hyperadrenergic Activity

Induction of hyperadrenergic activity was experimentally achieved in urethane-anesthetized rats using epinephrine (adrenaline). Acute administration of epinephrine (100 microg/kg) for 2 hours induced several cardiac disorders and vasomotor dysfunction. Pretreatment with natural wild honey (5 g/kg) for 1 hour prior to the injection with epinephrine (100 mug/kg) protected the anesthetized normal rats from the incidence of epinephrine-induced cardiac disorders and vasomotor dysfunction. Moreover, posttreatment with natural wild honey (5 g/kg) following the injection with epinephrine (100 microg/kg) for 1 hour showed several ameliorative outcomes to the electrocardiographic parameters and vasomotor dysfunction of anesthetized stressed rats. Furthermore, natural wild honey preserved the positive inotropic effect of epinephrine in both cases. Also, the total antioxidant capacity (AOC) of natural wild honey was found to be very pronounced. Levels of both reduced glutathione and ascorbic acid (vitamin C) were considered relatively high in natural wild honey. Activity of superoxide dismutase (SOD) was also high, whereas catalase activity was relatively low, especially when compared to the value of SOD activity. It would appear from the results of the present study that natural wild honey may exert its cardioprotective and therapeutic effects against epinephrine-induced cardiac disorders and vasomotor dysfunction directly, via its very pronounced total AOC and its great wealth of both enzymatic and nonenzymatic antioxidants involved in cardiovascular defense mechanisms, besides its substantial quantities of mineral elements such as magnesium, sodium, and chlorine, and/or indirectly, via the enhancement of the endothelium-derived relaxing factor nitric oxide release through the influence of ascorbic acid (vitamin C).

Cytochrome P450 2C9 is involved in flow‐dependent vasodilation of peripheral conduit arteries in healthy subjects and in patients with chronic heart failure

Flow-mediated dilation (FMD) of human conduit arteries is, in part, related to shear stress-induced release of endothelium-derived nitric oxide (NO). However, NO synthase inhibitors do not completely abolish this FMD-response. Recently, a cytochrome P450 (CYP) epoxygenase of the 2C family was linked to NO- and prostacyclin-independent relaxation of conduit arteries. We therefore evaluated the contribution of CYP 2C9 to FMD in humans. FMD of the radial artery was determined in 12 healthy volunteers by high-resolution ultrasound and analyzed before and after intra-arterial infusion of sulfaphenazole, a specific CYP 2C9 inhibitor, L-NMMA (NO synthase inhibitor) and co-infusion of both. Endothelium-independent vasodilation was characterized after intra-arterial infusion of SNP. FMD was reduced after sulfaphenazole (11.5+/-0.87% vs. 7.4+/-0.95%, p<0.01), after L-NMMA (6.0+/-0.71%; p<0.01), and after co-infusion 3.9+/-0.73% (p<0.05 vs. L-NMMA; p<0.01 vs. sulfaphenazole). Sulfaphenazole had no effect on endothelium-independent vasodilation. In patients with chronic heart failure, the portion of FMD blocked by sulfaphenazole was not affected. CYP 2C was detected by immunohistochemistry in radial artery samples obtained from patients undergoing coronary bypass surgery. FMD in human conductance arteries is reduced after inhibition of CYP 2C9, supporting the concept that CYP 2C metabolites contribute to endothelium-mediated vasodilation of peripheral conduit arteries in vivo. In patients with heart failure, the CYP-dependent FMD appears to be preserved.

Pulmonary arterial reactivity during induced infection of single lung allografts☆

Infection is a major cause of mortality in the first year following single lung transplantation and is a distinct risk factor for the development of obliterative bronchiolitis. However, little is known about changes in pulmonary vascular activity in the setting of infection, which might affect and limit function of the graft. Therefore, the aim of this study was to determine how acute infection altered pulmonary arterial reactivity in single lung allografts. Such information could help to develop better diagnostic and therapeutic targets to improve outcome when grafts become infected. Following single lung transplantation, dogs were immunosuppressed with methylprednisolone acetate, cyclosporine and azathioprine. On postoperative day 5, infection was induced in one group of dogs by endobronchial inoculation of antibiotic resistant Eschericia coli (infection group, n=5); in the second group, the same amount of culture media without bacteria was flushed into the bronchus (control group, n=4). All animals were medicated under the same drug protocol. On postoperative day 8, lungs were removed, reviewed for histological assessment, pulmonary arteries were isolated, cut into rings and suspended for pharmacological characterization in organ chambers. With acute infections, contractions to phenylephrine and angiotensin-1, but not endothelin-1, were reduced in pulmonary arteries with but not without endothelium. Inhibition of nitric oxide synthase with N(G)-monomethyl-L-arginine, monoacetate salt (L-NMMA) restored these contractions. Endothelium-dependent relaxations to adenosine diphosphate and calcium ionophore, which stimulate release of endothelium-derived nitric oxide by a receptor and non-receptor mediated process, respectively, were not different between groups. Relaxations to nitric oxide also were not different between groups. These results suggest that acute infection selectively reduces contractions of pulmonary arteries in part through receptor-mediated release of nitric oxide from the endothelium. Inhibiting nitric oxide during episodes of acute infection may help to improve graft perfusion during episodes of acute infection.

Methylene Blue as a Treatment for Refractory Anaphylaxis

INTRODUCTION: Protamine is a polycationic protein added to insulin and used to reverse heparin-induced anticoagulation. We describe the case of a 71-year-old man who had anaphylaxis to protamine intraoperatively, which was refractory to conventional therapy.CASE REPORT: A 71-year-old diabetic man previously on insulin NPH was undergoing cardiac surgery. He was weaned off cardiopulmonary bypass and then received a slow infusion of 100 mg protamine for heparin reversal. He immediately developed systemic hypotension, pulmonary hypertension and diffuse urticaria. Resuscitative efforts with standard therapy for anaphylaxis were unsuccessful. Cardiopulmonary bypass was reinstituted and an intraaortic balloon pump was inserted. Based on a few published cases by a single group describing methylene blue as an effective treatment for protamine-induced and anaphylactoid reactions, the patient received 100 mg intravenous methylene blue. Within minutes, the patient's systemic blood pressure and pulmonary artery pressure normalized, and he was weaned off pressors and bypass without using protamine. Thirty days later, skin testing to protamine and protamine-containing insulin preparations was positive.CONCLUSIONS: The mechanism of protamine-induced reactions is uncertain. In addition to being IgE-mediated, it is speculated that protamine directly leads to severe vasodilation by causing release of endothelium-derived relaxing factor and nitric oxide, which is inhibited by methylene blue.Our case supports methylene blue as an adjunctive treatment for refractory anaphylaxis and suggests that it may be prudent to skin test diabetics with previous protamine exposure to exclude protamine allergy. Increased awareness of this diagnosis and potential therapy among allergists, anesthesiologists, and surgeons may prevent perioperative fatalities from refractory anaphylaxis. INTRODUCTION: Protamine is a polycationic protein added to insulin and used to reverse heparin-induced anticoagulation. We describe the case of a 71-year-old man who had anaphylaxis to protamine intraoperatively, which was refractory to conventional therapy. CASE REPORT: A 71-year-old diabetic man previously on insulin NPH was undergoing cardiac surgery. He was weaned off cardiopulmonary bypass and then received a slow infusion of 100 mg protamine for heparin reversal. He immediately developed systemic hypotension, pulmonary hypertension and diffuse urticaria. Resuscitative efforts with standard therapy for anaphylaxis were unsuccessful. Cardiopulmonary bypass was reinstituted and an intraaortic balloon pump was inserted. Based on a few published cases by a single group describing methylene blue as an effective treatment for protamine-induced and anaphylactoid reactions, the patient received 100 mg intravenous methylene blue. Within minutes, the patient's systemic blood pressure and pulmonary artery pressure normalized, and he was weaned off pressors and bypass without using protamine. Thirty days later, skin testing to protamine and protamine-containing insulin preparations was positive. CONCLUSIONS: The mechanism of protamine-induced reactions is uncertain. In addition to being IgE-mediated, it is speculated that protamine directly leads to severe vasodilation by causing release of endothelium-derived relaxing factor and nitric oxide, which is inhibited by methylene blue. Our case supports methylene blue as an adjunctive treatment for refractory anaphylaxis and suggests that it may be prudent to skin test diabetics with previous protamine exposure to exclude protamine allergy. Increased awareness of this diagnosis and potential therapy among allergists, anesthesiologists, and surgeons may prevent perioperative fatalities from refractory anaphylaxis.

Activation of endothelial Ca2+‐activated potassium channels can improve endothelial function in basilar arteries from diabetic rats

Endothelium-dependent relaxation of isolated rat basilar artery to acetylcholine (ACh) can be fully accounted for by the release of endothelium-derived NO. In addition, activation of endothelial Ca2+-activated potassium channels (KCa) plays a key role in modulating ACh-stimulated production of endothelium-derived nitric oxide (NO) in this artery. Thus, we have investigated whether activation of endothelial small (SKCa) and intermediate (IKCa) KCa by 1-ethyl-2-benzimidazolinone (1-EBIO) can improve endothelial function in basilar arteries from diabetic rats. Relaxations to ACh (0.01–30 μM) were significantly depressed in isolated basilar arteries from Streptozotocin-treated (STZ) diabetic rats compared to vessels from control animals. In contrast, relaxation of basilar arteries evoked by 1-EBIO (1–300 μM), was not significantly different in arteries from STZ and control rats. Application of low concentrations of 1-EBIO (1–5 μM) significantly enhanced ACh-evoked relaxations in basilar arteries from STZ-treated rats. Pre-incubation with L-NAME abolished relaxations to ACh both in the presence and absence of 1-EBIO indicating that the enhancement of ACh-evoked responses was due to increased production of NO. Quantitative real-time PCR analysis demonstrated that mRNA levels for both SKCa and IKCa were not significantly different in basilar arteries from control and STZ-treated rats. It is concluded that activation of KCa may significantly improve endothelial function in basilar arteries from STZ-treated diabetic rats. Supported by CIHR.

The role of L-arginine following trauma and blood loss

Vascular endothelial cells control vascular smooth muscle tone via the release of nitric oxide. Following adverse circulatory conditions, namely trauma and hemorrhage, endothelial cell dysfunction occurs, leading to a decrease in the release of endothelium-derived nitric oxide, which contributes to further alterations in tissue perfusion and organ function. Early administration of L-arginine (the precursor of nitric oxide) and the substrate for nitric oxide synthase in vascular endothelial cells has been found to restore the depressed organ blood flow and to reduce tissue injury following shock. This improvement in cardiovascular function was associated with restoration of the depressed cell-mediated immune responses and attenuation of the massive inflammatory response encountered under such conditions. Furthermore, the excessive infiltration of the liver with neutrophils following trauma-hemorrhage was decreased by L-arginine administration, thereby reducing hepatic injury. In addition, L-arginine treatment decreased the inflammatory response at the site of trauma and the improved wound-healing process following blood loss. Despite those promising results in animal models at present, none of the published clinical trials has demonstrated efficacy of L-arginine at doses above standard dietary practices on the outcome in critically ill surgical patients, besides the reduction in infectious complications.

Functional effects of nitric oxide-releasing aspirin on vein conduits of diabetic patients undergoing CABG

Background Type 2 diabetes mellitus (DM) is known to negatively affect biological properties of venous vasculature, and, particularly, to reduce endothelium-derived nitric oxide release. This condition might influence venous graft function following coronary artery bypass surgery (CABG). The aim of this study was to evaluate the functional effects of a NO-releasing aspirin (NORA) on vein grafts (VG) of diabetics and control patients undergoing elective CABG. Methods In 40 consecutive ischemic heart disease patients, the effects of NORA were tested on segments of saphenous vein conduits harvested during elective CABG. Twenty patients had type-2 DM (mean age 69 ± 2), whereas 20 patients had no DM (NDM) and represented the control group (mean age 67 ± 4). Functional responses were tested by exposing VGs to NORA and to standard vasoactive agents in an organ-bath preparation. Histological features of VGs were also assessed by light and electronic microscopy. Results Significant impairment of endothelial-dependent vasodilation (acetylcholine induced) was documented in VGs of DM subjects. NORA induced a significant and comparable vascular relaxation in all venous segments of NDM and DM patients (56 ± 12% of maximal relaxation vs 61 ± 11% in the control group, respectively). Histology showed variable extent of vascular layer and cellular abnormalities in VGs of diabetics (intimal hyperplasia, calcific deposition, endothelial cell degeneration) likely responsible of the endothelial functional impairment, whereas control group VG showed preserved structures. Conclusions This preliminary study confirms the impairment of endothelium-dependent vasodilative property of VGs in DM patients. It also indicates that NORA effectively induces vasodilation of VGs which was effective also in DM patients thereby representing a promising therapy for diabetics undergoing CABG with the use of VGs, although further studies are mandatory to conclusively assess the safety and benefits of this pharmacological agent.

Translocation of endothelial nitric oxide synthase: Another feat of amlodipine, a cardiovascular jack-of-all-trades

See article by Batova et al. [12] (pages 478–485) in this issue. The antihypertensive drug amlodipine (Norvasc®) is a long-acting, third-generation Ca2+ channel blocker that has been shown to limit the progression of atherosclerosis and to decrease the incidence of cardiovascular events in humans [1]. It is generally well tolerated and often used in combination with diuretics, angiotensin-converting enzyme (ACE) inhibitors, angiotensin II receptor antagonists and statins [2–4]. Amlodipine exhibits nanomolar affinity to dihydropyridine as well as benzothiazepine and phenylalkylamine binding sites of voltage-dependent L-type Ca2+ channels [5]. It is several-fold more lipophilic than the first- and second-generation Ca2+ channel blockers and adopts a well-defined position in the bilayer membrane [6]. It has been suggested that partitioning of amlodipine to membranes results in beneficial modification of the structure of cholesterol-rich membranes of vascular smooth muscle cells, an effect that might contribute to atheroprotection [7]. Besides the well-documented inhibition of L-type Ca2+ channels, amlodipine exerts additional effects that contribute to the improvement of vascular function in hypertension and ischemic heart disease [8]. There is both in vitro and in vivo evidence indicating that dihydropyridines including amlodipine act as antioxidants preventing the progression of atherosclerosis by interfering with lipid peroxidation. However, amlodipine has pharmacological properties distinct from other Ca2+ channel blockers. Probably most relevant to the antihypertensive and antiplatelet effects of the drug is the release of endothelium-derived nitric oxide (NO). …

Experimental Study of Effect of Fontan Circuit on Pulmonary Microcirculation

Pulsatile pulmonary blood flow plays an important role in regulating shear-stress-mediated release of endothelium-derived nitric oxide and endothelin-1, and it reduces pulmonary vascular resistance by passive capillary recruitment. The aim of this study was to demonstrate changes in pulmonary capillary structure and endothelial function induced by the chronic nonpulsatile flow of the Fontan circulation. A canine model with nonpulsatile flow in the right lung was established, and sacrificed 3 months later. Compared to the left lung, wall thickness of the pulmonary arterioles was thinner, endothelin-1 expression was weaker, endothelial nitric oxide synthase activity was stronger, and there was a good correlation between the histomorphometric and immunohistochemical findings. These data indicate that long-term nonpulsatile flow can lead to endothelial dysfunction, which may be involved in distention and vascular structure remodeling due to the increase in pulmonary vascular resistance; but it also can lead to increased patency of the arteriovenous shunt, which might be at least partly involved in pulmonary arteriovenous fistula development and exercise limitation after the Fontan operation.

Exploring Vascular Benefits of Endothelium-Derived Nitric Oxide

Although the regulation of arterial blood flow has been a subject of intensive medical research, the precise circulatory mechanisms involved are still not fully understood. It has been increasingly recognized that the endothelium plays a vital role in regulating vascular tone, structure, and function. A seminal discovery was made with the identification of endothelium-derived relaxing factor, a key mediator of vasodilation, which was later identified as nitric oxide (NO). Nitric oxide is synthesized from the amino acid L-arginine in the endothelium. Decreased bioavailability of NO is associated with arterial stiffness, hypertension, atherosclerosis, and cardiovascular disease (CVD). Nebivolol is a novel beta-blocker that is highly selective for beta1-adrenergic receptors. Nebivolol also causes vasodilation through a mechanism involving endothelium-derived NO. In clinical studies in hypertensive subjects, nebivolol significantly improves vasodilator responses to endothelium-dependent agonists such as acetylcholine. In addition, nebivolol significantly reduces pulse wave velocity (PWV), a measure of arterial stiffness, whereas the beta-blocker atenolol has no effect on PWV. Because endothelial dysfunction and arterial stiffness play an integral part in the early atherosclerotic process and are associated with poor outcomes and increased mortality, independent of blood pressure, the ability of nebivolol to enhance release of endothelium-derived NO may have significant clinical implications for the use of this agent in the treatment of hypertension and CVD.

MECHANISMS UNDERLYING RELAXATION OF RABBIT AORTA BY BAY 41‐2272, A NITRIC OXIDE‐INDEPENDENT SOLUBLE GUANYLATE CYCLASE ACTIVATOR

1. The compound BAY 41-2272 (5-cyclopropyl-2-[1-(2-fluoro-benzyl)-1H-pyrazolo[3,4-b]pyridin-3-yl]-pyrimidin-4-ylamine) has been described as a potent, nitric oxide (NO)-independent, stimulator of soluble guanylate cyclase. In the present study, the mechanisms underlying the relaxant effect of BAY 41-2272 in endothelium-intact and -denuded precontracted rabbit aortic rings were investigated. 2. Male New Zealand white rabbits were anaesthetized with pentobarbital sodium. Aortic rings were transferred to 10 mL organ baths containing oxygenated and warmed Krebs' solution. Tissues were connected to force-displacement transducers and changes in isometric force were recorded. Aortic rings were precontracted submaximally with phenylephrine (1 micromol/L). 3. The addition of BAY 41-2272 (0.01-10 micromol/L) to the organ bath produced concentration-dependent relaxations of the aortic rings with a higher potency in endothelium-intact (pEC50 6.59 +/- 0.05) compared with endothelium-denuded (pEC50 6.19 +/- 0.04; P < 0.05) preparations. No differences in maximal responses were observed in either preparation. The NO synthesis inhibitor NG-nitro-L-arginine methyl ester (100 micromol/L) produced a 2.1-fold rightward shift in endothelium-intact (P < 0.01) rings, but had no effect in endothelium-denuded rings. The soluble guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ; 1 micromol/L) caused significant rightward shifts of the concentration-response curves to BAY 41-2272 of 4.9- and 2.6-fold in endothelium-intact and -denuded rings, respectively. The phosphodiesterase-5 inhibitor sildenafil (0.1 micromol/L) significantly potentiated the relaxant effects of BAY 41-2272 in both endothelium-intact and -denuded rings. 4. At 1 micromol/L, BAY 41-2272 significantly elevated the aortic cGMP content above basal levels in both endothelium-intact and -denuded rings. Furthermore, ODQ reduced BAY 41-2272-elicited increases in cGMP content by 17 and 90% in endothelium-intact and -denuded rings, respectively (P < 0.01). 5. In conclusion, BAY 41-2272 potently relaxes endothelium-intact and -denuded rabbit aortic rings. The basal release of endothelium-derived NO enhances BAY 41-2272-induced relaxations, suggesting a synergistic effect of BAY 41-2272 and NO on soluble guanylate cyclase. In addition, the endothelium-independent relaxation involves both GMP-dependent and -independent mechanisms.