Endothelium-derived Relaxing Factor Activity Research Articles

A paradigm shift for local blood flow regulation

a remarkable example of physiological regulation is the coordination between metabolic rate and local blood flow in microscopic volumes of an organ. In skeletal muscle an increase in oxygen consumption (Vo2) over a wide range evokes a proportional (∼5 to 6 × Vo2) increase in blood flow ([29][

World Health Organization Pulmonary Hypertension Group 2: Pulmonary hypertension due to left heart disease in the adult—a summary statement from the Pulmonary Hypertension Council of the International Society for Heart and Lung Transplantation

Pulmonary hypertension associated with left heart disease is the most common form of pulmonary hypertension encountered in clinical practice today. Although frequently a target of therapy, its pathophysiology remains poorly understood and its treatment remains undefined. Pulmonary hypertension in the context of left heart disease is a marker of worse prognosis and disease severity, but whether its primary treatment is beneficial or harmful is unknown. An important step to the future study of this important clinical problem will be to standardize definitions across disciplines to facilitate an evidence base that is interpretable and applicable to clinical practice. In this current statement, we provide an extensive review and interpretation of the current available literature to guide current practice and future investigation. At the request of the Pulmonary Hypertension (PH) Council of the International Society for Heart and Lung Transplantation (ISHLT), a writing group was assembled and tasked to put forth this document as described above. The review process was facilitated through the peer review process of the Journal of Heart and Lung Transplantation and ultimately endorsed by the leadership of the ISHLT PH Council.

Hydrogen Sulfide as Endothelium-Derived Hyperpolarizing Factor Sulfhydrates Potassium Channels

Nitric oxide, the classic endothelium-derived relaxing factor (EDRF), acts through cyclic GMP and calcium without notably affecting membrane potential. A major component of EDRF activity derives from hyperpolarization and is termed endothelium-derived hyperpolarizing factor (EDHF). Hydrogen sulfide (H(2)S) is a prominent EDRF, since mice lacking its biosynthetic enzyme, cystathionine γ-lyase (CSE), display pronounced hypertension with deficient vasorelaxant responses to acetylcholine. The purpose of this study was to determine if H(2)S is a major physiological EDHF. We now show that H(2)S is a major EDHF because in blood vessels of CSE-deleted mice, hyperpolarization is virtually abolished. H(2)S acts by covalently modifying (sulfhydrating) the ATP-sensitive potassium channel, as mutating the site of sulfhydration prevents H(2)S-elicited hyperpolarization. The endothelial intermediate conductance (IK(Ca)) and small conductance (SK(Ca)) potassium channels mediate in part the effects of H(2)S, as selective IK(Ca) and SK(Ca) channel inhibitors, charybdotoxin and apamin, inhibit glibenclamide-insensitive, H(2)S-induced vasorelaxation. H(2)S is a major EDHF that causes vascular endothelial and smooth muscle cell hyperpolarization and vasorelaxation by activating the ATP-sensitive, intermediate conductance and small conductance potassium channels through cysteine S-sulfhydration. Because EDHF activity is a principal determinant of vasorelaxation in numerous vascular beds, drugs influencing H(2)S biosynthesis offer therapeutic potential.

Effects of Glutathione-depleting Drug Buthionine Sulfoximine and Aging on Activity of Endothelium-derived Relaxing and Contracting Factors in Carotid Artery of Sprague–Dawley Rats

The role of the antioxidant glutathione (GSH) in mediating endothelial (dys)function, and how that role may depend on age, is unclear. The main purpose of the current study was to investigate the effect of 10-day treatment with the GSH-depleting drug l-buthionine sulfoximine (BSO) on endothelium-derived relaxing factor and endothelium-derived contracting factor activities in the isolated common carotid artery (CCA) of Adult and Aging animals. CCA blood pressure and flow were unaffected by age or BSO. Endothelium-derived relaxing factor activity, examined in precontracted CCA as relaxation to cumulative acetylcholine (ACh), was largely nitric oxide synthase (NOS) mediated and was not different between Adult and Aging animals at lower ACh; however, at higher ACh, relaxation was blunted in Aging CCA, an effect abolished by cyclooxygenase (COX) inhibition but not by NOS inhibition nor by the reactive oxygen species (ROS) inhibitors 4-hydroxy-TEMPO or Mn(III)tetrakis(1-methyl-4-pyridyl)porphyrin,tetratosylate,hydroxide. Specific examination of endothelium-derived contracting factor activity in quiescent NOS-inhibited CCA established that higher ACh elicited a contractile response, ∼3.5-fold greater in Aging versus Adult CCA, which was abolished by COX-1-specific inhibition but unaffected by ROS inhibitors. Aging was unrelated to changes in liver or vascular tissue GSH or ROS content. BSO was effective in significantly decreasing GSH and increasing ROS content in both animal cohorts. However, NOS-mediated endothelium-derived relaxing factor activity was well preserved and age-related COX-mediated endothelium-derived contracting factor activity was unaffected in response to these BSO-induced perturbations, as were exogenous H2O2-stimulated NOS/non-NOS-mediated relaxation and COX-mediated contractile activities. These data suggest that, regardless of age, chronic partial depletion of GSH in vivo does not necessarily cause endothelium-dependent vasomotor dysfunction.

NO control: nitric oxide directly regulates substrate delivery to NOS. Focus on “Nitric oxide can acutely modulate its biosynthesis through a negative feedback mechanism on l-arginine transport in cardiac myocytes”

although the amino acid arginine is commonly associated with nitric oxide (NO) production via NO synthase (NOS), it also participates in the synthesis of urea, creatine, creatinine, agmatine, polyamines, as well as overall protein synthesis. Furthermore, it also influences hormone release (insulin,

The role of nitric oxide in the pathophysiology of intimal hyperplasia

Since its discovery, nitric oxide (NO) has emerged as a biologically important molecule and was even named Molecule of the Year by Science magazine in 1992. Specific to our interests, NO has been implicated in the regulation of vascular pathology. This review begins with a summary of the molecular biology of NO, from its discovery to the mechanisms of endogenous production. Next, we turn our attention to describing the arterial injury response of neointimal hyperplasia, and we review the role of NO in the pathophysiology of neointimal hyperplasia. Finally, we review the literature regarding NO-based therapies. This includes the development of inhalational-based NO therapies, systemically administered L-arginine and NO donors, NO synthase gene therapy, locally applied NO donors, and NO-releasing prosthetic materials. By reviewing the current literature, we emphasize the tremendous clinical potential that NO-based therapies can have on the development of neointimal hyperplasia.

The lung in the balance: arginine, methylated arginines, and nitric oxide

the lung is a major source of nitric oxide (NO), be it from nitric oxide synthase (NOS) III in the endothelium of the vast pulmonary circulation, NOS II in the epithelium of the large surface area of the airways, or NOS I in the nonadrenergic noncholinergic nerves ([6][1]). NO produced in the lung

The effects of Δ9‐tetrahydrocannabinol in rat mesenteric vasculature, and its interactions with the endocannabinoid anandamide

1 Delta9-tetrahydrocannabinol (THC) produces varying effects in mesenteric arteries: vasorelaxation (third-order branches, G3), modest vasorelaxation (G2), no effect (G1) and vasoconstriction (the superior mesenteric artery, G0). 2 In G3, vasorelaxation to THC was inhibited by pertussis toxin, but was unaffected by the CB1 receptor antagonist, AM251 (1 microM), incubation with the TRPV1 receptor agonist capsaicin (10 microM, 1 h), the TRPV1 receptor antagonist capsazepine (10 microM) or de-endothelialisation. 3 In G3, vasorelaxation to THC was inhibited by high K+ buffer, and by the following K+ channel inhibitors: charybdotoxin (100 nM), apamin (500 nM) and barium chloride (30 microM), but not by 4-aminopyridine, glibenclamide or tertiapin. 4 In G3, THC (10 and 100 microM) inhibited the contractile response to Ca2+ in a Ca2+-free, high potassium buffer, indicating that THC blocks Ca2+ influx. 5 In G0, the vasoconstrictor responses to THC were inhibited by de-endothelialisation and SR141716A (100 nM), but not by the endothelin (ET(A)) receptor antagonist FR139317 (1 microM).THC (1 and 10 microM) antagonised vasorelaxation to anandamide in G3 but not G0. THC did not antagonise the noncannabinoid verapamil, capsaicin or the CB1 receptor agonist CP55,940. 6 THC (10 and 100 microM) inhibited endothelium-derived relaxing factor (EDHF)-mediated responses to carbachol in a manner similar to the gap junction inhibitor 18alpha-glycyrrhetinic acid. 7 These data show that THC causes vasorelaxation through activation of K+ channels and inhibition of Ca2+ channels, and this involves non-CB1, non-TRPV1 but G-protein-coupled receptors. In G0, THC does not cause relaxation and at high concentrations causes contractions. Importantly, THC antagonises the effects of anandamide, possibly through inhibition of EDHF activity.

Con: Intraoperative use of nitric oxide for treatment of pulmonary hypertension in patients with congenital heart disease is not effective

PATIENTS with pulmonary hypertension (PHTN) have had no effective treatment available because of the lack of a selective pulmonary vasodilator. In 1980, a labile humoral factor produced by endothelial cells (endothelium-derived relaxing factor) was shown to mediate vascular dilation. 1 Palmer RM Ferrige AG Moncada S Nitric oxide release accounts for the biological activity of endothelium-derived relaxing factor. Nature. 1987; 327: 524-526 Crossref PubMed Scopus (9259) Google Scholar Nitric oxide (NO) has been shown to be biologically identical to endothelium-derived relaxing factor. Once released, NO diffuses into vascular smooth muscle cells; activates soluble guanylate cyclase, which increases the concentration of cyclic guanosine monophosphate and initiates a cascade of events that results in smooth muscle relaxation. 2 Fiscus RR Molecular mechanisms of endothelium-mediated vasodilation. Semin Thromb Hemost. 1988; 14: 12-22 PubMed Google Scholar In vitro and in vivo evidence indicate that NO may be an important mediator of pulmonary vascular tone. 2 Fiscus RR Molecular mechanisms of endothelium-mediated vasodilation. Semin Thromb Hemost. 1988; 14: 12-22 PubMed Google Scholar , 3 Furchgott RF Vanhoutte PM Endothelium-derived relaxing and contracting factors. FASEB J. 1989; 3: 2007-2018 Crossref PubMed Scopus (1669) Google Scholar Many reports have appeared evaluating the effect of NO as a selective pulmonary vasodilator in the treatment of PHTN. It has not been clearly established, however, whether NO is effective for patients with congenital heart disease during cardiac surgery and whether outcome is improved when NO is used.

Neuronal and endothelial nitric oxide synthase gene knockout mice.

Targeted disruption of the neuronal nitric oxide synthase (nNOS) and endothelial nitric oxide synthase (eNOS) genes has led to knockout mice that lack these isoforms. These animal models have been useful to study the roles of nitric oxide (NO) in physiologic processes. nNOS knockout mice have enlarged stomachs and defects in the inhibitory junction potential involved in gastrointestinal motility. eNOS knockout mice are hypertensive and lack endothelium-derived relaxing factor activity. When these animals are subjected to models of focal ischemia, the nNOS mutant mice develop smaller infarcts, consistent with a role for nNOS in neurotoxicity following cerebral ischemia. In contrast, eNOS mutant mice develop larger infarcts, and show a more pronounced hemodynamic effect of vascular occlusion. The knockout mice also show that nNOS and eNOS isoforms differentially modulate the release of neurotransmitters in various regions of the brain. eNOS knockout mice respond to vessel injury with greater neointimal proliferation, confirming that reduced NO levels seen in endothelial dysfunction change the vessel response to injury. Furthermore, eNOS mutant mice still show a protective effect of female gender, indicating that the mechanism of this protection cannot be limited to upregulation of eNOS expression. The eNOS mutant mice also prove that eNOS modulates the cardiac contractile response to ss-adrenergic agonists and baseline diastolic relaxation. Atrial natriuretic peptide, upregulated in the hearts of eNOS mutant mice, normalizes cGMP levels and restores normal diastolic relaxation.

Nitric Oxide: A Unique Endogenous Signaling Molecule in Vascular Biology (Nobel Lecture).

"Endothelium-derived relaxing factor (EDRF) must be nitric oxide." Although there was much experimental evidence that effects on or actions of EDRF were identical to those of nitric oxide, proposing this hypothesis certainly meant going out on a limb. However, once this discovery was made increased investigation indicated the physiological significance of nitric oxide in vascular biology.

Estradiol increases rat aorta endothelium-derived relaxing factor (EDRF) activity without changes in endothelial NO synthase gene expression: possible role of decreased endothelium-derived superoxide anion production.

Estradiol is known to exert a protective effect against atherosclerosis, but the mechanism(s) whereby this protection is mediated is/are unclear. However, estradiol-treated castrated animals exhibit increased activity of endothelium-derived relaxing factor (EDRF), which could contribute to vasculoprotection. In the present work, we investigated the molecular mechanism(s) of the enhancement of EDRF activity in the thoracic aorta of oophorectomized female rats given 17 beta-estradiol (E2, 2 or 40 micrograms/kg/day) compared to those given a placebo. The abundance in the thoracic aorta of NO synthase I, II and III mRNA (using RT-PCR) and of NO synthase I, II and III immunoreactive protein (using Western blotting) was unaltered by E2. NO synthase activity (based on arginine/citrulline conversion) in thoracic aorta homogenates did not differ significantly among the three groups, suggesting that NO production was not enhanced by E2. In contrast, lucigenin-enhanced chemiluminescence of aorta from the E2 group was decreased compared to that of the placebo group. Desendothelialization and exogenously added superoxide dismutase suggested that this difference was due to a decrease in extracellular endothelium-derived production of superoxide anion (O2-.). Experiments in cultured bovine aortic endothelial cells confirmed a decreased extracellular production of O2-. in response to ethinylestradiol (1 nM) using both lucigenin-enhanced chemiluminescence and ESR spectroscopy. Luminol-enhanced chemiluminescence revealed that ethinylestradioltreated cultured endothelial cells generated less peroxynitrite (the byproduct of NO-. and O2-. interaction) than control cells. Estradiol increases rat aorta EDRF activity in the absence of changes in endothelial NO synthase gene expression. The decreased endothelium-derived generation of O2-. in response to estrogens could account for enhanced EDRF-NO bioactivity and decreased peroxynitrite release. All of these effects could contribute to the vascular protective properties of estrogens.

Differential regulation of hepatic arterial and portal venous vascular resistance by nitric oxide and carbon monoxide in rats

Nitric oxide (NO), a gaseous mediator that accounts for the biological activity of endothelium-derived relaxing factor, has been shown to play an important role in the reduction of basal vascular tone in multiple vascular beds, including the hepatic circulation. On the other hand, recent studies have provided first evidence that endogenously generated carbon monoxide (CO) may exert vasodilatory effects in the hepatic portal vein and within sinusoids. Thus, we defined the differential role of NO and CO in the regulation of vascular resistance in the two inflows to the liver in the normal rat in vivo. Male Sprague-Dawley rats were anesthetized with pentobarbital sodium and surgically instrumented in order to study the change in hepatic arterial (R ha and portal venous vascular resistance (R pv) in response to intravenous bolus administration of either the NO-synthase inhibitor N ω-nitro-L-arginine methyl ester (L-NAME) (1 mg/kg; n = 7 animals) or of tin protoporphyrin-IX (SnPP-IX) (50 μmol/kg), a specific inhibitor of the CO-generating enzyme heme oxygenase (n = 8 animals). While L-NAME caused a substantial increase in R ha, R pv increased only slightly under these conditions. In sharp contrast, SnPP-IX did not affect R ha, but caused a profound increase in R pv. In conclusion, R ha and R pv are differentially regulated by NO and CO in the normal rat liver in vivo, i.e., NO serves as a potent vasodilator in the hepatic arterial circulation, but exerts only a minor vasodilatory effect in the portal venous vascular bed. In contrast, while there is no intrinsic CO-mediated vasodilation in the hepatic artery, CO acts to maintain portal venous vascular tone in a relaxed state.

Homocyst(e)ine decreases bioavailable nitric oxide by a mechanism involving glutathione peroxidase.

Hyperhomocyst(e)inemia is believed to injure endothelial cells in vivo through a number of mechanisms, including the generation of hydrogen peroxide (H2O2). Earlier in vitro studies demonstrated that homocyst(e)ine (Hcy) decreases the biological activity of endothelium-derived relaxing factor and that this decrease can be reversed by preventing the generation of hydrogen peroxide. Here we show that Hcy treatment of bovine aortic endothelial cells leads to a dose-dependent decrease in NOx (p = 0.001 by one-way analysis of variance) independent of endothelial nitric-oxide synthase activity or protein levels and nos3 transcription, suggesting that Hcy affects the bioavailability of NO, not its production. We hypothesized that, in addition to increasing the generation of H2O2, Hcy decreases the cell's ability to detoxify H2O2 by impairing intracellular antioxidant enzymes, specifically the intracellular isoform of glutathione peroxidase (GPx). To test this hypothesis, confluent bovine aortic endothelial cells were treated with a range of concentrations of Hcy, and intracellular GPx activity was determined. Compared with control cells, cells treated with Hcy showed a significant reduction in GPx activity (up to 81% at 250 microM Hcy). In parallel with the decrease in GPx activity, steady-state GPx mRNA levels were also significantly decreased compared with control levels after exposure to Hcy, which appeared not to be a consequence of message destabilization. These data suggest a novel mechanism by which Hcy, in addition to increasing the generation of hydrogen peroxide, may selectively impair the endothelial cell's ability to detoxify H2O2, thus rendering NO more susceptible to oxidative inactivation.

Selective inhibitors of neuronal nitric oxide synthase--is no NOS really good NOS for the nervous system?

It is now ten years since NO was shown to account for the biological activity of endothelium-derived relaxing factor (EDRF). It is also the tenth anniversary of the identification of L-NG monomethyl arginine (L-NMMA) as the very first inhibitor of NO biosynthesis. That EDRF and NO were one and the same sparked an explosion of interest in the biochemistry and pharmacology of NO which has yet to subside. In contrast, the first ever nitric oxide synthase (NOS) inhibitor slipped seamlessly into the literature virtually without comment at the time. Over the following decade, L-NMMA (and like NOS inhibitors) have proved invaluable as tools for probing the biological roles of NO in health and disease and, in particular, have increased our understanding of the function of NO in the nervous system. Further advances in this important area now require the development of inhibitors selective for the neuronal isoform of NOS (nNOS). Here, Philip Moore and Rachel Handy provide an up-to-date account of the literature regarding the biochemical and pharmacological characterization of NOS inhibitors with particular reference to compounds with greater selectivity for the nNOS isoform.

N-acetylcysteine does not influence the activity of endothelium-derived relaxing factor in vivo.

Nitric oxide forms complexes with an array of biomolecular carriers that retain biological activity. This reactivity of nitric oxide in physiological systems has led to some dispute as to whether endothelium-derived relaxing factors nitric oxide or a closely related adduct thereof, such as a nitrosothiol. In vitro bioassays used to address this question are limited by the exclusion of biological thiols that are requisite for nitrosothiol formation. Thus, the purpose of this study was to obtain insight into the identity of endothelium-derived relaxing factor in vivo. We reasoned that if endothelium-derived relaxing factor in nitric oxide, infusion of physiological concentrations of thiol would potentiate its bioactivity by analogy with effects seen in vitro, whereas nitrosothiol would be resistant to such modulation. We used venous-occlusion plethysmography to study forearm blood flow in normal subjects. Methacholine (0.3 to 10 micrograms/min) and nitroglycerin (1 to 30 micrograms/min) were infused via the brachial artery to elicit endothelium-dependent and endothelium-independent vasodilation, respectively. Dose-response determinations were made for each drug before and after an intra-arterial infusion of the reduced thiol, N-acetylcysteine, at rates estimated to achieve a physiological concentration of 1 mmol/L. Methacholine increased forearm blood flow in a dose-dependent manner. Infusion of N-acetylcysteine did not change the sensitivity (ED50, 1.7 versus 1.7 micrograms/min, P = NS) or maximal response to methacholine. In contrast, thiol increased the sensitivity to nitroglycerin (ED50, 4.7 versus 2.8 micrograms/min, P < .01). Thus, conflicting with reports in vitro, thiol does not modulate endothelium-derived relaxing factor responses in vivo. These data indicate that sulfhydryl groups are not a limiting factor for endothelium-derived relaxing factor responses in forearm resistance vessels in normal humans and are in keeping with reports that nitrosothiol contributes to endothelium-derived relaxing factor bioactivity in plasma and vascular smooth muscle. Potentiation of the effects of nitroglycerin by N-acetylcysteine can be attributed to its enhanced biotransformation to an endothelium-derived relaxing factor equivalent, such as nitrosothiol. These observations support the notion of an equilibrium between nitric oxide and nitrosothiol in biological systems that may be influenced by redox state.

Endothelial dysfunction and atherothrombotic occlusive disease.

The endothelium plays a crucial role in the regulation of vascular function through the release of locally important molecular effectors such as endothelium-derived relaxing factor (EDRF) and prostacyclin. Endothelial cells also regulate vascular patency and tissue perfusion by inhibiting platelet aggregation and thrombosis, suppressing intimal proliferation, and maintaining vascular tone. Disturbances of the regulatory functions of the endothelium contribute to the pathophysiology of various disease states, including cardiovascular disease and stroke. Most studies focused on endothelial control of vasomotion and, in particular, on the action of EDRF; many studies have also emphasised that altered endothelial control of fibrinolysis and intimal growth influence the clinical expression of atherothrombotic disease. Importantly, understanding the pathophysiological role of endothelial dysfunction may lead to new therapeutic approaches for disease states caused by vascular disease.

Genetic analysis of nitric oxide synthase isoforms: targeted mutation in mice.

Since the discovery that nitric oxide is an endogenous vasodilator responsible for endothelium-derived relaxing factor activity, nitric oxide has been found in many different cell types and implicated in many diverse biological processes. Because pharmacological blockade does not distinguish between the three major isoforms of nitric oxide synthase, the tissue and enzyme source of nitric oxide is unclear in many situations. Targeted disruption of the genes for the various isoforms of nitric oxide synthase offers a useful genetic approach to study the roles of each isoform and to examine the effects of their deletion on physiological processes in intact animals. Here we review the phenotypes of the various nitric oxide synthase mutant mice and examine what they reveal about the complexities of the nitric oxide signaling system and about molecular and physiological compensations brought into play in the absence of individual isoforms.

Comparison of endothelium-derived relaxing factor activity between nonpregnant and pregnant rats

The tension of isolated ring preparation of aorta from nonpregnant and pregnant rats was measured isometrically to study the effect of pregnancy on endothelium-derived relaxing factor activity. Contraction in response to norepinephrine and potassium chloride was greater in aortae from nonpregnant rats than in those from pregnant rats. The endothelium-dependent relaxation that was caused by acetylcholine (10 −10−3 × 10 −9 M) in aortae precontracted with norepinephrine was significantly enhanced in aortae from pregnant rats compared with the relaxation in those from nonpregnant rats. N G -nitro-L-arginine methyl ester ( l-NAME) inhibited the endothelium-dependent relaxation in both aorta from pregnant and nonpregnant rats. l-Arginine reversed the inhibition of l-NAME. Those results suggest that the enhanced endothelium-derived relaxing factor activity in rats aortae is associated with pregnancy.

Cigarette smoke-modified low density lipoprotein impairs endothelium-dependent relaxation in isolated rabbit arteries

1. 1. We investigated the effects of cigarette smoke-modified low-density lipoprotein (CS-LDL) on endothelium-dependent relaxations in various isolated rabbit arteries, and compared them with those of oxidized LDL (Ox-LDL). 2. 2. In aorta rings, acetylcholine-induced endothelium-dependent relaxations were attenuated by prein-cubation with CS-LDL and Ox-LDL. Endothelium-independent relaxations induced by sodium nitroprus-side were not modified. Similar changes were observed in rings of coronary and basilar arteries. 3. 3. These findings indicate that CS-LDL, as well as Ox-LDL, may decrease the release or activity of endothelium-derived relaxing factor in vascular endothelial cells.