Vascular endothelial cells synthesize nitric oxide from L-arginine.
TL;DR: It is demonstrated that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture and the strict substrate specificity of this reaction suggests that L- arginine is the precursor for NO synthesis in vascular endothelium cells.
Abstract: Nitric oxide (NO) released by vascular endothelial cells accounts for the relaxation of strips of vascular tissue1 and for the inhibition of platelet aggregation2 and platelet adhesion3 attributed to endothelium-derived relaxing factor4. We now demonstrate that NO can be synthesized from L-arginine by porcine aortic endothelial cells in culture. Nitric oxide was detected by bioassay5, chemiluminescence1 or by mass spectrometry. Release of NO from the endothelial cells induced by bradykinin and the calcium ionophore A23187 was reversibly enhanced by infusions of L-arginine and L-citrulline, but not D-arginine or other close structural analogues. Mass spectrometry studies using 15N-labelled L-arginine indicated that this enhancement was due to the formation of NO from the terminal guanidino nitrogen atom(s) of L-arginine. The strict substrate specificity of this reaction suggests that L-arginine is the precursor for NO synthesis in vascular endothelial cells.
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TL;DR: It is proposed that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO under pathological conditions by preventing the formation of peroxynitrite.
Abstract: Superoxide dismutase reduces injury in many disease processes, implicating superoxide anion radical (O2-.) as a toxic species in vivo. A critical target of superoxide may be nitric oxide (NO.) produced by endothelium, macrophages, neutrophils, and brain synaptosomes. Superoxide and NO. are known to rapidly react to form the stable peroxynitrite anion (ONOO-). We have shown that peroxynitrite has a pKa of 7.49 +/- 0.06 at 37 degrees C and rapidly decomposes once protonated with a half-life of 1.9 sec at pH 7.4. Peroxynitrite decomposition generates a strong oxidant with reactivity similar to hydroxyl radical, as assessed by the oxidation of deoxyribose or dimethyl sulfoxide. Product yields indicative of hydroxyl radical were 5.1 +/- 0.1% and 24.3 +/- 1.0%, respectively, of added peroxynitrite. Product formation was not affected by the metal chelator diethyltriaminepentaacetic acid, suggesting that iron was not required to catalyze oxidation. In contrast, desferrioxamine was a potent, competitive inhibitor of peroxynitrite-initiated oxidation because of a direct reaction between desferrioxamine and peroxynitrite rather than by iron chelation. We propose that superoxide dismutase may protect vascular tissue stimulated to produce superoxide and NO. under pathological conditions by preventing the formation of peroxynitrite.
7,027 citations
TL;DR: The discovery that mammalian cells generate nitric oxide, a gas previously considered to be merely an atmospheric pollutant, is providing important information about many biologic processes.
Abstract: The discovery that mammalian cells generate nitric oxide, a gas previously considered to be merely an atmospheric pollutant, is providing important information about many biologic processes. Nitric oxide is synthesized from the amino acid L-arginine by a family of enzymes, the nitric oxide synthases, through a hitherto unrecognized metabolic route -- namely, the L-arginine-nitric oxide pathway1–8. The synthesis of nitric oxide by vascular endothelium is responsible for the vasodilator tone that is essential for the regulation of blood pressure. In the central nervous system nitric oxide is a neurotransmitter that underpins several functions, including the formation of memory. . . .
6,464 citations
TL;DR: The two hypotheses to explain the pathogenesis of atherosclerosis, the "incrustation" hypothesis and the "lipid" hypothesis, are now known.
Abstract: IN the 19th century there were two major hypotheses to explain the pathogenesis of atherosclerosis: the "incrustation" hypothesis and the "lipid" hypothesis. The incrustation hypothesis of von Rokitansky,1 proposed in 1852 and modified by Duguid,2 suggested that intimal thickening resulted from fibrin deposition, with subsequent organization by fibroblasts and secondary lipid accumulation. The lipid hypothesis, proposed by Virchow3 in 1856, suggested that lipid in the arterial wall represented a transduction of blood lipid, which subsequently formed complexes with acid mucopolysaccharides; lipid accumulated in arterial walls because mechanisms of lipid deposition predominated over those of removal. The two hypotheses are now . . .
3,779 citations
TL;DR: The functional regulation of the endothelium by local hemodynamic shear stress provides a model for understanding the focal propensity of atherosclerosis in the setting of systemic factors and may help guide future therapeutic strategies.
Abstract: Atherosclerosis, the leading cause of death in the developed world and
nearly the leading cause in the developing world, is associated with systemic
risk factors including hypertension, smoking, hyperlipidemia, and diabetes
mellitus, among others. Nonetheless, atherosclerosis remains a geometrically
focal disease, preferentially affecting the outer edges of vessel bifurcations.
In these predisposed areas, hemodynamic shear stress, the frictional force
acting on the endothelial cell surface as a result of blood flow, is weaker
than in protected regions. Studies have identified hemodynamic shear stress
as an important determinant of endothelial function and phenotype. Arterial-level
shear stress (>15 dyne/cm2) induces endothelial quiescence and
an atheroprotective gene expression profile, while low shear stress (<4
dyne/cm2), which is prevalent at atherosclerosis-prone sites, stimulates
an atherogenic phenotype. The functional regulation of the endothelium by
local hemodynamic shear stress provides a model for understanding the focal
propensity of atherosclerosis in the setting of systemic factors and may help
guide future therapeutic strategies.
3,246 citations
TL;DR: A method for simultaneous evaluation of nitrate and nitrite concentrations in a microtiter plate format is developed and S-Nitrosothiols and L-arginine derivatives were found to be potential interfering agents.
3,170 citations
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TL;DR: It is demonstrated that relaxation of isolated preparations of rabbit thoracic aorta and other blood vessels by ACh requires the presence of endothelial cells, and that ACh, acting on muscarinic receptors of these cells, stimulates release of a substance(s) that causes relaxation of the vascular smooth muscle.
Abstract: Despite its very potent vasodilating action in vivo, acetylcholine (ACh) does not always produce relaxation of isolated preparations of blood vessels in vitro. For example, in the helical strip of the rabbit descending thoracic aorta, the only reported response to ACh has been graded contractions, occurring at concentrations above 0.1 muM and mediated by muscarinic receptors. Recently, we observed that in a ring preparation from the rabbit thoracic aorta, ACh produced marked relaxation at concentrations lower than those required to produce contraction (confirming an earlier report by Jelliffe). In investigating this apparent discrepancy, we discovered that the loss of relaxation of ACh in the case of the strip was the result of unintentional rubbing of its intimal surface against foreign surfaces during its preparation. If care was taken to avoid rubbing of the intimal surface during preparation, the tissue, whether ring, transverse strip or helical strip, always exhibited relaxation to ACh, and the possibility was considered that rubbing of the intimal surface had removed endothelial cells. We demonstrate here that relaxation of isolated preparations of rabbit thoracic aorta and other blood vessels by ACh requires the presence of endothelial cells, and that ACh, acting on muscarinic receptors of these cells, stimulates release of a substance(s) that causes relaxation of the vascular smooth muscle. We propose that this may be one of the principal mechanisms for ACh-induced vasodilation in vivo. Preliminary reports on some aspects of the work have been reported elsewhere.
11,871 citations
TL;DR: NO released from endothelial cells is indistinguishable from EDRF in terms of biological activity, stability, and susceptibility to an inhibitor and to a potentiator.
Abstract: Endothelium-derived relaxing factor (EDRF) is a labile humoral agent which mediates the action of some vasodilators. Nitrovasodilators, which may act by releasing nitric oxide (NO), mimic the effect of EDRF and it has recently been suggested by Furchgott that EDRF may be NO. We have examined this suggestion by studying the release of EDRF and NO from endothelial cells in culture. No was determined as the chemiluminescent product of its reaction with ozone. The biological activity of EDRF and of NO was measured by bioassay. The relaxation of the bioassay tissues induced by EDRF was indistinguishable from that induced by NO. Both substances were equally unstable. Bradykinin caused concentration-dependent release of NO from the cells in amounts sufficient to account for the biological activity of EDRF. The relaxations induced by EDRF and NO were inhibited by haemoglobin and enhanced by superoxide dismutase to a similar degree. Thus NO released from endothelial cells is indistinguishable from EDRF in terms of biological activity, stability, and susceptibility to an inhibitor and to a potentiator. We suggest that EDRF and NO are identical.
10,739 citations
TL;DR: It is demonstrated that EDRF is protected from breakdown by superoxide dismutase (SOD) and Cu2+, but not by catalase, and is inactivated by Fe2+.
Abstract: Endothelium-derived vascular relaxing factor (EDRF) is a humoral agent that is released by vascular endothelium and mediates vasodilator responses induced by various substances including acetylcholine and bradykinin. EDRF is very unstable, with a half-life of between 6 and 50 s, and is clearly distinguishable from prostacyclin. The chemical structure of EDRF is unknown but it has been suggested that it is either a hydroperoxy- or free radical-derivative of arachidonic acid or an unstable aldehyde, ketone or lactone. We have examined the role of superoxide anion (O-2) in the inactivation of EDRF released from vascular endothelial cells cultured on microcarrier beads and bioassayed using a cascade of superfused aortic smooth muscle strips. With this system, we have now demonstrated that EDRF is protected from breakdown by superoxide dismutase (SOD) and Cu2+, but not by catalase, and is inactivated by Fe2+. These findings indicate that O-2 contributes significantly to the instability of EDRF.
2,434 citations
TL;DR: An L-arginine-dependent biochemical pathway synthesizing L-citrulline and nitrite, coupled to an effector mechanism, is shown to cause this pattern of metabolic inhibition in cytotoxic activated macrophages.
Abstract: Previous studies have shown that cytotoxic activated macrophages cause inhibition of DNA synthesis, of mitochondrial respiration, and of aconitase activity in tumor target cells. An L-arginine-dependent biochemical pathway synthesizing L-citrulline and nitrite, coupled to an effector mechanism, is now shown to cause this pattern of metabolic inhibition. Murine cytotoxic activated macrophages synthesize L-citrulline and nitrite in the presence of L-arginine but not D-arginine. L-Citrulline and nitrite biosynthesis by cytotoxic activated macrophages is inhibited by NG-monomethyl-L-arginine, which also inhibits this cytotoxic effector mechanism. This activated macrophage cytotoxic effector system is associated with L-arginine deiminase activity, and the imino nitrogen removed from the guanido group of L-arginine by the deiminase reaction subsequently undergoes oxidation to nitrite. L-Homoarginine, an alternative substrate for this deiminase, is converted to L-homocitrulline with concurrent nitrite synthesis and similar biologic effects.
1,549 citations
TL;DR: It is likely that interactions between prostacyclin and NO released by the endothelium play a role in the homeostatic regulation of platelet‐vessel wall interactions.
Abstract: 1. The interactions between endothelium-derived nitric oxide (NO) and prostacyclin as inhibitors of platelet aggregation were examined. 2. Porcine aortic endothelial cells treated with indomethacin and stimulated with bradykinin (10-100 nM) released NO in quantities sufficient to account for the inhibition of platelet aggregation attributed to endothelium-derived relaxing factor (EDRF). 3. In the absence of indomethacin, stimulation of the cells with bradykinin (1-3 nM) released small amounts of prostacyclin and EDRF which synergistically inhibited platelet aggregation. 4. EDRF and authentic NO also caused disaggregation of platelets aggregated either with collagen or with U46619. 5. A reciprocal potentiation of both the anti- and the dis-aggregating activity was also observed between low concentrations of prostacyclin and authentic NO or EDRF released from endothelial cells. 6. It is likely that interactions between prostacyclin and NO released by the endothelium play a role in the homeostatic regulation of platelet-vessel wall interactions.
1,167 citations