Regular ArticleInhibition of Purified Nitric Oxide Synthase from Rat Cerebellum and Macrophage by L-Arginine Analogs
Abstract
The inhibition of nitric oxide synthase (NOS) activity by a variety of L-arginine-related compounds has been investigated. The inhibitory properties of NG-amino-, NG-methyl-, NG-hydroxy-, NG-ethyl-, NG-allyl-, NG,NG-dimethyl-, NG-methoxy-L-arginine, and several other L-arginine derivatives were compared in NOS purified from both macrophage and rat cerebellum, Also, these compounds were tested for their potential as alternate substrates by determining their ability to elicit NADPH consumption by NOS. NG-Methoxy-L-arginine appears to be an alternate substrate for NOS, whereas most other L-arginine analogs, except for the biosynthetic intermediate NG-hydroxy-L-arginine, do not elicit significant enzyme turnover.
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Nitric oxide interaction with insect nitrophorins and possibilities for the electron configuration of the {feno} <sup>6</sup> complex
2008, The Smallest Biomolecules: Diatomics and their Interactions with Heme ProteinsThe nitrophorins are NO-carrying heme proteins that are found in the saliva of two species of blood-sucking insects, the kissing bug (Rhodnius prolixus) and the bedbug (Cimex lectularius). In both insects NO is bound to the ferric form of the protein, which gives rise to Kds in the micromolar to nanomolar range, and thus upon injection of the saliva into the tissues of the victim the NO can dissociate to cause vasodilation and inhibition of platelet aggregation. The structures of the proteins from each of these insects are unique, and each has a large component of β-sheet structure, which is unusual for heme proteins. While the R. prolixus nitrophorins increase the effectiveness of their NO-heme proteins by also binding histamine, secreted by the victim in response to the bite, to the heme, the Cimex nitrophorin does not bind histamine but rather binds two molecules of NO reversibly, one to the heme and the other to the cysteine thiolate which serves as the heme ligand in the absence of NO. This requires homolytic cleavage of the Fe—S–Cys bond, which produces an EPR-active Fe(II)—NO complex having the {FeNO}7 electron configuration and a Cys—SNO. This reaction is reversible at low pH. Investigations of the R. prolixus nitrophorins and their axial ligand complexes by NMR and vibrational spectroscopies have allowed detailed characterization of the complexes, and investigation of site-directed mutants by spectroelectrochemistry and NMR spectroscopy has allowed pinpointing the importance of certain charged, distal pocket and belt residues in stabilizing the Fe(III) form of the protein that allows NO to be released into the tissues of the victim. For the Rhodnius nitrophorins, the heme of the {FeNO}6 stable NO complex could have the limiting electron configurations Fe(III)—NO• or Fe(II)—NO+. While vibrational spectroscopy suggests the latter and Mössbauer spectroscopy cannot differentiate between a purely diamagnetic Fe(II) center and a strongly antiferromagnetically coupled Fe(III)—NO• center, the strong ruffling of the heme (with alternate meso-carbons shifted significantly above and below the mean plane of the porphyrin, and concomitant shifts of the β-pyrrole carbons above and below the mean plane of the ring, to produce a very nonplanar porphyrin macrocycle) may suggest at least an important contribution of the latter. The strong ruffling would help to stabilize the (dxz, dyz)4 (dxy)1 electron configuration of low-spin Fe(III) (but not low-spin Fe(II)), and the dxy orbital does not have correct symmetry for overlap with the half-filled π* orbital of NO. This Fe(III)—NO• electron configuration would facilitate reversible dissociation of NO.
Partial nicotinic receptor blockade unmasks a modulatory role of nitric oxide on urethral striated neuromuscular transmission
2005, Nitric Oxide - Biology and ChemistryThe objective of this study was to investigate the possible modulatory role of endogenous nitric oxide (NO) production on the urethral striated muscle (USM) function in the sheep urethra. Significant NO synthase (NOS) activity was measured in both the particulate and cytosolic fractions of USM homogenates. NOS activity was calcium-dependent and showed greater inhibition by NOS inhibitors selective of the neural NOS isoform (nNOS). nNOS immunoreactivity was present in intramural nerves as well as in the sarcolemma of some striated fibers, being denser at the neuromuscular junction (NMJ). Double immunolabeling showed co-localization of nNOS with both α-bungarotoxin and choline acetyltransferase, at the USM endplates. For the first time, functional data support a role of NO on the USM contractility “in vitro,” which became evident following partial nicotinic receptor inactivation with low concentrations of D-tubocurarine. Only under D-tubocurarine (0.25 μM) treatment, different NOS inhibitors, specially NG-propyl-l-arginine, as well as the guanylate cyclase inhibitor ODQ, all showed a significant enhancing effect on contractions induced by electrical field stimulation of intrinsic somatic nerves. These data suggest that local production of NO at the urethral NMJ may modulate release and/or action of acetylcholine on motor endplates by cyclic GMP-mediated effects. This modulatory action could be especially relevant when neuromuscular transmission at the USM is impaired.
Nitric oxide interaction with insect nitrophorins and thoughts on the electron configuration of the {FeNO}<sup>6</sup> complex
2005, Journal of Inorganic BiochemistryThe nitrophorins are NO-carrying heme proteins that are found in the saliva of two species of blood-sucking insects, the kissing bug (Rhodnius prolixus) and the bedbug (Cimex lectularius). In both insects the NO is bound to the ferric form of the protein, which gives rise to Kds in the micromolar to nanomolar range, and thus upon injection of the saliva into the tissues of the victim the NO can dissociate to cause vasodilation and inhibition of platelet aggregation. The structures of the proteins from each of these insects are unique, and each has a large component of β-sheet structure, which is unusual for heme proteins. While the Rhodnius nitrophorins increase the effectiveness of their NO–heme proteins by also binding histamine, secreted by the victim in response to the bite, to the heme, the Cimex nitrophorin does not bind histamine but rather binds two molecules of NO reversibly, one to the heme and the other to the cysteine thiolate which serves as the heme ligand in the absence of NO. This requires homolytic cleavage of the Fe–S–Cys bond, which produces an EPR-active Fe(II)–NO complex having the {FeNO}7 electron configuration. For the Rhodnius nitrophorins, the heme of the {FeNO}6 stable NO complex could have the limiting electron configurations Fe(III)–NO or Fe(II)–NO+. While vibrational spectroscopy suggests the latter and Mössbauer spectroscopy cannot differentiate between a purely diamagnetic Fe(II) center and a strongly antiferromagnetically coupled Fe(III)–NO center, the strong ruffling of the heme (with alternate meso-carbons shifted significantly above and below the mean plane of the porphyrin, and concomitant shifts of the β-pyrrole carbons above and below the mean plane of the porphyrin ring, to produce a very nonplanar porphyrin macrocycle) may suggest at least an important contribution of the latter. The strong ruffling would help to stabilize the (dxz, dyz)4(dxy)1 electron configuration of low-spin Fe(III) (but not low-spin Fe(II)), and the dxy orbital does not have correct symmetry for overlap with the half-filled π* orbital of NO. This Fe(III)–NO electron configuration would facilitate reversible dissociation of NO.
Accelerated functional recovery and neuroprotection by agmatine after spinal cord ischemia in rats
2000, Neuroscience LettersTreatment with agmatine, decarboxylated arginine, proved to be non-toxic and to exert neuroprotective effects in several models of neurotoxic and ischemic brain and spinal cord injuries. Here we sought to find out whether agmatine treatment would also prove beneficial in a rat spinal cord ischemia model (balloon occlusion of the abdominal aorta bellow the branching point of the left subclavian artery for 5 min). Agmatine was injected (100 mg/kg, i.p.) 5 min after beginning of re-perfusion and again once daily for the next 3 post-operative days. Motor performance (‘combined motor score’) was recorded for up to 17 days post-operative and motoneuron cell counts (in representative spinal cord sections) performed on the 17th post-operative day. Agmatine treatment was found to accelerate recovery of motor deficits and to prevent the loss of motoneurons in the spinal cord after transient ischemia. Together, the present and previous findings demonstrate that agmatine is an efficacious neuroprotective agent and that this naturally occurring non-toxic compound should be tried for therapeutic use after neurotrauma and in neurodegenerative diseases.
Modulation of Polymorphonuclear Leukocytes Function by Nitric Oxide
2000, Thrombosis ResearchRecognition of the endothelium-derived relaxation factor as nitric oxide (NO) gave rise to an impression that NO was synthesised only by the endothelial lining of the vessel wall. Later it was found that NO is synthesized constitutively by the enzyme nitric oxide synthase (NOS) in various cells. However, inflammatory cytokines can induce NOS (known as inducible NOS [iNOS]) activity in all the somatic cells. Blood cells, such as eosinophils, platelets, neutrophils, monocytes, and macrophages, also synthesize NO. Among them, polymorphonuclear leukocytes (PMNs) constitute an important proportion and are also the major participants in a number of pathological conditions with suggestive involvement of NO. PMNs can synthesize NO at rates similar to endothelial cells, thus suggesting the importance of PMN-derived NO in various physiological and pathological conditions. Most of the studies so far focus on the peripheral PMNs, while studies on PMNs after emigration are limited, thus warranting systematic studies on PMNs from both sources. The role of the endothelial NOS (eNOS) and functions of NO derived from the endothelial cells has been studied extensively. However, understanding of the PMNs NOS and its regulatory role in their function is unraveling. The present review summarizes the modulatory role of NO on PMNs functions and points out the discrepancies relating to presence of NOS in PMNs. This information will be helpful in understanding the importance of NO in physiological and pathological conditions associated with PMNs.
Selective inhibitors of neuronal nitric oxide synthase - Is no NOS really good NOS for the nervous system?
1997, Trends in Pharmacological SciencesIt 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.