Cytosolic and membrane-bound nitric oxide synthase.

Cytotoxic activated macrophages were sonicated and centrifuged. The activity of nitric oxide (NO) synthase was present in the supernatant and independent of Ca2+. The pellets were washed three times and treated with buffer containing 0.1% Triton X-100 or buffer alone, followed by centrifugation. The supernatant containing Triton X-100 showed NO synthase activity that was dependent on Ca2+, whereas the supernatant without the detergent had little activity. These data suggest that there are two forms of NO synthase: cytosolic and membrane-bound enzymes.     

Abundance of low molecular weight phosphotyrosine protein phosphatase in the nerve-ending fraction in the brain.

The distribution of low molecular weight phosphotyrosine protein phosphatase (LMW-PTP) in subcellular fractions of rat brain tissue was investigated by immunoblotting analysis using anti-LMW-PTP antibody. The enzyme was detected in the 105000 g precipitate in addition to the supernatant of brain homogenate, even after the precipitate was extensively washed, and was abundant in the particulate fraction of nerve endings. Nerve ending LMW-PTP was effectively solubilized by 1% Triton X-100 or 1% deoxycholate, though the enzyme was solubilized by thorough sonication. Two forms of LMW-PTP, designated as LMW-PTP-I and -II, were separated from the nerve ending-rich fraction by chromatofocusing. Nerve endings PTP-I and -II were different in molecular weight, isoelectric point and susceptibility to activators and inhibitors. The properties of nerve endings LMW-PTP-I and -II were similar to those of cytosolic LMW-PTP-I and -II. The abundance of LMW-PTP in nerve endings as well as in the cytosol suggests that this enzyme plays an important role in synaptic function.     

Partial characterization of neurotoxic esterase of human placenta

Neurotoxic esterase (NTE), the putative target for organophosphorus-induced delayed axonopathy, has been found in preparations of human placenta. The activity was primarily found in membrane-enriched fractions rather than high-speed supernatant. NTE was solubilized from a mixture of mitochondrial and microsomal membranes with Triton X-100. The crude and solubilized activities had inhibitor characteristics similar to preparations from avian brain. Because of the similarities to NTE from brain and ready availability, human placenta may be an ideal source for the bulk purification of a human form of the enzyme.     

Differential contributions of NOS isoforms in the rostral ventrolateral medulla to cardiovascular responses associated with mevinphos intoxication in the rat

The organophosphate poison mevinphos (Mev) elicits cardiovascular responses via nitric oxide (NO) produced on activation of M2 muscarinic receptors (M2R) in the rostral ventrolateral medulla (RVLM), where sympathetic vasomotor tone originates. This study further evaluated the contribution of nitric oxide synthase (NOS) isoforms at the RVLM to this process, using adult Sprague-Dawley rats. Bilateral co-microinjection into the RVLM of the selective NOS I inhibitor (250 pmol), 7-nitroindazole or N(omega)-propyl-L-arginine antagonized the initial sympathoexcitatory cardiovascular responses to Mev (10 nmol). Co-administration of a selective NOS II inhibitor, N6-(1-iminoethyl)-L-lysine (250 or 500 pmol) further enhanced these cardiovascular responses and reversed the secondary sympathoinhibitory actions of Mev. A potent NOS III inhibitor, N5-(1-iminoethyl)-L-ornithine (46 or 92 nmol) was ineffective. We also found that M2R co-localized only with NOS I- or NOS II-immunoreactive RVLM neurons. Furthermore, only NOS I or II in the ventrolateral medulla exhibited an elevation in mRNA or protein levels during the sympathoexcitatory phase, with further up-regulated synthesis of NOS II during the sympathoinhibitory phase of Mev intoxication. We conclude that whereas NOS III is not engaged, NO produced by NOS I and II in the RVLM plays, respectively, a sympathoexcitatory and sympathoinhibitory role in the cardiovascular responses during Mev intoxication.     

Novel agents targeting nitric oxide

Nitric oxide (NO) is a soluble gas continuously synthesized from the amino acid L-arginine in endothelial cells by the constitutive calcium-calmodulin-dependent enzyme nitric oxide synthase (NOS). Endothelial dysfunction has been identified as a major mechanism involved in all the stages of atherogenesis. Evaluation of endothelial function seems to have a predictive role in humans, and therapeutic interventions improving nitric oxide bioavailability in the vasculature, may improve the long-term outcome in healthy individuals, high-risk subjects or patients with advanced atherosclerosis. Several therapeutic strategies (including statins, angiotensin converting enzyme inhibitors/angiotensin receptors blockers, insulin sensitizers, antioxidant compounds) are now available, targeting both the synthesis and oxidative inactivation of NO in human vasculature, reversing in that way endothelial dysfunction which is enhanced by the release of nitric oxide from the endothelium.     

Inducible nitric oxide synthase and inflammation

Nitric oxide (NO), derived from L-arginine (L-Arg) by the enzyme nitric oxide synthase (NOS), is involved in acute and chronic inflammatory events. In view of the complexity associated with the inflammatory response, the dissection of possible mechanisms by which NO modulates this response will be profitable in designing novel and more efficacious NOS inhibitors. In this review we describe the consequences associated with the induction of inducible nitric oxide synthase (iNOS) and its therapeutic implications.     

Characterization of nitric oxide synthases in non-adrenergic non-cholinergic nerve containing tissue from the rat anococcygeus muscle.

Tissue homogenates prepared from rat anococcygeus muscle converted L-arginine to L-citrulline indicating the presence of nitric oxide (NO) synthase. NO synthase activity was also found in crude and partially-purified soluble and particulate fractions prepared from the homogenates. Both soluble and particulate NO synthase were dependent on NADPH, 5,6,7,8-tetrahydrobiopterin and calcium, and inhibited by NG-nitro-L-arginine. Tissue homogenates or crude cytosolic and membrane fractions from rat vas deferens, which does not contain NO releasing non-adrenergic non-cholinergic neurones, had no NO synthase activity.     

Biochemical analysis of solubilized angiotensin II receptors from murine neuroblastoma N1E-115 cells by covalent cross-linking and affinity purification

Angiotensin II (Ang-II) receptors were solubilized from differentiated N1E-115 neuroblastoma cell membranes with the zwitterionic detergent 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), whereas other detergents, such as digitonin, sodium cholate, and Triton X-100, were much less effective. Binding of 125I-Ang-II or the antagonist 125I-Sar1,Ile8-Ang-II to 1% CHAPS-solubilized membranes was saturable and of high affinity. Moreover, these solubilized receptors retained the pharmacological specificity characteristic of particulate receptors. Covalent cross-linking of 125I-Ang-II to either particulate or solubilized membrane fractions, with the homobifunctional cross-linker disuccinimidyl suberate, followed by size exclusion chromatography or sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis, resulted in the identification of the same two distinct 125I-Ang-II binding entities, with approximate molecular masses of 111 kDa and 68 kDa. The estimated molecular weights of the Ang-II binding sites in differentiated N1E-115 cells are in good agreement with the molecular weights obtained previously from solubilized rat brain membranes, suggesting that the N1E-115 Ang-II receptors are similar to those present in the brain. Finally, solubilized N1E-115 membranes could be purified by Ang-II affinity chromatography, resulting in only a single protein (66 kDa), which retained its ability to specifically bind 125I-Ang-II.     

Phasic cardiovascular responses to mevinphos are mediated through differential activation of cGMP/PKG cascade and peroxynitrite via nitric oxide generated in the rat rostral ventrolateral medulla by NOS I and II isoforms

The organophosphate insecticide mevinphos (Mev) acts on the rostral ventrolateral medulla (RVLM), where sympathetic vasomotor tone originates, to elicit phasic cardiovascular responses via nitric oxide (NO) generated by NO synthase (NOS) I and II. We evaluated the contribution of soluble guanylyl cyclase (sGC)/cyclic guanosine monophosphate (cGMP)/protein kinase G (PKG) cascade and peroxynitrite in this process. PKG expression in ventrolateral medulla of Sprague-Dawley rats manifested an increase during the sympathoexcitatory phase (Phase I) of cardiovascular responses induced by microinjection of Mev bilaterally into the RVLM that was antagonized by co-administration of 7-nitroindazole or Nomega-propyl-L-arginine, two selective NOS I inhibitors or 1-H-[1,2,4]oxadiaolo[4,3-a]quinoxalin-1-one (ODQ), a selective sGC antagonist. Co-microinjection of ODQ or two PKG inhibitors, KT5823 or Rp-8-Br-cGMPS, also blunted the Mev-elicited sympathoexcitatory effects. However, the increase in nitrotyrosine, a marker for peroxynitrite, and the sympathoinhibitory circulatory actions during Phase II Mev intoxication were antagonized by co-administration of S-methylisothiourea, a selective NOS II inhibitor, Mn(III)-tetrakis-(4-benzoic acid) porphyrin, a superoxide dismutase mimetic, 5,10,15,20-tetrakis-N-methyl-4'-pyridyl)-porphyrinato iron (III), a peroxynitrite decomposition catalyst, or L-cysteine, a peroxynitrite scavenger. We conclude that sGC/cGMP/PKG cascade and peroxynitrite formation may participate in Mev-induced phasic cardiovascular responses as signals downstream to NO generated respectively by NOS I and II in the RVLM.     

The effect of divalent cations on neuronal nitric oxide synthase activity.

Neuronal nitric oxide synthase (NOS I) is a Ca(2+)/calmodulin-binding enzyme that generates nitric oxide (NO*) and L-citrulline from the oxidation of L-arginine, and superoxide (O(2)*(-)) from the one-electron reduction of oxygen (O(2)). Nitric oxide in particular has been implicated in many physiological processes, including vasodilator tone, hypertension, and the development and properties of neuronal function. Unlike Ca(2+), which is tightly regulated in the cell, many other divalent cations are unfettered and can compete for the four Ca(2+) binding sites on calmodulin. The results presented in this article survey the effects of various divalent metal ions on NOS I-mediated catalysis. As in the case of Ca(2+), we demonstrate that Ni(2+), Ba(2+), and Mn(2+) can activate NOS I to metabolize L-arginine to L-citrulline and NO*, and afford O(2)*(-) in the absence of L-arginine. In contrast, Cd(2+) did not activate NOS I to produce either NO* or O(2)*(-), and the combination of Ca(2+) and either Cd(2+), Ni(2+), or Mn(2+) inhibited enzyme activity. These interactions may initiate cellular toxicity by negatively affecting NOS I activity through production of NO*, O(2)*(-) and products derived from these free radicals.     

Mechanism of CGRP-induced vasodilation in the rat isolated perfused kidney

We investigated the intracellular mechanisms involved in calcitonin gene-related peptide (CGRP)-induced vasodilation in rat isolated perfused kidney. CGRP-1 receptor antagonist, CGRP-8-37, abolished the responses. Endothelial denudation by Triton X-100 or nitric oxide (NO) synthase inhibition by NG-nitro-L-arginine attenuated the maximum dilation by about 63 and 55%, respectively. Protein kinase A inhibitor, KT-5720, caused an about 72% inhibition in CGRP-induced maximum dilation. Soluble guanylate cyclase inhibitor, ODQ, and ATP-sensitive potassium channel blocker, glibenclamide, inhibited the CGRP-induced maximum responses by 75 and 55%, respectively. Cyclooxygenase inhibitor, indomethacin, had no effect. Our data suggest that CGRP-1 receptors, endothelium, NO synthase, protein kinase A, soluble guanylate cyclase, and ATP-sensitive potassium channels, but not the cyclooxygenase pathway, may play a role in CGRP-induced vasodilation in rat isolated perfused kidney.     

Minimal amidine structure for inhibition of nitric oxide biosynthesis

Pharmacological modulation of nitric oxide synthase activity has been achieved using structural analogs of arginine. In the present studies, we demonstrated that the minimal amidine structure required for enzymatic inhibition is formamidine. We found that the production of nitric oxide by primary cultures of rat hepatocytes and several mouse and human cell lines, including RAW 264.7 macrophages, PAM 212 keratinocytes, G8 myoblasts, S180 sarcoma, CX-1 human colon cells, and GH3 rat pituitary cells, was inhibited in a concentration- and time-dependent manner by formamidine. Formamidine was 2- to 6-fold more effective in inhibiting nitric oxide production in cells expressing inducible nitric oxide synthase (NOS2) than in a cell line expressing calcium-dependent neuronal nitric oxide synthase (NOS1). Whereas formamidine had no effect on gamma-interferon-induced expression of nitric oxide synthase protein, its enzymatic activity was blocked. Kinetic analysis revealed that formamidine acts as a simple competitive inhibitor with respect to arginine (K(i) formamidine approximately 800 microM). Using a polarographic microsensor to measure real-time flux of nitric oxide release from RAW 264.7 macrophages, formamidine was found to require 30-90 min to inhibit enzyme activity, suggesting that cellular uptake of the drug may limit its biological activity. Our data indicate that formamidine is an effective inhibitor of nitric oxide production. Furthermore, its low toxicity may make it useful as a potential therapeutic agent in diseases associated with the increased production of nitric oxide.     

Separation of paraoxon and mipafox sensitive esterases by sucrose density gradient sedimentation

Paraoxon- and mipafox-sensitive phenyl valerate (PV) hydrolases found in chicken nervous tissue were solubilized by Triton X-100 and separated by sucrose density gradient centrifugation. Enzyme activity from chick embryo brain with properties of neurotoxic esterase (NTE, insensitive to 40 microM paraoxon, sensitive to 50 microM mipafox) migrated in a 9S peak.     

一氧化氮合酶基因治疗在心血管疾病中的应用

一氧化氮(NO)为内皮细胞舒血管因子(EDRF)，是心血管系统重要的调节因子，NO功能异常在许多心血管系统疾病的发病中起重要作用。催化NO合成的酶称为一氧化氮合酶(NOS)，用转基因方法转移重组NOS基因，重建内源性NO功能，是治疗心血管系统疾病的新策略，本文就重组NOS转基因的研究进行综述。     

Role of nitric oxide on in vitro human eosinophil migration.

Eosinophils purified from the rat peritoneal cavity have been found to contain nitric oxide synthase (NOS) functionally coupled to a cyclic GMP transduction pathway that is involved in in vitro eosinophil migration, but no studies on cell locomotion have been done with purified human eosinophils. Therefore, this study was carried out to investigate the effects of N(omega) -nitro-L-arginine methyl ester (L-NAME; a non-selective NOS inhibitor), 1-(2-trifluoromethylphenyl) imidazole (TRIM; a type I/type II NOS inhibitor), 2-amino-5,6-dihydro-6-methyl-4H-1,3-thiazine (AMT; a selective type II NOS inhibitor), and 1H-[1,2,4]-oxidiazolo[4,3-a] quinoxalin-1-one (ODQ; a soluble guanylate cyclase inhibitor) on human eosinophil migration induced by N-formyl-methionyl-leucyl-phenylalanine (fMLP). Human eosinophils were purified from peripheral blood of healthy volunteers using a Percoll gradient followed by an immunomagnetic cell separator. Chemotaxis was evaluated using a 48-well microchemotaxis chamber. The fMLP (1.0 x 10(-7) M)-induced eosinophil migration was reduced significantly by l-NAME (0.1 and 1.0 mM), whereas the inactive enantiomer N(omega)-nitro-D-arginine methyl ester (D-NAME) had no effect. The inhibition by l-NAME was restored by sodium nitroprusside (0.25 mM). The NOS inhibitors AMT and TRIM (0.05 to 0.25 mM each) also markedly attenuated fMLP-induced chemotaxis. Additionally, ODQ (0.01 to 0.5 mM) concentration-dependently inhibited fMLP-induced migration, and the inhibition was restored by 2.0 mM dibutyryl cyclic GMP. In conclusion, this study demonstrates that human eosinophils present a nitric oxide-cyclic GMP pathway that is involved in the in vitro locomotion of this cell type.     

晚期糖基化终末产物对人脐静脉内皮细胞分泌NO功能的影响

【摘要】 目的 探讨晚期糖基化终产物（AGEs）对人脐静脉内皮细胞一氧化氮分泌功能的影响。方法 培养人脐静脉内皮细胞,制备晚期糖基化终末产物（AGEs-HSA）。将体外培养的人脐静脉内皮细胞随机分为AGEs-HSA组和HSA（人血白蛋白）组, 分别用不同浓度的AGEs-HSA、HSA刺激内皮细胞24 h。然后用酶联免疫法分别测定细胞培养上清液中的NO含量、NOS活性、SOD活性和MDA含量。结果 1与HSA组相比,AGEs-HSA组在浓度为100、200、400 mg/L 时能够明显抑制内皮细胞NO的产生和NOS的活性,并呈浓度依赖性（P <0.05）。2与HSA组相比,AGEs-HSA组在浓度为25、50、100、200、400 mg/L 时均能够明显抑制内皮细胞SOD的活性和促进MDA含量的增加,并呈浓度依赖性（P <0.05）。3 AGEs-HSA组中在AGEs-HSA刺激浓度为400 mg/L时, NO浓度和NOS活性、SOD活性呈正相关（P <0.01）,而与MDA含量呈负相关（P <0.01）。NOS的活性与SOD活性呈正相关（P <0.01）,与MDA含量呈负相关（P <0.01）。结论 晚期糖基化终末产物能够抑制人脐静脉内皮细胞NO的分泌。其机制之一可能是AGEs介导内皮细胞发生氧化损伤致使NOS活性降低进而导致内皮细胞NO分泌下降。      

Antinociceptive properties and nitric oxide synthase inhibitory action of new ruthenium complexes

This study evaluates the actions of the new ruthenium complexes trans-[RuCl2(nic)4] (Complex I) and trans-[RuCl2(i-nic)4] (Complex II) as antinociceptives, and their interaction with nitric oxide isoenzymes and with acetylcholine-induced relaxation of rat and rabbit aorta. Complex II inhibited, in a graded manner, neuronal and inducible nitric oxide (NO) synthase, and was about two fold more effective in inhibiting the neuronal NO synthase than the inducible form of the enzyme. Complex I was inactive. Both complexes failed to interfere with constitutive endothelial nitric oxide synthase because they did not change the mean arterial blood pressure of rats. The vasorelaxant effect of acetylcholine was markedly antagonised by the Complexes I and II in rings of both rat and rabbit aorta. Complexes I and II, given intraperitoneally, like N(omega)-nitro-L-arginine methyl ester (L-NAME) and N(G)-nitro-L-arginine (L-NOARG), inhibited, in a graded manner, both phases of the pain response induced by formalin. The actions of L-NAME, L-NOARG and Complex II, but not that of Complex I, were largely reversed by L-arginine. Both complexes failed to affect the motor response of animals in the rota-rod test and had no effect in the hot-plate assay. Together, these findings provide indications that the new ruthenium complexes, especially Complex II and its derivatives, might be of potential therapeutic benefit in the management of pain disorders.     

Role of nitric oxide/cyclic GMP in myocardial adenosine A1 receptor-inotropic response

In this study we have determined the different signalling pathways involved in adenosine A(1)-receptor (A(1)-receptor)-dependent inhibition of contractility in rat isolated atria. N-cyclopentyladenosine (CPA) stimulation of A(1)-receptor exerts: negative inotropic response, inositol phosphates accumulation, stimulation of nitric oxide synthase (NOS), increased production of nitric oxide (NO) and cyclic GMP. Inhibitors of phospholipase C (PLC), protein kinase C (PKC), calcium/calmodulin, NOS and guanylate cyclase shifted the dose-response curve of CPA on contractility to the right. Those inhibitors also attenuated the A(1)-receptor-dependent increase in cyclic GMP and activation of NOS. These results suggest that CPA activation of A(1)-receptors exerts a negative inotropic effect associated with increased production of nitric oxide and cyclic GMP. The mechanism appears to occur secondarily to stimulation of phosphoinositide turnover via PLC activation. This, in turn, triggers cascade reactions involving calcium/calmodulin and PKC, leading to activation of NOS and soluble guanylate cyclase.     

Insulin induces internalization of the plasma membrane 5-hydroxytryptamine2A (5-HT2A) receptor in the isolated human endothelium-denuded saphenous vein via the phosphatidylinositol 3-kinase pathway

The aim of this study was to investigate the relaxant effect of insulin on the 5-hydroxytryptamine (5-HT)-induced constriction of the human endothelium-denuded saphenous vein (SV) and its signal transduction pathway. During the 5-HT-induced sustained constriction of vessels, insulin induced vasorelaxation in a concentration-dependent manner. This insulin-induced vasorelaxation was partially attenuated by L-NAME, a nitric oxide synthase (NOS) inhibitor, and was abolished by wortmannin, a phosphatidylinositol 3-kinase (PI3-K) inhibitor. Insulin increased the Ser(473) phosphorylation of Akt. Endothelial NOS and inducible NOS protein expressions were observed in SV smooth muscle when insulin induced relaxation of SV vessels preconstricted with 5-HT. Although insulin did not affect the total protein level of 5-HT(2A) receptors, it decreased the particulate protein level and reciprocally increased the soluble protein level of 5-HT(2A) receptors in a concentration-dependent manner. These results demonstrate that insulin can induce the internalization of 5-HT(2A) receptors from the plasma membrane to the cytoplasm. The insulin-induced internalization of 5-HT(2A) receptors was abolished by wortmannin but was not affected by L-NAME. These results suggest that the relaxant effect of insulin on 5-HT-induced vasoconstriction is mediated in part by the internalization of plasma membrane 5-HT(2A) receptors and the production of nitric oxide via the PI3-K/Akt pathway.