Selective substrate-based inhibitors of mammalian dimethylarginine dimethylaminohydrolase

The enzyme DDAH metabolizes methylarginines that are inhibitors of nitric oxide synthase (NOS). Substrate-based inhibitors of mammalian DDAH have been synthesized, with optimization to give selective inhibition of DDAH with no significant direct effect on NOSs. These are the first examples of reversible DDAH inhibitors with significant activity and selectivity. In vivo administration increases plasma ADMA levels, giving proof of concept that these inhibitors can be used to probe the physiological effects of DDAH inhibition, with potential for pharmaceutical use of DDAH inhibitors in diseases where excess NO production is implicated.     

Regulation of nitric oxide synthesis in the liver

Nitric oxide signalling during the past two decades has been one of the most rapidly growing areas in biology. This simple free radical gas can regulate an ever-growing list of biological processes. Here the regulation of NO synthesis in the liver is reviewed. The biogenesis of nitric oxide (NO) is catalysed by nitric oxide synthases (NOS). These enzymes catalyse the oxidation of one of the guanidino nitrogens of l-arginine by molecular oxygen to form NO and citrulline. Three NOS have been identified: two constitutive (cNOS: type 1 or neuronal and type 3 or endothelial) and one inducible (iNOS: type 2). As to the liver, cNOS activity is normally detectable in Kupffer cells, whereas no cNOS is ever encoded in hepatocytes. However, hepatocytes, Kupffer and stellate cells (the three main types of liver cells) are prompted to express an intense iNOS activity once exposed to effective stimuli such as bacterial lipopolysaccharide and cytokines. This review is focused mainly on two aspects: regulation of NOS activity and expression by endogenous and exogenous compounds. Because NO production has beneficial and detrimental effects, understanding the molecular mechanisms that govern NOS is critical to developing strategies to manipulate NO production in liver diseases.     

Regulation of endothelial nitric oxide synthase and asymmetric dimethylarginine by matrine attenuates isoproterenol-induced acute myocardial injury in rats

Objectives This study was designed to investigate the cardioprotective effects of matrine on regulation of endothelial nitric oxide synthase (eNOS) and asymmetric dimethylarginine (ADMA) in isoproterenol‐induced acute myocardial ischaemic rats. Methods Male Sprague–Dawley rats were pretreated with matrine (200, 100 and 50 mg/kg) orally for 10 days. Acute myocardial injury was induced in rats by subcutaneous injection of isoproterenol. Serum and haemodynamic parameters, histopathological variables and expression of protein levels were analysed. Key findings Oral administration of matrine (200, 100 and 50 mg/kg) significantly attenuated isoproterenol‐induced cardiac necrosis and left ventricular dysfunction. Matrine treatment restored impaired ventricular Akt and eNOS protein expression with concomitant increased phosphorylation of Akt (Ser473) and eNOS (Ser1177), and also restored glycogen synthase kinase 3β activity, as indicated by increased phosphorylation at Ser 9. Moreover, treatment with matrine had no effect on the isoproterenol‐induced elevated protein arginine methyltransferase 1 protein expression, but could significantly normalize the reduced dimethylarginine dimethylaminohydrolase 2 expression and attenuate the increased serum level of ADMA. The expression of catechol‐o‐methyltransferase and monoamine oxidase did not differ among all groups (all P > 0.05). Conclusions Our results suggested that matrine protects against isoproterenol‐induced myocardial ischaemia via eNOS and ADMA pathway.     

Calycosin and formononetin from astragalus root enhance dimethylarginine dimethylaminohydrolase 2 and nitric oxide synthase expressions in Madin Darby Canine Kidney II cells

Nitric oxide (NO) is a crucial vasodilator produced by nitric oxide synthase (NOS). Asymmetric dimethylarginine (ADMA) is an endogenous NOS inhibitor and mainly catabolized by dimethylarginine dimethylaminohydrolase (DDAH). As we reported, the antihypertensive effect of shichimotsukokato (SKT), a formula of Japanese traditional kampo medicine consisting of 7 crude drugs, in 5/6 nephrectomized rats, is mediated by the DDAH-ADMA-NO pathway. Our present study aimed to explore the effective compounds of SKT using Madin Darby Canine Kidney (MDCK) II cells. We isolated two isoflavones, calycosin and formononetin from astragalus root, one of the components of SKT, which can promote DDAH2 protein and mRNA expressions in MDCK II cells. The neuronal NOS levels were also upregulated by the treatment of calycosin and formononetin. These results suggest that calycosin and formononetin could be the active ingredients of astragalus root and SKT that cause antihypertensive effects. The increased levels of DDAH2 and NOS may enhance NO production, decrease ADMA level and improve endothelial and cardiovascular dysfunction.     

Nebivolol reduces asymmetric dimethylarginine in endothelial cells by increasing dimethylarginine dimethylaminohydrolase 2 (DDAH2) expression and activity

Asymmetric dimethylarginine (ADMA) has been reported to affect the synthesis of nitric oxide (NO) in endothelial cells by inhibiting endothelial NO synthase (eNOS) activity and to cause endothelial dysfunction in humans. This study was conducted in human umbilical vein endothelial cells (HUVECs) to evaluate the effect of nebivolol, a selective beta1-adrenergic receptor antagonist, on ADMA concentration and on dimethylarginine dimethylaminohydrolase (DDAH2), the enzyme that regulates ADMA catabolism. Nebivolol dose-dependently decreased ADMA/symmetric dimethylarginine (SDMA) ratio (p from <0.01 to <0.001). This was parallelled by a dose-dependent increase in DDAH2 mRNA (p from <0.01 to <0.001) and protein expression (p from <0.01 to <0.001) and activity (p from <0.01 to <0.001). The small interference RNA (siRNA)-mediated knockdown of DDAH2 abolished the modification of DDAH2 expression (p<0.001) and ADMA/SDMA ratio (p<0.001) induced by nebivolol. In conclusion, the results of this study demonstrate that nebivolol reduces ADMA concentration by increasing DDAH2 expression and activity. Our in vitro findings describe a novel vascular effect of nebivolol and clearly identify this compound as the first antihypertensive agent that modulates DDAH2 in endothelial cells.     

兔肾脏DDAH活性测定方法的研究

目的　建立日本大耳白家兔肾脏二甲基精氨酸二甲基氨基水解酶 (DDAH)活性的检测方法。方法　空气栓塞处死正常日本大耳白家兔后 ,取出肾脏并制备匀浆 ,以非对称性二甲基精氨酸 (ADMA)为底物 ,用UV 2 6 5型分光光度计在 5 2 5nm下测定生成L 胍氨酸 (L Cit)的量 ,不同条件下比较 ,筛选最适条件 ,并计算DDAH降解ADMA的酶促反应动力学参数。结果　DDAH降解ADMA的酶促反应动力学参数为Km =(0 2 8± 0 10 )mmol·L-1,Vm =(1 36±0 4 2 )mmol·L-1·g-1·min-1。本实验选定的兔肾DDAH活性检测最适条件为 :酶蛋白浓度 12g·L-1、缓冲液最适pH7 4、ADMA终浓度 2mmol·L-1、反应时间 30min。结论　本法简便易行 ,且以Km 值作为确定酶促反应底物浓度的依据 ,有利于准确检测该酶活性     

Screening for dimethylarginine dimethylaminohydrolase inhibitors reveals ebselen as a bioavailable inactivator

Dimethylarginine dimethylaminohydrolase (DDAH) is an endogenous regulator of nitric oxide production and represents a potential therapeutic target. However, only a small number of biologically useful inhibitors have been reported, and many of these are substrate analogs. To seek more diverse scaffolds, we developed a high-throughput screening (HTS) assay and queried two small libraries totaling 2446 compounds. The HTS assay proved to be robust, reproducible and scalable, with Z' factors ≥ 0.78. One inhibitor, ebselen, is structurally divergent from substrate and was characterized in detail. This selenazole covalently inactivates DDAH in vitro and in cultured cells. The rate constant for inactivation of DDAH (44,000 ± 2,400 M(-1)s(-1)) is greater than those reported for any other target, suggesting this pathway is an important aspect of ebselen's total pharmacological effects.     

Regulation of prostaglandin production by nitric oxide; an in vivo analysis.

1. Endotoxin E. Coli lipopolysaccharide (LPS)-treatment in conscious, restrained rats increased plasma and urinary prostaglandin (PG) and nitric oxide (NO) production. Inducible cyclo-oxygenase (COX-2) and nitric oxide synthase (iNOS) expression accounted for the LPS-induced PG and NO release since the glucocorticoid, dexamethasone inhibited both effects. Thus, LPS (4 mg kg-1) increased the plasma levels of nitrite/nitrate from 14 +/- 1 to 84 +/- 7 microM within 3 h and this rise was inhibited to 35 +/- 1 microM by dexamethasone. Levels of 6-keto PGF1 alpha in the plasma were below the detection limit of the assay (< 0.2 ng ml-1). However, 3 h after the injection of LPS these levels rose to 2.6 +/- 0.2 ng ml-1 and to 0.7 +/- 0.01 ng ml-1 after LPS in rats that received dexamethasone. 2. The induced enzymes were inhibited in vivo with selective COX and NOS inhibitors. Furthermore, NOS inhibitors, that did not affect COX activity in vitro markedly suppressed PG production in the LPS-treated animals. For instance, the LPS-induced increased in plasma nitrite/nitrate and 6-keto PGF1 alpha at 3 h was decreased to 18 +/- 2 microM and 0.5 +/- 0.02 ng ml-1, 23 +/- 1 microM and 0.7 +/- 0.01 ng ml-1, 29 +/- 2 microM and 1 +/- 0.01 ng ml-1 in rats treated with LPS in the presence of the NOS inhibitors NG-monomethyl-L-arginine, NG-nitro arginine methyl ester and aminoguanidine, respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

Regulation of baseline vascular resistance in the canine diaphragm by nitric oxide.

1. The role played by nitric oxide (NO) in the regulation of blood flow to the canine isolated hemidiaphragm was evaluated by determining (a) the effects of the L-arginine analogues NG-nitro-L-arginine methyl ester (L-NAME), NG-nitro-L-arginine (L-NOARG), and argininosuccinic acid (ArgSA) on baseline vascular resistance and of the latter two agents on endothelium-dependent (acetylcholine, ACh) and endothelium independent (sodium nitroprusside, SNP) vasodilatation; (b) the effects of L- and D-arginine on baseline vascular resistance; and (c) the effects of L-glutamine, an inhibitor of intracellular recycling of L-citrulline to L-arginine, on baseline resistance and on the response to ACh and SNP. 2. L-NAME, L-NOARG and ArgSA (6 x 10(-4) M final concentration) increased baseline diaphragmatic vascular resistance to a similar extent (28.6 +/- 4.2%, 26.7 +/- 4.3% and 32.8 +/- 4.6% respectively). L-NOARG and ArgSA reversed the vasodilator effect of ACh but not of SNP. 3. L- and D-arginine had no effect on vascular resistance. 4. L-Glutamine (10(-3) M) increased baseline vascular resistance by 10 +/- 1.9% (P < 0.05) but did not alter responses to either ACh or SNP. 5. Basal NO release plays a role in the regulation of baseline diaphragmatic vascular resistance. L-Arginine analogues tested potently and specifically inhibited this process. Moreover, extracellular L-arginine appears to have no effect on baseline diaphragmatic vascular resistance.     

Feedback inhibition of nitric oxide synthase activity by nitric oxide.

1. A murine macrophage cell line, J774, expressed nitric oxide (NO) synthase activity in response to interferon-gamma (IFN-gamma, 10 u ml-1) plus lipopolysaccharide (LPS, 10 ng ml-1). The enzyme activity was first detectable 6 h after incubation, peaked at 12 h and became undetectable after 48 h. 2. The decline in the NO synthase activity was not due to inhibition by stable substances secreted by the cells into the culture supernatant. 3. The decline in the NO synthase activity was significantly slowed down in cells cultured in a low L-arginine medium or with added haemoglobin, suggesting that NO may be involved in a feedback inhibitory mechanism. 4. The addition of NO generators, S-nitroso-acetyl-penicillamine (SNAP) or S-nitroso-glutathione (GSNO) markedly inhibited the NO synthase activity in a dose-dependent manner. The effect of NO on the enzyme was not due to the inhibition of de novo protein synthesis. 5. SNAP directly inhibited the inducible NO synthase extracted from activated J774 cells, as well as the constitutive NO synthase extracted from the rat brain. 6. The enzyme activity of J774 cells was not restored after the removal of SNAP by gel filtration, suggesting that NO inhibits NO synthase irreversibly.     

Regulation of inducible nitric oxide synthase by cAMP-elevating phospho-diesterase inhibitors

Among the numerous genes controlled by cyclic adenosine monophosphate (cAMP)/protein kinase A signalling machinery is the gene encoding the inducible nitric oxide synthase (iNOS), an enzyme catalyzing the synthesis of a highly reactive free radical nitric oxide (NO). While being a major microbicidal and tumoricidal molecule, iNOS-derived NO has also been implicated in tissue destruction, as well as in regulation of inflammatory/immune cell function in various disorders associated with excessive inflammation. A feasible way for cAMP-dependent therapeutic control of inflammation, including iNOS-mediated NO synthesis, could involve the administration of drugs that block the enzymatic activity of cAMP-degrading phosphodiesterases (PDE). Indeed, cAMP-elevating PDE inhibitors can influence iNOS activation in different cell types in vitro, and their potent anti-inflammatory effects in experimental disease models and clinical studies were frequently accompanied with profound modulation of NO production. A set of conflicting data has been generated over the years, ranging from strong suppression to marked enhancement of NO release by cAMP-increasing PDE inhibitors, depending on cell-type, iNOS stimuli, and/or the agents used. The present review summarizes the data on iNOS modulation by cAMP-elevating PDE inhibitors and possible mechanisms behind it, speculating on its contribution to the therapeutic effects of these drugs.     

Inhibition of nitric oxide synthesis in vascular smooth muscle by retinoids.

1. These studies examine the effect of retinoids on interleukin 1 beta (IL-1 beta)-induced nitric oxide synthase (NOS) activity in cultured rat aortic vascular smooth muscle (VSM) cells and isolated rat aortic rings. 2. All-trans-retinoic acid (all-trans-RA, 0.1-10 microM) and its active analogues produced concentration-dependent inhibition of IL-1 beta (0.1-10 ng ml-1)-induced nitrite production in cultured VSM cells. In contrast, the inactive retinoid, Ro 14-6113 (0.1-10 microM), had no effect on IL-1 beta-induced nitrite production. 3. Since some of the actions of retinoids are mediated by induction of transforming growth factor beta (TGF-beta), its effect on inducible NOS activity in VSM cells was examined. TGF-beta produced concentration-dependent (0.1-10 ng ml-1) inhibition of IL-1 beta-induced nitrite production and the maximum effect (approximately 90% inhibition) was significantly greater than that seen with all-trans-RA (approximately 70% with 10 microM). However, an anti-TGF-beta antibody (50 micrograms ml-1) which blocked the effect of exogenous TGF-beta (5 ng ml-1) did not significantly reverse the inhibitory action of all-trans-RA (10 microM). 4. In addition to inhibiting IL-1 beta-induced nitrite production, all-trans-RA (10 microM) reduced substantially inducible NOS mRNA and protein levels in IL-1 beta-induced VSM cells (P < 0.01). 5. Incubation of isolated rat aortic rings with IL-1 beta (10 ng ml-1) caused a progressive resistance of the rings to the vasoconstrictor action of phenylephrine (10 nM to 10 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

内皮源性超极化因子对内皮一氧化氮合酶基因表达的调节

目的　以内皮细胞产生NO的关键酶———eNOS(内皮一氧化氮合酶 )为研究目标 ,探讨外源性内皮源性超极化因子EDHF(EETs)对内皮细胞合成NO的影响。方法　在原代培养 3～ 4代以内的牛主动脉内皮细胞中 ,分别加入不同浓度 (5 0～ 2 0 0nmol·L-1)的 8,9 EET、11,12 EET、14 ,15 EET ,作用 1h后用不同的方法收获细胞。用WesternBlot以及NorthernBlot方法检测EETs对eNOS基因表达的影响 ;同时通过检测L [3 H] 精氨酸转化为L [3 H] 瓜氨酸的量研究EETs对NOS活性的影响。结果　显示 8,9 EET、11,12 EET、14 ,15 EET均呈浓度依赖性地增加eNOS蛋白质的表达 ,并提高eNOSmRNA表达水平以及NOS酶活性。结论　外源性EDHF对eNOS基因表达是一种正反馈调节作用 ,从而能够促进内皮细胞NO的产生 ,通过药物调节内皮表氧化酶进而促进eNOS基因表达可作为防治心血管疾病的新策略     

Dimethylarginine dimethylaminohydrolase: a new therapeutic target for the modulation of nitric oxide and angiogenesis

Nitric oxide (NO) has a key role in promoting angiogenesis by increasing vasodilation, vascular permeability, endothelial cell proliferation and migration, and by modifying the activities of angiogenic mediators. NO is also critical for the mobilization of endothelial progenitor cells from the bone marrow which promotes vasculogenesis and angiogenesis. Studies have shown that the enzymes catalyzing NO synthesis are inhibited by the endogenously generated inhibitor asymmetric dimethylarginine (ADMA). Pharmacological agents targeted to modulate dimethyl-arginine dimethylaminohydrolase, the key enzyme metabolizing ADMA, may offer a potential strategy for developing novel pro- and anti-angiogenic therapies.     

Regulation of programmed cell death in neuronal cells by nitric oxide

Nitric oxide (NO), produced from L-arginine and molecular oxygen in a reaction catalyzed by one of three NO synthase isoenzymes, can prevent or induce neuronal apoptosis depending on its concentration and cellular redox state. This molecule affords neuroprotection by post-translational S-nitrosylation of NMDA receptor, caspases and p21ras, and increases the expression of cytoprotective genes such as HSP70, heme oxygenase and Bcl-2. Moreover, the NO/cGMP pathway activates the anti-apoptotic serine/threonine kinase Akt by protein kinase G-dependent activation of phosphatidylinositol 3-kinase. A high concentration of NO and peroxynitrite, a reaction product of NO with superoxide anion, can promote apoptotic pathways in neuronal cells through the indirect activation of caspases. We review the molecular mechanism by which NO exerts both pro- and anti-apoptotic actions in neuronal cells and the clinical implications for regulating neuronal apoptosis.     

Antioxidants and endothelial nitric oxide synthesis

Oxidative stress in the vasculature has been suggested to contribute to the development of endothelial dysfunction via different mechanisms including LDL oxidation, nitric oxide (NO) scavenging, or oxidation of tetrahydrobiopterin, a critical cofactor of endothelial NO synthase (eNOS). Antioxidants may interfere with these processes and protect NO formed in the endothelium. In particular, ascorbic acid at high concentrations seems to be a prerequisite for sufficient NO bioavailability. Moreover, there is accumulating evidence that ascorbic acid improves tetrahydrobiopterin availability in the vasculature most probably via recycling oxidized tetrahydrobiopterin back to the fully reduced pterin. In addition, ascorbic acid may reduce the α-tocopheroxyl radical and may be required for beneficial vascular effects of α-tocopherol. Recent data have shown that apart from indirect protection of NO from inactivation, α-tocopherol exerts a direct stimulatory effect on eNOS activation via serine 1177 phosphorylation. This effect was amplified by ascorbic acid suggesting that both compounds may act synergistically in optimizing endothelial NO synthesis. The data obtained in cell culture and animal studies are promising, but human long-term studies are needed to determine whether the described mechanisms are active in vivo and may provide a rationale for optimizing dietary or supplementary intake of antioxidant vitamins in certain subsets of patients.

     

Teratological consequences of nitric oxide synthesis inhibition

Nitric oxide (NO) is generated by a family of NO synthase (NOS) enzymes, including endothelial (eNOS), inducible (iNOS) and neuronal (nNOS). NO is an important bioregulator of a wide variety of physiological processes. Recent experimental evidence indicates that inhibition of NO synthesis can lead to teratogenesis. The current review focuses on this aspect of NOS. Exposure of pregnant rodents to non-selective NOS inhibitors, such as N(G)-nitro-L-arginine-methyl ester (L-NAME) and N(G)-nitro-L-arginine (L-NNA), has been linked to limb reduction defects. The teratogenic phenotype, characterized by hemorrhage and transverse terminal tissue destruction, has been regarded to be compatible with a vascular origin. The critical time for teratogenic response was traced to advanced stages of gestation. Similar limb reduction defects have been described in mice deficient in eNOS, but not in other NOS isoforms. Several observations have led to the proposal that hypoxia and possible consequential generation of reactive oxygen species are involved in the causation of NOS inhibitors induced limb defects.     

Inhibition of inducible nitric oxide synthesis by catalposide from Catalpa ovata

Catalposide (1) and two related iridoids were isolated from the stem of Catalpa ovata (Bignoniaceae) by bioassay guided fractionation. Catalposide (1) significantly inhibited the production of nitric oxide (NO) in lipopolysaccharide (LPS)-stimulated RAW 264.7 macrophages in a dose-dependent manner. RT-PCR and Western blot analyses demonstrated that catalposide (1) suppressed the expression of inducible nitric oxide synthase (iNOS) gene and iNOS protein. Catalposide (1) also inhibited the activation of LPS-induced NF-kappaB as analyzed by electrophoretic mobility shift assay (EMSA). In addition to the inhibitory effect on NO production in LPS-stimulated RAW 264.7 cells, catalposide (1) significantly inhibited the NO production in cytokine-stimulated human DLD-1 and rat vascular smooth muscle (VSM) cells in a dose-dependent manner.