Impaired nitric oxide-mediated vasodilation in transgenic sickle mouse

Transgenic sickle mice expressing human beta(S)- and beta(S-Antilles)-globins show intravascular sickling, red blood cell adhesion, and attenuated arteriolar constriction in response to oxygen. We hypothesize that these abnormalities and the likely endothelial damage, also reported in sickle cell anemia, alter nitric oxide (NO)-mediated microvascular responses and hemodynamics in this mouse model. Transgenic mice showed a lower mean arterial pressure (MAP) compared with control groups (90 +/- 7 vs. 113 +/- 8 mmHg, P < 0.00001), accompanied by increased endothelial nitric oxide synthase (eNOS) expression. N(G)-nitro-L-arginine methyl ester (L-NAME), a nonselective inhibitor of NOS, caused an approximately 30% increase in MAP and approximately 40% decrease in the diameters of cremaster muscle arterioles (branching orders: A2 and A3) in both control and transgenic mice, confirming NOS activity; these changes were reversible after L-arginine administration. Aminoguanidine, an inhibitor of inducible NOS, had no effect. Transgenic mice showed a decreased (P < 0.02-0.01) arteriolar dilation in response to NO-mediated vasodilators, i.e., ACh and sodium nitroprusside (SNP). Indomethacin did not alter the responses to ACh and SNP. Forskolin, a cAMP-activating agent, caused a comparable dilation of A2 and A3 vessels ( approximately 44 and 70%) in both groups of mice. Thus in transgenic mice, an increased eNOS/NO activity results in lower blood pressure and diminished arteriolar responses to NO-mediated vasodilators. Although the increased NOS/NO activity may compensate for flow abnormalities, it may also cause pathophysiological alterations in vascular tone.     

Inhaled nitric oxide does not reduce systemic vascular resistance in mice

Inhaled nitric oxide (NO) is a highly selective pulmonary vasodilator. It was recently reported that inhaled NO causes peripheral vasodilatation after treatment with a NO synthase (NOS) inhibitor. These findings suggested the possibility that inhibition of endogenous NOS uncovered the systemic vasodilating effect of NO or NO adducts absorbed via the lungs during NO inhalation. To learn whether inhaled NO reduces systemic vascular resistance in the absence of endothelial NOS, we studied the systemic vascular effects of NO breathing in wild-type mice treated without and with the NOS inhibitor N(omega)-nitro-l-arginine methyl ester and in NOS3-deficient (NOS3(-/-)) mice. During general anesthesia, the cardiac output, left ventricular function, and systemic vascular resistance were not altered by NO breathing at 80 parts/million in both genotypes. Breathing NO in air did not alter blood pressure and heart rate, as measured by tail-cuff and telemetric methods, in either awake wild-type mice (whether or not they were treated with N(omega)-nitro-l-arginine methyl ester), or in awake NOS3(-/-) mice. Our findings suggest that absorption of NO or adducts during NO breathing is insufficient to cause systemic vasodilation in mice, even when endogenous endothelial NO production is congenitally absent.     

Inhibitors of nitric oxide synthase attenuate nerve growth factor-mediated increases in choline acetyltransferase expression in PC12 cells

NGF can regulate nitric oxide synthase (NOS) expression and nitric oxide (NO) can modulate NGF-mediated neurotrophic responses. To investigate the role of NO in NGF-activated expression of cholinergic phenotype, PC12 cells were treated with either the nonselective NOS inhibitor L-NAME (N (omega)-nitro-L-arginine methylester) or the inducible NOS selective inhibitor MIU (s-methylisothiourea), and the effect on NGF-stimulated ChAT mRNA levels and ChAT specific activity was determined. NGF increased steady-state levels of mRNA and protein for both inducible and constitutive isozymes of NOS in PC12 cells, and led to enhanced NOS activity and NO production. MIU and, to a lesser extent, L-NAME blocked neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells. Both L-NAME and MIU attenuated NGF-mediated increases in choline transferase (ChAT)-specific activity and prevented the increase in expression of ChAT mRNA normally produced by NGF treatment of PC12 cells. The present study indicates that NO may be involved in the modulation of signal transduction pathways by which NGF leads to increased ChAT gene expression in PC12 cells.     

Role of nitric oxide in heparin-induced attenuation of hypoxic pulmonary vascular remodeling.

Heparin and nitric oxide (NO) attenuate changes to the pulmonary vasculature caused by prolonged hypoxia. Heparin may increase NO; therefore, we hypothesized that heparin may attenuate hypoxia-induced pulmonary vascular remodeling via a NO-mediated mechanism. In vivo, rats were exposed to normoxia (N) or hypoxia (H; 10% O(2)) with or without heparin (1,200 U x kg-1 x day-1) and/or the NO synthase (NOS) inhibitor Nomega-nitro-L-arginine methyl ester (L-NAME; 20 mg x kg-1 x day-1) for 3 days or 3 wk. Heparin attenuated increases in pulmonary arterial pressure, the percentage of muscular pulmonary vessels, and their medial thickness induced by 3 wk of H. Importantly, although L-NAME alone had no effect, it prevented these effects of heparin on vascular remodeling. In H lungs, heparin increased NOS activity and cGMP levels at 3 days and 3 wk and endothelial NOS protein expression at 3 days but not at 3 wk. In vitro, heparin (10 and 100 U x kg-1 x ml-1) increased cGMP levels after 10 min and 24 h in N and anoxic (0% O2) endothelial cell-smooth muscle cell (SMC) coculture. SMC proliferation, assessed by 5-bromo-2'-deoxyuridine incorporation during a 3-h incubation period, was decreased by heparin under N, but not anoxic, conditions. The antiproliferative effects of heparin were not altered by L-NAME. In conclusion, the in vivo results suggest that attenuation of hypoxia-induced pulmonary vascular remodeling by heparin is NO mediated. Heparin increases cGMP in vitro; however, the heparin-induced decrease in SMC proliferation in the coculture model appears to be NO independent.     

NO参与玉米幼苗对盐胁迫的应答

以玉米幼苗为材料,研究盐胁迫下其內源NO含量、NR和NOS活性的变化;NOS专一性抑制剂L-NAME和NR非专一性抑制剂NaN3对玉米幼苗內源NO含量的影响;利用激光共聚焦显微技术观测盐胁迫下玉米幼苗根部NO含量的变化及其分布特点。结果表明,盐胁迫下玉米幼苗根尖和叶片中NO含量有猝发现象,NOS活性也随之显著提高,NR活性则显著降低;L-NAME或NaN3均可降低盐胁迫所引起的玉米幼苗NO水平的增加,L-NAME对NO含量的影响比NaN3更显著。推测,NO参与玉米幼苗对盐胁迫的应答,NOS途径是盐胁迫下玉米幼苗內源NO合成的主要途径。     

Nitric oxide synthase activity is critical for the preovulatory epidermal growth factor-like cascade induced by luteinizing hormone in bovine granulosa cells

In rabbits and rodents, nitric oxide (NO) is generally considered to be critical for ovulation. In monovulatory species, however, the importance of NO has not been determined, nor is it clear where in the preovulatory cascade NO may act. The objectives of this study were (1) to determine if nitric oxide synthase (NOS) enzymes are regulated by luteinizing hormone (LH) and (2) to determine if and where endogenous NO is critical for expression of genes essential for the ovulatory cascade in bovine granulosa cells in serum-free culture. Time– and dose–response experiments demonstrated that LH had a significant stimulatory effect on endothelial NOS (NOS3) mRNA abundance, but in a prostaglandin-dependent manner. NO production was stimulated by LH before a detectable increase in NOS3 mRNA levels was observed. Pretreatment of cells with the NOS inhibitor L-NAME blocked the effect of LH on the epidermal growth factor (EGF)-like ligands epiregulin and amphiregulin, as well as prostaglandin–endoperoxide synthase-2 mRNA abundance and protein levels. Similarly, EGF treatment increased mRNA encoding epiregulin, amphiregulin, and the early response gene EGR1, and this was inhibited by pretreatment with L-NAME. Interestingly, pretreatment with L-NAME had no effect on either ERK1/2 or AKT activation. Taken together, these results suggest that endogenous NOS activity is critical for the LH-induced ovulatory cascade in granulosa cells of a monotocous species and acts downstream of EGF receptor activation but upstream of the EGF-like ligands.     

Nitric oxide mediates the mitogenic effects of insulin and vascular endothelial growth factor but not of leptin in endothelial cells

The regulation of vascular wall homeostasis by nitric oxide (NO) generated by endothelium is being intensively studied. In the present paper, the involvement of NO in the vascular endothelial growth factor (VEGF), insulin or leptin-stimulated proliferation of human endothelial cells (HUVEC) was measured by [3H]thymidine or bromodeoxyuridine incorporation. VEGF and insulin, but not leptin, increased NO generation in HUVEC, as detected with ISO-NO electrode. Proliferation of HUVEC induced by leptin was not changed or was higher in the presence of N(omega)-nitro-L-arginine methyl ester (L-NAME) a nitric oxide synthase (NOS) inhibitor. In contrast, L-NAME blunted the proproliferative effect of VEGF and insulin. Furthermore, we demonstrated that, in human arterial smooth muscle cells (hASMC) transfected with endothelial NOS (eNOS) gene, the generation of biologically active VEGF protein was NO-dependent. Inhibition of NO generation by L-NAME decreased the synthesis of VEGF protein and attenuated HUVEC proliferation induced by conditioned media from transfected hASMC. Endothelium-derived NO seems to participate in VEGF and insulin, but not leptin, mitogenic activity. Additionally, the small amounts of NO released from endothelial cells, as mimicked by eNOS transfection into hASMC, may activate generation of VEGF in sub-endothelial smooth muscle cells, leading to increased synthesis of VEGF protein necessary for turnover and restitution of endothelial cells.     

Nitric oxide mediates the interleukin-1beta- and nicotine-induced hypothalamic-pituitary-adrenocortical response during social stress.

We investigated the role of nitric oxide (NO) in the interleukin 1beta (IL-1beta) and nicotine induced hypothalamic-pituitary-adrenal axis (HPA) responses, and a possible significance of CRH and vasopressin in these responses under basal and social stress conditions. Male Wistar rats were crowded in cages for 7 days prior to treatment. All compounds were injected i.p., nitric oxide synthase (NOS) inhibitors, alpha-helical CRH antagonist and vasopressin receptor antagonist 15 min before IL-1beta or nicotine. Identical treatment received control non-stressed rats. Plasma ACTH and serum corticosterone levels were measured 1 h after IL-1beta or nicotine injection. L-NAME (2 mg/kg), a general nitric oxide synthase (NOS) inhibitor, considerably reduced the ACTH and corticosterone response to IL-1beta (0.5 microg/rat) the same extent in control and crowded rats. CRH antagonist almost abolished the nicotine-induced hormone responses and vasopressin antagonist reduced ACTH secretion. Constitutive endothelial eNOS and neuronal nNOS inhibitors substantially enhanced the nicotine-elicited ACTH and corticosterone response and inducible iNOS inhibitor, aminoguanidine, did not affect these responses in non-stressed rats. Social stress significantly attenuated the nicotine-induced ACTH and corticosterone response. In crowded rats L-NAME significantly deepened the stress-induced decrease in the nicotine-evoked ACTH and corticosterone response. In stressed rats neuronal NOS antagonist did not alter the nicotine-evoked hormone responses and inducible NOS inhibitor partly reversed the stress-induced decrease in ACTH response to nicotine. These results indicate that NO plays crucial role in the IL-1beta-induced HPA axis stimulation under basal and social stress conditions. CRH and vasopressin of the hypothalamic paraventricular nucleus may be involved in the nicotine induced alterations of HPA axis activity. NO generated by eNOS, but not nNOS, is involved in the stress-induced alterations of HPA axis activity by nicotine.     

Contributions of nitric oxide synthase isozymes to exhaled nitric oxide and hypoxic pulmonary vasoconstriction in rabbit lungs

We investigated the source(s) for exhaled nitric oxide (NO) in isolated, perfused rabbits lungs by using isozyme-specific nitric oxide synthase (NOS) inhibitors and antibodies. Each inhibitor was studied under normoxia and hypoxia. Only nitro-L-arginine methyl ester (L-NAME, a nonselective NOS inhibitor) reduced exhaled NO and increased hypoxic pulmonary vasoconstriction (HPV), in contrast to 1400W, an inhibitor of inducible NOS (iNOS), and 7-nitroindazole, an inhibitor of neuronal NOS (nNOS). Acetylcholine-mediated stimulation of vascular endothelial NOS (eNOS) increased exhaled NO and could only be inhibited by L-NAME. Selective inhibition of airway and alveolar epithelial NO production by nebulized L-NAME decreased exhaled NO and increased hypoxic pulmonary artery pressure. Immunohistochemistry demonstrated extensive staining for eNOS in the epithelia, vasculature, and lymphatic tissue. There was no staining for iNOS but moderate staining for nNOS in the ciliated cells of the epithelia, lymphoid tissue, and cartilage cells. Our findings show virtually all exhaled NO in the rabbit lung is produced by eNOS, which is present throughout the airways, alveoli, and vessels. Both vascular and epithelial-derived NO modulate HPV.     

NOS1-derived nitric oxide facilitates macrophage uptake of low-density lipoprotein

Foam cell formation is a hallmark event during atherosclerosis. The current paradigm is that lipid uptake by a scavenger receptor in macrophages initiates necrosis core formation that characterizes atherosclerosis. We report that NOS1-derived nitric oxide (NO) facilitates low-density lipoprotein (LDL) uptake by macrophages independent of the inflammatory response. LDL uptake could be dramatically suppressed by NOS1 specific inhibitor 1-(2-trifluoromethylphenyl) imidazole (TRIM). Importantly, the notion that NOS1 can mediate uptake of lipoproteins suggests that the foam cell formation is regulated by NOS1-derived NO-mediated mechanism. This is a novel study involving NOS1 as a critical player of foam cell formation and reveals much about the key molecular proteins involved in atherosclerosis. Targeting NOS1 would be a useful strategy in reducing LDL uptake by macrophages and hence dampening the atherosclerosis progression.     

Dilatory responses to estrogenic compounds in small femoral arteries of male and female estrogen receptor-beta knockout mice

The objectives of this study were to determine whether acute dilatory responses to estrogen receptor agonists are altered in isolated arteries from estrogen receptor beta-deficient mice (beta-ERKO) and to gain insight into the role of nitric oxide (NO) in these responses. Femoral arteries (approximately 250 microm) from male and female beta-ERKO mice and wild-type (WT) littermates (26 female, 13 in each group; and 24 male, 12 in each group) were mounted on a Multi-Myograph. Concentration-response curves to 17beta-estradiol (17beta-E2) and the selective estrogen receptor-alpha (ER-alpha) agonist propyl-[1H]-pyrazole-1,3,5-triy-trisphenol (PPT) were obtained before and after NO synthase (NOS) inhibition [Nomega-nitro-L-arginine methyl ester (L-NAME), 0.1 mM] in arteries preconstricted with U-46619 (a thromboxane analog). In WT mice, responses to the potent estrogen receptor-beta (ER-beta) agonist 2,3-bis(4-hydroxyphenyl)-propionitrile (DPN) and the contribution of NO were also assessed. Concentration-response curves to 17beta-E2 and PPT were similar in arteries from WT and -ERKO mice of both genders, but NO-mediated relaxation was different, since L-NAME reduced 17-E2 mediated relaxation in arteries from male and female beta-ERKO but not WT mice (P < 0.05). NOS inhibition reduced dilation to PPT in arteries from male and female WT mice, as well as arteries from female beta-ERKO mice (P < 0.05). Responses to DPN in arteries from WT female and male mice did not differ after NOS inhibition. The acute dilatory responses to estrogenic compounds are similar in WT and beta-ERKO mice but differ mechanistically. Because NO appeared to contribute to responses to 17beta-E2 in arteries from beta-ERKO but not WT mice, the presence of ER- apparently inhibits ER--mediated NO relaxation.     

Facilitatory role of NO in neural norepinephrine release in the rat kidney

We examined modulation by nitric oxide (NO) of sympathetic neurotransmitter release and vasoconstriction in the isolated pump-perfused rat kidney. Electrical renal nerve stimulation (RNS; 1 and 2 Hz) increased renal perfusion pressure and renal norepinephrine (NE) efflux. Nonselective NO synthase (NOS) inhibitors [N(omega)-nitro-L-arginine methyl ester (L-NAME) or N(omega)-nitro-L-arginine], but not a selective neuronal NO synthase inhibitor (7-nitroindazole sodium salt), suppressed the NE efflux response and enhanced the perfusion pressure response. Pretreatment with L-arginine prevented the effects of L-NAME on the RNS-induced responses. 2-(4-Carboxyphenyl)-4,4,5,5-tetramethylimidazoline-1-oxyl-3-oxide (carboxy-PTIO), which eliminates NO by oxidizing it to NO(2), suppressed the NE efflux response, whereas the perfusion pressure response was less susceptible to carboxy-PTIO. 8-Bromoguanosine cGMP suppressed and a guanylate cyclase inhibitor [4H-8-bromo-1,2,4-oxadiazolo(3,4-d)benz(b)(1,4)oxazin-1-one] enhanced the RNS-induced perfusion pressure response, but neither of these drugs affected the NE efflux response. These results suggest that endogenous NO facilitates the NE release through cGMP-independent mechanisms, NO metabolites formed after NO(2) rather than NO itself counteract the vasoconstriction, and neuronal NOS does not contribute to these modulatory mechanisms in the sympathetic nervous system of the rat kidney.     

Constitutive eNOS-derived nitric oxide is a determinant of endothelial junctional integrity

Basal vascular endothelial permeability is normally kept low in part by the restrictiveness of interendothelial junctions (IEJs). We investigated the possible role of nitric oxide (NO) in controlling IEJ integrity and thereby regulating basal vascular permeability. We determined the permeability of continuous endothelia in multiple murine vascular beds, including lung vasculature, of wild-type mice, endothelial nitric oxide synthase (eNOS) null mice, and mice treated with NOS inhibitor N-nitro-L-arginine methyl ester (L-NAME). Light and electron microscopic studies revealed that L-NAME treatment resulted in IEJs opening within a few minutes with a widespread response within 30 min. We observed a 35% increase in transendothelial transport of albumin, using as tracer dinitrophenylated albumin in mouse lungs and other organs studied. To rule out the involvement of blood cells in the mechanism of increased endothelial permeability, vascular beds were flushed free of blood, treated with L-NAME, and perfused with the tracer. The open IEJs observed in these studies indicated a direct role for NO in preserving the normal structure of endothelial junctions. We also used the electron-opaque tracer lanthanum chloride to assess vascular permeability. Lanthanum chloride was presented by perfusion to various vascular beds of mice lacking NO. Open IEJs were seen only in capillary and venular endothelial segments of mice lacking NO, and there was a concomitant increase in vascular permeability to the tracer. Together, these data demonstrate that constitutive eNOS-derived NO is a crucial determinant of IEJ integrity and thus serves to maintain the low basal permeability of continuous endothelia.     

Physical conditioning modulates rat cardiac vascular endothelial growth factor gene expression in nitric oxide-deficient hypertension

Many individuals with cardiac diseases undergo periodic physical conditioning with or without medication to improve cardiovascular health. Therefore, this study investigated the interaction of physical training and chronic nitric oxide synthase (NOS) inhibitor (nitro-L-arginine methyl ester, L-NAME) treatment on blood pressure (BP), cardiac vascular endothelial factor (VEGF) gene expression, and nitric oxide (NO) systems in rats. Fisher 344 rats were divided into four groups and treated as follows: (1) sedentary control, (2) exercise training (ET) for 8 weeks, (3) L-NAME (10mg/kg, s.c. for 8 weeks), and (4) ET+L-NAME. BP was monitored with tail-cuff method. The animals were sacrificed 24h after last treatments and hearts were isolated and analyzed. Physical conditioning significantly increased respiratory exchange ratio, cardiac NO levels, NOS activity, endothelial eNOS, and inducible iNOS protein expression as well as VEGF gene expression. Training also caused depletion of cardiac malondialdehyde (MDA) levels indicating the beneficial effects of the training. Chronic L-NAME administration resulted in a depletion of cardiac NO level, NOS activity, and eNOS, nNOS, and iNOS protein expressions, as well as VEGF gene expression (2-fold increase in VEGF mRNA). Chronic L-NAME administration also enhanced cardiac MDA levels indicating cardiac oxidative injury. These biochemical changes were accompanied by increases in BP after L-NAME administration. Interaction of training and NOS inhibitor treatment resulted in normalization of BP and up-regulation of cardiac VEGF gene expression. The data suggest that physical conditioning attenuated the oxidative injury caused by chronic NOS inhibition by up-regulating the cardiac VEGF and NO levels and lowering the BP in rats.     

The role of nitric oxide in ischaemia/reperfusion injury of isolated hearts from severely atherosclerotic mice

Nitric oxide (NO) may play an essential role for maintenance of cardiac function and perfusion, while endothelial dysfunction of atherosclerotic vessels may aggravate ischaemia/reperfusion injury. This paper investigates the role of nitric oxide in ischaemia/reperfusion injury in hearts with coronary atherosclerosis. Hearts of apolipoprotein E/LDL receptor double knockout (ApoE/LDLr KO) mice fed an atherogenic diet for 7-9 months were isolated and Langendorff-perfused with 40 minutes of global ischaemia and 60 minutes reperfusion, and funtion and infarction compared with hearts of C57BL/6 controls in the prescence or abscence of the NO-donor SNAP or the NOS inhibitor L-NAME. Hearts of animals with atherosclerosis were more susceptible to ischaemia/reperfusion injury than hearts of animals with healthy vessels, evident as more impaired left ventricular performance. SNAP protected function and reduced infarct size in atherosclerotic hearts, but the same concentration of SNAP was detrimental in normal hearts, perhaps due to NO-overproduction and peroxynitrite formation demonstrated immunohistochemically as increased formation of nitrosylated tyrosine. A low concentration of SNAP protected against ischaemia/reperfusion dysfunction in normal hearts. L-NAME decreased left ventricular performance in atherosclerotic hearts. These findings suggest that impaired endothelium dependent function contributes to reperfusion injury in coronary atherosclerosis.     

Endothelial nitric oxide synthase is predominantly involved in angiotensin II modulation of renal vascular resistance and norepinephrine release

Nitric oxide (NO) is mainly generated by endothelial NO synthase (eNOS) or neuronal NOS (nNOS). Recent studies indicate that angiotensin II generates NO release, which modulates renal vascular resistance and sympathetic neurotransmission. Experiments in wild-type [eNOS(+/+) and nNOS(+/+)], eNOS-deficient [eNOS(-/-)], and nNOS-deficient [nNOS(-/-)] mice were performed to determine which NOS isoform is involved. Isolated mice kidneys were perfused with Krebs-Henseleit solution. Endogenous norepinephrine release was measured by HPLC. Angiotensin II dose dependently increased renal vascular resistance in all mice species. EC(50) and maximal pressor responses to angiotensin II were greater in eNOS(-/-) than in nNOS(-/-) and smaller in wild-type mice. The nonselective NOS inhibitor N(omega)-nitro-L-arginine methyl ester (L-NAME; 0.3 mM) enhanced angiotensin II-induced pressor responses in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. In nNOS(+/+) mice, 7-nitroindazole monosodium salt (7-NINA; 0.3 mM), a selective nNOS inhibitor, enhanced angiotensin II-induced pressor responses slightly. Angiotensin II-enhanced renal nerve stimulation induced norepinephrine release in all species. L-NAME (0.3 mM) reduced angiotensin II-mediated facilitation of norepinephrine release in nNOS(-/-) and wild-type mice but not in eNOS(-/-) mice. 7-NINA failed to modulate norepinephrine release in nNOS(+/+) mice. (4-Chlorophrnylthio)guanosine-3', 5'-cyclic monophosphate (0.1 nM) increased norepinephrine release. mRNA expression of eNOS, nNOS, and inducible NOS did not differ between mice strains. In conclusion, angiotensin II-mediated effects on renal vascular resistance and sympathetic neurotransmission are modulated by NO in mice. These effects are mediated by eNOS and nNOS, but NO derived from eNOS dominates. Only NO derived from eNOS seems to modulate angiotensin II-mediated renal norepinephrine release.     

Role of DDAH-1 in lipid peroxidation product-mediated inhibition of endothelial NO generation

Altered nitric oxide (NO) biosynthesis is thought to play a role in the initiation and progression of atherosclerosis and may contribute to increased risk seen in other cardiovascular diseases. It is hypothesized that altered NO bioavailability may result from an increase in endogenous NO synthase (NOS) inhibitors, asymmetric dimethly araginine (ADMA), and N(G)-monomethyl arginine, which are normally metabolized by dimethyarginine dimethylamine hydrolase (DDAH). Lipid hydroperoxides and their degradation products are generated during inflammation and oxidative stress and have been implicated in the pathogenesis of cardiovascular disorders. Here, we show that the lipid hydroperoxide degradation product 4-hydroxy-2-nonenal (4-HNE) causes a dose-dependent decrease in NO generation from bovine aortic endothelial cells, accompanied by a decrease in DDAH enzyme activity. The inhibitory effects of 4-HNE (50 microM) on endothelial NO production were partially reversed with L-Arg supplementation (1 mM). Overexpression of human DDAH-1 along with antioxidant supplementation completely restored endothelial NO production following exposure to 4-HNE (50 microM). These results demonstrate a critical role for the endogenous methylarginines in the pathogenesis of endothelial dysfunction. Because lipid hydroperoxides and their degradation products are known to be involved in atherosclerosis, modulation of DDAH and methylarginines may serve as a novel therapeutic target in the treatment of cardiovascular disorders associated with oxidative stress.     

An inhibitor of macrophage arginine transport and nitric oxide production (CNI-1493) prevents acute inflammation and endotoxin lethality.

BACKGROUND: Nitric oxide (NO), a small effector molecule produced enzymatically from L-arginine by nitric oxide synthase (NOS), is a mediator not only of important homeostatic mechanisms (e.g., blood vessel tone and tissue perfusion), but also of key aspects of local and systemic inflammatory responses. Previous efforts to develop inhibitors of NOS to protect against NO-mediated tissue damage in endotoxin shock have been unsuccessful, largely because such competitive NOS antagonists interfere with critical vasoregulatory NO production in blood vessels and decrease survival in endotoxemic animals. Accordingly, we sought to develop a pharmaceutical approach to selectively inhibit NO production in macrophages while sparing NO responses in blood vessels. MATERIALS AND METHODS: The process of cytokine-inducible L-arginine transport and NO production were studied in the murine macrophage-like cell line (RAW 264.7). A series of multivalent guanylhydrazones were synthesized to inhibit cytokine-inducible L-arginine transport. One such compound (CNI-1493) was studied further in animal models of endothelial-derived relaxing factor (EDRF) activity, carrageenan inflammation, and lethal lipopolysaccharide (LPS) challenge. RESULTS: Upon activation with cytokines, macrophages increase transport of L-arginine to support the production of NO by NOS. Since endothelial cells do not require this additional arginine transport to produce NO, we reasoned that a competitive inhibitor of cytokine-inducible L-arginine transport would not inhibit EDRF activity in blood vessels, and thus might be effectively employed against endotoxic shock. CNI-1493, a tetravalent guanylhydrazone, proved to be a selective inhibitor of cytokine-inducible arginine transport and NO production, but did not inhibit EDRF activity. In mice, CNI-1493 prevented the development of carrageenan-induced footpad inflammation, and conferred protection against lethal LPS challenge. CONCLUSIONS: A selective inhibitor of cytokine-inducible L-arginine transport that does not inhibit vascular EDRF responses is effective against endotoxin lethality and significantly reduces inflammatory responses.     

Opposing interplay between Neuropeptide FF and nitric oxide in antinociception and hypothermia

This study examined the ability of the anti-opioid Neuropeptide FF (NPFF) to modify the endogenous activity of nitric oxide (NO). Antinociceptive and hypothermic effects of 1DMe (D.Tyr-Leu-(n.Me)Phe-Gln-Pro-Gln-Arg-Phe-NH(2)), an NPFF agonist, and of L-NAME (N(omega)nitro-L-arginine methyl ester), an inhibitor of nitric oxide synthase, were investigated in mice. Intraperitoneal (i.p.) injection of L-NAME induced, in the hot plate test, a dose-dependent antinociception not reversed by naloxone, an opioid antagonist, but inhibited by L-Arg, the NO synthesis precursor. Intracerebroventricular (i.c.v.) injections of 1DMe inhibit the antinociceptive activity of L-NAME in a dose-dependent manner. On the contrary, L-NAME markedly potentiated hypothermia induced by 1DMe injected in the third ventricle. These data show that Neuropeptide FF receptors exert a dual effect on endogenous NO functions and could modulate pain transmission independently of opioids.     

Dual role of endogenous nitric oxide in development of dextran sodium sulfate-induced colitis in rats.

The role of nitric oxide (NO) in the etiology of ulcerative colitis is controversial with reports of the improvement and aggravation of colonic lesions by inducible NO synthase (iNOS) inhibitors. In the present study, we compared the effect of the selective iNOS inhibitor aminoguanidine and the nonselective NOS inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) on a dextran sulfate sodium (DSS)-induced model of colitis in rats. Experimental colitis was induced by a 3% DSS-solution added to drinking water for 7 days. Aminoguanidine (5 approximately 20 mg/kg) and L-NAME (10 mg/kg) were administered p.o. twice daily for the first 3 days, the last 3 days or all 6 days of DSS treatment. Body weight and severity of colitis (diarrhea, bloody feces) were observed over a period of 7 days. DSS treatment resulted in severe colonic lesions, accompanied by diarrhea, bloody feces, decrease of body weight and colon shortening. All of the parameters investigated improved significantly with aminoguanidine treatment at 20 mg/kg for 6 days or the last 3 days of DSS-treatment, but L-NAME did not significantly affect the colitis during these periods. When L-NAME or aminoguanidine was given in the first 3 days of DSS treatment, the colonic lesions were slightly aggravated by L-NAME but not affected by aminoguanidine. The expression of iNOS mRNA was observed from the 3(rd) day of DSS treatment. These results suggested that endogenous NO exerts a biphasic influence on DSS-induced colitis, depending on the NOS isoenzyme; a beneficial effect of NO derived from constitutive NOS and a detrimental effect of NO produced by iNOS in the development of colitis.