Two new nitric oxide synthase inhibitors: pyridoxal aminoguanidine and 8-quinolinecarboxylic hydrazide selectively inhibit basal but not agonist-stimulated release of nitric oxide in rat aorta.

Structural modification at one of the guanidine nitrogens of L-arginine has led to the development of a number of compounds N(G)-monomethyl-L-arginine (L-NMMA), N(G)-nitro-L-arginine (L-NOARG), N(G)-nitro-L-arginine methyl ester (L-NAME) that competitively inhibit nitric oxide synthase (NOS). It was reported that another chemically related compound known as a glycation inhibitor, aminoguanidine also inhibits NOS. Recently, two new glycation inhibitors, structurally related to aminoguanidine (AG), pyridoxal aminoguanidine (PLAG) and 8-quinoline carboxylic hydrazide (8Q) were synthesized. In this study, the effects of these two inhibitors on responses mediated by constitutive nitric oxide (NO) were investigated in vitro. For this purpose, in the present study vascular responses to phenylephrine and acetylcholine in isolated aortas were evaluated. Incubation (15 min) with PLAG and 8Q (10(-4)M for each) induced potentiation of phenylephrine-induced contraction in endothelium intact but not in endothelium denuded rings of rat aorta. The ability of PLAG or 8Q to augment phenylephrine-induced tone in endothelium containing rings was completely prevented by preincubation with L-arginine (1mM), but not with D-arginine. Both compounds (PLAG, 8Q) did not affect acetylcholine-induced relaxation. These results suggest that both of the new compounds produced a selective inhibition of basal but not agonist stimulated production of nitric oxide in rat aorta.     

Differential sensitivity of basal and acetylcholine-stimulated activity of nitric oxide to destruction by superoxide anion in rat aorta.

1. In this study we compared the ability of superoxide anion to destroy the relaxant activity of basal and acetylcholine (ACh)-stimulated activity of NO in isolated rings of rat aorta. 2. Superoxide dismutase (SOD, 1-300 u ml-1) induced a concentration-dependent relaxation of phenylephrine (PE)-induced tone in endothelium-containing rings which was blocked by NG-nitro-L-arginine (L-NOARG, 30 microM), but had no effect on endothelium-denuded rings. It was likely therefore that the relaxant action of SOD resulted from protection of basally produced NO from destruction by superoxide anion, generated either within the tissue or in the oxygenated Krebs solution. 3. In contrast, a concentration of SOD (50 u ml-1) which produced almost maximal enhancement of basal NO activity, had no effect on ACh (10 nM-3 microM)-induced relaxation. 4. In the presence of catalase (3000 u ml-1) to prevent the actions of hydrogen peroxide, superoxide anion generation using hypoxanthine (HX, 0.1 mM)/xanthine oxidase (XO, 16 mu ml-1) produced an augmentation of PE-induced tone in endothelium-containing but not endothelium-denuded rings. This was likely to have resulted from removal of the tonic vasodilator action of basally-produced NO by superoxide anion, since it was blocked in tissues treated with SOD (250 u ml-1), NG-monomethyl-L-arginine (L-NMMA, 30 microM) or L-NOARG (30 microM). Pyrogallol (0.1 mM) had a similar action to HX/XO, but produced an additional augmentation of tone by an endothelium-independent mechanism.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

Differential sensitivity of basal and acetylcholine-induced activity of nitric oxide to blockade by asymmetric dimethylarginine in the rat aorta

Background and purpose:

Previous work has shown that N^G-monomethyl-l-arginine (l-NMMA) paradoxically inhibits basal, but not ACh-stimulated activity of nitric oxide in rat aorta. The aim of this study was to determine if the endogenously produced agent, asymmetric N^G, N^G-dimethyl-l-arginine (ADMA), also exhibits this unusual selective blocking action.

Experimental approach:

The effect of ADMA on basal nitric oxide activity was assessed by examining its ability to enhance phenylephrine (PE)-induced tone in endothelium-containing rings. Its effect on ACh-induced relaxation was assessed both in conditions where ADMA greatly enhanced PE tone and where tone was carefully matched with control tissues at a range of different levels.

Key results:

ADMA (100 µM) potentiated PE-induced contraction, consistent with inhibition of basal nitric oxide activity. Higher concentrations (300–1000 µM) had no greater effect. Although ADMA (100 µM) also appeared to block ACh-induced relaxation when it enhanced PE tone to maximal levels, virtually no block was seen at intermediate levels of tone in the presence of ADMA. Even ADMA at 1000 µM had no effect on the maximal relaxation to ACh, although it produced a small (two- to threefold) reduction in sensitivity. ADMA and l-NMMA, like l-arginine (all at 1000 µM), protected ACh-induced relaxation against blockade by l-NAME (30 µM).

Conclusions and implications:

In the rat aorta, ADMA, like l-NMMA, blocks basal activity of nitric oxide, but has little effect on that stimulated by ACh. Further studies are required to explain these seemingly anomalous actions of ADMA and l-NMMA.     

Lack of inhibition of endothelial nitric oxide synthase in the isolated rat aorta by doxorubicin.

Besides inducing cardiotoxicity, doxorubicin also affects the vasculature. Recent observations in cultured endothelial cells indicated that the endothelial form of nitric oxide synthase might be inhibited by doxorubicin thereby seriously interfering with vascular function. We have investigated the effect of doxorubicin on the relaxation induced by the muscarinic agonist carbachol in the isolated rat aorta. It was found that doxorubicin at concentrations up to 50 microM does not alter the relaxant response to carbachol. Direct measurement of nitrite, the metabolite of NO*, by the Griess assay confirmed our observation that NO*)production is not inhibited by doxorubicin.     

前列腺素而非一氧化氮是鳟主动脉内皮细胞舒血管因子(英文)

AIM:To identify the type of prostanoids produced by endothelial cells of trout aorta and to determine whether or not the smooth muscle responds to nitric oxide. METHODS:Ventral aortas, with and without endotheli-um from rainbow trout (S gairdneri), were incubated in a buffered salt solution. RESULTS:Addition of the calcium ionophore A23187 caused a significant increase in prostaglandin E's and a consistent increase in the stable metabolite of prostacyclin (6-keto-prostaglandin F1a) in the incubation media only when the endothelium was present. This production was inhibited by methylene blue (10umol/L). In rings of trout aorta without endothelium suspended for the measurement of isometric force in organ chambers,prostacyclin and prostaglandin E1 but not prostaglandin E2 caused concentration-dependent decreases in tension when the rings were contracted with acetyl-choline. The smooth muscle did not relax to nitric oxide but did so to sodium nitroprusside. Relaxations to the latter nitrovasodilator were no     

Effects of superoxide dismutase mimetics on the activity of nitric oxide in rat aorta.

A number of structurally distinct superoxide dismutase (SOD) mimetics were examined to determine if they shared the ability of authentic Cu/Zn SOD to produce endothelium-dependent relaxation of rings of rat aorta by protecting basal nitric oxide from destruction by endogenously produced superoxide anion. MnCl2 (10 nM-100 microM), CuSO4 (100 nM-1 mM) and CuDIPS (Cu [II]-[diisopropylsalicylate]2; 100 nM-30 microM) each mimicked the ability of Cu/Zn SOD (0.1-300 u ml(-1)) to produce relaxation of phenylephrine-precontracted aortic rings in a manner inhibited by endothelial removal or treatment with N(G)-nitro-L-arginine methyl ester (L-NAME, 100 microM). In contrast, MnTMPyP (Mn [III] tetrakis [1-methyl-4-pyridyl] porphyrin; 10 nM-30 microM) augmented phenylephrine-induced contraction and this was blocked by endothelial removal or treatment with L-NAME (100 microM), consistent with destruction rather than protection of basal nitric oxide activity. Pretreatment with Cu/Zn SOD (250 u ml(-1)) blocked this augmentation suggesting that it arose paradoxically through destruction of nitric oxide by superoxide anion. The spin trap agents tiron (100 nM-1 mM), tempol (100 nM-1 mM) and PTIYO (4-phenyl-2,2,5,5-tetramethyl imidazolin-1-yloxy-5-oxide; 100 nM-300 microM) all failed to promote endothelium-dependent relaxation. In fact, the last two augmented phenylephrine-induced tone and this was blocked by endothelial removal or treatment with L-NAME (100 microM), consistent with destruction of basal nitric oxide activity. This destruction was unaffected by pretreatment with Cu/Zn SOD (250 u ml(-1)) and probably reflected the direct ability of tempol and PTIYO to destroy nitric oxide. Thus, the ideal SOD mimetic for protection of nitric oxide activity in conditions of oxidant stress still awaits development.     

Differential effects of L-arginine on the inhibition by NG-nitro-L-arginine methyl ester of basal and agonist-stimulated EDRF activity.

1. An isolated, buffer-perfused rabbit ear preparation was used to investigate the influence of NG-nitro-L-arginine methyl ester (L-NAME) on endothelium-dependent vasodiltation and modulation of vasoconstrictor responses and vascular conductance. 2. Acetylcholine (0.55 pmol-1.6 nmol) caused dose-related vasodilatation of preparations constricted by the combination of 5-hydroxytryptamine and histamine (both 1 microM), with an ED50 = 31.1 +/- 7.8 pmol and a maximum dilatation of 69.9 +/- 4.3%. In the presence of 10 microM L-NAME the dose-response for vasodilator effects was shifted significantly (P less than 0.001) to the right (ED50 = 3.07 +/- 1.18 nmol) and there was a significant (P less than 0.01) depression of the maximum response (Rmax = 44.3 +/- 4.0%). The higher concentration of 100 microM L-NAME completely abolished vasodilatation to acetylcholine. L-Arginine (10 mM) did not reverse the inhibitory actions of L-NAME at either concentration. 3. L-NAME 100 microM, augmented vascular tone induced by 1 microM 5-hydroxytryptamine and 1 microM histamine, thus altering the characteristics of both pressure/flow and conductance/flow relationships such that conductance was reduced at all flow rates. The augmentation of constrictor tone was reversed in a concentration-dependent manner by L-arginine (10 microM-10 mM) and the effect of L-NAME on the conductance/flow relationships was similarly reversed by 10 mM L-arginine. The augmentation of tone was endothelium-dependent as it did not occur following functional destruction of the endothelium by perfusion of the vascular bed with the detergent CHAPS (0.3%) for 150s.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inhibition of human inducible nitric oxide synthase by S-alkyl-L-isothiocitrulline-containing dipeptides

The aim of this study was to investigate the structure-activity relationship of S-alkyl-L-isothiocitrulline-containing dipeptides towards three partially purified recombinant human nitric oxide synthase (NOS) isozymes, as well as the effects of these compounds on cytokine-induced NO production by human DLD-1 cells. In an in vitro assay, S-methyl-L-isothiocitrulline (L-MIT) was slightly selective for human neuronal NOS (nNOS) over the inducible (iNOS) or endothelial (eNOS) isozyme, but the combination of a hydrophobic L-amino acid (L-Phe, L-Leu or L-Trp) with L-MIT dramatically altered the inhibition pattern to give selective iNOS inhibitors. Introduction of a hydroxy, nitro, amino or methoxy group at the para position of the aromatic ring of L-MIT-L-Phe (MILF) decreased the selectivity and inhibitory potency. A longer or larger S-alkyl group also decreased the selectivity and potency. Dixon analysis showed that all of the dipeptides were competitive inhibitors of the three isoforms of human NOS. The enzymatic time course curves indicated that MILF was a slow binding inhibitor of human iNOS. These results suggest that the human NOS isozymes have different-sized cavities in the binding site near the position to which the C-terminal of L-arginine binds, and the cavity of iNOS is hydrophobic. Interestingly, L-MIT-D-Phe (MIDF) showed little inhibitory activity or selectivity, suggesting that the cavity of human iNOS is located in a well-defined direction from the alpha carbon atom. NO production in cytokine-stimulated human DLD-1 cells was measured with a fluorescent indicator, DAF-FM. MILF, L-MIT-L-Trp(-CHO) (MILW) and L-MIT-L-Tyr (MILY) showed more potent activity than L-MIT in this whole-cell assay. Thus, S-alkyl-L-isothiocitrulline-containing dipeptides are selective inhibitors of human iNOS, and work efficiently in cell-based assay.     

Endogenous nitric oxide attenuates beta-adrenoceptor-mediated relaxation in rat aorta

1. Divergent evidence suggests that the intracellular signalling pathways for beta-adrenoceptor-mediated vascular relaxation involves either cAMP/protein kinase (PK) A or endothelial nitric oxide (NO) release and subsequent activation of cGMP/PKG. The present study identifies the relative roles of NO and cAMP, as well as dependence on the endothelium for beta-adrenoceptor-mediated relaxation of rat isolated aortas. 2. Cumulative concentration-response curves to isoprenaline (0.01-3 micromol/L) in phenylephrine (0.1 micromol/L)-preconstricted endothelium-intact and -denuded aortas were constructed. Isoprenaline-mediated relaxation was partially reduced by endothelium removal and the presence of the NO synthase inhibitor N(G)-monomethyl-L-arginine (0.1 mmol/L), but not by the cAMP antagonist (Rp)-cyclic adenosine-3',5'-monophosphorothioate (Rp-cAMPS; 0.5 mmol/L). 3. In contrast, in endothelium-denuded aortas, the isoprenaline-mediated relaxation was inhibited by Rp-cAMPS and this inhibition was lost in the presence of the NO donor sodium nitroprusside (1 nmol/L). This effect was not due to phosphodiesterase (PDE) activity because the non-selective PDE inhibitor 3-isobutyl-1-methylxanthine (1 micromol/L) failed to affect the isoprenaline vasorelaxant response. 4. The K(+) channel blocker tetraethylammonium (TEA; 1 mmol/L) attenuated isoprenaline-induced relaxation in endothelium-denuded aorta, but its effect was non-additive with Rp-cAMPS, suggesting that the K(+) channel component may involve cAMP. In endothelium-intact aortas, TEA but not Rp-cAMPS reduced isoprenaline relaxation, suggesting an additional non-cAMP component. 5. These findings suggest that beta-adrenoceptors induce vascular smooth muscle relaxation by acting through the NO-cGMP pathway and, when that is disrupted by endothelium removal or the presence of an NO synthase inhibitor, the cAMP pathway in smooth muscles is used. The lack of cAMP participation in endothelium-intact vessels may be because NO suppresses or overrides the cAMP effect.     

Acute inhibition of inducible nitric oxide synthase but not its absence suppresses asthma-like responses

In the present study we investigated the lymphocytes infiltration and other parameters of allergic lung inflammation comparing mice submitted to acute suppression of nitric oxide synthesis with mice deficient in inducible nitric oxide synthase (NOS2-/-) gene. At weekly intervals C57Bl/6 mice, wild type and NOS2-/- were sensitized twice with ovalbumin-alumen and challenged twice with ovalbumin aerosol and lungs examined 24 h later. In wild type mice, treatment with nitric oxide synthase inhibitor, N(omega)-nitro-L-arginine-methyl-ester (L-NAME) or aminoguanidine (i.p., 30 min before each ovalbumin challenge) caused a significant decrease in bronchoalveolar lavage cell number: eosinophils (90%), lymphocytes NK1.1+ (70%), Tgammadelta+ (50%), CD4+ (55%), CD8+ (60%) and B220+ (65%). Both inhibitors abolished airway hyperreactivity and significantly reduced mucus secretion (L-NAME 64%; aminoguanidine 58%). Surprisingly, in NOS2-/- mice these parameters of allergic lung inflammation were not significantly different when compared with wild type mice. In addition, treatment of NOS2-/- mice with L-NAME or aminoguanidine did not affect these parameters. Thus, acute inhibition of NOS2 activity inhibits asthma-like responses but absence of NOS2 has no affect.     

Selective inhibition of inducible nitric oxide synthase prevents ischaemic brain injury.

1. The aim of this study was to investigate the effect of N-(3-(aminomethyl)benzyl)acetamidine (1400W), a selective inhibitor of inducible calcium-independent nitric oxide synthase (iNOS), on the functional and histopathological outcomes of experimental transient focal cerebral ischaemia in rats. 2. Transient ischaemia was produced by the occlusion for 2 h of both the left middle cerebral artery and common carotid artery. Treatments with 1400W (20 mg kg(-1)) or vehicle were started 18 h after occlusion of the arteries and consisted in seven subcutaneous injections at 8 h interval. Ischaemic outcomes and NOS activities (constitutive and calcium-independent NOS) were evaluated 3 days after ischaemia. 3. 1400W significantly reduced ischaemic lesion volume by 31%, and attenuated weight loss and neurological dysfunction. 4. 1400W attenuated the calcium-independent NOS activity in the infarct by 36% without affecting the constitutive NOS activity. 5. These findings suggest that iNOS activation contributes to tissue damage and that selective inhibitors of this isoform may be of interest for the treatment of stroke.     

Platelet aggregation responses are critically regulated in vivo by endogenous nitric oxide but not by endothelial nitric oxide synthase

Background and purpose:

Although exogenous nitric oxide (NO) clearly modifies platelet function, the role and the source of endogenous NO in vivo remain undefined. In addition, endothelial NO synthase (NOS-3) critically regulates vessel tone but its role in modulating platelet function is unclear. In this paper we have investigated the roles of endogenous NO and NOS-3 in regulating platelet function in vivo and determined the functional contribution made by platelet-derived NO.

Experimental approach:

We used a mouse model for directly assessing platelet functional responses in situ in the presence of an intact vascular endothelium with supporting in vitro and molecular studies.

Key results:

Acute NOS inhibition by N_ω-nitro-L-arginine methyl ester hydrochloride (L-NAME) enhanced platelet aggregatory responses to thrombin and platelets were shown to be regulated primarily by NO sources external to the platelet. Elevation of endogenous NOS inhibitors to mimic effects reported in patients with cardiovascular diseases did not enhance platelet responses. Platelet responsiveness following agonist stimulation was not modified in male or female NOS-3^−/− mice but responses in NOS-3^−/− mice were enhanced by L-NAME.

Conclusions and implications:

Platelets are regulated by endogenous NO in vivo, primarily by NO originating from the environment external to the platelet with a negligible or undetectable role of platelet-derived NO. Raised levels of endogenous NOS inhibitors, as reported in a range of diseases were not, in isolation, sufficient to enhance platelet activity and NOS-3 is not essential for normal platelet function in vivo due to the presence of bioactive NO following deletion of NOS-3.     

Protection by nitric oxide of morphine-induced inhibition of rat submandibular gland function.

The effects of morphine, l -arginine (nitric oxide precursor) and l -NAME (nitric oxide synthesis inhibitor ) and their concurrent therapy on rat submandibular secretory function were studied. Pure submandibular saliva was collected intraorally by micro polyethylene cannula from anaesthetized rats using pilocarpine as secretagogue. Single intraperitoneal injection of morphine (6 mg kg(-1)) to rats induced significant (P< 0.01) inhibition of salivary flow rate (28%), total protein (12%) and calcium concentrations (27%). Sodium output was increased (23%, P< 0.01). Single intraperitoneal administration of l -arginine (100 mg kg(-1)) and l -NAME (10 mg kg(-1)) affected salivary gland function. Saliva flow rate was reduced by l -NAME (23%, P< 0.01). The total protein concentration of saliva was increased by l -arginine (21%, P< 0.05) and decreased by l -NAME (19%, P< 0.01). Calcium concentration of saliva was increased by l -arginine (25%, P< 0.01) and reduced by l -NAME (21%, P< 0.01). In combination treatment, l -arginine prevented (P< 0.01) morphine-induced reduction of flow rate while l -NAME potentiated it (P< 0.01). The secretion of total protein and calcium were influenced in a similar trend by concurrent therapy. l -NAME potentiated morphine-induced decrease of total protein and calcium concentrations (P< 0.01) while l -arginine restored (P< 0.01) them to levels close to control and morphine groups respectively. It is concluded that morphine inhibits salivary gland function and nitric oxide (NO) plays a positive role in this system. Also it is confirmed that morphine inhibitory effects on submandibular function are somewhat restored by l -arginine and expanded by l -NAME. The modulatory effect of the l -arginine/NO system on salivary gland function is suggested.     

Inhibition of hypoxia-induced relaxation of rabbit isolated coronary arteries by NG-monomethyl-L-arginine but not glibenclamide.

1. This study describes the in vitro interaction of the muscarinic ligand McNeil-A-343 with two 5-hydroxytryptamine (5-HT) receptor subtypes, the 5-HT4 and 5-HT3 receptors, using functional as well as radioligand binding studies. 2. In the rat oesophageal muscularis mucosae, precontracted with carbachol, McNeil-A-343 was a competitive antagonist (pA2 6.2) of the 5-HT4 receptor which mediates the relaxation induced by 5-HT. The compound per se relaxed the oesophagus at high concentration only (> or = 10 microM), an effect unchanged by desensitization of the 5-HT4 receptor with 10 microM 5-methoxytryptamine. In the same preparation in the absence of tone, McNeil-A-343 displaced the carbachol concentration-response curve to the right, yielding an apparent affinity (pA2) of 4.9 for muscarinic receptors. 3. In the rat isolated superior cervical ganglion preparation, after blockade of muscarinic and nicotinic receptors, McNeil-A-343 caused a concentration-dependent depolarization that was unaffected by 100 nM ondansetron. The concentration-fast depolarization curve to 5-HT, mediated by the 5-HT3 receptor, was displaced to the right by McNeil-A-343, which showed an apparent affinity (pA2) of 4.8 for the 5-HT3 subtype. 4. In binding studies, McNeil-A-343 recognized a single population of 5-HT4 receptors in pig caudate nucleus, with a pKI of 5.9. The binding affinity of McNeil-A-343 for 5-HT3 receptors in NG 108-15 cells was approximately four times lower (pKI 5.3). Binding affinities (pKI) for muscarinic receptor subtypes in rat tissues were 5.3 (M1, cortex), 5.2 (M2, heart) and 4.9 (M3, submandibular glands), respectively.(ABSTRACT TRUNCATED AT 250 WORDS)     

A flavonoid-rich diet increases nitric oxide production in rat aorta

1. Red wine intake is associated with a low risk of cardiovascular disease. This effect has been partly attributed to the action of polyphenolic compounds, which decrease the oxidation of plasma low density lipoproteins. Moreover, nitric oxide ((*)NO) is a vasodilator and polyphenolic compounds induce endothelium-dependent vasorelaxation in vitro. 2. Here we studied whether a diet rich in dealcoholated red wine (DRW) increases acetylcholine-induced vasorelaxation and whether ingestion of DRW-, quercetin- or catechin-rich diets modifies the (*)NO-cyclic guanosine-3',5'-monophosphate (cyclic GMP) pathway and superoxide anion (O2(.-)) release in aorta in a resting state in rats fed semi-purified diets containing either 35% (v w(-1)) DRW, 0.3% (w w(-1)) quercetin or 0.3% (w w(-1)) catechin for 10 days. 3. (*)NO-mediated vasorelaxation induced by acetylcholine was greater in rats fed the DRW-rich diet than in those that received the control diet. 4. Expression of endothelial (*)NO synthase (eNOS) was similar in the four dietary groups. The aortic rings of rats fed either the DRW-, quercetin-, or catechin-rich diets showed higher NOS activity, (*)NO production and cyclic GMP content than those of rats fed the control diet. No changes were observed in O2(.-) production. 5. In summary, diets rich in either DRW, quercetin or catechin induced endothelium-dependent vasorelaxation in rat aorta in a resting state through the enhancement of (*)NO production, without modifying O2(.-) generation, thus the bioavailability of (*)NO was increased. The increase in the (*)NO-cyclic GMP pathway explains the beneficial effect of flavonoids at vascular level.     

Effects of oral propylthiouracil treatment on nitric oxide production in rat aorta.

The effects of oral propylthiouracil (PTU) treatment on vascular nitric oxide (NO) production were studied in the rat aorta. Rats were fed a standard low fat diet with or without 0.1% PTU, for 2 or 4 weeks, or for 2 weeks with additional thyroxine injections. Concentration response curves were then constructed to phenylephrine (PE) in both endothelium-intact and denuded aortic rings from these animals and after incubation with 0.1 mM L-N(G)nitroarginine (L-NOARG). In addition, expression of nitric oxide synthase (NOS) was analysed in sections of aorta from PTU-treated and control rats using rabbit polyclonal antibodies to both inducible NOS (iNOS) and endothelial NOS (eNOS). Oral PTU treatment resulted in a significant reduction in both the maximum response (control, 0.53+/-0.02; 2 week PTU, 0.20+/-0.07; 4 week PTU, 0.07+/-0.02 g mg(-1)) and vessel sensitivity (EC50 values: control, 9.10x10(-8)+/-0.67; 2 week PTU, 7.45x10(-7)+/-1.15; 4 week PTU, 9.73x10(-7)+/-0.45 M) to PE in endothelium-intact vessel rings, as compared to controls (P<0.05). Both endothelial removal and incubation with L-NOARG restored the maximum response after 2, but not 4 weeks, although, in general, vessel sensitivity was not altered by either treatment. Vessels from PTU-treated rats given thyroxine injections showed no significant differences between any of the dose response curve parameters. Immunohistochemical analysis suggested that labelling for eNOS may be increased after PTU treatment as compared to control animals, whereas iNOS antibody immunoreactivity was not different between the two groups. These results suggest that the hyporesponsiveness to PE observed after oral PTU treatment is, in part, due to enhanced nitric oxide (NO) production by the endothelium, and demonstrate for the first time that thyroid hormones may play a role in the regulation of eNOS activity in the rat aorta.     

Tetrahydrobiopterin synthesis inhibitors induce nitric oxide synthesis in rat aorta

I. Incubation of rato aortic rings with tetrahydrobiopterin synthesis inhibitors (NAS or DAHP) significantly decreased contractions to phenylephrine. These two compounds significantly potentiated the vascular hyporeactivity induced by endotoxin. Inhibitors of nitric oxide synthesis (NLA or MLA) restored the contractile responses to this α₁-agonist in NAS- or DAHP-treated control rings and abolished the NAS- or DAHP-induced increased hyporeactivity to PE in endotoxin-treated aortic rings. These observations suggest that treatment of isolated blood vessels with BH₄ synthesis inhibitors induces the production of NO' synthesis, resulting in turn in a vascular hyporeactivity to PE potentiated in endotoxin-treated preparations.     

Role of adventitial nitric oxide in vascular hyporeactivity induced by lipopolysaccharide in rat aorta

This study was designed to elucidate the role of the adventitia in NO-mediated vascular effects of lipopolysaccharide (LPS). After incubation of rat aorta with LPS, the adventitia generated 3.5 times more nitrite plus nitrate than a corresponding segment of media. Control media covered by adventitia from LPS-treated aortic rings exhibited a 4 fold elevated level of cyclic GMP. Medial layers from LPS-treated aortic rings (like LPS-treated adventitia-intact rings) exhibited a decrease in sensitivity to noradrenaline (NA) that was reversed by 1H-(1,2,4)oxadiazolo(4,3-a)quinoxalin-1-one (1 μM) or N^ω-nitro-L-arginine methylester (0.3 mM). However, in contrast to LPS-treated adventitia-intact rings, medial layers showed no reduction in maximal contraction to NA and virtually no relaxation to L-arginine. These data indicate that in blood vessels exposed to LPS, the adventitia is a more powerful source of NO than the media. The adventitia-derived NO can reach soluble guanylyl cyclase in the medial layer and contribute greatly to vascular hyporeactivity and L-arginine-induced relaxation observed in blood vessels exposed to LPS.