Lack of correlation between the observed stability and pharmacological properties of S-nitroso derivatives of glutathione and cysteine-related peptides.

S-Nitrosothiols (RSNOs) have been widely studied as donors of nitric oxide. In general, RSNOs are considered to be somewhat unstable; however, they are both potent vasodilators and inhibitors of platelet aggregation. In order to improve our understanding of the factors that determine the biological activity of RSNOs, the chemical stability and pharmacological activity of a series of RSNOs was determined. Results show that millimolar solutions of S-nitrosocysteine (SNOCys) and S-nitroso-L-cysteinylglycine (SNOCysGly) were the least stable, whereas S-nitroso-3-mercaptopropionic acid (SNOPROPA) and S-nitroso-N-acetyl-L-cysteine (SNONAC) were the most stable of the compounds tested. Recent evidence suggests that RSNOs, such as SNONAC, are as unstable as SNOCys at micromolar concentrations. The decomposition of certain RSNOs is catalysed by trace amounts of copper (II) ions, with this phenomenon being particularly evident for SNOCys and SNOCysGly. The decomposition of the more stable RSNOs, including S-nitroso-L-glutathione (SNOGSH) and L-gamma-glutamyl-L-cysteine (SNOGluCys), were not as sensitive to copper ions. The decomposition of the stable RSNO, SNOGSH, was more rapid in the presence of excess thiol, whereas the decay of the unstable RSNO, SNOCys, was reduced with added thiol. All RSNOs tested inhibited platelet aggregation, relaxed vascular smooth muscle, and inhibited cell growth in the nanomolar range, but their order of potency did not correlate with their chemical stability of millimolar solutions. It is apparent that the potency of an RSNO in a physiological situation will depend on the concentration of the compound present, the presence of trace metal ions such as copper, and the occurrence of transnitrosation reactions.

Inhibitory effect of cystic fibrosis sputum on adenovirus-mediated gene transfer in cultured epithelial cells.

Effective gene transfer to the airway epithelial cells of individuals with cystic fibrosis (CF) requires gene therapy vectors to effectively penetrate the mucous lining of the airways of these patients. In this study, we examined the effects of the aqueous sol fraction of sputum recovered from CF patients (CF sol) on adenovirus (Ad)-mediated gene transfer to cultured epithelial cells. Sputum collected from patients with CF was separated into aqueous sol and gel fractions by ultracentrifugation and the sol fraction from different individuals was pooled. To determine if CF sol affects Ad-mediated transfection, Fisher rat thyroid (FRT) epithelial cells or normal human bronchial epithelial (NHBE) cells were infected with an Ad encoding beta-galactosidase (Ad2/betagal-2) in the presence or absence of the pooled CF sol. Transfection efficiency was determined by measuring beta-Gal activity. CF sol significantly inhibited Ad2-mediated gene transfer in a dose-dependent manner when the vector was incubated with CF sol prior to exposure to the cells. In contrast, preincubation of the cells with the sol was without effect. The inhibition of Ad-mediated gene transfer by CF sol was not related to its low pH, was abrogated by preadsorption with an Ad2 serotype vector, and was neutralized by heat treatment, but was not affected by treatment with protease inhibitors. Analysis of CF sol fractions from seven different individuals with CF showed inhibition of Ad-mediated gene transfer in four of the seven samples tested and, further, the inhibitory effect was correlated with the presence of Ad-specific antibodies. We conclude that preexisting adenovirus-specific antibodies present in some of the patient samples were the predominant factor inhibiting Ad-mediated gene transfer.

Vascular endothelial cell activation associated with increased plasma asymmetric dimethyl arginine in children and young adults with hypertension: a basis for atheroma?

The mechanism behind the development of vascular complications of hypertension in the young human remains unclear. To explore the role of vascular endothelium-generated nitric oxide (a known mediator of leucocyte-platelet-endothelial interactions) in this context, we investigated markers of endothelial activation (soluble vascular cell adhesion molecule-1 (VCAM-1), intercellular adhesion molecule-1 (ICAM-1), P-selectin, E-selectin), and von Willebrand factor and the plasma level of the endogenous nitric oxide inhibitor asymmetric dimethyl arginine (ADMA) in a group of 31 (17 male, mean age 9.4 years) hypertensive and 9 (4 male, mean age 9.1 years) healthy, normotensive children and young adults. We found raised levels of ADMA (mean (SEM) 235 (32) n mol/l) and VCAM-1 (median (range) 1237 (675-2700) ng/ml) in the plasma of hypertensive subjects compared with those of normotensives (ADMA, 103 (7) n mol/l and VCAM-1, 1005 (425-1650) ng/ml, respectively). Furthermore, in hypertensive subjects, higher VCAM-1 concentrations (r = 0.66, p < 0.001) and vWF concentrations (r = 0.37, p = 0.04) were significantly associated with a higher plasma ADMA level. Therefore, an isolated increase in plasma VCAM-1 in hypertensives in association with raised ADMA may signify a selective "non-inflammatory" endothelial activation triggered by endothelial nitric oxide synthase inhibition. Since VCAM-1 is implicated in the origins of atherosclerosis, ADMA may be an important contributory factor in increasing the risk of atheroma formation in hypertensive children and young adults.

Infarct size and nitric oxide synthase in murine myocardium.

Controversy surrounds the involvement of nitric oxide (NO) in myocardial ischaemia-reperfusion injury and the balance between deleterious and beneficial effects. NO synthase (NOS) is expressed constitutively as two isoforms: endothelial (eNOS) and neuronal (nNOS). Knockout mice lacking the gene for either eNOS (eNOS KO) or nNOS (nNOS KO), were compared with wild-types (WT) during a protocol of global ischaemia-reperfusion injury. Thirty-six mouse hearts (12 from each group) were isolated and the aorta cannulated for Langendorff perfusion with modified Krebs solution at constant pressure. An apical suture connected the left ventricle to a force transducer via a light weight coupling rod. Following stabilization hearts were subjected to 30 min of global ischaemia at 37 degrees C. During 30 min reperfusion, the recovery of baseline force-rate product (F%) was recorded. Hearts were then stained with tetrazolium, frozen, sliced, and fixed with formalin. Slices were compressed between plexiglas plates, and a magnified video image digitized to allow planimetry for infarct size (as percentage of ventricular volume I/R). Although recovery of contractile function did not differ between groups, eNOS KOs suffered significantly larger infarcts than WT or nNOS KOs (41 v 33 and 30% respectively, P<0.05 for both comparisons). A protective role for eNOS against global ischaemia-reperfusion injury has been demonstrated for the first time in murine myocardium. This may have important clinical implications for future pharmacotherapy to enhance myocardial protection.

Use of NO donors in biological systems.

Owing to the increased interest in the biological roles of nitric oxide (NO) the use of NO donors is a desired method of delivering NO to the tissues of interest. This article gives an overview of the most commonly used classes of NO donors and their biotransformation to release NO. A major consideration when choosing an NO donor is the preparation and handling of the compounds. A method has been outlined for the preparation of S-nitrosothiols which eliminates the problem of the overall instability of these compounds both as a solid and in solution. The main aim of this article is to outline the methods used in assessing the ability of NO donors to elicit a biological response in vitro in particular relaxation of vascular smooth muscle and inhibition of platelet aggregation. In addition a method is described for assessing the toxicological potential of NO donors in vitro.

Recombinant human Monocyte/Neutrophil elastase inhibitor protects rat lungs against injury from cystic fibrosis airway secretions.

Human monocyte/neutrophil elastase inhibitor (M/NEI) is a fast-acting stoichiometric inhibitor of neutrophil elastase (NE), cathepsin-G, and proteinase-3. Recombinant M/NEI (rM/NEI) was evaluated with a rat model of NE-induced lung damage. rM/NEI was found to protect against pulmonary injury caused by instilled human NE or by a preparation from airway secretions (sputum) of cystic fibrosis patients (CF sol). Human NE instilled into rat lungs produced dose-dependent hemorrhage and increased epithelial permeability, whereas NE incubated in vitro with rM/NEI did neither. Similarly, hemorrhage was induced by CF sol, but not by CF sol incubated in vitro with rM/NEI. To examine its distribution and survival time in airways, rM/NEI was labeled with the fluorochrome Texas Red (rM/NEI-TR) and instilled into rat lungs. Confocal microscopy showed that rM/NEI-TR could be detected on large airways (300 microm) at 5 min, 1 h, 4 h, and 24 h after instillation. Pretreating rats with rM/NEI was found to provide extended protection upon subsequent NE challenge, reducing hemorrhage by 98, 96, and 73%, respectively, at 1, 4, and 24 h after rM/NEI pretreatment. Pretreating rats with rM/NEI similarly conferred protection against subsequent exposure to CF sol, reducing hemorrhage by 95, 86, and 87%, respectively, at 1, 4, and 24 h after pretreatment. The findings that rM/NEI (1) mitigates protease-induced lung injury and (2) remains present and active in the lungs for 24 h after instillation strongly support its potential for treating patients with neutrophil protease-induced inflammatory lung damage, such as occurs in CF and other diseases.

Nitric oxide and the haemodynamic profile of endotoxin shock in the conscious mouse.

1. The release of cytokines following administration of endotoxin and the contribution of nitric oxide (NO) to the subsequent haemodynamic profile were investigated in the conscious mouse. 2. Administration of endotoxin (E. Coli, 026:B6, 12.5 mg kg(-1), i.v.) elevated the concentration of tumour necrosis factor-alpha (TNF-alpha) in the plasma within 0.5 h, reaching a maximum at 2 h and returning to control concentrations by 4 h. In addition, the concentration of interleukin-6 (IL-6) in the plasma was also elevated within 1 h, reaching a maximum at 3 h and remaining elevated throughout the 12 h of study. 3. Endotoxin (12.5 mg kg(-1), i.v.) induced the expression of a Ca2+-independent (inducible) NO synthase in the mouse heart and elevated the concentrations of nitrite and nitrate in the plasma within 4 h, reaching a maximum at 12 h. This was accompanied by a progressive fall in blood pressure over the same period. 4. The vasopressor effect of noradrenaline (0.5-4 microg kg(-1) min(-1), i.v.) administered as a continuous infusion was significantly attenuated 7 h after endotoxin (12.5 mg kg(-1), i.v). 5. The NO synthase inhibitor NG-monomethyl-L-arginine HCl (L-NMMA; 1-10 mg kg(-1), i.v. bolus) reversed the fall in blood pressure when administered 7 h after endotoxin (12.5 mg kg(-1), i.v.). 6. In an attempt to maintain a constant blood concentration, L-NMMA was administered as a continuous infusion (10 mg kg(-1) h(-1), i.v.), beginning 4 h after a lower dose of endotoxin (6 mg kg(-1), i.v.). Such treatment prevented the fall in blood pressure and the elevation of nitrite and nitrate in the plasma throughout the 18 h of observation. 7. The fall in blood pressure following endotoxin (3 mg kg(-1), i.v.) was significantly reduced throughout the 18 h of observation in homozygous mutant mice lacking the inducible NO synthase. 8. In summary, we have developed a model of endotoxin shock in the conscious mouse in which an overproduction of NO by the inducible NO synthase is associated with the haemodynamic disturbances. This model, which exhibits many of the characteristics of septic shock in man, will enable the study of the pathology of this condition in more detail and aid the investigation of potential therapeutic agents both as prophylactics and, more importantly, as treatments.

Sex differences in the abundance of endothelial nitric oxide in a model of genetic hypertension.

A deficiency of nitric oxide may be responsible for the increased vascular resistance associated with human essential hypertension and that seen in animal models of hypertension. Premenopausal females are relatively protected from hypertension and cardiovascular complications. Levels of superoxide can influence the availability of nitric oxide. We hypothesize that there are differences in nitric oxide availability between stroke-prone spontaneously hypertensive rats (SHRSP) and Wistar-Kyoto rats (WKY) and that superoxide may be responsible for at least some of these differences. We studied vascular reactivity in endothelium-intact aortic rings from WKY and SHRSP. We measured nitric oxide synthase activity in endothelial cells removed from aortas and also measured circulating nitrite/nitrate levels. We found the response to N(G)-nitro-L-arginine methyl ester to be significantly greater in WKY compared with SHRSP (95% CI: 20 to 174; P=.015) and in females compared with males in WKY (95% CI: 143 to 333; P=.00004) and SHRSP (95% CI: 70 to 224; P=.0006). Endothelial nitric oxide synthase activity was significantly greater in SHRSP compared with WKY (95% CI: 2.3 to 17.6; P=.016). The EC50 for relaxation to carbachol was significantly greater in male rats compared with female rats (95% CI: -1.1 to -0.2; P=.003) within the SHRSP strain. The maximum relaxation to carbachol was significantly attenuated in stroke prone spontaneously hypertensive compared with Wistar-Kyoto rats (95% CI: 1.7 to 14.4; P=.015). Diethyldithiocarbamate had a significantly greater effect on the stroke prone spontaneously hypertensive rats' carbachol response than that of Wistar-Kyoto rats (95% CI: 14.3 to 47.0; P=.0008). We conclude that superoxide may be responsible for strain differences in vascular reactivity, whereas nitric oxide availability may be responsible for sex differences independently of endothelial nitric oxide synthase activity and superoxide.

Nitric oxide synthase inhibitors and hypertension in children and adolescents.

OBJECTIVE: To establish the role played by the circulating nitric oxide synthase inhibitors N(G)-monomethyl-L-arginine (L-NMMA), asymmetrical dimethyl arginine (ADMA) and symmetric dimethyl arginine (SDMA) and its association with hypertension of children and adolescents. DESIGN: We measured plasma concentrations of L-NMMA, ADMA and SDMA in 38 hypertensives (median age 7.7 years) and in nine healthy normotensive controls (median age 8.2 years) using high-performance liquid chromatography. In addition, their plasma renin activity was determined. The subjects' glomerular filtration rates were calculated from plasma creatinine and height measurements. To determine the vasoactive potency of the arginine analogues, concentration-response curves were plotted for the responses in isolated endothelium-intact and endothelium-denuded mouse aortic rings that had been pre-contracted by administration of a threshold concentration of phenylephrine. RESULTS: Plasma ADMA and SDMA concentrations in members of the hypertensive group [0.23 +/- 0.03 and 1.37 +/- 0.06 micromol/l, respectively (means +/- SEM)] were significantly higher than those in members of the control group (ADMA 0.10 +/- 0.01 micromol/l and SDMA 1.18 +/- 0.06 micromol/l). Plasma concentrations of L-NMMA were similar in members of the hypertensive (0.21 +/- 0.01 micromol/l) and control (0.18 +/- 0.02 micromol/l) groups. The glomerular filtration rate of the hypertensive group was below normal [70.4 +/- 5.4 ml/min per 1.73 m2 (mean +/- SEM)] and was significantly associated with elevated plasma concentrations of ADMA (r = -0.77, P < 0.001), SDMA (r = -0.38, P = 0.02) and L-NMMA (r = 0.35, P = 0.03). Higher plasma ADMA concentrations were associated with a lower plasma renin activity (r = -0.36, P = 0.04). The vasoactive potencies of ADMA (concentration for half-maximal effect with the endothelium intact 25.4 +/- 7.1 micromol/l) and L-NMMA (concentration for half-maximal effect with the endothelium intact 8.2 +/- 2.9 micromol/l) was significantly (P < 0.05) greater than that of SDMA. Both ADMA and L-NMMA (at 3 micromol/l concentrations) initiated a significant vasocontractile response from baseline (P = 0.03 and P < 0.001, respectively). These effects were absent after the endothelium had been removed. SDMA had no effect. CONCLUSIONS: Plasma ADMA and SDMA levels are increased in hypertensive children. By inference from in-vitro data, ADMA appears to attain sufficient concentrations to produce a significant change in vascular tone and hence might play a role in the pathophysiology of childhood hypertension.

Nitric oxide activity in childhood hypertension.

OBJECTIVES: To investigate nitric oxide (NO) activity in childhood hypertension using nitrite and nitrate (NOx) concentrations in plasma as an index of nitric oxide generation. DESIGN: Cross sectional study. SETTING: Tertiary care paediatric centre and district general hospitals in the UK. PATIENTS: Children attending the above centre for treatment of hypertension. The control subjects were normotensive healthy children attending district general hospitals for minor medical and surgical disorders. INTERVENTIONS: None. MAIN OUTCOME MEASURES: Plasma (P) and urinary (U) NOx concentrations, blood pressure, and glomerular filtration rate. RESULTS: Sixteen normal children (mean age 6.9 years), 13 children with renovascular hypertension (mean age 7.8 years), and 25 children with hypertension associated with renal parenchymal disease (mean age 10.7 years) were studied. Mean (SD) PNOx values of children with hypertension with renovascular disease (15.3 (11.4) mumol/l) and renal parenchymal disease (18.3 (11.4) mumol/l) were significantly above that of normal children (11.9 (5.9) mumol/l) after accounting for age and glomerular filtration rate influences. Higher concentrations of PNOx in normal children were associated with younger age, but not in the children with hypertension. Higher PNOx concentrations were also associated with a lower glomerular filtration rate in normal children and children with hypertension with renal parenchymal disease, but not in the children with hypertension with renovascular disease. UNOx excretion expressed as a ratio against urine creatinine (Ucreat) excretion was not statistically different among the study groups. CONCLUSIONS: PNOx is increased in children with hypertension even after statistical elimination of the glomerular filtration rate and age influences. This suggests a normal or increased NO synthase activity in childhood hypertension in contrast with adults with hypertension in whom it is described as reduced.

Inhibition of neutrophil elastase in CF sputum by L-658,758.

Elastases in cystic fibrosis (CF) pulmonary fluids damage lung tissue and perpetuate cycles of infection, inflammation and injury. Elastases from three different sources may be present in CF airways: neutrophils, macrophages and Pseudomonas. We measured how well the cephalosporin-based antielastase L-658,758 blocks the activity of human neutrophil elastase (NE), human proteinase-3, human macrophage metalloelastase, mouse macrophage metalloelastase and Pseudomonas aeruginosa elastase. We also examined the ability of L-658,758 to block elastases in CF sputum in vitro. Sputum samples from adult CF patients were fractionated to obtain the aqueous sol phase. These were then studied individually or pooled. Elastinolytic activity, which ranged from 3.2 microg elastin degraded/ml sol/min to 26.3 microg elastin degraded/ml sol/min, was measurable in every individual sol sample and in the pooled sol. L-658,758 effectively inhibited elastinolysis by NE, proteinase-3 and the pooled sol but did not inhibit the activity of the metalloelastases, human and mouse macrophage metalloelastase and Pseudomonas elastase. Secretory leukoprotease inhibitor, which inhibited NE but did not inhibit proteinase-3, blocked 90% of sol elastinolytic activity; this suggests that the majority of this activity in the pooled sol derived from NE. L-658,758 was an effective inhibitor of sol elastase, blocking more than 97% of elastinolytic activity in the individual sol samples. We conclude that L-658,758 is an effective inhibitor of NE, proteinase-3 and CF sputum sol elastase.

Effects of cystic fibrosis airway secretions on rat lung: role of neutrophil elastase.

Products of inflammatory cells present in pulmonary secretions may compromise lung structure and function. To investigate the pathogenic potential of cystic fibrosis (CF) airway secretions, we instilled CF sputum sol into the lungs of healthy rats and measured the resulting lung injury and inflammation. The ability of a neutrophil elastase (NE) inhibitor, L-680,833, to mitigate these responses was also investigated. CF sputum sol instilled into rat lungs induced hemorrhage, an increase in epithelial permeability, and neutrophil recruitment to the airspaces. However, when sputum sol was preincubated with NE inhibitor before instillation, hemorrhage was completely prevented, suggesting that NE within the CF airway secretions was responsible for the observed hemorrhagic injury. NE-inhibitor treatment had no effect on the observed increases in bronchoalveolar lavage albumin level or neutrophil numbers. Rats treated orally with NE inhibitor before instillation of sputum sol were also protected from hemorrhagic injury. These results demonstrate that NE within CF airway secretions causes lung tissue damage and that animals can be protected from such damage with an oral anti-elastase.

Sequential induction of nitric oxide synthase by Corynebacterium parvum in different organs of the mouse.

1. The ability of Corynebacterium parvum (C. parvum) to induce nitric oxide (NO) synthase in the macrophage, spleen, liver, aorta, heart and brain, and to elevate plasma NO2-/NO3- in the mouse was investigated. In addition, the relationship between NO synthase activity and blood pressure was studied. 2. C. parvum (100 mg kg-1, i.p.) induced a time-dependent expression of a Ca(2+)-independent NO synthase in the macrophage, spleen, liver, aorta and heart. The time course of induction of the NO synthase varied such that the maximum enzyme activity was at day 8 in the macrophage and liver, day 12 in the spleen and heart and day 16 in the aorta. 3. There was no significant induction of a Ca(2+)-independent NO synthase in the brain, nor was there any change in the Ca(2+)-dependent enzyme in this organ, during the study period. 4. C. parvum produced a gradual decrease in blood pressure, with a maximum fall at day 16 (from 108 +/- 1 mmHg to 79 +/- 3 mmHg), which recovered gradually by day 28. 5. Plasma NO2-/NO3- was significantly elevated between days 8 and 24, with a maximum increase at day 12. 6. These results show that C. parvum induces a Ca(2+)-independent NO synthase in a number of tissues and that this induction occurs initially in macrophages and the liver. This suggests that induction of the NO synthase in the other tissues is secondary and probably the result of activation of macrophages and some cells of the liver. 7. Furthermore, the decrease in blood pressure induced by C. parvum is associated with the induction of NO synthase in the vasculature, whereas the increased concentration of plasma NO2-/NO3- seems to result from the generation of NO by a number of tissues.

S-nitroso-glutathione inhibits platelet activation in vitro and in vivo.

1. The effect of S-nitroso-glutathione (GSNO), a stable nitrosothiol, on platelet activation was examined in vitro and in vivo. 2. The adhesion of human platelets to fibrillar collagen and human endothelial cell monolayers was inhibited by GSNO. 3. GSNO caused a concentration-dependent inhibition of collagen-induced platelet aggregation in vitro and decreased ADP-induced aggregation in the conscious rat. 4. Inhibition of platelet aggregation in vitro correlated with the increase in intraplatelet cyclic GMP levels. 5. The release of NO from GSNO was enhanced by platelet lysate, native glutathione and ascorbate. 6. The results show that GSNO is a carrier of NO and therefore has pharmacological activity as an inhibitor of platelet activation.

Protective and pathological roles of nitric oxide in endotoxin shock.

OBJECTIVE: The aim was to investigate the effects of NG-monomethyl-L-arginine (L-NMMA), an inhibitor of both the constitutive (Ca2+ dependent) and inducible (Ca2+ independent) nitric oxide (NO) synthases, or of pretreatment with the glucocorticoid dexamethasone, an inhibitor of the induction of the Ca2+ independent NO synthase, on lipopolysaccharide induced shock in the anaesthetised rabbit. METHODS: Mean arterial blood pressure, and blood flow in the portal vein, hepatic artery, and hindquarter vascular beds were measured in 49 halothane anaesthetised New Zealand White rabbits given lipopolysaccharide (Salmonella minnesota, 500 micrograms.kg-1 intravenously). The effects of pre- or post-lipopolysaccharide treatment with L-NMMA (300 mg.kg-1 intravenously) and of pretreatment with dexamethasone (3 mg.kg-1 intravenously) were determined. The effect of the NO donor S-nitroso-N-acetyl-penicillamine (SNAP, 300 micrograms.kg-1.h-1 intravenously) in animals treated with lipopolysaccharide and L-NMMA was also studied. RESULTS: Lipopolysaccharide elicited an initial transient fall in mean arterial pressure and decreases in blood flow in the vascular beds, followed by a progressive fall in mean arterial pressure. L-NMMA when given either before or after lipopolysaccharide markedly exacerbated its effects and resulted in severe hypotension, intense vasoconstriction, and increased mortality. Pretreatment with dexamethasone had no effect on the initial haemodynamic changes following lipopolysaccharide, but prevented the subsequent fall in mean arterial pressure observed in animals treated with lipopolysaccharide alone. Dexamethasone failed, however, to protect animals also treated with L-NMMA before lipopolysaccharide. Animals pretreated with L-NMMA and SNAP showed reduced haemodynamic changes when compared with controls (lipopolysaccharide only) or lipopolysaccharide and L-NMMA treated animals. CONCLUSIONS: Inhibition of both constitutive and inducible NO synthases during endotoxaemia is deleterious. This can be overcome by replacing NO intravenously with a donor of NO. Selective inhibition of the inducible NO synthase may, however, be beneficial in shock.

Development and mechanism of a specific supersensitivity to nitrovasodilators after inhibition of vascular nitric oxide synthesis in vivo.

The mechanism of the increased sensitivity to nitrovasodilators after removal of endothelial nitric oxide (NO) was investigated in vitro and in vivo. The vasoconstrictor potency of phenylephrine and the force of contraction of rat isolated aortic rings were significantly enhanced after endothelium removal or treatment with inhibitors of endothelial NO synthase. Furthermore, these procedures led to a significant decrease in the basal levels of cGMP in the vascular rings. Moreover, the potency of glyceryl trinitrate (n3Gro) and sodium nitroprusside (SNP) as relaxing agents and the ability of SNP to induce increases in cGMP in aortic rings were significantly enhanced in those rings denuded of endothelium or treated with the inhibitors. These procedures did not affect the vasodilator actions of isoprenaline or 8-bromo-cGMP. In the anesthetized rat, treatment with the inhibitors enhanced significantly the hypotensive responses to n3Gro without affecting those to isoprenaline. These data indicate that the removal of the basal NO-mediated vasodilator tone in the cardiovascular system leads, at the level of the soluble guanylate cyclase, to a specific supersensitivity to nitrovasodilators in vivo. The existence of such a phenomenon has important implications for understanding the local physiological control of blood flow, its pathological disturbances, and the mechanism of action of nitrovasodilators.

Dexamethasone prevents the induction by endotoxin of a nitric oxide synthase and the associated effects on vascular tone: an insight into endotoxin shock.

The relationship between vascular tone and the induction by endotoxin of a nitric oxide (NO) synthase was studied in vitro in rings of rat thoracic aorta. In rings with and without endothelium there was a time-dependent induction of NO synthase accompanied by both spontaneous and L-arginine-induced relaxation and by reduced contractility to phenylephrine. These effects, which were attributable to the presence of endotoxin in the Krebs' buffer, were attenuated by cycloheximide, polymyxin B and inhibitors of NO synthase. Furthermore, dexamethasone inhibited the induction of NO synthase and the consequent effects on vascular tone. These findings indicate that prevention of the induction of NO synthase by glucocorticoids may be an important component of their therapeutic action.

Characterization of three inhibitors of endothelial nitric oxide synthase in vitro and in vivo.

1. Three analogues of L-arginine were characterized as inhibitors of endothelial nitric oxide (NO) synthase by measuring their effect on the endothelial NO synthase from porcine aortae, on the vascular tone of rings of rat aorta and on the blood pressure of the anaesthetized rat. 2. NG-monomethyl-L-arginine (L-NMMA), N-iminoethyl-L-ornithine (L-NIO) and NG-nitro-L-arginine methyl ester (L-NAME; all at 0.1-100 microM) caused concentration-dependent inhibition of the Ca2(+)-dependent endothelial NO synthase from porcine aortae. 3. L-NMMA, L-NIO and L-NAME caused an endothelium-dependent contraction and an inhibition of the endothelium-dependent relaxation induced by acetylcholine (ACh) in aortic rings. 4. L-NMMA, L-NIO and L-NAME (0.03-300 mg kg-1, i.v.) induced a dose-dependent increase in mean systemic arterial blood pressure accompanied by bradycardia. 5. L-NMMA, L-NIO and L-NAME (100 mg kg-1, i.v.) inhibited significantly the hypotensive responses to ACh and bradykinin. 6. The increase in blood pressure and bradycardia produced by these compounds were reversed by L-arginine (30-100 mg kg-1, i.v.) in a dose-dependent manner. 7. All of these effects were enantiomer specific. 8. These results indicate that L-NMMA, L-NIO and L-NAME are inhibitors of NO synthase in the vascular endothelium and confirm the important role of NO synthesis in the maintenance of vascular tone and blood pressure.