Inhibition of poly (ADP-ribose) synthetase attenuates neutrophil recruitment and exerts antiinflammatory effects.

A cytotoxic cycle triggered by DNA single-strand breakage and poly (ADP-ribose) synthetase activation has been shown to contribute to the cellular injury during various forms of oxidant stress in vitro. The aim of this study was to investigate the role of poly (ADP-ribose) synthetase (PARS) in the process of neutrophil recruitment and in development of local and systemic inflammation. In pharmacological studies, PARS was inhibited by 3-aminobenzamide (10-20 mg/kg) in rats and mice. In other sets of studies, inflammatory responses in PARS-/- mice were compared with the responses in corresponding wild-type controls. Inhibition of PARS reduced neutrophil recruitment and reduced the extent of edema in zymosan- and carrageenan-triggered models of local inflammation. Moreover, inhibition of PARS prevented neutrophil recruitment, and reduced organ injury in rodent models of inflammation and multiple organ failure elicited by intraperitoneal injection of zymosan. Inhibition of PARS also reduced the extent of neutrophil emigration across murine mesenteric postcapillary venules. This reduction was due to an increased rate of adherent neutrophil detachment from the endothelium, promoting their reentry into the circulation. Taken together, our results demonstrate that PARS inhibition reduces local and systemic inflammation. Part of the antiinflammatory effects of PARS inhibition is due to reduced neutrophil recruitment, which may be related to maintained endothelial integrity.

Endothelial dysfunction in a rat model of endotoxic shock. Importance of the activation of poly (ADP-ribose) synthetase by peroxynitrite.

DNA single strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) contribute to peroxynitrite-induced cellular injury. We investigated the role of PARS activation in the pathogenesis of endothelial dysfunction. In human umbilical vein endothelial cells (HUVEC), DNA strand breakage (alkaline unwinding assay), PARS activation (incorporation or radiolabeled NAD+ into proteins), mitochondrial respiration [conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide to formazan] and apoptotic index (cytoplasmatic release of histones) were measured. Endotoxin shock was induced in rats by bacterial lipopolysaccharide. Vascular reactivity of thoracic aortic rings were measured in organ chambers. In HUVEC, peroxynitrite caused a dose-dependent suppression of mitochondrial respiration, induced DNA strand breakage and caused an activation of PARS. Pharmacological inhibition of PARS reduced the acute and delayed suppression of mitochondrial respiration when cells were exposed to intermediate, but not high doses of peroxynitrite. Similarly, protection against the intermediate, but not high doses of peroxynitrite was seen in fibroblasts from the PARS-/- mice, when compared to wild-type controls. These data suggest that PARS plays a role in peroxynitrite-induced cytotoxicity, but at very high levels of oxidant exposure, PARS-independent cytotoxic mechanisms become predominant. Peroxynitrite-induced apoptosis was not affected by PARS inhibition. Vascular rings exposed to peroxynitrite and rings taken from rats subjected to endotoxic shock exhibited reduced endothelium-dependent relaxant responses in response to acetylcholine. The development of this endothelial dysfunction was ameliorated by the PARS inhibitor 3-aminobenzamide. Activation of PARS by peroxynitrite, therefore, may be involved in the development of endothelial dysfunction in endotoxemia.

Crucial role of endogenous interleukin-10 production in myocardial ischemia/reperfusion injury.

BACKGROUND: The anti-inflammatory cytokine interleukin-10 (IL-10) has been detected in the plasma of patients with myocardial ischemia/reperfusion. The aim of our study was to investigate the role of endogenously produced IL-10 in myocardial ischemia/reperfusion. METHODS AND RESULTS: In the present study, we used wild-type and IL-10-deficient mice subjected to myocardial ischemia/reperfusion. Significant levels of IL-10 were produced in wild-type mice at 2 to 6 hours after myocardial reperfusion. The genetic deletion of IL-10 enhanced neutrophil infiltration into the reperfused tissues at 6 hours after reperfusion and increased infarct size and myocardial necrosis. Furthermore, in the absence of IL-10, an enhancement of the inflammatory response was seen, as demonstrated by increased plasma levels of tumor necrosis factor-alpha, nitrite/nitrate (breakdown products of NO), and increased tissue expression of intercellular adhesion molecule-1. Reperfusion for 24 hours was associated with a 75% mortality rate in IL-10-deficient mice, whereas no deaths occurred in the wild-type animals. CONCLUSIONS: The present findings provide the first direct evidence that endogenous IL-10 inhibits the production of tumor necrosis factor-alpha and NO and serves to protect the ischemic and reperfused myocardium through the suppression of neutrophil recruitment.

Protection against myocardial ischemia and reperfusion injury by 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase.

OBJECTIVE: Peroxynitrite and hydroxyl radical, reactive oxidants produced during reperfusion, are potent triggers of DNA single strand breakage. DNA injury triggers the activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS), which contributes to cellular energetic depletion. Using 3-aminobenzamide, an inhibitor of PARS, we investigated the role of PARS in the pathogenesis of myocardial reperfusion injury in a rat model. METHODS AND RESULTS: Occlusion of the left main coronary artery (one hour) followed by reperfusion (one hour) in the anesthetized rat caused severe cardiac necrosis, neutrophil infiltration, and increased plasma creatine phosphokinase activity. There was significant peroxynitrite production during reperfusion, as indicated by a massive increase in nitrotyrosine in the necrotic myocardium. Reperfusion was also associated with a significant loss of myocardial ATP. In vivo administration of the PARS inhibitor 3-aminobenzamide (10 mg/kg i.v.) to rats subjected to myocardial ischemia and reperfusion, reduced myocardial infarct size and blunted the increase in plasma creatine phosphokinase activity and myeloperoxidase activity in infarcted hearts. In addition, 3-aminobenzamide partially preserved the myocardial ATP levels. In vitro, pharmacological inhibition of PARS also ameliorated peroxynitrite-induced cytotoxicity in rat cardiac myocytes and human endothelial cells. CONCLUSION: 3-aminobenzamide has significant protective effects in myocardial reperfusion injury. We hypothesize that activation of PARS activation plays a role in the pathophysiology of acute myocardial infarction.

Protective effects of isohelenin, an inhibitor of nuclear factor kappaB, in endotoxic shock in rats.

Recent in vitro studies have shown that isohelenin, a sesquiterpene lactone, inhibits the NF-kappaB pathway. This study examines the effect of isohelenin in endotoxic shock induced by administration of Escherichia coli endotoxin in male Wistar rats. A group of rats received isohelenin (2 mg/kg intraperitoneally) 15 min before endotoxin. In vehicle-treated rats, administration of endotoxin caused severe hypotension, which was associated with a marked hyporeactivity to norepinephrine and acetylcholine in ex vivo aortas. Elevated levels of plasma nitrate/nitrite, metabolites of nitric oxide (NO), were also found. These inflammatory events were preceded by cytosolic degradation of inhibitor-kappaBalpha (IkappaBalpha) and activation of nuclear factor-kappaB (NF-kappaB) in the lung within 15 min of endotoxin administration. Treatment with isohelenin resulted in hemodynamic improvement and reduced plasma levels of NO metabolites. Nuclear translocation of NF-kappaB was inhibited by isohelenin treatment in the lung, whereas degradation of IkappaBalpha was unchanged. In a separate set of experiments, treatment with isohelenin significantly improved survival in mice challenged with endotoxin. We conclude that isohelenin exerts beneficial therapeutic effects during endotoxic shock through inhibition of NF-kappaB.

Antiinflammatory effects of mercaptoethylguanidine, a combined inhibitor of nitric oxide synthase and peroxynitrite scavenger, in carrageenan-induced models of inflammation.

In vitro studies have demonstrated that mercaptoethylguanidine (MEG), a selective inhibitor of the inducible NO synthase (iNOS), is also effective as a scavenger of peroxynitrite (a potent cytotoxic oxidant produced by the reaction of NO and superoxide). In the present study, we evaluated the antiinflammatory potential of MEG treatment in two models of acute inflammation (carrageenan-induced paw edema and pleurisy), where oxyradicals, NO, and peroxynitrite play a crucial role in the inflammatory process. Our data show that MEG (given at 25 microg/paw in the paw edema model or 10 mg/kg in the pleurisy model) inhibits the inflammatory response (paw swelling, pleural exudate formation, mononuclear cell infiltration, histological injury) in both models. Furthermore, MEG reduced nitrite/nitrate concentrations in the exudate and reduced the activity of the inducible isoform of NO synthase in the lung ex vivo. MEG also reduced the appearance of nitrotyrosine immunoreactivity in the inflamed tissues. Taken together, the present results demonstrate that MEG exerts potent antiinflammatory effects. Part of these antiinflammatory effects may be related to an inhibition of the expression/activity of the inducible NO synthase, another part may be related to oxyradical and peroxynitrite scavenging.

Beneficial effects of Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), a superoxide dismutase mimetic, in carrageenan-induced pleurisy.

Peroxynitrite, a potent cytotoxic oxidant formed by the reaction of NO with superoxide anion, has been proposed to have major pathogenetic role in inflammatory process. Here we have investigated the therapeutic efficacy of Mn(III)tetrakis (4-benzoic acid) porphyrin (MnTBAP), a novel superoxide dismutase mimetic that possesses peroxynitrite scavenging effect, in rats subjected to carrageenan-induced pleurisy. In vivo treatment with MnTBAP (3 and 10 mg/kg 5 min before carrageenan) prevented in a dose-dependent manner the carrageenan-induced the degree of pleural exudation, polymorphonuclear migration in rats subjected to carrageenan-induced pleurisy. Lung myeloperoxidase (MPO) activity and histological organ injury was significantly reduced by MnTBAP. However, MnTBAP did not inhibit the inducible NO synthase in lung samples. Immunohistochemical analysis for nitrotyrosine, a footprint of peroxynitrite, revealed a positive staining in lungs from carrageenan-treated rats. No positive nitrotyrosine staining was found in the lungs of the carrageenan-treated rats that received MnTBAP (10 mg/kg) treatment. In addition, in vivo MnTBAP treatment significantly reduced in a dose-dependent manner peroxynitrite formation as measured by the oxidation of the fluorescent dye dihydrorhodamine 123, prevented the appearance of DNA damage, the decrease in mitochondrial respiration and partially restored the cellular level of NAD+ in ex vivo macrophages harvested from the pleural cavity of rats subjected to carrageenan-induced pleurisy. Our study demonstrates that the MnTBAP exerts multiple protective effects in carrageenan-induced pleurisy. We suggest peroxynitrite produced during the inflammatory process trigger DNA strand breakage and subsequent cellular dysfunction. Part of these anti-inflammatory effects may be related to: (1) reduction of superoxide formation due to the superoxide dismutase-like activity of the compound and (2) scavenging of peroxynitrite.

Reduction of myocardial infarct size in rat by IRFI-048, a selective analogue of vitamin E.

The effects of IRFI-048 (2,3-dihydro-5-methoxy-4,6,7-trimethyl-2-benzofuranyl acetic acid), a selective analogue of Vitamin E, on myocardial tissue injury were examined in anaesthetized rats subjected to 60-min occlusion of the left coronary artery followed by 60-min reperfusion. Infarct size (Evan's blue and tetrazolium stain), serum creatinphosphokinase (CPK), plasma malonaldehyde (MAL), cardiac myeloperoxidase (MPO) activity, and ST-segment of electrocardiogram (ECG) and survival rate were evaluated. Postischaemic reperfusion produced severe cardiac necrosis, caused neutrophil (PMNs) infiltration (evaluated by MPO activity) in the jeopardized tissue, increased serum CPK and plasma MAL, raised ST-segment of ECG, and decreased survival rate. IRFI-048, (200 and 400 mg/kg o.s.) given to the rats 6 h before occlusion, caused a reduction of necrotic area expressed as a percentage of either the area at risk or the total left ventricle, decreased MPO activity both in the area at risk (from 3.2 +/- 0.3 U x 10(-3)/g tissue to 1.1 +/- 0.4 U x 10(-3)/g tissue; p < .005) and in the necrotic area (from 5.7 +/- 0.9 U x 10(-3)/g tissue to 1.8 +/- 0.5 U x 10(-3)/g tissue; p < .001), attenuated the rise of ST-segment of ECG (from 0.51 +/- 0.14 mV in the vehicle group to 0.28 +/- 0.11 mV in the treated group; p < .005), reduced the increase of plasma MAL and serum CPK during reperfusion (from 42 +/- 5.3 nmol/ml to 15 +/- 3.1 nmol/ml and 139 +/- 13 IU/100 ml to 58 +/- 7.5 IU/100 ml, respectively; p < .001).(ABSTRACT TRUNCATED AT 250 WORDS)

Evidence that nitric oxide modulates drinking behaviour.

The involvement of the L-arginine-nitric oxide (NO) pathway in brain, in the regulation of drinking behaviour, has been evaluated by injecting L-arginine and N omega-nitro-L-arginine methyl ester (L-NAME) into the lateral cerebral ventricle (i.c.v.). L-Arginine (5 and 10 micrograms/rat), but not D-arginine, was antidipsogenic when administered to 24 hr water-deprived rats but did not change the intake of water in normally hydrated rats. However, L-NAME (5 and 10 micrograms/rat) did antagonize the effect of L-arginine in water-deprived animals but, by itself, did not increase thirst. L-Arginine (100 ng), when injected into the preoptic area significantly reduced water deprivation-induced drinking. The same dose was unaffective when given intraventricularly. Finally, L-arginine (5 and 10 micrograms/rat, i.c.v.) inhibited drinking induced by intraventricular injection of angiotensin II (250 ng/rat). The effect was dose-dependent. The results indicate that: (1) NO acts as an inhibitory mechanism when thirst is stimulated by water deprivation or by angiotensin II; (2) the preoptic area might be one of the central sites of antidipsogenic action of NO and (3) nitric oxide synthase might be inhibited during water deprivation.

Food deprivation increases brain nitric oxide synthase and depresses brain serotonin levels in rats.

We studied nitric oxide (NO) synthase activity and serotonin content in the diencephalon of 24 hr food deprived rats. NO synthase activity was significantly increased whereas serotonin levels together with those of tryptophan and 5-hydroxyindoleacetic acid (5-HIAA) were reduced in food deprived rats when compared to control rats. NG-Nitro-L-arginine (L-NO Arg), an inhibitor of NO synthase, was used as a tool to study the role of NO in food deprivation. Twenty-four hr food deprived male Sprague-Dawley rats were intraperitoneally (i.p.) administered L-NO Arg (12.5, 25 and 50 mg/kg) before food presentation. Control rats received a NaCl (0.9%) solution. Food consumption was monitored 1 and 2 hr after food presentation. L-NO Arg administration produced a dose-dependent reduction in food intake. Pretreatment with metergoline (2 mg/kg) but not with ritanserin (1 mg/kg) antagonized the anorectic effect of L-NO Arg. Moreover, in the diencephalon L-NO Arg significantly reduced NO synthase activity whereas it increased serotonin levels. Our data indicate that NO might have a physiological role in the regulation of food intake and suggest that brain NO may modulate the central serotoninergic system.

Modulation of lipopolysaccharide-induced tumor necrosis factor-alpha and nitric oxide production by dopamine receptor agonists and antagonists in mice.

The effects of various agonist and antagonists of dopamine D1 and D2 receptors on lipopolysaccharide (LPS)-induced tumor necrosis factor-alpha (TNF-alpha) and nitric oxide (NO) production was investigated in mice. Pretreatment of animals with bromocryptine or quinpirole, agonists of dopamine D2 receptors caused a blunting of both the TNF-alpha and NO responses to LPS injected intraperitoneally. Sulpiride, an antagonist of dopamine D2 receptors, decreased the LPS-induced TNF-alpha plasma levels in a dose-dependent manner and inhibited the LPS-induced NO production by peritoneal macrophages. Bromocryptine or quinpirole blunted both the TNF-alpha and NO response to LPS. SCH-23390, an antagonist of dopamine D1 receptors did not alter LPS-induced TNF-alpha production, but inhibited LPS-induced NO production. These results indicate that while the D2 subtype of dopamine receptors is involve in the modulation of both LPS-induced TNF-alpha and NO production, dopamine D1 receptors only regulate the production of NO. Since several drugs possess effect on dopamine D2 receptors, the present observations may be of clinical relevance.

Mediation by nitric oxide formation in the preoptic area of endotoxin and tumour necrosis factor-induced inhibition of water intake in the rat.

1. Drinking was induced in rats by 24 h of water deprivation. Water intake (ml) was evaluated for a 1 h period. 2. NG-nitro-L-arginine methyl ester (L-NAME, 5-10 micrograms, i.c.v., 50-100 ng into the preoptic area (POA)), an inhibitor of nitric oxide (NO) synthase, and methylene blue (50-100 ng into POA), an inhibitor of guanylate cyclase activation, antagonized the inhibition of drinking induced by E. coli endotoxin (LPS, 640 micrograms kg-1, i.v.) and tumour necrosis factor (TNF alpha, 40 ng, i.c.v.) in 24 h water-deprived rats. 3. L-Arginine (25, 50 and 100 ng), the precursor amino acid of NO, but not the stereoisomer D-arginine (100 ng), inhibited drinking induced by water deprivation when injected into the POA 30 min before water presentation (74.4% of inhibition with the highest dose). A dose of 12.5 ng L-arginine into the POA did not exhibit antidipsogenic effects. 4. TNF alpha (20 and 40 ng, i.c.v.; 1.25, 2.5 and 5 ng into the POA) showed a dose-dependent and powerful inhibition of drinking behaviour in water-deprived rats (70.4% and 80.8%, i.c.v. and into POA, with the highest doses, respectively). A dose of 10 ng of TNF alpha given i.c.v. had no effect on the intake of water. 5. LPS and TNF alpha, given at doses (160 micrograms kg-1, i.v. and 10 ng, i.c.v., respectively) that did not influence drinking in water-deprived rats, exhibited a strong antidipsogenic effect in water-deprived rats treated with a dose of L-arginine (12.5 ng, into the POA) which did not modify drinking by itself. (LPS + L-arginine:53.6% of inhibition; TNFalpha + L-arginine: 52.0% of inhibition).6. These results suggest that NO into the POA: (1) acts as an inhibitory mechanism on thirst and (2)plays a role in the antidipsogenic effect of LPS and TNFalpha.

The potential role of peroxynitrite in the vascular contractile and cellular energetic failure in endotoxic shock.

1. Peroxynitrite is a toxic oxidant species produced from nitric oxide (NO) and superoxide. We have recently observed that the cell-permeable superoxide dismutase mimetic Mn(III)tetrakis(4-benzoic acid) porphyrin (MnTBAP) inhibits the suppression of mitochondrial respiration elicited by authentic peroxynitrite in vitro. Here we have investigated the relative potency of MnTBAP and a range of related compounds in terms of inhibition of peroxynitrite-induced oxidation and cytotoxicity. In addition, we tested the effects of MnTBAP on the vascular and the cellular energetic failure in rodent models of endotoxic shock. 2. We observed a dose-related inhibition of the peroxynitrite-induced oxidation of dihydrorhodamine 123 to rhodamine by MnTBAP, ZnTBAP and FeTBAP, but not by MnTMPyP [(5,10,15,20-tetrakis(N-methyl-4'-pirydyl)porphinato)-mangan ese (III)]. In addition, MnTBAP, ZnTBAP and FeTBAP, but not MnTMPyP prevented the suppression of mitochondrial respiration by authentic peroxynitrite in cultured J774 macrophages. 3. In rat cultured aortic smooth muscle cells, MnTBAP protected against the suppression of mitochondrial respiration in response to authentic peroxynitrite, immunostimulation and nitric oxide (NO) donor compounds. MnTBAP slightly reduced the amount of nitrite/nitrate produced in response to immunostimulation in these cells. 4. Administration of MnTBAP, 15 mg kg-1 i.v., before the administration of endotoxin (15 mg kg-1, i.v.) to rats ameliorated the development of vascular hyporeactivity and the development of endothelial dysfunction in the thoracic aorta ex vivo. 5. MnTBAP also prevented the endotoxin-induced decrease in mitochondrial respiration, the development of DNA single strand breaks, and the depletion of intracellular NAD+ in peritoneal macrophages ex vivo. 6. MnTBAP did not inhibit the expression by endotoxin of the inducible NO synthase in lung samples. 7. MnTBAP did not alter survival rate in mice challenged with high dose endotoxin. 8. Our findings, taken together with previous data demonstrating protective effects of NO synthase inhibitors against the endotoxin-induced contractile and energetic failure in the models of shock used in the current study, and with the known ability of peroxynitrite to cause cellular energy depletion, suggest a role for peroxynitrite in the pathogenesis of cellular energetic failure and contractile dysfunction in endotoxin shock.

Beneficial effects of 3-aminobenzamide, an inhibitor of poly (ADP-ribose) synthetase in a rat model of splanchnic artery occlusion and reperfusion.

1. Peroxynitrite, a potent cytotoxic oxidant formed by the reaction of nitric oxide with superoxide anion, and hydroxyl radical, formed in the iron-catalysed Fenton reaction, are important mediators of reperfusion injury. In in vitro studies, DNA single strand breakage, triggered by peroxynitrite or by hydroxyl radical, activates the nuclear enzyme poly (ADP-ribose) synthetase (PARS), with consequent cytotoxic effects. Using 3-aminobenzamide, an inhibitor of PARS, we investigated the role of PARS in the pathogenesis of splanchnic artery occlusion shock. 2. Splanchnic artery occlusion and reperfusion shock (SAO/R) was induced in rats by clamping both the superior mesenteric artery and the coeliac trunk for 45 min, followed by release of the clamp (reperfusion). At 60 min after reperfusion, animals were killed for histological examination and biochemical studies. 3. SAO/R rats developed a significant fall in mean arterial blood pressure, significant increase of tissue myeloperoxidase activity and marked histological injury to the distal ileum. SAO/R was also associated with a significant mortality (0% survival at 2 h after reperfusion). 4. There was a marked increase in the oxidation of dihydrorhodamine 123 to rhodamine (a marker of peroxynitrite-induced oxidative processes) in the plasma of the SAO/R rats, starting early after reperfusion, but not during ischaemia alone. Immunohistochemical examination demonstrated a marked increase in the immunoreactivity to nitrotyrosine, a specific 'footprint' of peroxynitrite, in the necrotic ileum in shocked rats, as measured at 60 min after the start of reperfusion. 5. In addition, in ex vivo studies in aortic rings from shocked rats, we found reduced contractions to noradrenaline and reduced responsiveness to a relaxant effect to acetylcholine (vascular hyporeactivity and endothelial dysfunction, respectively). 6. In a separate set of studies, using a 4000 Dalton fluorescent dextran tracer, we investigated the changes in epithelial permeability associated with SAO/R. Ten minutes of reperfusion, after 30 min of splanchnic artery ischaemia, resulted in a marked increase in epithelial permeability. 7. There was a significant increase in PARS activity in the intestinal epithelial cells, as measured 10 min after reperfusion ex vivo. 3-Aminobenzamide, a pharmacological inhibitor of PARS (applied at 10 mg kg(-1), i.v., 5 min before reperfusion, followed by an infusion of 10 mg kg(-1) h(-1)), significantly reduced ischaemia/reperfusion injury in the bowel, as evaluated by histological examination. Also it significantly improved mean arterial blood pressure, improved contractile responsiveness to noradrenaline, enhanced the endothelium-dependent relaxations and reduced the reperfusion-induced increase in epithelial permeability. 8. 3-Aminobenzamide also prevented the infiltration of neutrophils into the reperfused intestine, as evidenced by reduced myeloperoxidase activity. It improved the histological status of the reperfused tissues, reduced the production of peroxynitrite in the late phase of reperfusion and improved survival. 9. In conclusion, our study demonstrates that the PARS inhibitor 3-aminobenzamide exerts multiple protective effects in splanchnic artery occlusion/reperfusion shock. We suggest that peroxynitrite and/or hydroxyl radical, produced during the reperfusion phase, trigger DNA strand breakage, PARS activation and subsequent cellular dysfunction. The vascular endothelium is likely to represent an important cellular site of protection by 3-aminobenzamide in SAO shock.

The inhibitory effects of mercaptoalkylguanidines on cyclo-oxygenase activity.

1. It has been proposed that in inflammatory conditions, in which both the inducible isoforms of nitric oxide synthase (iNOS) and cyclo-oxygenase (COX-2) are induced, inhibition of NOS also results in inhibition of arachidonic acid metabolism. In the present study we have investigated whether mercaptoalkylguanidines, a novel class of selective iNOS inhibitors, may also influence the activity of cyclo-oxygenase (COX). Therefore, the effect of mercaptoethylguanidine (MEG) and related compounds on the activity of the constitutive (COX-1) and the inducible COX (COX-2) was investigated in cells and in purified enzymes. Aminoguanidine, NG-methyl-L-arginine (L-NMA) and NG-nitro-L-arginine methyl ester (L-NAME) were also studied for comparative purposes. 2. Western blot analysis demonstrated a significant COX-1 activity in unstimulated J774 macrophages and in unstimulated human umbilical vein endothelial cells (HUVEC). Immunostimulation of the J774 macrophages by endotoxin (lipopolysaccharide of E. coli, LPS 10 micrograms ml-1) and interferon gamma (IFN gamma, 100 u ml-1) for 6 h resulted in a significant induction of COX-2, and a down-regulation of COX-1. No COX-2 immunoreactivity was detected in unstimulated HUVEC or unstimulated J774 cells. Therefore, in subsequent studies, the effect of mercaptoalkylguanidines on COX-1 activity was studied in HUVEC stimulated with arachidonic acid for 6 h, and in J774 cells stimulated with arachidonic acid for 30 min. The effect of mercaptoalkylguanidines on COX-2 activity was studied in immunostimulated J774 macrophages, both on prostaglandin production by endogenous sources, and on prostaglandin production in response to exogenous arachidonic acid stimulation. In addition, the effect of mercaptoalkylguanidines on purified COX-1 and COX-2 activities was also studied. 3. In experiments designed to measure COX-1 activity in HUVEC, the cells were stimulated by arachidonic acid (15 microM) for 6 h. This treatment induced a significant production of 6-keto-prostaglandin F1 alpha (6-keto-PGF1 alpha, the stable metabolite of prostacyclin), while nitrite production was undetectable by the Griess reaction. MEG (1 microM to 3 mM) caused a dose-dependent inhibition of the accumulation of 6-keto-PGF1 alpha, with an IC50 of 20 microM. However, aminoguanidine, L-NAME or L-NMA (up to 3 mM) did not affect the production of 6-keto-PGF1 alpha in this experimental system. In experiments designed to measure COX-1 activity in J774.2 macrophages, the cells were stimulated by arachidonic acid (15 microM) for 30 min; this also induced a significant production of 6-keto-PGF1 alpha and MEG (1 microM to 3 mM), aminoguanidine (at 1 and 3 mM), but neither L-NAME nor L-NMA inhibited the production of prostaglandins. 4. In experiments designed to measure prostaglandin production by COX-2 with endogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h in the absence or presence of various inhibitors. In experiments designed to measure prostaglandin production by COX-2 with exogenous arachidonic acid, J774.2 cells were immunostimulated for 6 h, followed by a replacement of the culture medium with fresh medium containing arachidonic acid and various inhibitors. Both of these treatments induced a significant production of 6-keto-PGF1 alpha. Nitrite production, an indicator of NOS activity, was moderately increased after immunostimulation. MEG (1 microM to 3 mM) caused a dose-dependent inhibition of the accumulation of COX metabolites. Similar inhibition of LPS-stimulated 6-keto PGF1 alpha production was shown by other mercaptoalkylguanidines (such as N-methyl-mercaptoethylguanidine, N,N'-dimethyl-mercaptoethylguanidine, S-methyl-mercaptoethylguanidine and guanidino-ethyldisulphide), with IC50 values ranging between 34-55 microM. However, aminoguanidine, L-NAME and L-NMA (up to 3 mM) did not affect the production of prostaglandins.5. In comparative experiments indomethacin, a non selective COX inhibitor, and NS-398, a selective COX-2 inhibitor, reduced (LPS) stimulated 6-keto-PGF1alpha production in J774 macrophages in a dose-dependent manner without affecting nitrite release. Indomethacin, but not NS-398, inhibited 6-keto-PGF1alpha production in the HUVECs. 6.The inhibitory effect of MEG was due to direct inhibition of the catalytic activity of COX as indicated in experiments with purified COX-1 and COX-2. MEG dose-dependently inhibited the purified COX-1 and COX-2 activity with IC50 values of 33microM and 36microM, respectively. Aminoguanidine (at the highest concentrations) inhibited the formation of COX-1 metabolites, without affecting COX-2 activity. High doses of L-NAME (3mM) decreased COX-1 activity only, while L-NMA (up to 3mM) had no effect on the activity of either enzyme. 7.These results suggest that MEG and related compounds are direct inhibitors of the constitutive and the inducible cyclo-oxygenases, in addition to their effects on the inducible NOS. The additional effect of mercaptoalkylguanidines on COX activity may contribute to the beneficial effects of these agents in inflammatory conditions where both iNOS and COX-2 are expressed.

Effect of L-buthionine-(S,R)-sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase on peroxynitrite- and endotoxic shock-induced vascular failure.

1. Peroxynitrite, a cytotoxic oxidant formed from the reaction of nitric oxide (NO) and superoxide is a mediator of cellular injury in ischaemia/reperfusion injury, shock and inflammation. Here we investigated whether L-buthionine-(S,R)-sulphoximine (BSO), an inhibitor of gamma-glutamylcysteine synthetase, alters endothelial and vascular smooth muscle injury in response to peroxynitrite in vitro and during endotoxic shock in vivo. 2. In human umbilical vein endothelial cells and in rat aortic smooth muscle cells, BSO (1 mM, for 24 h) enhanced, whereas glutathione (3 mM) or glutathione ethyl ester (3 mM) attenuated the peroxynitrite (100-1000 microM)-induced suppression of mitochondrial respiration (measured by the conversion of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) to formazan), formation of nitrotyrosine (detected by Western blotting), protein oxidation (measured by detection of 2,4 dinitrophenylhydrazine-reactive carbonyls), and DNA single strand breakage and activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS) (measured by the incorporation of radiolabelled NAD+ into nuclear proteins and by the alkaline unwinding assay, respectively). Glutathione ethyl ester treatment reduced the BSO-induced enhancement of peroxynitrite-induced cytotoxicity. 3. In rat isolated thoracic aortic rings, BSO treatment (in vivo, at 1 g kg(-1) intraperitoneally (i.p.) for 24 h) enhanced, whereas pretreatment with glutathione (in vitro, 3 mM) attenuated the peroxynitrite-induced reduction of the contractions to noradrenaline, and the peroxynitrite-induced impairment of the endothelium-dependent relaxations to acetylcholine. 4. In BSO-pretreated rats, treatment with bacterial lipopolysaccharide (LPS, 15 mg kg(-1), i.p., for 6 h) caused a more pronounced vascular hyporeactivity and endothelial dysfunction ex vivo. BSO pretreatment also increased the degree of nitrotyrosine staining (detected by imunohistochemistry) in the aorta after LPS treatment. 5. In conclusion, our results demonstrate that L-buthionine-(S,R)-sulphoximine, an inhibitor of gamma-glutamylcysteine synthetase enhances peroxynitrite- and endotoxic shock-induced vascular failure. Based on these findings, we suggest that endogenous glutathione plays an important protective role against peroxynitrite- and LPS-induced vascular injury.

Role of peroxynitrite and activation of poly (ADP-ribose) synthase in the vascular failure induced by zymosan-activated plasma.

1. Zymosan is a wall component of the yeast Saccharomyces Cerevisiae. Injection of zymosan into experimental animals is known to produce an intense inflammatory response. Recent studies demonstrated that the zymosan-induced inflammatory response in vivo can be ameliorated by inhibitors of nitric oxide (NO) biosynthesis. The cytotoxic effects of NO are, in part, mediated by the oxidant preoxynitrite and subsequent activation of the nuclear enzyme poly (ADP-ribose) synthetase (PARS). In the present in vitro study, we have investigated the cellular mechanisms of vascular failure elicited by zymosan-activated plasma and the contribution of peroxynitrite production and activation of PARS to the changes. 2. Incubation of rat aortic smooth muscle cells with zymosan-activated plasma (ZAP) induced the production of nitrite, the breakdown product of NO, due to the expression of the inducible isoform of NO synthase (iNOS) over 6 24 h. In addition, ZAP triggered the production of peroxynitrite in these cells, as measured by the oxidation of the fluorescent dye dihydrorhodamine 123 and by nitrotyrosine Western blotting. 3. Incubation of the smooth muscle cells with ZAP induced DNA single strand breakage and PARS activation. These effects were reduced by inhibition of NOS with NG-methyl-L-arginine (L-NMA, 3 mM), and by glutathione (3 mM), a scavenger of peroxynitrite. The PARS inhibitor 3-aminobenzamide (1 mM) inhibited the ZAP-induced activation of PARS. 4. Incubation of thoracic aortae with ZAP in vitro caused a reduction of the contractions of the blood vessels to noradrenaline (vascular hyporeactivity) and elicited a reduced responsiveness to the endothelium-dependent vasodilator acetylcholine (endothelial dysfunction). 5. Preincubation of the thoracic aortae with L-NMA (1 mM), glutathione (3 mM) or by the PARS inhibitor 3-aminobenzamide (1 mM) prevented the development of vascular hyporeactivity in response to ZAP. Moreover, glutathione and 3-aminobenzamide treatment protected against the ZAP-induced development of endothelial dysfunction. The PARS-related loss of the vascular contractility was evident at 30 min after incubation in endothelium-intact, but not in endothelium-denuded vessels and also manifested at 6 h after incubation with ZAP in endothelium-denuded rings. The acute response is probably related, therefore, to peroxynitrite formation (involving the endothelial NO synthase), whereas the delayed response may be related to the expression of iNOS in the smooth muscle. 6. The data obtained suggest that zymosan-activated plasma causes vascular dysfunction by inducing the simultaneous formation of superoxide and NO. These radicals combine to form peroxynitrite, which, in turn causes DNA injury and PARS activation. The protective effect of 3-aminobenzamide demonstrates that PARS activation contributes both to the development of vascular hyporeactivity and endothelial dysfunction during the vascular failure induced by ZAP.

Spontaneous rearrangement of aminoalkylisothioureas into mercaptoalkylguanidines, a novel class of nitric oxide synthase inhibitors with selectivity towards the inducible isoform.

1. The generation of nitric oxide (NO) from L-arginine by NO synthases (NOS) can be inhibited by guanidines, amidines and S-alkylisothioureas. Unlike most L-arginine based inhibitors, however, some guanidines and S-alkylisothioureas, in particular aminoethylisothiourea (AETU), show selectivity towards the inducible isoform (iNOS) over the constitutive isoforms (endothelial, ecNOS and brain isoform, bNOS) and so may be of therapeutic benefit. In the present study we have investigated the effects of AETU and other aminoalkylisothioureas on the activities of iNOS, ecNOS and bNOS. 2. AETU, aminopropylisothiourea (APTU) and their derivatives containing alkyl substituents on one of the amidino nitrogens, potently inhibit nitrite formation by immunostimulated J774 macrophages (a model of iNOS activity) with EC50 values ranging from 6-30 microM (EC50 values for NG-methyl-L-arginine (L-NMA) and NG-nitro-L-arginine were 159 and > 1000 microM, respectively). The inhibitory effects of these aminoalkylisothioureas (AATUs) were attentuated by L-arginine in the incubation medium, indicating that these agents may complete with L-arginine for its binding site on NOS. 3. The above AATUs undergo chemical conversion in neutral or basic solution (pH 7 or above) as indicated by (1) the disappearance of AATUs from solution as measured by h.p.l.c., (2) the generation of free thiols not previously present and (3) the isolation of species (as picrate and flavianate salts) from neutral or basic solutions of AATUs that are different from those obtained from acid solutions. 4. Mercaptoalkylguanidines (MAGs) were prepared and shown to be potent inhibitors of iNOS activity with EC50s comparable to those of their isomeric AATUs. 5. These findings suggest that certain AATUs exert their potent inhibitory effects through intramolecular rearrangement to mercaptoalkylguanidines (MAGs) at physiological pH. Those AATUs not capable of such rearrangement do not exhibit the same degree of inhibition of iNOS. 6. In contrast to their potent effects on iNOS, some AATUs and MAGs were 20-100 times weaker than NG-methyl-L-arginine and NG-nitro-L-arginine as inhibitors of ecNOS as assessed by their effects on the conversion of L-arginine to L-citrulline in homogenates of bovine endothelial cells and by their pressor effects in anaesthetized rats. Thus mercaptoalkylguanidines represent a new class of NOS inhibitors with preference towards iNOS. 7. AETU and mercaptoethylguanidine (MEG), when given as infusions, gave slight decreases in MAP in control rats. However, infusions of AETU or MEG to endotoxin-treated rats caused an increase in MAP and restored 80% of the endotoxin-induced fall in MAP. 8. High doses of MEG (30-60 mg kg-1) caused a decrease in MAP of normal rats. This depressor effect may be a consequence of the in vivo oxidation of MEG to the disulphide, guanidinoethyldisulphide (GED), which caused pronounced, transient hypotensive responses in anaesthetized rats and caused endothelium-independent vasodilator responses in precontracted rat aortic rings in vitro. 9. In some cases, slight differences were observed in the activities of AATUs and the corresponding MAGs. These may be explained by the formation of other species from AATUs in physiological media. For example, AETU can give rise to small amounts of the potent ecNOS inhibitor, 2-aminothiazoline, in addition to MEG. This may account for the differences in the in vitro and in vivo effects of AETU and MEG. 10. In conclusion, the in vitro and in vivo effects of AETU and related aminoalkylisothioureas can be explained in terms of their intramolecular rearrangement to generate mercaptoalkylguanidines, a novel class of selective inhibitors of iNOS.

Participation of tumour necrosis factor and nitric oxide in the mediation of vascular dysfunction in splanchnic artery occlusion shock.

1. Splanchnic artery occlusion (SAO) shock is characterized by irreversible circulatory failure. Tumour necrosis factor (TNF-alpha) may affect the L-arginine/nitric oxide (NO) pathway, thus contributing to the cardiovascular derangements of circulatory shock. 2. We investigated the contribution of both TNF-alpha and the L-arginine/nitric oxide pathway to the vascular dysfunction of SAO shock. Anaesthetized rats, subjected to total occlusion of the superior mesenteric artery and the coeliac trunk for 45 min developed a severe shock state (SAO shock) resulting in a fatal outcome within 75-90 min after the release of occlusion. Sham operated animals were used as controls. SAO shocked rats had also a marked hypotension and enhanced macrophage and serum levels of TNF-alpha. Furthermore, aortic rings from shocked rats showed a marked hyporeactivity to phenylephrine (PE 1 nM-10 microM) and reduced responsiveness to acetylcholine (ACh 10 nM-10 microM). Endothelium-denuded aortic rings had also a marked hyporeactivity to phenylephrine, which was restored to control values by in vitro administration of NG nitro-L-arginine-methyl ester (L-NAME 10 microM). 3. In vivo administration of cloricromene (2 mg kg-1, i.v.), an inhibitor of TNF-alpha biosynthesis, increased survival, enhanced mean arterial blood pressure and reduced macrophage and serum levels of TNF-alpha. Furthermore, aortic rings from shocked rats treated with cloricromene exhibited a greater contractile response to phenylephrine and improved responsiveness to ACh when compared to aortic rings from vehicle-treated SAO shocked rats. 4. Our results suggest that TNF-alpha alters both endothelial and muscular L-arginine/nitric oxide pathways which in turn produce vascular dysfunction in SAO shock.

Pharmacological characterization of guanidinoethyldisulphide (GED), a novel inhibitor of nitric oxide synthase with selectivity towards the inducible isoform.

1. Guanidines, amidines, S-alkylisothioureas, and recently, mercaptoalkylguanidines have been described as inhibitors of the generation of nitric oxide (NO) from L-arginine by NO synthases (NOS). We have recently demonstrated that guanidinoethyldisulphide (GED), formed from the dimerisation of mercaptoethylguanidine (MEG), is a novel inhibitor of nitric oxide synthases. Here we describe the pharmacological properties of GED on purified NOS isoforms, various cultured cell types, vascular ring preparations, and in endotoxin shock. 2. GED potently inhibited NOS activity of purified inducible NOS (iNOS), endothelial NOS (ecNOS), and brain NOS (bNOS) enzymes with Ki values of 4.3, 18 and 25 microM, respectively. Thus, GED has a 4 fold selectivity for iNOS over ecNOS at the enzyme level. The inhibitory effect of GED on ecNOS and iNOS was competitive vs. L-arginine and non-competitive vs. tetrahydrobiopterin. 3. Murine J774 macrophages, rat aortic smooth muscle cells, murine lung epithelial cells, and human intestinal DLD-1 cells were stimulated with appropriate mixtures of pro-inflammatory cytokines or bacterial lipopolysaccharide to express iNOS. In these cells, GED potently inhibited nitrite formation (EC50 values: 11, 9, 1 and 30 microM, respectively). This suggests that uptake of GED may be cell type and species-dependent. The inhibitory effect of GED on nitrite production was independent of whether GED was given together with immunostimulation or 6 h afterwards, indicating that GED does not interfere with the process of iNOS induction. 4. GED caused relaxations in the precontracted vascular ring preparations (EC50: 20 microM). Part of this relaxation was endothelium-dependent, but was not blocked by methylene blue (100 microM), an inhibitor of soluble guanylyl cyclase. In precontracted rings, GED enhanced the acetylcholine-induced, endothelium-dependent relaxations at 10 microM and caused a slight inhibition of the relaxations at 100 microM. The vascular studies demonstrate that the inhibitory potency of GED on ecNOS in the ring preparations is considerably lower than its potency against iNOS in the cultured cells. These data suggest that the selectivity of GED towards iNOS may lie, in part, at the enzyme level, as well as differential uptake by cells expressing the various isoforms of NOS. 5. In a rat model of endotoxin shock in vivo, administration of GED, at 3 mg kg-1 bolus followed by 10 mg kg-1 h-1 infusion, starting at 90 min after bacterial lipopolysaccharide (LPS, 15 mg kg-1, i.v.), prevented the delayed fall in mean arterial blood pressure, prevented the development of the vascular hyporeactivity to noradrenaline of the thoracic aorta ex vivo and protected against the impairment of the endothelium-dependent relaxations associated with this model of endotoxaemia. The same bolus and infusion of the inhibitor did not alter blood pressure or ex vivo vascular reactivity in normal animals over 90 min. 6. Administration of GED (10 mg kg-1, i.p.) given at 2 h after LPS (120 mg kg-1, i.p.) and every 6 h thereafter caused a significant improvement in the survival rate in a lethal model of endotoxin shock in mice between 12 and 42 h. 7. In conclusion, we found that GED is a competitive inhibitor of iNOS activity. Its selectivity towards iNOS may lie both at the enzyme level and at the level of cell uptake. GED has beneficial effects in models of endotoxin shock that are driven by iNOS. GED or its derivatives may be useful tools in the experimental therapy of inflammatory conditions associated with NO overproduction due to iNOS expression.