Asymmetric dimethylarginine endogenous inhibition of nitric oxide synthase causes differential vasculature effects

Background: Asymmetric dimethylarginine (ADMA), produced during protein metabolism, is an endogenous inhibitor of nitric oxide synthase, but little is known about its direct vasoactive properties in different arterial beds. Material/Methods: Segments of canine coronary, renal, and femoral arteries were pretreated with increasing concentrations of ADMA, and endothelial function was evaluated in organ chambers. Results: In precontracted canine coronary arteries, the highest concentrations of ADMA inhibited endothelium-dependent relaxation mediated by acetylcholine (n=7), but no concentration of ADMA inhibited receptor-independent relaxation mediated by calcium ionophore (n=7) (P<.001). The effect of ADMA on acetylcholine-mediated relaxation was shown to be competitive inhibition of the nitric oxide synthase pathway, because the addition of L-arginine (10(-3) M), but not D-arginine (101 M), reversed the effect produced by 10(-5) M ADMA. Further, ADMA did not alter endothelium-independent relaxation mediated by sodium nitroprusside (10(-9) to 10(-6) M; n=7). Femoral arteries (n=7) and renal arteries (n=7) were more sensitive to ADMA than were coronary arteries, and they demonstrated significant ADMA inhibition to receptor dependent relaxation induced by acetylcholine (P=.03 and P=.01, respectively) and to receptor-independent relaxation induced by calcium ionophore (P=.02 and P=.01, respectively). Conclusions: Endothelium-dependent relaxation mediated by ADMA is more marked in femoral and renal arteries than in coronary arteries. The response in coronary arteries may be overall protective. Considering these different effects in various artery types, the role of ADMA as a confiable and specific cardiovascular risk factor is questioned.

Subfornical organ mediates pressor effect of angiotensin: Influence of nitric oxide synthase inhibitors, AT(1) and AT(2) angiotensin antagonist's receptors

Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)

Inhibitors of the proteasome pathway interfere with induction of nitric oxide synthase in macrophages by blocking activation of transcription factor NF-kappa B.

The objective of this study was to elucidate the role of the proteasome pathway or multicatalytic proteinase complex in the induction of immunologic nitric oxide (NO) synthase (iNOS) in rat alveolar macrophages activated by lipopolysaccharide. Macrophages were incubated in the presence of lipopolysaccharide plus test agent for up to 24 hr. Culture media were analyzed for accumulation of stable oxidation products of NO (NO2- + N03-, designated as NOX-), cellular RNA was extracted for determination of iNOS mRNA levels by Northern blot analysis, and nuclear extracts were prepared for determination of NF-kappa B by electrophoretic mobility-shift assay. Inhibitors of calpain (alpha-N-acetyl-Leu-Leu-norleucinal; N-benzyloxycarbonyl-Leu-leucinal) and the proteasome (N-benzyloxycarbonyl-Ile-Glu-(O-t-Bu)-Ala-leucinal) markedly inhibited or abolished the induction of iNOS in macrophages. The proteinase inhibitors interfered with lipopolysaccharide-induced NOX- production by macrophages, and this effect was accompanied by comparable interference with the appearance of both iNOS mRNA and NF-kappa B. Calpain inhibitors elicited effects at concentrations of 1-100 microM, whereas the proteasome inhibitor was 1000-fold more potent, producing significant inhibitory effects at 1 nM. The present findings indicate that the proteasome pathway is essential for lipopolysaccharide-induced expression of the iNOS gene in rat alveolar macrophages. Furthermore, the data support the view that the proteasome pathway is directly involved in promoting the activation of NF-kappa B and that the induction of iNOS by lipopolysaccharide involves the transcriptional action of NF-kappaB.

Vascular hyporeactivity to angiotensin II induced by Escherichia coli endotoxin is reversed by Nω-Nitro-L-Arginine, an inhibitor of nitric oxide synthase

Septic shock or sepsis is reported to be one of the major causes of death when followed by systemic infectious trauma in humans and other mammals. Its development leads to a large drop in blood pressure and a reduction in vascular responsiveness to physiological vasoconstrictors which, if not contained, can lead to death. It is proposed that this vascular response is due to the action of bacterial cell wall products released into the bloodstream by the vascular endothelium and is considered a normal response of the body's defenses against infection. A reduction in vascular reactivity to epinephrine and norepinephrine is observed under these conditions. In the present study in rats, the aim was to assess whether those effects of hypotension and hyporeactivity are also related to another endogenous vasoconstrictor, angiotensin II (AII). We evaluated the variation in the power of this vasoconstrictor over the mean arterial pressure in anesthetized rats, before and after the establishment of hypotension by Escherichia coli endotoxin (Etx). Our results show that in this model of septic shock, there is a reduction in vascular reactivity to AII and this reduction can be reversed by the inhibitor of nitric oxide synthase, Nω-Nitro-L- Arginine (NωNLA). Our results also suggest that other endogenous factors (not yet fully known) are involved in the protection of rats against septic shock, in addition to the L-arginine NO pathway.

Role of the different isoforms of cyclooxygenase and nitric oxide synthase during gastric ulcer healing in cyclooxygenase-1 and -2 knockout mice

Traditional NSAIDs, selective cyclooxygenase (COX)-2 inhibitors, and inhibitors of nitric oxide synthase (NOS) impair the healing of preexisting gastric ulcers. However, the role of COX-1 (with or without impairment of COX-2) and the interaction between COX and NOS isoforms during healing are less clear. Thus we investigated healing and regulation of COX and NOS isoforms during ulcer healing in COX-1 and COX-2 deficiency and inhibition mouse models. In this study, female wild-type COX-1(-/-) and COX-2(-/-) mice with gastric ulcers induced by cryoprobe were treated intragastrically with vehicle, selective COX-1 (SC-560), COX-2 (celecoxib, rofecoxib, and valdedoxib), and unselective COX (piroxicam) inhibitors. Ulcer healing parameters, mRNA expression, and activity of COX and NOS were quantified. Gene disruption or inhibition of COX-1 did not impair ulcer healing. In contrast, COX-2 gene disruption and COX-2 inhibitors moderately impaired wound healing. More severe healing impairment was found in dual (SC-560 + rofecoxib) and unselective (piroxicam) COX inhibition and combined COX impairment (in COX-1(-/-) mice with COX-2 inhibition and COX-2(-/-) mice with COX-1 inhibition). In the ulcerated repair tissue, COX-2 mRNA in COX-1(-/-) mice, COX-1 mRNA in COX-2(-/-) mice, and, remarkably, NOS-2 and NOS-3 mRNA in COX-impaired mice were more upregulated than in wild-type mice. This study demonstrates that COX-2 is a key mediator in gastric wound healing. In contrast, COX-1 has no significant role in healing when COX-2 is unimpaired but becomes important when COX-2 is impaired. As counterregulatory mechanisms, mRNA of COX and NOS isoforms were increased during healing in COX-impaired mice.

Lipocortin 1 mediates the inhibition by dexamethasone of the induction by endotoxin of nitric oxide synthase in the rat.

Administration of Escherichia coli lipopolysaccharide (LPS; 10 mg/kg i.v.) to male Wistar rats caused within 240 min (i) a sustained fall (approximately 30 mmHg) in mean arterial blood pressure, (ii) a reduction (> 75%) in the pressor responses to norepinephrine (1 microgram/kg i.v.), and (iii) an induction of nitric oxide synthase (iNOS) as measured in the lung. Dexamethasone (1 mg/kg i.p. at 2 h prior to LPS) attenuated the hypotension and the vascular hyporeactivity to norepinephrine and reduced (by approximately 77%) the expression of iNOS in the lung. These effects of dexamethasone were prevented by pretreatment of LPS-treated rats with a neutralizing antiserum to lipocortin 1 (anti-LC1; 60 mg/kg s.c. at 24 h prior to LPS) but not by a control nonimmune sheep serum. Stimulation of J774.2 macrophages with LPS (1 microgram/ml for 24 h) caused the expression of iNOS and cyclooxygenase 2 (COX-2) protein and significantly increased nitrite generation; this was prevented by dexamethasone (0.1 microM at 1 h prior to LPS), which also increased cell surface lipocortin 1. Pretreatment of J774.2 cells with anti-LC1 (1:60 dilution at 4 h prior to LPS) also abolished the inhibitory effect of dexamethasone on iNOS expression and nitrite accumulation but not that on COX-2 expression. A lipocortin 1 fragment (residues 1-188 of human lipocortin 1; 20 micrograms/ml at 1 h prior to LPS) also blocked iNOS in J774.2 macrophages activated by LPS (approximately 78% inhibition), and this too was prevented by anti-LC1. We conclude that the extracellular release of endogenous lipocortin 1 (i) mediates the inhibition by dexamethasone of the expression of iNOS, but not of COX-2, and (ii) contributes substantially to the beneficial actions of dexamethasone in rats with endotoxic shock.

Effects of Partial Liver Ischemia Followed by Global Liver Reperfusion on the Remote Tissue Expression of Nitric Oxide Synthase: Lungs and Kidneys

Hepatic ischemia followed by reperfusion (IR) results in mild to severe remote organ injury. Oxidative stress and nitric oxide (NO) seem to be involved in the IR injury. Our aim was to investigate the effects of liver I/R on hepatic function and lipid peroxidation, leukocyte infiltration and NO synthase (NOS) immunostaining in the lung and the kidney. We randomized 24 male Wistar rats into 3 groups: 1) control; 2) 60 minutes of partial (70%) liver 1 and 2 hours of global liver R; and 3) 60 minutes of partial (70%) liver I and 6 hours of global liver R. Groups 2 and 3 showed significant increases in plasma alanine and aspartate aminotransferase levels and in tissue malondialdehyde and myeloperoxidase contents. In the kidney, positive endothelial NOS (eNOS) staining was significantly decreased in group 3 compared with group 1. However, staining for inducible NOS (iNOS) and neuronal NOS (nNOS) did not differ among the groups. In the lung, the staining for eNOS and iNOS did not show significant differences among the groups; no positive nNOS staining was observed in any group. These results suggested that partial liver I followed by global liver R induced liver, kidney, and lung injuries characterized by neutrophil sequestration and increased oxidative stress. In addition, we supposed that the reduced NO formation via eNOS may be implicated in the moderate impairment of renal function, observed by others at 24 hours after liver I/R.

Immunolocalization of nitric oxide synthase isoforms in human archival and rat tissues, and cultured cells

Nitric oxide (NO) exerts important physiological and pathological roles in humans. The study of NO requires the immunolocalization of its synthesizing enzymes, neuronal, endothelial and inducible NO synthases (NOS). NOS are labile to formalin-fixation and paraffin-embedding, which are used to prepare human archival tissues. This lability has made NOS immunohistochemical studies difficult, and a detailed protocol is not yet available. We describe here a protocol for the immunolocalization of NOS isoforms in human archival cerebellum and non-nervous tissues, and in rat tissues and cultured cells. Neuronal NOS antigenicity in human archival and rat nervous tissue sections was microwave-retrieved in 50 mM Tris-HCl buffer, pH 9.5, for 20 min at 900W. Neuronal NOS was expressed in stellate, basket, Purkinje and granule cells in human and rat cerebellum. Archival and frozen human cerebellar sections showed the same neuronal NOS staining pattern. Archival cerebellar sections not subjected to antigen retrieval stained weakly. Antigenicity of inducible NOS in human lung was best retrieved in 10 mM sodium citrate buffer, pH 6.0, for 15 min at 900W. Inflammatory cells in a human lung tuberculoma were strongly stained by anti-inducible NOS antibody. Anti-endothelial NOS strongly stained kidney glomeruli. Cultured PC12 cells were strongly stained by anti-neuronal NOS without antigen retrieving. The present immunohistochemistry protocol is easy to perform, timeless, and suitable for the localization of NOS isoforms in nervous and non-nervous tissues, in human archival and rat tissues. It has been extensively used in our laboratory, and is also appropriate for other antigens. (C) 2011 Elsevier B.V. All rights reserved.

Vascular changes after cardiopulmonary bypass and ischemic cardiac arrest: roles of nitric oxide synthase and cyclooxygenase

Cardiac surgery involving ischemic arrest and extracorporeal circulation is often associated with alterations in vascular reactivity and permeability due to changes in the expression and activity of isoforms of nitric oxide synthase and cyclooxygenase. These inflammatory changes may manifest as systemic hypotension, coronary spasm or contraction, myocardial failure, and dysfunction of the lungs, gut, brain and other organs. In addition, endothelial dysfunction may increase the occurrence of late cardiac events such as graft thrombosis and myocardial infarction. These vascular changes may lead to increased mortality and morbidity and markedly lengthen the time of hospitalization and cost of cardiac surgery. Developing a better understanding of the vascular changes operating through nitric oxide synthase and cyclooxygenase may improve the care and help decrease the cost of cardiovascular operations.

Effect of inhibition of nitric oxide synthase on blood pressure and renal sodium handling in renal denervated rats

The role of sympathetic nerve activity in the changes in arterial blood pressure and renal function caused by the chronic administration of NG-nitro-L-arginine methyl ester (L-NAME), an inhibitor of nitric oxide (NO) synthesis, was examined in sham and bilaterally renal denervated rats. Several studies have demonstrated that sympathetic nerve activity is elevated acutely after L-NAME administration. To evaluate the role of renal nerve activity in L-NAME-induced hypertension, we compared the blood pressure response in four groups (N = 10 each) of male Wistar-Hannover rats weighing 200 to 250 g: 1) sham-operated vehicle-treated, 2) sham-operated L-NAME-treated, 3) denervated vehicle-treated, and 4) denervated L-NAME-treated rats. After renal denervation or sham surgery, one control week was followed by three weeks of oral administration of L-NAME by gavage. Arterial pressure was measured weekly in conscious rats by a tail-cuff method and renal function tests were performed in individual metabolic cages 0, 7, 14 and 21 days after the beginning of L-NAME administration. L-NAME (60 mg kg-1 day-1) progressively increased arterial pressure from 108 ± 6.0 to 149 ± 12 mmHg (P<0.05) in the sham-operated group by the third week of treatment which was accompanied by a fall in creatinine clearance from 336 ± 18 to 222 ± 59 µl min-1 100 g body weight-1 (P<0.05) and a rise in fractional urinary sodium excretion from 0.2 ± 0.04 to 1.62 ± 0.35% (P<0.05) and in sodium post-proximal fractional excretion from 0.54 ± 0.09 to 4.7 ± 0.86% (P<0.05). The development of hypertension was significantly delayed and attenuated in denervated L-NAME-treated rats. This was accompanied by a striking additional increase in fractional renal sodium and potassium excretion from 0.2 ± 0.04 to 4.5 ± 1.6% and from 0.1 ± 0.015 to 1.21 ± 0.37%, respectively, and an enhanced post-proximal sodium excretion compared to the sham-operated group. These differences occurred despite an unchanged creatinine clearance and Na+ filtered load. These results suggest that bilateral renal denervation delayed and attenuated the L-NAME-induced hypertension by promoting an additional decrease in tubule sodium reabsorption in the post-proximal segments of nephrons. Much of the hypertension caused by chronic NO synthesis inhibition is thus dependent on renal nerve activity.

Morphometric evaluation of nitric oxide synthase isoforms and their cytokine regulators predict pulmonary dysfunction and survival in systemic sclerosis

Because histopathological changes in the lungs of patients with systemic sclerosis (SSc) are consistent with alveolar and vessel cell damage, we presume that this interaction can be characterized by analyzing the expression of proteins regulating nitric oxide (NO) and plasminogen activator inhibitor-1 (PAI-1) synthesis. To validate the importance of alveolar-vascular interactions and to explore the quantitative relationship between these factors and other clinical data, we studied these markers in 23 cases of SSc nonspecific interstitial pneumonia (SSc-NSIP). We used immunohistochemistry and morphometry to evaluate the amount of cells in alveolar septa and vessels staining for NO synthase (NOS) and PAI-1, and the outcomes of our study were cellular and fibrotic NSIP, pulmonary function tests, and survival time until death. General linear model analysis demonstrated that staining for septal inducible NOS (iNOS) related significantly to staining of septal cells for interleukin (IL)-4 and to septal IL-13. In univariate analysis, higher levels of septal and vascular cells staining for iNOS were associated with a smaller percentage of septal and vascular cells expressing fibroblast growth factor and myofibroblast proliferation, respectively. Multivariate Cox model analysis demonstrated that, after controlling for SSc-NSIP histological patterns, just three variables were significantly associated with survival time: septal iNOS (P=0.04), septal IL-13 (P=0.03), and septal basic fibroblast growth factor (bFGF; P=0.02). Augmented NOS, IL-13, and bFGF in SSc-NSIP histological patterns suggest a possible functional role for iNOS in SSc. In addition, the extent of iNOS, PAI-1, and IL-4 staining in alveolar septa and vessels provides a possible independent diagnostic measure for the degree of pulmonary dysfunction and fibrosis with an impact on the survival of patients with SSc.

Daily cycling of nitric oxide synthase (NOS) in the hippocampus of pigeons (C. livia)

Abstract: Background: Nitric oxide synthase (NOS) is essential for the synthesis of nitric oxide (NO), a non-conventional neurotransmitter with an important role in synaptic plasticity underlying processes of hippocampus-dependent memory and in the regulation of biological clocks and circadian rhythms. Many studies have shown that both the NOS cytosolic protein content and its enzymatic activity present a circadian variation in different regions of the rodent brain, including the hippocampus. The present study investigated the daily variation of NOS enzymatic activity and the cytosolic content of nNOS in the hippocampus of pigeons. Results: Adult pigeons kept under a skeleton photoperiod were assigned to six different groups. Homogenates of the hippocampus obtained at six different times-of-day were used for NOS analyses. Both iNOS activity and nNOS cytosolic protein concentrations were highest during the subjective light phase and lowest in the subjective dark phase of the circadian period. ANOVA showed significant time differences for iNOS enzymatic activity (p < 0.05) and for nNOS protein content (p < 0.05) in the hippocampus. A significant daily rhythm for both iNOS and nNOS was confirmed by analysis with the Cosinor method (p < 0.05). The present findings indicate that the enzymatic activity of iNOS and content of nNOS protein in the hippocampus of pigeons exhibit a daily rhythm, with acrophase values occurring during the behavioral activity phase. Conclusions: The data corroborate the reports on circadian variation of NOS in the mammalian hippocampus and can be considered indicative of a dynamic interaction between hippocampus-dependent processes and circadian clock mechanisms.

Daily cycling of nitric oxide synthase (NOS) in the hippocampus of pigeons (C. livia)

Background: Nitric oxide synthase (NOS) is essential for the synthesis of nitric oxide (NO), a non-conventional neurotransmitter with an important role in synaptic plasticity underlying processes of hippocampus-dependent memory and in the regulation of biological clocks and circadian rhythms. Many studies have shown that both the NOS cytosolic protein content and its enzymatic activity present a circadian variation in different regions of the rodent brain, including the hippocampus. The present study investigated the daily variation of NOS enzymatic activity and the cytosolic content of nNOS in the hippocampus of pigeons. Results: Adult pigeons kept under a skeleton photoperiod were assigned to six different groups. Homogenates of the hippocampus obtained at six different times-of-day were used for NOS analyses. Both iNOS activity and nNOS cytosolic protein concentrations were highest during the subjective light phase and lowest in the subjective dark phase of the circadian period. ANOVA showed significant time differences for iNOS enzymatic activity (p < 0.05) and for nNOS protein content (p < 0.05) in the hippocampus. A significant daily rhythm for both iNOS and nNOS was confirmed by analysis with the Cosinor method (p < 0.05). The present findings indicate that the enzymatic activity of iNOS and content of nNOS protein in the hippocampus of pigeons exhibit a daily rhythm, with acrophase values occurring during the behavioral activity phase. Conclusions: The data corroborate the reports on circadian variation of NOS in the mammalian hippocampus and can be considered indicative of a dynamic interaction between hippocampus-dependent processes and circadian clock mechanisms.