Synergistic action of advanced glycation end products and endogenous nitric oxide leads to neuronal apoptosis in vitro: A new insight into selective nitrergic neuropathy in diabetes

Aims/hypothesis We have previously shown that in diabetes nitrergic neurones innervating the urogenital and gastrointestinal organs undergo a selective degenerative process. This comprises an initial insulin-reversible decrease in neuronal nitric oxide synthase (nNOS) in the axons, followed by apoptosis of the nitrergic neurones, a process that is not reversible by insulin. Since apoptosis was independent of serum glucose concentrations, and advanced glycation endproducts (AGEs) have been implicated in the pathogenesis of diabetic complications, we have now measured AGEs in the serum and penis, pyloric sphincter and pelvic ganglia of diabetic animals at different times after streptozotocin treatment. Furthermore, we have studied their effect in vitro on human neuroblastoma (SH-SY5Y) cells in the presence or absence of nNOS expression.Methods Serum AGEs were measured using fluorometry and ELISA. Accumulation of AGEs in the tissues was evaluated with immunohistochemistry. The viability, apoptosis and oxidative stress in SH-SY5Y cells were measured upon exposure to AGEs or high concentrations of glucose.Results AGEs increased gradually in the serum and tissues of streptozotocin-induced diabetic rats; this process was not affected by delayed insulin treatment. In SH-SY5Y cells, AGEs, but not high glucose concentrations, increased the reactive oxygen species and caspase-3-dependent apoptosis in a synergistic fashion with endogenous nitric oxide (NO). Apoptosis was prevented by treatment with a NOS inhibitor, a pan-caspase inhibitor, a soluble receptor of AGEs or an anti-oxidant, but not an inhibitor of soluble guanylate cyclase.Conclusions/interpretation The synergistic actions of NO and AGEs account for the irreversible nitrergic degeneration in diabetes.Abbreviations AGEs advanced glycation endproducts - eNOS endothelial nitric oxide synthase - HSA-AGEs advanced glycated human serum albumin - iNOS inducible nitric oxide synthase - L-NAME N^G-nitro-L-arginine methyl ester (NOS inhibitor) - MPG major pelvic ganglion - NAC N-acetyl-L-cysteine - NO nitric oxide - NOS nitric oxide synthase - nNOS neuronal nitric oxide synthase - ODQ 1H-[1,2,4]oxadiazolo[4,3,-a]quinoxalin-1-one (sGC inhibitor) - RA retinoic acid - ROS reactive oxygen species - sRAGE soluble receptor of advanced glycation endproduct - STZ streptozotocin - Z-VAD-FMK Z-Val-Ala-Asp(OMe)-CH₂F (pan-caspase inhibitor)     

脂多糖对大鼠肺泡巨噬细胞分泌一氧化氮和氧化应激的影响

目的探讨细菌内毒素脂多糖（LPS）对SD大鼠肺泡巨噬细胞产生一氧化氮（NO）和氧化应激的影响。方法采用支气管肺泡灌洗和细胞差速贴壁的方法分离大鼠肺泡巨噬细胞（AM）,分别测定AM培养上清液NO含量、一氧化氮合酶（NOS）活性、丙二醛（MDA）含量和超氧化物歧化酶（SOD）活性。结果在5,10,20,50mg/LLPS分别干预下,大鼠AM培养上清液NO含量、NOS活性和MDA含量均显著升高,SOD活性显著降低,并且具有浓度依赖性。结论LPS促进大鼠AM分泌NO,并诱导AM脂质过氧化损伤,这可能是内毒素诱发肺部炎症反应不易控制和急性肺损伤的机制之一。     

Apocynin restores endothelial dysfunction in streptozotocin diabetic rats through regulation of nitric oxide synthase and NADPH oxidase expressions

AimIncreased production of reactive oxygen species (ROS) in the diabetic vasculature results in the impairment of nitric oxide (NO)-mediated relaxations leading to impaired endothelium-dependent vasodilation. An important source of ROS is nicotinamide adenine dinucleotide phosphate (NADPH) oxidase, and the inhibition of this enzyme is an active area of interest. This study aimed to investigate the effects of apocynin, an NADPH oxidase inhibitor, on endothelial dysfunction and on the expression of NO synthase (NOS) and NADPH oxidase in thoracic aorta of diabetic rats.MethodStreptozotocin (STZ)-diabetic rats received apocynin (16 mg/kg per day) for 4 weeks. Endothelium-dependent and -independent relaxations were determined in thoracic aortic rings. Western blotting and RT-PCR analysis were performed for NOSs and NADPH oxidase in the aortic tissue.ResultsAcetylcholine-induced relaxations and l-NAME-induced contractions were decreased in diabetic aorta. The decrease in acetylcholine and l-NAME responses were prevented by apocynin treatment without a significant change in plasma glucose levels. Endothelial NOS (eNOS) protein and mRNA expression exhibited significant decrease in diabetes, while protein and/or mRNA expressions of inducible NOS (iNOS) as well as p22^phox and gp91^phox subunits of NADPH oxidase were increased, and these alterations were markedly prevented by apocynin treatment.ConclusionNADPH oxidase expression is increased in diabetic rat aorta. NADPH oxidase-mediated oxidative stress is accompanied by the decreased eNOS and increased iNOS expressions, contributing to endothelial dysfunction. Apocynin effectively prevents the increased NADPH oxidase expression in diabetic aorta and restores the alterations in NOS expression, blocking the vicious cycle leading to diabetes-associated endothelial dysfunction.     

Expression of endothelial nitric oxide synthase and inducible nitric oxide synthase in synovium of rheumatoid arthritis]

OBJECTIVES: To examine the localization and distribution of endothelial nitric oxide synthase (eNOS) and inducible nitric oxide synthase (iNOS), which participate in nitric oxide (NO) production, in synovium of rheumatoid arthritis (RA). MATERIALS AND METHODS: Immunohistochemical analysis for eNOS and iNOS in synovial tissues obtained from 10 patients with RA who were underwent total knee replacement. Synovial tissues of osteoarthritis (OA) were used as control. The percentage of cells that were positive for eNOS and iNOS was estimated in five hundred endothelial cells, synovial lining cells and interstitial cells, respectively. And mRNA expression of NOS was confirmed by in situ hybridization. In addition, to test NO production, nitration of tyrosines was assessed by immunohistochemistry. RESULTS: Not only endothelial cells but also synovial lining cells and interstitial cells exhibited immune-reactive both eNOS and iNOS. Cells which were seemed immune-reactive eNOS and iNOS expressed nitrotyrosin. By in situ hybridization, we detected mRNA expression for eNOS and iNOS. CONCLUSIONS: Endothelial cells, synovial lining cells and interstitial cells expressed both eNOS and iNOS with high frequency in RA synovium compared with OA synovium. It seemed to correlate with NO production. These results suggest that expression of iNOS may be involved in the induction of arthritis and eNOS may be participated in augmentation of inflammation in RA.     

一氧化氮及其合酶在哮喘发病机制中的作用

探讨一氧化氮及其合酶在哮喘发病机制中的作用。方法 采用哮喘豚鼠模型，将豚鼠分为４组；１．哮喘组，用１０％卵白蛋白腹腔注射１ｍｌ致敏，２周后用１％卵白蛋白超声雾化吸入致其哮喘发作．２；肾上腺皮质激素预防组；诱喘同哮喘组，在每次诱喘前腹腔滴注地塞米松０．５ｍｇ／ｋｇ。３．硝基精氨酸甲酯预防组；诱喘同哮喘组，每次诱喘产腹腔注射ＬＮＮＡ０．４ｍｇ／ｋｇ。４．正常对照组；用生理盐水代替诱喘剂。每组分别测定其     

Inducible nitric oxide synthase contributes to ventilator-induced lung injury

RATIONALE: Inducible nitric oxide synthase (iNOS) has been implicated in the development of acute lung injury. Recent studies indicate a role for mechanical stress in iNOS and endothelial NOS (eNOS) regulation. OBJECTIVES: This study investigated changes in lung NOS expression and activity in a mouse model of ventilator-induced lung injury. METHODS: C57BL/6J (wild-type [WT]) and iNOS-deficient (iNOS(-/-)) mice received spontaneous ventilation (control) or mechanical ventilation (MV; VT of 7 and 20 ml/kg) for 2 hours, after which NOS gene expression and activity were determined and pulmonary capillary leakage assessed by the Evans blue albumin assay. RESULTS: iNOS mRNA and protein expression was absent in iNOS(-/-) mice, minimal in WT control mice, but significantly upregulated in response to 2 hours of MV. In contrast, eNOS protein was decreased in WT mice, and nonsignificantly increased in iNOS(-/-) mice, as compared with control animals. iNOS and eNOS activities followed similar patterns in WT and iNOS(-/-) mice. MV caused acute lung injury as suggested by cell infiltration and nitrotyrosine accumulation in the lung, and a significant increase in bronchoalveolar lavage cell count in WT mice, findings that were reduced in iNOS(-/-) mice. Finally, Evans blue albumin accumulation in lungs of WT mice was significant (50 vs. 15% increase in iNOS(-/-) mice compared with control animals) in response to MV and was prevented by treatment of the animals with the iNOS inhibitor aminoguanidine. CONCLUSION: Taken together, our results indicate that iNOS gene expression and activity are significantly upregulated and contribute to lung edema in ventilator-induced lung injury.     

乙醇诱导脂肪肝过程中肝组织一氧化氮合酶的表达及作用

目的　观察乙醇诱导脂肪肝过程中肝组织一氧化氮合酶 (NOS)的表达 ,探讨不同亚型NOS表达与脂质过氧化的关系。方法　在大鼠饮水中加入乙醇建立乙醇性脂肪肝模型 ,采用免疫组化和逆转录多聚酶链反应 (RT PCR)的方法动态观察肝组织中NOS的蛋白及基因表达 ,同时检测肝组织中一氧化氮 (NO)、丙二醛 (MDA)含量。结果　成功建立乙醇性脂肪肝大鼠模型。与正常对照组相比 ,造模大鼠肝组织在第 4周时诱导型一氧化氮合酶 (iNOS)表达已显著增加 (P <0 .0 5 ) ,第 12周达高峰 ,而后有所下降 ,第 2 0周时又明显上升 (P <0 .0 1)。内皮型一氧化氮合酶 (eNOS)在第 4周时表达无明显改变 (P >0 .0 5 ) ,随造模时间延长而逐渐降低 (P <0 .0 5 )。肝组织中NO水平在第 4周时无明显升高 (P>0 .0 5 ) ,以后显著增加 ,第 12周达高峰 ,而后有所下降 ,第 2 0周时又明显上升 (P <0 .0 1)。肝组织中MDA含量在第 4周已显著升高 (P <0 .0 5 ) ,随饮用乙醇时间延长进行性增加。肝组织中NO、MDA含量与iNOS表达成正相关 (P <0 .0 1) ,与eNOS表达成负相关 (P <0 .0 1)。结论　乙醇诱导脂肪肝过程中肝组织NO水平升高主要与iNOS的活化有关 ,iNOS产生的NO可能与脂质过氧化损伤有关 ,而eNOS产生的NO可能起抗脂质过氧化损伤的保护作用。     

不同HO-1表达水平对糖尿病模型大鼠血管舒张功能及NOS的影响

目的 探讨改变血红素加氧酶-1(HO-1)表达水平对糖尿病(DM)大鼠血管舒张功能的影响及与一氧化氮合酶(NOS)/一氧化氮(NO)的关系.方法 以链脲佐菌素(STZ)诱导DM大鼠模型.SD大鼠分成4组:对照组、DM组、正铁血红素(HO-1诱导剂)组、锌原卟啉(HO-1抑制剂)组.应用离体血管张力检测技术观察胸主动脉舒张功能变化;RT-PCR法及比色法分别检测血管组织和血清中诱生型NOS(iNOS)及内皮型NOS(eNOS)的表达和NO含量.结果 与DM组相比,正铁血红素组血管环对乙酰胆碱舒张百分率有所提高,而锌原卟啉组血管舒张反应继续下降.应用正铁血红素可在提高DM大鼠血管和血清eNOS表达的同时降低iNOS/NO表达;而锌原卟啉组血清中iNOS活性及其在血管组织表达均增高.结论 提高HO-1的表达水平有益于改善DM大鼠血管舒张反应失调,这种保护作用与抑制iNOS/NO的生成、上调eNOS表达水平有关.     

Atorvastatin causes depressor and sympatho-inhibitory effects with upregulation of nitric oxide synthases in stroke-prone spontaneously hypertensive rats

OBJECTIVE: Recent studies have suggested that statins decrease blood pressure in hypertensive animals and upregulate endothelial nitric oxide synthase (eNOS) expression. However, the effects of statins on the expression of nitric oxide synthase (NOS) in the brain and the sympathetic nervous system remain to be elucidated. The aim of this study was thus to examine the effects of atorvastatin on blood pressure, sympathetic nerve activity, and the expression of NOS in stroke-prone spontaneously hypertensive rats (SHRSP) as well as in Wistar-Kyoto (WKY) rats. METHODS: The animals received atorvastatin (50 mg/kg per day) for 30 days. Systolic blood pressure and heart rate were evaluated using the tail-cuff method. Urinary norepinephrine excretion was measured for 24 h. The expression of eNOS, neuronal NOS (nNOS), and inducible NOS (iNOS) in the brain (cortex, cerebellum, hypothalamus and brainstem), aorta and heart were determined by Western blot analysis. RESULTS: Systolic blood pressure and 24-h urinary norepinephrine excretion were significantly decreased in SHRSP, but not in WKY, after the treatment with atorvastatin. The eNOS and iNOS expression in the brain and aorta was significantly increased in atorvastatin-treated SHRSP and WKY. However, the nNOS expression in the brain was not altered in the atorvastatin-treated group. CONCLUSIONS: These results suggest that atorvastatin decreases blood pressure, at least in part via the reduction of sympathetic nervous system activity in SHRSP. They also suggest that this sympatho-inhibitory effect may be mediated by an increase in NO production, with the upregulation of eNOS expression in the brain.     

Endothelial,but not the inducible,nitric oxide synthase is detectable in normal and portal hypertensive rats

BACKGROUND: Chronic portal hypertension is accompanied by a nitric oxide (NO) dependent vasodilation. Three isoforms of NO producing synthases (NOS) are characterized: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Sources of increased NO levels in chronic hypertension is disputed. METHODS: To determine eNOS and iNOS expression in different organs of portal hypertensive and control rats, we divided Sprague-Dawley rats in 6 groups: (1). Partial portal vein ligated rats, (2). Bile duct ligated rats, (3). Carbon tetrachloride treated rats, (4). Sham operated rats, (5). Untreated control rats, and (6). LPS treated rats. Immunohistochemistry (IHC) and immunoblotting (IB) using antibodies against eNOS or iNOS were carried out on samples from thymus, aorta, heart, lung, oesophagus, liver, spleen, kidney, pancreas, small and large intestine. RESULTS: IHC revealed an even eNOS expression in all groups. Expression of iNOS was restricted to macrophages in organs of LPS treated and the thymus of rats. IB mirrored these results. CONCLUSION: In chronic portal hypertension, the main source for NO production depends on eNOS activity.     

Cystic fibrosis lung disease: the role of nitric oxide

This review summarizes current knowledge about the role of nitric oxide (NO) in cystic fibrosis (CF) lung disease. NO is endogenously produced by a group of enzymes, the NO synthases (NOSs). There are three isoforms of NOS, each encoded by different genes: neuronal (nNOS), immune or inducible (iNOS), and endothelial (eNOS) nitric oxide synthase.(1) They all form NO and L-citrulline by enzymatic oxidation of L-arginine. This reaction requires a number of cosubstrates, including molecular oxygen and tetrahydrobiopterin. It is now known whether all three isoenzymes are constitutively expressed in cells of the respiratory tract and that their gene expression is inducible.(2,3) NO production by iNOS, the "high-output" NOS, is stimulated by bacterial lipopolysaccharide (LPS) as well as proinflammatory cytokines such as interleukin (IL)-1gamma, IL-2, interferon (IFN)-gamma, and tumor necrosis factor (TNF). In contrast to nNOS and eNOS, activation of iNOS does not require an increase in intracellular Ca(2+) concentration.     

Caloric restriction improves endothelial dysfunction during vascular aging: Effects on nitric oxide synthase isoforms and oxidative stress in rat aorta

Aging is characterized by activation of inducible over endothelial nitric oxide synthase (iNOS and eNOS), impaired antioxidant activity and increased oxidative stress, which reduces nitric oxide bioavailability and causes endothelial dysfunction. Caloric restriction (CR) blunts oxidative stress. We investigated whether CR impacts endothelial dysfunction in aging and the underlying mechanisms. Aortas from young (YC, 6 months of age) and old (OC, 24 months of age) rats ad-libitum fed and from old rats caloric-restricted for 3-weeks (OR, 26%) were investigated. Endothelium-dependent vasorelaxation was impaired in OC, associated with reduced eNOS and increased iNOS expression (P < 0.05). Aortic nitrite was similar in OC and YC, but the contribution of calcium-independent NOS to total NOS activity was increased whereas that of calcium-dependent NOS was reduced (p ≤ 0.0003). Plasma thiobarbituric acid-reactive substances (TBARS) were elevated in OC as well as aortic nitrotyrosine (P < 0.05). Expression of manganese superoxide dismutase (MnSOD) and total SOD activity were impaired in OC (P < 0.05 vs. YC), whereas copper-zinc (CuZn) SOD expression was similar in OC and YC. CR restored endothelial dysfunction in old rats, reduced iNOS expression, total nitrite and calcium-independent NOS activity in aorta (P < 0.05) without changes in eNOS expression and calcium-dependent NOS activity. Sirtuin-1 expression did not differ among groups. Plasma TBARS and aortic nitrotyrosine were reduced (P < 0.05) in OR compared with OC. In OR CuZnSOD protein and SOD activity increased (P < 0.05) without changes in MnSOD expression. Short-term CR improves age-related endothelial dysfunction. Reversal of altered iNOS/eNOS ratio, reduced oxidative stress and increased SOD enzyme activity rather than enhanced NO production appear to be involved in this effect.     

Role of nitric oxide in posthypoxic contractile dysfunction of diabetic cardiomyopathy

We investigated the role of nitric oxide synthase (NOS) in the contractile dysfunction of diabetic cardiomyopathy, comparing streptozotocin-treated (60 mg/kg) diabetic Wistar rats with matched non-diabetic controls. Isolated isovolumic heart function was studied during normoxia and in response to brief hypoxia-reoxygenation. Diabetic hearts had significantly lower left-ventricular pressure and slower isovolumic relaxation than controls (relaxation time constant, T 40.2+/-2.3 vs. 27.7+/-0.9 ms; P<0.05) and a blunted response to hypoxia. These abnormalities were unaffected by NOS inhibition. Upon reoxygenation after brief hypoxia, diabetic hearts exhibited substantial worsening of LV relaxation compared to normal hearts (T 69.1+/-3.3 vs. 56.6+/-7.9 ms; P<0.05). This post-hypoxic diastolic dysfunction was significantly attenuated either by the non-selective NOS inhibitor L-NAME, the iNOS inhibitor L-NIL, or the reactive-oxygen-species (ROS) scavenger thiourea. Only diabetic hearts expressed iNOS protein, whereas eNOS expression was similar in both groups. In conclusion, diabetic hearts exhibit markedly abnormal post-hypoxic relaxation, which is attributable to both ROS and NO derived from iNOS.     

The presence of citrulline in salivary glands is evidence that nitric oxide is mediator of inflammation in Sjögren acinar epithelia

Aim: The present study addresses the question whether nitric oxide (NO) plays a role in inflammation of salivary glands in Sjögren's syndrome. Methods: The expression of inducible nitric oxide synthase (iNOS) and the presence of citrulline, as a monitor of NO activity were studied by immunohistochemistry, in minor salivary glands from 24 patients with primary Sjögren syndrome. An equal number of control tissues were included. The presence of mRNA of eNOS (endothelial NOS) and iNOS in tissues was studied by fluorescent in situ hybridization. Results: All salivary glands displayed eNOS along the ductal epithelia, blood vessels and acini. In Sjögren disease iNOS enzyme was widely expressed along ductal epithelia, acini and in foci of lymphocyte infiltration; therefore by the extensive citrulline presence and iNOS, we infer that NO is related to inflammation. Control biopsies were negative for iNOS and citrulline. Conclusion: The present data suggest that local production of NO should contribute to salivary gland inflammation in Sjögren disease.     

Effect of chronic insulin treatment on NO production and endothelium-dependent relaxation in aortae from established STZ-induced diabetic rats

The hypothesis that the impaired endothelial function seen in streptozotocin (STZ)-induced diabetic rats may result from an increased nitric oxide (NO) metabolism was tested. Acetylcholine (ACh) increased the nitrite NO(2-) and nitrate (NO(3-)) levels in the perfusates from both control and diabetic aortic strips, although the level of NO(2-) was significantly lower in diabetic rats while the NO(3-) level was significantly higher. Both effects (decrease in NO(2-) and increase in NO(3-)) were ameliorated by chronic administration of insulin to diabetic rats but NOx (NO(2-) plus NO(3-)) was increased. The expression of endothelial nitric oxide synthase (eNOS) was significantly increased by chronic administration of insulin to diabetic rats. A decrease in NO(2-) and an increase in NO(3-) occurred following treatment of control aortae with hypoxanthine/xanthine oxidase. Incubating diabetic aortic strips with superoxide dismutase (SOD) normalized the production of both NO(2-) and NO(3-). Both the basal and the ACh-stimulated production of O(2)(-) were significantly higher in diabetic rats than in controls. These results demonstrate that the ACh-induced relaxation of aortic strips was significantly impaired in diabetic rats and that this impairment may be due to an abnormal oxidative metabolism of NO, rather than to a decrease in NOS mRNA and NO production.     

KMUP-1 inhibits hypertension-induced left ventricular hypertrophy through regulation of nitric oxide synthases,ERK1/2, and calcineurin

Hypertension can induce left ventricular hypertrophy (LVH), and the nitric oxide (NO) pathway plays an important role in the pathogenesis of cardiac hypertrophy. This study aimed to examine whether KMUP-1, a novel xanthine-based derivative, could inhibit LVH in spontaneously hypertensive rats (SHRs) and to investigate potential mechanisms underlying its antihypertrophic effects. Two groups of animals with chronic or subacute LVH were treated. In the chronic LVH group, KMUP-1 (10 or 30 mg/kg/d orally) was administered for 28 days to both normotensive rats and SHRs. In the subacute LVH group, KMUP-1 (0.5 mg/kg/d intraperitoneally) or sildenafil (0.7 mg/kg/d intraperitoneally) was administered for 10 days with or without co-treatment with the nitric oxide synthase (NOS) inhibitor N-omega-nitro-l-arginine (L-NNA; 20 mg/L orally). After treatment, the effects of KMUP-1 or sildenafil on hypertension, cardiac hypertrophy, survival, expression of the NO/soluble guanylate cyclase (sGC)/protein kinase G (NO/sGC/PKG) pathway in the aorta andleft ventricle, and calcineurin A/extracellular signal-regulated kinase 1/2 (ERK1/2) signaling in the left ventricle were examined. In the chronic LVH group, the SHRs developed hypertension with LVH over the 28 days. KMUP-1 attenuated the hypertension and LVH, increased survival rate, enhanced endothelial NOS/cyclic guanosine monophosphate/PKG (eNOS/cGMP/PKG) and decreased inducible NOS (iNOS) expression in the aorta and left ventricle of the SHRs. In the subacute LVH group, both KMUP-1 and sildenafil administered for 10 days attenuated the LVH in SHRs, with enhanced eNOS/cGMP/PKG and suppressed iNOS/calcineurin A/ERK1/2 expression in the left ventricle. In addition, both KMUP-1 and sildenafil attenuated L-NNA-induced LVH. KMUP-1 inhibition of hypertension-induced LVH with associated upregulation of eNOS, downregulation of iNOS in both the aorta and left ventricle, and attenuation of calcineurin A and ERK1/2 signaling in the left ventricle.     

Endothelial,but not the inducible,nitric oxide synthase is detectable in normal and portal hypertensive rats

Abstract: Background: Chronic portal hypertension is accompanied by a nitric oxide (NO) dependent vasodilation. Three isoforms of NO producing synthases (NOS) are characterized: neuronal NOS (nNOS), endothelial NOS (eNOS) and inducible NOS (iNOS). Sources of increased NO levels in chronic hypertension is disputed. Methods: To determine eNOS and iNOS expression in different organs of portal hypertensive and control rats, we divided Sprague-Dawley rats in 6 groups: (1) Partial portal vein ligated rats, (2) Bile duct ligated rats, (3) Carbon tetrachloride treated rats, (4) Sham operated rats, (5) Untreated control rats, and (6) LPS treated rats. Immunohistochemistry (IHC) and immunoblotting (IB) using antibodies against eNOS or iNOS were carried out on samples from thymus, aorta, heart, lung, oesophagus, liver, spleen, kidney, pancreas, small and large intestine. Results: IHC revealed an even eNOS expression in all groups. Expression of iNOS was restricted to macrophages in organs of LPS treated and the thymus of rats. IB mirrored these results. Conclusion: In chronic portal hypertension, the main source for NO production depends on eNOS activity.     

Chronic administration of aminoguanidine reduces vascular nitric oxide production and attenuates liver damage in bile duct-ligated rats.

BACKGROUND: Nitric oxide (NO) has been implicated in the pathogenesis of liver cirrhosis. This study investigated the activity of nitric oxide synthase (NOS) in cirrhosis induced by bile duct-ligation (BDL) with NOS inhibitors. METHOD: Three days after operation, rats were randomized to receive aminoguanidine (AG, 25 mg/kg/day) or L-N(G)-nitro-L-arginine methyl ester (L-NAME, 10 mg/kg/day) for 21 days. RESULTS: Vascular NO production, which was increased in BDL cirrhotic rats, was reduced by 75% with AG but not L-NAME chronic administration. AG treatment attenuated liver damage, while L-NAME aggravated it. AG significantly suppressed inducible NOS (iNOS) expression in aorta of BDL rats at both mRNA and protein level, but much less efficient in reducing it in liver. In contrast, endothelial NOS (eNOS) expression was not markedly affected. Calcium-independent NOS activity, which was dramatically increased in aorta of BDL rats, was abolished by AG treatment. In liver, however, both calcium-dependent and -independent NOS activity were increased by AG treatment. CONCLUSION: Chronic administration of AG could reduce systemic NO levels as well as suppress iNOS expression and activity in aorta of BDL rats. It also improved liver function, possibly because of its ability to increase hepatic NOS activity, and to correct the systemic hemodynamic disorders by decreasing vascular NO production.     

Role of nitric oxide (NO) in pulmonary dysfunction associated with experimental cirrhosis

We examined the functional role of nitric oxide (NO) and nitric oxide synthase (NOS) isoforms in the pulmonary dysfunction seen in cirrhosis. Lungs were isolated from control and carbon tetrachloride (CCl(4))-induced cirrhotic rats and perfused at constant flow with a whole blood mixture. Ventilation with hypoxic gas resulted in attenuated hypoxic pulmonary vasoconstriction (HPV) in lungs from cirrhotic animals. Administration of the non-selective NOS inhibitor N-omega-Nitro-L-Arginine (L-NNA) resulted in HPV responses that were not different between groups. However, inhibition of inducible nitric oxide synthase (iNOS) did not restore cirrhotic HPV responses. Lungs from cirrhotic rats demonstrated enhanced endothelial-dependent vasodilation to vasopressin when preconstricted with hypoxia but not when preconstricted with thromboxane mimetic. Western blot analysis failed to demonstrate differences in pulmonary endothelial NOS (eNOS) or iNOS levels between groups. Our data suggest that, while NO may play a role in mediating the reduced pulmonary vasoreactivity observed in cirrhosis, other vasoactive factors are likely also important modulators of the pulmonary dysfunction seen in this disease.