Enhanced pause correlates with airway neutrophils and airway-epithelial injury in asthmatic mice treated with dexamethasone

Objective: To investigate the correlations among airway inflammation, airway epithelial injury and airway hyperresponsiveness (AHR) in asthmatic mice treated with dexamethasone. Methods: Female BALB/c mice were sensitized with intraperitoneal and hypodermic injections of ovalbumin (OVA) and aluminum on days 0, 7 and 14, challenged with OVA starting on day 21 for 10 days, and treated with dexamethasone via intraperitoneal injection starting on day 28 for 3 days. Female C57BL/6 mice were treated intranasally with house dust mite (HDM) on days 1 and 14, challenged intranasally with HDM on days 21, 23, 25, 27 and 29, and treated with sivelestat (a selective neutrophil elastase inhibitor) via intraperitoneal injection after each challenge. Following the final challenge, enhanced pause (Penh) and differential cell counts in the broncho-alveolar lavage fluid were measured and the correlations were analyzed. Results: Compared with OVA-challenged BALB/c mice, the counterpart mice treated with dexamethasone showed reduced Penh and shedding of airway epithelial cells. In addition, we found that Penh 50 (an indicator of AHR) had positive correlations with airway neutrophils and shedding of airway epithelial cells, but no correlation with eosinophils, lymphocytes or macrophages. Moreover, shedding of airway epithelial cells had positive correlations with airway neutrophils, but no correlation with eosinophils, lymphocytes or macrophages. Further, sivelestat decreased Penh 50 and shed airway-epithelial cells in HDM-challenged C57BL/6 mice. Conclusions: Collectively, our findings suggest that airway neutrophils and excessive shedding of airway epithelial cells, but not eosinophils, lymphocytes or macrophages, may be involved in AHR in asthmatic mice treated with dexamethasone.     

Myocardial reperfusion injury in neuronal nitric oxide synthase deficient mice

BACKGROUND: A substantial amount of data suggesting that endothelial cell nitric oxide synthase (eNOS) plays a cardioprotective role in animal models of ischemia-reperfusion injury has amassed. We have previously demonstrated that eNOS-deficient (-/-) mice exhibit significantly larger myocardial infarcts than do wild-type mice. Few investigations have examined the neuronal form of nitric oxide synthase in the heart. The two constitutive isoforms have been demonstrated to play differing roles in studies of cerebral ischemia-reperfusion. OBJECTIVE: To characterize the role of neuronal nitric oxide synthase (nNOS) in myocardial ischemia-reperfusion injury. METHODS: Wild-type and nNOS -/- mice were subjected to 20 min of coronary artery occlusion and 120 min of reflow. RESULTS: We found no significant difference between the two groups in terms of infarct size. Microscopic cross-sections from both groups were examined for infiltration of polymorphonuclear leukocyte. Hearts of nNOS -/- mice exhibited significantly (P < 0.05) more polymorphonuclear leukocytes than did hearts of wild-type mice. CONCLUSION: Despite the fact that eNOS plays a cardioprotective role in the ischemic-reperfused myocardium, we observed no change in size of myocardial infarcts when nNOS was genetically disrupted.     

Development of lyme arthritis in mice deficient in inducible nitric oxide synthase

Nitric oxide (NO) is a powerful antimicrobial agent and an important regulatory molecule of the innate immune response. To determine if NO has a role in experimental Lyme disease, arthritis-resistant DBA/2J and arthritis-susceptible C3H/HeJ mice were bred to be genetically deficient for inducible NO synthase (iNOS). Following footpad injection of Borrelia burgdorferi, arthritis was similar between iNOS-deficient and control animals regardless of their genetic background. Histologic examination and arthritis severity scores of ankles revealed no differences in arthritis development between iNOS-deficient and control animals. Despite being deficient in a key antimicrobial agent, iNOS-deficient mice had tissue levels of B. burgdorferi similar to those in control mice. Thus, NO does not have a critical role in susceptibility to Lyme arthritis through tissue damage via an overexuberant inflammatory response, nor is it required in resistance through the clearance of spirochetes from tissues.     

Role of inducible nitric oxide synthase in trinitrobenzene sulphonic acid induced colitis in mice

BACKGROUND: Studies using inhibitors of nitric oxide synthase (NOS) to date are inconclusive regarding the role of inducible NOS (iNOS) in intestinal inflammation. AIMS: (1) To examine the role of iNOS in the development of chronic intestinal inflammation; (2) to identify the cellular source(s) of iNOS. METHODS: Colitis was induced by an intrarectal instillation of trinitrobenzene sulphonic acid (TNBS, 60 mg/ml, 30% ethanol), in wild type (control) or iNOS deficient mice. Mice were studied over 14 days; the colons were scored for injury and granulocyte infiltration was quantified. Blood to lumen leakage of (51)Cr-EDTA was measured as a quantitative index of mucosal damage. RESULTS: At 24 and 72 hours, iNOS deficient mice had significantly increased macroscopic inflammation compared with wild type mice. Granulocyte infiltration increased significantly at 24 hours and remained elevated in iNOS deficient mice at 72 hours, but significantly decreased in controls. However, by seven days post-TNBS macroscopic damage, microscopic histology, granulocyte infiltration, and mucosal permeability did not differ between wild type and iNOS deficient mice. A four- to fivefold increase in iNOS mRNA was observed in wild type mice at 72 hours and seven days post-TNBS and was absent in iNOS deficient mice. Immunohistochemistry techniques showed that iNOS expression was predominantly localised in neutrophils, with some staining also in macrophages. CONCLUSIONS: These results suggest that leucocyte derived iNOS ameliorates the early phase, but does not impact on the chronic phase of TNBS induced colitis despite the presence of iNOS.     

Effect of neutropenia on gastric mucosal integrity and mucosal nitric oxide synthesis in the rat

The present study examined the interaction between neutrophils and gastric mucosal nitric oxide (NO) synthesis in the regulation of gastric mucosal integrity in the rat. Male Sprague-Dawley rats were made neutropenic by intraperitoneal injections of methotrexate (MT; 2.5mg/kg) or antineutrophil serum (ANS; 100 l). Control rats were treated with saline. Neutropenia was confirmed by circulating neutrophil counts and tissue myeloperoxidase activity. Neutropenic or control rats were given 2 ml of ethanol (EtOH; 40% w/v intragastrically). Neutropenic rats were less susceptible to EtOH-induced mucosal damage when compared to control rats. Mucosal NO synthesis was increased in neutropenic rats. EtOH instillation reduced NO synthase in control rats. However, in MT-treated rats the reduced NO synthase activity was not different from that observed in untreated control rats. Conclusions: (1) EtOH-mediated gastric mucosal injury appears to be a neutrophil-mediated process. (2) Neutropenia results in an increase in mucosal NO synthesis. (3) MT treatment augments the degree of mucosal integrity. The increase in integrity is associated with a reduction in mucosal neutrophil infiltration as well as maintenance of NO synthase activity. ANS appears to influence mucosal integrity primarily by a reduction in circulating neutrophils.Supported by the Medical Research Council of Canada: MT6426.     

Enhanced colonic nitric oxide generation and nitric oxide synthase activity in ulcerative colitis and Crohn's disease.

Recent studies have suggested that nitric oxide (NO.), the product of nitric oxide synthase in inflammatory cells, may play a part in tissue injury and inflammation through its oxidative metabolism. In this study the colonic generation of oxides of nitrogen (NOx) and nitric oxide synthase activity was determined in ulcerative colitis and Crohn's disease. Colonic biopsy specimens were obtained from inflammatory bowel disease patients and from normal controls. Mucosal explants were cultured in vitro for 24 hours and NOx generation was determined. Nitric oxide synthase activity was monitored by the conversion of [3H]-L-arginine to citrulline. Median NOx generation by inflamed colonic mucosa of patients with active ulcerative colitis and Crohn's colitis was 4.2- and 8.1-fold respectively higher than that by normal human colonic mucosa. In ulcerative colitis and Crohn's colitis nitric oxide synthase activity was 10.0- and 3.8-fold respectively higher than in normal subjects. Colonic NOx generation is significantly decreased by methylprednisolone and ketotifen. The decrease in NOx generation by cultured colonic mucosa induced by methylprednisolone suggests that NO synthase activity is induced during the culture and the steroid effect may contribute to its therapeutic effect. Enhanced colonic NOx generation by stimulated nitric oxide synthase activity in ulcerative colitis and Crohn's disease may contribute to tissue injury.     

Enhanced gastric nitric oxide synthase activity in duodenal ulcer patients.

Nitric oxide, the product of nitric oxide synthase in inflammatory cells, may have a role in tissue injury through its oxidative metabolism. Nitric oxide may have a role in the pathogenesis of duodenal ulcer and may be one of the mechanisms responsible for the association between gastric infection with Helicobacter pylori and peptic disease. In this study, calcium independent nitric oxide synthase activity was detected in human gastric mucosa suggesting expression of the inducible isoform. In 17 duodenal ulcer patients gastric antral and fundic nitric oxide synthase activity was found to be two and 1.5-fold respectively higher than its activity in the antrum and fundus of 14 normal subjects (p < 0.05). H pylori was detected in the antrum of 15 of 17 duodenal ulcer patients and only in 7 of 14 of the control subjects. Antral nitric oxide synthase activity in H pylori positive duodenal ulcer patients was twofold higher than in H pylori positive normal subjects (p < 0.05). In duodenal ulcer patients antral and fundic nitric oxide synthase activity resumed normal values after induction of ulcer healing with ranitidine. Eradication of H pylori did not further affect gastric nitric oxide synthase activity. These findings suggest that in duodenal ulcer patients stimulated gastric mucosal nitric oxide synthase activity, though independent of the H pylori state, may contribute to the pathogenesis of the disease.     

幽门螺杆菌感染时胃黏膜一氧化氮合酶活性和脂质过氧化

目的探讨幽门螺杆菌（Ｈｐ）感染时胃黏膜一氧化氮（ＮＯ）与脂质过氧化损伤的关系。方法测定１２名正常对照、１４例Ｈｐ阴性、２２例Ｈｐ阳性慢性胃炎和２７例Ｈｐ阳性消化性溃疡患者胃窦黏膜ＮＯ合酶（ＮＯＳ）活性、铜锌超氧化物歧化酶（ＣｕＺｎＳＯＤ）和丙二醛（ＭＤＡ）含量。结果Ｈｐ阳性胃炎和溃疡患者ＮＯＳ活性和ＭＤＡ含量显著高于、ＣｕＺｎＳＯＤ含量低于Ｈｐ阴性者，溃疡ＮＯＳ活性高于Ｈｐ阳性胃炎；Ｈｐ阳性患者ＮＯＳ活性与胃炎积分和ＭＤＡ呈正相关，ＣｕＺｎＳＯＤ与ＭＤＡ呈负相关；ＮＯＳ抑制剂ＬＮＭＭＡ可显著抑制Ｈｐ阳性患者ＮＯＳ活性，而乙二醇双四乙酸对ＮＯＳ活性无影响。Ｈｐ根除后，胃炎积分、ＮＯＳ活性和ＭＤＡ含量均显著下降、ＣｕＺｎＳＯＤ含量增高。结论Ｈｐ感染时胃黏膜ＮＯＳ活性增高与来源于炎症细胞产生的诱生型ＮＯＳ增多有关；ＮＯ参与了胃黏膜脂质过氧化损伤的病理过程     

Effects of L-arginine on serum nitric oxide, nitric oxide synthase and mucosal Na^＋-K^＋-ATPase and nitric oxide synthase activity in segmental small-bowel autotransplantation model

AIM:To explore a simple method to create intestinal autotransplantation in rats and growing pigs and to investigate the effect of L-arginine supplementation on serum nitric oxide (NO), nitric oxide synthase (NOS) and intestinal mucosal NOS and Na+-K+-ATPase activity during cold ischemia-reperfusion (IR) in growing pigs. METHODS: In adult Wistar rat models of small bowel autotransplantation, a fine tube was inserted into mesenteric artery via the abdominal aorta. The superior mesenteric artery and vein were occluded. Isolated terminal ileum segment was irrigated with Ringer's solution at 4℃ and preserved in the same solution at 0-4℃ for 60 min. Then, the tube was removed and reperfusion was established. In growing pig models, a terminal ileum segment, 50 cm in length, was isolated and its mesenteric artery was irrigated via a needle with lactated Ringer's solution at 4℃. The method and period of cold preservation and reperfusion were described above. Ten white outbred pigs were randomly divided into control group and experimental group. L-arginine (150 mg/kg) was continuously infused for 15 min before reperfusion and for 30 min after reperfusion in the experimental group. One, 24, 48, and 72 h after reperfusion, peripheral vein blood was respectively collected for NO and NOS determination. At the same time point, intestinal mucosae were also obtained for NOS and Na+-K+-ATPase activity measurement. RESULTS: In adult rat models, 16 of 20 rats sustained the procedure, three died of hemorrhage shock and one of deep anesthesia. In growing pig models, the viability of small bowel graft remained for 72 h after cold IR in eight of 10 pigs. In experimental group, serum NO level at 1 and 24 h after reperfusion increased significantly when compared with control group at the same time point (152.2±61.4μmol/L /s60.8±31.6μmol/L, t=2.802, P=0.02<0.05; 82.2±24.0μmol/L vs 54.0±24.3μmol/L, t=2.490, P=0.04<0.05). Serum NO level increased significantly at 1 h post-reperfusion when compared with the same group before cold IR, 24 and 48 h post-reperfusion (152.2±61.4μmol/L vs 75.6±16.2μmol/L,t=2.820, P=0.02<0.05,82.2±24.0μmol/L,t=2.760, P= 0.03<0.05, 74.2±21.9μmol/L, t=2.822, P= 0.02<0.05). Serum NOS activity at each time point had no significant difference between two groups. In experimental group, intestinal mucosal NOS activity at 1 h post-reperfusion reduced significantly when compared with pre-cold IR (0.79±0.04 U/mg vs 0.46±0.12 U/mg, t = 3.460, P= 0.009<0.01). Mucosal NOS activity at 24, 48, and 72 h post-reperfusion also reduced significantly when compared with pre-cold IR (0.79±0.04 U/mg vs 0.57±0.14 U/mg, t= 2.380, P=0.04 <0.05, 0.61±0.11 U/mg, t= 2.309, P = 0.04<0.05, 0.63±0.12U/mg, t = 2.307, P= 0.04<0.05). In control group, mucosal NOS activity at 1 and 24 h post-reperfusion was significantly lower than that in pre-cold IR (0.72±0.12 U/mg vs 0.60±0.07 U/mg, t= 2.320, P= 0.04<0.05, 0.58±0.18 U/mg, t=2.310, P= 0.04<0.05). When compared to the normal value, Na+-K+-ATPase activity increased significantly at 48 and 72 h post-reperfusion in experimental group (2.48±0.59μmol/mg vs 3.89±1.43μmol/mg, t=3.202, P= 0.04<0.05, 3.96±0.86μmol/mg, t=3.401, P= 0.009 <0.01) and control group (2.48±0.59μmol/mg vs 3.58±0.76 μmol/mg, t=2.489, P= 0.04<0.05, 3.67±0.81μmol/mg, t= 2.542, P= 0.03<0.05). CONCLUSION: This novel technique for intestinal autotransplantation provides a potentially consistent and practical model for experimental studies of graft cold preservation. L-arginine supplementation during cold IR may act as a useful adjunct to preserve the grafted intestine.     

Responses to Leishmania donovani in mice deficient in both phagocyte oxidase and inducible nitric oxide synthase

Mice deficient in phagocyte oxidase (phox) and inducible nitric oxide synthase (iNOS), which are primary macrophage killing mechanisms, generated tissue granulomas but showed unrestrained Leishmania donovani visceral replication and suboptimal initial responsiveness to antimony treatment. Nevertheless, visceral infection was controlled post-treatment and did not recur. A phox/iNOS-independent macrophage mechanism, which was not triggered by L. donovani, emerges after chemotherapy.     

Distribution of constitutive nitric oxide synthase in the jejunum of adult rat

AIM: To study the distribution of the constitutive nitric oxidesynthase (NOS) in the jejunum of adult mt.METHODS: The distribution of endothelial NOS (eNOS) wasdetected by immunohistochemistry. Immunofluorescencehistochemical dual staining technique were used forstudying the distribution of neuronal NOS (nNOS) andeNOS. The dual stained slides were observed under aconfocal laser scanning microscopeRESULTS: Positive neuronal NOS (nNOS) and endothelialNOS (eNOS) cells were found to be distributed in laminapropria of villi, and the epithelial cell was not stained. eNOSwas mainly located in subrnucosal vascular endothelia,while nNOS was mainly situated in myenteric plexus. Somecells in the villi had both nNOS and eNOS. More than 80 %of the cells were positive for both nNOS and eNOS, the restcells were positive either for nNOS or for eNOS.CONCLUSION: The two constitutive nitric oxide synthasesam distributed differently in the jejunum of rat. nNOSdistributed in myenteric plexus is a neurotransmitter in thenon-adrenergic non-cholinergic (NANC) inhibitory nerves.eNOS distributed in endothelial and smooth muscle cells ofblood vessesels plays vasodilator role. eNOS and nNOS arecoexpressed in some cells of lamina propria of villi. NOgeneratedl by those NOS is very important in thephysiological and pathological process of small intestine.     

Distribution of constitutive nitric oxide synthase in the jejunum of adult rat

AIM：To study the distribution of the constitutive nitric oxide synthase(NOS) in the jejunom of adult rat.METHODS:The distribution of endothelial NOS(eNOS) was detected by immunohistochemistry.Immunofluorescence histochemical dual stainging technique were used for studying the distribution of neuronal NOS( nNOS) and eNOS,The dual stained slides were observed under a confocal laser scanning microscope.RESULTS:Positive neuronal NOS(nNOS) and endothelial NOS(eNOS) cells were found to be distributed in lamina propria of villi,and the epithelial cell was not stained,eNOS was mainly located in submucosal vascular endothelia while nNOS was mainly sityated in myenteric plexus.Some cells in the villi had both nNOS and eNOS.More than 80% of the cells were positive for both nNOS and eNOS,the rest cells were positive either for nNOS or for eNOS.CONCLUSION:The two constitutive nitric oxide synthases are distributed differently in the jejunum of rat.nNOS distributed in myenteric plexus is a neurotransmitter in the non-adrenergic non-cholinergic(NANC)inhibitory nerves eNOS distributed in endothelial and smooth muscle cells of blood vessels plays vasodilator role .eNOS and nNOS are coexpressed in some cells of lamina propria of villi.NO genearted y those NOS is very important in the physiological and pathological process of small intestine.     

Structure of cerebral arterioles in mice deficient in expression of the gene for endothelial nitric oxide synthase

We examined effects of pharmacological inhibition of nitric oxide synthase (NOS) and genetic deficiency of the endothelial isoform of NOS (eNOS) on structure and mechanics of cerebral arterioles. We measured pressure, diameter, and cross-sectional area (CSA) of the vessel wall (histologically) in maximally dilated cerebral arterioles in mice that were untreated or treated for 3 months with the NOS inhibitor, N(G)-nitro-L-arginine methyl ester (L-NAME; 10 mg/kg per day in drinking water). Treatment with L-NAME increased systemic arterial mean pressure (SAP; 143+/-4 versus 121+/-4 mm Hg, P<0.05) and CSA (437+/-27 versus 310+/-34 microm2, P<0.05). These findings suggest that hypertension induced in mice by NOS inhibition is accompanied by hypertrophy of cerebral arterioles. To determine the role of the eNOS isoform in regulation of cerebral vascular growth, we examined mice with targeted disruption of one (heterozygous) or both (homozygous) genes encoding eNOS. Wild-type littermates served as controls. SAP and CSA were significantly increased in homozygous (SAP, 141+/-5 versus 122+/-3 mm Hg in wild-type mice, P<0.05; CSA, 410+/-18 versus 306+/-15 microm2 in wild-type mice, P<0.05), but not in heterozygous (SAP, 135+/-4 mm Hg; CSA, 316+/-32 microm2) eNOS-deficient mice. Carotid ligation normalized cerebral arteriolar pulse pressure did not prevent increases in CSA in homozygous eNOS-deficient mice. Thus, cerebral arterioles undergo hypertrophy in homozygous eNOS-deficient mice, even in the absence of increases in arteriolar pulse pressure. These findings suggest that eNOS plays a major role in regulation of cerebral vascular growth.     

A magnetic resonance imaging study of intestinal dilation in Trypanosoma cruzi-infected mice deficient in nitric oxide synthase

Infection with Trypanosoma cruzi causes megasyndromes of the gastrointestinal (GI) tract. We used magnetic resonance imaging (MRI) to monitor alterations in the GI tract of T. cruzi-infected mice, and to assess the role of nitric oxide (NO) in the development of intestinal dilation. Brazil strain-infected C57BL/6 wild-type (WT) mice exhibited dilatation of the intestines by 30 days post-infection. Average intestine lumen diameter increased by 72%. Levels of intestinal NO synthase (NOS) isoforms, NOS2 and NOS3, were elevated in infected WT mice. Inflammation and ganglionitis were observed in all infected mice. Intestinal dilation was observed in infected WT, NOS1, NOS2, and NOS3 null mice. This study demonstrates that MRI is a useful tool to monitor intestinal dilation in living mice and that these alterations may begin during acute infection. Furthermore, our data strongly suggests that NO may not be the sole contributor to intestinal dysfunction resulting from this infection.     

Maternal endothelial nitric oxide synthase genotype influences offspring blood pressure and activity in mice

Deficiencies in maternal endothelial NO synthase (eNOS) have been associated with pregnancy complications, intrauterine growth retardation, and altered vascular function in offspring. In the present study, we investigated the influence of the maternal eNOS genotype on offspring's blood pressure, heart rate, and locomotor activity. The effect of maternal eNOS genotype was made by comparing telemetered blood pressure and activity between 2 groups of 13- to 16-week-old male heterozygous eNOS knockout mice, 1 produced by a cross of eNOS knockout (eNOS-/-) mothers and wild-type (eNOS+/+) fathers (eNOS(+/-MAT) offspring, N=11), the other by a cross of eNOS+/+ mothers and eNOS-/- fathers (eNOS(+/-PAT) offspring, N=10). Data were also collected for homozygous eNOS-/- and eNOS+/+ mice (N=15 each). Heterozygous eNOS knockout mice exhibited blood pressures that were intermediate to the eNOS+/+ and eNOS-/- groups. Relative to eNOS(+/-PAT) mice, eNOS(+/-MAT) mice exhibited significant increases in nocturnal diastolic arterial pressure and diurnal variations (dark-light difference) in systolic, mean, and diastolic arterial pressure. In addition, indices of spontaneous nocturnal locomotor activity, including both the proportion of time spent active and the intensity of activity when active, were also significantly increased. Heart rate did not differ between the groups. Our results suggest that the maternal eNOS genotype influences both blood pressure and behavior of offspring, possibly as a consequence of developmental programming associated with intrauterine growth retardation.     

幽门螺杆菌阳性胃炎患者胃粘膜-氧化氮合酶活力变化的意义

目的探讨幽门螺杆菌(Hp)感染时胃粘膜内一氧化氮合酶(NOS)活性和一氧化氮(NO)含量与细胞凋亡改变的意义.方法选择慢性活动性胃炎病人27例,其中Hp阳性者15例,Hp阴性者12例正常对照组10例.应用生物化学方法和切口末端标记法(Tunel)检测胃粘膜组织中一氧化氮产物N0-2水平、NOS活性及细胞凋亡指数.结果Hp感染时,胃粘膜NOS活性、NO-2水平及细胞凋亡指数明显升高,较Hp阴性组差异显著(P＜0.01),胃炎积分数与胃粘膜组织中NOS活性和NO-2水平呈明显正相关(r=0.66和0.84,P＜0.01).当根除Hp后,胃粘膜组织NOS活性,NO-2水平及细胞凋亡指数则显著降低,细胞凋亡指数与NOS活性和N0-2水平呈明显正相关(r=0.68和0.79,P＜0.01).结论Hp感染时可引起胃粘膜组织NOS活化,NO过量产生,细胞凋亡增加,这从又一方面说明Hp感染在胃腺癌发病机制中作用.     

Relative contributions of advanced glycation and nitric oxide synthase inhibition to aminoguanidine-mediated renoprotection in diabetic rats

Summary Advanced glycation end products (AGEs) have previously been shown to be increased in the diabetic kidney. Aminoguanidine, an inhibitor of advanced glycation, has been shown to attenuate the development of AGEs as well as the progression of renal disease in experimental diabetes. However, the precise mechanisms through which aminoguanidine acts remain to be elucidated since it is also able to act as an inhibitor of nitric oxide synthase (NOS). This study has therefore compared the effects of aminoguanidine with the effects of two other inhibitors of NOS, L -NAME and methylguanidine, on the development of experimental diabetic nephropathy. Diabetic rats were randomised to receive no treatment, aminoguanidine (1 g/l in drinking water), L -NAME (5 mg/l in drinking water) or methylguanidine (1 g/l in drinking water). Diabetic rats had increased levels of albuminuria and urinary nitrite/nitrate excretion when compared to control rats. Renal AGEs measured by fluorescence as well as by a carboxymethyllysine reactive radioimmunoassay, were elevated in diabetic rats. No changes in inducible NOS (iNOS) protein expression were detected in experimental diabetes nor did aminoguanidine affect iNOS expression. Aminoguanidine did not affect blood glucose or HbA_1c but it did prevent increases in albuminuria, urinary nitrites/nitrates and renal AGE levels as measured by fluorescence and radioimmunoassay. L -NAME and methylguanidine did not retard the development of albuminuria, nor did they prevent increases in renal AGE levels, as assessed by fluorescence. However, these treatments did prevent increases in AGEs, as measured by radioimmunoassay. This study indicates that the renoprotective effect of aminoguanidine in experimental diabetes cannot be reproduced by L -NAME or methylguanidine. It is likely that the effect of aminoguanidine is mediated predominantly by decreased AGE formation rather than via NOS inhibition. It also raises the possibility that inhibition of fluorescent AGE formation may be more renoprotective than inhibition of the formation of carboxymethyllysine-containing AGEs. [Diabetologia (1997) 40: 1141–1151] Received: 10 April 1997 and in revised form: 18 June 1997     

Aortic smooth muscle cell proliferation and endothelial nitric oxide synthase activity in fructose-fed rats

The aim of this study was to evaluate the proliferative behavior of vascular smooth muscle cells in primary culture (pC-SMC) and the endothelial nitric oxide synthase (eNOS) activity in the endothelial lining of the aorta of fructose-fed rats (FFR). This is an experimental model of syndrome X, a cluster of cardiovascular risk factors including hyperinsulinemia, insulin resistance, and hypertension that has been suggested to be of pathophysiologic importance for the development of atherosclerosis.Male Wistar rats were used: Control (n = 12) and FFR (n = 12). After receiving fructose in drinking water (10% w/v) during 8 weeks, biochemical parameters, systolic blood pressure (SBP) and relative heart weight (RHW) were determined. The proliferative effect of 10% fetal calf serum (FCS) was examined in aortic pC-SMC by [³H]thymidine incorporation and by cell counting. Ca²⁺/calmodulin-dependent NOS activity was estimated in aortic endothelial lining and in heart tissue homogenates by conversion of [³H]arginine into [³H]citrulline.Fructose-fed rats showed hyperinsulinemia (P = .0263), altered glucose tolerance test (P < .001), higher SBP (P < .0001), and RHW (P = .0145), compared to control rats. These animals also showed an increase of 10% FCS-induced [³H]thymidine incorporation (P < .0001) and cell number of aortic pC-SMC (P = .0049) and decreased eNOS activity in both aortic endothelium (P = .0147) and cardiac tissue (P < .0001).These data support the hypothesis that syndrome X is associated to changes in SMC proliferation and endothelial dysfunction, which could be involved in the onset or progression of the atherogenic process.