辛伐他汀对脂多糖诱导肺损伤大鼠的一氧化氮合酶的影响

目的 探讨辛伐他汀对脂多糖(LPS)诱导急性肺损伤大鼠的一氧化氮合酶(NOS)的影响.方法 30只SD大鼠随机分为对照组、LPS组和辛伐他汀治疗组,治疗组又分1 h、3 d和7 d组,每组6只.治疗组大鼠在实验前经胃管分别给予辛伐他汀10 mg/kg(4 ml/kg)治疗1 d,3 d,7 d,每天1次;对照组和LPS组则给予蒸馏水(4 ml/kg),每天1次,连续7 d.在最后一次给药1 h后,LPS组和治疗组大鼠从尾静脉注射LPS 5 mg/kg(2.5 ml/kg),对照组则予注射无菌生理盐水(2.5 ml/kg).观察6 h,处死大鼠,取样,比色法测定血清和肺匀浆诱导型一氧化氮合酶(iNOS)、构成型一氧化氮合酶(cNOS)和一氧化氮(NO)水平,免疫组化法检测肺组织iNOS和内皮型一氧化氮合酶(eNOS)的表达.结果 LPS组血清和肺匀浆NO均高于对照组(p<0.001),治疗组则低于LPS组(P<0.05);LPS组血清和肺匀浆iNOS活力均高于对照组而cNOS活力则降低(P<0.05),治疗组肺匀浆iNOS活力显著低于LPS组而eNOS显著升高(P<0.001);免疫组化显示:LPS组肺组织iNOS表达强于对照组,而eNOS表达则显著减弱,治疗组的iNOS表达弱于LPS组,eNOS表达则显著增强,与其减轻肺损伤相关.结论 辛伐他汀可逆转LPS诱导的肺组织iNOS活性的升高和eNOS活性的下降,减轻内毒素性肺损伤.     

Rapid up-regulation of endothelial nitric-oxide synthase in a mouse model of Escherichia coli lipopolysaccharide-induced bladder inflammation

Increases in the signaling molecule nitric oxide (NO) during inflammation may be linked not only to inducible nitric-oxide synthase (iNOS) but also to endothelial (e)NOS. Escherichia coli lipopolysaccharide (LPS) induces an inflammatory response in the bladder and rapidly increases phosphorylation of Akt/protein kinase B (Akt), a key enzyme regulating proliferation, apoptosis, and inflammation. Activated Akt phosphorylates human eNOS at serine 1177 and subsequently increases NOS activity. Because Akt and eNOS are both localized in the bladder urothelium, phosphorylation of eNOS by Akt provides an attractive mechanism for rapid increases in urinary NO production. Female mice were intraperitoneally injected with LPS (25 mg/kg) or pyrogen-free water (control). Four hours before LPS injection, some mice were injected with wortmannin, which inhibits Akt phosphorylation. Levels of urinary cyclic GMP, a downstream product of NO, increase 75% within 1 h after intraperitoneal injection of LPS, and this increase is blocked by wortmannin. Bladder eNOS and phosphorylated eNOS protein increase 94 and 151%, respectively, 1 h after LPS treatment, whereas iNOS was not detected. Wortmannin decreases eNOS phosphorylation by 60%. Furthermore, bladder Ca(2+)-dependent NOS activity (eNOS, neuronal NOS) is increased 79 +/- 20% 1 h after LPS treatment, whereas there is no increase in Ca(2+)-independent (iNOS) activity (n = 4). Increases in urinary cyclic GMP, NOS activity, and eNOS protein and phosphorylation 1 h after induction of inflammation with LPS, indicate that eNOS plays a role in the early response to bladder inflammation.     

The modulation of hepatic injury and heat shock expression by inhibition of inducible nitric oxide synthase after hemorrhagic shock.

The role of nitric oxide (NO) in maintaining homeostasis and regulating organ function during hemorrhagic shock is complex. The inducible NO synthase (iNOS) has been hypothesized to play a critical role in the pathophysiologic consequences of severe hemorrhage. Heat shock protein (HSP) expression is increased by hemorrhage and is a marker of the magnitude of ischemic injury in the liver. HSP induction is protective against injury in animal models of inflammation and is regulated by NO in hepatocytes. To clarify the role of iNOS in hepatic injury and its relationship to HSP expression in hemorrhagic shock, NOS was inhibited with L-N-6-(1-iminoethyl) lysine (L-NIL), which is reported to be a selective inhibitor of the inducible NOS isoform. Doses of 50 microg/kg or 150 microg/kg were infused over 1 h at the end of compensated shock. Plasma ornithine carbamoyltransferase (OCT), a specific marker of liver injury, was significantly reduced after hemorrhage with low-dose L-NIL (7.1+/-1.5 IU/L) compared to saline-treated control rats (13.0+/-1.5 IU/L, P < 0.005), while high-dose L-NIL significantly increased OCT release (35.9+/-7.2 IU/L, P< 0.05 versus shock alone) despite a greater MAP after resuscitation. HSP expression (HSP-72 and HSP-32) after hemorrhage was increased by L-NIL treatment at the highest dose. We conclude that excessive NO production from iNOS contributes to shock-induced hepatic injury. Our data suggest HSP expression may reflect the degree of ischemic injury after hemorrhage.     

Effect of NOS inhibition on rat gastric matrix metalloproteinase production during endotoxemia

Matrix metalloproteinases (MMPs) degrade the extracellular matrix and contribute to LPS-induced gastric injury. MMPs are closely modulated by their activators, membrane type-MMP (MT-MMPs) and their endogenous inhibitors, the tissue inhibitors of metalloproteinases (TIMPs). As LPS-induced gastric injury is mediated in part by iNOS, and NO modulates MMP production in vitro, we hypothesized that NOS inhibition would similarly modulate LPS-induced gastric MMP production. Therefore, the purpose of these studies was to compare the effects of selective and nonselective NOS inhibition on LPS-induced gastric MMP production. METHODS: Sprague-Dawley rats were given either the nonselective NOS inhibitor NG-nitro-L-arginine methyl ester (L-NAME; 5 mg/kg, s.c.), a selective iNOS inhibitor, aminoguanidine (45 mg/kg, i.p.) or L-N-iminoethyl-lysine (L-NIL; 10 mg/kg, i.p.), or vehicle 15 min before saline or LPS (20 mg/kg, i.p.) and killed 24 h after LPS administration. Stomachs were assessed for macroscopic injury (computed planimetry), and gastric mucosal MMP production was assessed by gelatin zymography, in situ zymography, and Western analysis for MMP-2, MT1-MMP, and TIMP-2. (n > or = 4/group; ANOVA). RESULTS: Aminoguanidine treatment decreased LPS-induced macroscopic gastric injury as well as MMP-2 and MT1-MMP protein production while having no effect on TIMP-2 protein levels. L-NIL similarly attenuated the induction of MMP-2 and MT1-MMP by LPS. L-NAME failed to attenuate LPS induced gastric injury or MT1-MMP protein induction and increased MMP-2 levels. L-NAME similarly had no effect on gastric TIMP-2 production. CONCLUSIONS: Selective iNOS inhibition decreases gastric MMP-2 activity after LPS administration, whereas nonselective inhibition increases MMP-2 levels. The ability of selective iNOS inhibition to ameliorate LPS-induced gastric injury may be due in part to its inhibition of active MMP-2 production, whereas nonselective NOS inhibitors increase MMP-2 levels and maintain gastric injury after LPS administration.     

Presence of nitrotyrosine with minimal inducible nitric oxide synthase induction in lipopolysaccharide-treated pigs

The production of large amounts of nitric oxide (NO) by the inducible form of nitric oxide synthase (iNOS) and the subsequent production of peroxynitrite (OONO-) are believed to be major factors in the hemodynamic abnormalities of sepsis. This finding is based on data from rats and mice but has not been established in other species. Therefore, we examined the role of iNOS in lipopolysaccharide (LPS)-treated pigs, which have a hemodynamic pattern with sepsis that is more similar to humans than rats. Pigs were anesthetized, ventilated, and given LPS (n = 12), 20 microg/kg over 2 h, or saline (n = 7). They were killed after 2 (n = 8 LPS, 7 control) or 4 h (4 LPS). We measured cardiac output (CO), mean arterial (Part), and pulmonary and central venous pressures. We evaluated NO production by measuring expired NO, and plasma nitrate/nitrite concentration, NOS activity (in lung tissue), and iNOS protein by Western analysis, and immunohistochemistry (lung and liver), as well as iNOS mRNA by Northern analysis (liver and lung). We also measured nitrotyrosine as evidence of OONO- production by slot blot, Western analysis, and immunohistochemistry. By 2 h, Part fell and CO did not change so that systemic vascular resistance decreased from 21.5+/-2.9 to 12.7+/-3.1 mmHg x L(-1) x min (P < 0.05) and remained at 11.3+/-1.7 mmHg x L(-1) x min in the animals observed for 4 h. Plasma nitrate/nitrite, expired NO, and NOS activity did not change. We found no iNOS in tissues by Western analysis with 5 different antibodies but detected a small amount of iNOS by immunohistochemistry in inflammatory cells and small vessels. There was a small increase in iNOS mRNA in liver and lung. Despite the minimal increase in iNOS, nitrotyrosine was increased in small vessels and in inflammatory cells. In conclusion, caution should be used when extrapolating the septic response in rodents to other species, for the pattern of iNOS induction is very different.     

Ultrasound at 27 kHz increases tissue expression and activity of nitric oxide synthases in acute limb ischemia in rabbits

Transcutaneous low-frequency ultrasound (US) preserves myocardial and skeletal muscle viability by increasing tissue perfusion through an undefined nitric oxide (NO)-dependent mechanism. We have examined whether US increases tissue expression and activity of the three nitric oxide synthase (NOS) isoforms: endothelial (eNOS), neuronal (nNOS) and inducible (iNOS). The two femoral arteries of four New Zealand rabbits were ligated for a total of 120 min. After 60 min of ligation, transcutaneous low-frequency US (27 kHz, 0.13 W/cm2) was applied for 60 min to one thigh, while the contra-lateral artery served as a control (total ischemia time=120 min). Calcium-dependent (cNOS) and -independent (ciNOS) NOS activity, and concentration of total eNOS, ser-1177 phosphorylated eNOS (P-eNOS), nNOS and iNOS were then determined in the gracilis muscle. Compared with the control, US application significantly increased cNOS activity [3.34+/-0.28 versus 3.87+/-0.10x1000 counts per minute (cpm), respectively, p=0.031] and ciNOS activity (1.99+/-0.09 versus 3.26+/-0.68 cpm, respectively, p<0.001). Western immunoblotting revealed a significant increase in protein content of both iNOS (184.5+/-1.08%; p<0.0001) and P-eNOS (381.5+/-2.47%; p<0.001), with only a small increase in total eNOS and nNOS expression. In conclusion, application of transcutaneous low-frequency US to ischemic muscular tissue significantly increases both cNOS and ciNOS activity by increasing eNOS phosphorylation and iNOS expression, respectively.     

Increased blood pressure in rats after long-term inhibition of the neuronal isoform of nitric oxide synthase.

In the kidney, nitric oxide synthase (NOS) of the neuronal isoform (nNOS) is predominantly located in the macula densa cells. Unspecific chronic NOS inhibition in rats leads to elevated blood pressure (P(A)), associated with increased renal vascular resistance. This study was designed to examine the effect of chronic selective inhibition of nNOS with 7-nitro indazole (7-NI) on P(A), GFR, and the tubuloglomerular feedback (TGF) system. P(A) was repeatedly measured by a noninvasive tail-cuff technique for 4 wk in rats treated orally with 7-NI, and in control rats. After treatment, the animals were anesthetized and renal excretion rates, GFR, and TGF activity were determined. After 1 wk of 7-NI treatment P(A) was increased from 129+/-4 to 143 2 mmHg. GFR (1.85+/-0.1 vs. 1.97+/-0.2 ml/min in controls) was unchanged, but micropuncture studies revealed a more sensitive TGF than in controls. After 4 wk of 7-NI treatment P(A) was 152+/-4 mmHg, but no change in GFR (1.90+/-0.5 ml/min) or TGF sensitivity was detected. Acute administration of 7-NI to nontreated rats did not affect P(A), but decreased GFR (1.49+/-0.1 ml/min) and increased TGF sensitivity. In conclusion, chronic nNOS inhibition leads to increased P(A). Our results suggest that the elevated P(A) could be caused by an initially increased TGF sensitivity, leading to decreased GFR and an increased body fluid volume.     

Dual inhibition of nitric oxide and prostaglandin production contributes to the antiinflammatory properties of nitric oxide synthase inhibitors.

We have recently put forward the hypothesis that the dual inhibition of proinflammatory nitric oxide (NO) and prostaglandins (PG) may contribute to the antiinflammatory properties of nitric oxide synthase (NOS) inhibitors. This hypothesis was tested in the present study. A rapid inflammatory response characterized by edema, high levels of nitrites (NO2-, a breakdown product of NO), PG, and cellular infiltration into a fluid exudate was induced by the administration of carrageenan into the subcutaneous rat air pouch. The time course of the induction of inducible nitric oxide synthase (iNOS) protein in the pouch tissue was found to coincide with the production of NO2-. Dexamethasone inhibited both iNOS protein expression and NO2- synthesis in the fluid exudate (IC50 = 0.16 mg/kg). Oral administration of N-iminoethyl-L-lysine (L-NIL) or NG-nitro-L-arginine methyl ester (NO2Arg) not only blocked nitrite accumulation in the pouch fluid in a dose-dependent fashion but also attenuated the elevated release of PG. Finally, carrageenan administration produced a time-dependent increase in cellular infiltration into the pouch exudate that was inhibited by dexamethasone and NOS inhibitors. At early times, i.e., 6 h, the cellular infiltrate is composed primarily of neutrophils (98%). Pretreatment with colchicine reduced both neutrophil infiltration and leukotriene B4 accumulation in the air pouch by 98% but did not affect either NO2- or PG levels. In conclusion, the major findings of this paper are that (a) selective inhibitors of iNOS are clearly antiinflammatory agents by inhibiting not only NO but also PG and cellular infiltration and (b) that neutrophils are not responsible for high levels of NO and PG produced.     

八肽缩胆囊素对脂多糖诱导血管内皮细胞诱生型一氧化氮合酶表达变化的抑制作用

目的探讨八肽缩胆囊素（CCK-8）对脂多糖（LPS）诱导血管内皮细胞诱生型一氧化氯合酶（iNOS）表达变化的影响。方法培养人脐静脉内皮细胞株ECV-304细胞。用0．01、0．1和1mg／L LPS处理2～24h，用生理盐水、10mol／LCCK-8和0．1mg／L LPS＋10^-8、10^-7、10^-8mol／L CCK-8处理16h；用比色法检测培养液中一氧化氮（NO）含量、细胞NOS活性，免疫细胞化学及蛋白质免疫印迹法检测iNOS蛋白表达。结果与生理盐水处理的对照组比较，LPS诱导培养液NO含量增多、细胞NOS活性增高、iNOS蛋白表达上调；CCK-8剂量依赣性抑制LPS的上述效应。而单独作用对iNOS蛋白表达、NOS活性和NO含量均无明显影响。结论CCK-8可以明显抑制LPS引起ECV-304细胞iNOS蛋白表达上调。减少NO生成。     

Increased cardiac endothelial nitric oxide synthase expression in patients taking angiotensin-converting enzyme inhibitor therapy

BACKGROUND: The efficacy of angiotensin-converting enzyme (ACE) inhibitors has been demonstrated in large clinical trials, but knowledge of the underlying mechanisms remains incomplete. Therefore, this study investigated the impact of ACE inhibitor therapy on cardiac nitric oxide (NO) synthases in patients with coronary artery disease (CAD) or heart failure. PATIENTS AND METHODS: The mRNA expression was quantified by standard calibrated competitive RT-PCR, protein expression by Western blotting and NOS activity by monitoring the conversion of [3H]arginine to [3H]citrulline during enzymatic formation of NO in tissue homogenates of myocardium of patients with, or without, ACE inhibitor treatment before elective coronary artery bypass grafting or heart transplantation. RESULTS: The mRNA expression (amol microg(-1) RNA) of endothelial NO synthase (eNOS) was higher (22.5 +/- 4.8, n = 23) in the atrial myocardium of patients taking ACE inhibitor treatment, before elective coronary artery bypass grafting, compared with patients not taking this therapy (8.9 +/- 0.7, n = 33, P < 0.0001). The ACE inhibitor therapy increased eNOS protein expression from [(9 +/- 0.7) relative units (RUs) to (12 +/- 0.9) RUs, P < 0.05, respectively] and cardiac NOS activity from 17.6 +/- 1.3 to 23.7 +/- 1.1 pmol mg protein(-1) min(-1) (P < 0.001, respectively). Inducible and neuronal NO synthase expression was not changed by the ACE inhibition. A similar up-regulation of eNOS by ACE inhibition was found in the left ventricles of patients with heart failure. The augmented endothelial NOS expression and activity was not the result of differences in clinical characteristics and concomitant therapy between the patient groups. CONCLUSION: Increased eNOS expression and activity might contribute to the beneficial effects of ACE inhibitor therapy in the treatment of CAD and heart failure.     

Induction of diaphragmatic nitric oxide synthase after endotoxin administration in rats: role on diaphragmatic contractile dysfunction.

Nitric oxide (NO), a free radical that is negatively inotropic in the heart and skeletal muscle, is produced in large amounts during sepsis by an NO synthase inducible (iNOS) by LPS and/or cytokines. The aim of this study was to examine iNOS induction in the rat diaphragm after Escherichia Coli LPS inoculation (1.6 mg/kg i.p.), and its involvement in diaphragmatic contractile dysfunction. Inducible NOS protein and activity could be detected in the diaphragm as early as 6 h after LPS inoculation. 6 and 12 h after LPS, iNOS was expressed in inflammatory cells infiltrating the perivascular spaces of the diaphragm, whereas 12 and 24 h after LPS it was expressed in skeletal muscle fibers. Inducible NOS was also expressed in the left ventricular myocardium, whereas no expression was observed in the abdominal, intercostal, and peripheral skeletal muscles. Diaphragmatic force was significantly decreased 12 and 24 h after LPS. This decrease was prevented by inhibition of iNOS induction by dexamethasone or by inhibition of iNOS activity by N(G)-methyl-L-arginine. We conclude that iNOS was induced in the diaphragm after E. Coli LPS inoculation in rats, being involved in the decreased muscular force.     

Expression of nitric oxide synthase,aquaporin 1 and aquaporin 5 in rat after bleomycin inhalation

OBJECTIVE: Nitric oxide (NO) and aquaporins (AQPs) are believed to play an important role in the pathogenesis of pulmonary inflammation and edema. The aim of this study was to investigate the role of NO synthase (NOS) and AQP in acute lung injury (ALI) lung following bleomycin inhalation in rats. DESIGN AND SETTING: A prospective controlled trial in a university research laboratory. ANIMALS AND INTERVENTIONS: Sprague-Dawley rats were treated by inhalation of 10 U/kg bleomycin hydrochloride in 5 ml of normal saline. Control rats were treated with 5 ml normal saline alone. The animals (6-8 rats per group) were killed on days 4, 7 or 14. MEASUREMENTS AND RESULTS: We analyzed the change in expression of inducible NOS (iNOS), neuronal NOS (nNOS), endothelial NOS (eNOS), aquaporin 1 (AQP1) and aquaporin 5 (AQP5) over time by Western blot. Nitrate and nitrite concentrations were measured in bronchoalveolar lavage fluid (BALF) using a modified Griess reaction. The nitrite and nitrate concentrations in BALF from rats 4 days after bleomycin exposure were greater than those from saline-treated rats. Immunoblotting studies demonstrated increased levels of eNOS in the rat lung at 4, 7 and 14 days and iNOS at 7 and 14 days after bleomycin inhalation. However, nNOS expression was unaltered. Although AQP1 expression was decreased in rats at 4 days, AQP5 expression was increased at 4, 7 and 14 days. CONCLUSIONS: This study demonstrates that NO metabolites increase along with eNOS and iNOS expression during the acute exudative phase in ALI, and that AQP and NOS are regulated independently in bleomycin-induced pulmonary edema.     

Modulation of hypoxic pulmonary vasoconstriction is time and nitric oxide dependent in a peritonitis model of sepsis

Objective This study assessed modulation of hypoxic pulmonary vasoconstriction (HPV) in isolated perfused rat lungs during sepsis induced by cecal ligation and perforation (CLP) at different times and its relationship to nitric oxide synthases (NOS).Design and setting Prospective controlled trial in a university research laboratory.Subjects 102 male Sprague-Dawley rats.Interventions Groups 1–3 received sham laparotomy 6 h before lung isolation: group 1, only laparotomy; group 2, concurrently l-N ⁶-(1-iminoethyl)-lysine (L-NIL, 3 mg/kg); group 3, concurrently N -nitro-l-arginine methylester (L-NAME, 5 mg/kg). Groups 4–6 received CLP 6 h before lung isolation: group 4, only CLP; group 5, concurrently L-NIL; group 6, concurrently L-NAME. The same experiments were carried out with sham and CLP treatment for 24 h (groups 7–12). Exhaled NO from rats lungs was measured after anesthesia and tracheostomy. After the pulmonary circuit was isolated and perfused, angiotensin II (0.1 µg) was injected into the inflow tract. The lungs were ventilated with the hypoxic mixture (HPV, 3% O₂) for 10 min and then again with the normoxic mixture (21% O₂) for an equal period. Changes in perfusion pressure were measured. Endothelial (eNOS) and inducible NOS (iNOS) expression of the lungs was determined.Measurements and results Treatment with L-NAME but not L-NIL increased HPV in sham lungs. HPV was unaltered after CLP 6 h and decreased after CLP 24 h compared to sham. In CLP animals eNOS protein expression was reduced whereas iNOS expression was increased compared to sham animals. Exhaled NO, reflecting NOS activity was twice as high in the CLP 24 h group than in the CLP 6 h group.Conclusions In the CLP sepsis model modulation of HPV was time-dependent. In addition, vasoconstriction to hypoxic stimuli was dependent on NOS activity.L.G. F. is supported by Innovative Medizinische Forschung Münster, Germany (Fi-1-2000-4)     

Autocrine inhibition of Na+/K(+)-ATPase by nitric oxide in mouse proximal tubule epithelial cells.

An inducible nitric oxide synthase has recently been described in proximal tubule epithelium. To investigate the effects of proximal tubule NO on Na+/K(+)-ATPase, we induced NO production in mouse proximal tubule epithelial cells by treatment with lipopolysaccharide (LPS) and interferon-gamma (IFN gamma) followed by determinations of ouabain-sensitive ATPase activity. Na+/K(+)-ATPase activity decreased after 4 h of LPS/IFN gamma treatment, reaching maximal inhibition after 24 h (34% reduction in activity). The inhibition of Na+/K(+)-ATPase activity by LPS/IFN gamma was prevented by simultaneous incubation with N omega-nitro L-arginine and markedly blunted by removal of L-arginine from the medium. The NO donors sodium nitroprusside and SIN-1 also inhibited Na+/K(+)-ATPase activity to a similar extent than LPS/IFN gamma. However, treatment with 8-pCPT-cGMP only modestly reduced Na+/K(+)-ATPase activity. Interestingly, superoxide dismutase prevented the inhibitory effects of NO on Na+/K(+)-ATPase activity, suggesting a role for peroxynitrite in this inhibition. We conclude that NO generated by mouse proximal tubule epithelial cell iNOS inhibits Na/K ATPase activity in an autocrine fashion and that this inhibition is accompanied by a reduction in Na-dependent solute transport.     

Burn-induced lung damage in rat is mediated by a nitric oxide/cGMP system

This study was conducted to demonstrate the burn-induced lung neutrophil deposition and damage in rats is affected by the nitric oxide (NO)-dependent downstream cGMP signaling. In experiment 1, 1H-[1,2,4] oxadiazolo [4,3-alpha] quinoxalin-1-one (ODQ) was given (20 mg/kg i.p.) to specific pathogen-free Sprague-Dawley rats immediately postburn to suppress the guanylate cyclase (GC) activity. At 8 h after burn, blood was assayed for the peroxynitrite-mediated dihydrorhodamine 123 (DHR 123) oxidation and lung tissues were harvested for myeloperoxidase (MPO) determination and histological studies. Pulmonary microvascular dysfunction was quantified by measuring the extravasations of Evans blue dye. In experiment 2, Sodium nitroprusside (SNP) was given (2 mM, i.p.) to elevate cGMP levels and ODQ (20 mg/kg, i.p.) or methylene blue (100 microM, i.p.) or saline was given. The animals were sacrificed 4 h after injection and lung tissues were harvested for iNOS mRNA study. The MPO activity in lung, blood DHR 123 oxidation level, and lung permeability increased up to 2-fold, 4-fold, and 2.5-fold after burn. Inhibition of GC by ODQ administration significantly decreased MPO activity, blood DHR 123 oxidation, and lung permeability by 55%, 66%, and 53%, respectively, and markedly decreased the thermal injury-induced perivascular and interstitial inflammatory cell infiltration and septum edema. The protective effects of ODQ were comparable to the use of selective iNOS inhibitor as demonstrated previously. Furthermore, ODQ decreased the burn or SNP-induced iNOS mRNA levels at 4 h after burn. These findings suggest that burn-induced lung dysfunction is mediated by the NO/cGMP system because it is abolished by application of either iNOS inhibitor or GC inhibitor. Also, the beneficial effect of ODQ is partly due to the attenuation of burn-induced iNOS expression by GC inhibition.     

Hypotension and reduced nitric oxide-elicited vasorelaxation in transgenic mice overexpressing endothelial nitric oxide synthase.

Nitric oxide (NO), constitutively produced by endothelial nitric oxide synthase (eNOS), plays a major role in the regulation of blood pressure and vascular tone. We generated transgenic mice overexpressing bovine eNOS in the vascular wall using murine preproendothelin-1 promoter. In transgenic lineages with three to eight transgene copies, bovine eNOS-specific mRNA, protein expression in the particulate fractions, and calcium-dependent NOS activity were confirmed by RNase protection assay, immunoblotting, and L-arginine/citrulline conversion. Immunohistochemical studies revealed that eNOS protein was predominantly localized in the endothelial cells of aorta, heart, and lung. Blood pressure was significantly lower in eNOS-overexpressing mice than in control littermates. In the transgenic aorta, basal NO release (estimated by Nomega-nitro-L-arginine-induced facilitation of the contraction by prostaglandin F2alpha) and basal cGMP levels (measured by enzyme immunoassay) were significantly increased. In contrast, relaxations of transgenic aorta in response to acetylcholine and sodium nitroprusside were significantly attenuated, and the reduced vascular reactivity was associated with reduced response of cGMP elevation to these agents as compared with control aortas. Thus, our novel mouse model of chronic eNOS overexpression demonstrates that, in addition to the essential role of eNOS in blood pressure regulation, tonic NO release by eNOS in the endothelium induces the reduced vascular reactivity to NO-mediated vasodilators, providing several insights into the pathogenesis of nitrate tolerance.     

Lipopolysaccharide-induced enterocyte-derived nitric oxide induces intestinal monolayer permeability in an autocrine fashion

Studies indicate that endotoxin (LPS) causes intestinal injury, increases inducible nitric oxide synthase (iNOS) activity, leads to increased NO production, and promotes bacterial translocation (BT). To investigate the mechanism by which LPS causes gut injury and to test the hypothesis that NO produced by enterocytes promotes gut injury in an autocrine fashion, rat intestinal epithelial cell (IEC-6) monolayers were tested. IEC-6 monolayers grown in a bicameral system were incubated with media or with LPS (25 microg/mL) and tested for permeability to phenol red, BT, and nitrate/nitrite (NO2/NO3) production. To determine the direct effect of NO on permeability, monolayers were incubated with the NO donor S-nitroso-acetylpenicillinamide (SNAP; 1 mM) and tested for permeability. Next, the protective effects of two NOS inhibitors (L-NMMA and L-NIL) were tested. Finally, to determine if LPS-induced permeability occurs via a poly (ADP-ribose) synthetase- (PARS) dependent pathway, monolayers incubated with LPS alone or with the PARS inhibitor, INH2BP (100 microM) were tested. LPS significantly increased IEC-6 permeability to phenol red, as well as increased NO2/NO3 by 20-fold (P < 0.001) and increased BT 10-fold (P < 0.001). SNAP mimicked the effect of LPS and significantly increased both permeability to phenol red and BT. Inhibition of iNOS significantly decreased the LPS-induced increase in monolayer permeability and BT (P < 0.05). Monolayers incubated with INH2BP had significantly decreased permeability to phenol red and BT, suggesting that LPS-induced NO production increases monolayer permeability at least in part via a PARS-dependent mechanism. In summary, LPS-induced disruption of monolayer barrier function appears to be related, at least in part, to enterocyte produced NO. This supports the hypothesis that NO produced by LPS-stimulated enterocytes promotes injury in an autocrine fashion and highlights the fact that enterocytes can be a target as well as a producer of NO.