Vascular endothelial growth factor/vascular permeability factor produces nitric oxide-dependent hypotension. Evidence for a maintenance role in quiescent adult endothelium

In vitro studies suggest that vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) may stimulate release of nitric oxide (NO) from endothelial cells. To investigate the hemodynamic consequences of recombinant VEGF/VPF administered in vivo, recombinant human VEGF/VPF was administered as a bolus dose of 500 micrograms to anesthetized (n = 6) or conscious (n = 5) New Zealand White rabbits, as well as anesthetized rabbits with diet-induced hypercholesterolemia (HC; n = 7). Anesthetized Yorkshire farm pigs (no specific dietary pretreatment) were studied before and after receiving 500 micrograms intravenous (IV; n = 5) or intracoronary (IC; n = 5) VEGF/VPF. In anesthetized, normal rabbits, mean arterial pressure (MAP) fell by 20.5 +/- 1.4% (P < .05 versus baseline) within 3 minutes after IV VEGF/VPF. Pretreatment with N omega-nitro-L-arginine caused a significant inhibition of VEGF/VPF-induced hypotension. In conscious, normal rabbits, VEGF/VPF produced a consistent though lesser reduction in MAP. The fall in MAP induced by VEGF/VPF in anesthetized, HC rabbits (21.5 +/- 2.5% from baseline) was no different from that observed in normal anesthetized rabbits. In pigs, both IV and IC administration of VEGF/VPF produced a prompt reduction in MAP. Heart rate increased, while cardiac output, stroke volume, left atrial pressure, and total peripheral resistance all declined to a similar, statistically significant degree in both IV and IC groups. Epicardial echocardiography disclosed neither global nor segmental wall motion abnormalities in response to VEGF/VPF. We conclude that (1) VEGF/VPF-stimulated release of NO, previously suggested in vitro, occurs in vivo; (2) this finding suggests that functional VEGF/VPF receptors are present on quiescent adult endothelium, consistent with a maintenance function for VEGF/VPF, which may include regulation of NO; and (3) the preserved response of HC rabbits suggests that endothelial cell receptors for VEGF/VPF are spared in the setting of hypercholesterolemia.     

Recycling of L-citrulline to sustain nitric oxide-dependent enteric neurotransmission

Neurons that synthesize nitric oxide from arginine produce stoichiometric amounts of citrulline. We investigated whether nitric oxide-releasing enteric neurons have the capacity to recycle citrulline to arginine and thereby sustain nitrergic neurotransmission. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity, enzymes capable of citrulline to arginine conversion, were both localized in discrete populations of myenteric and submucosal neurons in the canine proximal colon. Argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity co-localized with neuronal beta-nicotinamide adenine dinucleotide phosphate diaphorase staining, a marker for nitric oxide synthase. The functional significance of argininosuccinate synthetase-like immunoreactivity and argininosuccinate lyase-like immunoreactivity was shown by testing the effects of exogenous citrulline on responses to enteric inhibitory nerve stimulation, which were assessed by measuring contractions, inhibitory junction potentials and electrical slow waves. As shown previously, arginine analogues (L-nitroarginine methyl ester or L-nitroarginine; 100 microM) inhibited nitric oxide-dependent responses, and excess L-arginine restored inhibitory responses. Citrulline alone (0.1-2 mM) had no effect on nitrergic transmission under control conditions, but in the presence of L-nitroarginine methyl ester or L-nitroarginine, citrulline (0.1-2 mM) restored nitrergic transmission in a concentration-dependent manner. Other neutral amino acids (L-serine, L-leucine) did not mimic the effects of citrulline. Taken together, these data suggest that enteric nitrergic neurons have the enzymatic apparatus and functional capability of recycling citrulline to arginine.     

Kinetic isotope effects and electron transfer in the reduction of xanthine oxidoreductase with 4-hydroxypyrimidine. A comparison between oxidase and dehydrogenase forms

Isolated from bovine milk, xanthine oxidase (XO) and xanthine dehydrogenase (XDH) are two interconvertible forms of the same protein, differing in the number of protein cysteines versus cystines. Most differences between XO and XDH are localized to the FAD center, the site at which the oxidizing substrates NAD and molecular oxygen react. A comparative study of the reduction of XO and XDH has been performed to assess differences in reactivity of the molybdopterin site, as well as subsequent electron-transfer events from molybdenum to 2Fe/2S and FAD centers. The compound 4-hydroxypyrimidine (4-OH-P) was chosen as reducing substrate because its higher Km value raised the possibility of binding weak enough to measure kinetically, and its high kcat value could allow detection of intramolecular electron-transfer reactions. As measured by stopped flow spectrophotometry, XO and XDH react with the first equivalent of 4-OH-P via similar mechanisms, differing in the magnitude of rate and dissociation constants. Using [2-2H]4-OH-P as substrate, a D(k/Kd) isotope effect of 1.9 to 2.3 suggests that movement of the hydrogen abstracted from substrate appreciably limits the rate of initial enzyme reduction from Mo(VI) to Mo(IV). Monitoring the visible spectrum of the enzymes, the first observed step is reduction of a single 2Fe/2S center and presumably re-oxidation of Mo(IV) to Mo(V). This suggests a common pathway for electron transfer involving reduction of a 2Fe/2S center prior to reduction of the second 2Fe/2S and FAD centers. Rates of the first electron transfer from molybdenum to the 2Fe/2S center are rapid, 290 s-1 with XO and 180 s-1 with XDH, and are consistent with rates measured by flash photolysis (Walker, M. C., Hazzard, J. T., Tollin, G., and Edmondson, D. E. (1991) Biochemistry 30, 5912-5917) allowing discrete observation of the electron-transfer reactions that occur during turnover. This step also exhibits a modest primary kinetic isotope effect of 1.5 to 1.6 when [2-2H]4-OH-P is used, possibly due to deprotonation of the molybdenum center prior to electron transfer. A second one-electron transfer, presumably oxidizing Mo(V) to Mo(VI), follows in a step coincident with product dissociation, consistent with a role for product release in controlling electron transfer events. The kinetics of this complex system are described and interpreted quantitatively in models that are consistent with all the data.     

Reduced intracellular oxidative metabolism promotes firm adhesion of human polymorphonuclear leukocytes to vascular endothelium under flow conditions

The interaction of polymorphonuclear leukocytes (PMN) with the vascular endothelium and their subsequent extravasation to the tissues is a key step during different physiological and pathological processes. In certain of these pathologies the oxygen tension becomes very low, leading to reduced cellular oxidative status. To evaluate the effect of lowering the intracellular redox status in the interaction of PMN with the endothelium, exposure to hypoxic conditions as well as treatment with different antioxidant agents was carried out. PMN exposure to hypoxia enhanced beta2 integrin-dependent adhesion to intercellular adhesion molecule-1-coated surfaces, concomitant with a decrease in the intracellular redox status of the cell. As occurs with hypoxia, treatment with antioxidants produced a decrease in the oxidation state of PMN. These agents enhanced adhesion of PMN to human umbilical vein endothelial cells stimulated with tumor necrosis factor-alpha (TNF-alpha), and this effect was also mediated by beta2 integrins LFA-1 and Mac-1. Adhesion studies under defined laminar flow conditions showed that the antioxidant treatment induced an enhanced adhesion mediated by beta2 integrins with a decrease in the fraction of PMN rolling on TNF-alpha-activated endothelial cells. The up-regulated PMN adhesion was correlated to an increase in the expression and activation of integrin Mac-1, without loss of L-selectin surface expression. Altogether, these results demonstrate that a reduction in the intracellular oxidative state produces an enhanced beta2 integrin-dependent adhesion of PMN to stimulated endothelial cells under conditions of flow.     

Tautomerism of xanthine and alloxanthine: a model for substrate recognition by xanthine oxidase

Tautomerism of neutral xanthine and alloxanthine has been examined both in the gas phase and in aqueous solution. The tautomeric preference in the gas phase has been studied by means of semiempirical and ab initio quantum-mechanical computations with inclusion of correlation effects at the M?ller-Plesset level, and from density-functional calculations. The influence of solvent on the relative stability between tautomers has been estimated from self-consistent reaction field calculations performed with different models. The results provide a detailed picture of tautomerism for these biologically relevant purine bases. The functional implications in the recognition by xanthine oxidase are analyzed from inspection of the interaction patterns of the most stable tautomeric forms. A model for the recognition of these purine derivatives in the enzyme binding site is discussed.     

Effects of aminosalicylates and immunosuppressive agents on nitric oxide-dependent N-nitrosation reactions

Recent studies have demonstrated that nitric oxide (NO) rapidly and spontaneously decomposes in oxygenated solutions to generate potent N-nitrosating agents. These electrophilic substances have been shown to mediate mutagenesis and carcinogenesis via the formation of aliphatic and aromatic nitrosamines. We have also demonstrated that extravasated neutrophils and macrophages produce significant amounts of N-nitrosating agents derived exclusively from NO. During the course of these studies, we found that certain antioxidants, including 5-aminosalicylic acid (5-ASA), inhibited the leukocyte-mediated N-nitrosation reaction. Because 5-ASA and other anti-inflammatory and immunosuppressive drugs are used to treat inflammatory bowel disease, we wondered if any of these other compounds might also modulate N-nitrosation reactions in vitro. Therefore, the objectives of this study were to assess the ability of aminosalicylates and certain immunosuppressive agents to inhibit NO-dependent N-nitrosation of a model aromatic amine (2,3-diaminonaphthalene) and to determine whether this inhibitory activity correlated with their oxidation potential. We found that the concentrations necessary to inhibit the N-nitrosation reaction by 50% (IC50) were 25, 50 and 100 microM for 5-ASA, olsalazine (dimeric 5-ASA) and sulfasalazine, respectively. In contrast, sulfapyridine, 4-ASA, N-acetyl-5-ASA, 6-mercaptopurine, azathioprine, and methotrexate were either much less effective or inactive at inhibiting the N-nitrosation reaction. Although 5-ASA was able to fully scavenge the stable free radical 1,1-diphenyl-2-picrylhydrazyl, neither olsalazine nor sulfasalazine was found to be effective at scavenging this weak oxidant. We did find that olsalazine possessed an oxidation potential substantially less than that of sulfasalazine, suggesting that it may, in fact, scavenge more potent oxidizing agents such as the N-nitrosating agent. We conclude that 5-ASA and olsalazine inhibit NO-dependent N-nitrosation reactions by scavenging or decomposing the nitrosating agent(s). We propose that the secondary nitrogen unique to sulfasalazine interacts with the nitrosating agent to yield a secondary nitrosamine, thereby competing for N-nitrosation of our detector.     

Generation of nitric oxide by a nitrite reductase activity of xanthine oxidase: a potential pathway for nitric oxide formation in the absence of nitric oxide synthase activity

The effects of substrate, L-Arg and cofactors, (6R)-L-erythro-5,6,7,8-tetrahydrobiopterin (H4B) and calmodulin (CaM), on chiral discrimination by rat neuronal nitric oxide synthase (nNOS) for binding the enantiomers of 1-(1-naphthyl)ethylamine (ligand I), 1-cyclohexylethylamine (ligand II), and 1-(4-pyridyl)ethanol (ligand III) were studied under anaerobic conditions by optical absorption spectroscopy. The ratio of the dissociation constant (Kd) values for the S- and R-enantiomers of ligand I (S/R) was 30, while the S/R ratio for ligand II and the R/S ratio for ligand III were 1.8 and < 0.14, respectively, in the presence of 0.15 microM H4B. However, in the presence of 1 mM L-Arg, the S/R ratio of the Kd values for ligand I was decreased down to 5.9. In the presence of both 1 mM L-Arg and 0.1 mM H4B, the S/R ratios for ligands I and II and the R/S ratio for ligand III were enormously increased up to 29, > 80, and 60, respectively. These and other spectral observations strongly suggest that strict chiral recognition at the active site of nNOS during catalysis is exhibited only in the presence of the active effector.     

Functions and dysfunctions of the vascular endothelium

Endothelium represents a large paracrine gland with an enormous reactive surface. By means of its numerous vasodilation and vasospastic factors it manages the basal and working tonus of vessels and thus also the regional flow and the access of target tissues to hormones and metabolic substrates. It manages also the proliferation and migration of myocytes of the vascular wall and thus its adaptation to overload. The dysfunctional states of endothelium are observed in arterial hypertensions, diabetes, dyslipoproteinaemia, and they grow with age. They are the first stage of atherothrombogenic processes. They manifest themselves by a decreased vasodilation reserve of the vascular wall to strain, insulin and many other stimuli. On the contrary, quite frequently they paradoxically react to physical strain, acetylcholine, histamine, ATP etc. by vascular spasms which can determine vasospastic and microvascular angina pectoris including spasms and occlusions of e.g. coronary arteries in sites of insignificant stenoses with the origin of infarctions. The damaged endothelium, so to explain, conceives these stimuli in accordance with the encoded programme as a stimulus to the protection from haemorrhage during stress (fight or flight) and develops "suicidal" defensive reaction against them which we are nowadays able to modulate by administration of ACE-inhibitors, beta-blockers, hypolipidaemic drugs, inhibitors of cyclooxygenase-1 (30-100 mg of aspirin), Ca-antagonists and antioxidants including numerous non-pharmacological procedures. We can retard or halt the process of atherothrombogenesis and avoid or lower thus the number of sudden vascular ventricular as well as brain episodes, including the congestive heart failures, limb amputations and ischaemic damage of the brain. (Fig. 4, Ref. 70.)     

Enhancement of oxidative cell injury and antitumor effects of localized 44 degrees C hyperthermia upon combination with respiratory hyperoxia and xanthine oxidase

Polymerase chain reaction (PCR) assays were designed to amplify 56- and 99-base regions of the pmoA gene from Methylosinus trichosporium OB3b and Methylomicrobium albus BG8, two species of methanotrophic bacteria that are of interest for monitoring bioremediation activity. The PCR product sizes are in a mass range that is accessible to analysis by MALDI-TOF mass spectrometry. A rapid purification procedure using commercially available reversed-phase cartridges was applied prior to MALDI-TOF analysis. A small aliquot (1.5%, 1.5 microL) from a single 100-microL PCR reaction was sufficient for reliable detection. No cross-amplification products were observed when primers designed for one bacterial species were used with genomic DNA of the other species. The methodology described here has potential to allow less expensive and faster characterization of the ability of microbial populations to destroy pollutants in groundwater and soil at contaminated industrial sites.     

Role of the hepatic xanthine oxidase in thyroid dysfunction: effect of thyroid hormones in oxidative stress in rat liver

Temporary vocal fold immobility resolving in 4 weeks or less is considered a transient immobility. Many different disorders may lead to this type of laryngeal motion impairment. It is important for otolaryngologists to be familiar with the differential diagnosis for transient vocal fold immobility in order to optimize the management of these unusual cases.     

Fever-range hyperthermia enhances L-selectin-dependent adhesion of lymphocytes to vascular endothelium

The L-selectin leukocyte adhesion molecule plays an important role in controlling leukocyte extravasation in peripheral lymph nodes and at sites of tissue injury or infection. Although febrile responses during infection and inflammation are associated with enhanced immune activity, the contribution of fever-range temperatures to controlling lymphocyte recruitment to tissues has not been previously examined. In this report we provide evidence that direct exposure of lymphocytes to fever-range temperatures (38-41 degrees C) in vitro for 9 to 24 h resulted in a >100% increase in L-selectin-dependent adhesion of these cells to lymph node high endothelial venules (HEV). Moreover, culture of lymphocytes under hyperthermia conditions markedly enhanced the ability of these cells to traffic in an L-selectin-dependent manner to peripheral lymph nodes, mesenteric lymph nodes, and Peyer's patches. In contrast, febrile temperatures did not increase LFA-1 function as assessed by measuring lymphocyte adhesion to ICAM-1-3T3 transfectants. Fever-range hyperthermia further did not increase L-selectin surface density on lymphocytes or L-selectin-dependent recognition of soluble carbohydrate substrates; however, a marked increase in ultrastructural immunogold-labeling of L-selectin was observed in response to thermal stimuli. These results suggest that elevated temperatures enhance L-selectin adhesion and/or avidity through the regulation of L-selectin conformation or organization in the plasma membrane. Finally, the observed thermal effects on L-selectin adhesion were attributed to soluble factors in the conditioned medium of heat-treated cells. Taken together, these data provide new insight into the potential physiologic role of the febrile response in enhancing lymphocyte recruitment to tissues through the regulation of L-selectin adhesion.     

Role of xanthine oxidase and eicosanoids in development of pancreatic ischemia-reperfusion injury

The implication of different eicosanoids and oxygen free radicals in the development of pancreatic injury after an ischemia-reperfusion process has been evaluated. For this purpose we have compared the effect of allopurinol and indomethacin administration on the pancreatic levels of eicosanoids in a rat model of pancreatic ischemia-reperfusion. After 60 min of pancreatic ischemia and 2 h of reperfusion, significant increases in 6-keto-PGF1 alpha, PGE2, and LTB4 in pancreas tissue were detected. Allopurinol before the ischemic period reduced 6-keto-PGF1 alpha, PGE2, and LTB4 levels to the range of basal values, while prior indomethacin treatment significantly reduced 6-keto-PGF1 alpha and PGE2 levels, with LTB4 remaining unmodified. Increased postischemic plasma lipases were also significantly reduced by allopurinol to the range of sham-operated animals whereas indomethacin did not modify these levels. The data suggest a role for lipoxygenase metabolites in the development of pancreatic injury and the importance of the enzyme xanthine oxidase as an inductor of eicosanoid biosynthesis.     

Effect of glutathione depletion on the conversion of xanthine dehydrogenase to oxidase in rat liver

The ability of endogenous glutathione (GSH) to modify the activity of the enzyme xanthine oxidase (XO) in rat liver was investigated. The effect of hepatic GSH depletion on the conversion of xanthine dehydrogenase (XDH) (EC 1.1.1.204) to XO (EC 1.1.3.22) was determined 10 min after i.p. administration of different amounts of diethylmaleate to fasted rats. After administration of 400 mg/kg, total hepatic non-protein GSH (reduced + oxidized GSH) decreased significantly to 14% of controls. In this condition the level of oxidized GSH was unchanged and no lipid peroxidation was observed, while a significant increase of reversible XO and a minor increase of the irreversible form of the enzyme was detected.     

Role of gender and vascular endothelium in rat aorta response to 17 beta-estradiol

Estrogen supplementation in postmenopausal women offers significant cardiovascular protection. The mechanism for this benefit is unclear but may be due to an interaction of estrogen with the blood vessel wall (vascular smooth muscle and endothelium). We examined the response of weight-matched female and male endothelium-intact and -denuded aortae to 17 beta-estradiol, its interaction with noradrenaline, and the effect of N-nitro-L-arginine. Estradiol produced relaxation responses that were significantly greater in female endothelium-intact preparations. This response was sensitive to N-nitro-L-arginine, while the response to 17 beta-estradiol in male endothelum-intact and both female and male endothelum-denuded preparations was resistant. Estradiol also inhibited contractions to noradrenaline, which was more pronounced in the female endothelium-intact aortic rings. These data imply that estradiol interacts preferentially with the female vascular endothelium, but there exists an endothelium-independent process that can also be activated in the male aorta. Further studies are warranted to elucidate these differential mechanisms.     

Interaction of thrombin and platelets with the vascular endothelium

Thrombin is a potent stimulus for prostacyclin (PGI2) release from the vascular endothelium. Treatment of the endothelium with high concentrations of aspirin to block PGI2 formation was associated with increased platelet adherence in a system employing thrombin and 51Cr-labeled platelets. Addition of exogenous PGI2 to the aspirin-treated endothelium restored platelet adherence to the low baseline values. After an initial exposure of the endothelium to thrombin, the cultured endothelium was unable to respond to a second thrombin stimulus with release of PGI2. During this refractory period, the absence of PGI2 was associated with increased platelet adherence. Thus thrombin, an active coagulant and thrombogenic substance, has the capability to release PGI2, the most potent inhibitor of platelet aggregation known to exist in vivo.     

Toxicological effects of beryllium on platelets and vascular endothelium

The crystal structure of rabbit muscle pyruvate kinase complexed with Mn2+, K+, and pyruvate revealed a binding site of K+ [T. M. Larsen, L. T. Laughlin, H. M. Holden, I. Rayment, and G. H. Reed (1994) Biochemistry 33, 6301-6309]. Sequence comparisons of rabbit muscle pyruvate kinase and pyruvate kinases from Corynebacterium glutamicum and Escherichia coli, which do not exhibit a requirement for activation by monovalent cations, indicate that the only substitutions in the K+ binding site are conservative. Glu 117 in the rabbit muscle enzyme, which is close to the K+ site, is, however, replaced by Lys in these two bacterial pyruvate kinases. The proximity of Glu 117 to K+ in the structure of the rabbit enzyme and conservation of the binding site in the bacterial enzymes which lack a dependence on monovalent cations suggested that a protonated epsilon-amino group of Lys 117 in these bacterial enzymes may provide an "internal monovalent cation." Site-specific mutant forms of the rabbit enzyme corresponding to E117K, E117A, E117D, and E117K/K114Q pyruvate kinase were examined to test this hypothesis. The E117K pyruvate kinase exhibits 12% of the activity of the fully activated wild-type enzyme but is > 200-fold more active than the wild-type enzyme in the absence of activating monovalent cations. Moreover, the activity of E117K pyruvate kinase exhibits no stimulation by monovalent cations in the assay mixtures. Both E117A and E117D pyruvate kinases retain activation by monovalent cations but have reduced activities relative to wild type. The results are consistent with the hypothesis that pyruvate kinases that do not require activation by monovalent cations supply an internal monovalent cation in the form of a protonated epsilon-amino group of Lys. The results also support the assignment of the monovalent cation in the active site of pyruvate kinase.     

玉米须中抑制黄嘌呤氧化酶活性组分的筛选及其作用

目的:提取玉米须中具有抑制黄嘌呤氧化酶(XOD)作用的活性组分,为进一步开发治疗痛风的中药新药提供依据.方法:通过测定黄嘌呤-黄嘌呤氧化酶体系产生尿酸和超氧离子的生成量,得出玉米须不同溶剂提取物(乙醚提取物、乙酸乙酯提取物、正丁醇提取物和水提取物)及不同溶剂(水和10%、30%、50%、70%、90%乙醇)纯化得到的活性组分对XOD活性抑制率,以确定活性组分,并将其与阳性对照药别嘌醇进行XOD活性半数抑制率(IC50)比较.结果:乙酸乙酯提取物XOD活性抑制率最高,为(25.60±1.01)%;与其他各组比较,经50%乙醇纯化得到的活性组分XOD活性抑制率最高,为(60.90±1.27)%.与阳性药别嘌醇进行比较,通过测定尿酸生成量得出50%乙醇纯化得到的活性组分对XOD的IC50为6.00 mg·L-1,别嘌呤醇对XOD的IC50为1.10 mg·L-1;而通过测定超氧离子生成量得出50%乙醇纯化得到的活性组分对XOD的IC50为4.50 mg·L-1,别嘌呤醇对XOD的IC50为1.05 mg·L-1.结论:玉米须中抑制XOD的活性组分为经50%乙醇纯化得到的活性组分.     

In vitro oxidation of 6-MP and its metallo complexes by xanthine oxidase

Xanthine oxidase oxidized pure 6-MP, and its bismuth complex to 6-thiouric acid, in vitro. The enzyme did not oxidize the palladium complex of 6-MP under identical conditions.     

Anterograde signaling by nitric oxide: characterization and in vitro reconstitution of an identified nitrergic synapse

Nitric oxide (NO) is recognized as a signaling molecule in the CNS where it is a candidate retrograde neurotransmitter. Here we provide direct evidence that NO mediates slow excitatory anterograde transmission between the NO synthase (NOS)-expressing B2 neuron and an NO-responsive follower neuron named B7nor. Both are motoneurons located in the buccal ganglia of the snail Lymnaea stagnalis where they participate in feeding behavior. Transmission between B2 and B7nor is blocked by inhibiting NOS and is suppressed by extracellular scavenging of NO. Furthermore, focal application of NO to the cell body of the B7nor neuron causes a depolarization that mimics the effect of B2 activity. The slow interaction between the B2 and B7nor neurons can be re-established when the two neurons are cocultured, and it shows the same susceptibility to NOS inhibition and NO scavenging. In cell culture we have also examined spatial aspects of NO signaling. We show that before the formation of an anatomical connection, the presynaptic neuron can cause depolarizing potentials in the follower neuron at distances up to 50 micro(m). The strength of the interaction increases when the distance between the cells is reduced. Our results suggest that NO can function as both a synaptic and a nonsynaptic signaling molecule.     

Upregulation of xanthine oxidase by lipopolysaccharide, interleukin-1, and hypoxia. Role in acute lung injury

LPS and selected cytokines upregulate xanthine dehydrogenase/xanthine oxidase (XDH/XO) in cellular systems. However, the effect of these factors on in vivo XDH/XO expression, and their contribution to lung injury, are poorly understood. Rats were exposed to normoxia or hypoxia for 24 h after treatment with LPS (1 mg/kg) and IL-1beta (100 microg/kg) or sterile saline. Lungs were then harvested for measurement of XDH/XO enzymatic activity and gene expression, and pulmonary edema was assessed by measurement of the wet/dry lung weight ratio (W/D). Although treatment with LPS + IL-1beta or hypoxia independently produced a 2-fold elevation (p < 0. 05 versus exposure to normoxia and treatment with saline) in lung XDH/XO activity and mRNA, the combination of LPS + IL-1beta and hypoxia caused a 4- and 3.5-fold increase in these values, respectively. XDH/XO protein expression was increased 2-fold by hypoxia alone and 1.3-fold by treatment with LPS + IL-1beta alone or combination treatment. Compared with normoxic lungs, W/D was significantly increased by exposure to hypoxia, LPS + IL-1beta, or combination treatment. This increase was prevented by treatment of the animals with tungsten, which abrogated lung XDH/XO activity. In conclusion, LPS, IL-1beta, and hypoxia significantly upregulate lung XDH/XO expression in vivo. The present data support a role for this enzyme in the pathogenesis of acute lung injury.