Lysosomal enzyme leakage during the hypoxanthine/xanthine oxidase reaction

Impairment of lysosomal stability due to reactive oxygen species generated during the oxidation of hypoxanthine by xanthine oxidase was studied in rat liver lysosomes isolated in a discontinuous Nycodenz gradient. Production of O ₂ ^⋅ and H₂O₂ during the hypoxanthine/xanthine oxidase reaction occurred for at least 5 min, while lysosomal damage, indicated by the release of N-acetyl-β-glucosaminidase, occurred within 30 s, there being no further damage to these organelles thereafter. The extent of lysosomal enzyme release increased with increasing xanthine oxidase concentration. Superoxide dismutase and catalase did not prevent lysosomal damage during the hypoxanthine/xanthine oxidase reaction. Lysosomes reduced xanthine oxidase activity, as assessed in terms of O₂ consumption, only slightly but substantially inhibited in a competitive manner the O ₂ ^⋅ -mediated reduction of cytochrome c. This inhibition was almost completely reversed by potassium cyanide, thus pointing to the presence of a cyanide-sensitive Superoxide dismutase in the lysosomal fraction. However, potassium cyanide did not affect the hypoxanthine/xanthine oxidase-mediated lysosomal damage, thus suggesting an inability of the lysosomal superoxide dismutase to protect the organelles. Negligible malondialdehyde formation was observed in the lysosomes either during the hypoxanthine/xanthine oxidase reaction or with different selective experimental approaches known to produce lipid peroxidation in other organelles such as microsomes and mitochondria. These results are interpreted in terms of a possible lysosomal membrane permeability to O ₂ ^⋅ causing organelle impairment by a process that, though leading to enzyme-marker leakage, does not involve lipid peroxidation.     

Effects of reactive oxygen metabolites on norepinephrine-induced vasoconstriction

Aortic rings, 4 mm in length, were obtained from rats and placed on isometric force transducers in oxygenated Krebs buffer. Following a period of stabilization, the cumulative dose response relationship to norepinephrine was assessed. The vessels were washed and allowed to return to baseline in Krebs buffer containing xanthine (0.5 mM). Xanthine oxidase (0.1 U/ml) was then added to the bath and vessels incubated for 30 min. The vessels were resuspended in Krebs buffer and cumulative dose-response curves to norepinephrine reevaluated. The results indicate that generation of reactive oxygen metabolites by xanthine/xanthine oxidase decreases the pD₂ from 7.80 ± 0.04 to 7.40 ± 0.09 with the endothelium intact. Removal of the endothelium did not attenuate the contractile dysfunction, indicating that endothelial-derived metabolites were not mediating the loss of vasoconstrictor effectiveness. Maximal tension development did not differ between normal and oxidized vessel rings. Introduction of oxypurinol (0.2 mg/ml) to the bath prevented the loss of constrictor responsiveness, thereby confirming that all of the oxidants were derived from the xanthine/xanthine oxidase reaction. Superoxide dismutase (200 U/ml) partially prevented the loss of norepinephrine responsiveness produced by xanthine oxidase-derived radicals. The pD₂ in the SOD + xanthine/xanthine oxidase-treated vessels rings (7.19 ± 0.11) was significantly lower tan control vessel rings (7.49 ± 0.04) and significantly higher than xanthine/xanthine oxidase-treated vessels (6.89 ± 0.06). Catalase (1000 U/ml) also partially attenuated the loss of vascular norepinephrine responsiveness. The pD₂ for the catalase + xanthine/xanthine oxidase-treated vessels (7.15 ± 0.02) was significantly lower than control vessels (7.39 ± 0.07)and significantly higher than the xanthine/xanthine oxidase-treated vessels (6.82 ± 0.11). The pD₂ of vessels treated with a combination of SOD and catalase (7.40 ± 0.10) did not differ from control vessels (7.49 ± 0.12). The results of this study indicate that reactive species produced by the interaction of xanthine with xanthine oxidase depress norepinephrine-induced vasoconstriction. The loss of vasoconstrictor responsiveness appears to involve both superoxide and hydrogen peroxide.     

Synergic action of insulin and genistein on Na+/K+/2Cl- cotransporter in renal epithelium

Transepithelial Cl(-) secretion in polarized renal A6 cells is composed of two steps: (1) Cl(-) entry step across the basolateral membrane mediated by Na(+)/K(+)/2Cl(-) cotransporter (NKCC) and (2) Cl(-) releasing step across the apical membrane via cystic fibrosis transmembrane conductance regulator (CFTR) Cl(-) channel. We estimated CFTR Cl(-) channel activity and transcellular Cl(-) secretion by measuring 5-nitro 2-(3-phenylpropylamino)benzoate (NPPB, a blocker of CFTR Cl(-) channel)-sensitive transepithelial conductance (Gt) and short-circuit current (Isc), respectively. Pretreatment with 1 microM insulin for 24 h had no effects on NPPB-sensitive Gt or Isc. On the other hand, in A6 cells treated with carbobenzoxy-L-leucyl-leucyl-L-leucinal (MG132; 100 microM for 2 h) that inhibits endocytosis of proteins at the plasma membrane into the cytosolic space, insulin pretreatment increased the NPPB-sensitive Isc with no effects on NPPB-sensitive Gt. Genistein (100 microM) induced sustained increases in NPPB-sensitive Gt and Isc, which were diminished by brefeldin A (a blocker of protein translocation to Golgi apparatus from endoplasmic reticulum). Co-application of insulin and genistein synergically stimulated the NPPB-sensitive Isc without any effects on NPPB-sensitive Gt. These observations suggest that: (1) insertion and endocytosis of NKCC are stimulated by insulin, (2) the insulin-induced stimulation of NKCC insertion into the basolateral membrane is offset by the stimulatory action on NKCC endocytosis from the basolateral membrane, (3) genistein stimulates insertion of both CFTR Cl(-) channel into the apical membrane and NKCC into the basolateral membrane, and (4) insulin and genistein synergically stimulated NKCC insertion into the basolateral membrane.     

Generation of superoxide radicals in alkaline solutions of hydrogen peroxide and the effect of superoxide dismutase on this system

Superoxide radicals in high concentrations were generated from alkaline H₂O₂ without using catalysts or irradiation. The dependence of the intensity and parameters of the superoxide radical EPR spectrum on pH, temperature, viscosity and H₂O₂ concentration were studied. The observed changes are explained on the base of matrix effects. The addition of superoxide dismutase to alkaline H₂O₂ led initially to a drop in the EPR spectrum intensity, followed by an increase in the concentration of superoxide radicals.     

Superoxide Radical-Mediated Alteration of Synaptosome Membrane Structure and High-Affinity γ-[14C]Aminobutyric Acid Uptake

Mouse cortical synaptosomal structure and function are altered when exposed to hypoxanthine/xanthine oxidase (HPX/XOD)-generated active oxygen/free radical species. The structure of both the synaptic vesicle and plasma membrane systems are altered by HPX/XOD treatment. The alteration of synaptic vesicle structure is exhibited by a significant increase in the cumulative length of nonsynaptic vesicle membrane per nerve terminal. With respect to the nerve terminal plasma membrane, the length of the perimeter of the synaptosome is increased as the membrane pulls away from portions of the terminal in blebs. The functional lesion generated by HPX/XOD treatment results in a reduction in selective high-affinity gamma-[14C]aminobutyric acid (GABA) uptake. Kinetic analysis of the reduction in high-affinity uptake reveals that the Vmax is significantly altered whereas the Km is not. Preincubation with specific active oxygen/free radical scavengers indicates that the super-oxide radical is directly involved. This radical, most probably in the protonated perhydroxyl form, initiates lipid peroxidative damage of the synaptosomal membrane systems. Low-affinity [14C]GABA transport is unaltered by the HPX/XOD treatment. The apparent ineffectiveness of free radical exposure on low-affinity [14C]GABA transport coupled with its effectiveness in reducing high-affinity transport supports the idea that two separate and different amino acid uptake systems exist in CNS tissue, with the high-affinity being more sensitive (lipid-dependent) and/or more energy-dependent (Na+,K+-ATPase) than the low-affinity system.     

The antioxidant action of N-acetylcysteine: Its reaction with hydrogen peroxide, hydroxyl radical, superoxide, and hypochlorous acid

N-acetylcysteine has been widely used as an antioxidant in vivo and in vitro. Its reaction with four oxidant species has therefore been examined. N-acetylcysteine is a powerful scavenger of hypochlorous acid (H---OCl); low concentrations are able to protect ₁-antiproteinase against inactivation by HOCl. N-acetylcysteine also reacts with hydroxyl radical with a rate constant of 1.36 × 10¹⁰ M⁻¹s⁻¹, as determined by pulse radiolysis. It also reacts slowly with H₂O₂, but no reaction of N-acetylcysteine with superoxide (O₂⁻) could be detected within the limits of our assay procedures.     

Posttranslational ruling of xanthine oxidase activity in bovine milk by its substrates

The aims of this study were to test the hypothesis that the substrates of xanthine oxidase (XO), xanthine and hypoxanthine, are consumed while the milk is stored in the gland between milkings, and to explore how XO activity responds to bacteria commonly associated with subclinical infections in the mammary gland. Freshly secreted milk was obtained following complete evacuation of the gland and induction of milk ejection with oxytocin. In bacteria-free fresh milk xanthine and hypoxanthine were converted to uric acid within 30 min (T1/2 approximately 10 min), which in turn provides electrons for formation of hydrogen peroxide and endows the alveolar lumen with passive protection against invading bacteria. On the other hand, the longer residence time of milk in the cistern compartment was not associated with oxidative stress as a result of XO idleness caused by exhaustion of its physiological fuels. The specific response of XO to bacteria species and the resulting bacteria-dependent nitrosative stress further demonstrates that it is part of the gland immune system.     

Mechanisms of H2O2-induced oxidative stress in endothelial cells

Hydrogen peroxide, produced by inflammatory and vascular cells, induces oxidative stress that may contribute to endothelial dysfunction. In smooth muscle cells, H₂O₂ induces production of O₂⁻ by activating NADPH oxidase. However, the mechanisms whereby H₂O₂ induces oxidative stress in endothelial cells are poorly understood. We examined the effects of H₂O₂ on O₂⁻ levels on porcine aortic endothelial cells (PAEC). Treatment with 60 μmol/L H₂O₂ markedly increased intracellular O₂⁻ levels (determined by conversion of dihydroethidium to hydroxyethidium) and produced cytotoxicity (determined by propidium iodide staining) in PAEC. Overexpression of human manganese superoxide dismutase in PAEC reduced O₂⁻ levels and attenuated cytotoxicity resulting from treatment with H₂O₂. L-NAME, an inhibitor of nitric oxide synthase (NOS), and apocynin, an inhibitor of NADPH oxidase, reduced O₂⁻ levels in PAEC treated with H₂O₂, suggesting that both NOS and NADPH oxidase contribute to H₂O₂-induced O₂⁻ in PAEC. Inhibition of NADPH oxidase using apocynin and NOS rescue with L-sepiapterin together reduced O₂⁻ levels in PAEC treated with H₂O₂ to control levels. This suggests interaction-distinct NOS and NADPH oxidase pathways to superoxide. We conclude that H₂O₂ produces oxidative stress in endothelial cells by increasing intracellular O₂⁻ levels through NOS and NADPH oxidase. These findings suggest a complex interaction between H₂O₂ and oxidant-generating enzymes that may contribute to endothelial dysfunction.     

Hyperpnea-induced production of TBHP-initiated chemiluminescence in guinea pigs

Antioxidants attenuate hyperpnea-induced airway constriction. It was hypothesized that this type of airway constriction is closely related to reactive oxygen species (ROS). However, there is no direct evidence of an increase in ROS during or right after the course of hyperpnea. To detect ROS production induced by hyperpnea, forty one guinea pigs were divided into four groups: control; control with 95% O2-5% CO2; hyperpnea with 95% air-5% CO2; and hyperpnea with 95% O2-5% CO2. Three minutes following hyperpnea or at the equivalent time, we obtained bronchoalveolar lavage (BAL) and measured its chemiluminescence (CL) counts. In addition, hyperpnea with 95% O2-5% CO2 gas mixture was carried out and BAL was collected 3 minutes after the hyperpnea in an additional forty animals. We measured CL counts in BAL samples before and after the treatments of the following ROS scavenger(s) or saline in vitro: control (saline); superoxide dismutase (SOD); catalase; dimethylthiourea (DMTU); and SOD+catalase+DMTU. Hyperpnea with 95% O2-5% CO2, but not with 95% air-5% CO2, gas mixture induced significant increase in t-butyl hydroperoxide-initiated CL counts, which were inhibited by DMTU, catalase, or SOD in vitro. Our data suggest that hyperpnea with a 95% O2-5% CO2, but not with 95% air-5% CO2, gas mixture induced an increase in ROS production.     

A fluctuation analysis study of the development of amiloride-sensitive Na+ transport in the skin of larval bullfrogs (Rana catesbeiana)

In the presence of the Na⁺-channel blocker amiloride, the short-circuit current across the skins of bullfrog tadpoles in metamorphic stages XIX–XXIV was subjected to fluctuation analysis. The resulting power spectra contained a Lorentzian component of which the plateau value ($\text{S}{}_0$) decreased while the corner frequency ($\text{f}{}_{\mathrm{<mtext>c</mtext>}}$) increased as the mucosal amiloride concentration was increased from 0.5 to 24 μM. From the linear relationship between the $\text{f}{}_{\mathrm{<mtext>c</mtext>}}$ values and the amiloride concentrations it was possible to determine the binding ($\text{k′}{}_{01}$) and unbinding ($\text{k}{}_{10}$) constants for amiloride to its receptor on the Na⁺ channel. With these parameters as well as short-circuit current and $\text{S}{}_0$ values, the current through the individual Na⁺ channels ($\text{i}$) was calculated (average 0.58 pA). It did not increase significantly during late metamorphosis. The density of Na⁺ channels ($\text{M}$) in the apical membrane, on the other hand, increased significantly. It would appear that the increase in short-circuit current which occurs at this time is due primarily to an increase in amiloride-blockable Na⁺ channels. Unexpectedly, a Lorentzian component could be fitted to power spectra in amiloride-treated skins (stages XIX–XXI) which showed no amiloride-sensitive short-circuit current. Moreover, the typical increase in $\text{f}{}_{\mathrm{<mtext>c</mtext>}}$ with the amiloride concentration did not occur in these animals.     

Specificity and sodium dependence of the active nucleoside transport system in choroid plexus

The transport of [3H]deoxyuridine by the active nucleoside transport system into the isolated rabbit choroid plexus was measured in vitro under various conditions. Choroid plexuses were incubated in artificial CSF containing 1 microM [3H]deoxyuridine and 1 microM nitrobenzylthioinosine for 5 min under 95% O2-5% CO2 at 37 degrees C and the accumulation of [3H]deoxyuridine measured. Nitrobenzylthioinosine was added to the artificial CSF at a concentration (1 microM) that did not inhibit the active nucleoside transport system but did inhibit the separate, saturable nucleoside efflux system. The active transport of deoxyuridine into the choroid plexus depended on Na+ in the medium, as ouabain, substitution of Li+ and choline for Na+, and poly-L-lysine all inhibited deoxyuridine transport. Thiocyanate in place of chloride and penetrating sulfhydryl reagents also inhibited the active transport of deoxyuridine into choroid plexus. The active transport of deoxyuridine into choroid plexus, which is inhibited by naturally occurring ribo- and deoxyribonucleosides (IC50 = 7-21 microM), was not inhibited (IC50 much greater than 150 microM) by nucleosides with certain alterations on the 2', 3', or 5' positions in D-ribose or 2-deoxy-D-ribose (e.g., adenine arabinoside, 3'-deoxyadenosine, xylosyladenosine); or the pyrimidine or purine rings (e.g., 6-azauridine, xanthosine, 7-methylinosine, or 8-bromoadenosine). Other analogues were effective (IC50 = 8-26 microM; e.g., 5-substituted pyrimidine nucleosides, 7-deazaadenosine, 6-mercaptoguanosine) or less effective (IC50 = 46-145 microM; e.g., 5-azacytidine, 3-deazauridine) inhibitors of deoxyuridine transport into the isolated choroid plexus.     

Generation of hydrogen peroxide,superoxide anion and the hydroxyl free radical from polyphenols and active benzene metabolites: Their possible role in mutagenesis

Benzene is strongly suspected of being an animal and human carcinogen, but the mechanisms by which it induces tumors of lymphoid and hematopoietic organs are unknown. Production of active oxygen species from benzene metabolites [hydroquinone (HQ), catechol and 1,2,4-benzenetriol (1,2,4-BT) and related polyphenols (resorcinol, pyrogallol and phloroglucinol) are investigated. Pyrogallol and 1,2,4-BT can produce H₂O₂, O ₂ ^– and^·OH simultaneously, and have powerful mutagenic potential. Resorcinol and phloroglucinol cannot produce all of the active oxygen species, and show no mutagenic effects. Catechol can produce H₂O₂, but cannot produce O ₂ ^– and^·OH, and has no mutagenic activity. These data strongly support the hypothesis that benzene metabolites can cause mutagenicity via the generation of oxygen radicals. Although HQ produces H₂O₂ only, and less than produced by pyrogallol and 1,2,4-BT, the mutagenicity of HQ is higher. The results indicate that HQ may act via another mechanism to cause mutagenicity. In the presence of trace metal ions, the reactivity of polyphenols is increased. The biological significance of these phenomena are investigated and discussed.     

Application of a trityl-based radical probe for measuring superoxide

The use of triarylmethyl (trityl) free radical, TAM OX063, for detection of superoxide in aqueous solutions by electron paramagnetic resonance (EPR) spectroscopy was investigated. TAM is paramagnetic (EPR active), highly soluble in water and exhibits a single sharp EPR peak in aqueous media. It is also highly stable in presence of many oxidoreductants such as ascorbate and glutathione that are present in the biological systems. TAM reacts with superoxide with an apparent second order rate constant of 3.1 × 10³ M⁻¹ s⁻¹. The specific reactivity of TAM with superoxide, which leads to loss of EPR signal, was utilized to detect the generation of superoxide in various chemical (light/riboflavin/electron/donor), enzymatic (xanthine/xanthine oxidase), and cellular (stimulated neutrophils) model systems. The changes in the EPR line-width, induced by molecular oxygen, were utilized in the simultaneous determination of consumption of oxygen in the model systems. The effects of flux of superoxide and concentration of TAM on the efficiency of detection of superoxide were studied. The use of TAM for detection of superoxide offers unique advantages namely, (i) the utilization of very low concentration of the probe, (ii) its stability to bioreduction, and (iii) its use in the simultaneous determination of concentrations of superoxide and oxygen.     

Enzymatic and Non-Enzymatic Oxygenation of Tyrosine

Tyrosinase isolated from cultured human melanoma cells was studied for tyrosine oxygenation activity. l -Tyrosine and d -tyrosine were used as substrates and dopa was measured with HPLC and electrochemical detection as the product of oxygenation. Incubations were performed in the presence or absence of dopamine as co-substrate. Oxygenation of l -tyrosine occurred only in the presence of dopamine as co-substrate. No oxygenation of d -tyrosine was found, and we conclude that human tyrosinase is characterised by exclusive specificity for the l -isomer of tyrosine in its oxygenase function. It has recently been suggested that superoxide anion is a preferential oxygen substrate for human tyrosinase. Incubations were therefore performed with l - and d -tyrosine, human tyrosinase, and xanthine/xanthine oxidase in the system, generating superoxide anion and hydrogen peroxide. Considerable formation of dopa was observed, but the quantity was the same irrespective of whether d -tyrosine or l -tyrosine was used as the substrate. Furthermore, formation of dopa occurred in a xanthine/xanthine oxidase system when bovine serum albumin (BSA) was substituted for tyrosinase. Our results provide no evidence that superoxide anion is an oxygen substrate for human tyrosinase. In the incubate containing xanthine/xanthine oxidase, catalase completely inhibited dopa formation, and superoxide dismutase and mannitol each strongly inhibited dopa formation. The results are compatible with hydroxyl radicals being responsible for the formation of dopa, since such radicals may be secondarily formed in the presence of superoxide anion and hydrogen peroxide.     

Production of superoxide/H2O2 by dihydroorotate dehydrogenase in rat skeletal muscle mitochondria

Dehydrogenases that use ubiquinone as an electron acceptor, including complex I of the respiratory chain, complex II, and glycerol-3-phosphate dehydrogenase, are known to be direct generators of superoxide and/or H₂O₂. Dihydroorotate dehydrogenase oxidizes dihydroorotate to orotate and reduces ubiquinone to ubiquinol during pyrimidine metabolism, but it is unclear whether it produces superoxide and/or H₂O₂ directly or does so only indirectly from other sites in the electron transport chain. Using mitochondria isolated from rat skeletal muscle we establish that dihydroorotate oxidation leads to superoxide/H₂O₂ production at a fairly high rate of about 300 pmol H₂O₂·min⁻¹·mg protein⁻¹ when oxidation of ubiquinol is prevented and complex II is uninhibited. This H₂O₂ production is abolished by brequinar or leflunomide, known inhibitors of dihydroorotate dehydrogenase. Eighty percent of this rate is indirect, originating from site II_F of complex II, because it can be prevented by malonate or atpenin A5, inhibitors of complex II. In the presence of inhibitors of all known sites of superoxide/H₂O₂ production (rotenone to inhibit sites in complex I (site I_Q and, indirectly, site I_F), myxothiazol to inhibit site III_Qo in complex III, and malonate plus atpenin A5 to inhibit site II_F in complex II), dihydroorotate dehydrogenase generates superoxide/H₂O₂, at a small but significant rate (23 pmol H₂O₂·min⁻¹·mg protein⁻¹), from the ubiquinone-binding site. We conclude that dihydroorotate dehydrogenase can generate superoxide and/or H₂O₂ directly at low rates and is also capable of indirect production at higher rates from other sites through its ability to reduce the ubiquinone pool.     

Simultaneous use of electrochemistry and chemiluminescence to detect reactive oxygen species produced by human neutrophils

A novel approach for the simultaneous optical and electrochemical detection of biologically produced reactive oxygen species has been developed and applied. The set-up consists of a luminol-dependent chemiluminescence assay combined with two amperometric biosensors sensitive to superoxide anion radicals (O(2)(-)) and hydrogen peroxide (H(2)O(2)), respectively. The method permits direct, real-time in vitro determination of both extra- and intracellular O(2)(-) and H(2)O(2) produced by human neutrophil granulocytes. The rate of O(2)(-) production by stimulated neutrophils was calculated to about 10(-17)mol s(-1) per single cell. With inhibited NADPH oxidase, a distinct extracellular release of H(2)O(2) instead of O(2)(-) was obtained from stimulated neutrophils with the rate of about 3 x 10(-18)mol s(-1) per single cell. When the H(2)O(2) release was discontinued, fast H(2)O(2) utilisation was observed. Direct interaction with and possibly attachment of neutrophils to redox protein-modified gold electrodes, resulted in a spontaneous respiratory burst in the population of cells closely associated to the electrode surface. Hence, further stimulation of human neutrophils with a potent receptor agonist (fMLF) did not significantly increase the O(2)(-) sensitive amperometric response. By contrast, the H(2)O(2) sensitive biosensor, based on an HRP-modified graphite electrode, was able to reflect the bulk concentration of H(2)O(2), produced by stimulated neutrophils and would be very useful in modestly equipped biomedical research laboratories. In summary, the system would also be appropriate for assessment of several other metabolites in different cell types, and tissues of varying complexity, with only minor electrode modifications.     

Phosphate increases mitochondrial reactive oxygen species release

The effects of inorganic phosphate (P_i), the main intracellular membrane permeable anion capable of altering mitochondrial pH gradients (ΔpH), were measured on mitochondrial H₂O₂ release. As expected, P_i decreased ΔpH and increased the electric membrane potential (ΔΨ). Mitochondrial H₂O₂ release was stimulated by P_i and also by its structural analogue arsenate. However, acetate, another membrane-permeable anion, did not stimulate mitochondrial H₂O₂ release. The stimulatory effect promoted by P_i was prevented by CCCP, which decreases transport of P_i across the inner mitochondrial membrane, indicating that P_i must be in the mitochondrial matrix to stimulate H₂O₂ release. In conclusion, we found that P_i and arsenate stimulate mitochondrial reactive oxygen release, an effect that may contribute towards oxidative stress under conditions such as ischemia/reperfusion, in which high-energy phosphate bonds are hydrolyzed.     

Confirmation of Superoxide Generation via Xanthine Oxidase in Streptozotocin-induced Diabetic Mice

Reactive oxygen species (ROS) may play key roles in vascular inflammation and atherogenesis in patients with diabetes. In this study, xanthine oxidase (XO) system was examined as a potential source of superoxide in mice with streptozotocin (STZ)-induced experimental diabetes. Plasma XO activity increased 3-fold in diabetic mice (50±33?μU/ml) 2 weeks after the onset of diabetes, as compared with non-diabetic control mice (15±6?μU/ml). In vivo superoxide generation in diabetic mice was evaluated by an in vivo electron spin resonance (ESR)/spin probe method. Superoxide generation was significantly enhanced in diabetic mice, and the enhancement was restored by the administration of superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), which was reported to scavenge superoxide. Pretreatment of diabetic mice with XO inhibitors, allopurinol and its active metabolite oxipurinol, normalized the increased superoxide generation. In addition, there was a correlation (r=0.78) between the level of plasma XO activity and the relative degree of superoxide generation in diabetic and non-diabetic mice. Hence, the results of this study strongly suggest that superoxide should be generated through the increased XO seen in the diabetic model mice, which may be involved in the pathogenesis of diabetic vascular complications.     

Confirmation of superoxide generation via xanthine oxidase in streptozotocin-induced diabetic mice

Reactive oxygen species (ROS) may play key roles in vascular inflammation and atherogenesis in patients with diabetes. In this study, xanthine oxidase (XO) system was examined as a potential source of superoxide in mice with streptozotocin (STZ)-induced experimental diabetes. Plasma XO activity increased 3-fold in diabetic mice (50±33 μU/ml) 2 weeks after the onset of diabetes, as compared with non-diabetic control mice (15±6 μU/ml). In vivo superoxide generation in diabetic mice was evaluated by an in vivo electron spin resonance (ESR)/spin probe method. Superoxide generation was significantly enhanced in diabetic mice, and the enhancement was restored by the administration of superoxide dismutase (SOD) and 4,5-dihydroxy-1,3-benzene disulfonic acid (Tiron), which was reported to scavenge superoxide. Pretreatment of diabetic mice with XO inhibitors, allopurinol and its active metabolite oxipurinol, normalized the increased superoxide generation. In addition, there was a correlation (r=0.78) between the level of plasma XO activity and the relative degree of superoxide generation in diabetic and non-diabetic mice. Hence, the results of this study strongly suggest that superoxide should be generated through the increased XO seen in the diabetic model mice, which may be involved in the pathogenesis of diabetic vascular complications.     

Change of ruminal sodium transport in sheep during dietary adaptation

Rumen adaptation plays an important role in the productive cycle of dairy cattle. In this study, the time course of functional rumen epithelium adaptation after a change from hay feeding (ad libitum) to a mixed hay/concentrate diet was monitored by measuring Na⁺ transport rates in Ussing chamber experiments. A total of 18 sheep were subjected to different periods of mixed hay/concentrate feeding ranging from 0 weeks (control; hay ad libitum) to 12 weeks (800 g hay plus 800 g concentrate per day in two equal portions). For each animal, the net absorption of sodium was measured following the mixed hay/concentrate feeding period. Net Na transport, J_net, significantly rose from 2.15 ± 0.43 (control) to 3.73 ± 1.02 μeq · cm^?2 · h^?1 after one week of mixed hay/concentrate diet, reached peak levels of 4.55 ± 0.50 μEq · cm^?2 · h^?1 after four weeks and levelled out at 3.92 ± 0.36 μeq · cm^?2 · h^?1 after 12 weeks of mixed feeding. Thus, 73% of functional adaptation occurred during the first week after diet change. This is in apparent contrast to findings that morphological adaptation takes approximately six weeks to reach peak levels. Hence, early functional adaptation to a mixed hay/concentrate diet is characterised by enhanced Na absorption rates per epithelial cell. Absorption rates are likely to be further enhanced by proliferative effects on the rumen epithelium (number and size of papillae) when concentrate diets are fed over longer periods of time. Early functional adaptation without surface area enlargement of the rumen epithelium appears to be the first step in coping with altered fermentation rates following diet change.