Showing papers on "Xanthine published in 1988"

Role of xanthine oxidase and granulocytes in ischemia-reperfusion injury

Abstract: In this lecture, evidence is presented to support the following hypothesis regarding the roles of xanthine oxidase-derived oxidants and granulocytes in ischemia-reperfusion-induced microvascular injury. During the ischemic period, ATP is catabolized to yield hypoxanthine. The hypoxic stress also triggers the conversion of NAD-reducing xanthine dehydrogenase to the oxygen radical-producing xanthine oxidase. During reperfusion, molecular oxygen is reintroduced into the tissue where it reacts with hypoxanthine and xanthine oxidase to produce a burst of superoxide anion and hydrogen peroxide. In the presence of iron, superoxide anion and hydrogen peroxide react via the Haber-Weiss reaction to form hydroxyl radicals. This highly reactive and cytotoxic radical then initiates lipid peroxidation of cell membrane components and the subsequent release of substances that attract, activate, and promote the adherence of granulocytes to microvascular endothelium. The adherent granulocytes then cause further endothelial cell injury via the release of superoxide and various proteases.

Excitatory amino acid release from rat hippocampal slices as a consequence of free-radical formation.

Abstract: The release of D-[3H]aspartate, [3H]noradrenaline, and of endogenous glutamate and aspartate from rat hippocampal slices was significantly increased when the slices were incubated with xanthine oxidase plus xanthine to produce superoxide and hydroxyl free radicals locally. Allopurinol, a specific xanthine oxidase inhibitor, the hy-droxyl-radical scavenger D-mannitol, or the superoxide-radical scavenger system formed by superoxide dismutase plus catalase prevented this release. These results suggest that endogenous excitatory amino acids are released consequent to the formation of free radicals. The excess of glutamate and aspartate released by this mechanism could be one of the factors contributing to the death of neurons after anoxic or ischemic injuries.

Mitochondria contain a proteolytic system which can recognize and degrade oxidatively-denatured proteins.

Abstract: When incubated with mitochondria in an air atmosphere, menadione and doxorubicin (which redox cycle with the respiratory chain to produce oxygen radicals), as well as xanthine oxidase plus xanthine (which generate superoxide and H2O2), stimulated the degradation of newly-synthesized [( 3H]leucine-labelled) mitochondrial polypeptides. No stimulation was observed in an N2 atmosphere, ATP was not required, and xanthine oxidase was not effective without xanthine. Various forms of oxidative stress induced varying degrees of protein cross-linking, protein fragmentation and proteolysis, as judged by gel electrophoresis and amino acid analysis. To learn more about the proteolytic enzymes involved in degradation, we undertook studies with purified protein substrates which had been exposed to oxidative stress (OH or H2O2) in vitro. Despite mitochondrial contamination with acid proteases of lysosomal (and other) origin, pH profiles revealed distinct proteolytic activities at both pH 4 and pH 8. The pH 8 activity preferentially degraded the oxidatively-denatured forms of haemoglobin, albumin and superoxide dismutase; was unaffected by digitonin; and exhibited a several-fold increase in activity upon mitochondrial disruption (highest activity being found in the matrix). In contrast, the pH 4 activity was dramatically decreased by digitonin treatment (to reduce lysosomal contamination); was unaffected by mitochondrial disruption; and showed no preference for oxidatively-denatured proteins. The pH 8 activity was not stimulated by ATP, but was inhibited by EDTA, haemin and phenylmethylsulphonyl fluoride. In contrast, the contaminating pH 4 activity was only inhibited by pepstatin and leupeptin. Thus, our experiments reveal a distinct mitochondrial (matrix) proteolytic pathway which can preferentially degrade oxidatively-denatured proteins.

Effect of superoxide radical on Ca2+ pumps of coronary artery.

Abstract: The effect of superoxide radical on the azide-insensitive ATP-dependent Ca2+-transport by a plasma membrane (PM)-enriched fraction (F2) and an endoplasmic reticulum (ER)-enriched fraction (F3) isolated from pig coronary artery was examined using xanthine oxidase plus xanthine to generate superoxide ions. A preincubation with xanthine oxidase plus xanthine at 37 degrees C preferentially inactivated the oxalate-stimulated Ca2+ uptake by the F3 fraction rather than the phosphate-stimulated uptake by the F2 fraction, indicating that the Ca2+ pump in the ER was more susceptible to this free radical. The inactivation of the Ca2+ uptake depended on the concentrations of xanthine oxidase and xanthine in the preincubation mixture as well as on the preincubation time. Furthermore, the inclusion of superoxide dismutase in the preincubation mixture prevented the inactivation. Thus the inactivation was caused by superoxide radical. Preincubation with xanthine oxidase plus xanthine, however, altered the half-life of efflux of Ca2+ from these vesicles only marginally. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the F3 fraction showed formation of a Ca2+-dependent acid stable phosphoenzyme at 0 degree C predominantly at a protein band corresponding to 100 kDa. The level of the 100-kDa acylphosphate intermediate was inhibited in parallel with the inhibition of the Ca2+ uptake by preincubation with xanthine oxidase plus xanthine. We conclude that superoxide radical inactivates the ER Ca2+ transport by lowering the level of the phosphoenzyme.

Adenine nucleotide tissue concentrations and liver allograft viability after cold preservation and warm ischemia.

Abstract: The relation between adenine nucleotide liver concentrations and the viability of liver allografts after cold preservation and warm ischemia was studied. A rat model was used with storage times defined in terms of allograft viability. Livers were excised and stored for 4 hr at 4 degrees C or 1 hr at 37 degrees C (viable if transplanted) or for 8 hr at 4 degrees C or 2 hr at 37 degrees C (not viable if transplanted) in a solution containing 0.9% NaCl and 2 mM CaCl2. Adenine nucleotide, malondialdehyde, and glutathione concentrations were measured in liver biopsies at the end of the storage periods and in control livers. During cold preservation, ATP concentrations decline, but degradation is largely halted at AMP, and this is independent of the length of storage or viability of the allograft. Graft failure is not due to lack of availability of intramitochondrial substrate (AMP) for rephosphorylation to adenosine triphosphate (ATP), nor is it likely that provision of such substrate will be helpful. On the other hand, with warm ischemia, degradation to inosine, hypoxanthine and xanthine occurs and nonviable livers develop higher levels of xanthine than viable ones; in fact, xanthine concentrations provide 100% discrimination between viable and nonviable warm preserved livers. Malondialdehyde concentrations were also significantly greater in the warm preserved nonviable livers, indicating that some lipid peroxidation may occur even before reperfusion of allografts. Glutathione concentrations were similar in all experimental groups.

An all-purine precursor of nucleic acids

Abstract: The theory is proposed that the pyrimidines in extant nucleic acids are postenzymatic substitutes for their isoelectronic and isogeometric position 3-bonded purine analogs xanthine and isoguanine, which were sibling products in a preenzymatic de novo purine pathway.

Role of hydrogen peroxide in the cytotoxicity of the xanthine/xanthine oxidase system.

Abstract: 1. The survival of mammalian epithelial cells exposed in vitro to the xanthine/xanthine oxidase system in phosphate-buffered saline (PBS) or serum-containing medium (SCMEM) was investigated. 2. The cytotoxic effect observed depended on the composition of the medium in which the enzymic reaction was carried out; a surviving fraction of 5 x 10(-5) was found for cells exposed in PBS and 5.2 x 10(-1) for those in SCMEM. 3. The cytotoxic product(s) formed by the xanthine/xanthine oxidase system was relatively stable in PBS; survival of cells incubated after completion of the enzymic reaction was always less than that found for cells exposed during the reaction in the same system. 4. Superoxide dismutase or mannitol present during the enzymic reaction did not inhibit the cytotoxic effect. 5. NaN3 (a single-oxygen quencher and a catalase inhibitor) added to the system in SCMEM caused a reduction in survival to the level observed for cells exposed to the enzymic reaction in PBS. 6. Catalase completely protected cells, but no protection was observed when both catalase and NaN3 were present in the reaction mixture. 7. A similar cytotoxic effect was produced when cells were treated with H2O2 alone. 8. The rate of H2O2 decomposition in medium was accelerated by the presence of serum, but this was completely inhibited by NaN3. 9. It is concluded that H2O2 is the major cytotoxic product formed by the xanthine/xanthine oxidase system.

Inhibitors of Xanthine Oxidase from Alpinia galanga

Abstract: Xanthine oxidase (XO) inhibitors were isolated from the rhizomes of Alpinia galanga (Zingiberaceae), and were identified as trans-p-coumaryl diacetate (1), trans-coniferyl diacetate (2), [1'S]-1'-acetoxychavicol acetate (3), [1'S]-1'-acetoxyeugenol acetate (4) and 4-hydroxybenzaldehyde (5). The type of inhibition by either 1 or 3 with respect to xanthine as a substrate was uncompetitive.

Multiple components of the A1 adenosine receptor-adenylate cyclase system are regulated in rat cerebral cortex by chronic caffeine ingestion.

Abstract: The effects of chronic caffeine on the A1 adenosine receptor-adenylate cyclase system of rat cerebral cortical membranes were studied. Caffeine treatment significantly increased the number of A1 adenosine receptors as determined with the A1 adenosine receptor antagonist radioligand [3H]xanthine amine congener (XAC). R-PIA (agonist) competition curves constructed with [3H]XAC were most appropriately described by a two affinity state model in control membranes with a KH of 2.1 +/- 0.8 and a KL of 404 +/- 330 nM with 50 +/- 4% of receptors in the high affinity state (%RH). In contrast, in membranes from treated animals, there was a marked shift towards the high affinity state. In three of seven animals all of the receptors were shifted to a unique high affinity state which was indistinguishable from the KH observed in membranes from control animals. In four of seven animals the %RH increased from 50 to 69% with KH and KL indistinguishable from the control values. Thus, the agonist specific high affinity form of the receptor was enhanced following caffeine treatment. Maximal inhibition of adenylate cyclase activity in cerebral cortical membranes by R-PIA (1 microM) was significantly increased by 28% following caffeine treatment, consistent with an increased coupling of receptor-Gi protein with adenylate cyclase. Importantly, the quantity of Gi (alpha i) in rat cerebral cortex, determined by pertussis toxin-mediated labeling, was also increased to 133% of control values by this treatment. Thus, multiple components and interactions of the A1 adenosine receptor-adenylate cyclase complex are regulated by caffeine. These changes are likely compensatory measures to offset blockade of A1 receptors in vivo by caffeine and lead to a sensitization of this inhibitory receptor system.

Adenosine antagonists as potential therapeutic agents

Abstract: The methylxanthine caffeine has been identified in more than 60 plant species and has been in human use for its various therapeutic actions for many hundreds of years and perhaps, with the exception of aspirin and related compounds, is the most widely consumed drug today. Pharmacologically, the xanthines are prototypic inhibitors of the enzyme, cyclic nucleotide phosphodiesterase, are calcium mobilizers and have been reported to inhibit the enzymes, monoamine oxidase and cyclooxygenase as well as affect uptake of the putative neuromodulator, adenosine. However, many of the therapeutic effects ascribed to caffeine are due to its selective ability to antagonize the actions of adenosine. Many xanthines, especially those substituted in the 8-position with a phenyl derivative, are potent and selective adenosine antagonists. The xanthine adenosine antagonists have mild psychostimulant, analgesic adjuvant, diuretic, cardiotonic and antiasthmatic activity. Adenosine antagonists also have nootropic activity. A major limiting factor to the development of this class of compound has been in the lack of selectivity for either of the major classes of adenosine receptor. Several non-xanthines including the pyrazolopyrimidine, DJB-KK, the pyrazoloquinoline, CGS 8216 and the pyrazolopyridine, etazolate have been shown to have adenosine antagonist activity. The triazoloquinazoline, CGS 15943 A has been identified as the first, potent (IC50 = 3 nM) nonxanthine, A2-selective adenosine antagonist while the phenylquinazoline, HTQZ, has 25-fold selectivity for the A2 receptor. The availability of such novel entities may permit the development of a new class of therapeutic agents able to affect neuromodulator, as opposed to neurotransmitter, function.

Xanthine and uric acid levels in rat brain following focal ischemia.

Abstract: Changes of the xanthine and uric acid (UA) levels in rat forebrain following focal cerebral ischemia were studied by reversed-phase HPLC with electrochemical detection. Focal ischemia was induced by occluding the left middle cerebral artery in the rat. The xanthine level in the normal group was 11.50 nmol/g tissue. In the ischemic group, the xanthine concentration in the ischemic hemisphere progressively increased after occlusion and reached a maximum value of 59.42 nmol/g tissue 4 h after operation. The UA level in the normal group was 2.20 nmol/g tissue, whereas in the ischemic group the UA concentration in the ischemic hemisphere gradually increased after occlusion, reaching a value of 38.53 nmol/g tissue 24 h after ischemia. The concentration of UA remained elevated in the ischemic hemisphere until 48 h after occlusion, and reached a maximum value of 38.98 nmol/g tissue. The xanthine and UA levels in the contralateral hemisphere remained unchanged. The xanthine and UA concentrations in the sham-operated group did not show a significant increase after operation. The time course of the xanthine and UA levels suggests that in ischemic forebrain UA is formed from xanthine as a product of purine metabolism.

Lesch‐Nyhan syndrome and its pathogenesis: Purine concentrations in plasma and urine with metabolite profiles in CSF

Abstract: Purine metabolism in the Lesch-Nyhan syndrome has been re-examined in 10 patients. Hypoxanthine and xanthine concentrations in plasma and CSF and urinary excretion have been studied, on and off allopurinol treatment, using high performance liquid chromatographic methods. Accumulation of the substrate, hypoxanthine, of the missing hypoxanthine guanine phosphoribosyltransferase (HPRT) enzyme, is more marked in urine and in CSF than in plasma. The greater increase in CSF is consistent with the most metabolically active tissue, brain, showing the most marked functional changes. The function of HPRT seems to be the recycling of hypoxanthine which is released from tissues in increasing quantities as energy use, ATP 'turnover', in the tissue increases. The existing screening method for HPRT deficiency, the ratio of the urinary concentration of urate to that of creatinine, shows overlap between the values in severe HPRT deficiency and in controls; this overlap is not found with a urinary hypoxanthine/creatinine molar concentration ratio.

Effects of deoxycoformycin on adenosine, inosine, hypoxanthine, xanthine, and uric acid release from the hypoxemic rat cerebral cortex.

Abstract: The effects of the adenosine deaminase inhibitor, deoxycoformycin, on purine release from the rat cerebral cortex were studied with the cortical cup technique. Deoxycoformycin (5 and 500 μg/kg i.v.) enhanced the hypoxia/ischemia-evoked release of adenosine from the cerebral cortex, indicating a marked rise in the adenosine content of interstitial fluid in the cerebral cortex. Inosine and hypoxanthine release were attenuated at the higher dose of deoxycoformycin, acid release into the cortical perfusates was enhanced at the higher dose level. These results demonstrate that low doses of deoxycoformycin can be used to elevate interstitial levels of adenosine in the brain during hypoxia, and to depress the formation of some of its metabolites. The elevation of hypoxia/ischemia-evoked adenosine levels can account for the previously reported potentiation of hypoxia-evoked increases in rat cerebral blood flow after deoxycoformycin administration. The potential therapeutic utility of these findings is discussed.

A kinetic study of hypoxanthine oxidation by milk xanthine oxidase

Abstract: The course of the reaction sequence hypoxanthine----xanthine----uric acid catalysed by xanthine:oxygen oxidoreductase from milk was investigated on the basis of uv spectra taken during the course of hypoxanthine and xanthine oxidations It was found that xanthine accumulated in the reaction mixture when hypoxanthine was used as a substrate The time course of the concentrations of hypoxanthine, xanthine intermediate and uric acid product was simulated numerically The mathematical model takes into account the competition of substrate, intermediate and product and the accumulation of the intermediate at the enzyme This type of analysis permits the kinetic parameters of the enzyme for hypoxanthine and xanthine to be obtained

Quantitative identification of superoxide anion as a negative inotropic species.

Abstract: Reduced oxygen intermediates have been shown to directly depress cardiac muscle function at the subcellular, tissue, and whole animal levels. The exact species of reduced oxygen intermediate [superoxide anion radical (O2-.), H2O2, hydroxyl free radical (HO.)] and the concentrations necessary to depress cardiac muscle function have not been quantified. To better understand the role of O2-. and H2O2, we have studied rabbit right ventricular papillary muscle function in the presence of these reduced oxygen intermediates generated by a xanthine-xanthine oxidase system at 37 degrees C. In the presence of xanthine (0.1 mM) and xanthine oxidase (0.02 U/ml), 57.5 +/- 0.85 nmol.l-1.s-1 O2-. and 69.25 +/- 5.3 nmol.l-1.s-1 H2O2 were produced. In the presence of superoxide dismutase (SOD), O2-. was eliminated and H2O2 concentration increased. Catalase effectively eliminated the accumulation of H2O2 without significantly changing the rate of O2-. generation. When applied to isometrically contracting right ventricular papillary muscles, this system, with or without SOD and catalase, had no effect on peak developed tension or +/- dT/dt derived either from length-tension or force-frequency studies. However, when the xanthine oxidase concentration was increased to 0.112 U/ml, the rate of O2-. generation increased to 196.67 +/- 3.26 nmol.l-1.s-1 and H2O2 production increased to 142.19 +/- 9.3 nmol.l-1.s-1 with significant depression of papillary muscle tension development. SOD virtually eliminated O2-. production, whereas H2O2 production increased to 199.48 +/- 9.8 nmol.l-1.s-1 with no effect on papillary muscle tension development.(ABSTRACT TRUNCATED AT 250 WORDS)

The effect of oxygen free radicals on calcium permeability and calcium loading at steady state in cardiac sarcoplasmic reticulum.

Abstract: It has been proposed that oxygen free radical production is an important mediator of the myocardial dysfunction during the course of acute ischemia. We tested this hypothesis by characterizing the pathway of calcium efflux across sarcoplasmic reticulum (SR) membranes affected by oxygen free radicals. The effect of oxygen free radicals on the steady state calcium load, calcium permeability, and Ca,Mg-ATPase activity of isolated canine cardiac SR vesicles was investigated at pH 7.0. In vitro generation of oxygen free radicals by xanthine oxidase (0.09 units/ml), acting on xanthine in doses up to 50 microM as a substrate, increased the permeability of the SR vesicles to calcium, determined by measuring net efflux of calcium after stopping pump-mediated fluxes, and decreased total intravesicular calcium and free intravesicular calcium with no effect on Ca,Mg-ATPase activity. The effect of oxygen free radicals on calcium permeability was calcium gradient-dependent. Xanthine alone or xanthine plus denatured xanthine oxidase had no effect on this system. Superoxide dismutase (SOD, 56 units/ml), but not denatured SOD, significantly inhibited the effect of xanthine-xanthine oxidase reaction. The calcium permeability of the SR membrane decreased with decreasing calcium load. In addition, inasmuch as extravesicular calcium exerts only a slight effect on calcium permeability, the decrease in the permeability with calcium load is specifically related to the calcium load. Oxygen free radical-induced increase in calcium permeability was unaffected by Mg concentration between 2.1 and 21 mM. In summary, our data reveal that .O2- can produce a diminished level of accumulated calcium, which is reflected by the decreased calcium load and an increase in passive calcium permeability, and that the decreased calcium accumulation in the presence of the xanthine-xanthine oxidase system may not be mainly due to an inhibited calcium pump but due to an increased calcium permeability. Our results also suggest that increased SR membrane passive calcium permeability induced by oxygen free radicals is not carrier mediated. It is postulated that, with the oxygen free radical-mediated progressive increase in calcium permeability, free cytosolic calcium concentrations would increase in ischemic myocardium.

[3H]-8-cyclopentyl-1,3-dipropylxanthine binding to A1 adenosine receptors of intact rat ventricular myocytes.

Abstract: The purpose of the present study was the identification of A1 adenosine receptors in intact rat ventricular myocytes, which are thought to mediate the negative inotropic effects of adenosine. The adenosine receptor antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine was used as radioligand. Binding of the radioligand to intact myocytes was rapid, reversible, and saturable with a binding capacity of 40,000 binding sites per cell. The dissociation constant of the radioligand was 0.48 nM. The adenosine receptor antagonists 8-cyclopentyl-1,3-dipropylxanthine, "xanthine amine congener," and theophylline were competitive inhibitors with affinities in agreement with results obtained for A1 receptors in other tissues. Competition experiments using the adenosine receptor agonists R-N(6)-phenylisopropyladenosine, 5'-N-ethylcarboxamidoadenosine, and S-N(6)-phenylisopropyladenosine gave monophasic displacement curves with Ki values of 50 nM, 440 nM, and 4,300 nM, which agreed well with the GTP-inducible low affinity state in cardiac membranes. The low affinity for agonists was not due to agonist-induced desensitization, and correlated well with the corresponding IC50 values for the inhibition of cyclic AMP accumulation by isoprenaline. It is suggested that only a low affinity state of A1 receptors can be detected in intact rat myocytes due to the presence of high concentrations of guanine nucleotides in intact cells.

Differences in uricogenic effects of dietary purine bases, nucleosides and nucleotides in rats.

Abstract: The uricogenic effects of dietary free purines (adenine, guanine, hypoxanthine and xanthine), their nucleosides (adenosine, monophosphate, guanosine monophosphate and inosine monophosphate) were studied in rats. Casein-based diets (20% protein) supplemented with 30 mmol/kg diet of each of the free purine base, nucleoside or nucleotide were fed to male Sprague-Dawley rats (100 +/- 5 g) for 14 d. Addition of adenine resulted in less weight gain than in controls, greater kidney weight, greater urine volume and higher levels of blood urea nitrogen, serum uric acid, creatinine and allantoin but lower urinary levels of allantoin, uric acid and creatinine. The adenine diet also caused nephropathy characterized by nephromegaly and deposition of crystals. A microscopic examination of the kidneys revealed deposition of crystals mainly in the lumen of convoluted tubules of the cortex. Feeding of diets containing other purine bases, nucleosides and nucleotides had no adverse effects on kidney weight or structure, urine volume, serum uric acid or creatinine. Urinary allantoin excretion, however, was greater in rats fed hypoxanthine, xanthine, nucleoside and nucleotide diets than in control rats. Adenine produced adverse effects only when fed in the free form and not when fed as the nucleoside or nucleotide, suggesting a metabolic significance for free adenine in predicting hyperuricemic effects of foods.

125I-labeled 8-phenylxanthine derivatives: antagonist radioligands for adenosine A1 receptors.

Abstract: A series of 8-phenylxanthine derivatives has been synthesized with oxyacetic acid on the para phenyl position to increase aqueous solubility and minimize nonspecific binding and iodinatable groups on the 1- or 3-position of the xanthine ring. The structure-activity relationship for binding of these compounds to A1 adenosine receptors of bovine and rat brain and A2 receptors of human platelets was examined. The addition of arylamine or photosensitive aryl azide groups to the 3-position of xanthine had little effect on A1 binding affinity with or without iodination, whereas substitutions at the 1-position caused greatly reduced A1 binding affinity. The addition of an aminobenzyl group to the 3-position of the xanthine had little effect on A2 binding affinity, but 3-aminophenethyl substitution decreased A2 binding affinity. Two acidic 3-(arylamino)-8-phenylxanthine derivatives were labeled with /sup 125/I and evaluated as A1 receptor radioligands. The new radioligands bound to A1 receptors with KD values of 1-1.25 nM. Specific binding represented over 80% of total binding. High concentrations of NaCl or other salts increased the binding affinity of acidic but not neutral antagonists, suggesting that interactions between ionized xanthines and receptors may be affected significantly by changes in ionic strength. On the basis of binding studies withmore » these antagonists and isotope dilution with the agonist (/sup 125/I)N6-(4-amino-3-iodobenzyl)adenosine, multiple agonist affinity states of A1 receptors have been identified.« less