Showing papers on "Xanthine published in 1985"

Identification of hypoxanthine transport and xanthine oxidase activity in brain capillaries.

Abstract: Microvessel segments were isolated from rat brain and used for studies of hypoxanthine transport and metabolism. Compared to an homogenate of cerebral cortex, the isolated microvessels were 3.7-fold enriched in xanthine oxidase. Incubation of the isolated microvessels with labeled hypoxanthine resulted in its rapid uptake followed by the slower accumulation of hypoxanthine metabolites including xanthine and uric acid. The intracellular accumulation of these metabolites was inhibited by the xanthine oxidase inhibitor allopurinol. Hypoxanthine transport into isolated capillaries was inhibited by adenine but not by representative pyrimidines or nucleosides. Similar results were obtained when blood to brain transport of hypoxanthine in vivo was measured using the intracarotid bolus injection technique. Thus, hypoxanthine is transported into brain capillaries by a transport system shared with adenine. Once inside the cell, hypoxanthine can be metabolized to xanthine and uric acid by xanthine oxidase. Since this reaction leads to the release of oxygen radicals, it is suggested that brain capillaries may be susceptible to free radical mediated damage. This would be most likely to occur in conditions where the brain hypoxanthine concentration is increased as following ischemia.

Failure of the xanthine oxidase inhibitor allopurinol to limit infarct size after ischemia and reperfusion in dogs.

Abstract: During the acute phase of myocardial ischemia, adenine nucleotides are degraded to nucleosides and bases, especially inosine and hypoxanthine. Simultaneously, xanthine dehydrogenase is converted to xanthine oxidase, an enzyme that converts hypoxanthine to xanthine, and xanthine to uric acid, producing a superoxide anion for each molecule of hypoxanthine or xanthine oxidized. To determine if free radicals via this enzymatic source contribute to cell death in myocardial ischemia, we determined whether allopurinol, an inhibitor of xanthine oxidase, could limit infarct size in a reperfusion preparation of myocardial infarction. The circumflex coronary artery of each of 34 dogs was occluded for 40 min, followed by reperfusion for 4 days. Infarct size then was measured by histologic methods and was related to major baseline predictors of infarct size, including anatomic area at risk and collateral blood flow. Infarct size was larger (NS) in the allopurinol (n = 8) than in the control (n = 11) group, a trend that was related to slightly higher (NS) collateral blood flow in the control group. We conclude that allopurinol has no beneficial effect in this preparation of experimental myocardial infarction. The results oppose the hypothesis that free radicals, produced via the xanthine oxidase reaction, are an important contributing factor in myocardial ischemic cell death.

Xanthine derivatives as antagonists at A1 and A2 adenosine receptors.

Abstract: A variety of alkylxanthines has been comparatively examined as antagonists of A1 adenosine receptors in rat fat cells, rat and bovine cerebral cortex and of A2 adenosine receptors in human platelets. With few exceptions all xanthine derivatives with 7-position substituents such as diprophylline, proxyfylline, pentoxifylline and etofylline were less potent antagonists than xanthine itself which hadK i-values of 170 μmol/l (A1) and 93 μmol/l (A2). Theophylline, caffeine and 3-isobutyl-1-methylxanthine were more potent than xanthine but nearly equipotent antagonists at both receptor subtypes. 8-Phenyl substituents considerably increased the antagonist potency at A1 and A2 receptors. 1,3-Diethyl-8-phenylxanthine was the most potent A2 antagonist (K i 0.2 μmol/l) in human platelets. At A1 receptors 1,3-dipropyl-8-(2-amino-4-chlorophenyl)xanthine (PACPX) was the most potent antagonist in all three tissues withK i-values from 0.3 to 8.6 nmol/l. Several 8-phenylxanthine derivatives were remarkably selective antagonists at A1 receptors. 8-Phenyltheophylline was approximately 700 times more potent as antagonist at A1 receptors (bovine brain) than at A2 receptors (human platelets), and PACPX was even 1,600 times more potent as A1 adenosine receptor antagonist. These compounds offer a possibility for a subtype-selective blockade of adenosine receptors.

Effects of oxygen radicals on cerebral arterioles

Abstract: Xanthine oxidase and xanthine, a combination that produces hydrogen peroxide and superoxide anion radical, applied topically in anesthetized cats equipped with cranial windows caused arteriolar dilation during application, sustained dilation 1 h after washout, and reduced reactivity to the vasoconstrictive effects of arterial hypocapnia, discrete lesions of the endothelium, and morphological abnormalities of the vascular smooth muscle by electron microscopy. Similar effects were seen in small, but not in large, arterioles during topical application of hydrogen peroxide or hydrogen peroxide plus ferrous sulfate, a combination that produces free hydroxyl radical. The functional changes caused by xanthine oxidase plus xanthine were inhibited completely by superoxide dismutase plus catalase. Superoxide dismutase or catalase, each by itself, eliminated the residual effects seen after washout and reduced the dilation during application of xanthine oxidase. The results show that superoxide anion radical and hydrogen peroxide produce reversible arteriolar dilation and that consistent vascular damage is produced in the presence of both superoxide anion radical and hydrogen peroxide.

Functionalized congeners of 1,3-dialkylxanthines: preparation of analogues with high affinity for adenosine receptors.

Abstract: Alkylxanthines, with theophylline (1) as the prototype, represent the major class of antagonists for adenosine receptors.1 Although theophylline and caffeine are relatively weak adenosine antagonists, with affinity constants in the 10–50 µM range, they undoubtedly owe many of their pharmacological effects to blockade of adenosine-mediated functions. Two classes of adenosine receptors have been characterized.1 The A1-adenosine receptor is inhibitory to adenylate cyclase and appears involved in antilipolytic, cardiac depressant, and central depressant effects of adenosine analogues. The A2-adenosine receptor is stimulatory to adenylate cyclase and appears involved in hypotensive, vasodilatory, antithrombotic, and endocrine effects of adenosine analogues. Xanthines, by blockade of adenosine binding at such receptors, would have the opposite effects. Some xanthines, such as 3-iso-butyl-1-methylxanthine, not only block adenosine receptors but also have potent inhibitory effects on phosphodiesterases.2a The presence of an 8-phenyl group greatly enhances the potency of theophylline as an adenosine antagonist.2a,b Even more potent antagonists result from the replacement of the 1,3-methyl groups of 8-phenyl-theophylline with n-propyl groups and by situating uncharged electron-donating para substituents on the 8-phenyl ring. Affinity constants (Ki) of less than 1 nM at A1-adenosine receptors have been attained.3,22 In addition to high potency, some selectivity toward the A1 subclass of adenosine receptors has been obtained with 1,3-dipropyl-8-phenylxanthines.4 1,3-Dipropyl-8-(p-hydroxyphenyl)xanthine (2b) was chosen as a suitable lead compound in an effort to develop potent and selective functionalized congeners as antagonists for adenosine receptors. An N6-phenyladenosine was chosen as a suitable lead compound for a parallel effort to develop potent and selective agonists for A1-adenosine receptors.5 In the design of active, covalent conjugates of drugs, the goals of the functionalized congener approach are several, including increasing the potency, prolonging the duration of action, and/or changing the specificity. This approach has been applied to isoproterenol and has lead to analogues with activity at β-adrenergic receptors as much as 4 orders of magnitude greater than that of isoproterenol.6–8 Nontherapeutic applications of active, functionalized drugs include receptor probes,5,9,10 immobilized ligands for affinity chromatography,11 and radio-labeled analogues. We report here the synthesis of a series of highly potent analogues of theophylline and 1,3-dipropylxanthine, some of which contain groups designed for radiolabeling through the introduction of radioisotopes of iodine. The functionalized congeners are also suitable for the preparation of affinity columns. A biotinylated conjugate is intended for use as a molecular probe through linkage to avidin.12a Recently, biotinylated derivatives of peptide hormones have been described.12b,c

Free radical damage to cultured porcine aortic endothelial cells and lung fibroblasts: modulation by culture conditions.

Abstract: Culture conditions modulating cell damage from xanthine plus xanthine oxidase-derived partially reduced oxygen species were studied. Porcine thoracic aorta endothelial cells and porcine lung fibroblasts were maintained in monolayer culture. Cells were prelabeled with51Cr before xanthine plus xanthine oxidase exposure. Endothelial cells showed 30 to 100% more lysis than fibroblasts and thus seemed more sensitive to this oxidant stress. The effect of cell culture age, as indicated by population doubling level (PDL), was examined. Response of low PDL endothelial cells and fibroblasts subjected to oxidant stress was compared with the response of PDL 15 cells. Both low PDL endothelial cells and fibroblasts responded differently to the lytic effect of xanthine oxidase-derived free radicals than did higher PDL cells. Specific activities of the antioxidant enzymes catalase, managanese superoxide dismutase, copper-zinc superoxide dismutase, glutathione peroxidase, and glucose-6-phosphate dehydrogenase were measured in both low and high PDL fibroblasts and endothelial cells. Antioxidant enzyme specific activities could only partially explain the differences in response to oxidant stress between fibroblasts and endothelial cells and between low and high PDL cells. Cell culture medium composition modulated the rate of production, and relative proportions of xanthine plus xanthine oxidase-derived partially reduced species of oxygen, i.e. superoxide, hydrogen peroxide, and hydroxyl radical. Serum content of medium was important in modulating free radical generation; superoxide production rates decreased 32%, H2O2 became undetectable, and hydroxyl radical generation decreased 54% in the presence of 10% serum. The medium protein and iron content also modulated free radical generation. The data suggest that cell culture media constituents, cell type, and cell culture age greatly affect in vitro response of cells subjected to oxidant stress.

Calcium enhances in vitro free radical-induced damage to brain synaptosomes, mitochondria, and cultured spinal cord neurons

Abstract: Preincubation of rat brain synaptosomes with xanthine and xanthine oxidase (X/XO) in Ca2+-free Krebs buffer resulted in a 27% inhibition of synaptosomal gamma-aminobutyric acid (GABA) uptake. Addition of 1.5 mM CaCl2 increased the inhibition with X/XO to 46%, and inhibition was essentially complete when the calcium ionophore A23187 also was included. In other studies, preincubation of purified rat brain mitochondria with the combination of X/XO and 4 microM CaCl2 produced a significant (38%) decrease in state 3 respiration with glutamate/malate as substrate that was not seen with either X/XO or Ca2+ alone. Similar results were obtained using cultured mouse spinal cord neurons in which incubation with X/XO/ADP/FeCl2 and A23187 produced membrane damage as assessed by a 32% reduction of neuronal Na+, K+-ATPase activity. Neither X/XO/ADP/FeCl2 nor A23187 alone caused detectable inhibition. These results demonstrate the synergistic damaging effect of free radicals and Ca2+ on membrane function. In addition, they suggest that free radical-induced peroxidation of membrane lipid, occurring focally during complete or nearly complete ischemia in vivo, could result in intense cellular perturbation when coupled with increased intracellular Ca2+.

Interaction of lipid peroxidation and calcium in the pathogenesis of neuronal injury.

Abstract: The interactions between lipid peroxidation and calcium in mediating damage to central nervous system membranes have been examined in several in vitro systems. Using isolated rat brain synaptosomes, brain mitochondria, or cultured fetal mouse spinal cord neurons, Ca2+ was found to markedly enhance lipid peroxidation-induced disruption of membrane function. Gamma-aminobutyric acid (GABA) uptake by synaptosomes was inhibited 25% by either lipid peroxidation (induced with xanthine and xanthine oxidase) or Ca2+ alone, whereas inhibition was 46% with their combination. Ca2+ enhancement of lipid peroxidation-induced damage to synaptosomes was intensified by the Ca2+ ionophore, A23187, and was partially blocked by the Ca2+ channel blocker, verapamil. Similarly, inhibition of state 3 respiration in isolated rat brain mitochondria was observed with Ca2+ and a free radical generating system (xanthine and xanthine oxidase) under conditions where either insult alone failed to cause detectable damage. Na+,K+-ATPase activity of cultured fetal mouse spinal cord neurons was inhibited 32% when cells were incubated for 30 minutes in the presence of both A23187 and a free radical generating system. However, Na+,K+-ATPase was not affected during a 30 minute incubation with either A23187 or radical generating system alone. In further studies, peroxidation of rat brain synaptosomes by ferrous iron (Fe2+) and H2O2 was coupled with a rapid and large (2-7-fold) uptake of Ca2+ by synaptosomes. Fe2+ also enhanced Ca2+ uptake by spinal cord neurons in culture, an effect that was coincident with peroxidation of neuronal membranes and the release of arachidonic acid from cells. Iron-induced Ca2+ uptake was blocked by high concentrations of either desferrioxamine or methylprednisolone, whereas Ca2+ channel blockers did not affect Ca2+ uptake induced by Fe2+. Finally, peroxidation of membrane lipids by Fe2+ was stimulated by Ca2+. Concentrations of Ca2+ as low as 10(-9) M increased peroxidation reactions within brain synaptosomal membranes. The results of these studies indicate that lipid peroxidation and Ca2+ can synergistically act to damage biologic membranes. The findings suggest that Ca2+ and lipid peroxidation cannot be considered as separate entities in the pathophysiology of CNS trauma. A hypothesis proposing an inseparable interplay between lipid peroxidation and Ca2+ in the pathogenesis of traumatic and ischemic cell injury is presented.

Use of adenosine derivatives as anti-allergic compounds and pharmaceutical compositions containing them

Abstract: The present invention is concerned with the use of adenosine derivatives for the treatment of allergic diseases, as well as for bronchosphatic and broncho-constrictive reactions brought about by inflammation. The above adenosine derivatives can be used alone or together with xanthine derivatives. The invention is further concerned with compositions containing compounds of adenosine derivatives and optionally compounds of xanthine derivatives together with appropriate pharmaceutical carriers.

Superoxide anion selectively attenuates catecholamine-induced contractile tension in isolated rabbit aorta

Abstract: Xanthine oxidase-derived oxygen metabolites caused a selective loss of norepinephrine-induced contractile tension in rings and helical strips from rabbit aorta. Phenylephrine-induced tension was not affected. The relaxation was selectively and completely blocked by superoxide dismutase but not by catalase. Isoproterenol-induced relaxation was also reversed by xanthine oxidase-derived oxygen metabolites. These observations are consistent with the chemical reaction of superoxide anion with catecholamines and suggest that the reaction may have significance at physiological concentrations of norepinephrine. The time course of the effects of superoxide generation on contractile tension was consistent with the properties of the chemical reaction (measured spectrophotometrically) and with the dependence of tone on norepinephrine concentration. These results indicate that superoxide anion, in situations at which submicromolar concentrations of this reduced oxygen metabolite are present, will selectively oxidize catecholamines, which may attenuate local adrenergic regulation.

Inhibition of Xanthine Oxidase by Flavonoids

Abstract: The influence of nineteen flavonoids on cow’s milk xanthine oxidase (xanthine: oxygen oxidoreductase, EC 1.2.3.2) was investigated. The enzyme activity was estimated by measuring the increase in absorbance at 290 nm due to uricate formation as well as by a colorimetric method assaying hydrogen peroxide generated from uricate by uricase. Among the flavonoids tested, myricetin, kaempferol, quercetin, fisetin, quercitrin, and morin inhibited the enzyme strongly at 50 μm; the concentrations which gave 50% inhibition (ID50) were 2, 2, 3, 7, 15, and 19μm, respectively. The inhibition mode of the former three compounds was of mixed type and the kinetic parameters were determined. Chrysin and naringenin were moderately inhibitory, while other flavonoids showed weak to no inhibition.

Protonation of hypoxanthine, guanine, xanthine, and caffeine

Abstract: The protonation equilibria of three hydroxypurines, hypoxanthine, guanine, and xanthine, and of related caffeine have been studied by 1H and 13C nmr and uv spectroscopies in aqueous sulfuric or perchloric acids. The results have been interpreted on the basis of the excess acidity method. The values and the protonation sites are discussed and comparisons are made with results of recent theoretical calculations.

Adenosine deaminase inhibition and myocardial purine release during normoxia and ischaemia

Abstract: Quantitative determination of myocardial adenosine formation and breakdown is necessary to gain insight into the mechanism and regulation of its physiological actions. Deamination of adenosine was studied in isolated perfused rat hearts by infusion of adenosine (1 to 20 μmol·litre-1). All catabolites in the perfusates (inosine, hypoxanthine, xanthine and uric acid) were measured, as well as unchanged adenosine. Apparent uptake of adenosine was determined; it increased linearly with the concentration of adenosine infused. Adenosine was predominantly deaminated, even at low (1 μmol·litre-1) concentration. The inhibitory capacity of the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA) was determined, while 5 μmol·litre-1 adenosine was infused. EHNA inhibited the apparent adenosine deaminase activity for 62 and 92% at 5 and 50 μmol·litre-1, respectively. When 50 μmol·litre-1 EHNA was infused into normoxic hearts, release of adenosine was significantly elevated, as was coronary flow. Induction of ischaemia increased total purine release four- to fivefold. Infusion of EHNA into ischaemic hearts did not alter total purine release, but adenosine release increased from 15 to 60% of total purines. However, when EHNA was present, a large part of total purine release still existed of inosine, hypoxanthine, xanthiner and uric acid. This was 83% during normoxia and 40% during ischaemia. These results suggest significant contribution of IMP and GMP breakdown to purine release from isolated perfused rat hearts.

ATP degradation products after ischemic exercise Hereditary lack of phosphorylase or carnitine palmityltransferase

Abstract: We measured purine degradation products of ATP in plasma after ischemic exercise in eight normal subjects, one patient with myo-phosphorylase deficiency (McArdle9s disease), and one with carnitine palmityltransferase deficiency. Normal subjects increase hypoxanthine and inosine, but not xanthine. Plasma purine levels were elevated above the normal range after ischemic exercise in McArdle9s disease and in carnitine palmityltransferase deficiency after fasting. Those changes implied abnormally accelerated ATP degradation in these two myopathies.

Mechanisms of ischemic injury in the heart.

Abstract: Cardiac ischemia is characterized by rapid deterioration of cardiac function, which has been linked to the fall in intracellular pH, increased levels of inorganic phosphate and reduction in free energy change of ATP-hydrolysis. Biochemical events responsible for irreversible myocardial injury involve various mechanisms which change the properties of the cardiac cell membrane (disorders in lipid metabolism, free radical formation). Recent evidence suggests that in the heart, xanthine oxidase is a major source of free radical formation. During ischemia, adenine-nucleotide breakdown in the cardiomyocyte proceeds only to the stage of inosine. Due to the localisation of nucleoside phosphorylase and xanthine-oxidase in vascular endothelium, further degradation of inosine to hypoxanthine, xanthine and uric acid occurs predominantly in the vascular space. It is therefore conceivable that the primary site of reperfusion injury in the ischemic heart may be the coronary endothelium damaged by free radicals.

Rapid assay for detection of Eschenchia coli xanthine-guanine phosphoribosyltransferase activity in transduced cells

Abstract: Cultured mammalian cells transduced with the Escherichia coli gene, Ecogpt, synthesize the bacterial enzyme xanthine-guanine phosphoribosyl transferase (XGPT) (1). This paper describes a method for measuring XGPT activity in crude cell extracts by following the conversion of 14C-xanthine (X) to 14C-xanthine monophosphate (XMP) and 14C-xanthosine (XR) by thin layer chromatography. The method is rapid, easy to use, sensitive and linear over a wide range of XGPT activity and has been useful for detecting XGPT in cells that were transiently transfected or stably transformed with Ecogpt. During our studies, we have found that a human cell line (XP20S) converts xanthine to XMP. This activity is probably catalyzed by a variant hypoxanthine-guanine phosphoribosyltransferase (HGPT) since the low activity is readily inhibited by hypoxanthine. A low level of conversion of X to XMP may explain why some cell lines are not killed in a medium containing mycophenolic acid and X.

Methylxanthine discrimination in the rat: possible benzodiazepine and adenosine mechanisms.

Abstract: Rats were trained to discriminate either caffeine or theophylline from saline using a two-lever discrimination paradigm. Since methylxanthines have been found to interfere with agonist binding at both adenosine and benzodiazepine (BDZ) receptors, chlordiazepoxide (CDP) and L-PIA (an adenosine analog) were tested for generalization to and blockade of both xanthine cues. Neither L-PIA nor CDP generalized to either xanthine cue, although both produced dose-related decreases in response rate. CDP, but not L-PIA, produced dose-related decreases in drug-lever responses when combined with training doses of caffeine or theophylline. Response rates indicated a complex interaction between the xanthines and both L-PIA and CDP. When combined with the caffeine training dose, pentobarbital also produced a dose-dependent decrease in response rate but not in drug lever choices. Finally, papaverine generalized to the caffeine cue in a dose-dependent fashion. In a second experiment, rats trained to discriminate CDP from saline showed no generalization in L-PIA tests. CDP-appropriate responding was not significantly affected when the CDP training dose was combined with caffeine. These data indicate that: (a) methylxanthine interactions with L-PIA and CDP on response rate likely involve blockade of adenosine mechanisms; (b) the xanthine cue does not appear to depend on interactions with adenosine receptors; and (c) the xanthine cue may involve effects on cyclic AMP activity and/or interaction with the BDZ/GABA receptor complex.

Free radicals and protein degradation in human red blood cells.

Abstract: Human red blood cells (RBC) were exposed to oxygen-based free-radicals, and other activated oxygen species generated during incubation with xanthine plus xanthine oxidase (X+XO). Oxygen-radical exposure induced up to 30 fold increases in human RBC protein degradation, compared to 12 fold increases in rabbit RBC protein degradation. Protein degradation increased as a function of X+XO, but demonstrated saturation kinetics at higher XO concentrations. The presence or absence of an energy substrate (glucose) had no effect on protein degradation, indicating the possible role of ATP-independent proteinolytic systems. It is proposed that human RBC proteins can be oxidatively damaged by certain free-radicals, and that the oxidized proteins are specifically recognized and degraded by intracellular proteinolytic systems.

Analgesic, anti-inflammatory and skeletal muscle relaxant compositions

Abstract: Pharmaceutical compositions and methods of using same comprising at least one non-steroidal anti-inflammatory drug other than aspirin, acetaminophen and phenacetin, in combination with at least one skeletal muscle relaxant, and optionally xanthine or a xanthine derivative, such as caffeine. The xanthine or xanthine derivative has a two-fold benefit; it enhances the effect of the non-steroidal anti-inflammatory drug and its stimulant effect counteracts the sedative effect of the skeletal muscle relaxant.