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• W1974539684 abstract "While the free radical-generating enzyme xanthine oxidase is a central mechanism of injury in postischemic tissues, questions remain regarding how xanthine oxidase-mediated radical generation is triggered during ischemia and reperfusion. There is controversy regarding whether radical generation is caused by enzyme formation or that of its substrates xanthine and hypoxanthine. Therefore, studies were performed in isolated rat hearts correlating the magnitude and time course of radical generation with alterations in xanthine oxidase and its substrates. Radical generation was measured by electron paramagnetic resonance spectroscopy and correlated with spectrophotometric assays of tissue xanthine oxidase activity and chromatographic measurement of tissue and effluent concentrations of xanthine oxidase substrates and products. Xanthine oxidase was present in preischemic hearts and slightly increased during 30-min global ischemia. Hypoxanthine and xanthine were not present prior to ischemia but accumulated greatly during ischemia due to ATP degradation. These substrate concentrations rapidly declined over the first 5 min of reperfusion matching the observed time course of radical generation, whereas xanthine oxidase activity was largely unchanged. Both substrates were also observed in the coronary effluent during the first 5 min of reflow along with the product uric acid. Thus, the burst of xanthine oxidase-mediated free radical generation upon reperfusion is triggered and its time course controlled by a large increase in substrate formation that occurs secondary to the degradation of ATP during ischemia. While the free radical-generating enzyme xanthine oxidase is a central mechanism of injury in postischemic tissues, questions remain regarding how xanthine oxidase-mediated radical generation is triggered during ischemia and reperfusion. There is controversy regarding whether radical generation is caused by enzyme formation or that of its substrates xanthine and hypoxanthine. Therefore, studies were performed in isolated rat hearts correlating the magnitude and time course of radical generation with alterations in xanthine oxidase and its substrates. Radical generation was measured by electron paramagnetic resonance spectroscopy and correlated with spectrophotometric assays of tissue xanthine oxidase activity and chromatographic measurement of tissue and effluent concentrations of xanthine oxidase substrates and products. Xanthine oxidase was present in preischemic hearts and slightly increased during 30-min global ischemia. Hypoxanthine and xanthine were not present prior to ischemia but accumulated greatly during ischemia due to ATP degradation. These substrate concentrations rapidly declined over the first 5 min of reperfusion matching the observed time course of radical generation, whereas xanthine oxidase activity was largely unchanged. Both substrates were also observed in the coronary effluent during the first 5 min of reflow along with the product uric acid. Thus, the burst of xanthine oxidase-mediated free radical generation upon reperfusion is triggered and its time course controlled by a large increase in substrate formation that occurs secondary to the degradation of ATP during ischemia. Oxygen free radical generation has been proposed to be an important mechanism of cellular injury in ischemic and reperfused tissues(1Kukreja R.C. Hess M.L. Cardiovasc. Res. 1992; 26: 641-655Crossref PubMed Scopus (379) Google Scholar, 2Simpson P.J. Lucchesi B.R. J. Lab. Clin. Med. 1987; 110: 13-30PubMed Google Scholar). Studies in a variety of tissues, including heart, lung, kidney, and brain, have demonstrated that intravascular administration of antioxidant enzymes or free radical-scavenging drugs can prevent reperfusion damage and improve postischemic function(3Cerutti P.A. Fridovich I. McCord J.M. Oxy-radicals in Molecular Biology and Pathology. Alan R. Liss, New York1988: 1-563Google Scholar, 4Taylor A.E. Matalon S. Ward P.A. Physiology of Oxygen Radicals. William and Wilkins, Baltimore1986: 1-287Google Scholar). These studies have provided indirect evidence of free radical generation in postischemic tissues. Recently, free radical generation has been measured in ischemic tissues with electron paramagnetic resonance (EPR) spectroscopy. Both direct and spin trapping EPR techniques have demonstrated that there is a burst of oxygen free radical generation in the heart after postischemic reperfusion(5Zweier J.L. Flaherty J.T. Weisfeldt M.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1404-1407Crossref PubMed Scopus (1026) Google Scholar, 6Zweier J.L. J. Biol. Chem. 1988; 263: 1353-1357Abstract Full Text PDF PubMed Google Scholar, 7Zweier J.L. Kuppusamy P. Williams R. Rayburn B.K. Smith D. Weisfeldt M.L. Flaherty J.T. J. Biol. Chem. 1989; 264: 18890-18895Abstract Full Text PDF PubMed Google Scholar, 8Arroyo C.M. Kramer J.H. Dickens B.F. Weglicki W.B. FEBS Lett. 1987; 221: 101-104Crossref PubMed Scopus (308) Google Scholar). It was further demonstrated that the administration of copper-zinc superoxide dismutase, solely upon reperfusion, can prevent contractile dysfunction and quench free radical generation(6Zweier J.L. J. Biol. Chem. 1988; 263: 1353-1357Abstract Full Text PDF PubMed Google Scholar, 9Ambrosio G. Becker L.C. Hutchins G.M. Weisman H.F. Weisfeldt M.L. Circulation. 1986; 74: 1424-1433Crossref PubMed Scopus (303) Google Scholar). Although several mechanisms have been demonstrated to be involved in the generation of oxygen free radicals, xanthine oxidase has been proposed to be a central mechanism in a variety of postischemic cells and tissues(10McCord J.M. N. Engl. J. Med. 1985; 312: 159-163Crossref PubMed Scopus (4999) Google Scholar, 11Roy R.S. McCord J.M. Greenwald R.A. Cohen G. Oxy Radicals and Their Scavenger Systems. Elsevier Science Publishing Co., Inc., New York1983: 145-153Google Scholar, 12Zweier J.L. Kuppusamy P. Lutty G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4046-4050Crossref PubMed Scopus (561) Google Scholar). Although there has been controversy regarding whether this mechanism occurs in human cells and tissues, recent studies have shown that the enzyme is present in human endothelial cells and is responsible for free radical generation in reoxygenated human endothelial cells(13Zweier J.L. Broderick R. Kuppusamy P. Thompson-Gorman S.L. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar). In reperfused tissues, xanthine oxidase in the presence of its substrates hypoxanthine or xanthine reduces molecular oxygen to O2− and H2O2, which can further react to form the more reactive OH(14Kuppusamy P. Zweier J.L. J. Biol. Chem. 1989; 264: 9880-9884Abstract Full Text PDF PubMed Google Scholar, 15Thompson-Gorman S.L. Zweier J.L. J. Biol. Chem. 1990; 265: 6656-6663Abstract Full Text PDF PubMed Google Scholar). The OH and O2− radicals produced by the enzyme can then in turn react with cellular proteins and membranes inducing cellular injury. In particular, it was hypothesized that in ischemic tissues xanthine dehydrogenase, which reduces NAD to NADH, is converted via proteolytic cleavage to xanthine oxidase(10McCord J.M. N. Engl. J. Med. 1985; 312: 159-163Crossref PubMed Scopus (4999) Google Scholar, 16Parks D.A. Granger D.N. Acta Physiol. Scand. 1986; 548: 87-99Google Scholar). This increase in xanthine oxidase activity has been proposed to play a key role in triggering postischemic free radical generation. It has been demonstrated that the enzyme and its substrates are present and give rise to a burst of free radical generation upon postischemic reperfusion in the isolated rat heart, as well as in bovine and human endothelial cells, but considerable controversy still remains regarding the process by which ischemia and reperfusion triggers xanthine oxidase-mediated free radical generation(12Zweier J.L. Kuppusamy P. Lutty G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4046-4050Crossref PubMed Scopus (561) Google Scholar, 13Zweier J.L. Broderick R. Kuppusamy P. Thompson-Gorman S.L. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar, 15Thompson-Gorman S.L. Zweier J.L. J. Biol. Chem. 1990; 265: 6656-6663Abstract Full Text PDF PubMed Google Scholar). Despite extensive study over the last decade, the regulation and control of this important mechanism of free radical generation and cellular injury is largely unknown. Questions remain regarding whether it is enzyme or substrate formation that triggers the burst in free radical generation which occurs upon postischemic reperfusion(10McCord J.M. N. Engl. J. Med. 1985; 312: 159-163Crossref PubMed Scopus (4999) Google Scholar, 16Parks D.A. Granger D.N. Acta Physiol. Scand. 1986; 548: 87-99Google Scholar, 17Chambers D.E. Parks D.A. Patterson G. Roy R. McCord J.M. Yoshida S. Parmely L.F. Downey J.M. J. Mol. Cell Cardiol. 1985; 17: 145-152Abstract Full Text PDF PubMed Scopus (562) Google Scholar, 18Adkison D. Hollwarth M.E. Benoit J.N. Parks D.A. McCord J.M. Granger D.N. Acta Physiol. Scand. 1986; 548: 101-107Google Scholar, 19Stirpe F. Dellas Corte E. J. Biol. Chem. 1969; 244: 3855-3863Abstract Full Text PDF PubMed Google Scholar, 20Frideriks W.M. Marx F. Kooij A. Int. J. Exp. Pathol. 1993; 74: 21-26PubMed Google Scholar, 21Abd-Elfattah A.S. Jessen M.E. Lekven J. Doherty III, N.E. Brunsting L.A. Wechsler A.S. Circulation. 1988; 78 (Suppl. III–): III224-III235PubMed Google Scholar, 22Abd-Elfattah A.S. Jessen M.E. Hanan S.A. Tuchy G. Wechsler A.S. Circulation. 1990; 80 (Suppl. IV–): IV341-IV350Google Scholar). The precise effect of alterations in myocardial enzyme and substrate concentrations in controlling the time course of free radical generation is also unknown. Therefore, controversy has remained regarding the role of xanthine oxidase and its radical-generating substrates in triggering the presence and time course of free radical generation in the postischemic heart. In the present study, experiments were performed to evaluate the role of alterations in xanthine oxidase and its substrates hypoxanthine and xanthine during myocardial ischemia and reperfusion in triggering the presence and controlling the time course of free radical generation in the postischemic rat heart. Experiments were performed correlating the time course of free radical generation, with alterations in xanthine oxidase activity and the concentrations of its free radical-generating substrates, during the process of myocardial ischemia and reperfusion. These studies indicate that substrate availability is the primary factor in triggering and controlling xanthine oxidase-mediated free radical generation in the postischemic heart. Female Sprague-Dawley rats (250-300 g) were heparinized and anesthetized with intraperitoneal pentobarbital. The hearts were excised, the aorta was cannulated, and retrograde perfusion was initiated. Hearts were perfused at a constant pressure of 80 mm Hg using Krebs bicarbonate buffer consisting of 120 mM NaCl, 17 mM glucose, 25 mM NaHCO3, 5.9 mM KCl, 1.2 mM MgCl2, 2.0 mM CaCl2, and bubbled with 95% O2 and 5% CO2 gas at 37°C as described previously(15Thompson-Gorman S.L. Zweier J.L. J. Biol. Chem. 1990; 265: 6656-6663Abstract Full Text PDF PubMed Google Scholar). After 15 min of equilibration with normal perfusate, the hearts were subjected to 30-min global ischemia and variable time of reflow according to the experimental purpose. Studies were performed using the spin trap 5,5-dimethyl-1-pyrroline-N-oxide (DMPO). 1The abbreviations used are: DMPO5,5-dimethyl-1-pyrroline-N-oxidePMSFphenylmethylsulfonyl fluorideDTTdithiothreitolHPLChigh performance liquid chromatography. The DMPO (>97% pure) was purchased from Aldrich and further purified by double distillation. DMPO was infused through a sidearm located just proximal to the heart perfusion cannula with a final concentration of 40 mM(5Zweier J.L. Flaherty J.T. Weisfeldt M.L. Proc. Natl. Acad. Sci. U. S. A. 1987; 84: 1404-1407Crossref PubMed Scopus (1026) Google Scholar, 15Thompson-Gorman S.L. Zweier J.L. J. Biol. Chem. 1990; 265: 6656-6663Abstract Full Text PDF PubMed Google Scholar). Spin trap-containing effluent was collected in 20-s aliquots prior to ischemia (control) and at different times of reflow. 5,5-dimethyl-1-pyrroline-N-oxide phenylmethylsulfonyl fluoride dithiothreitol high performance liquid chromatography. EPR spectra were recorded in flat cells at room temperature with a Bruker-IBM ER 300 spectrometer operating at X-band with a TM 110 cavity using a modulation frequency of 100 kHz, modulation amplitude of 0.5 G, microwave power of 20 milliwatts, microwave frequency of 9.77 GHz, and acquisition of 10 1-min scans. Quantitation of the free radical signals was performed by comparing the double integral of the observed signal to that of a known concentration of the 2,2,6,6-tetramethylpiperidinoxy free radical in aqueous solution as described previously(12Zweier J.L. Kuppusamy P. Lutty G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4046-4050Crossref PubMed Scopus (561) Google Scholar, 13Zweier J.L. Broderick R. Kuppusamy P. Thompson-Gorman S.L. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar). Control hearts were perfused for 15 min and then immediately frozen in liquid nitrogen. Ischemic and reperfused hearts were subjected to global ischemia for 30 min followed by variable duration of reflow and then frozen in liquid nitrogen. Frozen hearts were finely ground under liquid nitrogen and homogenized in 5 ml of 50 mM potassium phosphate buffer, pH 7.8, containing 1 mM phenylmethylsulfonyl fluoride (PMSF) and 10 mM dithiothreitol (DTT), which prevented the in vitro conversion of xanthine dehydrogenase to xanthine oxidase. A small amount of homogenate was removed for assay of total protein content by Lowry assay(23Lowry O.H. Rosebrough N.J. Farr A.L. Randall R.L. J. Biol. Chem. 1951; 193: 265-275Abstract Full Text PDF PubMed Google Scholar). Homogenates were spun at 600 × g for 20 min at −4°C, and the supernatants were centrifuged at 105,000 × g for 60 min at 4°C. The lipid layer was removed, and the supernatants were passed through a Sephadex G-25 column (Phamarcia Biotech Inc.) equilibrated with the phosphate buffer. The processed effluent was assayed spectrophotometrically at 295 nm for uric acid production in the presence of 60 μM xanthine using a Hewlett-Packard 8452A diode array spectrophotometer as described previously(13Zweier J.L. Broderick R. Kuppusamy P. Thompson-Gorman S.L. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar, 15Thompson-Gorman S.L. Zweier J.L. J. Biol. Chem. 1990; 265: 6656-6663Abstract Full Text PDF PubMed Google Scholar). The reaction mixture contained 0.2 ml of effluent, in 50 mM phosphate buffer containing PMSF and DTT, and 60 μM xanthine in a 1-ml cuvette at 37°C for the measurement of xanthine oxidase activity. For determination of total xanthine dehydrogenase plus oxidase activity, measurements were performed after the addition of 0.6 mM NAD+ to the reaction mixture. Enzyme activity was expressed in milliunits/g of protein, where 1 unit of activity equals 1 μmol of substrate converted to uric acid per min. After 15-min perfusion with equilibration of contractile function, hearts were frozen in liquid nitrogen as a preischemic control. For ischemic and reperfusion measurements, hearts were subjected to further 30-min periods of global ischemia or 30 min ischemia followed by variable duration of reflow. The hearts were immediately frozen in liquid nitrogen at the desired time point and then ground under liquid nitrogen. Then the frozen tissue was transferred to a glass homogenizer with a Teflon pastel and allowed to thaw with homogenization in 10 ml of 0.5 M perchloric acid at 4°C. Acid extraction continued on ice for 15 min, at which time cellular debris was pelleted by centrifugation at 14,000 × g for 1 min. The acid extract was neutralized by mixing with 20 ml of Freon/trioctylamine (4:1) for 30 s. The mixture was centrifuged at 14,000 × g for 1 min, and the upper aqueous layer was recovered and passed through a 0.45-μm Millipore filter, then stored at −80°C for analysis. A separate series of hearts was studied with collection of effluent perfusate prior to ischemia and after reflow to determine the time course of xanthine oxidase substrate and product release in the coronary effluent. 30-s collections of perfusate were performed at each sampling time, and these perfusate samples were simply passed through 0.45-μm Millipore filters prior to HPLC analysis. Reversed-phase HPLC was performed using a procedure similar to that of Hull-Ryde et al.(24Hull-Ryde E.A. Lewis W.R. Veronee C.D. Jowe J. J. Chromatogr. Biomed. Appl. 1986; 377: 165-174Crossref Scopus (76) Google Scholar) using a Waters μBondapak C18 column and a Waters the HPLC system (Waters Associates, Milford, MA) with a model 484 UV detector, two model 510 reciprocating pumps, and Maxima software, as described previously(13Zweier J.L. Broderick R. Kuppusamy P. Thompson-Gorman S.L. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar). Data are expressed as mean ± S.E. The statistical significance of the differences between the groups were calculated using Student's t test for independent means. Two-tailed values of p < 0.05 were considered significant. It has been reported previously that a burst of free radical generation occurs in the isolated rat heart upon postischemic reperfusion, which can be measured using the spin trap DMPO, and this radical generation was shown to be inhibited by the xanthine oxidase blocker oxypurinol(15Thompson-Gorman S.L. Zweier J.L. J. Biol. Chem. 1990; 265: 6656-6663Abstract Full Text PDF PubMed Google Scholar). It has also been reported that there is a temporal window of ischemic duration which results in maximum free radical generation after reperfusion, with maximum radical generation observed after an ischemic duration of 30 min(7Zweier J.L. Kuppusamy P. Williams R. Rayburn B.K. Smith D. Weisfeldt M.L. Flaherty J.T. J. Biol. Chem. 1989; 264: 18890-18895Abstract Full Text PDF PubMed Google Scholar). Therefore, experiments were performed measuring the time course of cardiac free radical generation following 30-min ischemia. The coronary effluent containing the spin trap DMPO was collected in 20-s aliquots during both preischemic and postischemic infusion and EPR measurements were performed. No significant signal was observed prior to ischemia; however, a prominent spectrum was seen immediately after the onset of reflow (Fig. 1). This spectrum consisted of a large 1:2:2:1 quartet signal, aH = aN = 14.9 G, indicative of DMPO-OH, and a small 1:1:1:1:1:1 sextet signal, aH = 16.0 and aN = 22.0 G, indicative of DMPO-R. Maximum signal intensity was observed at 20-40 s of reflow and then this signal decreased over the next 5 min of reperfusion. After 5 min of reflow, the amplitude of observed free radical signals was decreased to less than 15% of the maximum observed signal. No significant signal was observed after 10 min of reperfusion (Fig. 2). In a series of four hearts similar magnitude and time course of free radical generation were seen to that shown in Fig. 1, with peak radical concentrations observed in the range of 180-240 nM occurring over the first minute of reperfusion followed by a rapid decline over the next 5 min.Figure 2:Graph of the time course of total free radical generation in the pre- and postischemic rat heart. Data were obtained from the intensity of the EPR spectra observed from the coronary effluent of hearts perfused with 40 mM DMPO as described in the legend to Fig. 1. Data are shown with a break in the reperfusion time axis between 120 and 300 s, after which the scale is condensed in order to depict the complete time course of free radical generation after the 30-min period of ischemia.View Large Image Figure ViewerDownload Hi-res image Download (PPT) In order to evaluate the contribution of alterations in myocardial xanthine oxidase activity in triggering and controlling the time course of free radical generation, spectrophotometric assays were performed to measure the activity of this enzyme in preischemic and postischemic hearts. DTT together with the protease inhibitor PMSF was used to prevent the proteolytic or oxidative conversion of xanthine dehydrogenase to xanthine oxidase during tissue processing. In order to assure that the DTT did not convert xanthine oxidase back to xanthine dehydrogenase, additional experiments were performed in homogenates prepared in the absence of DTT. As reported previously(15Thompson-Gorman S.L. Zweier J.L. J. Biol. Chem. 1990; 265: 6656-6663Abstract Full Text PDF PubMed Google Scholar), identical xanthine oxidase activities were observed in the presence or absence of DTT, indicating that the DTT did not significantly convert xanthine oxidase back to xanthine dehydrogenase. Both control and postischemic heart tissue contained significant xanthine oxidase and xanthine dehydrogenase activity. As shown in the representative tracings (Fig. 3), prominent xanthine oxidase activity was present and corresponded to about 7% of the total xanthine oxidase plus xanthine dehydrogenase activity. After 30-min ischemia and 10-min reperfusion the activity of xanthine oxidase was increased by about 30%. A series of hearts were studied to characterize the time course of alterations in xanthine oxidase and xanthine dehydrogenase activity during ischemia and reflow (Fig. 4). In preischemic hearts, the activity of xanthine oxidase and xanthine dehydrogenase were 8.4 ± 1.2 and 130 ± 28 milliunits/g of protein, respectively. After 30 min of ischemia, there was no significant change in total xanthine oxidase and xanthine dehydrogenase activity. However, the activity of xanthine oxidase markedly increased to 16.8 ± 1.2 (p < 0.05, versus preischemia) after 1-min reperfusion. Over the subsequent 45-min period of reperfusion the activity of xanthine oxidase remained 30-50% higher than that observed in preischemic control hearts. While some decrease in the activity was seen between 2 and 10 min of reflow, overall the activity of xanthine dehydrogenase was not significantly altered during the time course of reflow. In plots of the ratio of xanthine oxidase activity to xanthine oxidase plus xanthine dehydrogenase activity it was observed that the percent xanthine oxidase increased from base-line values of 7% prior to ischemia to peak values of 11% after reflow. Since only a small increase was seen, this indicates that at most only a small fraction of the xanthine dehydrogenase may have been converted to xanthine oxidase. While the increases in xanthine oxidase activity observed during reperfusion could contribute to the increase in free radical generation which occurs upon reperfusion, these changes are relatively modest and cannot explain the absence of radical generation prior to ischemia or the time course which occurs.Figure 4:Graphs showing the time course of alterations in xanthine oxidase (XO), xanthine dehydrogenase (XD), and the ratio of XO to (XO+XD) in the rat hearts prior to ischemia and after 30-min ischemia with different durations of reperfusion. Data are shown with a break in the reperfusion time axis between 120 and 300 s, after which the scale is condensed in order to depict the complete time course throughout the 45-min period of reflow.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Since free radical generation from xanthine oxidase is also controlled by the concentration of the available substrates, xanthine and hypoxanthine, experiments were performed to measure the presence and magnitude of the alterations in the concentrations of these substrates in the postischemic heart. Reversed-phase HPLC of heart extracts was performed to measure the intracellular adenine nucleotide pool of normal and postischemic hearts. As shown in Fig. 5a, and reported previously(13Zweier J.L. Broderick R. Kuppusamy P. Thompson-Gorman S.L. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar), each of the cellular nucleotides and their metabolites have unique retention times allowing measurement and quantitation. In the preischemic heart, ATP comprises a major part of total adenine pool, as well as ADP and AMP. As shown in Fig. 5b, prior to ischemia, a large ATP peak and smaller ADP peak are seen, whereas no xanthine or hypoxanthine peaks are observed. After 30-min ischemia the ATP peak is greatly decreased, with a large increase in the AMP peak and the appearance of prominent xanthine and hypoxanthine peaks. After 5 min of reflow the xanthine and hypoxanthine peaks are decreased and are again almost undetectable. In a series of 27 hearts, 3 for each time point, HPLC measurements were performed, and the concentration of each metabolite was quantitated from these chromatograms and values were normalized for protein content (Fig. 6). At the end of 30 min of ischemia, the ATP level decreased to 7.14 ± 0.4 nmol/mg of protein compared with a preischemic value of 33.4 ± 0.5 nmol/mg of protein (p < 0.01). During reperfusion, ATP concentrations showed a slight recovery but remained depressed compared with preischemic values (Fig. 6, top panel). No hypoxanthine, xanthine, or uric acid were detected in preischemic hearts (Fig. 5b and 6). After 30 min of ischemia, however, significant hypoxanthine and xanthine formation and accumulation occurred (Fig. 5c), with average concentrations of 2.18 ± 0.27 and 2.41 ± 0.22 nmol/mg of protein, respectively. After the onset of reflow, hypoxanthine and xanthine levels rapidly decreased. After 5 min of reperfusion, no significant hypoxanthine and xanthine peaks were observed in the myocardium (Fig. 6).Figure 6:Time course of the alterations in ATP, hypoxanthine, and xanthine concentrations in isolated rat hearts during ischemia and reperfusion. Data are shown from preischemic hearts, hearts subjected to 30-min global ischemia, and hearts subjected to different durations of reflow. The data are graphed with a break in the reperfusion time axis between 120 and 300 s, after which the scale is condensed in order to depict the entire time course throughout the 45-min period of reflow. Each point corresponds to the mean ± S.E. values obtained from three hearts.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Two possible pathways may be involved in the rapid decline of hypoxanthine and xanthine in the myocardium during the early period of reperfusion. One possibility is that hypoxanthine and xanthine were converted by xanthine oxidase to form uric acid with accompanying oxygen free radical generation. Another possibility is that reflow may wash out these substrates from the heart. To determine the release of xanthine oxidase substrates and products, coronary effluent was collected in 30-s intervals prior to ischemia and after postischemic reperfusion. 33 samples were studied from a series of 3 hearts, and HPLC measurements were performed on each sample. Representative chromatograms of the effluent from a heart prior to and after reflow are shown in Fig. 7. Prior to ischemia no hypoxanthine and xanthine peaks were seen, and no significant uric acid peak was detected in coronary effluent. After reflow, however, large hypoxanthine and xanthine peaks were observed along with a large peak from the xanthine oxidase product uric acid (Fig. 7b). Maximum substrate and product peaks were seen over the first 30 s of reflow and then peak height declined over the next 5 min until only trace peaks were seen. The amount of xanthine, hypoxanthine, and uric acid in each 30 s effluent sample was quantitated. In the effluent from the first 30 s of reflow, average values of 1.1 ± 0.3 and 3.1 ± 0.8 nmol were observed for hypoxanthine and xanthine, respectively, with a value of 2.5 ± 0.5 nmol for uric acid. The observed amounts of both the substrates hypoxanthine and xanthine as well as the product uric acid rapidly decreased in the following 2 min of reflow, until after 5-min reperfusion, no hypoxanthine, xanthine, or uric acid was measured in coronary effluent (Fig. 8). The time course observed for xanthine oxidase substrate and product formation in the heart tissue and coronary effluent closely correlated with that observed in the EPR spin trapping measurements of free radical generation, suggesting that the presence and time course of free radical generation was triggered and controlled primarily by the availability of enzyme substrates.Figure 8:Graph of the amount of xanthine, hypoxanthine, and uric acid measured in the coronary effluent from preischemic and reperfused hearts by HPLC. Data are shown with a break in the reperfusion time axis between 150 and 300 s, after which the scale is condensed in order to depict the complete time course before ischemia and throughout the 45-min period of reflow. Each point corresponds to the mean ± S.E. value obtained from measurements of three hearts.View Large Image Figure ViewerDownload Hi-res image Download (PPT) Oxidative reperfusion injury is thought to be a central mechanism of the cellular damage affecting all organs and tissues after ischemia; however, the mechanisms which trigger and modulate this damage are still only partially characterized. Xanthine oxidase has been proposed to be a major primary source of free radical generation. This hypothesis was initially supported by many studies which demonstrated that administration of the xanthine oxidase inhibitors allopurinol or oxypurinol reduced infarct size and improved functional recovery(17Chambers D.E. Parks D.A. Patterson G. Roy R. McCord J.M. Yoshida S. Parmely L.F. Downey J.M. J. Mol. Cell Cardiol. 1985; 17: 145-152Abstract Full Text PDF PubMed Scopus (562) Google Scholar, 25Chambers D.J. Braimbridge M.V. Hearse D.J. Ann. Thorac. Surg. 1987; 44: 291-297Abstract Full Text PDF PubMed Scopus (49) Google Scholar); however, subsequently these results have been questioned by other laboratories who have questioned whether the enzyme xanthine oxidase or its substrates are present and whether it can cause reperfusion injury (26Reimer K.A. Jennings R.B. Circulation. 1985; 71: 1069-1975Crossref PubMed Scopus (143) Google Scholar, 27Eddy L.J. Stewart J.R. Jones H.P. Engerson T.D. McCord J.M. Downey J.M. Am. J. Physiol. 1987; 253: H709-H711PubMed Google Scholar). More recently, further direct evidence for the presence of xanthine oxidase-mediated free radical generation in postischemic cells and tissues has been provided by EPR studies(12Zweier J.L. Kuppusamy P. Lutty G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4046-4050Crossref PubMed Scopus (561) Google Scholar, 13Zweier J.L. Broderick R. Kuppusamy P. Thompson-Gorman S.L. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar, 15Thompson-Gorman S.L. Zweier J.L. J. Biol. Chem. 1990; 265: 6656-6663Abstract Full Text PDF PubMed Google Scholar). It has been demonstrated that a burst of xanthine oxidase-mediated radical generation occurs upon reperfusion and is the major source of free radicals in the isolated rat heart. This burst of radical generation can be blocked by the xanthine oxidase inhibitor oxypurinol (15Thompson-Gorman S.L. Zweier J.L. J. Biol. Chem. 1990; 265: 6656-6663Abstract Full Text PDF PubMed Google Scholar). In the present study we observe that the burst of radical generation peaks during the first minute of reperfusion with little, if any, radical generation continuing after 5 min. A number of studies have shown that endothelial cells from animal species as well as humans contain xanthine oxidase and similarly give rise to the formation of oxygen free radicals at the time of reoxygenation(12Zweier J.L. Kuppusamy P. Lutty G.A. Proc. Natl. Acad. Sci. U. S. A. 1988; 85: 4046-4050Crossref PubMed Scopus (561) Google Scholar, 13Zweier J.L. Broderick R. Kuppusamy P. Thompson-Gorman S.L. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar, 15Thompson-Gorman S.L. Zweier J.L. J. Biol. Chem. 1990; 265: 6656-6663Abstract Full Text PDF PubMed Google Scholar). In human aortic endothelial cells we have demonstrated that free radical generation occurs due to xanthine oxidase substrate formation in the presence of the enzyme. However, in these isolated cellular preparations the observed free radical generation persists for more than 30 min after reoxygenation. Therefore, while xanthine oxidase-mediated free radical generation has been observed both in isolated endothelial cells and whole tissues such as the heart, clear differences in the time course of radical generation are present that could not be explained previously. Although there is considerable evidence that xanthine oxidase-mediated free radical generation occurs in reperfused tissues, the precise nature of the enzyme and substrate control of the magnitude and time course of this process had not been elucidated. Both the formation and modulation of tissue concentrations of xanthine oxidase and its requisite substrates are factors that would be expected to limit and regulate the generation of free radicals in the pathogenesis of reperfusion injury. McCord (10McCord J.M. N. Engl. J. Med. 1985; 312: 159-163Crossref PubMed Scopus (4999) Google Scholar, 11Roy R.S. McCord J.M. Greenwald R.A. Cohen G. Oxy Radicals and Their Scavenger Systems. Elsevier Science Publishing Co., Inc., New York1983: 145-153Google Scholar) proposed that in ischemic tissues xanthine dehydrogenase undergoes proteolytic conversion to the oxidase that uses O2 as its electron acceptor with the formation of superoxide and hydrogen peroxide. This protease-mediated increase in xanthine oxidase activity was hypothesized to play a major role in triggering the generation of oxygen free radicals. Subsequently, it was proposed that this protease-dependent xanthine oxidase conversion was the major event triggering xanthine oxidase-mediated reperfusion injury (16Parks D.A. Granger D.N. Acta Physiol. Scand. 1986; 548: 87-99Google Scholar, 17Chambers D.E. Parks D.A. Patterson G. Roy R. McCord J.M. Yoshida S. Parmely L.F. Downey J.M. J. Mol. Cell Cardiol. 1985; 17: 145-152Abstract Full Text PDF PubMed Scopus (562) Google Scholar, 18Adkison D. Hollwarth M.E. Benoit J.N. Parks D.A. McCord J.M. Granger D.N. Acta Physiol. Scand. 1986; 548: 101-107Google Scholar). However, subsequent reports (28Engerson T.D. McKelvey T.G. Jones H.P. FASEB J. 1989; 3 (abstr.): 628Google Scholar, 29Coudray C. Pucheu S. Boucher F. de Leiris J. Favier A. Basic Res. Cardiol. 1992; 87: 478-488Crossref PubMed Scopus (18) Google Scholar) have shown little if any conversion of dehydrogenase to oxidase in perfused rat hearts following periods of global ischemia. Similarly, in the present study we did observe that xanthine oxidase is present in the preischemic rat heart, with 7% of the dehydrogenase plus oxidase pool present as oxidase. While a 50-100% increase in xanthine oxidase activity was observed upon reperfusion, still only a small proportion of the xanthine dehydrogenase appeared to be converted to xanthine oxidase. Our results clearly demonstrate that although xanthine oxidase activity is somewhat increased in the postischemic heart, these alterations of xanthine oxidase activity alone do not explain the time course of free radical generation that is observed in the normal and postischemic heart. Prior to ischemia, xanthine oxidase is present, but no free radical generation is observed. Although the enzyme activity is increased after ischemia, it is largely unchanged throughout the time course of reflow; however, free radical generation is only observed during the first 5 min of reperfusion. These data indicate that although the presence of xanthine oxidase is an important requisite factor in the process of free radical generation, alterations in xanthine oxidase activity are not sufficient to account for the observed time course of radical generation and are not the major limiting factor in the process of free radical generation during postischemic reperfusion. It was also well known that during ischemia ATP is degraded to ADP, AMP, and adenosine, which could be further metabolized to inosine, hypoxanthine, and xanthine(21Abd-Elfattah A.S. Jessen M.E. Lekven J. Doherty III, N.E. Brunsting L.A. Wechsler A.S. Circulation. 1988; 78 (Suppl. III–): III224-III235PubMed Google Scholar, 30Jennings R.B. Steenbergen Jr., C. Annu. Rev. Physiol. 1985; 47: 727-749Crossref PubMed Google Scholar). McCord (10McCord J.M. N. Engl. J. Med. 1985; 312: 159-163Crossref PubMed Scopus (4999) Google Scholar) also proposed that this process of increased substrate formation could be an important factor in triggering xanthine oxidase-mediated free radical generation. In general, however, the presence and importance of this substrate control has been less emphasized in the literature. Abd-Elfattah et al.(21Abd-Elfattah A.S. Jessen M.E. Lekven J. Doherty III, N.E. Brunsting L.A. Wechsler A.S. Circulation. 1988; 78 (Suppl. III–): III224-III235PubMed Google Scholar, 22Abd-Elfattah A.S. Jessen M.E. Hanan S.A. Tuchy G. Wechsler A.S. Circulation. 1990; 80 (Suppl. IV–): IV341-IV350Google Scholar, 31Abd-Elfattah A.S. Jessen M.E. Lekven J. Mask W.K. Brunsting L.A. Wechsler A.S. Cardiovasc. Drug Ther. 1987; 1: 210Google Scholar) have noted with in vivo surgical canine models of global ischemia and reflow that xanthine oxidase substrate formation is an important factor in the process of postischemic injury and have shown that pharmacological interventions aimed at decreasing substrate concentrations can decrease the severity of this injury. In the present study, we have observed that the alterations in myocardial xanthine oxidase substrate concentrations can explain the time course of radical generation which is observed. In preischemic hearts, no hypoxanthine and xanthine can be detected. During ischemia, hypoxanthine and xanthine accumulated due to the breakdown of ATP to AMP, adenosine, inosine, and finally hypoxanthine. At the end of ischemia, radical-generating substrate concentrations reach a maximal level in the myocardium. Upon reflow, O2 is reintroduced, then xanthine oxidase metabolizes xanthine and hypoxanthine with the formation of uric acid and the reduction of O2 to superoxide and hydrogen peroxide. Myocardial substrate levels then rapidly decrease after the start of reflow, due to both substrate metabolism and washout, with the presence of the product uric acid as well as the substrates hypoxanthine and xanthine in the coronary effluent. Therefore, substrate limitation results in the gradual decline of free radical generation that is observed, despite the presence of xanthine oxidase. These results demonstrate that the burst of xanthine oxidase-mediated free radical generation that occurs in the postischemic rat heart is largely triggered and controlled by substrate availability. From the data obtained in this study it is possible to consider the roles of in vivo substrate control in the process of postischemic free radical generation. Metabolite concentrations within the heart can be estimated from the measured number of micromoles per gram of cell water, to increase by from near 0, less than 0.5 μM, prior to ischemia, to values of 576 or 628 μM for hypoxanthine and xanthine, respectively, after 30-min ischemia. This more than 1000-fold increase would be expected to have a marked effect on radical generation from the enzyme. The Km of xanthine oxidase for xanthine has been reported to be in the range of 11-36 μM(33Massey V. Brumby P.E. Komai H. Palmer G. J. Biol. Chem. 1969; 244: 1682-1691Abstract Full Text PDF PubMed Google Scholar, 34Ackerman E. Brill A.S. Biochim. Biophys. Acta. 1962; 56: 397-412Crossref PubMed Scopus (8) Google Scholar, 35Mousson B. Desjacques P. Baltassat P. Enzyme. 1983; 29: 32-43Crossref PubMed Scopus (31) Google Scholar), therefore at least a 44-144-fold increase in radical generation would be expected. EPR spin trapping studies demonstrated that there was a marked increase in radical generation in the reperfused heart with more than a 40-fold increase seen. In studies of plasma concentrations of xanthine oxidase substrates in normal human plasma, similar low concentrations of hypoxanthine or xanthine have been reported with values in the range of 0.3-0.9 μM(36Ontyd J. Schrader J. J. Chromatogr. Biomed. Appl. 1984; 307: 404-409Crossref Scopus (128) Google Scholar, 37Lazzarino G. Raatikainen P. Nuutinen M. Nissinen J. Tavazzi B. Pierro D.D. Circulation. 1994; 90: 291-297Crossref PubMed Scopus (111) Google Scholar). Thus, in in vivo myocardial ischemia as occurs in human patients with coronary artery disease, xanthine oxidase substrate concentrations would be expected to have a similarly important role in modulating the process of radical generation from the enzyme. In the present experiments, myocardial flow was observed to be an important factor that effects the time course of xanthine oxidase-mediated free radical generation in the postischemic heart. Although it has been previously recognized that reflow is a critical event, since it results in reoxygenation and triggers the onset of oxygen free radical generation after the near anoxic conditions which occur during ischemia, it had not been considered that sustained flow could subsequently limit the duration of radical generation. In the present study, it was observed that myocardial flow is an important factor that effects the time course of substrate clearance and limits the duration of xanthine oxidase mediated free radical generation by decreasing the pool of available substrates. After postischemic reflow, large amounts of xanthine and hypoxanthine were present in the coronary effluent from the heart, and this substrate clearance paralleled the decrease in myocardial substrate concentration that was seen. This important effect of reflow may explain why xanthine oxidase-mediated free radical generation persists for a longer time in suspensions of reoxygenated endothelial cells than in the reperfused heart. In these static endothelial cell preparations, the high substrate concentrations observed (13Zweier J.L. Broderick R. Kuppusamy P. Thompson-Gorman S.L. Lutty G.A. J. Biol. Chem. 1994; 269: 24156-24162Abstract Full Text PDF PubMed Google Scholar) would be expected to remain elevated for prolonged periods of time in the absence of flow. Since substrate availability plays a limiting role in the process of free radical generation within postischemic myocardium, pharmacological approaches that decrease substrate formation may provide an alternative approach to that of radical scavenging compounds or direct xanthine oxidase blockers at preventing free radical generation and subsequent oxidant-mediated myocardial reperfusion injury. These approaches may also exert other beneficial effects, including salvage of high energy phosphates and increasing adenosine concentrations within the heart (32Pisarenko O.I. Lamkomkin V.L. Studneva I.M. Timoshin A.A. Kuzin A.I. Ruge E.K. Kapelko V.I. Biochem. Med. Metab. Biol. 1994; 51: 16-26Crossref PubMed Scopus (14) Google Scholar). Conversely, increased xanthine oxide substrate formation that would occur from increased ATP degradation due to increased severity of supply demand imbalance during myocardial ischemia would be expected to result in increased xanthine oxidase-mediated free radical generation and postischemic injury, further exacerbating the process of myocardial injury which occurs secondary to ATP depletion. In conclusion, we have characterized the presence and time course of xanthine oxidase-mediated free radical generation in the postischemic heart, as well as the nature of enzyme or substrate control which occurs. Xanthine oxidase activity was present in the preischemic heart and significantly increased during ischemia and reperfusion. However, the observed time course of radical generation was largely triggered and controlled by the formation of the radical-generating substrates hypoxanthine and xanthine that accumulated during ischemia. We thank Dr. P. Kuppusamy for helpful discussions and Dr. G. Khatchikian for assistance in some of the physiology experiments." @default.
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• W1974539684 title "Substrate Control of Free Radical Generation from Xanthine Oxidase in the Postischemic Heart" @default.
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