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Detection and characterization of hydroxyl radical generated during reperfusion of ischemic myocardium
J Mol
Cell
Cardiol21
367
DETECTION REPERFUSION
(Supplement
II) (1989)
AND CHARACTERIZATION OF OF ISCH!ZMIC MYDCARDIUM
HYDROXYL RADICAL J.E. Baker, “S.Y.H.
GENERATED
DURING
Tse, B. Kalyanaraman. Medical College of Wisconsin, Milwaukee, WI and *Georgetown University, Washington, DC. The spin trap 01 -phenyl-tert-butyl nitrone (PBN) has been used to detect radicals generated in the coronary effluentduring reperfusion of ischemic myocardium. The resulting PBN adduct was characterized unambiguosly using an independent synthesis. Isolated Krebsperfused hearts (n=6/group) were subjected to 15-20 min global normothermic ischemia followed Aerobic cardiac function was unaffected by by 10 min aerobic reperfusion with 3 mM PBN. 3 mM PBN. Coronary effluent was collected at 4OC end extracted with toluene. Electron Spin Resonance (ESR) measurements of the toiuene extract (at +22OC and -7OOC) revealed a spin adduct with hyperfine coupling constants of a&3.5 G and aH=1.75 G. Based upon solvent effects the adduct was assigned to a PBN-hydroxyl (PBN-OH) adduct. Our assignment was further verified by an independent synthesis. A Fenton system (Fez+ and H202) was The unstable PBN adduct was extracted with used to generate PBN-OH in phosphate buffer. toluene. ESR measurements of this authentic PBN-OH adduct in toluene gave the same spectra (at +22OC and -7OOC) as the reperfusion-derived adduct. Gas chromatography-mass spectrometry after trimethylsilyation of both the reperfusionand Fenton-derived adducts also resulted in fragmentation peaks consistent with that of trimethylsilyated PBN-OH adduct. We conclude that the PBN adduct generated during reperfusion in our model is derived predominantly from the hydroxyl radical.
368 EFFECTS OF FLUOROCARBONON VASCULARIZATION IN FETAL RAT HEART DURING PLACENTAL IN-
SUFFICIENCY. S.E. Campbell, K. Rakusan, N.S. Faithfull. Dept. of Physiology, U. of Ottawa, Ottawa, Canada and Dept. of Anesthesia, U. of Manchester, Manchester, UK. Fetal growth retardation resulting from placental insufficiency is an important obstetrical problem. Fetal hypoxia may have detrimental effects on the fetal heart itself, Oxygen-carrying plasma substitutes based on emulsified perfluorocarbons (e.g. FC-43) may alleviate the placental insufficiency by improving oxygen diffusion. Experimental placental insufficiency was unilaterally produced in pregnant rats on day 17 of gestation by ligation of the uterine artery. Fetuses from unligated horns served as controls. In seme animals, daily iv injections of 10 ml/kg of FC-43 were given in association with inhalation of air enriched with oxygen. Fetuses were recovered and weighed on day 21 of gestation, and heart weights determined. Fetal hearts were fixed in buffered glutaraldehyde and processed for quantitative analysis. FC-43 appears to be toxic to both mother and fetus as indicated by significantly lower birth weights and weight losses in mothers compared to controls. Uterine artery ligation and FC-43 treatment significantly decrease fetal heart weights. Preliminary results indicate that endomyocardial vascular capacity is increased in ligated fetuses compared to controls and ‘PC-43 tends to decrease the vascular response. Additionally, both experimental procedures seem to affect the maturation of the terminal vascular bed. Supported by the Ontario Heart and Stroke Foundation.
369
FREE RADICAL GENERATING SOLUTIONS ALTER IONIC CALCIUM LEVELS AND INCREASE CONJUGATED DIENES IN CULTURED NEONATAL RAT VENTRICULAR MYOCYTES. K.P. Burton, AC. Morris, K.D. Massey, L.M. Buja, H.K. Hagler. Depts Physiology and Pathology, Univ Texas Southwestern Med Ctr, Dallas TX. Oxygen-derived free radicals have been implicated in damage to membrane phospholibids leading to alterations in membrane permeability. Alterations in [Ca2+]i, cellular morphology and conjugated diene (CD) levels were monitored after exposure of cultured neonatal rat ventricular myocytes (M) to a free radical generafin system (FRGS) consisting of 2.3 mM purine, 0.01 U/ml xanthine oxidase and 2.4 uM iron-loaded transferrin (Fe 8-+ T). o ^ Tocopherol (Tot) was evaluated for its ability to prevent damage. M grown on coverslips for 3-4 days were preloaded with 3 pM fura-P/AM, placed in Sykes-Moore chambers and housed on a 37OC stage of an inverted fluorescence microscope interlaced with a TN FP-1000 microspectrofluorometer. Control M superfused with HEPES buffer or purine and Fe3+-T showed spontaneous Ca2+ transients. The control (n=9) fura340/380 ratio was 0.6+0.1 (mean+SE) and 1.5+0.1 at the minimum and maximum, respectively, of the Ca2+ transient after 1 hr perfusion. Exposure to FRGS (n=14) altered [Ca2+]l. The 3407380 minimum ratio was 402&51% of control value after approximately 30-70 min with cessation of normal Ca 2+ transients. Bleb development was associated with increased [Ca2+]i. M reperfused with control HEPES medium continued to exhibit an elevated minimum furaratio at 403~118% of control. M pretreated with 10 uM Tot (n=13) for 24 hrs did not exhibit increases in [Ca2+]l having a minimum 340/380 ratio of O&.0.1 after 60-90 min, resumed spontaneous Ca 2+ transients with control medium reperlusion and maintained normal structure. Exposure of M to FRGS (n=lO) resulted in a 133*8% increase in CD (ODimg protein) compared to control values after 2 hrs. Tot treated M (n=6) showed a 64~22% increase in CD levels. Thus, FRGS alter [Ca2+]l, CD and structure suggesting their involvement in myocardial membrane damage. Tot decreased free radical mediated injury. S.123
Cell
Cardiol21
367
DETECTION REPERFUSION
(Supplement
II) (1989)
AND CHARACTERIZATION OF OF ISCH!ZMIC MYDCARDIUM
HYDROXYL RADICAL J.E. Baker, “S.Y.H.
GENERATED
DURING
Tse, B. Kalyanaraman. Medical College of Wisconsin, Milwaukee, WI and *Georgetown University, Washington, DC. The spin trap 01 -phenyl-tert-butyl nitrone (PBN) has been used to detect radicals generated in the coronary effluentduring reperfusion of ischemic myocardium. The resulting PBN adduct was characterized unambiguosly using an independent synthesis. Isolated Krebsperfused hearts (n=6/group) were subjected to 15-20 min global normothermic ischemia followed Aerobic cardiac function was unaffected by by 10 min aerobic reperfusion with 3 mM PBN. 3 mM PBN. Coronary effluent was collected at 4OC end extracted with toluene. Electron Spin Resonance (ESR) measurements of the toiuene extract (at +22OC and -7OOC) revealed a spin adduct with hyperfine coupling constants of a&3.5 G and aH=1.75 G. Based upon solvent effects the adduct was assigned to a PBN-hydroxyl (PBN-OH) adduct. Our assignment was further verified by an independent synthesis. A Fenton system (Fez+ and H202) was The unstable PBN adduct was extracted with used to generate PBN-OH in phosphate buffer. toluene. ESR measurements of this authentic PBN-OH adduct in toluene gave the same spectra (at +22OC and -7OOC) as the reperfusion-derived adduct. Gas chromatography-mass spectrometry after trimethylsilyation of both the reperfusionand Fenton-derived adducts also resulted in fragmentation peaks consistent with that of trimethylsilyated PBN-OH adduct. We conclude that the PBN adduct generated during reperfusion in our model is derived predominantly from the hydroxyl radical.
368 EFFECTS OF FLUOROCARBONON VASCULARIZATION IN FETAL RAT HEART DURING PLACENTAL IN-
SUFFICIENCY. S.E. Campbell, K. Rakusan, N.S. Faithfull. Dept. of Physiology, U. of Ottawa, Ottawa, Canada and Dept. of Anesthesia, U. of Manchester, Manchester, UK. Fetal growth retardation resulting from placental insufficiency is an important obstetrical problem. Fetal hypoxia may have detrimental effects on the fetal heart itself, Oxygen-carrying plasma substitutes based on emulsified perfluorocarbons (e.g. FC-43) may alleviate the placental insufficiency by improving oxygen diffusion. Experimental placental insufficiency was unilaterally produced in pregnant rats on day 17 of gestation by ligation of the uterine artery. Fetuses from unligated horns served as controls. In seme animals, daily iv injections of 10 ml/kg of FC-43 were given in association with inhalation of air enriched with oxygen. Fetuses were recovered and weighed on day 21 of gestation, and heart weights determined. Fetal hearts were fixed in buffered glutaraldehyde and processed for quantitative analysis. FC-43 appears to be toxic to both mother and fetus as indicated by significantly lower birth weights and weight losses in mothers compared to controls. Uterine artery ligation and FC-43 treatment significantly decrease fetal heart weights. Preliminary results indicate that endomyocardial vascular capacity is increased in ligated fetuses compared to controls and ‘PC-43 tends to decrease the vascular response. Additionally, both experimental procedures seem to affect the maturation of the terminal vascular bed. Supported by the Ontario Heart and Stroke Foundation.
369
FREE RADICAL GENERATING SOLUTIONS ALTER IONIC CALCIUM LEVELS AND INCREASE CONJUGATED DIENES IN CULTURED NEONATAL RAT VENTRICULAR MYOCYTES. K.P. Burton, AC. Morris, K.D. Massey, L.M. Buja, H.K. Hagler. Depts Physiology and Pathology, Univ Texas Southwestern Med Ctr, Dallas TX. Oxygen-derived free radicals have been implicated in damage to membrane phospholibids leading to alterations in membrane permeability. Alterations in [Ca2+]i, cellular morphology and conjugated diene (CD) levels were monitored after exposure of cultured neonatal rat ventricular myocytes (M) to a free radical generafin system (FRGS) consisting of 2.3 mM purine, 0.01 U/ml xanthine oxidase and 2.4 uM iron-loaded transferrin (Fe 8-+ T). o ^ Tocopherol (Tot) was evaluated for its ability to prevent damage. M grown on coverslips for 3-4 days were preloaded with 3 pM fura-P/AM, placed in Sykes-Moore chambers and housed on a 37OC stage of an inverted fluorescence microscope interlaced with a TN FP-1000 microspectrofluorometer. Control M superfused with HEPES buffer or purine and Fe3+-T showed spontaneous Ca2+ transients. The control (n=9) fura340/380 ratio was 0.6+0.1 (mean+SE) and 1.5+0.1 at the minimum and maximum, respectively, of the Ca2+ transient after 1 hr perfusion. Exposure to FRGS (n=14) altered [Ca2+]l. The 3407380 minimum ratio was 402&51% of control value after approximately 30-70 min with cessation of normal Ca 2+ transients. Bleb development was associated with increased [Ca2+]i. M reperfused with control HEPES medium continued to exhibit an elevated minimum furaratio at 403~118% of control. M pretreated with 10 uM Tot (n=13) for 24 hrs did not exhibit increases in [Ca2+]l having a minimum 340/380 ratio of O&.0.1 after 60-90 min, resumed spontaneous Ca 2+ transients with control medium reperlusion and maintained normal structure. Exposure of M to FRGS (n=lO) resulted in a 133*8% increase in CD (ODimg protein) compared to control values after 2 hrs. Tot treated M (n=6) showed a 64~22% increase in CD levels. Thus, FRGS alter [Ca2+]l, CD and structure suggesting their involvement in myocardial membrane damage. Tot decreased free radical mediated injury. S.123