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Direct measurement of myocardial oxygen consumption during ischemia by spin label oximetry in the intact heart
Mechanisms of Ischcmia/Rcpeffusion Injury 10.9 DIRECT MEASUREMENTOF MYOCARDIALOXYGEN CONSUMPTION DURING ISCHEMIA BY SPIN LABEL OXIMETRY IN THE INTACT HEART J.E. Baker, W. Froncisz, and B. Kalyanaraman Med. Coll. Wisconsin, Milwaukee, WI 53226, U.S.A.
Measurement of myocardial oxygen consumption (MVO~) during ischemia by spin label oximetry has been-limited by ischemia-induced reduction of the neutral, water-soluble nitroxide TEMPONE. We have overcome this problem by encapsulating nitroxides within liposomes. Isolated rabbit hearts (n=8) were perfused aerobically within the cavity of a loop gap resonator with bicarbonate buffer containing an oxygen-sensitive, lipidsoluble nitroxide (ISN-TEMPO laurate in FC-43 perfluorocarbon micelles) and an oxygen-insensitive and positively charged nitroxide (14N-TEMPO choline in liposomes) as an internal standard. Hearts were subjected to 60 min of ischemia at 20%. Data (mean ± SD) expressed as extracellular oxygen concentration during ischemia. Ischemia (min) 0 15 45 60 [Oxygen] (~M) 1195±55 812±49a 352±25 a 61±5a a = p < 0.05, ischemia vs aerobic per±us±on The e l e c t r o n spin resonance (ESR) signal from TEMPO l a u r a t e broadened and increased w i t h the onset and progression o f ischemia, consistent with a decrease in e x t r a c e l l u l a r oxygen concentra t i o n . The ESR signal from TEMPO c h o l i n e was unchanged throughout aerobic per±us±on and ischemia. We conclude spin label oximetry can d i r e c t l y measure MVO2 during ischemia in the intact heart.
10.11
SALICYLATE AS A PROBE TO DIRECTLYUNK HYDROXYL RADICAL WITH MYOCARDIAL REPERFUSIONINJURY Dipak K. Das, Xuekun Uu, and Richard M. Engelman. University of Connecticut School of Medicine, Farmington, CT and Baystate Medical Center, Springfield, MA, U.S.A. Although many studies have shown the presence of hydroxyl radical (OH.), and thus linked the reactive oxygen metabolites with myocardial reperfusion injury, direct evidence of injury by free radicals has never been shown. In this study, the OH" generated during reperfusion of ischemic heart was chemically trapped by salicylate, which converted the cytotoxic OH' into innocent byproducts, thereby providing myocardial preservation. Two different protocols were used. In Group I, isolated rat heart was pre-perfused with 2 mM salicylate and then the heart was made ischemic for 30 min, which was followed by 30 min of reperfusion. In Group II, isolated rat heart was made ischemic, and then reperfused with buffer containing 2 mM salicylate. Hydroxylated end products were assayed by high pressure liquid chromatography to confirm the presence of OH' in the raparfused heart. In both groups, reperfusion of ischemic heart was associated with the generation of OH" 8alicylate also attenuated myocardial raper±us±oninjury in both groups as evidenced by reduced release of lactic acid debydrogenase and creatine kinase as well as by improved myocardial contractility and coronary flow during reperfusion. The results of the study indicate that salicylate can be used to directly demonstrate the presence of OH., and to preserve the heart dudng reperfus/on, thus providing direct evidence to link OH' with the raparfusion injury. (Supported by NIH H ~ 5 5 9 , HL33889 and HL34360).
H202 DOES NOT DIRECTLY ALTER NA, K, OR CA ION CURRENTS IN CAT VENTRICULAR MYOCYTES. Peggy L. Barrington and Ke Zhang Department of Pharmacology, Northwestern University, Chicago, IL 60611, U.S.A.
93 10.10
Exposure of cardiac myocytes to H202 results in a reproducible 3 stage sequence of changes in the cellular action potential (Barrington, et al.. 1988). This suggests that H202 acts by selectively modifying one or more of the ionic currents which contribute to the action potential. Enzymatically isolated feline myocytes were subjected to whole-cell voltage-clamp to determine if H202 altered the sodium (INa), calcium (Ica) or potassium (inward rectifier, IKI and transient outward, Ito ) currents. Each current was isolated by varying the holding potential and by addition of appropriate channel blockers to intracellular and extracellular solutions. The intracellular solutions for Ica , IKI and Ito included 5 mM EGTA which should prevent large fluctuations of [Ca2+]i . Currentvoltage relationships for each current were compared to time controls recorded from separate cells. Exposure of cells to i00 mM H202 for 20 minutes can cause increased action potential amplitude and duration but there were minimal effects on INa , ICa , IKI or Ito at this time. These results suggest that H202 does not alter the cellular action potential by direct modification of membrane channel proteins or adjacent ifpids. The results do not exclude possible modulation of channel function, i.e. by alteration of [Ca2+]i or of second messenger systems.
MARINE INVERTEBRATE MODELS OF REOXYG~qATION TISSUE INJURY James A. Dykens Biology, Grinnell College, Grinnell, IA 50112 Transient hypoxia is pathological for mammalian tissues susceptible to reoxygenation tissue damage (RTD). This is not true for intertidal invertebrates which thrive in spite of environmentally-imposed hypoxia/reoxygenation from the ebb and flow of tides; these animals are ideal comparative cases in which to study RTD. The intertidal mussel ~ edulis apparently avoids RTD by reduclng met~---l~--smduring hypoxia which conserves adenylates, and by having xanthine dehydrogenase (XDH) which resists conversion to xanthine oxidase (XOD). Conversely, the subtldal scallop Placopecten ma~ellanicus survives 24h hypoxia, but dies during 12h reoxygenation; this species undergoes adenylate depletion during hypoxia and shows XDD activity. Regardless, 02- flux from observed XOD activities appears inadequate to account for RTD unless oxidative defenses, such as superoxide dismutase (SOD), are concomitantly impaired during hypoxia. Indeed, mammalian and scallop SOD activities are diminished after reoxygenation. HOwever, in scallop this apparent impairment of SOD is paralleled by declines in G-6PDH, ODH, PGI and MDH activities, revealing cell necrosis, not SOD impairment. Given the inability of XOD to account for RTD, alternative sources of 02-, such as red/ox cycling of mitochondrial electron transport components, are being sought using esr. Supported by Bang (MBL, Woods Hole)) and Markey (Mt. Desert Isl. Hio. Lab) Fellowships; NSF Grant DCB-8815221.
10.12
Measurement of myocardial oxygen consumption (MVO~) during ischemia by spin label oximetry has been-limited by ischemia-induced reduction of the neutral, water-soluble nitroxide TEMPONE. We have overcome this problem by encapsulating nitroxides within liposomes. Isolated rabbit hearts (n=8) were perfused aerobically within the cavity of a loop gap resonator with bicarbonate buffer containing an oxygen-sensitive, lipidsoluble nitroxide (ISN-TEMPO laurate in FC-43 perfluorocarbon micelles) and an oxygen-insensitive and positively charged nitroxide (14N-TEMPO choline in liposomes) as an internal standard. Hearts were subjected to 60 min of ischemia at 20%. Data (mean ± SD) expressed as extracellular oxygen concentration during ischemia. Ischemia (min) 0 15 45 60 [Oxygen] (~M) 1195±55 812±49a 352±25 a 61±5a a = p < 0.05, ischemia vs aerobic per±us±on The e l e c t r o n spin resonance (ESR) signal from TEMPO l a u r a t e broadened and increased w i t h the onset and progression o f ischemia, consistent with a decrease in e x t r a c e l l u l a r oxygen concentra t i o n . The ESR signal from TEMPO c h o l i n e was unchanged throughout aerobic per±us±on and ischemia. We conclude spin label oximetry can d i r e c t l y measure MVO2 during ischemia in the intact heart.
10.11
SALICYLATE AS A PROBE TO DIRECTLYUNK HYDROXYL RADICAL WITH MYOCARDIAL REPERFUSIONINJURY Dipak K. Das, Xuekun Uu, and Richard M. Engelman. University of Connecticut School of Medicine, Farmington, CT and Baystate Medical Center, Springfield, MA, U.S.A. Although many studies have shown the presence of hydroxyl radical (OH.), and thus linked the reactive oxygen metabolites with myocardial reperfusion injury, direct evidence of injury by free radicals has never been shown. In this study, the OH" generated during reperfusion of ischemic heart was chemically trapped by salicylate, which converted the cytotoxic OH' into innocent byproducts, thereby providing myocardial preservation. Two different protocols were used. In Group I, isolated rat heart was pre-perfused with 2 mM salicylate and then the heart was made ischemic for 30 min, which was followed by 30 min of reperfusion. In Group II, isolated rat heart was made ischemic, and then reperfused with buffer containing 2 mM salicylate. Hydroxylated end products were assayed by high pressure liquid chromatography to confirm the presence of OH' in the raparfused heart. In both groups, reperfusion of ischemic heart was associated with the generation of OH" 8alicylate also attenuated myocardial raper±us±oninjury in both groups as evidenced by reduced release of lactic acid debydrogenase and creatine kinase as well as by improved myocardial contractility and coronary flow during reperfusion. The results of the study indicate that salicylate can be used to directly demonstrate the presence of OH., and to preserve the heart dudng reperfus/on, thus providing direct evidence to link OH' with the raparfusion injury. (Supported by NIH H ~ 5 5 9 , HL33889 and HL34360).
H202 DOES NOT DIRECTLY ALTER NA, K, OR CA ION CURRENTS IN CAT VENTRICULAR MYOCYTES. Peggy L. Barrington and Ke Zhang Department of Pharmacology, Northwestern University, Chicago, IL 60611, U.S.A.
93 10.10
Exposure of cardiac myocytes to H202 results in a reproducible 3 stage sequence of changes in the cellular action potential (Barrington, et al.. 1988). This suggests that H202 acts by selectively modifying one or more of the ionic currents which contribute to the action potential. Enzymatically isolated feline myocytes were subjected to whole-cell voltage-clamp to determine if H202 altered the sodium (INa), calcium (Ica) or potassium (inward rectifier, IKI and transient outward, Ito ) currents. Each current was isolated by varying the holding potential and by addition of appropriate channel blockers to intracellular and extracellular solutions. The intracellular solutions for Ica , IKI and Ito included 5 mM EGTA which should prevent large fluctuations of [Ca2+]i . Currentvoltage relationships for each current were compared to time controls recorded from separate cells. Exposure of cells to i00 mM H202 for 20 minutes can cause increased action potential amplitude and duration but there were minimal effects on INa , ICa , IKI or Ito at this time. These results suggest that H202 does not alter the cellular action potential by direct modification of membrane channel proteins or adjacent ifpids. The results do not exclude possible modulation of channel function, i.e. by alteration of [Ca2+]i or of second messenger systems.
MARINE INVERTEBRATE MODELS OF REOXYG~qATION TISSUE INJURY James A. Dykens Biology, Grinnell College, Grinnell, IA 50112 Transient hypoxia is pathological for mammalian tissues susceptible to reoxygenation tissue damage (RTD). This is not true for intertidal invertebrates which thrive in spite of environmentally-imposed hypoxia/reoxygenation from the ebb and flow of tides; these animals are ideal comparative cases in which to study RTD. The intertidal mussel ~ edulis apparently avoids RTD by reduclng met~---l~--smduring hypoxia which conserves adenylates, and by having xanthine dehydrogenase (XDH) which resists conversion to xanthine oxidase (XOD). Conversely, the subtldal scallop Placopecten ma~ellanicus survives 24h hypoxia, but dies during 12h reoxygenation; this species undergoes adenylate depletion during hypoxia and shows XDD activity. Regardless, 02- flux from observed XOD activities appears inadequate to account for RTD unless oxidative defenses, such as superoxide dismutase (SOD), are concomitantly impaired during hypoxia. Indeed, mammalian and scallop SOD activities are diminished after reoxygenation. HOwever, in scallop this apparent impairment of SOD is paralleled by declines in G-6PDH, ODH, PGI and MDH activities, revealing cell necrosis, not SOD impairment. Given the inability of XOD to account for RTD, alternative sources of 02-, such as red/ox cycling of mitochondrial electron transport components, are being sought using esr. Supported by Bang (MBL, Woods Hole)) and Markey (Mt. Desert Isl. Hio. Lab) Fellowships; NSF Grant DCB-8815221.
10.12