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Functional and electrophysiological effects of reactive oxygen intermediates on isolated rat ventricular muscle
J Mol
385
Cell
Cardiol
21 (Supplement
II) (1989)
EFFECTS OF MANGANESE-CONTAINING SUPEROXIDE DISMUTASE (in-SOD) ON REPERFUSION ARRHYTHMIA IN VITRO AND IN SITU. K.Yano, N.Makino, H. Matsui, H. Nakanishi, T. of Bioclimatology and Medicine, Medical Institute of Bio Hata, T. Yanaga. Dep. regulation, Kyushu University, Beppu, Japan Since there is good evidence for the involvement of oxygen-derived free radical we have examined the effects of Mn-SOD, which in reperfusion induced arrhythmia, on reperfusion induced arrhythmia is believed to have a half life of a long period, Reperfusion induced in isolated rat hearts and in anesthetized rats in situ. arrhythmias were observed when ischemia was induced by the occulusion of left coronary artery for 10 min and subsequently reperfused. After the reperfusion at 3 min, we have measured the Ca and the Na ion contents by the atomic absorption spectrometry and also isolated sarcolemmal membrane to determine the degree of the lipid per xidations [Malondialdehyde (MDA)]. In isolated herats, administration 9 U/L) prior to ischemia significantly prevented the occurrence of of Mn-SOD (10 ventricular arrhythmia and lowered Ca ion and MDA contents in reperfusion compared Mn-SOD. In anesthetized rat, the pretreatment with a single with rat hear s without 3 injection (IO U/Kg) prior to ischemia was also effective to prevent arrhythmias. These protective effects in situ was seen even when administration of Mn-SOD was at 2 hours before ischemia. These results suggest that oxygen free radical may play a role in the genesis of reperfusion induced arrhythmias and the administration of Mn-SOD may be beneficial effects in these types of arrhythmia.
386 FUNCTIONAL AND ELECTROPHYSIOLOGICAL EFFECTS OF REACTIVE OXYGEN INTERMEDIATES ON
ISOLATED RAT VENTRICULAR MUSCLE. Hiroshi Matsuuraand MichaelJ. Shatfock. Cardiovascular Research, The Rayne Insfifufe, St. Thomas’ Hospital, London SE1 7EH. The objective of this study was to investigate the mechanisms by which reactive oxygen intermediates can induce arrhythmias. Thin right ventricular papillary muscles were isolated from rat hearts and continuously superfused with Krebs-Henseleit bicarbonate bufferat 30°C. Muscles were stimulated at 0.5 Hz. and tension and membrane potential were recorded. Photosensitization of rose bengal (RB) was used to generate singlet oxygen and superoxide radicals. RB (0.1 nM-100 nM) without illumination, resulted in no changes in developed tension (DT) Upon illumination, however, atransientinotropywasobserved. In 1OnM RB,forexample, DTreachedamaximum of 193+26%ofcontrol(mean+sem; n=6) after 7.7~1.7 min of illumination. Approximately coincident with this transient inotropic response was an increase in resting tensionand theappearanceofafter-contractionsandoscillatoryafter-potentials.Thetimetoonsetofcontracture was 5.19.9 min in IO nM RR. The time-courses of all the effectsof RB were concentration-dependent with higher concentrations increasing, and lower concentrations decreasing, the rate of onset. These results are consistentwith the suggestion that reactiveoxygen intermediates induce cellular Ca overload. OscillatoryCa release fromthe sarcoplasmic reticulum (SR) is known to occur in Ca overloaded cells and this oscillatoryrise of [Cal. maylead to fluctuationsof tension and membrane potential. In order to assess the contribution of the SR to these R&induced changes, muscles were pretreated with caffeine(10 mM), and then superfused with solutions containing 10 nM RB (with illumination). Caffeine abolished the transient inotropyand theafter-contractionswhile hastening the onset ofcontracture to3.5kO.6 min. These results suggest that reactive oxygen intermediates maycause cellular Ca overload leading to oscillatory SR Ca release and potentially arrhythmogenic oscillations in membrane potential and developed tension.
387EFFECT OF HYDROGEN PEROXIDE ON CALCIUM BINDING AND CALCIUM TRANSPORT OF DOG HEART SARCOLEMMA. T.Yanagishita and K.J.Kako. University of Ottawa, Ottawa, Ontario. Recent evidence indicates that free radical species are formed in the ischemiareperfused heart. One of the precursors for reactive oxygen intermediates is H202. Our previous studies indicated that H202 accelerated net Ca uptake by isolated rat heart myocytes and this was attributed to an increased sarcolemmal Ca transport via Na/Ca exchange (Am.J.Physiol.,in press). However, it is not known whether or not H202 influences the amount of cell surface-bound Ca or other sarcolemmal functions. Therefore, we measured sarcolemmal Ca binding and Ca transport in this study. The sarcolemmal membrane of dog heart was isolated by the method of Slaughter et al. Na,K,ATPase activity of the preparation was 161 k 19 pmol Pi/mg protein.h. Ca binding and uptake were assessed by Millipore filtration techniques. The binding increased from 1.3 & 0.06 (n = 5, control) to 6.5 k 0.1 (n = 5) nmol Ca/mg protein with 0.1 &I F&13-ADP chelate. H202 (0.2 - 5 mM) did not increase further the Ca binding. By contrast, ATP-dependent Ca uptake by the sarcolemmal prepration was significantly decreased by Fe-ADP chelate plus 5 mM H202 [from 22.0 f 1.4 (n = 6), in control, to 16.2 2 0.6 (n = 3) nmol Ca/mg protein]. Thus, our results show that reactive oxygen species, derived from H202 and Fe-chelate, potentiated Ca binding to sarcolemmal membrane, whereas they inhibited sarcolemmal Ca pump function, and could eventually cause alterations in cell Ca homeostasis.
s.129
385
Cell
Cardiol
21 (Supplement
II) (1989)
EFFECTS OF MANGANESE-CONTAINING SUPEROXIDE DISMUTASE (in-SOD) ON REPERFUSION ARRHYTHMIA IN VITRO AND IN SITU. K.Yano, N.Makino, H. Matsui, H. Nakanishi, T. of Bioclimatology and Medicine, Medical Institute of Bio Hata, T. Yanaga. Dep. regulation, Kyushu University, Beppu, Japan Since there is good evidence for the involvement of oxygen-derived free radical we have examined the effects of Mn-SOD, which in reperfusion induced arrhythmia, on reperfusion induced arrhythmia is believed to have a half life of a long period, Reperfusion induced in isolated rat hearts and in anesthetized rats in situ. arrhythmias were observed when ischemia was induced by the occulusion of left coronary artery for 10 min and subsequently reperfused. After the reperfusion at 3 min, we have measured the Ca and the Na ion contents by the atomic absorption spectrometry and also isolated sarcolemmal membrane to determine the degree of the lipid per xidations [Malondialdehyde (MDA)]. In isolated herats, administration 9 U/L) prior to ischemia significantly prevented the occurrence of of Mn-SOD (10 ventricular arrhythmia and lowered Ca ion and MDA contents in reperfusion compared Mn-SOD. In anesthetized rat, the pretreatment with a single with rat hear s without 3 injection (IO U/Kg) prior to ischemia was also effective to prevent arrhythmias. These protective effects in situ was seen even when administration of Mn-SOD was at 2 hours before ischemia. These results suggest that oxygen free radical may play a role in the genesis of reperfusion induced arrhythmias and the administration of Mn-SOD may be beneficial effects in these types of arrhythmia.
386 FUNCTIONAL AND ELECTROPHYSIOLOGICAL EFFECTS OF REACTIVE OXYGEN INTERMEDIATES ON
ISOLATED RAT VENTRICULAR MUSCLE. Hiroshi Matsuuraand MichaelJ. Shatfock. Cardiovascular Research, The Rayne Insfifufe, St. Thomas’ Hospital, London SE1 7EH. The objective of this study was to investigate the mechanisms by which reactive oxygen intermediates can induce arrhythmias. Thin right ventricular papillary muscles were isolated from rat hearts and continuously superfused with Krebs-Henseleit bicarbonate bufferat 30°C. Muscles were stimulated at 0.5 Hz. and tension and membrane potential were recorded. Photosensitization of rose bengal (RB) was used to generate singlet oxygen and superoxide radicals. RB (0.1 nM-100 nM) without illumination, resulted in no changes in developed tension (DT) Upon illumination, however, atransientinotropywasobserved. In 1OnM RB,forexample, DTreachedamaximum of 193+26%ofcontrol(mean+sem; n=6) after 7.7~1.7 min of illumination. Approximately coincident with this transient inotropic response was an increase in resting tensionand theappearanceofafter-contractionsandoscillatoryafter-potentials.Thetimetoonsetofcontracture was 5.19.9 min in IO nM RR. The time-courses of all the effectsof RB were concentration-dependent with higher concentrations increasing, and lower concentrations decreasing, the rate of onset. These results are consistentwith the suggestion that reactiveoxygen intermediates induce cellular Ca overload. OscillatoryCa release fromthe sarcoplasmic reticulum (SR) is known to occur in Ca overloaded cells and this oscillatoryrise of [Cal. maylead to fluctuationsof tension and membrane potential. In order to assess the contribution of the SR to these R&induced changes, muscles were pretreated with caffeine(10 mM), and then superfused with solutions containing 10 nM RB (with illumination). Caffeine abolished the transient inotropyand theafter-contractionswhile hastening the onset ofcontracture to3.5kO.6 min. These results suggest that reactive oxygen intermediates maycause cellular Ca overload leading to oscillatory SR Ca release and potentially arrhythmogenic oscillations in membrane potential and developed tension.
387EFFECT OF HYDROGEN PEROXIDE ON CALCIUM BINDING AND CALCIUM TRANSPORT OF DOG HEART SARCOLEMMA. T.Yanagishita and K.J.Kako. University of Ottawa, Ottawa, Ontario. Recent evidence indicates that free radical species are formed in the ischemiareperfused heart. One of the precursors for reactive oxygen intermediates is H202. Our previous studies indicated that H202 accelerated net Ca uptake by isolated rat heart myocytes and this was attributed to an increased sarcolemmal Ca transport via Na/Ca exchange (Am.J.Physiol.,in press). However, it is not known whether or not H202 influences the amount of cell surface-bound Ca or other sarcolemmal functions. Therefore, we measured sarcolemmal Ca binding and Ca transport in this study. The sarcolemmal membrane of dog heart was isolated by the method of Slaughter et al. Na,K,ATPase activity of the preparation was 161 k 19 pmol Pi/mg protein.h. Ca binding and uptake were assessed by Millipore filtration techniques. The binding increased from 1.3 & 0.06 (n = 5, control) to 6.5 k 0.1 (n = 5) nmol Ca/mg protein with 0.1 &I F&13-ADP chelate. H202 (0.2 - 5 mM) did not increase further the Ca binding. By contrast, ATP-dependent Ca uptake by the sarcolemmal prepration was significantly decreased by Fe-ADP chelate plus 5 mM H202 [from 22.0 f 1.4 (n = 6), in control, to 16.2 2 0.6 (n = 3) nmol Ca/mg protein]. Thus, our results show that reactive oxygen species, derived from H202 and Fe-chelate, potentiated Ca binding to sarcolemmal membrane, whereas they inhibited sarcolemmal Ca pump function, and could eventually cause alterations in cell Ca homeostasis.
s.129