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Effect of hydrogen peroxide on mitochondrial function of rat heart myocytes
J Mol Cell Cardiol
23 (Supplement
III) (1991)
EXPOSURE OF ISOLATED ADULT RAT CARDIAC MYOCYTES TO OXYGEN-DERIVED FREE RADICALS INITIATES ALTERATIONS IN INTRACELLULAR LONIC CALCIUM LEVELS Karerfp. .BtM&, WmaymWimra~;+M5eff K’W&gfer. ‘D&partments of FWilbBgy antf$athoQy, Uhiversity~of Texas Southwestern Medical Center, Dallas, TX 75235 USA. Oxygen-derived free radicals .have been implicated in initiating ischemic membrane perturbations resulting in altered ion flux. Fura- was used to measure intracellular ionic calcium ([Ca++]i) levels in isolated adult rat cardiac myocytes (M) exposed to superoxide and hydroxyl radicals generated in a HEPES buffered physiological salt solution containing 2.3 mM purine, 2.4 PM iron-loaded transferrin (Fe-T) and 0.01 U/ml xanthine oxidase. M, stimulated at 1 Hz, were exposed to varying durations of free radical generation and graded concentrations of extracellular Ca++. Control M maintained in HEPES buffer (n=5) or the HEPES buffer containing purine and Fe-T (n=5) continued to stimulate, exhibited relatively uniform 340/380 ratios and maintained a rod shape for prolonged time periods. M exposed to the free radical generating solution (FRGS) were either exposed continuously or exposed for 5 or 10 min and then returned to the HEPES buffer. Continuous exposure to the FRGS, with 1.25 mM Ca ++, resulted in a significant increase in [Ca++]i (minimum 340/380 ratio was 171+20% of control). M became unresponsive to stimulation at 31~7 (SE) min (n=5) and eventually exhibited contracture. Exposure to the FRGS for 10 min (n=6) resulted in aiterations similar to continuous exposure. M exposed to the FRGS for 5 min exhibited regular Ca++ transients for 55+5 min (k5) (~~0.05). M exposed to the FRGS for 10 min and maintained in 2.5 mM Ca++ (n=6) accumulated significantly higher [Ca++]j versus 1.25 mM Ca ++. Thus, the potential of free radicals to peroxidize membrane lipids or.oxidize membrane protein sulfhydtyl groups may significantly affect the ability of M to maintain ion gradients and inay lead to the eventual development of Cat+ overload.
p-14'
EFFECT OF HMROGEN PEROXIDE ON HITOCHONDRIAL FUNCTION OF RAT HEART MYOCYTES. University Tetsuya Tatsumi, N. Konno, Y. Tanaka, K. .T. Kako. Department of Physiology, of Ottawa, Ottawa, Ontario, KlH 8M5, Canada. Available evidence indicates that reactive oxygen intermediates are detected during reperfusion of the previously ischemic heart. Although the precise localization of the active intermediates is not known, one possibility is that H,O, formed by the mitochondrial electron transport chain overcomes the cellular defense mechanism which is weakened by hypoxia. Therefore, in this study we investigated the action of H,O, on mitochondrial oligomycin-sensitive ATPase and adenine nucleotide translocase. These two enzymes were selected because we observed previously that the mitochondrial membrane Adult rat heart potential was relatively well maintained under the oxidant stress. cells were isolated by collagenase digestion and incubated in the presence of 0.1 - 10 Activities of adenine nucleotide translocase were determined by mN HzO, for 30 min. following permeabilization with atractyloside-inhibitable ADP uptake of myocytes saponin, whereas oligomycin-sensitive ATPase activities were assayedwith myocytes after The dose/enzyme activity relationship indicated that the translocase was sonication. The results suggest that the mechanism more susceptible to H,Oz than the ATPase. responsible for the relative tolerance of mitochondrial transmembrane potential gradient in response to oxidant stress is the partial inhibition of transport of high energy phosphates across the inner membrane.
P-l -3 EFFICACYOFAHYDROXnRADICALSCAVENGER lNJURYFOLLOWINGGLOBALISCHf5MIA
(VF233)INPBE OFISOLATEDRABBITHEARTS
Mai Ding, Cornelius Dyke, Anwar Abd-E@zttah, Jon Lenman, Andrew Wechsler, Dept. Virgiini4 Richmond, V4 USA 23298.
VENTlNGREPERFUSION
of Surgery,Medical
College
of
This study was designed to test the hypothesis that 3,4,5,-trihydroxybenzamidoxine (VF 233), which has been demonstrated degrees of iron chelating activities and a hydroxyl radical (OH) scavenging abilities, may attenuate reperfusion injury mediated by OH’ and improve LV function after &hernia and reperfusion. Eighteen isolated perfused rabbit hearts with intracavitary balloons were subjected to normothermic (37OC),global ischemia until the initiation of ischemic contracture (4mmHg rise in intracavitary pressure). Ventricular function was measured by peak develop pressure (PDP) and adenine nucleotide was determined by HPLC prior to ischemia and at 15 minute intervals during reperfusion. In the experimental group (n=9), a 5ml bolus of 1mM VF 233 was given immediately prior to reperfusion and followed by a continuous infusion (125 &l/ min). Controls (n=9) received the l$myofPDP(%Preiscbemia) 60' 3w 45' vehicle solution at identical times. Results from this pie 67.2t6.4 66.3t6.5 study demonstrate that administration of VF 233 g;’ ~~:~~$ ~;~~s& 76.h9.4 82.3t10.7' moderately improves ventricular function, but there are pcO.OSANOVA mean*SDM no significant differences in adenine nucleotide metabolism at the end of ischemia and during reperfusion. s.40
23 (Supplement
III) (1991)
EXPOSURE OF ISOLATED ADULT RAT CARDIAC MYOCYTES TO OXYGEN-DERIVED FREE RADICALS INITIATES ALTERATIONS IN INTRACELLULAR LONIC CALCIUM LEVELS Karerfp. .BtM&, WmaymWimra~;+M5eff K’W&gfer. ‘D&partments of FWilbBgy antf$athoQy, Uhiversity~of Texas Southwestern Medical Center, Dallas, TX 75235 USA. Oxygen-derived free radicals .have been implicated in initiating ischemic membrane perturbations resulting in altered ion flux. Fura- was used to measure intracellular ionic calcium ([Ca++]i) levels in isolated adult rat cardiac myocytes (M) exposed to superoxide and hydroxyl radicals generated in a HEPES buffered physiological salt solution containing 2.3 mM purine, 2.4 PM iron-loaded transferrin (Fe-T) and 0.01 U/ml xanthine oxidase. M, stimulated at 1 Hz, were exposed to varying durations of free radical generation and graded concentrations of extracellular Ca++. Control M maintained in HEPES buffer (n=5) or the HEPES buffer containing purine and Fe-T (n=5) continued to stimulate, exhibited relatively uniform 340/380 ratios and maintained a rod shape for prolonged time periods. M exposed to the free radical generating solution (FRGS) were either exposed continuously or exposed for 5 or 10 min and then returned to the HEPES buffer. Continuous exposure to the FRGS, with 1.25 mM Ca ++, resulted in a significant increase in [Ca++]i (minimum 340/380 ratio was 171+20% of control). M became unresponsive to stimulation at 31~7 (SE) min (n=5) and eventually exhibited contracture. Exposure to the FRGS for 10 min (n=6) resulted in aiterations similar to continuous exposure. M exposed to the FRGS for 5 min exhibited regular Ca++ transients for 55+5 min (k5) (~~0.05). M exposed to the FRGS for 10 min and maintained in 2.5 mM Ca++ (n=6) accumulated significantly higher [Ca++]j versus 1.25 mM Ca ++. Thus, the potential of free radicals to peroxidize membrane lipids or.oxidize membrane protein sulfhydtyl groups may significantly affect the ability of M to maintain ion gradients and inay lead to the eventual development of Cat+ overload.
p-14'
EFFECT OF HMROGEN PEROXIDE ON HITOCHONDRIAL FUNCTION OF RAT HEART MYOCYTES. University Tetsuya Tatsumi, N. Konno, Y. Tanaka, K. .T. Kako. Department of Physiology, of Ottawa, Ottawa, Ontario, KlH 8M5, Canada. Available evidence indicates that reactive oxygen intermediates are detected during reperfusion of the previously ischemic heart. Although the precise localization of the active intermediates is not known, one possibility is that H,O, formed by the mitochondrial electron transport chain overcomes the cellular defense mechanism which is weakened by hypoxia. Therefore, in this study we investigated the action of H,O, on mitochondrial oligomycin-sensitive ATPase and adenine nucleotide translocase. These two enzymes were selected because we observed previously that the mitochondrial membrane Adult rat heart potential was relatively well maintained under the oxidant stress. cells were isolated by collagenase digestion and incubated in the presence of 0.1 - 10 Activities of adenine nucleotide translocase were determined by mN HzO, for 30 min. following permeabilization with atractyloside-inhibitable ADP uptake of myocytes saponin, whereas oligomycin-sensitive ATPase activities were assayedwith myocytes after The dose/enzyme activity relationship indicated that the translocase was sonication. The results suggest that the mechanism more susceptible to H,Oz than the ATPase. responsible for the relative tolerance of mitochondrial transmembrane potential gradient in response to oxidant stress is the partial inhibition of transport of high energy phosphates across the inner membrane.
P-l -3 EFFICACYOFAHYDROXnRADICALSCAVENGER lNJURYFOLLOWINGGLOBALISCHf5MIA
(VF233)INPBE OFISOLATEDRABBITHEARTS
Mai Ding, Cornelius Dyke, Anwar Abd-E@zttah, Jon Lenman, Andrew Wechsler, Dept. Virgiini4 Richmond, V4 USA 23298.
VENTlNGREPERFUSION
of Surgery,Medical
College
of
This study was designed to test the hypothesis that 3,4,5,-trihydroxybenzamidoxine (VF 233), which has been demonstrated degrees of iron chelating activities and a hydroxyl radical (OH) scavenging abilities, may attenuate reperfusion injury mediated by OH’ and improve LV function after &hernia and reperfusion. Eighteen isolated perfused rabbit hearts with intracavitary balloons were subjected to normothermic (37OC),global ischemia until the initiation of ischemic contracture (4mmHg rise in intracavitary pressure). Ventricular function was measured by peak develop pressure (PDP) and adenine nucleotide was determined by HPLC prior to ischemia and at 15 minute intervals during reperfusion. In the experimental group (n=9), a 5ml bolus of 1mM VF 233 was given immediately prior to reperfusion and followed by a continuous infusion (125 &l/ min). Controls (n=9) received the l$myofPDP(%Preiscbemia) 60' 3w 45' vehicle solution at identical times. Results from this pie 67.2t6.4 66.3t6.5 study demonstrate that administration of VF 233 g;’ ~~:~~$ ~;~~s& 76.h9.4 82.3t10.7' moderately improves ventricular function, but there are pcO.OSANOVA mean*SDM no significant differences in adenine nucleotide metabolism at the end of ischemia and during reperfusion. s.40