Studies of Langendorff-perfused rat hearts have revealed a biphasic response of the mitochondrial respiratory chain to global ischaemia. The initial effect is a 30-40% increase in the rate of glutamate/malate oxidation after 10 min of ischaemia, owing to an increase in the capacity for NADH oxidation. This effect is followed by a progressive decrease in these oxidative activities as the ischaemia is prolonged, apparently owing to damage to Complex I at a site subsequent to the NADH dehydrogenase component. This damage is exacerbated by reperfusion, which causes a further decrease in Complex I activity and also decreases the activities of the other complexes, most notably of Complex III. Perfusion for up to 1 h with anoxic buffer produced only the increase in NADH oxidase activity, and neither anoxia alone, nor anoxia and reperfusion, caused loss of Complex I activity. Perfusing for 3-10 min with anoxic buffer before 1 h of global ischaemia had a significant protective effect against the ischaemia-induced damage to Complex I.
A diminished mitochondrial capacity to generate ATP after a period of ischaemia has been demonstrated in several perfused heart models in vitro. The cause of this apparent damage has been suggested to be free radicals [ 11, excessive Ca2 uptake upon reperfusion or the preferential uptake of Ca2+ rather than ATP-formation [2]. We were interested to examine the mitochondrial respiratory chain to determine if there was specific damage to the components of this system. Furthermore, we wanted to know whether such damage occurs during the ischaemic episode or is a consequence of reoxygenation, i.e. reperfusion damage.We studied the effects of global ischaemia, with and without reperfusion, and of anoxia on the function of mitochondrial fractions prepared from rat hearts perfused in a Langendorff apparatus with a Krebs-Henseleit buffer containin 11 mwglucose, continuously gassed with 0 2 / C 0 , (19:lf Aft er 15 rnin perfusion to equilibrate the tissue, one of the following protocols was imposed: continuous perfusion with oxygenated buffer for 15-60 rnin (Controls); global ischaemia induced for 6 0 rnin by clamping off the perfusate supply without reperfusion (Ischaemics) or with 5 rnin reperfusion with oxygenated buffer (Reperfused); 5 rnin perfusion with anoxic buffer to purge the tissue of oxygen before 6 0 min global ischaemia and 5 rnin reperfusion with anoxic buffer (N reperfused); or continuous perfusion for 60 rnin with anoxic buffer (Anoxic). Immediately after the end of each of the above treatments, the hearts were removed into ice-cold buffer and mitochondria prepared by a Nagarse digestion/Potter homogenization procedure. Oxygen uptake was measured in intact mitochondria with 5 mM-glutamate/S mwmalate as substrates and in lysed mitochondria (three freeze-thaw cycles) with 250 ,LAM-NADH as a substrate. The four respiratory chain complex activities were assayed using exogenous electron acceptors: ubiquinone-1 for complex 1; ubiquinone-1 and DCPIP for complex 11; ubiquinol-2 and cytochrome c for complex I11 and ascorbate/TMPD for complex IV ( polarographic). Specific mitochondrial marker enzyme activities, namely citrate synthase for the matrix, and NADH-ferricyanide reductase for the inner membrane, were also measured to compare the quality of the different preparations. All results are shown as the mean values of four to five hearts as a percentage of the mean control value ( n = 13).Ischaemia caused a marked decrease in state 3 glutamate/ malate oxidation (41% of controls) and 54% loss of NADH oxidase activity. This effect was worse in reperfused hearts (30% and 37%, respectively), suggesting some exacerbation of the damage by reoxygenation. This was indirectly supported by the effects of 'purging' the tissue of oxygen before the ischaemia, which protected against the effects of ischaemia alone (68% and 70% of controls, respectively). Furthermore, anoxia without ischaemia did not decrease these two activities. Rather, there was a slight stimulation in the activity of glutamate/malate ( 119'/0) and NADH (1 13...
Previous studies have shown that ischaemia and hypoxia induced a loss of Complex I (NADHubiquinone reductase) activity in cardiac mitochondria [l-31. We have confirmed the loss of Complex I activity after 60 min of ischaemia (with and without reperfusion), but we also reported that, by contrast, 60 min of anoxia resulted in a small increase in respiratory capacity [4]. Other groups have reported increased mitochondrial respiration after 10 rnin of hypoxia [5] and 30 min of hypoperfusion [6], the latter postulating a specific effect on Complex I. This report describes our studies on respiration and Complex I activity after short periods of ischaemia and anoxia.Male Wistar rats (200-25Og) were anaesthetised by i.p. Nembutal and the hearts were perfused in a Langendorff apparatus with a modified Krebs-Henseleit buffer containing l l m M glucose, continually gassed with either 95 % 0 2 / 5 % C02 (normoxic) or 95 % N2/5 % C 0 2 (anoxic). After 15 rnin of normoxic perfusion control hearts were perfused for a further 15-30 min in normoxic conditions, or global ischaemia was induced for 15 min by clamping off the perfusate supply, or hearts were perfused for 30 min with anoxic buffer. The ventricles were separated from the hearts and mitochondrial fractions prepared by a Nagarse digestion/ Potter homogenisation procedure. Oxygen uptake was measured in a Clark-type electrode in intact mitochondria with 5 mM glutamate/5 mM malate or 10 mM succinate (+ 2 Fg/ml rotenone) as substrates with 0.25 mM ADP, and in broken mitochondria with 250 pM NADH after three freeze-thawing cycles.The effects of ischaemia and anoxia on the respiration rates are shown in Table 1. Both ischaemia and anoxia increased glutamate/malate oxidation, by about 45%. Furthermore, these differences persisted when the mitochondria were lysed by freeze-thawing to assay NADH oxidation, which was increased by 36 % and 41 % in mitochondria from ischaemic and anoxic hearts respectively (Table 1). In contrast, the succinate rate was not different in mitochondria from ischaemic hearts. The oxidation rates were also assayed spectrophotometrically in intact mitochondria from control and anoxic hearts as the antimycin-sensitive reduction of ferricyanide [71. In this system, glutamate/malate oxidation was also increased after anoxia (689 * 25 vs. 462 f 36 nmoles FeCN.min-1
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