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Human peripheral blood leukocytes, activated by phorbol myristate acetate, disrupt canine sarcoplasmic reticulum calcium transport, in vitro, by an oxygen-derived free radical mechanism. Activated leukocytes significantly depress Ca++ uptake activity and Ca++ -stimulated, Mg++ -dependent ATPase activity. The depression is completely inhibited by sodium-azide (0.1 mM) or the combination of superoxide dismutase (10 micrograms/ml) and catalase (10 micrograms/ml). Exogenous hydrogen peroxide (0.441-4.41 mM) uncoupled Ca++ uptake activity from ATP hydrolysis, and this effect was inhibited by catalase. Mannitol alone did not inhibit the effects of activated leukocytes, but superoxide plus mannitol (20-100 mM) resulted in normal ATPase activity, while Ca++ uptake remained depressed. In the presence of indomethacin and ibuprofen, activated leukocytes depressed Ca++ uptake and had no effect on ATPase activity. 2-Amino-methyl-4-t-butyl-6-iodophenol (MK-447) further depressed Ca++ uptake and partially inhibited the effect on ATPase activity. Indomethacin plus catalase completely inhibited the effects of activated leukocytes on cardiac sarcoplasmic reticulum. We conclude, first, that activated leukocytes depress canine cardiac sarcoplasmic reticulum Ca++ transport by an oxygen-free radical mechanism with the generation of hydrogen peroxide and hydroxyl radical. In addition to the classical membrane NADPH oxidase system, significant oxygen radical generation can occur through the cyclooxygenase pathway of arachidonic acid metabolism, and seems to be responsible for the generation of the hydroxyl radical.

The effect of ischemia on the function of cardiac sarcoplasmic reticulum (SR) was assessed by the calcium uptake rate of rat whole-heart homogenates in the presence of 10 mM oxalate. Previous studies have shown that this uptake is restricted to the SR. The contribution of the ryanodine-sensitive fractions of the SR to the total homogenate uptake was assessed by using 20 microM ruthenium red and 625 microM ryanodine to close the SR calcium release channel under previously established optimal conditions. Global ischemia of 10, 15, 30, and 60 minutes depressed homogenate calcium uptake rate 19 +/- 2%, 50 +/- 6%, 65 +/- 3%, and 81 +/- 5%, respectively. This decrease was not observed when the uptake rates were measured after closure of the calcium channel with ryanodine or ruthenium red. Similar results were obtained with a Langendorff in vitro perfusion preparation, in which calcium uptake was decreased 35 +/- 5%, 37 +/- 8%, 58 +/- 7%, and 64 +/- 4% after 10, 15, 30, and 60 minutes of ischemia, but no significant decrease was observed when homogenate uptake rates were measured in the presence of ryanodine. Thus, ischemia caused a depression in the calcium uptake rate of cardiac SR only when this activity was measured in the absence of SR calcium channel blockers. Reperfusion of ischemic hearts in a Langendorff preparation resulted in recovery of homogenate calcium uptake activity that correlated well with the return to sinus rhythm of the reperfused hearts. These reperfused hearts showed no change in the calcium uptake rate measured in the presence of ryanodine. These results suggest that the decrease in homogenate calcium uptake caused by ischemia is not due to a defect in calcium pumping capabilities but is due to an increased efflux through the ryanodine-sensitive calcium release channel of cardiac SR.

Acute myocardial ischemia results in a decrease in developed tension and an increase in resting tension. A breakdown of the excitation–contraction coupling system can explain the behavior of ischemic muscle at a subcellular level. We have identified a specific defect in the sarcoplasmic reticulum (SR) from the ischemic myocardium; i.e., the uncoupling of calcium transport from ATP hydrolysis. The mediators of this excitation–contraction uncoupling process have not been identified. It is now established that the intracellular pH of the ischemic myocardium is in the range of 6.4 but the role of protons and potential role of free radicals have not been identified. We have hypothesized that protons and free radicals may interact to produce the excitation–contraction uncoupling of the ischemic myocardium. Cardiac SR was isolated from the wall of canine left ventricle and calcium uptake velocity and Ca2+-stimulated Mg2+-dependent ATPase activity determined. Increasing proton concentration between pH 7.0 and 6.4 significantly reduced calcium uptake rates (pH 7.0 = 0.95 ± 0.02; 6.4 = 0.50 ± 0.02 μmol Ca2+∙mg protein−1∙min−1; p < 0.01) with no effect on ATPase activity. Calculated coupling ratios (micromoles Ca2+/micromoles Pi) decreased from 0.87 ± 0.06 at pH 7.0 to 0.51 ± 0.05 at pH 6.4. At pH 7.0, the generation of exogenous free radicals from the xanthine (X) – xanthine oxidase (XO) system significantly depressed both calcium uptake rates (control = 0.95 ± 0.02; X + XO = 0.15 ± 0.02) and ATPase activity (control = 1.05 ± 0.02; X + XO = 0.30 ± 0.01 μmol Pi∙mg protein−1∙min−1; p < 0.01). The decreases in calcium uptake and in ATPase activity were completely reversible with superoxide dismutase (SOD). At pH 6.4 in the presence of xanthine and xanthine oxidase, there is a further depression of calcium uptake rates (control = 0.50 ± 0.02; X + XO = 0.11 ± 0.01; p < 0.05) but there is no SOD-reversible component. The addition of SOD + 20 mM mannitol normalized calcium transport at pH 6.4. The calculated coupling ratio at pH 6.4 in the presence of free radicals was 0.13. This value is identical with that reported for the ischemic myocardium. In the absence of an exogenous free-radical generating system, preincubation of whole heart homogenate at pH 6.4 resulted in a significant depression of calcium transport. At 1 min preincubation at pH 6.4, the return of the pH to 7.0 reversed the inhibiting effects of pH 6.4. At 5 min preincubation at pH 6.4, the system required both normalization to pH 7.0 and SOD to reverse the inhibiting effects of acidosis. At 15-min preincubation, the system required both SOD and 20 mM mannitol to reverse the depressant effects of acidosis. It is concluded that at pH 7.0, the xanthine – xanthine oxidase system generates superoxide anion [Formula: see text] which depresses both calcium uptake and ATPase activity. At pH 6.4, this system generates the more lethal hydroxylradical (∙OH) which uncouples calcium transport from ATP hydrolysis in a manner analogous to that observed in the ischemic myocardium. In the whole heart homogenate, acidosis results in a three phase reaction; an initial reversible effect of protons, followed by the production of [Formula: see text] and finally with continued acidosis, the generation of the ∙OH. Combining our observations with the mechanical function of the ischemic myocardium, it is hypothesized that free-radical generation, most probably the ∙OH, may form the link between the reversible and irreversible phase of myocardial ischemia.

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