NMR STUDIES OF THE MECHANISMS OF CELL INJURY

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This research is focused on the role of ionic alterations in the development of irreversible cell injury. We make use of vivo and in situ NMR methods on cell suspensions and perfused organs in order to determine the cytosolic concentrations of these ions and to monitor associated metabolic changes to which the NMR technique is sensitive. We have previously demonstrated that increases in cytosolic calcium (Ca-i, sodium (Na-i), magnesium (Mg-i), and hydrogen and a decrease in ATP occur during cell injury. Although a consensus appears to be developing that an increase in Ca-i precedes the onset of irreversible injury, this does not prove that an increase in Ca-i is necessary or responsible for the onset of irreversible cell injury. We therefore undertook studies to determine whether it was possible to manipulate experimental conditions to prevent or delay an increase in Ca-i. The perfused rat heart was studied under conditions of: total ischemia, potassium arrest, magnesium arrest, and pretreatment with 0.9 uM diltiazem to reduce but not abolish contractility. In all conditions tested, the increase in Ca-1 preceded lethal injury, as determined by enzyme release. Thus, we have not been able to dissociate the increase in calcium from irreversible cell injury. Using the NMR sensitive indicator 5FBAPTA to measure Ca-i, we have also demonstrated that amiloride significantly attenuates the increase in Ca-1 observed during ischemia. In addition, since ATP, measured by 31 p NMR, falls with a similar time course in the presence and absence of amiloride, we can therefore dissociate the increase in Ca-i from the decrease in ATP. We observed that the presence of amiloride during ischemia allows better recovery of contractile function during reflow after 20 minutes of ischemia. These data strongly suggest that the rise in Ca-i plays an important role in the contractile dysfunction which occurs after ischemia.