QUANTIFICATION OF [CA-2+]I IN PERFUSED HEARTS - CRITICAL-EVALUATION OF THE 5F-BAPTA AND NUCLEAR-MAGNETIC-RESONANCE METHOD AS APPLIED TO THE STUDY OF ISCHEMIA AND REPERFUSION

Calcium has been implicated as a mediator of cell injury in ischemia and reperfusion, but direct measurements of Ca2+ are required to refine this idea. We used nuclear magnetic resonance spectroscopy and the Ca2+ indicator 5F-BAPTA to measure [Ca2+](i) in perfused ferret hearts. Several lines of evidence are presented to show that loading with the acetoxymethyl ester of 5F-BAPTA is not significantly complicated by accumulation of partially de-esterified metabolites, compartmentalization into mitochondria, or disproportionate uptake into endothelial cells. During 20 minutes of total global ischemia at 30°C, time-averaged [Ca2+](i) increased significantly, reaching peak values roughly three times control at 15-20 minutes. Reperfusion resulted in a persistent elevation of [Ca2+](i) during the first 5 minutes, but not afterward. Although the nonlinear response of 5F-BAPTA to [Ca2+] leads to underestimation of the true time-averaged [Ca2+](i), the measured alterations of intracellular Ca2+ homeostasis during ischemia are large compared with the likely errors in quantification. Phosphorus nuclear magnetic resonance spectroscopy of 5F-BAPTA-loaded hearts reveals changes during ischemia similar to those recorded previously in hearts not containing a Ca2+ indicator. Developed pressure recovers to only 50% of control values during reflow, indicating that the presence of 5F-BAPTA in the cytosol does not protect against stunning, at least when the extracellular calcium concentration has been raised to 8 mM. We conclude that 5F-BAPTA provides useful measurements that reveal that time-averaged [Ca2+](i) rises during ischemia and returns to control levels soon after reperfusion.