Calcium concentration in rat liver mitochondria during anoxic incubation.

BACKGROUND AND PURPOSE The impairment of mitochondrial respiratory function is an important problem in anoxia-reoxygenation injury in animal cells. This study investigated the role of mitochondrial calcium ions (Ca2+) in a model of anoxic incubation in isolated rat liver mitochondria. METHODS Mitochondria isolated from Wistar rat liver were subjected to anoxic incubation for 5 to 60 minutes. Changes in oxygen consumption by anoxic mitochondria were recorded using polarography. Changes in NAD(P)H fluorescence were recorded simultaneously. Intramitochondrial matrix free calcium concentration ([Ca2+]) was measured using the fluo-3/AM loading method. Respiratory function in anoxic mitochondria was measured polarographically from oxygen consumption. RESULTS Mitochondrial state 3 respiration decreased progressively with increased duration of anoxic incubation, while state 4 respiration increased progressively. The decrease in the ADP/O ratio with the duration of anoxic incubation indicated that the efficiency of oxidative phosphorylation decreased with increased duration of anoxia. As the duration of anoxic incubation increased, the amplitude and rapidity of the oxido-reduction change in NAD(P)H fluorescence decreased. The respiratory control ratio showed that the functional integrity of mitochondria was linearly correlated with the free [Ca2+] in mitochondrial matrix (r2 = 0.977) and the total calcium concentration (r2 = 0.934). The free [Ca2+] in mitochondrial matrix significantly decreased with increasing anoxic duration. Total mitochondrial calcium concentration also decreased with increasing anoxic duration (p < 0.01). CONCLUSIONS The supply of NADH was reduced during anoxic incubation of isolated rat hepatocyte mitochondria, which resulted in impairment of mitochondrial respiratory function. The decrease in free [Ca2+] in the mitochondrial matrix with increased duration of anoxic incubation implies that calcium ions may be released into the cytosol during anoxia.