Calcium uptake and cytochrome c release from normal and ischemic brain mitochondria

Abstract

At abnormally elevated levels of intracellular Ca²⁺, mitochondrial Ca²⁺ uptake may compromise mitochondrial electron transport activities and trigger membrane permeability changes that allow for release of cytochrome c and other mitochondrial apoptotic proteins into the cytosol. In this study, a clinically relevant canine cardiac arrest model was used to assess the effects of global cerebral ischemia and reperfusion on mitochondrial Ca²⁺ uptake capacity, Ca²⁺ uptake-mediated inhibition of respiration, and Ca²⁺-induced cytochrome c release, as measured in vitro in a K⁺-based medium in the presence of Mg²⁺, ATP, and NADH-linked oxidizable substrates. Maximum Ca²⁺ uptake by frontal cortex mitochondria was significantly lower following 10 min cardiac arrest compared to non-ischemic controls. Mitochondria from ischemic brains were also more sensitive to the respiratory inhibition associated with accumulation of large levels of Ca²⁺. Cytochrome c was released from brain mitochondria in vitro in a Ca²⁺-dose-dependent manner and was more pronounced following both 10 min of ischemia alone and following 24 h reperfusion, in comparison to mitochondria from non-ischemic Shams. These effects of ischemia and reperfusion on brain mitochondria could compromise intracellular Ca²⁺ homeostasis, decrease aerobic and increase anaerobic cerebral energy metabolism, and potentiate the cytochrome c-dependent induction of apoptosis, when re-oxygenated mitochondria are exposed to abnormally high levels of intracellular Ca²⁺.

Keywords: Apoptosis; Cortex; Excitotoxicity; Hippocampus; Respiration.

Fig. 1. Fluorescent measurements of calcium uptake by cerebral cortex mitochondria

Synaptic plus non-synaptic mitochondria were isolated from non-ischemic frontal cortex and added at a final concentration of 0.5 mg protein ml⁻¹ to a KCl-based medium maintained at 37 °C and containing the respiratory substrates glutamate plus malate and the fluorescent calcium indicator Calcium Green 5N (0.1 μM). Mitochondria calcium uptake was measured by following the increase and subsequent decrease in fluorescence (506 nm excitation and 523 nm emission) upon addition of CaCl₂ aliquots. A. Mitochondrial uptake of 10 nmol Ca²⁺ mg⁻¹ protein aliquots in the absence of either ATP or Mg²⁺. B. Mitochondrial uptake of 300 nmol Ca²⁺ mg⁻¹ protein aliquots in the presence of 3 mM ATP and 4 mM Mg²⁺.

Fig. 2. Effects of ischemia and reperfusion on calcium uptake capacities of mitochondria isolated from cerebral cortex, striatum and hippocampus

The mean calcium uptake capacities ± s.e. for mitochondria isolated from; A. cerebral cortex, B. striatum, and C. hippocampus were measured using tissue obtained from non-ischemic controls, animals that underwent 10 min cardiac arrest alone, and animals that underwent 10 min cardiac arrest followed by 24 h reperfusion. Measurements were performed at 37 °C in the presence of the respiratory substrates glutamate and malate and both ATP and Mg²⁺, as shown in Fig. 1B. and explained in Materials and Methods.

Fig. 3. Western immunoblots of cytochrome c released from normal and ischemic canine cortex mitochondria in the absence and presence of calcium

Brain mitochondria were isolated from non-ischemic animals and from animals after 10 min cardiac arrest alone. Mitochondria were suspended in respiratory medium in the absence or presence of 0.3, 2.1, and 3.2 μmol Ca²⁺ mg⁻¹ protein. Following the 10 min incubation, a sample was removed and centrifuged at 13,000 × g for 3 min and the supernatant removed for cytochrome c western immunoblots. The immunoblot bands were compared to those generated with purified cytochrome c.

Fig. 4. Comparison of cytochrome c released from normal, ischemic, and ischemia/reperfused brains following incubation in the absence or presence of added calcium

Brain mitochondria were isolated from non-ischemic animals and from animals after 10 min cardiac arrest alone (n = 4/group). Mitochondria were suspended in respiratory medium in the absence or presence of 2.1 μmol Ca²⁺ mg⁻¹ protein. Release of cytochrome c was measured as described in Fig. 3. * Cytochrome c release was significantly greater in the presence of Ca²⁺ for all 3 animal groups in comparison to the absence of added Ca²⁺ (p < 0.001). # In the presence of Ca²⁺, cytochrome c release was significantly greater for mitochondria from ischemia or ischemia/reperfusion in comparison to release for non-ischemic, control brain mitochondria.