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Distinct Ca²⁺ thresholds determine cytochrome c release or permeability transition pore opening in brain mitochondria ¹

LORENZ SCHILD^*2, GERBURG KEILHOFF, WOLFGANG AUGUSTIN^*, GEORG REISER and FRANK STRIGGOW

^* Institute of Clinical Chemistry and Pathological Biochemistry, Department of Pathological Biochemistry,
Institute of Medical Neurobiology and
Institute of Neurobiochemistry, Medical Faculty, Otto-von-Guericke-University, Magdeburg, Germany

²Correspondence: Department of Pathological Biochemistry, Institute of Clinical Chemistry and Pathological Biochemistry, Otto-von-Guericke-University Magdeburg, Leipziger Str. 44, D-39120 Magdeburg, Germany. E-mail: lorenz.schild{at}medizin.uni-magdeburg.de

SPECIFIC AIMS

Mitochondria are of paramount importance for cellular life and death. This study aims to shed light on the mitochondrial involvement in key processes leading to cerebral cell death; that is, release of cytochrome c and opening of a permeability transition pore (PTP). In studying the permeability of the mitochondrial membrane by measuring membrane potential, mitochondrial swelling, and mitochondrial morphology, we considered the possibility that elevations of extramitochondrial Ca²⁺ ([Ca²⁺]_cyt) are sufficient to liberate cytochrome c or activate PTP opening. Cytochrome c release was determined by immunostaining.

PRINCIPAL FINDINGS

1. Low micromolar [Ca²⁺] above 1 µM inhibits mitochondrial respiration due to the release of cytochrome c
The respiration of isolated rat brain mitochondria in the presence of 4 µM extramitochondrial Ca²⁺ is shown in Figure 1 . This [Ca²⁺] caused an inhibition of the active (i.e., ADP-dependent) respiration (20 nmol O₂/mg/min versus 69 nmol O₂/mg/min; Fig. 1b ). This result might indicate a partial blockade or loss of molecular components of the respiratory chain, such as cytochrome c, which has an especially significant impact when the rate of oxygen consumption is high. Direct evidence for Ca²⁺-induced cytochrome c release from rat brain mitochondria was provided by immunostaining of cytochrome c in the supernatant after separating mitochondria by centrifugation (Fig. 2 ).

View larger version (13K): [in this window] [in a new window]   Figure 1. Respiration of rat brain mitochondria in the presence of low micromolar Ca2+. Rat brain mitochondria (0.5 mg protein/ml) were incubated at 30°C in a medium similar to the electrolyte composition of the cytosol (incubation medium) with 5 mM glutamate plus 5 mM malate as substrates. The additions were ADP-200 µM ADP, Ca2+-Ca,EGTA buffer with 4 µM Ca2+ final concentration, EGTA-10 mM EGTA, RR-10 µM ruthenium red, CGP3715–10 µM CGP3715, cyt c-30 µM cytochrome c. The number at the traces represent rates of respiration in nmol O2 min-1 mg-1. The experiments shown here and in Fig. 2 typify the five preparations of mitochondria.

View larger version (26K): [in this window] [in a new window]   Figure 2. Ca2+-induced cytochrome c release from rat brain mitochondria. Brain mitochondria (about 0.5 mg protein/ml) were incubated in the medium at 30°C with the additions indicated. After 10 min of incubation, cytochrome c was detected in the supernatant by Western blot analysis. The additions were cytochrome c-0.002 µg/µl cytochrome c, Ca2+-4 µM Ca2+, cyclosporin A-2 µM cyclosporin A, dextran-25% dextran, L-NAME-10 mM L-NAME.

2. Ca²⁺-dependent cytochrome c release from isolated rat brain mitochondria is reversible, requires the influx of Ca²⁺ into the mitochondrial matrix but is independent of NO generation
The addition of 10 µM extramitochondrial cytochrome c could restitute ADP-induced respiration in the presence of 4 µM extramitochondrial Ca²⁺ to a large degree (40 vs. 69 nmol O₂/mg/min, Fig. 1e ). This finding emphasizes the possibility that cytochrome c release by low micromolar [Ca²⁺] is reversible. Furthermore, complete prevention of Ca²⁺ influx into the mitochondrial matrix by combined inhibition of both the Ca²⁺-uniporter by ruthenium red and the Na⁺-Ca²⁺ exchanger by the specific inhibitor CGP37157 totally abolished the Ca²⁺-induced decrease in active respiration (Fig. 1d ). Thus, Ca²⁺ needs to enter the mitochondrial matrix to induce the inhibition of ADP-stimulated respiration. In contrast to other tissues such as liver mitochondria, a specific inhibitor of NO-synthase, L-NAME, did not prevent the release of cytochrome c, which indicates a NO-independent mechanism in brain (Fig. 2) .

3. The voltage-dependent anion channel (VDAC, porin pore) might mediate the passage of cytochrome c through the outer mitochondrial membrane
It has been speculated that cytochrome c might permeate through the mitochondrial outer membrane via VDAC. Because dextran is known to inhibit VDAC activity, we examined its effect on the appearance of cytochrome c in the extramitochondrial medium following the addition of 4 µM Ca²⁺. Indeed, dextran was a potent inhibitor of Ca²⁺-induced cytochrome c release, which confirms the involvement of VDAC (Fig. 2) . In contrast to dextran, cyclosporin A, the most prominent inhibitor of PTP, was ineffective. This finding suggests that mitochondrial permeability transition was not involved in cytochrome c release under the same experimental conditions (Fig. 2) .

4. High micromolar [Ca²⁺] is required to induce cytochrome c release following the opening of the PTP in rat brain mitochondria
Opening of the PTP is widely believed to underlie cytochrome c release. However, three separate experimental approaches clearly demonstrated a closed PTP at [Ca²⁺] in the low micromolar range despite cytochrome c release occurrence, whereas [Ca²⁺] 100 µM was necessary to stimulate a cyclosporin A-sensitive PTP opening. 1) In the presence of 4 µM Ca²⁺, brain mitochondria could generate a membrane potential that was detectable by the lipophilic cation tetraphenylphosphonium (TPP⁺). However, opening of the PTP should be accompanied by a complete breakdown of the mitochondrial membrane potential. 2) Opening of the PTP leads to mitochondrial swelling. Nevertheless, brain mitochondria did not swell when exposed to 4 µM Ca²⁺ as determined by light absorption at 546 nm. At 200 µM Ca²⁺ mitochondria did swell in a cyclosporin A-sensitive manner. 3) Electron microscopic analysis did not reveal changes of the characteristic cristae structure at 4 µM extramitochondrial Ca²⁺ that would be accompanied by the rupture of the mitochondrial outer membrane, which was observed at 200 µM Ca²⁺.

CONCLUSIONS

Disturbance of intracellular Ca²⁺ homeostasis is widely accepted as being associated with a wide range of neurodegenerative processes. There is also a growing body of evidence that acute as well as chronic neurological disorders are caused by apoptotic and/or necrotic cell death within the central nervous system. Mitochondria are crucial components of neuronal life and death. Whereas a critical breakdown of energy metabolism unavoidably leads to necrosis, the controlled release of mitochondrial cytochrome c represents a key step within the induction of apoptosis. However, cellular mechanisms linking a deregulation of the extramitochondrial Ca²⁺ concentration ([Ca²⁺]_cyt) to apoptotic or necrotic death have remained unclear.

We now demonstrate that a distinct threshold of the extramitochondrial [Ca²⁺] in the low micromolar range (1 µM) is sufficient to mediate the release of cytochrome c. We furthermore provide evidence that this event is reversible and is caused by influx of Ca²⁺ into the mitochondrial matrix. Recently, it has been shown that cytochrome c release in liver mitochondria is mediated by a mitochondrial NO synthase, which forms NO that is subsequently converted to peroxynitrite. Nevertheless, in here we suggest that cytochrome c is released by a NO-independent mechanism in brain.

PTP opening has been widely assumed to underlie the liberation of cytochrome c. However, our study revealed that Ca²⁺-triggered cytochrome c release appeared at low micromolar [Ca²⁺] from morphologically intact mitochondria and without PTP opening. On the contrary, only relatively high extramitochondrial [Ca²⁺] of 100 µM caused an opening of PTP. Such an extraordinarily high threshold is not consistent with the opening characteristics of PTP observed in tissues other than brain.

It has been reported that contact sites between the mitochondrial inner and outer membranes are of basic importance for the transport of compounds through the mitochondrial membrane. Furthermore, the voltage-dependent anion channel (VDAC), which is known to be involved in assembling such contact sites, can support the transport of cytochrome c through the mitochondrial outer membrane. It has been speculated that increasing Ca²⁺ concentrations can reduce the number of contact sites and thereby make VDAC available for cytochrome c transport. Consequently, a Ca²⁺-specific permeability pathway for cytochrome c transport through the morphologically intact outer membrane is provided, which was masked by the contact site complex. This possibility is supported by our finding that dextran, which is supposed to increase the number of contact sites, inhibits Ca²⁺-induced cytochrome c release.

A possible mechanism where Ca²⁺ dissociates cytochrome c release from PTP opening is schematically presented in Figure 3 . Under physiological conditions, cytochrome c is located in the intermembrane space and takes part in the electron transfer within the respiratory chain. Elevation of the extramitochondrial Ca²⁺ concentration into the low micromolar range causes a decrease in the number of contact sites between the mitochondrial inner and outer membranes. Ion channels within the outer membrane, such as VDAC, are freed to allow the passage of cytochrome c. Cytochrome c release from mitochondria results in a decrease in both mitochondrial ATP production and membrane potential. Because only a part of the mitochondrial cytochrome c pool is released into the extramitochondrial compartment, as indicated by the incomplete inhibition of ADP-stimulated respiration in Fig. 1b , ATP can still be provided by oxidative phosphorylation. In this way, cytochrome c can trigger the executioning part of apoptosis by caspase activation (Fig. 3 ; left side).

View larger version (37K): [in this window] [in a new window]   Figure 3. Schematic diagram of the effect of extramitochondrial Ca2+ on cytochrome c release and permeability transition in rat brain mitochondria. In the low micromolar concentration range, extramitochondrial Ca2+ induces decrease of the number of contact sites between the mitochondrial inner and outer membranes. Ion channels such as VDAC are freed for allowing cytochrome c release from morphologically intact mitochondria triggering the execution of apoptosis. At concentrations in the high micromolar range, Ca2+ induces swelling of the mitochondria and rupture of the outer membrane. This process is accompanied by complete energy breakdown resulting in necrosis.

The scenario described above, however, is significantly changed when [Ca²⁺]_cyt reaches the threshold of about 100 µM. Under such conditions, opening of PTP becomes relevant, which subsequently causes swelling of mitochondria and rupture of the mitochondrial outer membrane (Fig. 3 , right side). Because the membrane potential collapses and ATP levels dramatically decrease down to almost zero, cells undergo necrosis instead of performing a complex, well-organized, and energy-utilizing death program.

With respect to neurodegenerative processes, we postulate that, dependent on the strength of an insult, extramitochondrial [Ca²⁺] can define which type of cell death program will be executed: cytochrome c-triggered apoptosis or a complete and PTP-dependent failure of ATP generation leading to necrosis. We show that in contrast to other tissues an increase in [Ca²⁺] into the low micromolar range is sufficient to induce the liberation of cytochrome c from brain mitochondria, while opening of PTP requires relatively high micromolar [Ca²⁺]. Conceivably, these findings reflect a basic mechanism inherent to postmitotic neuronal tissue to protect cells with an extremely limited regenerative capacity against the devastating consequences of opening of PTP followed by necrotic and inflammatory processes.

FOOTNOTES

¹ To read the full text of this article, go to http://www.fasebj.org/cgi/doi/10.1096/fj.00-0551fje ; to cite this article, use FASEB J. (January 19, 2000) 10.1096/fj.00-0551fje

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