Exogenous Phospholipids Specifically Affect Transmembrane Potential of Brain Mitochondria and Cytochrome c Release

Abstract

Release of cytochrome c, a decrease of membrane potential (⌬ m ), and a reduction of cardiolipin (CL) of rat brain mitochondria occurred upon incubation in the absence of respiratory substrates. Since CL is critical for mitochondrial functioning, CL enrichment of mitochondria was achieved by fusion with CL liposomes. Fusion was triggered by potassium phosphate at concentrations producing mitochondrial permeability transition pore opening but not cytochrome c release, which was observed only at >10 mM. Cyclosporin A inhibited phosphate-induced CL fusion, whereas Pronase pretreatment of mitochondria abolished it, suggesting that mitochondrial permeability transition pore and protein(s) are involved in the fusion process. Phosphate-dependent fusion was enhanced in respiratory state 3 and influenced by phospholipid classes in the order CL > phosphatidylglycerol (PG) > phosphatidylserine. The probe 10-nonylacridine orange indicated that fused CL had migrated to the inner mitochondrial membrane. In state 3, CL enrichment of mitochondria resulted in a pH decrease in the intermembrane space. Cytofluorimetric analysis of mitochondria stained with 3,3-diexyloxacarbocyanine iodide and 5,5,6,6-tetrachloro-1,1,3,3-tetraethylbenzymidazolylcarbocyanine iodide showed ⌬ m increase upon fusion with CL or PG. In contrast, phosphatidylserine fusion required ⌬ m consumption, suggesting that ⌬ m is the driving force in mitochondrial phospholipid importation. Moreover, enrichment with CL and PG brought the low energy mitochondrial population to high ⌬ m values and prevented phosphate-dependent cytochrome c release.The role of mitochondria in neurodegenerative disease has been increasingly supported (1). Mitochondrial dysfunction can lead to deleterious cell injury due to free radical generation, intracellular calcium concentration imbalance, and activation of mitochondrial permeability transition pore (mPTP) 1 (2). In apoptosis, a caspase cascade often mediates cell death (3). Despite the complexity of the mechanisms involved, mitochondria appear to provide a link between the initiator caspases and the downstream effector caspases. Mitochondria release apoptosis-inducing factors that trigger DNA fragmentation of isolated nuclei (4, 5). In addition, Bax acts on mPTP, causing the release of cytochrome c into the cytosol (3, 6). Released cytochrome c binds to Apaf-1, thus initiating activation of caspase-1 and of downstream processes, leading to nuclear DNA fragmentation. Cytochrome c release is also stimulated by Bid (7, 8), whereas Bcl2 prevents it (9). The mechanism responsible for signaling molecule release from mitochondria is under extensive study. It is known that these molecules are bound to the inner mitochondrial membrane, whose integrity appears to be essential for mitochondrial functions. Cardiolipin (CL) is present only within the inner mitochondrial membrane, strictly associated to mitochondrial electron transport complexes and transporters (10). The specific requirement of heart cytochrome c oxidase for CL has been demonst...

Cardiolipin Is Not Required for Bax-mediated Cytochrome c Release from Yeast Mitochondria

“…Furthermore, spontaneous release of cytochrome c may also be dependent upon cell type. Indeed, many laboratories have reported cytochrome c in the supernatants of control incubations of mammalian (16,17,(35)(36)(37) and yeast (20) cells/mitochondria, so it is not surprising that more cytochrome c is spontaneously released from CL-deficient mitochondria. The same argument applies to explain the enhanced release of cytochrome c from ⌬CRD1 compared with WT mitochondria when both types of mitochondria are treated with Bax.…”

Rat Brain Cortex Mitochondria Release Group II Secretory Phospholipase A2 under Reduced Membrane Potential

“…Mitochondria were treated with trypsin or Pronase (25 g/mg mitochondrial protein) and then assayed for Ca 2ϩ -independent or -dependent activities. The choice of protease concentration was based on the observation that it did not affect mitochondrial membrane potential, as shown previously (46). The treatment with proteases caused a decrease of more than 50% in the activity of the Ca 2ϩ -independent enzyme, whereas it did not affect that of the Ca 2ϩ -dependent one (Fig.…”

“…The integrity of the outer mitochondrial membrane was in the range 90 -93%, as calculated by the latency in cytochrome c oxidase assay (45). In some experiments, mitochondria (2-3 mg of protein/ml of 0.32 M sucrose and 2 mM HEPES, pH 7.4) were incubated with 50 g of trypsin or Pronase at 30°C, a concentration known to be ineffective on mitochondrial functional parameters (46). After 30 min, aliquots were taken and mitochondria recovered by centrifugation at 10,000 ϫ g for 10 min.…”

Thematic Review Series: Glycerolipids. Phosphatidylserine and phosphatidylethanolamine in mammalian cells: two metabolically related aminophospholipids

“…In human gray matter, 22:6n-3 accounts for .36% of the fatty acyl species of PS (5-7). Because 22:6n-3 appears to be essential for the normal development and functioning of the nervous system (6,(8)(9)(10)(11), it is likely that PS plays an important role in the nervous system and in vision (reviewed in Ref. 4).…”