Abstract
Release of cytochrome c, a decrease of membrane potential (Deltapsi(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 Deltapsi(m) increase upon fusion with CL or PG. In contrast, phosphatidylserine fusion required Deltapsi(m) consumption, suggesting that Deltapsi(m) is the driving force in mitochondrial phospholipid importation. Moreover, enrichment with CL and PG brought the low energy mitochondrial population to high Deltapsi(m) values and prevented phosphate-dependent cytochrome c release.
Highlights
The role of mitochondria in neurodegenerative disease has been increasingly supported [1]
Since CL is critical for mitochondrial functioning, CL enrichment of mitochondria was achieved by fusion with CL liposomes
Fusion of CL Liposomes with Mitochondria—Since CL decreased in the resting state, we examined the conditions required to supply CL from exogenous sources
Summary
The role of mitochondria in neurodegenerative disease has been increasingly supported [1]. Localization in Mitochondria of Fused CL with the NAO Probe— Mitochondria (0.3 mg of protein) were incubated at 37 °C for 10 min in the presence of respiratory substrates and CL liposomes (20 nmol of lipid), in S/H buffer (total volume 0.45 ml). Swelling of Isolated Mitochondria—Mitochondrial swelling was determined by measuring the decrease of light scattering at 520 nm of a mitochondrial suspension (75 g of protein in 1.5 ml of S/H buffer) in the presence of potassium phosphate and respiratory substrates, with or without 15 M CCCP.
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