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

Abstract

Activation of brain mitochondrial phospholipase(s) A 2 (PLA 2 ) might contribute to cell damage and be involved in neurodegeneration. Despite the potential importance of the phenomenon, the number, identities, and properties of these enzymes are still unknown. Here, we demonstrate that isolated mitochondria from rat brain cortex, incubated in the absence of respiratory substrates, release a Ca 2؉ -dependent PLA 2 having biochemical properties characteristic to secreted PLA 2 (sPLA 2 ) and immunoreacting with the antibody raised against recombinant type IIA sPLA 2 (sPLA 2 -IIA). Under identical conditions, no release of fumarase in the extramitochondrial medium was observed. The release of sPLA 2 from mitochondria decreases when mitochondria are incubated in the presence of respiratory substrates such as ADP, malate, and pyruvate, which causes an increase of transmembrane potential determined by cytofluorimetric analysis using DiOC 6 (3) as a probe. The treatment of mitochondria with the uncoupler carbonyl cyanide 3-chlorophenylhydrazone slightly enhances sPLA 2 release. The increase of sPLA 2 specific activity after removal of mitochondrial outer membrane indicates that the enzyme is associated with mitoplasts. The mitochondrial localization of the enzyme has been confirmed by electron microscopy in U-251 astrocytoma cells and by confocal laser microscopy in the same cells and in PC-12 cells, where the structurally similar isoform type V-sPLA 2 has mainly nuclear localization. In addition to sPLA 2 , mitochondria contain another phospholipase A 2 that is Ca 2؉ -independent and sensitive to bromoenol lactone, associated with the outer mitochondrial membrane. We hypothesize that, under reduced respiratory rate, brain mitochondria release sPLA 2 -IIA that might contribute to cell damage.

Role of Calcium Independent Phospholipase A2 in Maintaining Mitochondrial Membrane Potential and Preventing Excessive Exocytosis in PC12 Cells

“…The finding is consistent with previous studies which showed the enzyme in mitochondria of myocardial cells [46] and rabbit proximal tubular cells [47]. Besides iPLA 2 , other PLA 2 isoforms such as cPLA 2 , and sPLA 2 -IIA, -III, -V and -X have been localized in PC12 cells [48][49][50][51][52][53]. sPLA 2 -IIA is present in mitochondria, whilst sPLA 2 -V showed mainly nuclear localization in these cells [49].…”

“…Besides iPLA 2 , other PLA 2 isoforms such as cPLA 2 , and sPLA 2 -IIA, -III, -V and -X have been localized in PC12 cells [48][49][50][51][52][53]. sPLA 2 -IIA is present in mitochondria, whilst sPLA 2 -V showed mainly nuclear localization in these cells [49]. Treatment of PC12 cells with BEL resulted in reduction of mitochondrial membrane potential, similar to that observed after treatment with the mitochondrial depolarizing agent FCCP.…”

Inhibitors of Brain Phospholipase A2Activity: Their Neuropharmacological Effects and Therapeutic Importance for the Treatment of Neurologic Disorders

“…However, it is proposed that at the molecular level, heparan sulfate, a glycosaminoglycan, may play an important role in internalization and attachment of PLA 2 isoforms to intracellular organelles Boilard et al, 2003). A reduction in the mitochondrial membrane potential causes the release of sPLA 2 and this sPLA 2 along with other PLA 2 isoforms may be involved in neural cell injury (Farooqui et al, 1997d;Macchioni et al, 2004).…”

“…In brain, Data summarized from Hirashima et al (1992); Matsuzawa et al (1996); Yang et al (1999);and Farooqui et al (2000b EFFECTS OF PLA 2 INHIBITORS 593 astrocytes express sPLA 2 , which can be induced in response to proinflammatory cytokines such as tumor necrosis factor-␣ and interleukin-1␤ (Lin et al, 2004). Mitochondrial fractions from rat brain, PC12, and U251 astrocytoma cell cultures contain significant sPLA 2 and iPLA 2 activities (Macchioni et al, 2004). The mechanism for a secretory protein (like sPLA 2 ) targeting an intracellular organelle (like mitochondria) remains unknown.…”

Effect of NGF on the Subcellular Localization of Group IIA Secretory Phospholipase A2 (GIIA) in PC12 Cells: Role in Neuritogenesis

“…GIIA, which is ubiquitously expressed in all areas of mammalian brain [16] and accounts for most of the PLA 2 activity detected in particulate fractions of rat brain [17]. In PC12 cells and in other neural cell lines GIIA has a cytosolic and mitochondrial localization [18]. Several lines of evidence indicate the involvement of this isoform in the onset and/or aggravation of various neurological diseases (for review see [13]).…”