Abstract
Our laboratory previously demonstrated Ca2+-independent phospholipase A2γ (iPLA2γ) is localized to mitochondria and that iPLA2 inhibition blocks cisplatin-induced caspase-mediated apoptosis. Whereas the mitochondrial permeability transition (MPT) is a key control point for apoptosis, the role of mitochondrial iPLA2γ in MPT has not been established. In the present study, we addressed this issue. Ca2+-induced renal cortex mitochondrial (RCM) swelling was blocked by the MPT inhibitor cyclosporine A. The R-isomer of bromoenol lactone (R-BEL), which enantiospecifically inhibits iPLA2γ, inhibited Ca2+-induced RCM MPT, whereas S-BEL (negative control) had no effect. Ca2+ treatment resulted in a significant increase in free arachidonic acid (AA) (>50 μM) in the RCM suspension that was blocked by pretreatment with BEL. No increases in free myristic, palmitic, stearic, oleic, linoleic, or docosahexaenoic acid were detected after Ca2+ treatment. The addition of AA (18 μM) to Ca2+-treated RCM with inhibited iPLA2γ activity restored MPT. We also determined that RCM iPLA2γ displays higher activity against plasmenylcholine with AA in the sn-2 position than oleic acid. Ca2+ exposure significantly increased RCM iPLA2γ activity; however, the Ca2+-induced activation of iPLA2γ was not the result of mitochondrial membrane potential dissipation, opening of the MPT pore, or mitochondrial swelling. Taken together these findings provide strong evidence that Ca2+-induced RCM MPT is mediated by iPLA2γ-catalyzed AA liberation.
Footnotes
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This research was supported by a National Institutes of Health (NIH) Grant DK-62028 (to R.G.S.). G.R.K. was supported by National Institute of Environmental Health Sciences (NIEHS), NIH Training Grant T32 ES-012878. Medical University of South Carolina animal facilities were funded by NIH Grant C06 RR015455. Its contents are solely the responsibility of the authors and do not represent the official views of the NIH.
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Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
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doi:10.1124/jpet.107.119545.
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ABBREVIATIONS: PLA2, cytosolic phospholipase A2; iPLA2, independent phospholipase A2; cPLA2, cytosolic phospholipase A2; MPT, mitochondrial permeability transition; RCM, renal cortex mitochondria; BEL, (E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one or bromoenol lactone; RPTC, rabbit renal proximal tubular cell; CsA, cyclosporine A; AA, arachidonic acid; OA, oleic acid; DPPD, N,N′-diphenyl-p-phenylenediamine; KATP, ATP-dependent K+ channel; FCCP, carbonyl cyanide p-trifluormethoxyphenyl-hydrazone; FAME, fatty acid methyl ester; GC-MS, gas chromatograph-mass spectrometry; PEG, polyethylene glycol; LC-NH2, liquid chromatograph amino propyl bonding.
- Received January 5, 2007.
- Accepted February 15, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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