Abstract
Herein, we demonstrate that calcium-independent phospholipase A(2)γ (iPLA(2)γ) is a critical mechanistic participant in the calcium-induced opening of the mitochondrial permeability transition pore (mPTP). Liver mitochondria from iPLA(2)γ(-/-) mice were markedly resistant to calcium-induced swelling in the presence or absence of phosphate in comparison with wild-type littermates. Furthermore, the iPLA(2)γ enantioselective inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one ((R)-BEL) was markedly more potent than (S)-BEL in inhibiting mPTP opening in mitochondria from wild-type liver in comparison with hepatic mitochondria from iPLA(2)γ(-/-) mice. Intriguingly, low micromolar concentrations of long chain fatty acyl-CoAs and the non-hydrolyzable thioether analog of palmitoyl-CoA markedly accelerated Ca(2+)-induced mPTP opening in liver mitochondria from wild-type mice. The addition of l-carnitine enabled the metabolic channeling of acyl-CoA through carnitine palmitoyltransferases (CPT-1/2) and attenuated the palmitoyl-CoA-mediated amplification of calcium-induced mPTP opening. In contrast, mitochondria from iPLA(2)γ(-/-) mice were insensitive to fatty acyl-CoA-mediated augmentation of calcium-induced mPTP opening. Moreover, mitochondria from iPLA(2)γ(-/-) mouse liver were resistant to Ca(2+)/t-butyl hydroperoxide-induced mPTP opening in comparison with wild-type littermates. In support of these findings, cytochrome c release from iPLA(2)γ(-/-) mitochondria was dramatically decreased in response to calcium in the presence or absence of either t-butyl hydroperoxide or phenylarsine oxide in comparison with wild-type littermates. Collectively, these results identify iPLA(2)γ as an important mechanistic component of the mPTP, define its downstream products as potent regulators of mPTP opening, and demonstrate the integrated roles of mitochondrial bioenergetics and lipidomic flux in modulating mPTP opening promoting the activation of necrotic and necroapoptotic pathways of cell death.
Highlights
The composition and regulation of the mitochondrial permeability transition pore are incompletely understood
Genetic Ablation of iPLA2␥ Results in Decreased Ca2ϩ-dependent PLA2 Activity in Mouse Liver Mitochondria—To examine whether iPLA2␥ was present in isolated murine liver mitochondria, we performed Western analyses of wild-type and independent phospholipase A2␥ (iPLA2␥)Ϫ/Ϫ liver mitochondrial proteins using a custom affinity-purified antibody directed against the C terminus of iPLA2␥ (CKINDWIKLKSDMYEGLPFF)
We examined whether iPLA2␥ loss of function impacted the expression levels of three extensively studied mitochondrial proteins implicated in the mitochondrial permeability transition: VDAC, ANT, and cyclophilin D
Summary
The composition and regulation of the mitochondrial permeability transition pore (mPTP) are incompletely understood. In support of these findings, cytochrome c release from iPLA2␥؊/؊ mitochondria was dramatically decreased in response to calcium in the presence or absence of either t-butyl hydroperoxide or phenylarsine oxide in comparison with wild-type littermates These results identify iPLA2␥ as an important mechanistic component of the mPTP, define its downstream products as potent regulators of mPTP opening, and demonstrate the integrated roles of mitochondrial bioenergetics and lipidomic flux in modulating mPTP opening promoting the activation of necrotic and necroapoptotic pathways of cell death. Long chain fatty acyl-CoA as well as fatty acid has previously been demonstrated to be a potent modulator facilitating the opening of the mitochondrial permeability transition pore, but their mechanisms of action are unknown [35,36,37]. The present study integrates alterations in mitochondrial bioenergetic function with mPTP opening in which iPLA2␥ plays a central mechanistic role
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