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Inhibition of mitochondrial calcium-independent phospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and is cardioprotective.

ABSTRACT: Calcium-independent phospholipase A(2) (iPLA(2)) is the predominant phospholipase A(2) present in myocardium, and its pathophysiological role in acute myocardial infarction has been suggested by the rapid increase in membrane-associated iPLA(2) activity during myocardial ischaemia and reperfusion (I/R). We therefore examined iPLA(2) in mitochondrial fractions prepared from Langendorff-perfused adult rabbit hearts. Our studies indicate that iPLA(2)beta is present in rabbit heart mitochondrial inner membranes with no apparent translocation during ischaemia, I/R or preconditioning. Mitochondrion-associated iPLA(2) was catalytically competent and exhibited 2-, 3- and 2.5-fold increases in measured iPLA(2) activity following ischaemia, I/R and preconditioning, respectively, when compared with the activity of iPLA(2) measured in mitochondria from control hearts. Mitochondrial phospholipids are essential for maintaining the ordered structure and function of the organelle. I/R resulted in a rapid overall decrease in phosphatidylcholine and phosphatidylethanolamine glycerophospholipid species, as determined by electrospray ionization MS, that was partially alleviated by pretreatment of hearts with the iPLA(2)-specific inhibitor, bromoenol lactone (BEL). Pretreatment of I/R hearts with 10 microM BEL significantly reduced the infarct size almost to that of continuously perfused hearts and was cardioprotective only when administered prior to ischaemia. Cardioprotection by BEL was reversed by the simultaneous perfusion of 100 microM 5-hydroxydecanoate, implicating the mitochondrial K(ATP) channel in BEL-mediated protection from I/R. Preconditioning also significantly reduced the infarct size in response to I/R but protection was lost by concurrent perfusion of 10 microM arachidonic acid. Taken together, these data strongly implicate mitochondria-associated iPLA(2) in the signal transduction of myocardial I/R injury.

SUBMITTER: Williams SD

PROVIDER: S-EPMC1222356 | biostudies-other | 2002 Feb

REPOSITORIES: biostudies-other

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Project description:Calcium-independent phospholipase A(2)? (iPLA(2)?) (PNPLA8) is the predominant phospholipase activity in mammalian mitochondria. However, the chemical mechanisms that regulate its activity are unknown. Here, we utilize iPLA(2)? gain of function and loss of function genetic models to demonstrate the robust activation of iPLA(2)? in murine myocardial mitochondria by Ca(2+) or Mg(2+) ions. Calcium ion stimulated the production of 2-arachidonoyl-lysophosphatidylcholine (2-AA-LPC) from 1-palmitoyl-2-[(14)C]arachidonoyl-sn-glycero-3-phosphocholine during incubations with wild-type heart mitochondrial homogenates. Furthermore, incubation of mitochondrial homogenates from transgenic myocardium expressing iPLA(2)? resulted in 13- and 25-fold increases in the initial rate of radiolabeled 2-AA-LPC and arachidonic acid (AA) production, respectively, in the presence of calcium ion. Mass spectrometric analysis of the products of calcium-activated hydrolysis of endogenous mitochondrial phospholipids in transgenic iPLA(2)? mitochondria revealed the robust production of AA, 2-AA-LPC, and 2-docosahexaenoyl-LPC that was over 10-fold greater than wild-type mitochondria. The mechanism-based inhibitor (R)-(E)-6-(bromomethylene)-3-(1-naphthalenyl)-2H-tetrahydropyran-2-one (BEL) (iPLA(2)? selective), but not its enantiomer, (S)-BEL (iPLA(2)? selective) or pyrrolidine (cytosolic PLA(2)? selective), markedly attenuated Ca(2+)-dependent fatty acid release and polyunsaturated LPC production. Moreover, Ca(2+)-induced iPLA(2)? activation was accompanied by the production of downstream eicosanoid metabolites that were nearly completely ablated by (R)-BEL or by genetic ablation of iPLA(2)?. Intriguingly, Ca(2+)-induced iPLA(2)? activation was completely inhibited by long-chain acyl-CoA (IC(50) ?20 ?m) as well as by a nonhydrolyzable acyl-CoA thioether analog. Collectively, these results demonstrate that mitochondrial iPLA(2)? is activated by divalent cations and inhibited by acyl-CoA modulating the generation of biologically active metabolites that regulate mitochondrial bioenergetic and signaling functions.

Project description:BackgroundAntimicrobial peptides derived from the natural processing of chromogranin A (CgA) are co-secreted with catecholamines upon stimulation of chromaffin cells. Since PMNs play a central role in innate immunity, we examine responses by PMNs following stimulation by two antimicrobial CgA-derived peptides.Methodology/principal findingsPMNs were treated with different concentrations of CgA-derived peptides in presence of several drugs. Calcium mobilization was observed by using flow cytometry and calcium imaging experiments. Immunocytochemistry and confocal microscopy have shown the intracellular localization of the peptides. The calmodulin-binding and iPLA2 activating properties of the peptides were shown by Surface Plasmon Resonance and iPLA2 activity assays. Finally, a proteomic analysis of the material released after PMNs treatment with CgA-derived peptides was performed by using HPLC and Nano-LC MS-MS. By using flow cytometry we first observed that after 15 s, in presence of extracellular calcium, Chromofungin (CHR) or Catestatin (CAT) induce a concentration-dependent transient increase of intracellular calcium. In contrast, in absence of extra cellular calcium the peptides are unable to induce calcium depletion from the stores after 10 minutes exposure. Treatment with 2-APB (2-aminoethoxydiphenyl borate), a store operated channels (SOCs) blocker, inhibits completely the calcium entry, as shown by calcium imaging. We also showed that they activate iPLA2 as the two CaM-binding factors (W7 and CMZ) and that the two sequences can be aligned with the two CaM-binding domains reported for iPLA2. We finally analyzed by HPLC and Nano-LC MS-MS the material released by PMNs following stimulation by CHR and CAT. We characterized several factors important for inflammation and innate immunity.Conclusions/significanceFor the first time, we demonstrate that CHR and CAT, penetrate into PMNs, inducing extracellular calcium entry by a CaM-regulated iPLA2 pathway. Our study highlights the role of two CgA-derived peptides in the active communication between neuroendocrine and immune systems.

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Inhibition of mitochondrial calcium-independent phospholipase A2 (iPLA2) attenuates mitochondrial phospholipid loss and is cardioprotective.

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