Calcium-independent phospholipase A2 in rabbit ventricular myocytes

Abstract

We have previously reported that the majority of phospholipase A2 (PLA2) activity in rabbit ventricular myocytes is membrane-associated, calcium-independent (iPLA2), selective for arachidonylated plasmalogen phospholipids and inhibited by the iPLA2-selective inhibitor bromoenol lactone (BEL). Here, we identified the presence of iPLA2 in rabbit ventricular myocytes, determined the full length sequences for rabbit iPLA2beta and iPLA2gamma and compared their homology to the human isoforms. Rabbit iPLA2beta encoded a protein with a predicated molecular mass of 74 kDa that is 91% identical to the human iPLA2beta short isoform. Full length iPLA2gamma protein has a predicated molecular mass of 88 kDa and is 88% identical to the human isoform. Immunoblot analysis of iPLA2beta and gamma in membrane and cytosolic fractions from rabbit and human cardiac myocytes demonstrated a similar pattern of distribution with both isoforms present in the membrane fraction, but no detectable protein in the cytosol. Membrane-associated iPLA2 activity was inhibited preferentially by the R enantiomer of bromoenol lactone [(R)-BEL], indicating that the majority of activity is due to iPLA2gamma.

FIGURE 1

Full-length rabbit iPLA2β cDNA sequence. Underlines indicate the conserved ATP-binding and lipase active sites. Potential transmembrane domains were predicted using the TMpred server and are indicated with boldface type. Potential PKC-phosphorylation sites were predicted using the NetPhosK 1.0 Server and are boxed.

FIGURE 2

Full-length rabbit iPLA₂γ cDNA sequence. Underlines indicate the conserved ATP-binding and lipase active sites. Potential PKC-phosphorylation sites were predicted using the NetPhosK 1.0 Server and are boxed.

FIGURE 3

Immunoblot analysis of the localization of iPLA₂β (Panel A) and iPLA₂γ (Panel B) in the cytosolic (C) and membrane (M) subcellular fractions isolated from rabbit and human cardiac myocytes. Proteins were separated by SDS-PAGE and transferred to polyvinylidene difluoride membranes. Membranes were probed with anti-iPLA₂β or anti-iPLA₂γ antibodies (1:1,000 dilution) and incubated with horseradish peroxidase-linked secondary antibody (1:50,000 dilution). Immunoblots were detected with enhanced chemiluminescence and exposure to film.

FIGURE 4

Membrane-associated iPLA₂ activity measured in the absence or presence of bromoenol lactone (BEL). Isolated membrane fractions were incubated with increasing concentrations of R-BEL (open circles), S-BEL (X) or racemic BEL (filled squares) for 10 minutes prior to assay of PLA₂ activity. PLA₂ activity was measured using 100 μM (16:0, [³H]18:1) plasmenylcholine (PlsCho) in the absence of Ca (4 mM EGTA). Values are means ± SEM for independent results from 6 separate animals. *p<0.05, **p<0.01 when compared to activity in the absence of BEL.