Bromoenol Lactone Inhibits Magnesium-dependent Phosphatidate Phosphohydrolase and Blocks Triacylglycerol Biosynthesis in Mouse P388D1 Macrophages

Jesús Balsinde,Edward A Dennis

doi:10.1074/jbc.271.50.31937

Jesús Balsinde, Edward A Dennis

Open Access

https://doi.org/10.1074/jbc.271.50.31937

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Abstract

Bromoenol lactone (BEL) has previously been identified as a potent, irreversible, mechanism-based phospholipase A2 (PLA2) inhibitor that possesses greater than 1000-fold selectivity for inhibition of Ca2+-independent PLA2 (iPLA2) versus the Ca2+-dependent ones. Thus, this compound has been used as a selective tool for studies aimed at elucidating the role of iPLA2 in certain cellular functions. Herein we report that BEL also inhibits cellular phosphatidic acid phosphohydrolase (PAP) activity in intact P388D1 macrophages with an IC50 of about 8 microM, which is very similar to that previously found for inhibition of iPLA2 under the same experimental conditions. This results in the blockage of the incorporation of exogenous arachidonate and palmitate into diacylglycerol and triacylglycerol. Thus, inhibition of PAP by BEL blocks triacylglycerol biosynthesis in P388D1 cells due to decreased diacylglycerol availability. Because two forms of PAP activity exist in mammalian cells, differential assays were performed to identify which of these forms was inhibited by BEL. The results of these experiments revealed that BEL selectively inhibits the cytosolic, Mg2+-dependent enzyme. No apparent effect of BEL on the membrane-bound Mg2+-independent PAP form could be detected. Collectively, the results reported herein establish that BEL inhibits two cellular phospholipases, namely iPLA2 and Mg2+-dependent PAP, with similar potency. Therefore, the inhibitory effect of BEL on Mg2+-dependent PAP might explain several cellular functions previously attributed to iPLA2.

Highlights

Current evidence indicates that phospholipase A2 (PLA2)1 is a major mediator of agonist-induced arachidonic acid (AA) release in most cell types
At high, micromolar levels of AA, abundant incorporation of fatty acid occurs via the de novo pathway, which leads to accumulation of AA in TAG [10, 19, 20]
The results presented in this paper demonstrate that, besides inhibiting independent PLA2 (iPLA2) [10], Bromoenol lactone (BEL) blocks phosphatidic acid phosphohydrolase (PAP) in P388D1 macrophage-like cells

Summary

Introduction

Current evidence indicates that phospholipase A2 (PLA2)1 is a major mediator of agonist-induced arachidonic acid (AA) release in most cell types (for review, see Ref. 1). We have found that BEL inhibits incorporation of AA into TAG, and that this is due to inhibition of the Mg2ϩ-dependent PA phosphohydrolase (PAP-1) present in these cells, an enzyme that converts PA into diacylglycerol (DAG).

Results

Conclusion