Macrophage arachidonate release via both the cytosolic Ca 2+-dependent and -independent phospholipases is necessary for cell spreading

Abstract

We have observed that phospholipase A 2 (PLA 2) activation and arachidonate (AA) release are essential for monocyte/macrophage adherence and spreading. In this study, we addressed the relationship between AA release and cell adherence/spreading in murine resident peritoneal macrophages, and the roles of specific PLA 2s in these processes. The PLA 2-specific inhibitors, ( E)-6-(bromomethylene)tetrahydro-3-(1-naphthalenyl)-2H-pyran-2-one (BEL, specific for the Ca 2+-independent PLA 2 (iPLA 2)) and methyl arachidonoyl fluorophosphonate (MAFP, specific for the Ca 2+-dependent phospholipase (cPLA 2)) inhibited AA release and cell spreading in a correlated fashion but only modestly decreased cell adherence. Cell spreading was normalized by the addition of AA to PLA 2-inhibited cells. AA release during spreading was also inhibited by Ca 2+ depletion or protein kinase C (PKC) inhibition, and was accompanied by increased (but transient) phosphorylation of cPLA 2. Inhibition of macrophage spreading, however, only partially inhibited AA release. Moreover, constitutive AA release was seen in fully spread macrophages which was inhibited by BEL, but not MAFP or Ca 2+ depletion. BEL also reversed the phenotype of fully spread cells. These data suggest that macrophage spreading requires the release of AA by the iPLA 2 (which appears to be constitutively active) and cPLA 2 (which appears to be stimulated by adherence/spreading). Maintenance of macrophage spreading, in contrast, appears to be principally dependent on the iPLA 2.