Independent functioning of cytosolic phospholipase A2 and phospholipase D1 in Trp-Lys-Tyr-Met-Val-D-Met-induced superoxide generation in human monocytes

Recently, a novel peptide (Trp-Lys-Tyr-Met-Val-D-Met, WKYMVm) has been shown to induce superoxide generation in human monocytes, The peptide stimulated phospholipase A(2) (PLA(2)) activity in a concentration- and time-dependent manner. Superoxide generation as well as arachidonic acid (AA) release evoked by treatment with WKYMVm could be almost completely blocked by pretreatment of the cells with cytosolic PLA(2) (cPLA(2))-specific inhibitors. The involvement of cPLA(2) in the peptide-induced AA release was further supported by translocation of cPLA(2) to the nuclear membrane of monocytes incubated with WKYMVm, WKYMVm-induced phosphatidylbutanol formation was completely abolished by pretreatment with PKC inhibitors. Immunoblot showed that monocytes express phospholipase D-1 (PLD1), but not PLD2, GF109203X as well as butan-1-ol inhibited peptide-induced superoxide generation in monocytes, Furthermore, the interrelationship between the two phospholipases, cPLA(2) and PLD1, and upstream signaling molecules involved in WKYMVm-dependent activation was investigated. The inhibition of cPLA(2) did not blunt peptide-stimulated PLD1 activation or vice versa, Intracellular Ca2+ mobilization was indispensable for the activation of PLD1 as well as cPLA(2), The WKYMVm-dependent stimulation of cPLA(2) activity was partially dependent on the activation of PKC and mitogen-activated protein kinase, while PKC activation, but not mitogen-activated protein kinase activation, was an essential prerequisite for stimulation of PLD,, Taken together, activation of the two phospholipases, which are absolutely required for superoxide generation, takes place through independent signaling pathways that diverge from a common pathway at a point downstream of Ca2+.