Independent functioning of cytosolic phospholipase A(2) and phospholipase D-1 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 show n to induce superoxide generation in human monocytes, The peptide stimulate d phospholipase A(2) (PLA(2)) activity in a concentration- and time-depende nt manner. Superoxide generation as well as arachidonic acid (AA) release e voked by treatment with WKYMVm could be almost completely blocked by pretre atment of the cells with cytosolic PLA(2) (cPLA(2))-specific inhibitors. Th e involvement of cPLA(2) in the peptide-induced AA release was further supp orted by translocation of cPLA(2) to the nuclear membrane of monocytes incu bated with WKYMVm, WKYMVm-induced phosphatidylbutanol formation was complet ely abolished by pretreatment with PKC inhibitors. Immunoblot showed that m onocytes 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 ac tivation was investigated. The inhibition of cPLA(2) did not blunt peptide- stimulated PLD1 activation or vice versa, Intracellular Ca2+ mobilization w as indispensable for the activation of PLD1 as well as cPLA(2), The WKYMVm- dependent stimulation of cPLA(2) activity was partially dependent on the ac tivation of PKC and mitogen-activated protein kinase, while PKC activation, but not mitogen-activated protein kinase activation, was an essential prer equisite for stimulation of PLD,, Taken together, activation of the two pho spholipases, which are absolutely required for superoxide generation, takes place through independent signaling pathways that diverge from a common pa thway at a point downstream of Ca2+.