Monosodium urate-crystal-stimulated phospholipase D in human neutrophils

Protein kinase C alpha (PKC alpha) and small GTPases of the Rho and ADP-rib osylation factor (Arf) family are implicated in the regulation of phospholi pase D1 (PLD1) activity. Although they are involved in fMet-Leu-Phe (fMLP)- mediated PLD activation, their role in monosodium urate (MSU)-stimulated PL D1 activity in human neutrophils is not clear. The translocation of PKC alp ha RhoA and Arf from the cytosol to the membranes was monitored. fMLP induc ed a cytochalasin B (CB)-dependent recruitment of Arf. RhoA and PKC alpha t o neutrophil membranes. CB also increased the activation of PLD 10-fold. In contrast with fMLP, MSU stimulated a sustained and time-dependent relocali zation of Arf and PKC alpha, but not of RhoA, to the membrane fraction. MSU -stimulated PLD was activated with a time course preceding membrane recruit ment of Arf and PKC alpha. in the absence of CB. Furthermore, MSU-induced P LD activation and the membrane recruitment of PKC alpha, but not that of Ar f, were inhibited by CB. An anti-Fc gamma RIIIB antibody, VIFcRIII, prevent ed the membrane relocalization of Arf and PKC alpha and the stimulation of the levels of tyrosine phosphorylation and of PLD activity induced by MSU. Erbstatin and ST-638, two inhibitors of tyrosine kinases, inhibited the MSU -induced translocation of Arf and PKC alpha but not MSU-induced tyrosine ph osphorylation and PLD activation. Furthermore MSU crystals did not cause th e tyrosine phosphorylation of PLD1. The present study indicates that solubl e and particulate agonists show selectivity in inducing the translocation o f RhoA in neutrophils and that the ability of MSU to increase PLD activatio n was independent of the membrane relocalization of Arf and PKC alpha.