Regulation of phospholipase D activity in synaptosomes permeabilized with Staphylococcus aureus alpha-toxin

In order to investigate the regulation of presynaptic phospholipase D (PLD) activity by calcium and G proteins, we established a permeabilization proc edure for rat cortical synaptosomes using Staphylococcus aureus alpha-toxin (30-100 mu g/ml). In permeabilized synaptosomes, PLD activity was signific antly stimulated when the concentration of free calcium was increased from 0.1 mu M to 1 mu M. This activation was inhibited in the presence of KN-62 (1 mu M), an inhibitor of calcium/calmodulin-dependent kinase II (CaMKII), but not by the protein kinase C inhibitor, Ro 31-8220 (1-10 mu M). Synaptos omal PLD activity was also stimulated in the presence of 1 mu M GTP gamma S , When Rho proteins were inhibited by pretreatment of the synaptosomes with Clostridium difficile toxin B (TcdB; 1-10 ng/ml), the effect of GTP gamma S was significantly reduced; in contrast, brefeldin A (10-100 mu M), an inh ibitor of ARF activation, was ineffective. Calcium stimulation of PLD was i nhibited by TcdB, but GTP gamma S-dependent activation was insensitive to K N-62, We conclude that synaptosomal PLD is activated in a pathway which seq uentially involves CaMKII and Rho proteins. (C) 1998 Federation of European Biochemical Societies.