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 procedure for rat cortical synaptosomes using Staphylococcus aureus alpha-toxin (30-100 microg/ml). In permeabilized synaptosomes, PLD activity was significantly stimulated when the concentration of free calcium was increased from 0.1 microM to 1 microM. This activation was inhibited in the presence of KN-62 (1 microM), an inhibitor of calcium/calmodulin-dependent kinase II (CaMKII), but not by the protein kinase C inhibitor, Ro 31-8220 (1-10 microM). Synaptosomal PLD activity was also stimulated in the presence of 1 microM GTPgammaS. When Rho proteins were inhibited by pretreatment of the synaptosomes with Clostridium difficile toxin B (TcdB; 1-10 ng/ml), the effect of GTPgammaS was significantly reduced; in contrast, brefeldin A (10-100 microM), an inhibitor of ARF activation, was ineffective. Calcium stimulation of PLD was inhibited by TcdB, but GTPgammaS-dependent activation was insensitive to KN-62. We conclude that synaptosomal PLD is activated in a pathway which sequentially involves CaMKII and Rho proteins.
Sarri
E
E
Department of Pharmacology, University of Mainz, Germany.
Böckmann
I
I
Kempter
U
U
Valeva
A
A
von Eichel-Streiber
C
C
Weichel
O
O
Klein
J
J
D000818:Animals; D001427:Bacterial Toxins; D002118:Calcium / Q000378:metabolism*; D002463:Cell Membrane Permeability / Q000187:drug effects; D004789:Enzyme Activation; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism*; D006460:Hemolysin Proteins; D066298:In Vitro Techniques; D008297:Male; D010739:Phospholipase D / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar; D013211:Staphylococcus aureus; D013574:Synaptosomes / Q000187:drug effects / Q000201:enzymology* / Q000378:metabolism
D001427:Bacterial Toxins; D006460:Hemolysin Proteins; C021091:staphylococcal alpha-toxin; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D010739:Phospholipase D; D002118:Calcium