Activation of astroglial phospholipase D activity by phorbol ester involves ARF and Rho proteins

Primary cultures of rat cortical astrocytes express phospholipase D (PLD) i soforms 1 and 2 as determined by RT-PCR and Western blot. Basal PLD activit y was strongly (10-fold) increased by 4 beta-phorbol-12 beta,13 alpha-dibut yrate (PDB) (EC50: 56 nM), an effect which was inhibited by Ro 31-8220 (0.1 -1 mu M), an inhibitor of protein kinase C (PKC), and by brefeldin A (10-10 0 mu g/ml), an inhibitor of ADP-ribosylating factor (ARF) activation. Pretr eatment of the cultures with Clostridium difficile toxin B-10463 (0.1-1 ng/ ml), which inactivates small G proteins of the Rho family, led to a breakdo wn of the astroglial cytoskeleton; concomitantly, PLD activation by PDB was reduced by up to 50%. In contrast, inactivation of proteins of the Ras fam ily by Clostridium sordellii lethal toxin 1522 did not affect PLD activatio n. In parallel experiments, serum-induced PLD activation was sensitive to b refeldin A, but not to Ro 31-8220 and not to clostridial toxins. We conclud e that, in astrocytes, the PLD isoform which is activated by phorbol eater requires PKC, ARF and Rho proteins for full activity and probably represent s PLD1. (C) 2000 Elsevier Science B.V. All rights reserved.