PHOSPHOLIPASE-C AND PHOSPHOLIPASE-D ARE ACTIVATED INDEPENDENTLY OF EACH OTHER IN CHEMOTACTIC PEPTIDE STIMULATED HUMAN NEUTROPHILS

When cytochalasin B-treated neutrophils were stimulated with fMet-Leu- Phe (fMLP) in the presence of Ca2+, phospholipase C (PLC) activity, as measured by inositol-1,4,5-trisphosphate (IP3) formation, preceded ph ospholipase D (PLD)-catalyzed breakdown of choline-containing phosphog lycerides to form choline and diradyl-sn-glycero-3-phosphate (phosphat idic acid), suggesting a possible link between PLC and PLD. However, i n the absence of cytochalasin B or extracellular Ca2+, PLC was fully a ctivated by fMLP with minimal activation of PLD, indicating that PLC a ctivation alone is not sufficient for PLD activation. Full activation of PLD by fMLP required the simultaneous presence of both Ca2+ and cyt ochalasin B, a condition that caused no further enhancement of PLC. Th is result suggests that PLD products are not involved in the regulatio n of PLC activation. Furthermore, under conditions of complete inhibit ion of PLC by phorbol 12-myristate 13-acetate (PMA), there was no inhi bition of PLD, showing that fMLP can activate PLD in the absence of PL C. Treatment of intact neutrophils with pertussis toxin inhibited both PLC and PLD, with PLC inhibition occurring at lower concentrations th an PLD inhibition. These differential effects of pertussis toxin and t he observed lack of inhibition of fMLP-stimulated PLD by PMA, which is believed to inactivate G-proteins involved in PLC activation, imply t hat PLC and PLD are linked to fMLP receptors through distinct G-protei ns. Taken together, these observations suggests that, in fMLP-stimulat ed neutrophils, PLC and PLD are activated through independent mechanis ms.