Membrane-associated phospholipase D activity in neural cell line PC12

Stimulation of PC12 cells with carbachol (Cch) or phorbol 12-myrisrate 13-acetate (PMA) induced [H-3]phosphatidylbutanol (PBut) production. Depletion of extracellular Ca2+ abolished the Cch-mediated phospholipase D (PLD) activation, indicating the requirement of Ca2+ influx. Two different types of PLD activity, oleate-dependent and GTP gamma S-dependent, were examined by using exogenous [H-3]phosphatidylcholine substrate. PLD activity of the membrane fraction of PC12 cells was highly dependent on oleate and independent of GTP gamma S. This profile is in sharp contrast to that observed in HL60 cells showing the profound GTP gamma S-induced activation of PLD. The oleate-dependent PLD activity of PC12 membrane was inhibited by high concentrations of Ca2+ and Mg2+. These results indicate that Ca2+ may not directly activate PLD but through some Ca2+-dependent mechanism(s) in Cch-stimulated cells.