Membrane-associated phospholipase D activity in neural cell line PC12

Stimulation of PC12 cells with carbachol (Cch) or phorbol 12-myristate 13-acetate (PMA) induced [³H]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 GTPγS-dependent, were examined by using exogenous [³H]phosphatidylcholine substrate. PLD activity of the membrane fraction of PC12 cells was highly dependent on oleate and independent of GTPγS. This profile is in sharp contrast to that observed in HL60 cells showing the profound GTPγS-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.