Calcium-dependent nerve growth factor-stimulated hydrolysis of phosphoinositides in PC12 cells

The effect of nerve growth factor (NGF) on the hydrolysis of phosphoinositides in PC12 cells was examined. Addition of NGF to PC12 cells prelabeled with [3H]-inositol resulted in an increase in the formation of labeled inositol trisphosphate ([3H]IP3), inositol bisphosphate ([3H]IP2), and inositol monophosphate ([3H]IP), an observation indicating that NGF stimulated hydrolysis of the polyphosphoinositides. The increase in these inositol phosphates was detected as early as 15 s after addition of NGF. In the presence of LiCl, the accumulation of [3H]IP was linear for at least 20 min. The NGF-stimulated accumulation of [3H]IP was dose-dependent with a Kact of 0.17 nM and was dependent on the presence of extracellular calcium. In a calcium-free buffer containing EGTA, the NGF-dependent increase in accumulation of [3H]IP was not seen, and the basal level of [3H]IP accumulation was lower than that observed in the presence of extracellular calcium. Lanthanum inhibited both the basal and NGF-stimulated accumulation of [3H]IP, whereas the calcium ionophore A23187, in the absence of NGF, stimulated an accumulation of [3H]IP. The maximal accumulation of [3H]IP in the presence of A23187 was the same as that observed in the presence of NGF. Incubation of the cells with both A23187 and NGF resulted in an accumulation of [3H]IP that was not significantly different from the effect of either agent alone. These results suggest that NGF rapidly stimulates the hydrolysis of phosphoinositides in PC12 cells and that this NGF-stimulated hydrolysis of phosphoinositides occurs by a calcium-dependent mechanism.