Nerve growth factor stimulates phospholipid methylation in growing neurites

Cultures of neurons from rat superior cervical ganglia were deprived of nerve growth factor, loaded with [methyl-3H]methionine, and then challenged with nerve growth factor for different periods of time. Growing neurites and perikarya were separated microsurgically and extracted with chloroform/methanol. Lipid-incorporated radioactivity in the extracts was measured and expressed on the basis of the amount of phospholipid present. The methylated species in the neurite fraction were identified by thin-layer chromatography as mono-, di-, and trimethylphosphatidylethanolamine (phosphatidylcholine). Furthermore, a small peak of lysophosphatidylcholine was detected. In the neurites, but not in the perikarya, phospholipid methylation was found to reach a peak at 10 sec after onset of stimulation. Stimulated levels were at least 4 times higher than levels of unstimulated controls. The peak was followed by rapid decline of phospholipid-incorporated radioactivity. Our result indicates that phospholipid methylation is part of a nerve-growth-factor-activated secondary messenger system in growing sympathetic neurites. The potential significance of this conclusion for directed neuritic growth and membrane expansion is discussed.