Endocytosis of nerve growth factor by 'differentiated' PC12 cells studied by quantitative ultrastructural autoradiography

The endocytosis of [¹²⁵I]nerve growth factor (NGF) by rat pheochromocytoma cells (PC12 line), previously exposed to the growth factor ('differentiated' or 'primed' cells), was studied by ultrastructural quantitative autoradiography. Cells previously grown in the presence of NGF were incubated at 37 °C with [¹²⁵I]NGF for periods of up to 24 h. Under these culture conditions, PC12 cells have a rich network of neurites. At the commencement of the experiment, after incubation of cells with [¹²⁵I]NGF for 1 min at room temperature, the plasma membranes of perikarya and processes showed similar levels of labeling by [¹²⁵I]NGF of 0.186 ± 0.03 grains/μm and 0.152 ± 0.013 grains/μm respectively. The density of grains per micron of plasma membrane of perikarya reached a plateau between 15 min to 2 h of incubation of cells at 37 °C with [¹²⁵I]NGF (0.58 ± 0.15 grains/μm and 0.65 ± 0.18 grains/μm, respectively). The endocytosis of [¹²⁵I]NGF in perikarya of cells incubated for 6 h at 37 °C was studied by the 'mask' analysis method of Salpeter et al.²². At this time, the greatest amount of endocytosis was observed, corresponding to 28.4% of total grain counts. The following optimized computed source densities, or relative specific activities ± standard errors of measurement (S.E.M.), were obtained: plasma membrane, 16.52 ± 0.86; multivesicular bodies, 9.58 ± 2.84; endosomes, 5.00 ± 0.97; smooth vesicles and tubules, 1.66 ± 0.38; lysosomes, 1.13 ± 0.20; mitochondria, 0.46 ± 0.10; nuclear membranes or envelopes, 0.32 ± 0.14; nuclei, 0.06 ± 0.01; the Golgi apparatus, 0.08 ± 0.06; and other cytoplasmic elements 0.07 ± 0.03. Our findings indicate that smooth vesicles and tubules, endosomes, multivesicular bodies and lysosomes are part of the pathway(s) of endocytosis of NGF, while all other cytoplasmic and nuclear elements, including the nuclear membrane, are not. The heavy plasma membrane labeling of NGF and the relatively low degree of its endocytosis are consistent with the hypothesis that the NGF action is mediated through plasma membrane activated second messenger(s).