Balance of two secretion pathways of nerve growth factor in PC12 cells changes during the progression of their differentiation, with a decrease in constitutive secretion in more differentiated cells

We have studied the development of the action potential Na+ channels in PC12 cells, an established line that has been useful as a model for neuronal differentiation. In continuous culture PC12 cells, although electrically inexcitable, nevertheless have a low level of Na+ channels as judged by the increase in 22Na+ uptake in the presence of veratridine and scorpion toxin. These two neurotoxins have been shown to promote activation of Na+ channels in a variety of electrically excitable cells. Following treatment with nerve growth factor (NGF), conditions which induce differentiation to an electrically excitably neuronal-cell type, the neurotoxin-activated 22Na+ uptake increases approximately 12-fold, on a per cell basis, reaching a maximum in 12–16 days. The dose–response curves for veratridine and scorpion toxin are unchanged by NGF treatment (K0.5 for veratridine, 18–14 μM; K0.5 for scorpion toxin, 120–96 nM). Na+ channels in both undifferentiated and differentiated cells are tetrodotoxin sensitive and NGF treatment has no effect on the inhibition constant (Ki, 10–12 nM). Na+ channel sites were measured directly by the specific binding of [3H]saxitoxin. In NGF-treated cells, the saxitoxin receptor density reaches 154 fmol/mg protein (Kd, 1.3 nM), a level comparable to other excitable cells. Levels in control cells were too low to measure accurately. These findings show that NGF treatment of PC12 cells leads to a substantial increase in the expression of neurotoxin-sensitive Na+ channels. Furthermore, these channels are pharmacologically similar, if not identical, to those which exist in undifferentiated cells and therefore do not appear to result from the conversion of preexisting channels.

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Long-term, heterologous down-regulation of the epidermal growth factor receptor in PC12 cells by nerve growth factor.

The Journal of Cell Biology ◽

10.1083/jcb.104.6.1611 ◽

1987 ◽

Vol 104 (6) ◽

pp. 1611-1621 ◽

Cited By ~ 40

Author(s):

P Lazarovici ◽

G Dickens ◽

H Kuzuya ◽

G Guroff

Keyword(s):

Nerve Growth Factor ◽

Growth Factor ◽

Epidermal Growth Factor ◽

Pc12 Cells ◽

Egf Receptor ◽

Down Regulation ◽

Nerve Growth ◽

Differentiated Cells ◽

Epidermal Growth

Cells of the rat pheochromocytoma clone PC12 possess receptors for both nerve growth factor (NGF) and epidermal growth factor (EGF), thus enabling the study of the interaction of these receptors in the regulation of proliferation and differentiation. Treatment of the cells with NGF induces a progressive and nearly total decrease in the specific binding of EGF beginning after 12 h and completed within 4 d. Three different measures of receptor show that the decreased binding capacity represents, in fact, a decreased amount of receptor: (a) affinity labeling of PC12 cell membranes by cross-linking of receptor-bound 125I-EGF showed a 60-90% decrease in the labeling of 170- and 150-kD receptor bands in cells treated with NGF for 1-4 d; (b) EGF-dependent phosphorylation of a src-related synthetic peptide or EGF receptor autophosphorylation with membranes from NGF-differentiated cells showed a decrease of 80 and 90% in the tyrosine kinase activity for the exogenous substrate and for receptor autophosphorylation, respectively; (c) analysis of 35S-labeled glycoproteins isolated by wheat germ agglutinin-Sepharose chromatography from detergent extracts of PC12 membranes showed a 70-90% decrease in the 170-kD band in NGF-differentiated cells. These findings permit the hypothesis that long-term heterologous down-regulation of EGF receptors by NGF in PC12 cells is mediated by an alteration in EGF receptor synthesis. It is suggested that this heterologous down-regulation is part of the mechanism by which differentiating cells become insensitive to mitogens.

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