g(m1)-ganglioside and Pheochromocytoma

We have investigated the ability of exogenous gangliosides to modulate nerve growth factor (NGF) signal transduction in PC12 cells. The effects of exogenous ganglioside GM1 on multiple protein kinase activities were assayed by analyzing site-specific serine phosphorylation of tyrosine hydroxylase (TyrOHase) by two-dimensional phosphopeptide mapping. In the presence of NGF, exogenous GM1 (1-10 microM) increased 32P incorporation into TyrOHase phosphopeptide T2, a Ca2+/calmodulin-dependent protein kinase substrate whose phosphorylation is not normally affected by NGF treatment. In the absence of NGF, GM1 treatment had no significant effects on TyrOHase phosphorylation. The removal of extracellular Ca2+ or blockade of dihydropyridine-sensitive Ca2+ channels prevented the GM1-induced increases in 32P incorporation into phosphopeptide T2. Exogenous GM1 also potentiated K+ depolarization-induced increases in the phosphorylation of TryOHase. These results suggest that the stimulatory effects of exogenous GM1 ganglioside on NGF actions may be due to its ability to potentiate a Ca(2+)-dependent signaling pathway.

Topics: Adrenal Gland Neoplasms; Animals; Calcium; Cholera Toxin; Enzyme Activation; G(M1) Ganglioside; In Vitro Techniques; Membrane Potentials; Nerve Growth Factors; Neurons; Peptide Mapping; Pheochromocytoma; Potassium; Protein Kinases; Rats; Signal Transduction; Tumor Cells, Cultured; Tyrosine 3-Monooxygenase

Rat clonal pheochromocytoma PC12h cells were found to bind beta-galactosidase modified with specific glycosides. The enzyme modified with p-aminophenyl beta-D-glucoside was most effectively bound to the cells, followed by alpha-D-mannoside and alpha-D-glucoside. The binding was dependent on the number of PC12h cells, the incubation interval, and the pH; the maximal binding at 4 degrees C was obtained by incubation with 75 micrograms of cell protein for 15 min at pH 4.0. The binding proved to be a saturable and receptor-mediated process, and the apparent Km value and the maximal binding capacity of the cells with beta-D-glucosylated beta-galactosidase were 1.03 +/- 0.06 microM and 333 +/- 24 pmol/min/mg of protein, respectively. When the cells were cultured in the presence of nerve growth factor (NGF), GM1, GM2, and a ganglioside mixture, marked morphological differentiation was observed in the presence of NGF, and the specificity of the binding was also affected. By supplementation of NGF in the culture medium, the cells lost the selectivity of the glycoside binding, whereas cells cultured with GM1 supplement showed increased binding of the specific glycosides.

Topics: Adrenal Gland Neoplasms; Animals; beta-Galactosidase; Clone Cells; G(M1) Ganglioside; G(M2) Ganglioside; Galactosidases; Gangliosides; Glucosides; Glycosides; Glycosylation; Kinetics; Nerve Growth Factors; Pheochromocytoma; Rats; Tumor Cells, Cultured

The effects of ganglioside supplementation of culture medium on monoamine oxidase (MAO) type A and B activities in a rat clonal pheochromocytoma cell line, PC12h, were examined. The MAO activity in PC12h cells proved to be mainly due to type A MAO, and type B MAO activity was negligible. After supplementation of the culture medium with ganglioside GM1, the PC12 cells were found to express type B MAO activity after 4 days of culture, and the amount of type B activity increased with the number of days of culture. After 3 weeks of culture in the presence of GM1, type B activity was about 10% of the total, whereas in control cells type B MAO activity was only about 0.6% of the total. By kinetic analyses of type A and B MAO in PC12h cells after 3 weeks of culture, the increase of type B MAO activity was found to be due to the increase in amount of type B MAO; the Km values were almost the same and only the Vmax values were increased in the cells supplemented with GM1. Among gangliosides tested GM1 was the most effective in causing expression of type B MAO activity, whereas nerve growth factor was not effective. These results suggest that GM1 and other gangliosides may be involved in the expression of type B MAO in nerve cells and in the regulation of levels of the biogenic amines in the brain.

Topics: Adrenal Gland Neoplasms; Animals; Cell Line; Clorgyline; G(M1) Ganglioside; G(M2) Ganglioside; Kinetics; Kynuramine; Monoamine Oxidase; Nerve Growth Factors; Pheochromocytoma; Rats; Selegiline

Topics: Adrenal Gland Neoplasms; Animals; Brain; Cell Line; G(M1) Ganglioside; Gangliosides; Nerve Degeneration; Nerve Growth Factors; Nerve Regeneration; Neurons; Peripheral Nerves; Pheochromocytoma; Rats

The incorporation of radioactive precursors into gangliosides and other glycolipids, glycoproteins, and total lipids has been studied in rat pheochromocytoma PC12 cells. Starting with the same PC12 cell pool, cultures displaying different degrees of neuritic expression in response to nerve growth factor (NGF) and combinations of serum ganglioside GM1 were produced. Attempts were then made to correlate neuritic regulation with biochemical performances of these cells. NGF stimulates the incorporation of [3H]galactose into gangliosides and other glycolipids and glycoproteins and [14C]acetate into total lipids, regardless of the serum concentration. NGF both increased their initial labeling rates and promoted additional and more extensive labeling from culture day 4 onward. Unexpectedly, exogenous GM1 also elicited an increase in ganglioside labeling as well as that of the other lipid classes, but not of glycoproteins. The GM1-induced increase was evident at higher serum concentrations (1%) regardless of the presence or absence of NGF, but not apparent in low (0.15%) serum. Serum levels themselves did not affect labeling patterns in the absence of NGF and GM1. GM1-induced stimulation of labeling reflects an increase in the synthetic activities of the cells, and not increased precursor uptake or reduced product degradation. For all constituents stimulated by GM1, concurrent treatment with NGF produces cumulative effects, suggesting independent mechanisms of action by the two molecules.

Topics: Acetates; Acetic Acid; Adrenal Gland Neoplasms; Animals; Cell Division; Cell Line; Dose-Response Relationship, Drug; G(M1) Ganglioside; Galactose; Gangliosides; Glycolipids; Lipids; Male; Mice; Nerve Growth Factors; Pheochromocytoma; Time Factors

PC12 pheochromocytoma cells in monolayer cultures secrete increased amounts of glycoproteins into the medium following the addition of nerve growth factor (NGF) or of brain gangliosides. After a 48-h incubation with 50 ng/ml NGF there is approximately a twofold increase in the total [14C]glucosamine-labeled, ethanol-precipitable cellular material released into the medium. Between 30 and 50% of the radioactivity is associated with a glycoprotein (Gpl) of molecular weight of 52,000; the remaining radioactivity is distributed between five and six major bands. Only a small amount (10%) is associated with a glycoprotein of Mr greater than 200,000 which might correspond to the NGF-induced large external glycoprotein. A substantial increase in the release of the glycoproteins is also seen on the addition of a variety of gangliosides including asialo GMl. This increase is independent of the presence of NGF. GMl and GDlb/GTlb but not GDla stimulate release above the levels seen in the presence of NGF. Addition of GDla (2 micrograms/ml) enhances selectively the release of various glycoproteins between 2.6- and 8-fold. The pattern of glycoprotein secretion is similar to that seen with NGF, although Gp2 (Mr 78,000) is more abundant. Stimulation of release by GDla is not accompanied by neurite outgrowth, suggesting that the glycoproteins are not directly associated with neuritogenesis. The release of these glycoproteins following the addition of NGF or gangliosides may relate to the neurotrophic properties that these two entirely different ligands exert on PC12 cells.

Topics: Adrenal Gland Neoplasms; Animals; Cell Line; Electrophoresis, Polyacrylamide Gel; G(M1) Ganglioside; Gangliosides; Glucosamine; Glycoproteins; Glycosphingolipids; Molecular Weight; Nerve Growth Factors; Pheochromocytoma; Rats; Tunicamycin

The effects of ganglioside GM1 on proliferation and neuritic growth of PC12 pheochromocytoma cells were studied in the presence and absence of nerve growth factor (NGF). In the absence of NGF, but not in its presence, a decrease in the total number of PC12 cells was first observed after 4-6 days of culture with 10(-6) M GM1 in 0.1% fetal calf serum, and with 10(-3) M GM1 on 10% serum. NGF, with or without GM1, limits cell growth to the first 4-6 days. GM1 enhanced neuritic recruitment with serum concentrations of 0.3% or more. Optimal neurite response varied from 10(-6) M GM1 with 0.3% serum to 10(-4) M GM1 with 10% serum. The influence of GM1 on neurites became more pronounced with increasing serum concentrations, becoming maximal with 1% or greater serum. Serum exhibited a concentration-dependent inhibitory influence (lag) on NGF-induced neuritic recruitment, which was abolished by GM1. Rates of neuritic recruitment following the lag were unaffected by GM1, while showing an inverse correlation with serum concentrations of 0.1-0.5%. Serum may delay the NGF-induced neuritic recruitment of PC12 cells by two independent mechanisms. These results suggest that GM1, in some manner, prevents the serum-induced delay in the onset of neuritic recruitment, rather than stimulating the rate at which it precedes.

Topics: Cell Differentiation; Cell Division; Cell Line; G(M1) Ganglioside; Gangliosides; Nerve Growth Factors; Neurons; Pheochromocytoma