Fucosylation of Glycolipids in PC12 Cells Is Dependent on the Sequence of Nerve Growth Factor Treatment and Adenylate Cyclase Activation

Abstract

Synthesis of complex glycosphingolipids was analyzed in cultures of PC 12 cells treated with nerve growth factor (NGF) and forskolin, either sequentially or simultaneously. For the sequential treatment, forskolin was added following 15 days of NGF treatment and cultures were continued for 3 additional days. For simultaneous treatment, cells maintained in medium with no additives received both agents and were then incubated for 3 additional days. Sequential NGF/forskolin treatment induced hypertrophy in PC12 cells and produced cells which had more neuron-like characteristics than cells treated with NGF alone. Simultaneous treatment initially accelerated the outgrowth of neurites and then reduced neurite elongation. Sequential treatment of PC12 cells resulted in a greater than 50% increase in the incorporation of fucose into neutral glycolipids and gangliosides compared to treatment with NGF alone. Galactose incorporation was unchanged, indicating that there was no net increase in glycolipid synthesis. The lack of an increase in total glycolipid accumulation was confirmed using monoclonal antibodies reacting with specific fucosylated and nonfucosylated PC12 cell glycolipids. However, TLC immunostaining analysis revealed that sequential NGF/forskolin treatment selectively altered the expression of some neutral glycolipid species. In contrast to the effects of the sequential treatment, simultaneous NGF and forskolin treatment was accompanied by a decrease in the incorporation of fucose and galactose into neutral glycolipids and gangliosides. These findings suggest that the timing of exposure to neuronotrophic factors and agents which activate adenylate cyclase might in part account for developmental regulated patterns of glycoconjugate expression and provide further evidence that fucosylation of glycolipids may be associated with maturation of sympathetic neurons.

Effect of Nerve Growth Factor and Forskolin on Glycosyltransferase Activities and Expression of a Globo‐Series Glycosphingolipid in PC12D Pheochromocytoma Cells

“…The extent of incorporation returned to the control level 24 h after administration of NGF . This observation is in contrast with Schwarting et al (1990) who reported that the incorporation of [ 1-l4C ]galactose into neutral GSLs of PC12 cells increased only after long-term (3 days) treatment with NGF alone . This discrepancy is most likely attributable to the different cellular responses to NGF by the two different cell lines ; that is, the response to NGF for neurite outgrowth is much quicker for PC12D than for PC 12 cells (Katoh-Semba et al ., 1987) .…”

Increase in β‐1,4‐Galactosyltransferase Activity During PC12 Cell Differentiation Induced by Forskolin and 2‐Chloroadenosine

“…Results reported in this article suggest that the increase in galactose incorporation into glycoproteins observed by Amos et al (1 990) may have been caused by an induction in the levels of GALTase within the Golgi apparatus. In contrast, Schwarting et al ( 1990) reported an increase in fucosylation in PC12 cells treated sequentially with NGF and forskolin, but no change in the incorporation of galactose into these cells. The effect of forskolin treatment alone on galactose incorporation in PC 12 cells was not investigated in this latter study and, therefore, direct comparison with the results observed in F9 cells is not possible.…”

“…In these studies, GALTase activity on the cell surface did not change upon cell differentiation induced by NGF. In contrast, recent studies have demonstrated that during PC 12 cell differentiation induced by NGF, galactose incorporation into both neutral glycolipids and gangliosides increased by greater than 100% (Schwarting et al, 1990). Addition of the cyclic AMP (CAMP)-stimulating agent, forskolin, to the NGF-induced cells, however, failed to increase galactose incorporation into these glycolipids.…”

“…Several reports (e.g., Amos et al, 1990;Schwarting et al, 1990) have indicated that carbohydrate content of glycolipids and glycoproteins can be altered by a variety of cell differentiating agents, including CAMPinducing agents, although the mechanism for this increase has not been determined. Amos et al (1990) reported an increased incorporation of galactose into glycoproteins in F9 cells that required the presence of both retinoic acid and dibutyryl CAMP.…”

Gal‐NCAM is a differentially expressed marker for mature sensory neurons in the rat olfactory system

“…The mouse mAb AG1 (IgG) recognizes the 140-and 180-kD molecular subtypes of rat NCAM (DiFiglia et al, 1989). The mouse mAbs CC1 and CC6 (IgMs), which recognize unique neuronal glycolipids, have been previously described (Schwarting et al, 1990(Schwarting et al, , 1994. Goat polyclonal antiserum (IgG) against olfactory marker protein (OMP: Hartman and Margolis, 1975) was kindly provided by Dr. F. Margolis.…”