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Nerve growth factor-induced neurite formation in PC12 cells is independent of endogenous cellular gangliosides
Introduction
Results
Discussion
Materials and methods
Acknowledgments
Abbreviations
References
Nerve growth factor-induced neurite formation in PC12 cells is independent of endogenous cellular gangliosides
The PC12 rat pheochromocytoma cell line is an established model for nerve growth factor (NGF)-induced neurite formation. It has been shown that when gangliosides are added to the culture medium of PC12 cells, NGF-induced neurite formation of PC12 cells is enhanced. To determine the role of endogenous cellular gangliosides themselves in NGF-elicited neurite formation, we depleted cellular gangliosides using the new specific glucosylceramide synthase inhibitor, d,l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol·HCl (PPPP). 0.5-2 µM PPPP rapidly inhibited ganglioside synthesis and depletedcellular gangliosides. Nonetheless, over a concentration range of 5-100 ng/ml NGF, in both low serum and serum-free medium, neurite formation was normal. Even pretreatment of PC12 cells for up to 6 days with 1 µM PPPP followed by cotreatment with PPPP and NGF for 10 days, still did not inhibit neurite formation. The conclusion that ganglioside depletion did not block neurite formation stimulated by NGF was supported by the lack of effect of PPPP, under these same conditions, on cellular acetylcholine esterase activity, a neuronal differentiation marker (73.8 ± 12.1 versus 67.2 ± 4.6 nmol/min/mg protein at 50 ng/ml NGF; control versus 1 µM PPPP). These findings, together with previous studies showing enhancement of NGF-induced neurite formation by exogenous gangliosides, underscore the vastly different effects that exogenous gangliosides and endogenous gangliosides may have upon cellular functions.
Key words: gangliosides/nerve growth factor/neurite formation/PC12 cells
Introduction
There has been great interest in the modulation of neurite formation in differentiating cells, with the ultimate goal of understanding the functions of neurons and the process of neuronal development. Since gangliosides are primarily present in the cell surface and are enriched in the central nervous system (Ledeen and Yu, 1982), they have been considered important for the differentiation of neuronal cells (Ledeen, 1984; Hakomori, 1990). Chemically, ganglioside molecules contain a hydrophobic tail (ceramide) and a hydrophilic head group (oligosaccharide chain). The ceramide is embedded in the external cell plasma membrane, and the carbohydrate faces the extracellular side, making it possible for these molecules to serve as either cell surface receptors or their modulators (Fishman and Brady, 1976; Hakomori and Igarashi, 1995).
Recent studies support a role of exogenous gangliosides in neurite formation. Addition of gangliosides to the culture medium can modify the biological responses of cells to several growth factors, including neurotrophic growth factors (Rabin and Mocchetti, 1995; Ferrari et al., 1995; Sakakura et al., 1996). Exogenously added gangliosides enhance neurite formation induced by nerve growth factor (NGF) in PC12 cells (Ferrari et al., 1983). Gangliosides also promote long-term survival of naive and NGF-pretreated PC12 cells in serum-free medium and prevent DNA internucleosomal cleavage (Ferrari et al., 1993), partially by causing Trk dimerization and autophosphorylation (Ferrari et al., 1995). GM1 has been found to bind to the NGF receptor TrkA and enhance NGF-induced TrkA phosphorylation and neurite formation in PC12 cells. The binding is specific in that GM1 does not bind to either the low affinity receptor, p75NGFR, or to the epidermal growth factor receptor (Mutoh et al., 1995). GM1 has also been found to enhance TrkA autophosphorylation and activate MAP kinase in C6-2B glioma derivative C6trk+ cells (Rabin and Mocchetti, 1995). Finally, a novel glycosphingolipid, plasmalopsychosine, purified from human brain, mimics the effect of NGF by activating its receptor kinase and MAP kinase in PC12 cells (Sakakura et al., 1996). What is common to all these studies is that the gangliosides were added to culture medium. While identifying certain pharmacologic effects of gangliosides, these studies have not answered the question of whether the activation of TrkA receptor by NGF is modulated by the interaction of endogenous gangliosides with receptors.
In a previous study, we showed that inhibition of endogenous gangliosides by d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP1) does not block neurite formation induced by retinoic acid or NGF in LAN-5 human neuroblastoma cells (Li and Ladisch, 1997). However, inhibition of ganglioside synthesis by D-PDMP also causes accumulation of ceramide (Radin et al., 1993), which in turn has been reported to induce neurite formation in Neuro2a neuroblastoma cells (Riboni et al., 1995). On the other hand, d,l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol·HCl (PPPP), a new specific enzyme inhibitor, inhibits glycosphingolipid synthesis without causing ceramide accumulation (Abe et al., 1995; Rani et al., 1995). The model, PC12 rat pheochromocytoma cells, is widely used for the study of NGF-induced neurite formation and differentiation. This cell line synthesizes fucose-containing and brain-type gangliosides (Ariga et al., 1987, Walton et al., 1988), and there is a large body of information on the effects of exogenous gangliosides on neurite formation and NGF-induced signal transduction. Thus, in the present study, we downregulated cell surface ganglioside expression in PC12 cells using PPPP, to further addressed the question of the role of endogenous cellular gangliosides in NGF-induced neurite formation. PPPP rapidly inhibited the synthesis of these PC12 cellular gangliosides, but this did not inhibit NGF-induced neurite formation.
Results
Gangliosides of PC12 cells
The ganglioside composition of PC12 rat pheochromocytoma cells was characterized by HPTLC (Figure 1). Nearly all the gangliosides (>90%) were highly polar, migrating on the HPTLC plate between GD1b and GT1b (Figure 1A). The PC12 gangliosides were previously identified as fucose-containing gangliosides including fucosyl-GM1 and fucosyl-GD1b, as well as trace amounts of brain-type gangliosides such as GM1 (Ariga et al., 1987; Mutoh et al., 1995). The ganglioside pattern was confirmed by metabolic radiolabeling, which revealed the same ganglioside pattern as that observed by chemical detection (Figure 1B). Quantification of cellular gangliosides of PC12 cells showed these cells to have a cellular ganglioside content of 6.0 ± 1.2 nmol/mg protein (mean ± SD, n = 3).
Figure 1. HPTLC analysis of PC12 cellular gangliosides.Total cellular gangliosides were isolated and purified from PC12 cells cultured in RPMI with 10% horse serum and 5% fetal bovine serum. (A) Gangliosides were stained as purple bands on the HPTLC plate with resorcinol·HCl reagent. Nearly all the gangliosides migrated between GD1b and GT1b. HBG, human brain gangliosides (8 nmol) used as standards. (B) HPTLC autoradiogram of PC12 cells. Gangliosides were metabolically radiolabeled with [3H]galactose and [3H]glucosamine·HCl as described in Materials and methods. RBG, [14C] labeled rat brain gangliosides prepared as described previously (Quarles and Brady, 1971). [3H]labeled PC12 cellular gangliosides (93,000 d.p.m.) and 1500 d.p.m. [14C]RBG were spotted on the HPTLC plate, which was exposed to the x-ray film for 2 weeks.
Inhibition of ganglioside synthesis by PPPP
To modulate ganglioside metabolism, we targeted glucosylceramide synthase. Glucosylceramide synthase is the key enzyme for glycosphingolipid synthesis. It catalyzes the synthesis of glucosylceramide from ceramide and glucose, which is the first step in the synthesis of neutral glycosphingolipids and thus gangliosides. PPPP is a newly discovered specific glucosylceramide synthase inhibitor (Abe et al., 1995), and unlike D-PDMP, which has been used to study the functional roles of glycosphingolipids (Radin et al., 1993; Felding-Habermann et al., 1990; Barbour et al., 1992; Fenderson et al., 1992), it does not cause ceramide accumulation at the concentration ([le]1.0 µM) which has potent inhibitory effects on ganglioside synthesis (Abe et al., 1995).
Exposure of PC12 cells to 1 µM PPPP rapidly inhibited ganglioside synthesis. As measured by metabolic radiolabeling, the rate of ganglioside synthesis fell from 14.4 ± 3.5 × 103 d.p.m./mg protein/h to 2.0 ± 0.1 × 103 d.p.m./mg protein/h within 1 day of treatment (Figure 2A). After 2 or more days of treatment, the rate of ganglioside synthesis was reduced to 5% of the control level. This blockade of ganglioside synthesis confirmed that PPPP is a potent inhibitor of ganglioside synthesis in PC12 cells.
Figure 2. ffects of PPPP on cellular gangliosides of PC12 cells. PC12 cells were treated with 1 µM PPPP for up to 6 days. The medium and the inhibitor were replaced on day 4. During the final 12 h, the [3H]labeled sugars were added to the culture medium. After harvesting the cells, gangliosides were purified and quantified either by ß-scintillation counting of the ganglioside-associated radioactivity (A) or by resorcinol assay as nanomoles of LBSA (B).
The effect of PPPP on cellular ganglioside content was also quantified. A rapid reduction of cellular gangliosides was observed (Figure 2B); after 24 h of treatment with PPPP, the cellular ganglioside content was reduced to 2.9 ± 0.2 nmol/mg protein, compared with 6.0 ± 1.2 nmol/mg protein in control cells. The cellular ganglioside level decreased to 1.2 nmol/mg protein by day 2, and eventually to 0.4 nmol/mg protein, or 6% of the normal control, by the end of the 6 day treatment. Thus, PPPP effectively inhibits ganglioside synthesis and depletes PC12 cells of their gangliosides, indicating that this experimental system should provide findings regarding the role of cellular gangliosides in NGF-induced neurite formation. Table I. 
Culture conditions
Cellular protein (mg/flask)
Control (0.1% ethanol)
4.55 ± 0.33 (4)
Days of exposure to 1 µM PPPP
1
4.54 ± 0.30 (4)
2
4.38 ± 0.04 (2)
3
3.79 ± 0.06 (2)
5
3.56 ± 0.02 (2)
6
4.13 ± 0.22 (2)
Table II.
| Culture conditions | Neurite bearing cells (%)a | AChE activityb (pmol/min/mg protein) |
| 0.1% Ethanol | 1 ± 1 | 19.4 ±1.2 |
| 0.5 µM PPPP | - | 16.9 ± 1.2 |
| 1 µM PPPP | 1 ± 1 | 16.8 ± 1.2 |
| 50 ng/ml NGF | 51 ±4 | 73.8 ± 12.1 |
| 50 ng/ml NGF +0.5 µM PPPP | - | 65.2 ± 3.7 |
| 50 ng/ml NGF +1 µM PPPP | 52 ± 7 | 67.2 ± 4.6 |
In view of this striking effect on ganglioside metabolism, it was important to establish whether PPPP affected PC12 cell growth.In fact, despite its potent inhibitory effect on ganglioside synthesis, PPPP had only a slight effect on cell growth. As shown in Table I, after treatment of PC12 cells with 1 µM PPPP for up to 6 days, the total cellular protein per flask was found to be almost the same as that of control cells, suggesting that there had been little if any inhibition of cell proliferation. For example, following treatment of PC12 cells with 1 µM PPPP for 6 days, the total protein content was 4.13 ± 0.22 mg/flask, compared with 4.55 ± 0.33 mg/flask for the control cells. These results confirmed that PPPP is a specific, potent inhibitor of ganglioside synthesis, without nonspecific toxic effects on the cells. This is of particular value in the study of the effects of depletion of cellular gangliosides on NGF-induced neurite formation and cellular differentiation in PC12 cells.
Effect of NGF and ganglioside depletion on neurite formation
In the first series of experiments evaluating the effect of NGF and short-term PPPP exposure on neurite formation, PC12 cells were cultured in serum-free RPMI-1640 for 3 days. In control cultures, fewer than 1% of the cells had neurites (Table II). The presence of 1 µM PPPP in the culture medium had no effect on the morphology of PC 12 cells. For example, exposure of the cells to NGF at a concentration of 50 ng/ml induced PC12 cells to differentiate with the appearance of neurites, as originally reported by Green and Tischler (Greene and Tischler, 1976). About half of the cells bore neurites (Table II). When PC12 cells were treated with both 50 ng/ml NGF and 1µM PPPP, the percentage of cells bearing neurites was identical, i.e., 52% of the cells had neurites (Table II). In a series of experiments conducted over a concentration range of 5-100 ng/ml NGF, in both serum-free medium and low serum medium (1% horse serum and 0.5% fetal bovine serum), neurite formation was consistently normal in the presence of 0.5-2 µM PPPP. These results suggested that PPPP-treatment in fact did not inhibit neurite formation induced by NGF in PC12 cells.
In a further experiment to delineate the effects of ganglioside depletion on NGF-induced neurite formation, we pretreated PC12 cells with 1 µM PPPP in RPMI-1640 with 10% horse serum and 5% fetal bovine serum for 4-6 days. The cells were then exposed to NGF for 10 days in the presence of PPPP to assure not only that ganglioside synthesis was inhibited but also that cellular ganglioside content was depleted. The long period of incubation was chosen because the cellular ganglioside content depends not only upon ganglioside synthesis but also upon catabolism and cell division (new membrane synthesis). It is the cell division that rapidly reduces the cellular ganglioside content when ganglioside synthesis is inhibited. As shown in Figure 3A, the control cells only developed some very short extensions, which generally were shorter than their soma diameter. 1 µM PPPP treatment alone did not have any effect on neurite formation (Figure 3B). NGF-treatment of PC12 cells caused striking neurite formation, as previously reported (Greene and Tischler, 1976). These neurites were long and very extensive (Figure 3C,E). Depletion of cellular gangliosides by PPPP did not have any apparent effects on the formation and growth of neurites (Figure 3D,F). In addition, PPPP treatment did not affect neurite branching, or arborization, as shown in Figure 3C-F. Therefore, even prolonged PPPP treatment of PC12 cells, with its even more complete ganglioside depletion, had no effect on NGF-induced neurite formation and growth.
Figure 3. ffect of prolonged PPPP treatment on NGF-induced neurite formation in PC12 cells.In this experiment, PC12 cells were first exposed to 1 µM PPPP (A, C, and E) or 0.1% ethanol (B, D, and F) for 4 days in RPMI-1640 with 10% horse serum and 5% fetal bovine serum and then were cultured for 10 days in low serum medium (1% horse serum and 0.5% fetal bovine serum) which contained 0.1% ethanol (A), 1 µM PPPP (B), 50 ng/ml NGF (C and E), or 50 ng/ml NGF +1 µM PPPP (D and F). Magnification was 400× for (A-D) and 100× for (E) and (F).
Effects of PPPP on NGF-induced acetylcholinesterase activity
To confirm the lack of an effect of PPPP on NGF-induced differentiation, we determined the NGF-induced cellular acetylcholinesterase activity. Induction of acetylcholinesterase activity is a neuronal differentiation marker. PC12 cells were first exposed to PPPP for 4 days in RPMI-1640 with 10% horse serum and 5% fetal bovine serum and then were cultured for 10 days in low serum medium (1% horse serum and 0.5% fetal bovine serum) containing PPPP with or without 50 ng/ml NGF. As shown in Table II, PC12 cells cultured in the presence of 0, 0.5, or 1 µM PPPP had similar acetylcholinesterase activity (19.4 ± 1.2, 16.9 ± 1.2, and 16.7 ± 1.2 nmol/min/mg protein, respectively). Exposure of PC12 cells to 50 ng/ml NGF caused a more than 3-fold increase in acetylcholinesterase activity, to 73.8 ± 12.1 nmol/min/mg protein. The cotreatment of PC12 cells with 1 µM PPPP as well as 50 ng/ml NGF did not significantly reduce the enzyme activity (67.2 ± 4.6 nmol/min/mg protein, p = 0.42, Student's t test, two-tailed). Thus, ganglioside depletion caused by PPPP does not affect the induction of activity of a neuronal differentiation enzyme marker, acetylcholinesterase by NGF.Overall, these results exclude a requisite role of endogenous cellular gangliosides in NGF-induced neurite formation of PC12 cells.
Discussion
PC12 rat pheochromocytoma cells have been extensively used as a model for studying neurite formation and differentiation. Upon treatment with nerve growth factor, PC12 cells stop dividing, form neurites, and undergo differentiation (Greene and Tischler, 1976). The interaction of NGF with its high affinity receptor TrkA in the cell membrane activates an intracellular signaling pathway (Klein et al., 1991; Kaplan et al., 1991). Trk receptors become activated by a two-step process that involves the ligand-mediated dimerization of receptor molecules at the cell surface followed by autophosphorylation of their tyrosine residues, a mechanism shared by all known tyrosine kinase receptors (Chao, 1992; Schlessinger and Ullrich, 1992; Jing et al., 1992). NGF signals at least in part through the well-known Ras/Raf/MAP kinase pathway (Egan and Weinberg, 1993).Addition of NGF to PC12 cells results in the rapid activation of Ras proteins (Thomas et al., 1992; Wood et al., 1992), as well as of the downstream Raf and MAP kinases (Schanen-King et al., 1991; Loeb et al., 1992; Thomas et al., 1992). Exogenously added gangliosides have been shown to enhance NGF-mediated signal transduction and neurite formation (Ferrari et al., 1983, 1995; Mutoh et al., 1995; Rabin and Mocchetti, 1995), and plasmalopsychosine, a glycosphingolipid purified from human brain, mimics the effect of NGF by activating its receptor kinase and MAP kinase in PC12 cells (Sakakura et al., 1996).
In addition to these effects of exogenous gangliosides on signal transduction, experimental and clinical studies in vivo are identifying a number of pharmacologic effects of exogenous gangliosides on the nervous system. For example, administration of GM1 ganglioside may have therapeutic effects in neurologic disorders and injury, e.g., acute stroke (Argentino et al., 1989), spinal cord injury (Geisler et al., 1991), and brain repair (Garofalo and Cuello, 1995). Systemic administration of GM1 enhances the effects of NGF in preventing vinblastine-induced sympathectomy in newborn rats (Vantini et al., 1988). Ganglioside GM1 also potentiates the effect of NGF in preventing rat cholinergic neuron death subsequent to cortical damage (Cuello et al., 1989; DiPatre et al., 1989) and enhancing neurite regeneration of chick sensory (dorsal root ganglion) neurons (Doherty et al., 1985). In primates with experimental parkinsonism, GM1 treatment ameliorated the parkinsonism-like symptoms (Schneider et al., 1992).
The above studies demonstrated activities of exogenous gangliosides both in cell systems and in vivo and made it important to delineate the role of endogenous cellular gangliosides, i.e., gangliosides naturally present in the cell membrane. Alteration of cellular ganglioside content, such as by gene transfer (Kojima et al., 1994; Ariga et al., 1995), or by treatment of the cells with antisense oligomers (Zeng et al., 1995), monoclonal antibodies (Ledeen et al., 1990; Wu et al., 1994), or sphingolipid synthesis inhibitors (Schwarz et al., 1995), has been shown to influence cellular differentiation and/or neurite formation in several cell lines. Using the newly discovered specific glucosylceramide synthase inhibitor, PPPP, here we approached the question of how inhibition of cellular ganglioside synthesis influences neurite formation and differentiation of NGF-treated PC12 cells. PPPP itself was highly active, rapidly causing the expected inhibition of ganglioside synthesis and depletion of cellular gangliosides. If endogenous cell surface gangliosides act to regulate or modulate NGF-mediated signaling, the depletion of cellular gangliosides should have altered NGF-elicited neurite formation. This was not observed: over a concentration range of 5-100 ng/ml NGF, in both serum free medium and low serum medium in the presence of PPPP, neurite formation was normal. Even prolonged pretreatment of PC12 cells with PPPP still did not inhibit the neurite formation. These results were confirmed by the lack of effects of PPPP on cellular acetylcholine esterase activity, a neuronal differentiation marker. Thus, the present study shows that ganglioside depletion did not block neurite formation elicited by NGF.
This conclusion is supported by some previous studies in other cell systems. Inhibition of glycosphingolipid synthesis by d-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP) did not affect fish embryo development (Fenderson et al., 1992) nor neurite formation in retinoic acid/NGF-treated neuroblastoma cells (Li and Ladisch, 1997). The loss of complex gangliosides in the GM2/GD2 synthase gene knockout mouse resulted in only subtle defects in nervous system function (Tarkamiya et al., 1996).It should be emphasized, however, that various cell lines, primary neuronal cell cultures, and organized neuronal tissues in vivo all may have very different susceptibility to gangliosides and to NGF. And, since the metabolic inhibitor, PPPP, cannot completely block glycosphingolipid synthesis, it seems that a glucosylceramide synthase knockout animal model would be the ideal tool for further studies.
Together with previous studies of enhancement of NGF-induced neurite formation by exogenously added gangliosides, the present study underscores the vastly different effects that exogenous gangliosides and endogenous gangliosides may have upon NGF-elicited neurite formation. Exogenously added gangliosides appear to be pharmacologically active, while depletion of endogenous gangliosides, normally found in the cell membrane in physiological circumstances, does not block NGF-elicited neurite formation in PC12 cells.
Materials and methods
Preparation of nerve growth factor and PPPP stock solutions
Nerve growth factor-[beta] (NGF-[beta]; mouse submaxillary glands, Sigma, St. Louis, MO)was dissolved in RPMI-1640 at the concentration of 20 ng/µl, and was diluted to the appropriate concentration before use and added to the culture medium. The stock solution was stored at -70°C. d,l-Threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol·HCl (PPPP; Matreya, Pleasant Gap, PA) was dissolved in ethanol at a concentration of 10-3 M and kept as a stock solution at -20°C. For each experiment, PPPP was directly added into the culture medium. The final concentration of ethanol in the culture medium was[le]0.1% (vol/vol).
Culture of PC12 cells
PC12 rat pheochromocytoma cells were purchased from American Type Culture Collection (Rockville, MD), and cultured routinely in RPMI-1640 (Biowhittaker, Walkersville, MD) with 10% horse serum (Life Technologies, Grand Island, NY) and 5% fetal bovine serum (Hyclone, Logan, UT). PC12 cells were cultured in Primaria tissue culture flasks (Becton Dickinson, Franklin Lakes, NJ) to improve their adherence. For differentiation assays, PC12 cells were cultured in low serum RPMI-1640 (1% horse serum and 0.5% fetal bovine serum) or in serum free RPMI-1640. The culture medium was changed every 3-4 days. Cell viability was assessed by trypan blue dye exclusion.
Differentiation of PC12 cells induced by NGF
PC12 cells were exposed to 5-100 ng/ml NGF in either low serum medium or in serum-free medium to induce neurite formation and cellular differentiation. Neurite formation was observed and photographed under phase-contrast microscopy (Greene and Tischler, 1976). For quantification of neurite outgrowth, 120-150 cells were counted. A cell was determined to be positive for neurite extension if it had at least one neurite that was longer than the soma diameter of the cell (Campbell and Neet, 1995). Cells cultured in medium containing 0.1% ethanol or 1 µM PPPP were used as controls.The specific acetylcholinesterase activity, a marker of neuronal differentiation, was used as a quantitative assay for PC12 cell differentiation (Tao-Cheng et al., 1995).
Metabolic radiolabeling of cellular gangliosides
PC12 cells were cultured in RPMI-1640 with 10% horse serum and 5% fetal bovine serum in the presence of 1 µM PPPP for up to 6 days. d-[6-3H]-Galactose (specific activity, 25 Ci/mmol) and d-[6-3H]-glucosamine·HCl (specific activity, 30 Ci/mmol, DuPont NEN, Boston, MA) were added to the culture medium (1 µCi ml-1) during the final 12 h to label the cellular gangliosides (Kemp and Stoolmiller, 1976). These radiolabeled cells were washed three times and harvested by trypsinization. The resulting cell suspension was centrifuged at 300 × g for 10 min, and the cell pellet was washed once with phosphate-buffered saline prior to processing for ganglioside purification (Li and Ladisch, 1992).
Ganglioside purification and quantitation
Total lipid extracts of the cell pellets and culture supernatant were obtained by extracting the lyophilized starting material twice with chloroform/methanol (1:1, by volume) for 18 h at 4°C with stirring. Gangliosides were isolated by partitioning the dried total lipid extract in diisopropyl ether/1-butanol/17 mM aqueous NaCl (6:4:5, by volume) (Ladisch and Gillard, 1985). Gangliosides, in the lower aqueous phase, were further purified by Sephadex G-50 gel filtration and quantified as nanomoles of lipid-bound sialic acid (LBSA) by the modified resorcinol method (Ledeen and Yu, 1982).Ganglioside-associated radioactivity was quantified by ß-scintillation counting (Betafluor; National Diagnostics, Manville, NJ).
High performance thin-layer chromatographic analysis
High performance thin-layer chromatographic (HPTLC) analysis of gangliosides was performed using 10 × 20 cm precoated silica gel 60 HPTLC plates (Merck, Darmstadt). The plates were developed in chloroform/methanol/0.25% aqueous CaCl2.2H20 (60:40:9, by volume) to separate gangliosides, which were stained as purple bands with resorcinol·HCl (Ledeen and Yu, 1982). Radiolabeled gangliosides were revealed by exposure of the XRP x-ray film (Eastman Kodak Co. Rochester, NY) to the HPTLC plate.
Acknowledgments
This work was supported by Grant CA61010 from the National Cancer Institute, by the Stewart Trust, and by the Discovery Fund from the Children's Research Institute.
Abbreviations
1D-PDMP, d-threo-1-phenyl-2-decanoylamino-3-morpholino-1- propanol; PPPP, d,l-threo-1-phenyl-2-hexadecanoylamino-3-pyrrolidino-1-propanol·HCl; HPTLC, high performance thin-layer chromatography; LBSA, lipid-bound sialic acid; NGF, nerve growth factor.
References
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