Endocytosis of cholera toxin in GERL-like structures of murine neuroblastoma cells pretreated with GM1 ganglioside. Cholera toxin internalization into Neuroblastoma GERL

Cholera toxin (CT), covalently attached to horseradish peroxidase (HRP), is a specific cytochemical marker for GM1 ganglioside (GM1) and retains the ability of the native toxin to raise levels of cyclic AMP in avian erythrocytes. Using a cytochemical stain for HRP, we found that 9% of control cultured murine neuroblastoma cells bound cholera toxin-horseradish peroxidase conjugates (CT-HRP) on their surfaces after incubations for 1 h at 4 degrees C. Exogenous GM1, the natural receptor of CT, becomes associated in the culture medium with the plasma membranes of these cells so that 96% of cells are stained. Cells preincubated with GM1 at 4 degrees C were exposed to CT-HRP for 1 h at 4 degrees C. After washing, cells were incubated at 37 degrees C for 30 min-24 h. Endocytosis of CT-HRP occurred within 30 min and CT-HRP remained, throughout the 24-h period, in tubules, vesicles, and cisternae often found near the Golgi apparatus; this aggregate of peroxidase-positive elements probably corresponds to Golgi apparatus-endoplasmic reticulum-lysosomes (GERL) of neurons. In metaphase cells, CT-HRP was observed in aggregates of vesicles and tubules clustered near the centriole. Conjugates of HRP with subunit B, the GM1 binding component of CT, were internalized by cells pretreated with GM1 as was CT-HRP. The 9% of neuroblastoma cells binding CT-HRP in the absence of exogenous GM1 internalized the ligand in a manner indistinguishable from that of the treated cells. These findings indicate that, in neuroblastoma cells, a system of vesicles, tubules, and cisternae, analogous to GERL of neurons, is the primary recipient of adsorptive endocytosis of CT bound to endogenous or exogenously introduced GM1.     

Incorporation of exogenous ganglioside GM1 into neuroblastoma membranes: Inhibition by calcium ion and dependence upon membrane protein

Since exogenous gangliosides are known to promote neuritogenesis, the incorporation of exogenous GM1 into neuroblastoma membranes was examined. Neuro-2A cells, synchronized in the G1/G0 phase, were suspended in HEPES buffered saline containing 10^–4 M [³H]GM1, and membrane incorporation was measured as radioactivity remaining with the cell pellet following incubation with serum-containing medium and trypsin. Calcium ion (0.01 to 10 mM) reduced incorporation of exogenous GM1, due to its interaction with GM1 micelles in solution. When cells were treated with proteases prior to incubation with GM1, the inhibitory effect of Ca²⁺ was lost and total incorporation into membranes was lowered by approximately one order of magnitude. Pretreatment of cells with 0.05% trypsin resulted in an inhibition of GM1 incorporation within 5 minutes. When trypsinized cells were resuspended in complete growth medium, the cells recovered the ability to incorporate GM1 with time, and this paralleled labeling of cellular protein with [³H]leucine. The role of membrane protein in the incorporation of exogenous GM1 could not be explained by the lytic release of cytosolic transfer proteins nor the artifactual coating of the cell surface by serum proteins. These results suggest that the incorporation of exogenous gangliosides into cellular membrane lipid bilayers cannot be fully explained by considerations of lipophilicity alone, and leads us to propose that initial recognition by membrane protein(s) is necessary.Abbreviations used GM1 H³NeuAc-GgOse₄Cer - HBS HEPES buffered saline - DMEM Dulbecco's modified Eagle's medium - FCS fetal calf serum     

Studies on the turnover and subcellular localization of membrane gangliosides in cultured neuroblastoma cells

To compare the subcellular distribution of endogenously synthesized and exogenous gangliosides, cultured murine neuroblastoma cells (N1E-115) were incubated in suspension for 22h in the presence ofd-[1-³H]galactose or [³H]GM1 ganglioside, transferred to culture medium containing no radioisotope for periods of up to 72 hr, and then subjected to subcellular fractionation and analysis of lipidsialic acid and radiolabeled ganglioside levels. The results indicated that GM2 and GM3 were the principal gangliosides in the cells with only traces of GM1 and small amounts of disialogangliosides present. About 50% of the endogenously synthesized radiolabelled ganglioside in the four major subcellular membrane fractions studied was recovered from plasma membrane and only 10–15% from the crude mitochondrial membrane fraction. In contrast, 45% of the exogenous [³H]GM1 taken up into the same subcellular membrane fractions was recovered from the crude mitochondrial fraction; less than 15% was localized in the plasma membrane fraction. The results are similar to those obtained from previously reported studies on membrane phospholipid turnover. They suggest that exogenous GM1 ganglioside, like exogenous phosphatidylcholine, does not intermix freely with any quantitatively major pool of endogenous membrane lipid.     

Rapid internalization of exogenous ganglioside GM3 and its metabolism to ceramide in human myelogenous leukemia HL-60 cells compared with control ganglioside GM1

Incorporation and metabolism of exogenous GM3 in human myelogenous leukemia HL-60 cells were analyzed using ³H-labeled GM3 ([³H]GM3). [³H]GM3 was rapidly internalized into the cells (trypsin-resistant fraction) 8 times more than the control, ³H-labeled GM1 ([³H]GM1). In addition, not only incorporation but also metabolism of [³H]GM3 was more rapid than [³H]GM1 in HL-60 cells. Moreover, one of the metabolites was found to co-migrate with ceramide in thin-layer chromatography analysis and ceramide formation from exogenous GM3 is more rapid than that from exogenous GM1. These results suggested that there would be some preferential mechanism to produce ceramide from differentiation-inducible GM3 in HL-60 cells rather than from non-inducing GM1.     

Localization of kappa opioid receptor binding sites in human forebrain using [3H]U69,593: Comparison with [3H]bremazocine

1. The autoradiographic distribution of kappa opioid receptor binding sites in human brain was examined using two radiolabeled probes, namely [3H]U69,593 and [3H]bremazocine. 2. [3H]U69,593 binding was performed in the absence of blockers for other sites, while [3H]bremazocine binding was investigated in the presence of saturating concentrations of mu and delta blockers to ensure selective labeling of kappa opioid receptors. 3. Our results show that the autoradiographic distribution of [3H]U69,593 and [3H]bremazocine (plus blockers) binding sites is identical, with high densities of sites found in deep cortical layers and claustrum. 4. This indicates that [3H]U69,593 is a highly selective ligand of the kappa opioid receptor type.     

Uptake and cellular transport of [11-3H] all-trans-retinoic acid in the liver of vitamin A-deficient hamsters

In this study, we examined, by ultrastructural autoradiography, the uptake and intracellular transport of [3H]all-trans-retinoic acid ([3H]RA) in the livers of vitamin A-deficient hamsters. Four-week-old animals were administered 25 microCi of [3H]RA by gavage, and, at different intervals thereafter, one animal was sacrificed. Their livers were excised and processed for autoradiography. Radioactive grains were observed to pass randomly through the plasma membrane by diffusion. No evidence of retinoid internalization by endocytosis was observed. Between 1 and 30 min after gavage, the radioactivity in parenchymal cells was associated mainly with rough endoplasmic reticulum (RER) and mitochondria. The labeling over nuclei was apparent at 1 min, remained relatively high up to 30 min, and subsequently decreased. At 2 and 5 hr, only a few grains were observed over nuclei, RER and mitochondria. At 24 hr, most of the labeling was associated with endothelial cells and sinusoidal spaces, indicating mobilization of [3H]RA from the liver. The results indicate that [3H]RA is transported through the plasma membrane by transmembrane diffusion without endocytosis and, after entering the cells, the ligand is rapidly translocated into nuclei.     

Cerebellar granule cells in culture exhibit a ganglioside-sialidase presumably linked to the plasma membrane

Cerebellar granule cells differentiated in culture were incubated with ganglioside [3H-Sph]GD1a in order to have it inserted into the plasma membrane, internalized by endocytosis, and metabolized. The metabolites formed included GM1, product of GD1a desialosylation. No GM1 or other metabolites were present in the incubation medium, whereas with the lysosomal apparatus blocked by chloroquine, or GD1a endocytosis prevented at 4 degrees C, the only metabolite formed was GM1. These results suggest that GD1a desialosylation did not occur either extracellularly or intracellularly but likely, at the membrane level. Similar results were obtained with [3H-Gal]GD1b, whereas no degradation of [3H-NeuAc]GM1 took place in the presence of chloroquine or at 4 degrees C. In conclusion, cerebellar granule cells express in vivo a sialidase, presumably located on the cell surface, that affects GD1a and GD1b but not GM1.     

Exchange kinetics and composition of endocytic membranes in terms of plasma membrane constituents: a morphometric study in macrophages

Intracellular membrane traffic, during endocytosis in mouse bone marrow-derived macrophages, was studied quantitatively by morphometric and kinetic analysis. Three functionally different markers were used: Horseradish peroxidase (HRP) served as a fluid-phase (FP) marker (1000 micrograms HRP/ml in the presence of mannan) or as a receptor-mediated (RM) membrane marker (25 micrograms HRP/ml) and, third, plasma membrane (PM) glycoconjugates, enzymatically labeled with [3H]galactose at the cell surface, served as a covalent membrane marker. The cell surface was labeled with [3H]galactose, followed by either FP or by RM uptake of HRP. The kinetics of the intracellular appearance of the markers were measured as the membrane area stained by HRP-reaction product and as the number of autoradiographic grains associated with these membranes. The following compartments were distinguished: PM, coated vesicles (VI), pinosomes or endosomes (VII), secondary lysosomes (VIII), and HRP-negative vesicles (EV). Tubular structures of VII became labeled with HRP only during RM uptake. The markers flowed first into VI and VII, and after 5 min into VIII. EV became labeled with the covalent membrane marker starting from 5 min. The ratio of autoradiographic grain number to HRP-stained membrane area remained constant with time although substantially different for the various compartments, viz. 100% (VI), 50% (VII and EV) and 30% (VIII) as compared to the PM (100%). This indicated that endosomes were only partially derived from internalized PM and that secondary lysosomes contained a substantial pool of PM constituents. The observed kinetics suggested that once every 30 to 40 min the entire PM was internalized, the bulk of which was recycled after 4 min from a prelysosomal compartment(s) leaving only 12 to 20% for recycling via membranes of secondary lysosomes after a residence time of 24 to 33 min.     

The plasma membrane-associated sialidase MmNEU3 modifies the ganglioside pattern of adjacent cells supporting its involvement in cell-to-cell interactions

We describe herein the enzyme behavior of MmNEU3, the plasma membrane-associated sialidase from mouse (Mus musculus). MmNEU3 is localized at the plasma membrane as demonstrated directly by confocal microscopy analysis. In addition, administration of the radiolabeled ganglioside GD1a to MmNEU3-transfected cells, under conditions that prevent lysosomal activity, led to its hydrolysis into ganglioside GM1, further indicating the plasma membrane topology of MmNEU3. Metabolic labeling with [1-(3)H]sphingosine allowed the characterization of the ganglioside patterns of COS-7 cells. MmNEU3 expression in COS-7 cells led to an extensive modification of the cell ganglioside pattern, i.e. GM3 and GD1a content was decreased to about one-third compared with mock-transfected cells. At the same time, a 35% increase in ganglioside GM1 content was observed. Mixed culture of MmNEU3-transfected cells with [1-(3)H]sphingosine-labeled cells demonstrates that the enzyme present at the cell surface is able to recognize gangliosides exposed on the membrane of nearby cells. Under these experimental conditions, the extent of ganglioside pattern changes was a function of MmNEU3 transient expression. Overall, the variations in GM3, GD1a, and GM1 content were very similar to those observed in the case of [1-(3)H]sphingosine-labeled MmNEU3-transfected cells, indicating that the enzyme mainly exerted its activity toward ganglioside substrates present at the surface of neighboring cells. These results indicate that the plasma membrane-associated sialidase MmNEU3 is able to hydrolyze ganglioside substrates in intact living cells at a neutral pH, mainly through cell-to-cell interactions.     

Autoradiographic Evidence of [3H]SCH 23390 Binding Site; in Human Prefrontal Cortex (Brodmann''s Area 9)

The distribution of dopamine D-1 receptors has been determined in human prefrontal cortex (Brodmann's area 9) by an in vitro light microscopic autoradiographic method. Dopamine D-1 receptors were localized by using [3H]SCH 23390 as a ligand. Our results indicated that [3H]SCH 23390 binding to slide-mounted tissue sections of human brain is specific, saturable, and of high affinity. Lamina Va contained the highest density of D-1 receptors, with a Bmax value of 11.2 +/- 1.3 fmol/mg tissue. The KD values for [3H]SCH 23390 in all laminae ranged from 2.6 to 3.2 nM. Competition studies performed with [3H]SCH 23390 indicated a pharmacologic profile consistent with labeling of the D-1 receptor.     

Surface distribution of monosialoganglioside GM1 on human blood cells and the effect of exogenous GM1 and neuraminidase on cholera toxin surface labeling. A quantitative immunocytochemical study

The cholera toxin-colloidal gold-labeled IgG-F(ab')2 anticholera toxin ultrastructural immunocytochemical procedure was used for the localization of GM1 monosialoganglioside on the surface of human blood cells. The number of gold particles per micron of cell surface was counted and the data subjected to statistical analysis. Cholera toxin (CT) binding characteristics assessed in several subjects showed consistent labeling patterns for the various hemic cells, although some quantitative differences were noted in surface labeling densities between subjects. Neutrophils were invariably the most heavily labeled of the hemic cells, while lymphocytes, erythrocytes, and platelets exhibited only limited CT labeling. Exposure of hemic cells to neuraminidase induced a major increase in surface CT labeling that proved to be directly related to cell type and differed in many respects with the CT labeling pattern noted in nonenzyme treated cells. Newly exposed CT binding sites attributed to "masked" GM1 and/or to neuraminidase-transformed GD1a or GT1 gangliosides, showed that the number of new binding sites were nearly twice as abundant on platelet and monocyte sufaces as on the surfaces of neutrophil, lymphocyte, and erythrocyte populations. However, ratios of new CT binding sites to those normally available for CT binding were approximately 10:1 for erythrocytes, approximately 3--7:1 for lymphocytes, monocytes, and platelets, and approximately 1:1 for the neutrophil group. Exogenous GM1 was incorporated into the cell surface of the hemic cells in a differential manner. Platelets showed a dramatic increase in surface CT labeling, viz. approximately 12- to 20-fold, compared to that of other hemic cells; however, neutrophil and erythrocyte GM1 uptake was limited. Our studies have demonstrated that distinct differences exist in the extent of surface CT labeling of the various types of blood cells. They further indicated that the ability of the cell surface to incorporate exogenous GM1 may represent a differential expression of the physiochemical properties of the surface of the individual cell types.     

Synthesis and uptake of gangliosides by choleragen-responsive human fibroblasts.

Human fibroblasts, cultured in medium containing 10% fetal calf serum, responded dramatically to choleragen with an increase in cyclic adenosine monophosphate content to greater than 48 times basal levels. Analysis of these cells for gangliosides indicated that the major ganglioside was N-acetylneuraminylgalactosylglucosylceramide (GM3) with trace amounts (less than or equal to 100 pmol/mg of protein) of other gangliosides including GM1, the putative choleragen receptor. Although the cells contained three glycosyltransferases required for ganglioside synthesis, the N-acetylgalactosaminyltransferase activity necessary for the conversion of GM3 to more complex gangliosides was not detected. When the cells were grown in medium containing [14C]galactose or N-acety[3H]mannosamine, however, all of the gangliosides became labeled, indicating that the cells can synthesize complex gangliosides. Although fetal calf serum contains gangliosides including GM1, [3H]GM1 was taken up poorly from the growth medium and uptake at the rate observed could have accounted for less than 2% of the GM1 content of the cells. When the cells were incubated in chemically defined medium containing [3H]GM1 at the concentrations present in fetal calf serum, rapid uptake of the ganglioside occurred and the total GM1 content of the cells increased threefold in less than 3 h. Thus, although the cells are capable of binding exogenous gangliosides, the gangliosides in fetal calf serum are in a form not readily available to the cells.     

The role of gangliosides in the interaction of human chorionic gonadotropin and cholera toxin with murine Leydig tumor cells

A clonal line of murine Leydig tumor cells (MLTC-1) bound both human chorionic gonadotropin (hCG) and cholera toxin (CT) with high affinity and accumulated cyclic AMP in response to either effector. The major cellular ganglioside was GM3 with small amounts of GM2, GM1, and GD1a. The gangliosides became labeled when the cells were grown in medium containing [3H] galactose or were exposed to galactose oxidase or NaIO4 followed by NaB3H4. CT specifically protected GM1 from surface labeling whereas hCG did not protect any gangliosides from being labeled. When the cells were exposed to sialidase, surface GD1a was eliminated, and GM1 increased with a corresponding increase in CT binding. When sialidase-treated cells were first incubated with the B component of CT, binding and action of CT was blocked. The cells, however, retained their ability to bind and respond to hCG. Addition of purified gangliosides to the medium effectively inhibited the binding and action of CT but not hCG. The cells incorporated the exogenous gangliosides and exhibited increased binding of and responsiveness to CT but not hCG. Both hCG- and CT-receptor complexes were extracted from the cells with nonionic detergent and analyzed by sucrose gradient centrifugation. The hCG-receptor complex had an apparent molecular weight of 190,000 whereas the CT-receptor complex sedimented only slightly faster than CT itself. MLTC-1 gangliosides were separated on thin layer chromatograms which were overlayed with either iodinated CT or hCG. The toxin bound to a ganglioside corresponding to GM1 whereas the hormone did not bind to any of the gangliosides. When the cells were incubated overnight with hCG, they lost their hCG receptors but exhibited an increase in CT binding and gangliosides. Our results indicate that GM1 is the specific receptor for CT whereas gangliosides are not involved in the binding and action of hCG.     

A new method for labeling and autoradiographic localization of androgen receptors

We have used a novel receptor labeling and autoradiographic technique to localize androgen receptors in the intact rat ventral prostate at the morphological level. Frozen slide-mounted prostate tissue sections (10 micron thick) were incubated with increasing concentrations of [3H]-R1881 in the absence and presence of excess unlabeled R1881. Tissue sections labeled in this way were subjected to concurrent biochemical and autoradiographic analysis. After incubation and washing to remove free [3H]-steroid, some of the sections were wiped from the slides for scintillation counting in order to characterize and quantitate [3H]-R1881 binding. Androgen receptors could indeed be labeled in slide-mounted tissue sections, and specific [3H]-R1881 binding to these receptors was high-affinity (Kd = 1 nM), saturable, and androgen-specific. All cellular androgen receptors appear to be retained, because receptor content in sections was comparable to the sum of receptors in subcellular fractions of homogenized tissue. Replicate labeled slide-mounted tissue sections were dried rapidly, apposed to dry emulsion-coated coverslips, and exposed in the dark for autoradiography. Silver grains were counted over nuclei or cytoplasm of epithelium or stroma to evaluate specific androgen receptor location. Autoradiographic analysis demonstrated androgen receptor localization almost exclusively in the epithelial nuclei, with little or none in the stroma. We discuss here the unique features and advantages of labeling androgen receptors in slide-mounted frozen tissue sections for autoradiographic localization.     

GM1 Ganglioside Treatment of PC 12 Cells Stimulates Ganglioside, Glycolipid, and Lipid, but Not Glycoprotein Synthesis Independently from the Effects of Nerve Growth Factor

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.     

Characteristic incorporation of ganglioside GM3, which induces monocytic differentiation in human myelogenous leukemia HL-60 cells

Using tritiated gangliosides [( 3H]-GM3 and [3H]-GM1), characteristic incorporation of exogenous GM3 to HL-60 cells was demonstrated in association with differentiation induction. [3H]-GM3 was bound 4-5 times more than [3H]-GM1 was. Scatchard analysis revealed high and low affinity patterns of binding to the cells. The concentration of GM3 that caused growth inhibition and cell differentiation corresponded well to that which showed the bi-phasic binding pattern. It was strongly suggested that GM3, which induces monocytic differentiation, was characteristically bound and incorporated to the cells around the concentration which caused growth inhibition and cell differentiation.     

Gangliosides modulate sodium transport in cultured toad kidney epithelia

Cultured A6 epithelial cells from toad kidney form confluent monolayers with tight junctions separating the apical and basolateral membranes. These two membrane domains have distinct compositions and functions. Thus, sodium is actively transported across the epithelia from the apical to basolateral surface via amiloride-inhibitable sodium channels located in the apical membrane. Sodium transport is stimulated by vasopressin, cholera toxin, and 8-bromo-cAMP applied to the basolateral surface where the receptors, adenylate cyclase, and Na+/K+-ATPase are located. In a previous study (Spiegel, S., Blumenthal, R., Fishman, P.H., and Handler, J.S. (1985) Biochim. Biophys. Acta 821, 310-318), we demonstrated that exogenous gangliosides inserted into the apical membrane of A6 epithelia do not redistribute to the basolateral membrane. With the ability to vary selectively the ganglioside composition of the apical membrane, we examined the effects of gangliosides on sodium transport in A6 epithelia. When the apical surface of A6 epithelia were exposed to exogenous gangliosides, sodium transport in response to vasopressin, cholera toxin, and 8-bromo-cAMP was enhanced compared to epithelia not exposed to gangliosides. The effect was observed with bovine brain gangliosides, NeuAc alpha 2----3Gal beta 1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GD1a) and Gal beta-1----3GalNAc beta 1----4[NeuAc alpha 2----3]Gal beta 1----4Glc beta 1----Cer (GM1), but not with the less complex ganglioside, Neu-Ac alpha 2----3Gal beta 1----4Glc beta 1----Cer (GM3). We examined A6 cells for endogenous gangliosides and found that, whereas GM3 was a major ganglioside, only trace amounts of GM1 and GD1a were present. Based on cell surface and metabolic labeling studies, these gangliosides were synthesized by the cells and were present on the apical as well as the basolateral surface. Bacterial sialidase, which hydrolyzes more complex gangliosides to GM1, was used to modify the endogenous gangliosides on the apical surface; after sialidase treatment, the epithelia were more responsive to vasopressin, cholera toxin, and 8-bromo-cAMP. Thus, gangliosides may be modulators of sodium channels present in the apical membrane of epithelial cells.     

GT3 Synthesis in the Proximal Golgi Occurs in a Compartment Different from Those for GD3 and GM3 Synthesis

Abstract: Previous studies from this laboratory have shown that synthesis of GT3, the precursor of c series gangliosides, occurs in proximal Golgi compartments, as has been shown for the synthesis of GM3 and GD3, the precursors of a and b series gangliosides, respectively. In this work we studied whether the synthesis of GM3, GD3, and GT3 occurs in the same or in different compartments of the proximal Golgi. For this, we examined in retina cells (a) the effect of monensin, a sodium ionophore that affects mostly the trans Golgi and the trans Golgi network function, on the metabolic labeling of glycolipids from [³H]Gal by cultured cells from 7- and 10-day chick embryos and (b) the labeling in vitro of endogenous glycolipids of Golgi membrane preparations from 7-day embryos incubated with UDP-[³H]Gal. In (a), 1 µM monensin produced a twofold accumulation of radioactive glucosylceramide and a decrease to ～50 and 20% of total ganglioside labeling in 7- and 10-day cells, respectively. At both ages, monensin produced a threefold accumulation of radioactive GM3 and an inhibition of >90% of GT3, GM1, GD1a, and GT1b synthesis. GD3 synthesis was inhibited ～30 and 70%, respectively, in 7- and 10-day cells. In (b), >80% of the [³H]Gal was incorporated into endogenous glucosylceramide to form radioactive lactosylceramide. About 90% of [³H]Gal-labeled lactosylceramide was converted into GM3, and most of this in turn into GD3 when unlabeled CMP-NeuAc was also present in the incubation system. Under the same conditions, however, <5% of labeled GD3 was converted into GT3. Golgi membranes incubated with CMP-[³H]NeuAc incorporated ～20% of [³H]NeuAc into endogenous GT3, and this percentage was not affected by 1 µM monensin. These results indicate that synthesis of GT3 is carried out in a compartment of the proximal Golgi different from those for lactosylceramide, GM3, and GD3 synthesis. Results from the experiments with monensin point to the cis/medial Golgi as the main compartment for coupled synthesis of lactosylceramide, GM3, and GD3 and to the trans Golgi as the main compartment for synthesis of GT3.     

Enzymatic and chemical oxidation of gangliosides in cultured cells: effects of choleragen.

Cell surface glycolipids of normal human fibroblasts and NCTC2071 cells (transformed mouse fibroblasts) were labeled by incubating the intact cells with either galactose oxidase or sodium periodate, followed by reduction of the oxidized sugar residues with NaB3H4. In intact human fibroblasts, incorporation of 3H was increased with increasing time of exposure to galactose oxidase prior to treatment with NaB3H4. Following limited exposure to galactose oxidase, more label was incorporated into the larger glycolipids. Although labeling of the monosialoganglioside GM1 was maximal by 16 h, not all of the GM1 in the intact cells appeared to be accessible to galactose oxidase, since 10 to 12 times more GM1 was labeled when cells were disrupted before incubation with the enzyme. The human fibroblasts contained approximately 8 X 10(6) molecules of GM1 per cell. Maximal binding of choleragen (5 X 10(5) molecules of [125I]choleragen per cell) completely prevented cholevented oxidation of GM1 in intact fibroblasts by galactose oxidase but only partially protected the sialic acid moiety of GM1 from oxidation by periodate. Choleragen had little effect on the enzymatic or chemical oxidation of other glycolipids. NCTC 2071 cells do not contain endogenous GM1 but incorporate exogenous GM1 from the culture medium. When bound to NCTC 2071 cells, exogenous GM1 was protected by choleragen from oxidation by galactose oxidase or whether endogenous or taken up from the incubation medium, are, after interaction with choleragen, less accessible to oxidation by periodate or galactose oxidase.     

Mechanism of Agonist-Induced Down-Regulation and Subsequent Recovery of Muscarinic Acetylcholine Receptors in a Clonal Neuroblastoma X Glioma Hybrid Cell Line

The mechanisms of carbachol-induced muscarinic acetylcholine receptor (mAChR) down-regulation, and recovery following carbachol withdrawal, were studied in the neuroblastoma x glioma hybrid NG108-15 cell line by specific ligand binding assays. N-[3H]Methylscopolamine ([3H]NMS) and [3H]quinuclidinyl benzilate ([3H]QNB) were used as the ligands for the cell surface and total cellular mAChRs, respectively. Exposure of cells to 1 mM carbachol for 16 h decreased the specific binding of [3H]NMS and [3H]QNB by approximately 80%. Bacitracin (1-4 mg/ml) and methylamine (1-15 mM), inhibitors of transglutaminase and of endocytosis, prevented agonist-induced loss of surface mAChRs. Pretreatment of cells with the antimicrotubular agents nocodazole (0.1-10 microM) and colchicine (1-10 microM) prevented carbachol-induced loss of [3H]QNB binding, but not that of [3H]NMS binding. These results indicate that agonist-induced mAChR down-regulation occurs by endocytosis, followed by microtubular transport of receptors to their intracellular degradation sites. When carbachol was withdrawn from the culture medium following treatment of cells for 16 h, receptors recovered and were incorporated to the surface membrane. This recovery process was antagonized by monovalent ionophores monensin (0.1 microM) and nigericin (40 nM), which interfere with Golgi complex function. Receptor recovery was also prevented by the antimicrotubular agent nocodazole. Thus, recovery of receptors appears to be mediated via Golgi complex and microtubular transport to the surface membrane.