Nerve Growth Factor and Gangliosides Stimulate the Release of Glycoproteins from PC 12 Pheochromocytoma Cells

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.     

Characterization of Xylamine Binding to Proteins of PC12 Pheochromocytoma Cells

PC12 pheochromocytoma cells take up 3,4-dihydroxyphenylethylamine (dopamine) and norepinephrine by a Na+-dependent, cocaine-sensitive system. The kinetics suggest that the same transporter functions for both substrates. Xylamine, a nitrogen mustard that blocks catecholamine uptake into neurons, irreversibly inhibited norepinephrine uptake into PC12 (IC50 = 15 microM). Pretreatment with 10 microM xylamine did not inhibit norepinephrine transport if 10 microM cocaine or 100 microM norepinephrine was also present during the pretreatment period or if Na+ was absent. These results indicate that xylamine must interact with the norepinephrine transporter to inhibit norepinephrine uptake. PC12 accumulated [3H]xylamine; this uptake had Na+-dependent and Na+-independent components. The Na+-dependent uptake was saturable (Km = 13 microM), and it was inhibited by cocaine (IC50 = 0.6 microM), desipramine (IC50 less than 1 nM), and norepinephrine (IC50 = 1 microM). Several proteins became prominently labeled when intact PC12 cells were incubated with [3H]xylamine; these proteins were enriched in a plasma membrane fraction and have molecular weights of 17,000, 24,000, 31,000, 33,000, 41,000, 42,000, 52,000, and 80,000. Other proteins were labeled less prominently. The labeling of all proteins was markedly decreased when the incubation with [3H]xylamine occurred in the presence of cocaine, desipramine, gramicidin D, or in a Na+-free buffer. These results indicate that xylamine must be transported into the cells for covalent binding to proteins to occur. [3H]Xylamine labeled essentially the same proteins when incubated with cell homogenates, but competition experiments with bretylium, desipramine, and cocaine failed to reveal which of the [3H]xylamine-labeled proteins is associated with the norepinephrine transporter.     

Glycosaminoglycan Composition of PC 12 Pheochromocytoma Cells: A Comparison with PC12D Cells, a New Subline of PC12 Cells

PC12D cells, a new subline of conventional PC12 cells, respond not only to nerve growth factor but also to cyclic AMP by extending their neurites. These cells are flat in shape and are similar in appearance to PC12 cells that have been treated with nerve growth factor for a few days. In both cell lines, we have characterized the glycosaminoglycans, the polysaccharide moieties of proteoglycans, which are believed to play an important role in cell adhesion and in cell morphology. Under the present culture conditions, only chondroitin sulfate was detected in the media from PC12 and PC12D cells, whereas both chondroitin sulfate and heparan sulfate were found in the cell layers. The levels of cell-associated heparan sulfate and chondroitin sulfate were about twofold and fourfold higher in PC12D cells than in PC12 cells, respectively. Compared to PC12 cells, the amounts of [35S]sulfate incorporated for 48 h into chondroitin sulfate were twofold lower but those into heparan sulfate were 35% higher in PC12D cells. The amount of chondroitin sulfate released by PC12D cells into the medium was about a half of that released by PC12 cells. The ratio of [35S]sulfate-labeled heparan sulfate to chondroitin sulfate was 6.2 in PC12D cells and 2.2 in PC12 cells. These results suggest that there may be some correlation between the increase in content of glycosaminoglycans and the change in cell morphology, which is followed by neurite outgrowth.     

Changes in Glycosaminoglycans During the Neuritogenesis in PC12 Pheochromocytoma Cells Induced by Nerve Growth Factor

Abstract: Previously, we had suggested that heparan sulfate (HS) makes some contribution to a flat-shaped morphology of PC12D cells. Therefore, we carried out quantitative and qualitative analyses of glycosaminoglycans (GAGs), the polysaccharide moiety of proteoglycans, during neuritogenesis in PC12 cells that is induced by nerve growth factor (NGF). (a) In PC12 cells, NGF induced a flat-shaped morphology with a few short processes after 3 days of culture, and then it elicited short and long neurites after 6 (in ～30% of cells) and 9 (in 60–70%) days of culture, respectively, (b) HS and chondroitin sulfate (CS) were detected in the cell layer at all times. Only CS was found in the medium at 3 and 6 days, whereas a low level of HS, in addition to CS, was detectable on day 9. (c) In the NGF-treated cultures, the amounts of cell-associated HS per cell were two to three times as high as those in the respective nontreated cultures at all times, whereas the amount based on phospholipid was about twofold higher after 3 days of culture. (d) The levels of HS labeled with [³⁵S]sulfate during the last 48 h of the culture were 1.5-to twofold higher in the NGF-treated cultures than in the respective controls at any time. (e) The amount of cell-associated CS per cell (or per unit of phospholipid), but not of labeled CS per cell, was transiently enhanced at 3 days in culture with or without NGF. At all times, NGF treatment caused an increase in the levels of total and [³⁵S]sulfate-labeled CS associated with the cells and released into the medium, (f) NGF enhanced the amount of N-sulfation of glucosamine residues of HS at all times, but it did not change the ratio of 4-sulfate units to 6-sulfate units in CS. (g) At 3 days in culture, the uptake of [³⁵S]sulfate by PC12 cells was lower in the NGF-treated culture than in the nontreated control. (h) In chase experiments, the percentage of unrecovered CS was about twofold higher in the NGF-treated culture than in the non-treated control. These results suggest that the enhanced synthetic activity and the accumulation of GAGs as well as the structural change of HS induced by NGF occur preceding the neurite elongation from PC12 cells. Also, it is suggested that the increase in content of HS is closely correlated with the morphological change from round to flat in PC12 cells.     

Multiple Pathways of N-Kinase Activation in PC12 Cells

Past work established a cell-free assay for a nerve growth factor (NGF)-activated protein kinase activity (designated N-kinase) that utilizes tyrosine hydroxylase and histone H1 as substrates and that is distinct from a variety of well-characterized kinases. This study explores the specificity and mechanistic pathway(s) by which N-kinase activity is regulated in PC12 rat pheochromocytoma cells. N-kinase is rapidly activated in these cells by treatment with NGF, epidermal growth factor (EGF), basic fibroblast growth factor (bFGF), phorbol ester, or dibutyryl cyclic AMP. Our data indicate that the stimulated activity is the same for each agent by several criteria: It exhibits the same characteristic biphasic elution pattern by Mono S fast protein liquid chromatography (FPLC), except for the case of dibutyryl cyclic AMP in which one of the activity peaks is somewhat shifted; it shows the same elution pattern by FPLC on a Superose 12 column; it possesses identical substrate specificity; and, except in the case of dibutyryl cyclic AMP, it does not show additivity when each agent is added simultaneously with NGF. The multiple forms of N-kinase are interconvertible in that rechromatography on a Mono S column yields a single peak of activity. Also, when NGF and dibutyryl cyclic AMP are simultaneously presented to cells, the chromatographic profile resembles that with NGF alone. Activation occurs through several independent initial pathways. Down-regulation of protein kinase C by phorbol ester pretreatment prevents N-kinase activation by phorbol ester, but not by the other agents. A PC12 cell-derived line deficient in cyclic AMP-dependent protein kinase II activity exhibits N-kinase activation by all treatments except dibutyryl cyclic AMP. The properties of N-kinase suggests that it is similar or identical to the ribosomal S6 protein kinase described by Blenis and Erikson. Additional experiments revealed that N-kinase activity can be stimulated in several cell lines in addition to PC12 cells. These findings indicate that the N-kinase can be activated via multiple second-messenger pathways and that it could therefore potentially play a significant role in mediating shared intracellular responses to various extracellular signals.     

Tetanus toxin receptor Specific cross-linking of tetanus toxin to a protein of NGF-differentiated PC 12 cells

A subclone of rat pheochromocytoma cells expresses high affinity receptors for tetanus toxin on differentiation with NGF [Walton, K.M., Sandberg, K., Rogers, T.B. and Schnaar, R.L. (1988) J. Biol. Chem. 263, 2055–2063]. In the presence of protein cross-linking agents, [¹²⁵I]tetanus toxin, bound to these cells at 0°C, forms a cross-linked product with apparent molecular weight of 120 kDa. The formation of [¹²⁵I]tetanus toxin conjugate involves the heavy chain of the toxin, is prevented by cold toxin and it is largely reduced by pretreating cells with proteases, The cross-linked product is formed only upon incubation of the toxin with NGF-differentiated cells. These results suggest that a protein with apparent molecular weight of 20 kDa is involved in the neurospecific binding of tetanus toxin.     

Control of Thy-1 Glycoprotein Expression in Cultures of PC12 Cells

The effects of nerve growth factor (NGF) and cholera toxin on the expression of the Thy-1 glycoprotein were examined in cultures of naive and primed PC12 cells using an enzyme-linked immunoadsorbent assay (ELISA). With primed PC12 cells, NGF induced a rapid increase in Thy-1 expression over a time course similar to that of neurite regeneration, with half-maximal and maximal increases apparent at 0.6 and 6 ng/ml NGF. Cholera toxin and dibutyryl cyclic AMP, but not B-cholera toxin or antibodies to the toxin receptor, were found to inhibit NGF-induced increases in Thy-1. Morphological differentiation of naive PC12 cells induced by NGF, but not cholera toxin, was also associated with increased expression of Thy-1. Despite showing a synergistic effect on morphological differentiation, cholera toxin was again found to inhibit NGF-induced increases in Thy-1 expression in cultures of naive PC12 cells. These data suggest that agents that interact directly or indirectly with adenylate cyclase may regulate the responsiveness of PC12 cells to NGF, and as such modulate the expression of the Thy-1 glycoprotein.     

Nerve Growth Factor-Stimulated Nuclear S6 Kinase in PC12 Cells

Abstract: Previous work has shown that nerve growth factor (NGF) stimulates the phosphorylation of the ribosomal protein S6 in PC12 cells. In this study, we show that S6 kinase activity is also present in purified PC12 cell nuclei. This activity was increased by treatment of the cells with NGF and, to a lesser extent, by treatment with epidermal growth factor. The NGF-stimulated activity was obtained from nuclear extracts and some of its characteristics described. The increase in activity was prevented by treatment of the cells with rapamycin or with wortmannin, and the overall activity could be precipitated by antibodies directed against the p85^S6K. These data indicate that p85^S6K is the NGF-stimulated S6 kinase in PC12 cell nuclei. The presence of S6 protein in the nucleus of PC12 cells has been confirmed and evidence is presented that suggests that it is identical to a protein called SMP reported some years ago.     

Effect of Nerve Growth Factor on Glucose Utilization and Nucleotide Content of Pheochromocytoma Cells (Clone PC12)

The effect of nerve growth factor (NGF) on the utilization and fate of uniformly labeled 14C glucose and on the content of several pyridine and purine nucleotides has been tested in the clonal cell line PC12. After incubation for 72 h with NGF, PC12 cells exhibit a 2.7-fold increase in glucose utilization and a 4.7-fold increase in CO2 release. During the same incubation period, all the nucleotides tested (NAD+, AMP, GMP, UDP-glucose, UDP-galactose, UDP, ADP, GDP, UTP, CTP, ATP, and GTP) underwent significant increments, varying from a minimum of 27% for ADP to a maximum of 90-120% for AMP, GMP, UDP-glucose, and UDP-galactose. These findings are discussed in connection with the trophic and differentiative effects of NGF in PC12 cells, which, in the presence of this factor, shifted from a neoplastic to a neuronal-like cell population.     

Cholera Toxin and Dibutyryl Cyclic AMP Inhibit the Expression of Neurofilament Protein Induced by Nerve Growth Factor in Cultures of Naive and Primed PC12 Cells

The effects of nerve growth factor (NGF), dibutyryl cyclic AMP (db cAMP), and cholera toxin on neurofilament protein expression in cultures of PC12 rat pheochromocytoma cells were examined using an enzyme-linked immunoadsorbent assay (ELISA). Morphological differentiation induced by NGF was associated with up to 30-fold increases in the level of neurofilament protein recognised by monoclonal antibody RT97. A more rapid response was apparent from primed as compared to naive PC12 cells. Cholera toxin and db cAMP both induced morphological differentiation of naive PC12 cells, but failed to promote neurite regeneration from primed cells. Neither response was associated with a significant induction of neurofilament protein. Both cholera toxin and db cAMP, but not B-cholera toxin nor antibodies to the toxin receptor, were found to inhibit the neurofilament protein response induced by NGF. Primed cells were more susceptible to this inhibition, and both cholera toxin and db cAMP inhibited neurite regeneration from these cells. These data suggest that increased intracellular cyclic AMP can suppress the expression of neuronal differentiation antigens induced by NGF, and are consistent with a role for neurofilament protein in promoting or facilitating the formation of a stable neuritic network.     

Specific Binding of Glycosylated β-Galactosidase to Rat Clonal Pheochromocytoma PC12h Cells: Effects of Nerve Growth Factor and Gangliosides

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.     

Insulin-Like Growth Factor I Receptor Expression and Function in Nerve Growth Factor-Differentiated PC12 Cells

Abstract: Receptors for insulin-like growth factor I (IGF-I) were studied on PC12EY cells, a subclone of PC12. Differentiation of PC12EY cells with nerve growth factor (NGF) did not alter either the number of IGF-I receptors nor their affinity for IGF-I. IGF-I receptors remained fully functional during differentiation, promoting increases in thymidine incorporation, glucose uptake, amino acid uptake, and the phosphorylation of the S6 protein of the ribosomes. IGF-I also increased the proportion of differentiated cells found in S-phase. But although the addition of IGF-I to naive cells caused an increase in cell number, there was no comparable increase when IGF-I was added to differentiated cells. Thus, although the receptor for IGF-I continues to be present and functional, IGF-I fails to induce cell proliferation in differentiated PC12 cells.     

The Effect of the B Subunit of Cholera Toxin on the Action of Nerve Growth Factor on PC12 Cells

Abstract: Exogenous gangliosides, especially ganglioside GM1 (GM1), seem to potentiate the action of nerve growth factor (NGF). We have examined the possible regulation of the NGF signaling pathway in PC12 cells by the B subunit of cholera toxin (CTB), which binds to endogenous GM1 specifically and with a high affinity. CTB treatment (1 μg/ml) enhanced NGF-induced neurite outgrowth from PC12 cells, NGF-induced activation of ribosomal protein S6 kinase, and NGF-induced stimulation of trk phosphorylation. CTB plus NGF also caused a greater inhibition of [³H]-thymidine incorporation into DNA than did NGF alone. These enhancing effects of CTB were blocked by the presence of cytochalasin B in the culture medium but were not affected by the presence of colchicine or by the depletion of Ca²⁺ in the medium. ¹²⁵I-NGF binding experiments revealed that CTB treatment did not affect the specific binding of NGF to the cells. These results strongly suggest that the binding of cell surface GM1 by CTB modulates the pathway of intracellular signaling initiated by NGF and that the association of CTB with a cytoskeletal component is essential for these effects.     

Down-Regulation of c-neu Receptors by Nerve Growth Factor in PC12 Cells

Abstract: A small number of p185^{c- neu} receptors have been found on PC12 cells. These receptors show some basal phosphorylation in quiescent cells. When the cells are treated with nerve growth factor (NGF) for a short time, some increase in phosphorylation is seen, mainly on serine and threonine residues, and this is accompanied by a slight shift in the apparent molecular weight. Epidermal growth factor (EGF) also increases the phosphorylation of p185^{c- neu} , in this case on tyrosine residues. Neither heregulin-β1 nor gp30 stimulates the tyrosine phosphorylation of p185^{c- neu} , and neither has a proliferative effect on the cells. Treatment of the cells with NGF for 5 days produces a 70–80% reduction in the number of p185^{c- neu} receptors. This down-regulation does not occur when PC12nnr5 cells, which lack the high-affinity NGF receptor, p140 ^trk , are treated with NGF.The level of p185^{c- neu} mRNA is not altered by NGF treatment, suggesting that the down-regulation is due to either a translational or a posttranslational alteration.     

The Action of Adenosine Analogs on PC12 Cells

Abstract: PC12 cells, a nerve growth factor–responsive clone of rat pheochromocytoma, contain a membrane–bound adenylate cyclase, which can be activated by adenosine analogs. The characteristics of the cyclase response indicate the presence of stimulatory adenosine receptors. Adenosine analogs also produce a marked increase in the ornithine decarboxylase levels of the cells, and the characteristics of this response suggest that it is linked to the adenylate cyclase–stimulatory adenosine receptors. The ornithine decarboxylase response elicited by 5'- N -ethyIcarboxamideadenosine (NECA), a potent stimulatory adenosine analog, is synergistic with that produced by nerve growth factor. Differentiation of the cells with nerve growth factor, however, does not substantially alter either the response of cyclase to the adenosine analog or the magnitude of the adenosine–evoked ornithine decarboxylase response. Treatment of the cells with NECA produces an increase in the phosphorylation of a specific non–histone nuclear protein. While causing little or no morphological alteration by itself, NECA is synergistic with nerve growth factor in producing neurite outgrowth in PC12 cells. NECA does not cause an induction of acetylcholinesterase in the cells, nor does it appear to affect the induction of this enzyme by nerve growth factor.     

A Nerve Growth Factor-Sensitive S6 Kinase in Cell-Free Extracts from PC 12 Cells

Soluble extracts from nerve growth factor (NGF)-stimulated PC12 cells prepared by alkaline lysis show a two- to 10-fold greater ability to phosphorylate the 40S ribosomal protein S6 than do extracts from control cells. The alkaline lysis method yields a preparation of much higher specific activity than does sonication. Half-maximal incorporation of 32P from [32P]ATP into S6 occurred after 4-7 min of NGF treatment. The partially purified NGF-sensitive S6 kinase has a molecular weight of 45,000. It is not inhibited by NaCl, chlorpromazine, or the specific inhibitor of cyclic AMP (cAMP)-dependent protein kinase, nor is it activated by addition of diolein plus phosphatidylserine. Trypsin treatment of either crude extracts or partially purified S6 kinase from control or NGF-treated cells was without effect. These data suggest that the S6 kinase stimulated by NGF is neither cAMP-dependent protein kinase or protein kinase C nor the result of tryptic activation of an inactive proenzyme. Treatment of intact cells with dibutyryl cAMP or 5'-N-ethylcarboxamideadenosine also increases the subsequent cell-free phosphorylation of S6. This observation suggests that cAMP-dependent protein kinase may be involved in the phosphorylation of S6 kinase.     

Nerve Growth Factor Stimulation of Arachidonic Acid Release from PC12 Cells: Independence from Phosphoinositide Turnover

The effect of nerve growth factor on the metabolism of arachidonic acid and the hydrolysis of phosphatidylinositol in PC12 cells was examined. Addition of nerve growth factor to PC12 cells isotopically labeled with [3H]arachidonic acid caused an increased release of radioactivity. In a similar manner, treatment of PC12 cells prelabeled with [3H]inositol increased inositol monophosphate accumulation in the presence of LiCl. Stimulation of [3H]arachidonic acid release by nerve growth factor was concentration dependent, attaining a maximum at 0.5 nM. Concentrations of nerve growth factor above 0.5 nM caused less than maximal stimulation. In contrast, nerve growth factor-stimulated accumulation of [3H]inositol monophosphate exhibited a sigmoidal dose-response curve with an apparent maximum at 8 nM. Increased accumulation of [3H]inositol monophosphate could be detected as early as 60 s after nerve growth factor addition, whereas nerve growth factor-stimulated release of [3H]arachidonic acid was not observed until 5 min after nerve growth factor treatment. The nerve growth factor-stimulated release of [3H]arachidonic acid was independent of extracellular calcium concentration. Increased [3H]inositol monophosphate accumulation elicited by nerve growth factor was dependent on the presence of extracellular calcium. These results suggest that the increased metabolism of arachidonic acid and the enhanced hydrolysis of phosphatidylinositol are separately regulated by nerve growth factor.     

Regulation of Nerve Growth Factor Action on Nsp100 Phosphorylation in PC12h Cells by Calcium

Previous work from these laboratories has shown that in PC12 cells the phosphorylation of a specific soluble protein is decreased by treatment with nerve growth factor. This protein, designated Nsp100, and its kinase have been separated and partially purified from PC12 cells. The present studies have been designed to investigate the role of calcium in this action of nerve growth factor. It is shown here, using PC12h cells, that A23187, a calcium ionophore, and high levels of K+, a depolarizing stimulus, also decrease phosphorylation of Nsp100. Furthermore, the actions of nerve growth factor as well as those of A23187 and high levels of K+ are prevented by treatment of the cells with the calcium chelator EGTA. It is also shown that agents that raise levels of cyclic AMP in the cells, specifically dibutyryl cyclic AMP and cholera toxin, also decrease phosphorylation of Nsp100 but, in addition, increase phosphorylation of tyrosine hydroxylase. The action of these latter agents on Nsp100 is blocked by EGTA, but their action on tyrosine hydroxylase is not, indicating that even agents such as cholera toxin act on Nsp100 through a Ca2+-dependent mechanism.     

Reevaluation of the Role of Gangliosides as Receptors for Tetanus Toxin

Binding of tetanus toxin to rat brain membranes was of lower affinity and capacity when binding was determined in 150 mM NaCl, 50 mM Tris-HCl (pH 7.4) than in 25 mM Tris-acetate (pH 6.0). Binding under both conditions was reduced by treating the membranes with neuraminidase. Pronase treatment, however, reduced toxin binding only in the Tris-saline buffer (pH 7.4). In addition, the concentration of gangliosides required to inhibit toxin binding was 100-fold higher in Tris-saline compared to Tris-acetate buffer. The toxin receptors in the membranes were analyzed by ligand blotting techniques. Membrane components were dissolved in sodium dodecyl sulfate, separated by polyacrylamide gel electrophoresis, and transferred to nitrocellulose sheets, which were overlaid with 125I-labeled toxin. Tetanus toxin bound only to material that migrated in the region of the dye front and was extracted with lipid solvents. Gangliosides isolated from the lipid extracts or other sources were separated by TLC on silica gel and the chromatograms were overlaid with labeled tetanus toxin. The toxin bound to areas where the major rat brain gangliosides migrated. When equimolar amounts of different purified gangliosides were applied to the chromatogram, binding of the toxin was in the order GD1b approximately equal to GT1b approximately equal to GQ1b greater than GD2 greater than GD3 much greater than GD1a approximately equal to GM1. Thus, the toxin appears to have the highest affinity for gangliosides with a disialyl group linked to the inner galactosyl residue. When binding of tetanus toxin to transfers and chromatograms was determined in the Tris-saline buffer (pH 7.4), the toxin bound to the same components but the extent of binding was markedly reduced compared with the low-salt and -pH conditions. Our results indicate that the interaction of tetanus toxin with rat brain membranes and gangliosides is greatly reduced under more physiological conditions of salt and pH and raise the possibility that other membrane components such as sialoglycoproteins may be receptors for the toxin under these conditions.     

Introduction of Macromolecules into Bovine Adrenal Medullary Chromaffin Cells and Rat Pheochromocytoma Cells (PC12) by Permeabilization with Streptolysin O: Inhibitory Effect of Tetanus Toxin on Catecholamine Secretion

Conditions are described for controlled plasma membrane permeabilization of rat pheochromocytoma cells (PC12) and cultured bovine adrenal chromaffin cells by streptolysin O (SLO). The transmembrane pores created by SLO invoke rapid efflux of intracellular 86Rb+ and ATP, and also permit passive diffusion of proteins, including immunoglobulins, into the cells. SLO-permeabilized PC12 cells release [3H]dopamine in response to micromolar concentrations of free Ca2+. Permeabilized adrenal chromaffin cells present a similar exocytotic response to Ca2+ in the presence of Mg2+/ATP. Permeabilized PC12 cells accumulate antibodies against synaptophysin and calmodulin, but neither antibody reduces the Ca2+-dependent secretory response. Reduced tetanus toxin, although ineffective when applied to intact chromaffin cells, inhibits Ca2+-induced exocytosis by both types of permeabilized cells studied. Omission of dithiothreitol, toxin inactivation by boiling, or preincubation with neutralizing antibodies abolishes the inhibitory effect. The data indicate that plasma membrane permeabilization by streptolysin O is a useful tool to probe and define cellular components that are involved in the final steps of exocytosis.