Effect of serum on ganglioside uptake and choleragen responsiveness of transformed mouse fibroblasts.

NCTC 2071 cells, transformed mouse fibroblasts, did not respond to choleragen when grown in chemically defined medium. When grown in medium containing 10 percent fetal calf serum, however, the cells accumulated cyclic AMP upon exposure to the toxin. Gangliosides isolated from the fetal calf serum were as effective as whole serum in inducing choleragen responsiveness in the cells. The putative choleragen receptor, the monosialo-ganglioside GM1, could not be detected by chemical analysis in cells exposed to serum. 3H-Labeled GM1 was detected in these cells, however, following sequential exposure to galactose oxidase and sodium borotritide. Thus, uptake of minute amounts of GM1 from serum by these cells sensitized them to choleragen.     

Properties of beta-adrenergic receptors in untreated and butyrate-treated Hela cells.

HeLa cells contain receptors on their surface which are beta-adrenergic in nature. The binding of (-)-[3H]dihydroalprenolol is rapid, reversible, stereospecific and of relatively high affinity. The HeLa cells also contain an adenylate cyclase which is activated by (-)-isoproterenol greater than (-)-epinephrine greater than (-)-norepinephrine. The adenylate cyclase of HeLa is also activated by guanyl-5'-ylimidodophosphate (Gpp(NH)p), a nonhydrolyzable analogue of GTP. Inclusion of both (-)-isoproterenol and Gpp(NH)p leads to approximately additive rather than synergistic activation of adenylate cyclase. After treatment of HeLa cells with 5mM sodium butyrate there is an increase in the number of beta-adrenergic receptors, but not in their affinity, which is reflected in an increased ability of (-)-isoproterenol to activate adenylate cyclase. Other properties of the beta-adrenergic receptor including association and dissociation rates, temperature optimum of adenylate cyclase and response to Gpp(NH)p are relatively unaffected by butyrate pretreatment of the cells.     

Modulation of the β-Adrenergic Receptor-Coupled Adenylate Cyclase by Chemical Inducers of Differentiation: Effects on β Receptors and the Inhibitory Regulatory Protein Gi

Abstract

Several drugs known to induce differentiation in tumor cells were analyzed for their effects on the β-adrenergic receptor-coupled adenylate cyclase system in two human carcinoma cell lines, HeLa and A431. Each of the drugs was tested alone or in combination with sodium butyrate (NaBu), a known inducer of this signal transduction system. Puromycine amino nucleoside (PMAN) caused the largest increase in β-adrenergic receptors in HeLa cells followed by hexamethylenebisacetamide (HMBA) whereas 5′-azacytidine (5AZC) was ineffective. In addition, PMAN but not the others acted together with NaBu to elevate receptor levels 12-fold over control values. In contrast, HMBA and 5AZC were much more effective on A431 cells, PMAN caused only a slight increase in β receptors and none of the drugs acted in concert with NaBu. The increase in β receptors was usually accompanied by a corresponding increase in isoproterenol-stimulated adenylate cyclase activity. These effects of the drugs appeared to require protein synthesis as they were blocked by cycloheximide. In addition, some of the drugs caused a substantial decrease in basal adenylate cyclase activity. This effect on basal activity was abolished in cells treated with pertussis toxin, which ADP-ribosylates the inhibitory GTP-binding protein, G_i. Both HeLa and A431 cells contained a 41 kDalton substrate for the toxin which corresponds to the α; subunit of G_i. The G_i subunit was ADP-ribosylated by the toxin to a similar extent in membranes from control and drug-treated cells. Thus, the drugs appear to induce quantitative changes in β-adrenergic receptors and qualitative changes in G_i which results in a highly responsive β-adrenergic-stimulated adenylate cyclase.     

Detergent extraction of cholera toxin and gangliosides from cultured cells and isolated membranes

Choleragen, when bound to various cultured cells, resisted extraction by Triton X-100 under conditions which retained the cytoskeletal framework of the cells. This resistance (> 75% of the bound toxin) was observed in Friend erythroleukemic, mouse neuroblastoma N18 and NB41A and rat glioma C6 cells even though the different cells varied over 1000-fold in the number of toxin receptors. The extent of extraction did not depend on whether the cells were in monolayer culture or in suspension or whether choleragen was bound at 0 or 37°C. A similar resistance to extraction was also observed in membranes isolated from toxin-treated cells. Using more drastic conditions and other non-ionic detergents, 90% of the bound choleragen was solubilized from cells and membranes. When rat glioma C6 cells, which bind only small amounts of choleragen, were incubated with the ganglioside G_M1, toxin binding was increased and the bound toxin was also resistant to extraction. When these cells were incubated with [³H]G_M1, up to 70% of the cell-associated G_M1 was extracted under the mild conditions. When the G_M1-labeled cells were incubated with choleragen or its B (binding) component, there was a significant reduction in the solubilization of G_M1. Similar results were obtained with isolated membranes. When choleragen-receptor complexes were isolated from N18 cells labeled with [³H]galactose by immunoadsorption, only labeled G_M1 was specifically recovered. These results suggest that it is the choleragen-ganglioside complex that is resistant to detergent extraction.     

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.     

Synthesis and characterization of N-parinaroyl ganglioside GM1: effect of choleragen binding on fluorescence anisotropy in model membranes

N-cis-Parinaroyl ganglioside GM1 and N-trans-parinaroyl ganglioside GM1 were synthesized and characterized by HPLC, TLC, component analysis, absorbance spectroscopy, and proton NMR spectroscopy. Steady-state fluorescence anisotropy of the purified compounds, incorporated into phosphatidylcholine liposomes, was measured in the presence and absence of choleragen (cholera toxin) and choleragenoid (cholera toxin B subunit). In gel-phase liposomes, anisotropy measurements indicated that the motion of the parinaroyl ganglioside was not affected by addition of choleragen or choleragenoid. In fluid-phase liposomes, however, addition of toxin resulted in increased anisotropy (decreased rotational motion) of the fluorescent gangliosides. This decreased motion was not observed with other parinaroyl lipid probes, such as phosphatidylcholine, glucosylceramide, or free fatty acids, indicating that the effect was due to specific ganglioside/toxin interactions. Varying the amount of ganglioside or the amount of toxin suggested that the effect of toxin on probe motion was saturable at approximately 1 choleragen (or choleragenoid) molecule/5 ganglioside molecules. These results are consistent with previous hypotheses regarding the ganglioside/choleragen interaction and indicate that parinaroyl ganglioside probes will be useful in elucidation of the molecular details of this interaction.     

Intoxication of cultured cells by cholera toxin: evidence for different pathways when bound to ganglioside GM1 or neoganglioproteins.

We previously reported that when the oligosaccharide of ganglioside GM1 is covalently attached to cell surface proteins of GM1-deficient rat glioma C6 cells, the cells bind large amounts of cholera toxin (CT) but their cAMP response to CT is not enhanced [Pacuszka, T., & Fishman, P. H. (1990) J. Biol. Chem. 265, 7673-7668]. We now report that when such cells were exposed to CT in the presence of chloroquine, an acidotropic agent, they accumulated cAMP. This raised the possibility that CT bound to cell surface "neoganglioproteins" may be entering the cells through a different pathway from that of CT-bound GM1. To further explore this phenomenon, we covalently attached GM1 oligosaccharide to human transferrin (Tf). The modified protein (GM1OS-Tf) bound with high affinity to Tf receptors on HeLa cells and increased the binding of CT to the cells. The bound CT, however, was unable to activate adenylyl cyclase as measured by cyclic AMP accumulation. By contrast, treatment of HeLa cells with GM1 increased both CT binding and stimulation of cyclic AMP accumulation. Control cells and cells treated with either GM1 or GM1OS-Tf were exposed to CT in the presence of chloroquine. Whereas chloroquine had little or no effect on the response of control or GM1-treated cells to CT, it made the cells treated with GM1OS-Tf responsive to the toxin. Our results indicate that CT bound to its natural receptor GM1 enters the cells through a pathway different from that of toxin bound to neoganglioproteins.     

Loss of choleragen receptors and ganglioside upon differentiation of 3T3-L1 preadipocytes

3T3-L1 preadipocytes differentiate in culture into cells having the enzymatic and morphological characteristics of adipocytes. Differentiation is accompanied by a decrease in total cellular ganglioside content; the ganglioside level is 1.8 to 2.5-fold higher in undifferentiated than in differentiated cells. Gangliosides GM3 and GD1a constitute a majority of total cell gangliosides in both cell types, while ganglioside GM1, the putative choleragen receptor, constitutes less than 5%. Differentiation results in a 75 to 85% decrease in ganglioside GM1. An inverse correlation exists between the percentage of adipocytes in the cell population and: 1) total ganglioside and ganglioside GM1 content, and 2) surface ganglioside GM1 as estimated by choleragen binding or fluorescent staining of bound choleragen. Nondifferentiating 3T3-C2 control cells do not exhibit changes in total ganglioside, ganglioside GM1, or choleragen binding that are observed with 3T3-L1 cells.     

Induction of catecholamine-responsive adenylate cyclase in HeLa cells by sodium butyrate. Evidence for a more efficient stimulatory regulatory component

HeLa cells, when exposed to 5 mM sodium butyrate, increased their responsiveness to isoproterenol and their number of beta-receptors. As untreated HeLa cells have a substantial number of receptors but respond poorly to isoproterenol, the effect of butyrate could be due to quantitative or qualitative changes in beta-receptors or other components of the adenylate cyclase system. Receptors were analyzed by membrane/membrane and membrane/cell fusion techniques. HeLa donor membranes, treated to inactivate regulatory and catalytic components of adenylate cyclase, were fused with Fc cells, which lack beta-receptors. Isoproterenol-stimulated adenylate cyclase activity in the fusates was proportional to the number of receptors present. There appeared to be only quantitative but not qualitative differences in beta-receptors from control and butyrate-treated HeLa. Prostaglandin E1 receptors from neuroblastoma cell membranes were similarly coupled to HeLa adenylate cyclase. The hybrid prostaglandin E1-stimulated activity was lower when acceptor membranes were from control HeLa than when they were from butyrate-treated HeLa cells. These results suggested that butyrate was altering the ability of the regulatory component to interact with receptors. HeLa membranes were extracted with sodium cholate and the extracts used to reconstitute effector-stimulated adenylate cyclase activity in S49 cyc- membranes, which lack a functional regulatory component. Whereas extracts from control and butyrate-treated HeLa were equally effective in restoring NaF-stimulated activity in cyc- membranes, extracts from control HeLa were less efficient in reconstituting isoproterenol- and prostaglandin E1-stimulated activities. We conclude that the poor response of control HeLa to beta-agonists is due to a limited activity of the regulatory component but not the receptor. Butyrate induces quantitative changes in the receptor and qualitative changes in the regulatory component that facilitate its ability to couple to receptors but do not alter its ability to interact with the catalytic component of adenylate cyclase.     

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.     

Properties of β-adrenergic receptors in untreated and butyrate-threated HeLa cells

HeLa cells contain receptors on their surface which are β-adrenergic in nature. The binding of (?)-[³H]dihydroalprenolol is rapid, reversible, stereo-specific and of relatively high affinity. The HeLa cells also contain an adenylate cyclase which is activated by (?)-isoproterenol > (?)-epinephrine > (?)-norepinephrine. The adenylate cyclase of HeLa is also activated by guanyl-5′-yl-imidodophosphate (Gpp(NH)p), a nonhydrolyzable analogue of GTP. Inclusion of both (?)-isoproterenol and Gpp(NH)p leads to approximately additive rathen than synergistic activation of adenylate cyclase. After treatment of HeLa cells with 5 mM sodium butyrate there is an increase in the number of β-adrenergic receptors, but not in their affinity, which is reflected in an increased ability of (?)-isoproterenol to activate adenylate cyclase. Other properties of the β-adrenergic receptor including association and dissociation rates, temperature optimum of adenylate cyclase and response to Gpp(NH)p are relatively unaffected by butyrate pretreatment of the cells.     

Generation of cell surface neoganglioproteins. GM1-neoganglioproteins are non-functional receptors for cholera toxin

GM1 (II3Neu5Ac-GgOse4Cer)-oligosaccharide was prepared from the ganglioside by ozonolysis and alkaline fragmentation, reductively aminated and coupled to the heterobifunctional cross-linker succinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate. The resulting derivative reacted with free sulfhydryl groups and readily cross-linked to cell surface components on rat glioma C6 cells which are GM1-deficient. Attachment of the GM1-oligosaccharide derivative, which was monitored by increased binding of 125I-cholera toxin to the cells, was both time- and concentration-dependent. Prior treatment of the cells with dithiothreitol enhanced the attachment by generating additional free sulfhydryl groups. The affinity of cholera toxin for cells treated with the GM1-oligosaccharide derivative or with GM1 was similar. The nature of the newly generated toxin receptors was determined by Western blotting. Membranes from derivatized cells were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and the resolved components were electrophoretically transferred to a nitrocellulose sheet which was overlain with 125I-cholera toxin. The toxin bound to a wide variety of membrane proteins, most of which were trypsin-sensitive. No such binding was observed using membranes from control cells. Although the GM1-neoganglioproteins newly generated on the surface of rat glioma C6 cells readily bound cholera toxin, the cells did not become more responsive to the toxin as measured by increased production of cyclic AMP or activation of adenylate cyclase. In contrast, cells exposed to GM1 became highly responsive to the toxin. Thus, neoganglioproteins on the cell surface appear to behave as nonfunctional receptors for cholera toxin.     

Modulation of the receptor-coupled adenylate cyclase system in HeLa cells by sodium butyrate

Exposure of HeLa cells to 5 mM sodium butyrate, but not 0.6 mM, resulted in a more efficient coupling between their beta-adrenergic receptors and the guanine nucleotide binding stimulatory (Ns) component of adenylate cyclase. Both concentrations of the fatty acid, however, caused an increase in receptor number. beta receptors from control and butyrate-treated cells had the same affinity for isoproterenol. Modulation of this affinity by GTP was greatly enhanced, however, in cells treated with 5 mM butyrate compared to untreated and 0.6 mM butyrate treated cells. The concentration of isoproterenol required to half-maximally stimulate adenylate cyclase (Kact) was reduced in cells treated with 5 mM butyrate. In addition, the Kact for GTP in the presence, but not the absence, of isoproterenol was reduced. The effect of butyrate on the coupling between beta receptors and Ns was analyzed in detail by monitoring the activation of Ns by guanine 5'-O-(3-thiotriphosphate) (GTP gamma S) in a two-step assay. In the absence of isoproterenol, Ns from control and 5 mM butyrate treated cells was activated to the same extent with the same time course and Kact for GTP gamma S. In the presence of isoproterenol, Ns from 5 mM butyrate treated cells was activated more rapidly and extensively than Ns from control cells. The Kact for both GTP gamma S and isoproterenol also was reduced. The rate of agonist-mediated activation of Ns was strongly dependent on temperature, which accentuated the differences between 5 mM butyrate treated and control cells. At 4 degrees C, the difference in rate was 8.8-fold.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fluorescent derivatives of ganglioside GM1 function as receptors for cholera toxin

A fluorescent derivative of ganglioside GM1 was prepared by oxidation of the sialic acid residue with sodium periodate and reaction of the resulting aldehyde with Lucifer yellow CH. The biological activity of the fluorescent derivative was compared with that of native GM1 using GM1-deficient rat glioma C6 cells. When the cells were exposed to either native or fluorescent GM1, their ability to bind 125I-labeled cholera toxin was increased to a similar extent. This increase in binding was directly proportional to the amount of ganglioside added to the medium. The affinity of the toxin for cells treated with either native or fluorescent GM1 also was similar. More importantly, the fluorescent GM1 was as effective as native GM1 in enhancing the responsiveness of the cells to cholera toxin. Thus, the ganglioside-treated cells exhibited a 9-fold increase in toxin-stimulated cyclic AMP production over cells not exposed to GM1. There was a similar increase in iodotoxin binding and toxin-stimulated cyclic AMP accumulation in cells treated with other GM1 derivatives containing rhodaminyl or dinitrophenyl groups. On the basis of these results, it is clear that these modified gangliosides retain the ability to function as receptors for cholera toxin. Consequently, fluorescent gangliosides are likely to be useful as probes for investigating the dynamics and function of these membrane components.     

Internalization and degradation of cholera toxin by cultured cells: relationship to toxin action

Using anticholeragen antibodies and 125I-protein A, we developed a specific and quantitative assay for measuring choleragen on the surfaces of cultured cells. When neuroblastoma cells containing bound toxin were incubated at 37 degrees C, surface toxin disappeared with a half-life of approximately 2 h and a significant loss was detected by 10 min. When cells were incubated with 125I-choleragen in order to measure toxin degradation, cell-associated radioactivity disappeared with time and a corresponding amount of TCA-soluble label appeared in the culture medium with a half-life of 4-6 h. No degradation was detected until 45 min. Although there was a lag of 15 min before bound choleragen activated adenylate cyclase, the enzyme became maximally activated between 45 and 60 min. Similar results were obtained with Friend erythroleukemia cells. Internalization, degradation, and activation all were blocked when the cells were maintained at 4 degrees C. At 22 degrees C, internalization and activation occurred, albeit at a slower rate, whereas degradation was effectively inhibited. These results indicated that choleragen does not have to be degraded by intact cells in order for it to activate adenylate cyclase. Some internalization of the toxin, however, appears to precede the activation process.     

Evidence for tight coupling of thyrotropin-releasing hormone receptors to stimulated inositol trisphosphate formation in rat pituitary cells

The effect of decreasing the concentration of receptors for thyrotropin-releasing hormone (TRH) on the surface of cloned rat pituitary (GH3) cells on TRH-stimulated inositol trisphosphate (Ins-P3) formation was investigated. Incubation of cells with dibutyryl cAMP (Bt2cAMP) for 16 h caused a decrease in [3H] TRH binding to intact cells to a minimum level 37 +/- 9.1% of control. Scatchard analysis of the concentration dependency of [3H]TRH binding showed that the effect of Bt2cAMP was to lower the receptor concentration without affecting its affinity for TRH. Similar decreases in [3H]TRH binding were found in cells incubated with 8-bromo-cAMP, cholera toxin, and sodium butyrate and, as shown previously, with TRH. In cells incubated with 1 mM Bt2cAMP for 16 h, but not for 1 h, the maximum TRH-induced increase in Ins-P3 was inhibited to 25 +/- 3.2% of that in control cells. Inhibition of TRH-induced Ins-P3 formation was also observed in cells treated with 8-bromo-cAMP, cholera toxin, and sodium butyrate for 16 h, and with TRH for 48 h. Inhibition of TRH-induced Ins-P3 formation and lowering of TRH receptor concentration caused by Bt2cAMP occurred in parallel with increasing doses of Bt2cAMP; at 16 h of exposure, half-maximal effects occurred with 0.3 mM Bt2cAMP. The concentration dependency of TRH-induced Ins-P3 formation was the same in control and Bt2cAMP-treated cells; half-maximal effects occurred with 10 nM TRH. These data demonstrate that decreases in TRH receptor concentration caused by several agents that act via different mechanisms are associated with reduced stimulation of Ins-P3 formation and suggest that the TRH receptor is tightly coupled to stimulation of hydrolysis of phosphatidylinositol 4,5-bisphosphate by a phospholipase C.     

Changes in cyclic nucleotide levels during erythroid differentiation in Friend leukemia cells

Changes in cyclic nucleotide levels were observed in Friend leukemia cells exposed to the inducers of erythroid differentiation dimethylsulfoxide (DMSO), sodium butyrate and hypoxanthine. The most extensive changes in cAMP levels occurred during the initial 24 h growth period. In uninduced cultures, cAMP concentration increased 35-fold compared with a 7-fold increase in the presence of inducer. During the same period. cGMP levels rose about 3-fold in uninduced cells compared with a 20-fold increase in induced cells.     

Adrenal cells in tissue culture the effects of choleragen and ACTH on steroid and cyclic-AMP metabolism.

Primary cultures of mouse adrenocortical tumors provide a sensitive system for investigating the effects of the enterotoxin of the V. cholerae (choleragen) on cyclic-AMP metabolism in the intact cell. Like ACTH, the toxin stimulates the synthesis and release of steroids from these cells but its mode of action differs from that of ACTH. The steroidogenic response to ACTH is immediate and of limited duration. The initial rate of steroidogenesis is the highest. In contrast, the steroidogenic response to choleragen is preceded by a 30-240 minute lag period which is inversely related to the concentration of the toxin. Whereas prolongation of the response to a single dose of ACTH requires hormone concentrations above those producing maximal initial steroidogenic activity, persistent steroidogenesis is induced at all levels of the toxin. Steroidogenic responses are detectable with 10 pg/ml of choleragen or less. The respective effects of ACTH and choleragen on cyclic-AMP synthesis and release into the medium parallel those on steroidogenesis. Intracellular cyclic-AMP levels in ACTH-treated cells reach a peak within 20-30 minutes and decline to normal levels within 2-4 hours. In choleragen-treated cells, after the lage period, the levels of intracellular cyclic-AMP remain above control levels indefinitely. The effects of ACTH and choleragen on cyclic-AMP biosynthesis are additive at all levels of the two compounds. The effects of choleragen are blocked by prior treatment of the toxin with a five-fold molar excess of ganglioside GM1, a presumed constituent of the toxin-binding site.     

Mechanism of action of cholera toxin: Studies on the lag period

Summary The lag period for activation of adenylate cyclase by choleragen was shorter in mouse neuroblastoma N18 cells than in rat glial C6 cells. N18 cells have 500-fold more toxin receptors than C6 cells. Treatment of C6 cells with ganglioside G_M1 increased the number of toxin receptors and decreased the lag phase. Choleragen concentration also effected the lag phase, which increased as the toxin concentration and the amount of toxin bound decreased. The concentration, however, required for half-maximal activation of adenylate cyclase depended on the exposure time; at 1.5, 24, and 48 hr, the values were 200, 1.1., and 0.35pm, respectively. Under the latter conditions, each cell was exposed to 84 molecules of toxin.The length of the lag period was temperature-dependent. When exposed to choleragen at 37, 24, and 20 °C, C6 cells began to accumulate cyclic AMP after 50, 90, and 180 min, respectively. In G_M1-treated cells, the corresponding times were 35, 60, and 120 min. Cells treated with toxin at 15 °C for up to 22 hr did not accumulate cAMP, whereas above this temperature they did. Antiserum to choleragen, when added prior to choleragen, completely blocked the activation of adenylate cyclase. When added after the toxin, the antitoxin lost its inhibitory capability in a time and temperature-dependent manner. Cells, however, could be preincubated with toxin at 15 °C, and the antitoxin was completely effective when added before the cells were warmed up. Finally, cells exposed to choleragen for >10 min at 37 °C accumulated cyclic AMP when shifted to 15 °C. Under optimum conditions at 37°C, the minimum lag period for adenylate cyclase activation in these cells was 10 min. These findings suggest that the lag period for cholerage action represents a temperature-dependent transmembrane event, during which the toxin (or its active component) gains access to adenylate cyclase.Abbreviations used: ganglioside nomenclature according to Svennerholm [32] (see Table 1 for structures) cAMP adenosine 35-monophosphate - MIX 3-isobutyl-1-methylxanthine - HEPES N-2-hydroxyethylpiperazine-N-2-ethane sulfonic acid - PBS phosphate-buffered saline (pH 7.4)     

Induction of alkaline phosphatase activity in HeLa cells by sodium butyrate.

The induction of HeLa cell alkaline phosphatase activity by sodium butyrate could be inhibited by the coadministration of caffeine or theophylline. The inhibitions were dose dependent, and at any given concentration the potency was theophylline greater than caffeine. Although the induction by sodium butyrate was more sensitive to the inhibition by the xanthines than was that produced by 5-iodo-2'-deoxyuridine, the magnitudes of the increases in cyclic AMP concentrations after treatment with the xanthines were similar in the inhibition of both types of induction. The induction of alkaline phosphatase activity by sodium butyrate also produced a shift in the thermostability pattern of the enzyme, with a proportionately greater increase in the heat-labile, rather than heat-stable, form of the activity.