Activation of rat liver adenylate cyclase by cholera toxin requires toxin internalization and processing in endosomes

Involvement of acidic cell compartments in processing and action of cholera toxin (CT) in rat liver has been examined using subcellular fractionation. Liver cell fractions prepared various times after CT injection display, after a lag phase, a progressive increase in adenylate cyclase activity, detectable earlier in Golgi-endosomal fractions (20 min) than in plasma membrane fractions (30 min), with a maximum (3-fold basal activity) achieved by 60-90 min. Endosomes containing in vivo internalized CT display a time-dependent increase in their ability to bind anti-A-subunit antibodies and to stimulate exogenous adenylate cyclase, which kinetically parallels the generation of A1 peptide, suggesting a translocation of A-subunit (or A1 peptide) across the endosomal membrane. In vivo chloroquine treatment inhibits endocytosis of CT taken up into the liver, lengthens the lag phase for adenylate cyclase activation by CT, and reduces by 3- to 10-fold the apparent affinity of the toxin for the enzyme. Incubation of endosomes containing internalized toxin at 37 degrees C under isotonic conditions results in a pH-dependent increase in generation of A1 peptide, membrane translocation of A-subunit (or A1 peptide), and degradation of toxin, with a maximum at pH 5. Addition of ATP, by decreasing the internal endosomal pH, stimulates both generation of the A1 peptide and degradation of toxin at pH 6-8. It is concluded that activation of adenylate cyclase by CT in intact liver requires association and subsequent processing of toxin in an acidic cell compartment, presumably endosomal.     

Stimulation of adenylate cyclase in washed pigeon erythrocyte membrane with cholera toxin and its subunits.

Cholera toxin was found to stimulate adenylate cyclase activity in washed membrane of pigeon erythrocytes in the presence of dithiothreitol and NAD. When tested with isolated cholera toxin components, the stimulatory activity was found with subunit A or polypeptide A1 derived from this subunit, but not with A2 or subunit B. On a molar basis, polypeptide A1 was approximately four times more active than cholera toxin. Dithiothreitol was not required in the action of polypeptide A1, suggesting that the reagent was needed only to release A1 from subunit A or the holotoxin for their action on adenylate cyclase. The single SH group in polypeptide A1 was not involved in the activity of the peptide, since chemical modification of the thiol group did not alter the stimulatory activity of the peptide. The presence of NAD was, however, essential for the activation of adenylate cyclase with cholera toxin, subunit A, or polypeptide A1. Elevation of the adenylate cyclase activity was also observed when the intact pigeon erythrocytes were incubated with polypeptide A1, although a 30-fold molar excess of A1 over that of holotoxin was required for the same level of activation.     

The mechanism of action of cholera toxin in pigeon erythrocyte lysates.

The adenylate cyclase activity of intact pigeon erythrocytes begins to rise after about 20 min of exposure to cholera toxin. The maximum rate at which the cyclase activity increases appears to be limited by the number of toxin molecules which can reach an intracellular target. If the erythrocytes are made permeable to the toxin by a bacterial hemolysin, no such limit exists, and adenylate cyclase activity starts to rise immediately upon the addition of toxin, and continues to rise to a maximum at an initially constant rate which is dependent upon the concentration of toxin. On lysed erythrocytes, the addition of cholera antitoxin immediately prevents any further rise in adenylate cyclase activity, but does not reverse any activation already achieved. Erythrocyte lysates may also be activated by isolated peptide A1 of cholera toxin, although activation of adenylate cyclase of intact erythrocytes requires the complete toxin molecule. In the intact cells, toxin first attaches by its Component B to surface receptors of which there are about 30 per erythrocyte. Subsequently, peptide A1 but not Component B is inserted into the erythrocyte. It takes only about 1 min at 37 degrees for peptide A1 to be sufficiently deep within the cell membrane to be inaccessible to extracellular antitoxin, but its complete transit through the membrane appears to take longer. The surface receptors are used only once, for they remain blocked by Component B. The number of receptors available on the surface may be increased by soaking cells in ganglioside GM1. Cholera toxin also decreases the rate of apparently spontaneous loss of adenylate cyclase activity and increases the response to epinephrine. Theophylline inhibits the action of cholera toxin.     

Short communications. Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes.

Intact cholera toxin and its purified subunit A both activate the adenylate cyclase of pigeon erythrocyte membranes, but subunit B does not. The activation by subunit A is unaffected by treatments that inhibit whole toxin by interfering with the binding of subunit B to cell membranes.     

Activation by cholera toxin of adenylate cyclase solubilized from rat liver.

Cholera toxin, or peptide A1 from the toxin, activates adenylate cyclase solubilized from rat liver with Lubrol PX, provided that cell sap, NAD+, ATP and thiol-group-containing compounds are present. The activation is abolished by antisera to whole toxin, but not to subunit B.     

Studies on the time course and rate-limiting steps in the activation of adenylate cyclase in rat liver by cholera toxin.

Cholera toxin stimulates adenylate cyclase in rat liver after intravenous injection. The stimulation follows a short latent period of 10min, and maximum stimulation was attained at 120min. Half-maximal stimulation was achieved at 35min. In contrast with this lengthy time course in the intact cell, adenylate cyclase in broken-cell preparations of rat liver in vitro were maximally stimulated by cholera toxin (in the presence of NAD+) in 20min with half-maximal stimulation in 8min. Binding of cholera toxin to cell membranes by the B subunits is followed by translocation of the A subunit into the cell or cell membrane, and separation of the A1 polypeptide chain from the A2 chain by disulphide-bond reduction, and finally activation of adenylate cyclase by the A1 chain and NAD+. As the binding of cholera toxin is rapid, two possible rate-limiting steps could be the determinants of the long time course of action. These are translocation of the A1 chain from the outside of the cell membrane to its site of action (this includes the time required for separation from the whole toxin) or the availability of NAD+ for activation. When NAD+ concentrations in rat liver were elevated 4-fold, by the administration of nicotinamide, no change in the rate of activation of adenylate cyclase by cholera toxin was observed. Thus the intracellular concentration of NAD+ is not rate-limiting and the major rate-limiting determinant in intact cells must be between the time of toxin binding to the cell membrane and the appearance of subunit A1 at the enzyme site.     

Essential role of GTP in the expression of adenylate cyclase activity after cholera toxin treatment.

Expression of activation of rat liver adenylate cyclase by the A1 peptide of cholera toxin and NAD is dependent on GTP. The nucleotide is effective either when added to the assay medium or during toxin (and NAD) treatment. Toxin treatment increases the Vmax for activation by GTP and the effect of GTP persists in toxin-treated membranes, a property seen in control membranes only with non-hydrolyzable analogs of GTP such as Gpp(NH)p. These observations could be explained by a recent report that cholera toxin acts to inhibit a GTPase associated with denylate cyclase. However, we have observed that one of the major effects of the toxin is to decrease the affinity of guanine nucleotides for the processes involved in the activation of adenylate cyclase and in the regulation of the binding of glucagon to its receptor. Moreover, the absence of lag time in the activation of adenylate cyclase by GTP, in contrast to by Gpp(NH)p, and the markedly reduced fluoride action after toxin treatment suggest that GTPase inhibition may not be the only action of cholera toxin on the adenylate cyclase system. We believe that the multiple effects of toxin action is a reflection of the recently revealed complexity of the regulation of adenylate cyclase by guanine nucleotides.     

Extracellular pH modulates Helicobacter pylori-induced vacuolation and VacA toxin internalization in human gastric epithelial cells

In this study we investigated whether an acidic extracellular pH may inhibit H. pylori-induced internalization of bacterial virulence factors by gastric epithelium, thus preventing ingestion of potentially dangerous luminal contents and resulting cellular damage. The interaction of H. pylori VacA toxin and ammonia (produced by H. pylori urease) with partly polarized gastric MKN 28 cells in culture was investigated at neutral and moderately acidic pH (6.2, compatible with cell viability) by means of neutral red dye uptake and ultrastructural immunocytochemistry. We found that acidic extracellular pH virtually abolished both VacA-dependent and ammonia-dependent cell vacuolation, as shown by the neutral red test, and caused a 50% decrease in VacA internalization into endosomal vesicles and vacuoles, as assessed by quantitation of immunogold particles. In addition, acidic pH blocked endosomal internalization of H. pylori outer membrane vesicles, a convenient indicator of endocytosis. Our data raise the possibility that suppression of gastric acid may increase H. pylori-induced gastric damage by enhancing epithelial internalization of H. pylori virulence factors through activation of endocytosis. Increased transmembrane diffusion of ammonia could also contribute to this process.     

Inhibition of murine lymphocyte proliferation by the B subunit of cholera toxin

Cholera toxin is known to inhibit lymphocyte activation in vitro, an effect attributed to its ability to activate adenylate cyclase and increase intracellular cyclic adenosine monophosphate. In these studies the effects of both cholera toxin (CT) and its purified binding subunit (CT-B) on lymphocyte proliferation in vitro was examined, using a variety of cell activators. We found that both CT and CT-B inhibited mitogen- and antigen-induced T cell proliferation and anti-IgM-induced B cell proliferation in a dose-dependent manner. However, only CT-inhibited lipopolysaccharide-induced B cell proliferation. Neither CT nor CT-B inhibited antigen uptake and presentation by macrophages. The CT-B preparation used was shown not to activate lymphocyte adenylate cyclase, although CT itself was a strong activator of this enzyme. Both molecules had to bind to the lymphocyte surface in order to inhibit. The time course of inhibition of both CT and CT-B was similar in that either could be added up to 24 hr after culture initiation and still inhibit substantially. The addition of excess human recombinant interleukin 2 to the cultures did not affect the inhibition by CT, and had only a partial affect on inhibition by CT-B. Similarly, CT was able to substantially inhibit recombinant interleukin 2-dependent T lymphoblast proliferation, whereas CT-B had only a small inhibitory effect. Inhibition was not major histocompatibility complex-restricted. We conclude that the binding of CT or CT-B to the lymphocyte surface membrane interferes in some way with the activation mechanism leading to proliferation. The inhibition mediated by CT-B does not involve the stimulation of intracellular adenylate cyclase. CT appears to inhibit both by binding via its B subunit and by activation of adenylate cyclase via its A subunit.     

Glucagon-mediated internalization of serine-phosphorylated glucagon receptor and Gsalpha in rat liver

To assess glucagon receptor compartmentalization and signal transduction in liver parenchyma, we have studied the functional relationship between glucagon receptor endocytosis, phosphorylation and coupling to the adenylate cyclase system. Following administration of a saturating dose of glucagon to rats, a rapid internalization of glucagon receptor was observed coincident with its serine phosphorylation both at the plasma membrane and within endosomes. Co-incident with glucagon receptor endocytosis, a massive internalization of both the 45- and 47-kDa Gsalpha proteins was also observed. In contrast, no change in the subcellular distribution of adenylate cyclase or beta-arrestin 1 and 2 was observed. In response to des-His(1)-[Glu(9)]glucagon amide, a glucagon receptor antagonist, the extent and rate of glucagon receptor endocytosis and Gsalpha shift were markedly reduced compared with wild-type glucagon. However, while the glucagon analog exhibited a wild-type affinity for endosomal acidic glucagonase activity and was processed at low pH with similar kinetics and rates, its proteolysis at neutral pH was 3-fold lower. In response to tetraiodoglucagon, a glucagon receptor agonist of enhanced biological potency, glucagon receptor endocytosis and Gsalpha shift were of higher magnitude and of longer duration, and a marked and prolonged activation of adenylate cyclase both at the plasma membrane and in endosomes was observed. The subsequent post-endosomal fate of internalized Gsalpha was evaluated in a cell-free rat liver endosome-lysosome fusion system following glucagon injection. A sustained endo-lysosomal transfer of the two 45- and 47-kDa Gsalpha isoforms was observed. Therefore, these results reveal that within hepatic target cells and consequent to glucagon-mediated internalization of the serine-phosphorylated glucagon receptor and the Gsalpha protein, extended signal transduction may occur in vivo at the locus of the endo-lysosomal apparatus.     

Translocation of phospholipid/Ca2+-dependent protein kinase in B-lymphocytes activated by phorbol ester or cross-linking of membrane immunoglobulin.

Treatment of hepatocytes with islet activating protein (pertussis toxin) from Bordetella pertussis blocked the ability of insulin to inhibit adenylate cyclase activity both in broken plasma membranes and in intact hepatocytes. Such treatment of intact hepatocytes with pertussis toxin did not prevent insulin from activating the peripheral plasma membrane cyclic AMP phosphodiesterase although it did inhibit the ability of insulin to activate the 'dense-vesicle' cyclic AMP phosphodiesterase. The ability of glucagon pretreatment of hepatocytes to block insulin's activation of the plasma membrane cyclic AMP phosphodiesterase was abolished in pertussis toxin-treated hepatocytes. It is suggested that the ability of insulin to manipulate cyclic AMP concentrations by inhibiting adenylate cyclase and activating the plasma membrane and 'dense-vesicle' cyclic AMP phosphodiesterases involves interactions with the guanine nucleotide regulatory protein system occurring in liver plasma membranes.     

Activity of covalently cross-linked cholera toxin with the adenylate cyclase of intact and lysed pigeon erythrocytes.

Reaction of cholera toxin with NN'-bis(carboximidomethyl)tartaramide dimethyl ester produced several cross-linked species that had subunit B (which binds to the cell surface) and peptides A1 (which activates adenylate cyclase) and A2 all covalently joined together. This cross-linded material had activity with pigeon erythrocytes that was comparable in all respects with that of native toxin. It activated the adenylate cyclase of whole cells, showing a characteristic lag phase, and this activation was increased if the cells had been preincubated with ganglioside GM1, but abolished if the protein had been preincubated with the ganglioside. It activated the enzyme in lysed cells more strongly and without the lag phase. These results show that the toxin is active even when peptide A1 cannot be released from the rest of the molecule.     

Human platelets are defective in processing of cholera toxin.

Cholera toxin is unable to elevate cyclic AMP levels in intact human platelets despite being very efficacious in this respect in other mammalian cells; in the presence of 0.5 mM-isobutylmethylxanthine, we found that 3-6nM-cholera toxin over 3h at 37 degrees C elevated platelet cyclic AMP from 33 +/- 13 to 39 +/- 12pmol/mg of protein (means +/- S.D.; n = 12). We have investigated the basis for this lack of response. 125I-labelled cholera toxin bound to platelets both saturably and with high affinity (Kd congruent to 60pM; Bmax. congruent to 50fmol/mg of protein). Incubation of platelets with the putative cholera toxin receptor monosialoganglioside GM1 enhanced 125I-labelled cholera toxin binding at least 40-fold but facilitated only a minimal (less than or equal to 3-fold) elevation of platelet cyclic AMP levels. In contrast, dithiothreitol-activated cholera toxin markedly stimulated adenylate cyclase activity in platelet membranes. Platelet cytosol both enhanced stimulation of adenylate cyclase activity by activated cholera toxin (A1 subunit) and supported stimulation by the A1-A2 subunit of cholera toxin. Neither GTP nor NAD+, both necessary for response to cholera toxin, was lacking in intact platelets. However, we found that platelets were unable to cleave cholera toxin to the active A1 subunit (as assessed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis). By contrast, murine S49 lymphoma cells were able to generate the A1 subunit with a time course that closely resembled the kinetics of toxin-mediated cyclic AMP accumulation in these cells. Thus we conclude that human platelets are defective in their ability to process surface-bound cholera toxin. These results indicate that binding of cholera toxin to surface receptors is necessary, but not sufficient, for expression of the toxin effect and the generation of the A1 subunit of the toxin may be rate-limiting for expression of cholera toxin response.     

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.     

Structure and function of cholera toxin and hormone receptors

The enterotoxin from Vibrio cholerae is a protein of 100,000 mol wt which stimulates adenylate cyclase activity ubiquitously. The binding of biologically active ¹²⁵I-labeled choleragen to cell membranes is of extraordinary affinity and specificity. The binding may be restricted to membrane-bound ganglioside G_MI. This ganglioside can be inserted into membranes from exogenous sources, and the increased toxin binding in such cells can be reflected by an increased sensitivity to the biological effects of the toxin. Features of the toxin-activated adenylate cyclase, including conversion of the enzyme to a GTP-sensitive state, and the increased sensitivity of activation by hormones, suggest analogies between the basic mechanism of action of choleragen and the events following binding of hormones to their receptors. The action of the toxin is probably not mediated through intermediary cytoplasmic events, suggesting that its effects are entirely due to processes involving the plasma membrane. The kinetics of activation of adenylate cyclase in erythrocytes from various species as well as in rat adipocytes suggest a direct interaction between toxin and the cyclase enzyme which is difficult to reconcile with catalytic mechanisms of adenylate cyclase activation. Direct evidence for this can be obtained from the comigration of toxin radioactivity with adenylate cyclase activity when toxin-activated membranes are dissolved in detergents and chromatographed on gel filtration columns. Agarose derivatives containing the “active” subunit of the toxin can specifically adsorb adenylate cyclase activity, and specific antibodies against the choleragen can be used for selective immunoprecipitation of adenylate cyclase activity from detergentsolubilized preparations of activated membranes. It is proposed that toxin action involves the initial formation of an inactive toxin-ganglioside complex which subsequently migrates and is somehow transformed into an active species which involves relocation within the two-dimensional structure of the membrane with direct pertubation of adenylate cyclase molecules (virtually irreversibly). These studies suggest new insights into the normal mechanisms by which hormone receptors modify membrane functions.     

Mechanism of action of choleragen andE. coli heat-labile enterotoxin: activation of adenylate cyclase by ADP-ribosylation

Summary Choleragen exerts its effects on cells through the activation of adenylate cyclase. The initial event appears to be the binding of the B subunit of the toxin to ganglioside G_M1 on the cell surface, following which there is a delay prior to activation of adenylate cyclase. Patching and capping of the toxin on the cell surface, perhaps involved in the internalization of the enzymatically active subunit, may be occuring during this time. The activation of adenylate cyclase, which is catalyzed by the A₁ peptide of choleragen, does not require the B subunit or ganglioside G_M1. The A₁ peptide catalyzes the transfer of ADP-ribose from NAD to an amino acid, probably arginine, in a 42 000 dalton membrane protein. This protein appears to be the GTP-binding component (or G/F factor) of the adenylate cyclase system and is cruical to the regulation of cyclase activity by hormones such as epinephrine. ADP-ribosylation of the G/F factor is enhanced by GTP and, in some systems, by a cytosolic factor. GTP is also required for stabilization and optimal catalytic function of the choleragen-activated cyclase. Calmodulin, a calcium-binding protein, is necessary for expression of catalytic activity of the toxin-activated adenylate cyclase in brain and other tissues. The ADP-ribosyltransferase activity required for activation of the cyclase is an intrinsic property of the A₁ peptide of choleragen which is expressed only after the peptide is released from the holotoxin by reduction of a single disulfide bond. In the absence of cellular components, choleragen catalyzes the ADP-ribosylation of small guanidino compounds such as arginine as well as peptides and proteins that contain arginine. It is assumed, therefore, that the site of ADP-ribosylation in the natural acceptor protein is an arginine or similar amino acid. When guanidino compounds are not present as ADP-ribose acceptors, choleragen hydrolyzes NAD to ADP-ribose and nicotinamide at a considerably slower rate. E. coli heat-labile enterotoxin (LT) is very similar to choleragen in structure and function. It consists of two types of subunits, A and B, with sizes comparable to those of the A and B subunits of choleragen. Binding of LT to the cell surface is enhanced by prior incorporation of G_M1 but not other gangliosides; the oligosaccharide of G_M1 specifically interacts with LT and its B subunit. The A subunit of LT exhibits ADP-ribosyltransferase activity following activation by thiol to release the A₁ peptide. The A subunit of LT can be isolated in an ‘unnicked’ form and thus requires, in addition to reduction by a thiol, proteolytic cleavage to generate the active A₁ peptide. Like choleragen, LT uses guanidino compounds as model ADP-ribose acceptors and catalyzes the ADP-ribosylation of a 42 000 dalton protein in cell membrane prepatations. ADP-ribosyltransferases that use arginine as ADP-ribose acceptors are not restricted to bacterial systems; such an enzyme has been purified to apparent homogeneity (>500 000-fold) from turkey erythrocytes. Based on a subunit molecular weight of 28 000, its turnover number with arginine as the ADP-ribose acceptor is considerably higher than that of either toxin. Although with low molecular weight guanidino derivatives the substrate specificity of the enzyme is similar to that of choleragen, with protein substrates it clearly differs. The physiological role of the turkey erythrocyte transferase remains to be established.     

Endosomal proteolysis of diphtheria toxin without toxin translocation into the cytosol of rat liver in vivo

A detailed proteolysis study of internalized diphtheria toxin (DT) within rat liver endosomes was undertaken to determine whether DT-resistant species exhibit defects in toxin endocytosis, toxin activation by cellular enzymes or toxin translocation to its cytosolic target. Following administration of a saturating dose of wild-type DT or nontoxic mutant DT (mDT) to rats, rapid endocytosis of the intact 62-kDa toxin was observed coincident with the endosomal association of DT-A (low association) and DT-B (high association) subunits. Assessment of the subsequent post-endosomal fate of internalized mDT revealed a sustained endo-lysosomal transfer of the mDT-B subunit accompanied by a net decrease in intact mDT and mDT-A subunit throughout the endo-lysosomal apparatus. In vitro proteolysis of DT, using an endosomal lysate, was observed at both neutral and acidic pH, with the subsequent generation of DT-A and DT-B subunits (pH 7) or DT fragments with low ADP-ribosyltransferase activity (pH 4). Biochemical characterization revealed that the neutral endosomal DT-degrading activity was due to a novel luminal 70-kDa furin enzyme, whereas the aspartic acid protease cathepsin D (EC 3.4.23.5) was identified as being responsible for toxin degradation at acidic pH. Moreover, an absence of in vivo association of the DT-A subunit with cytosolic fractions was identified, as well as an absence of in vitro translocation of the DT-A subunit from cell-free endosomes into the external milieu. Based on these findings, we propose that, in rat, resistance to DT may originate from two different mechanisms: the ability of free DT-A subunits to be rapidly proteolyzed by acidic cathepsin D within the endosomal lumen, and/or the absence of DT translocation across the endosomal membrane, which may arise from the absence of a functional cytosolic translocation factor previously reported to participate in the export of DT from human endosomes.     

Phosphorylation of the active, A1 component of cholera toxin by protein kinase.

Cholera toxin, an activator of adenylate cyclase in a wide variety of cells, is a substrate for the phosphotransferase reaction catalyzed by purified cyclic adenosine 5'-monophosphate dependent bovine cardiac muscle protein kinase and the protein associated with human erythrocyte membranes. Phosphorylation occurs when the toxin is dissociated with 5-20 mM dithiothreitol and is restricted to the A1 or "adenylate cyclase activating" subunit of the toxin.     

The activation of adenylate cyclase from small intestinal epithelium by cholera toxin

ADP-ribosylation of membrane proteins from rabbit small intestinal epithelium was investigated following incubation of membranes with [32P]NAD and cholera toxin. Cholera toxin catalyzes incorporation of 32P into three proteins of 40 kDA, 45 kDa and 47 kDa located in the brush-border membrane. In contrast, basal lateral membranes do not contain any protein which becomes labeled in a toxin-dependent manner when incubated with cholera toxin and [32P]NAD. The modification of membrane proteins from brush border occurred in spite of the virtual absence in these membranes of adenylate cyclase activatable either by cholera toxin, vasoactive intestinal peptide (VIP) or fluoride. The three agents activated adenylate cyclase when crude plasma membrane were used. Cholera toxin activated fivefold at 10 micrograms/ml. Vasoactive intestinal peptide activated at concentrations from 10-300 nM, the maximal stimulation being sixfold. Fluoride activated 10-fold at 10 mM. When basal lateral membranes were assayed for adenylate cyclase it was found that, with respect to the crude membranes, the specific activity of fluoride-activated enzyme was 3.3-fold higher, VIP stimulated enzyme was maintained while cholera-toxin-stimulated enzyme showed half specific activity. Moreover, while fluoride stimulated ninefold and VIP stimulated fivefold, cholera toxin only stimulated twofold at the highest concentration. The results suggest that the activation by cholera toxin of adenylate cyclase located at the basal lateral membrane requires ADPribosylation of proteins in the brush border membrane.     

The B subunit of cholera toxin enhances DNA synthesis in rat hepatocytes induced by insulin and epidermal growth factor.

The B subunit of cholera toxin, which binds to ganglioside GM1, enhanced DNA synthesis in rat hepatocytes in primary culture induced by insulin and/or epidermal growth factor. The effect was dose-dependent, and whole cholera toxin, activating adenylate cyclase, showed a higher effect than the B subunit alone. The B subunit acted additively with other agents that also increase cyclic AMP levels. A competitive antagonist of cyclic AMP could not suppress the effect of the B subunit completely. These data suggest that the effect is independent of the cyclic AMP signal pathway, and that GM1 plays a role in hepatocyte proliferation.