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.     

Inhibitors of protein synthesis block action of cholera toxin

Prior treatment of macrophages with cycloheximide blocked the activation of adenylate cyclase by choleragen. The effect of cycloheximide was time and dose dependent and also caused by puromycin. Toxin receptors and the catalytic and regulatory components of the cyclase were still present. As degradation and generation of the A₁ subunit of choleragen was also blocked, we propose the existence of a membrane component that mediates the translocation of choleragen across the membrane.     

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.     

Release of guanyl nucleotides from the regulatory subunit of adenylate cyclase

Choleragen and beta-adrenergic agonists, both of which activate turkey erythrocyte adenylate cyclase, have been reported to accelerate release of bound [3H]guanyl nucleotides from turkey erythrocyte membranes. We have now obtained evidence that choleragen- or isoproterenol-stimulated release reflects a change in the affinity of the regulatory subunit (G/F) of adenylate cyclase for guanyl nucleotides. Solubilized preparations of turkey erythrocytes that had bound radiolabeled GTP were chromatographed on Ultrogel AcA 34. The protein from which guanyl nucleotide was released upon incubation with choleragen or isoproterenol was co-eluted with G/F activity. Furthermore, this protein appears to be the same size as the complex containing the 42,000-dalton peptide, ADP*-ribosylated by choleragen, which is presumably a subunit of G/F. ADP ribosylation of the 42,000-dalton subunit of G/F by choleragen occurred with a half-time of about 5 min, whereas choleragen-stimulated release of guanyl nucleotides was much slower (t1/2 greater than or equal to 60 min). When membranes were treated with choleragen and NAD, the delay in activation of adenylate cyclase by guanylyl imidodiphosphate was decreased but not abolished, a finding consistent with the idea that release of endogenously bound nucleotide (and subsequent binding of the nonhydrolyzable GTP analog) occurs only slowly following ADP ribosylation. In contrast, activation of the adenylate cyclase of either toxin-treated or untreated membranes in the presence of isoproterenol and guanylyl imidodiphosphate was very rapid. These data support the hypothesis that isoproterenol and choleragen may activate adenylate cyclase, at least in part, by increasing the rate of release of guanyl nucleotides from G/F.     

Mechanism of action of choleragen. Evidence for ADP-ribosyltransferase activity with arginine as an acceptor.

Choleragen catalyzed the hydrolysis of NAD to ADP-ribose and nicotinamide; nicotinamide production was dramatically increased by L-arginine methyl ester and to a lesser extent by D- or L-arginine, but not by other basic amino acids. Guanidine was also effective. Nicotinamide formation in the presence of L-arginine methyl ester was greatest under conditions previously shown to accelerate the hydrolysis of NAD by choleragen (Moss, J., Manganiello, V. C., and Vaughan, M. (1976) Proc. Natl. Acad. Sci. U.S.A. 73, 4424-4427). After incubation of [adenine-U14C]NAD and L[3H]arginine with coleragen, a product was isolated by thin layer chromatography that contained adenine and arginine in a 1:1 ratio and has been tentatively identified as ADP-ribose-L-arginine. Parallel experiments with [carbonyl-14C]NAD have demonstrated that formation of the ADP-ribosyl-L-arginine derivative was associated with the production of [carbonyl-14C]nicotinamide. As guanidine itself was active and D- and L-arginine was equally effective in promoting nicotinamide production, whereas citrulline, which possesses a ureido rather than a guanidino function, was inactive, it seems probable that the guanidino group rather than the alpha-amino moiety participated in the linkage to ADP-ribose. Based on the assumption that the ADP-ribosylation of L-arginine by choleragen is a model for the NAD-dependent activation of adenylate cyclase by choleragen, it is proposed that the active A protomer of choleragen catalyzes the ADP-ribosylation of an arginine, or related amino acid residue in a protein, which is the cyclase itself or is critical to its activation by choleragen.     

Choleragen stimulates steroidogenesis and adenylate cyclase in cells lacking functional hormone receptors.

Choleragen stimulates steroid secretion and adenylate cyclase in three cell lines, adrenal tumor line (Y-1), a corticotropin-resistant mutant derived from Y-1 called OS-3, and a receptor-deficient Leydig tumor line (I-10). Sensitivity for half-maximal stimulation varies from 3 to 36 pM choleragen, the I-10 line being the most sensitive. Latency before the onset of steroidogenesis is longer in OS-3 and I-10 cells than in the Y-1 line. In both OS-3 and I-10 cells choleragen stimulates adenylate cyclase whether ITP or 5'-guanylylimidodiphosphate is the regulatory cofactor used. In addition to the responses of the receptor-deficient lines, choleragen does not, during its latency, block the response to corticotropin in Y-1 cells; corticotropin does not block binding of 125I-labeled choleragen to Y-1 cells; gangliosides do not interfere with the corticotropin-induced stimulation of Y-1 cells. We conclude that the corticotropin and choleragen receptors are different.     

Influence of termolabile enterotoxin of Enterobacter cloacae on phagocytic activity of mice peritoneal macrophages

Influence of termolabile enterotoxin (LT-enterotoxin) of Enterobacter cloacae on functional activity of mice peritoneal macrophages was studied and following combinations were used: LT-enterotoxin-producing E. cloacae, its isogenic pair--LT-enterotoxin non-producing E. cloacae, supernatantof broth culture containing LT-enterotoxin, and physiological salt solution (in control group). Data showing decrease in phagocytic and lysosomal activity, disorder in functions of hexosemonophosphate shunt enzymes in peritoneal phagocytes were obtained.     

Glucagon and choleragen stimulation of glycogenolysis in primary cultures of adult rat liver parenchymal cells: Lack of involvement of the glucocorticoids

The influence of glucagon, choleragen, and the adrenal glucocorticoids on glycogenolysis in primary cultures of adult rat liver parenchymal cells has been studied. Both glucagon and choleragen caused a twofold to threefold stimulation of glucose production from endogenous reserves of glycogen. The effect of glucagon on glucose production was noted at the earliest time point examined and the stimulation of glucose production was preceded by an elevation of cyclic AMP. Choleragen did not produce a significant stimulation of glucose production until 45 minutes after addition of the agent. Choleragen effects on glucose production were preceded by an elevation of cyclic AMP, but in contrast with glucagon, choleragen did not significantly elevate cyclic AMP until 30 minutes after addition to the culture. One ng of choleragen per ml of medium was sufficient to produce an effect on glucose production. Glucagon- or choleragen-treated cultures mobilized glycogen more rapidly than did untreated cultures incubated in glucose-free medium. In addition, both agents produced a stong inhibition of lactate production. Thus, the stimulation of glucose production by these agents was partially due to increased glycogen mobilization and partially due to redirection of carbon units from glycolysis. That glucose production in the hepatocytes is regulated in part by a cyclic AMP-dependent mechanism is strongly supported by the observation that both agents elevate cyclic AMP and cause an increase in glucose production and inhibition of lactate production. The possibility that the glucocorticoids participated in the regulation of glycogenolysis either in a direct or indirect (permissive) fashion was assessed. It was found that when the direct effect of the glucocorticoids on glycogenesis was taken into account, the glucocorticoids had no direct effects on glycogenolysis, nor did they alter the stimulation of glycogenolysis by glucagon or choleragen.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells.

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E1 also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E1 is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 muM isoproterenol is increased and approaches that produced by5.6 muM prostaglandin E1. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 muM prostaglandin E1 is reduced by as much as 50%, the concentration of prostaglandin E1 required to induce a maximal increase in cyclic AMP is 1/10 that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E1 can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparation from control or choleragen-treated cells. In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblast choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Choleragen stimulates steroidogenesis and adenylate cyclase in cells lacking functional hormone receptors

Choleragen stimulates steroid secretion and adenylate cyclase in three cell lines, adrenal tumor line (Y-1), a corticotropin-resistant mutant derived from Y-1 called OS-3, and a receptor-deficient Leydig tumor line (I-10). Sensitivity for half-maximal stimulation varies from 3 to 36 pM choleragen, the I-10 line being the most sensitive. Latency before the onset of steroidogenesis is longer in OS-3 and I-10 cells than in the Y-1 line. In both OS-3 and I-10 cells choleragen stimulates adenylate cyclase whether ITP or 5′-guanylylimidodiphosphate is the regulatory cofactor used. In addition to the responses of the receptor-deficient lines, choleragen does not during its latency, block the response to corticotropin in Y-1 cells; corticotropin does not block binding of ¹²⁵I-labeled choleragen to Y-1 cells; gangliosides do not interfere with the corticotropin-induced stimulation of Y-1 cells.We conclude that the corticotropin and choleragen receptors are different.     

Die blutgruppenassoziierten Elektrophoresetypen der alkalischen Serumphosphatase bei Gesunden und bei Patienten mit gastrointestinalen Erkrankungen

Zusammenfassung Mit Hilfe der Stärkegelelektrophorese wurden die phänotypischen Variationen der alkalischen Serumphosphatase bei 746 Gesunden, 62 Patienten mit Ulcus duodeni, 95 Patienten mit Ulcus ventriculi und 65 Patienten mit Magencarcinom untersucht. Die Beziehungen der quantitativen Variationen der langsamer wandernden B-Komponente zum ABO-Blutgruppensystem, zum Lewis-Blutgruppensystem, zum ABH-Ausscheidersystem und ihre Abhängigkeit von der Nahrungszufuhr werden bestätigt.Bei der Untersuchung von 637 nicht nüchternen Gesunden wurde eine positive Korrelation der B-Bande zu den Blutgruppen O, B und Le(a-/b+) und den Ausscheidern von ABH-Substanzen gefunden. Eine negative Korrelation der B-Bande fand sich zu den Blutgruppen A₁, Le(a+/b-) und den Nichtausscheidern von ABH-Substanzen.Bei einem Vergleichskollektiv von 109 nüchternen Gesunden wurde im Prinzip wieder die gleiche Korrelation gefunden. Die Häufigkeit und auch die Intensität der B-Bande war jedoch insgesamt geringer als bei den nicht nüchternen Gesunden.Nach Nahrungsaufnahme kommt es zu einem Anstieg der Intensität der B-Komponente der alkalischen Phosphatase im Serum, wie in einem Diätversuch bei drei Personen gezeigt werden konnte.Bei Patienten mit Ulcus duodeni, Ulcus ventriculi und Carcinoma ventriculi war sowohl die Häufigkeit als auch die Intensität der B-Bande geringer als bei Gesunden. Diese Abweichung wird auf eine Änderung der Ernährungsweise oder eine Störung im Bereich des Gastrointestinaltraktes zurückgeführt. Für einen Zusammenhang zwischen der B-Komponente der alkalischen Serumphosphatase und der bekannten ABO-Blutgruppenassoziation der drei untersuchten gastrointestinalen Erkrankungen ergab sich kein eindeutiger Hinweis.

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The alkaline serum phosphatase has been studied by starch gel electrophoresis in 746 healthy individuals, 62 patients with duodenal ulcer, 95 patients with stomach ulcer, and 65 patients with stomach cancer (Fig. 1, Table 1). The previously reported quantitative variations of the B-component of alkaline phosphatase, which is presumably identical with the intestinal component, has been confirmed. The variations of the B-component are associated with the ABO blood group system, the Lewis blood group system, and the ABH secretor status; they are also influenced by diet.In a sample of 637 healthy, nonfasting persons the B-component was positively correlated to the O, B and Le(a-/b+) blood groups and to ABH secretion. The B-component was negatively associated with the A₁ and Le(a+/b-) blood groups and the ABH non-secretor status (Table 2 and 3).In a sample of 109 fasting healthy individuals the B-component was less frequent and also less intense if present. Nevertheless, the same correlations to the ABO and Lewis blood groups and the ABH secretor status are found as in the non-fasting control (Table 5 and 6).In three persons the increase of the B-component of alkaline phosphatase in serum was followed after intake of a fatty meal (Table 7, Fig. 2 and 3).In patients with duodenal ulcer, stomach ulcer and stomach cancer the B-component is less frequent and less intense than in the control group of fasting healthy individuals. This deviation is related to differences in the diet or to disturbances of the gastrointestinal functions in these patients. There was no clear indication of a connection between the B-component of alkaline phosphatase and the ABO blood groups association with these gastrointestinal diseases (Table 8, 9, and 10).

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Mit Unterstützung durch die Deutsche Forschungsgemeinschaft, Bad Godesberg.     

Induction of cholera toxin receptors in cultured cells by butyric acid.

Exposure of HeLa cells to sodium butyrate caused an increase in choleragen (cholera toxin) receptors as measured by increased binding of 125I-choleragen to the intact cells. The process was dependent on time and butyrate concentration; maximal increases (over 40-fold) were observed at 48 h and 5 mM sodium butyrate. Other short chain fatty acids were less effective in elevating choleragen receptors in the order: butyrate greater than pentanoate greater than hexanoate greater than propionate. Acetate and isobutyrate had no effect. The increase in toxin receptors caused by butyrate was reversible and occurred in serum-free medium. The affinity of choleragen for control and butyrate-treated HeLa cells appeared to be similar. Butyrate also induced an elevation in choleragen receptors in rat C6 glial and Friend erythroleukemic cells but not in a butyrate-resistant HeLa mutant. The increase observed in Friend cells paralleled the increase in ganglioside GM1 (galactosyl-N-acetylgalactosaminyl-[N-acetylneuraminyl]-galactosylglucosylceramide), the reported choleragen receptor. Although no GM1 could be detected in untreated Hela cells, small amounts were found in cells exposed to butyrate.     

Mechanism of activation of adenylate cyclase by Vibrio cholerae enterotoxin. Relations to the mode of activation by hormones.

The influence of Vibrio cholerae enterotoxin (choleragen) on the response of adenylate cyclase to hormones and GTP, and on the binding of 125I-labeled glucagon to membranes, has been examined primarily in rat adipocytes, but also in guinea pig ileal mucosa and rat liver. Incubation of fat cells with choleragen converts adenylate cyclase to a GTP-responsive state; (-)-isoproterenol has a similar effect when added directly to membranes. Choleragen also increases by two- to fivefold the apparent affinity of (-)-isoproterenol, ACTH, glucagon, and vasoactive intestinal polypeptide for the activation of adenylate cyclase. This effect on vasoactive intestinal polypeptide action is also seen with the enzyme of guinea pig ileal mucosa; the toxin-induced sensitivity to VIP may be relevant in the pathogenesis of cholera diarrhea. The apparent affinity of binding of 125I-labeled glucagon is increased about 1.5- to twofold in choleragen-treated liver and fat cell membranes. The effects of choleragen on the response of adenylate cyclase to hormones are independent of protein synthesis, and they are not simply a consequence to protracted stimulation of the enzyme in vivo or during preparation of the membranes. Activation of cyclase in rat erythrocytes by choleragen is not impaired by agents which disrupt microtubules or microfilaments, and it is still observed in cultured fibroblasts after completely suppressing protein synthesis with diphtheria toxin. Choleragen does not interact directly with hormone receptor sites. Simple occupation of the choleragen binding sites with the analog, choleragenoid, does not lead to any of the biological effects of the toxin.     

The role of cyclic AMP in aldosterone production by isolated zona glomerulosa cells.

The role of cyclic AMP in the regulation of aldosterone production by adrenocorticotropic hormone (ACTH), angiotensin II (A II), potassium, and serotonin was examined in collagenase-dispersed adrenal glomerulosa cells. The ability of 8-bromo cyclic AMP and choleragen to stimulate maximum aldosterone production indicated that cyclic AMP could act as second messenger for certain of the aldosterone-stimulating factors. The actions of ACTH and choleragen on aldosterone and cyclic AMP production were correlated in dog and rat cells, and a similar relation was seen during stimulation of rat cells by serotonin. In contrast, A II and potassium did not cause changes in cyclic AMP formation while stimulating aldosterone production. Intracellular and receptor-bound cyclic AMP were increased 3-fold by 10(-7) M ACTH but not by A II. Addition of a phosphodiesterase inhibitor increased the magnitude of the cyclic AMP response to ACTH but did not change the lack of stimulation by A II or potassium. In dog cells, the effects of A II and potassium on aldosterone production were partially additive to those of ACTH, choleragen, and 8-bromo cyclic AMP. In contrast, no additivity was observed between A II and potassium, or between combinations of the cyclic AMP-dependent stimuli. These results indicate that the actions of ACTH on aldosterone secretion are mediated by cyclic AMP formation, whereas A II and potassium stimulate aldosterone production through an independent mechanism. The lack of additivity between steroid responses to A II and potassium suggests that these factors could share a common mode of action on steroidogenesis in zona glomerulosa cells.     

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.     

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)     

Cholera toxin and membrane gangliosides: Binding and adenylate cyclase activation in normal and transformed cells

Summary A virally transformed, ganglioside GM₁-deficient cell line binds 2% of the cholera toxin (choleragen) bound by the parent, line and is less responsive to choleragen with respect to adenylate cyclase stimulation. This biological response is maximal when 10% of choleragen-binding sites in the transformed line, or 0.5% in the parent line, are occupied. In contrast, in isolated fat cells saturation of binding and adenylate cyclase stimulation are seen at very similar concentrations.Incubation of ganglioside GM₁ with intact cells increases choleragen binding (defined here as ganglioside incorporation) in the transformed cell line but does not enhance the biological response to choleragen. Stimulation of adenylate cyclase is enhanced in isolated fat cells, however, by exogenous ganglioside GM₁. The binding and cyclase response in fat cells can be reduced by the addition of the inactive analog and competitive antagonist, choleragenoid, and there is recovery of the enzyme response and binding upon subsequent addition of exogenous GM₁. Failure of enhancement in the transformed cell line is explained by the presence of a five- to tenfold excess of binding sites over the number required for the full biological effect of choleragen. Cells with a large excess of toxin receptors are relatively refractory to the blocking effects of choleragenoid on biological responses. Notably, untransformed cells, which contain large quantities of toxin receptor, cannot incorporate exogenously added ganglioside GM₁. These findings suggest the possible existence in the cytoplasmic membrane of specific molecular structures, present in finite and limited number, for recognizing and accepting ganglioside molecules exposed to the external medium.     

Effects of choleragen on hormonal responsiveness of adenylate cyclase in human fibroblasts and rat fat cells

Choleragen increases cyclic AMP content of confluent human fibroblasts. Maximally effective concentrations of isoproterenol and prostaglandin E₁ also induce large increases in cyclic AMP content of human fibroblasts and in confluent cultures the effect of prostaglandin E₁ is much greater than that of isoproterenol. After incubation with choleragen, the increment in cyclic AMP produced by 2 μM isoproterenol is increased and approaches that produced by 5.6 μM prostaglandin E₁. Although the concentration of isoproterenol which produces a maximal increase in cyclic AMP is similar in both control and choleragen-treated cells, lower concentrations of isoproterenol are more effective in the choleragen-treated cells. In choleragen-treated cells, although the response to 5.6 μM prostaglandin E₁ is reduced by as much as 50%, the concentration of prostaglandin E₁ required to induce a maximal increase in cyclic AMP is $\text{1}\text{10}$ that required in control cells. Thus the capacities of intact human fibroblasts to respond to isoproterenol and prostaglandin E₁ can be altered independently during incubation of intact cells with choleragen. Differences in responsiveness to the two agonists were not demonstrable in adenylate cyclase preparations from control or choleragen-treated cells.In rat fat cells, the effects of choleragen on cyclic AMP content were much smaller than those in fibroblasts. In contrast to its effect on intact fibroblasts, choleragen treatment of rat fat cells did not alter the accumulation of cyclic AMP in response to a maximally effective concentration of isoproterenol. The responsiveness of adenylate cyclase preparations to isoproterenol was also not altered by exposure of fat cells to choleragen.     

Stimulation of bone resorption in organ culture by cholera toxin

Bone resorption in organ cultures of neonatal mouse calvaria was stimulated by choleragen (cholera enterotoxin) in a dose-related manner (0.5 to 5.0 ng/ml). Stimulation was potentiated by the cyclic nucleotide phosphodiesterase inhibitor isobutylmethylxanthine (4 μM) and was inhibited by human calcitonin (100 ng/ml), but not by indomethacin (0.7 μM), an inhibitor of the fatty acid cyclooxygenase. The action of choleragen on cyclic AMP accumulation and bone resorption was consistent with the known characteristics of this toxin: 1. choleragen increased cyclic AMP accumulation in bone cultures; 2. there was a lag period (20 – 120 min) prior to an increase in cyclic AMP accumulation following addition of choleragen; 3. incubation with choleragen for only 4 h stimulated bone resorption in the subsequent 44 h as much as did continuous incubation with choleragen for 48 h; and 4. choleragenoid, the biologically inactive toxoid, did not stimulate bone resorption in the concentration range in which choleragen was active. We conclude that activation of adenylyl cyclase and the subsequent increase in cyclic AMP production can stimulate bone resorption, and that cyclic AMP may, therefore, be involved in the enhanced bone resorption mediated by parathyroid hormone and other agents which increase cyclic AMP in bone.     

Requirement for both choleragen and pertussis toxin to obtain maximal activation of adenylate cyclase in cultured cells

NG108-15 cells contain both the inhibitory and stimulatory guanyl nucleotide-binding regulatory proteins of the cyclase system. Choleragen activates cyclase directly by ADP-ribosylating the stimulatory guanyl nucleotide-binding protein; prostaglandin E1 does not further increase activity of cells treated with maximally effective concentrations of choleragen. Including pertussis toxin during incubation with this concentration of choleragen, however, further augments both cyclase activity and cAMP accumulation by intact cells. These observations suggest that the inhibitory guanyl nucleotide-binding protein exerts basal inhibition on catalytic activity which cannot be overcome by maximally effective concentrations of choleragen, stimulatory hormones, or both.