INFECTION AND IMMUNITY, Oct. 1975, p. 768-771 Copyright ©) 1975 American Society for Microbiology

Vol. 12, No. 4 Printed in U.S.A.

Interaction of Cholera Enterotoxin with Cultured Adrenal Tumor Cells RODNEY M. WISHNOW,* ERIC LIFRAK,

AND

CHI-CHU CHEN

Departments of Medicine and Microbiology, University of California, Irvine 92664, and Veterans Administration Hospital, Long Beach California 90801 * Received for publication 28 May 1975

In the adrenal tumor cell system ganglioside GM, inhibited cholera enterotoxin (CT)-induced steroidogenesis if it was preincubated with the toxin or added to adrenal cells 10 min before CT. In the preincubation studies a molar ratio of GMI to toxin of 3:1 was necessary for half-maximal inhibition of steroidogenesis. On the other hand, horse serum anticholeragenoid neutralized the steroidogenic response to cell-bound CT by 50% if it was added to adrenal monolayer cultures 15 min after the toxin. Specific antiserum was able to neutralize 20% of the toxininduced activity even if it was added to adrenal cultures 2 h after CT. Phase contrast microscopy demonstrated that partial neutralization of the biochemical effect of CT by horse serum anticholeragenoid was accompanied by partial prevention of toxin-induced rounding of adrenal cells. Further studies showed that pretreatment of cultured adrenal cells with a maximal dose of CT increased cyclic adenosine 3'-5'-monophosphate formation in response to a maximal stimulating dose of adrenocorticotropin. This result suggested potentiation of hormonal activation of adenylate cyclase in intact adrenal tumor cells in response to CT.

Previous studies suggest that cholera enterotoxin (CT) and adrenocorticotropin (ACTH) interact with separate adrenal membrane receptors to stimulate cyclic adenosine 3'-5'-monophosphate (cAMP) formation and steroidogenesis in cultured adrenal tumor cells (3-5, 16, 17, 20). Firstly, the addition of ACTH to adrenal cells leads to a rapid increase in cAMP concentration, whereas in response to CT there is a lag phase of 45 min before any change in cAMP can be detected. Secondly, a mutant adrenal cell line unresponsive to ACTH can be maximally stimulated by CT. Thirdly, ACTH and CT have an additive effect on steroidogenesis, suggesting that they do not compete for the same membrane receptor. Furthermore, horse serum anticholeragenoid and a mixed ganglioside preparation completely block the steroidogenic effect of the toxin if added to adrenal cells before CT. but these agents have no effect on hormone-induced steroidogenesis. The present studies show that the steroidogenic effect of CT can be partially neutralized by horse serum anticholeragenoid but not by ganglioside GM1 after the toxin is fixed to specific membrane receptors. Furthermore, these studies suggest that pretreatment of adrenal cells with CT potentiates ACTH activation of adrenal adenylate cyclase. MATERIALS AND METHODS Cell culture procedures, steroid assay, and cAMP determination. The incubation procedures 768

and fluorometric steroid assay for Y-1 adrenal tumor cells have been described previously (20, 21). cAMP was measured in the cells and in the media after ethanol extraction by the Gilman competitive binding assay as described previously (10, 16). Adrenal monolayer morphology was observed under a phase contrast microscope (Nikon, MS). Ganglioside experiments. In these experiments ganglioside GM, was preincubated with CT, added to cells 10 min before toxin, or added 5 min after CT. Under the first conditions, 5 ng of CT plus from 25 pg to 5 ng of GM, were incubated at 24 C for 20 min in calcium- and magnesium-free phosphate-buffered saline. Then the mixture was added to duplicate confluent adrenal tumor cells in 2.0 ml of serum-free Eagle minimal essential medium (GIBCO, Grand Island, N.Y.) to give a final concentration of 2.5 ng of CT per ml plus 0.0125 to 2.5 ng of GMl per ml and incubated for 18 h before determining steroidogenesis. In the next series of experiments from 2.5 to 125 ng of GM, per ml was added to adrenal cells 10 min before 2.5 ng of CI' per ml, and steroidogenesis was determined at 18 h. In the third series of experiments, GMl (25 to 125 ng/ml) was added to adrenal cultures 5 min after the addition of CT (2.5 ng/ml), and steroidogenesis was measured at 18 h. Neutralization experiment. Confluent cultures were preincubated with CT (5 ng/ml) in Eagle minmal essential medium for 5 min, washed twice with 2.0 ml of phosphate-buffered saline, and incubated in Eagle minimal essential medium. At 15, 30, 60, 90, and 120 min of incubation horse serum anticholeragenoid (4 U/ml) was added and steroidogenesis in duplicate cultures was determined at the end of 24 h. An equal volume of horse serum without anticholeragenoid activity was added to duplicate cultures

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after pretreatment with CT to determine the control level of steroidogenesis. The results are expressed as a percentage of this control value. For the 0-min determination specific antiserum was added to duplicate cultures immediately after CT and steroidogenesis was determined at the end of 24 h. Cholera toxin plus ACTH incubation. Confluent monolayers were preincubated with CT (50 ng/ml) for 3 h and then ACTH (1 mU/ml) was added for 1 h, and intracellular and extracellular cAMP were determined in duplicate. In addition, duplicate cultures were preincubated with ACTH (1 mU/ml) for 1 h, and then CT (50 ng/ml) was added for 3 h and cAMP was determined. Control cultures were incubated with CT alone for 3 or 4 h and ACTH alone for 1 or 4 h. Reagents. ACTH (Armour, Chicago, Ill.), GM, brain ganglioside (gift of W. E. van Heyningen, Oxford, England). purified cholera enterotoxin, and horse serum anticholeragenoid ([8, 9] prepared under contract for National Institute of Allergy and Infectious Diseases by R. A. Finkelstein) were used in the tissue culture incubations. [3HIcAMP (24 Ci/mmol) (New England Nuclear Corp., Boston, Mass.) was used for cAMP determinations.

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GM, Ganglioside (ng/ml) FIG. 1. Inhibition of CT-induced steroidogenesis after preincubation of GMl plus CT. Toxin plus GM1 was preincubated for 20 min at 24 C, and then CT (25 ng/ml) and GmU (0.0125 to 25 ng/ml) were added to duplicate confluent adrenal cells. Steroidogenesis at 18 h was determined, and the mean steroid production is expressed as a percentage of toxin-treated control cultures.

100RESULTS Effect of ganglioside GM1 on toxin-induced 0 steroidogenesis. Purified ganglioside GM1 effec- 0c 80 tively prevented CT stimulation ofsteroidogene60sis when it was preincubated with the toxin for 20 min (Fig. 1). Under these conditions, half- 0 maximal inhibition of the toxin effect occurred ~0 at 0.15 ng of GM, per ml. In this experiment 2.5 0ou40 ng of GM, per ml maximally inhibited an equal 20weight of CT. The purified ganglioside itself had no effect on steroid production. Approxi---o mately 100 times more GM, was required for U') half-maximal inhibition if the ganglioside w.a 10 30 50 70 90 110 130 added to cultured adrenal cells 10 min before GMI Gonglioside (ng/mI) CT (Fig. 2). In this experiment 13.0 ng of GM, Inhibition of CT-induced steroidogenesis per ml was required for half-maximal inhibi- by FIG. 2. Adrenal were incubated with Gm, (25 to GM,. tion of CT-induced steroidogenesis, and 50 ng of 125 nglml) for 10cells and then CT ng/ml) was GM1 per ml maximally inhibited the toxin ef- added for an 18-hmin,incubation and (2.5 steroidogenesis fect. However, if CT was allowed to attach to its was determined. adrenal receptor for 5 min before the addition of GM,, then the ganglioside was ineffective in simultaneous addition of toxin. When antitoxin inhibiting CT activity. Under these incubation was added to adrenal cells 15 min after CT conditions, 125 ng of GM1 per ml reduced steroid there was a 50% neutralization of toxin induced production by 10%. steroidogenesis. This figure also shows that speEffect of horse serum anticholeragenoid cific antiserum was able to partially neutralize on CT-induced steroidogenesis. Previously, the steroidogenic response if it was added 2 h we have shown that 1.5 U of horse serum anti- after the toxin. Phase contrast microscopy demcholeragenoid per ml completely neutralized onstrated that neutralization of the toxin-inCT stimulation of steroidogenesis when it was duced biochemical effect was accompanied by a added to adrenal tumor cells before the toxin partial neutralization of the toxin-induced morbecame attached to its specific membrane recep- phological effect. In the toxin-treated control tor (20). In this experiment the ability of anti- cultures more than 95% of the cells changed toxin to neutralize the stimulatory effect of from flattened to spherical shaped cells at 4 h of membrane-bound CT was demonstrated. Fig- incubation. However, if antitoxin was added at ure 3 shows that 4 U of specific antiserum per 15 min only 20% of the cells became spherical, ml neutralized the steroidogenic effect of the and if antitoxin was added at 60 min approxi-

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WISHNOW, LIFRAK AND CHEN

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Time (min) FIG. 3. Effect ofhorse serum anticholeragenoid on CT-induced steroidogenesis. Adrenal cells were exposed to CT (5 nglml) for 5 min and washed, and antitoxin was added 15 to 120 min later. Each point represents the mean steroid determination, expressed as a percentage of CT-treated control cultures from two separate experiments.

mately 50% of the cells appeared rounded if observed at 4 h or 24 h of incubation. Previously we have demonstrated that adrenal morphology correlates with intracellular cAMP levels rather than steroidogenesis (15). Effect of ACTH plus CT on cAMP concentration. Preincubation of adrenal cells with a maximal dose of CT for 3 h potentiated the effect of a maximal dose of ACTH on cAMP formation (Fig. 4). The total cAMP concentration was two times the cAMP levels one would predict if ACTH plus toxin had an additive effect on cAMP concentration. On the other hand, preincubation of the cells with ACTH for 1 h followed by exposure to CT for 3 h did not enhance cAMP formation. In this experiment intracellular and extracellular cAMP were determined separately, and at the end of the incubation approximately 90% of the total cAMP was in the culture media.

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INFECT. IMMUN.

added to adrenal cells before the addition of enterotoxin. Once enterotoxin binds to its adrenal membrane receptor, GM, can no longer inhibit toxin-induced steroidogenesis. In the preincubation experiment 0.15 ng of GM, per ml inhibited toxin-induced steroidogenesis by 50%. If one assumes a molecular weight of 1,600 for GM, and 84,000 for CT, then a molar ratio of ganglioside to toxin of about 3:1 is necessary for half-maximal inhibition of CT in the adrenal cell assay system. Our results are very similar to the molar ratio of GM, to CT necessary to half-maximally inhibit CT-induced accumulation of cAMP in thymocytes (22). As in the adrenal system, preincubation of thymocytes with CT for 5 min prevented the ganglioside inhibitory effect. Donta and Viner have recently examined the effect of several gangliosides, including GM,, GDla, and GT1 on CT-induced steroidogenesis and has shown inhibition only by GM, (6). The ability of specific antiserum to neutralize toxin-induced steroidogenesis by 50% at 15 min and 20% at 2 h suggests that in the adrenal tumor cell system one can partially neutralize CT after it becomes fixed to a specific membrane receptor site but possibly before the active subunit traverses the membrane (19). The fact that anticholeragenoid can partially prevent the CT-induced morphological alteration of adrenal cells supports this view since spherical adrenal morphology correlates with elevated intracellular cAMP levels (15). Previously, Donta reported a 46% inhibition of steroidogenesis if antitoxin was added 1 h after CT (5). In ileal loop experiments investigators were unable to inhibit fluid accumulation if antitoxin was added after CT (18). On the other 7000 -

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DISCUSSION C T, 4hr ACTH CThr ACTH to be the The GM, brain ganglioside appears FIG. 4. Effect of CT plus ACTH on cAMP accumunatural membrane receptor for CT (12-14). Pre- lation. Duplicate adrenal cultures were preincubated viously, a mixed ganglioside preparation has with a maximal dose of CT for3 h and then ACTH (1 been shown to inhibit toxin-induced steroidogen- mU/ml) was added fori h and the total cAMP concenesis (20). The present results show that the tration was determined. The predicted value is the specific ganglioside GM1 can only block steroido- sum of cAMP determinations from cultures exposed genesis if it is preincubated with CT or if it is to CT or ACTH alone.

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CHOLERA ENTEROTOXIN AND ADRENAL CELLS

hand, Craig was able to partially neutralize CT activity in vivo (2). He used the vascular permeability factor assay to show that if antitoxin was administered intracardially 6 h after CT it could neutralize 20% of the activity of CT. Adrenal cells pretreated with a maximal dose of CT showed an increased sensitivity to a maximal dose of ACTH. Turkey erythrocyte ghost and rat liver membrane adenylate cyclase also shows potentiation of hormonal stimulation after pretreatment with CT (1, 7). It is possible that the irreversible binding of 125I-labeled toxin and its persistent biological effect may lead to stabilization of an activated adenylate cyclase complex. On the other hand, pretreatment of adrenal cells with a maximal dose of ACTH for 1 h followed by the addition of CT for 3 h did not potentiate the toxin's effect. This result could be related to the rapid dissociation of ACTH from its membrane receptor or possibly to hormonal stimulation of an adenylate cyclase inhibitor (11). ACKNOWLEDGMENTS This research was supported by Public Health Service grant CA 16868-01 from the National Cancer Institute and by the Medical Research Programs, Veterans Administration Hospital, Long Beach, Calif. We thank W. E. van Heyningen, Sir William Dunn School of Pathology, Oxford, England, for the gift of Ga,. LITERATURE CITED 1. Bennett, V., E. O'Keefe, and P. Cuatrecasas. 1975. Mechanism of action of cholera toxin and the mobile receptor theory of hormone receptor-adenylate cyclase interactions. Proc. Natl. Acad. Sci. U.S.A. 72:33-37. 2. Craig, J. P. 1970. Some observations on the neutralization of cholera vascular permeability factor in vivo. J. Infect. Dis. 121(Suppl.):100-S110. 3. Donta, S. T. 1974. Comparison of the effects of cholera enterotoxin and ACTH on adrenal cells in tissue culture. Am. J. Physiol. 227:109-113. 4. Donta, S. T. 1974. Differentiation between the steroidogenic effects ofcholera enterotoxin and adrenocorticotropin through use of a mutant adrenal cell line. J. Infect. Dis. 129:728-731. 5. Donta, S. T. 1974. Neutralization of cholera enterotoxin-induced steroidogenesis by specific antibody. J. Infect. Dis. 129:284-288. 6. Donta, S. T., and J. P. Viner. 1975. Inhibition of the

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steroidogenic effects of cholera and heat-labile Escherichia coli enterotoxins by Gm, ganglioside: evidence for a similar receptor site for the two toxins. Infect. Immun. 11:982-985. 7. Field, M. 1974. Mode of action ofcholera toxin: stabilization of catecholamine-sensitive adenylate cyclase in turkey erythrocytes. Proc. Natl. Acad. Sci. U.S.A. 71:3299-3303. 8. Finkelstein, R. A. 1970. Monospecific equine antiserum against cholera exoenterotoxin. Infect. Immun. 2:691-697. 9. Finkelstein, R. A., and J. J. Lo Spalluto. 1970. Production of highly purified choleragen and choleragenoid. J. Infect. Dis. 121(Suppl.):63-S72. 10. Gilman, A. G. 1970. Protein binding assay for adenosine 3'-5'-cyclic monophosphate. Proc. Natl. Acad. Sci. U.S.A. 67:305-312. 11. Ho, R. J., and E. W. Sutherland. 1975. Action of feedback regulator on adenylate cyclase. Proc. Natl. Acad. Sci. U.S.A. 72:1773-1777. 12. Holmgren, J. 1973. Comparison of the tissue receptors for Vibrio chokerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid. Infect. Immun. 8:851-859. 13. Holmgren, J., I. Lonnroth, and L. Svennerholm. 1973. Tissue receptor for cholera exotoxin: postulated structure from studies with GM, ganglioside and related glycolipids. Infect. Immun. 8:208-214. 14. King, C. A., and W. E. van Heyningen. 1972. Deactivation of cholera toxin by a sialidase-resistant monosialosylganglioside. J. Infect. Dis. 127:639-647. 15. Kwan, C. N., and R. M. Wishnow. 1974. Adenosine 3'5'-cyclic monophosphate and adrenal tumor cell morphology. In Vitro 10:268-273. 16. Kwan, C. N., and R. M. Wishnow. 1974. Cholera enterotoxin stimulation of cAMP in cultured adrenal tumor cells. Exp. Cell Res. 87:436-438. 17. Kwan, C. N., and R. M. Wishnow. 1974. Escherichia coli enterotoxin-induced steroidogenesis in cultured adrenal tumor cells. Infect. Immun. 10:146-151. 18. Pierce, N. F., W. B. Greenough, and C. C. J. Carpenter, Jr. 1971. Vibrio cholerae enterotoxin and its mode of action. Bacteriol. Rev. 35:1-13. 19. van Heyningen, S., and C. A. King. 1975. Subunit A from cholera toxin is an activator of adenylate cyclase in pigeon erythrocytes. Biochem. J. 146:269-271. 20. Wishnow, R. M., and P. Feist. 1974. The effect of cholera enterotoxin on steroidogenesis in cultured adrenal tumor cells. J. Infect. Dis. 129:690-695. 21. Wishnow, R. M., P. Feist, and M. Glowalla. 1974. The effect of 5-bromo-deoxyuridine on steroidogenesis in mouse adrenal tumor cells. Arch. Biochem. Biophys. 161:275-280. 22. Zenser, T. V., and J. F. Metzger. 1974. Comparison of the action of Escherichia coli enterotoxin on the thymocyte adenylate cyclase-cyclic adenosine monophosphate system to that of cholera toxin and prostaglandin E,. Infect. Immun. 10:503-509.

Interaction of cholera enterotoxin with cultured adrenal tumor cells.

INFECTION AND IMMUNITY, Oct. 1975, p. 768-771 Copyright ©) 1975 American Society for Microbiology Vol. 12, No. 4 Printed in U.S.A. Interaction of Ch...
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