INFECTION AND IMMUNITY, May 1976, p. 1479-1482 Copyright © 1976 American Society for Microbiology
Vol. 13, No. 5
Printed in U.SA.
Morphological and Steroidogenic Changes in Cultured Adrenal Tumor Cells Induced by a Subunit of Cholera Enterotoxin SAM T. DONTA,* SHELLEY R. KREITER,
AND
GWEN WENDELSCHAFER-CRABB
Department of Internal Medicine, University of Iowa,* and Veterans Administration Hospitals, Iowa City, Iowa 52242
Received for publication 23 October 1975
A purified subunit of the cholera enterotoxin molecule was found to have morphological and steroidogenic inducing effects similar to those induced by the native enterotoxin on monolayer tissue cultures of Y1 adrenal tumor cells, although 1,000 times more subunit than toxin (weight basis) was required for maximal effects. In contrast to the whole toxin, the effects of the active subunit could not be prevented by prior incubation with either G.,, ganglioside or with antibodies directed against choleragenoid (the binding subunit). These results suggest that different receptor sites may exist on cells for the binding and for the active subunits of cholera enterotoxin and/or that the active toxin fragment may exert its effects after gaining access to the intracellular compartment.
INTRODUCTION
Cholera enterotoxin induces adenylate cyclase-mediated events in a number of intestinal and extraintestinal model systems by mechanisms that remain to be defined (8). In all intact systems studied, there is a lag period of at least 20 to 30 min after the binding of toxin to cell membranes before the membrane-bound adenylate cyclase is activated. Initially, the toxin rapidly binds to a receptor site that is or resembles the monosialosyl, sialidase-resistant ganglioside GGnSLC (Gm,,) (12, 19). It has been suggested that subsequently the toxin, or a portion thereof, is translocated within the membrane to adenylate cyclase sites (1), with resultant activation of the enzyme and formation of intracellular cyclic adenosine 5'-monophosphate (AMP) from adenosine 5'-triphosphate. The enterotoxin molecule has been isolated and found to be a protein weighing 84,000 daltons (15); like diphtheria toxin, it is thought to be composed of an active fragment, A, and a binding portion, B. The B fragment has a molecular weight of 56,000 and consists of five or six subunits; the A fragment appears to consist of a subunit of approximately 23,000 daltons linked to a smaller 5,000-molecular-weight subunit through a sulfhydryl bridge, through which smaller subunit the B fragment may be associated by noncovalent interactions (9). A naturally occurring fermentation product of certain toxinogenic strains of Vibrio cholerae that is devoid of toxin activity in most systems
studied and thought to be functionally the B fragment has also been isolated and named choleragenoid (11). In several systems studied, choleragenoid has been found to inhibit binding of the whole toxin to G,Nu ganglioside and to inhibit the stimulation by the toxin of adenylate cyclase and cyclic AMP-associated events (7, 11, 16). One model system in which it has not been possible to demonstrate any inhibition of the effects of cholera enterotoxin by choleragenoid is that comprised of certain clonal lines of adrenal tumor cells in monolayer tissue culture (2, 3a, 14). These cells respond to picogram quantities of the enterotoxin by undergoing morphological changes (rounding) and increasing their production of M4,3-ketosteroids (4). The lack of inhibition of the effects of the toxin on the adrenal cells by choleragenoid might mean that there are different cellular receptor sites for choleragenoid (fragment B) and for the toxin, or for its active portion (fragment A). Recently, fragment (subunit) A has been isolated from highly purified enterotoxin by van Heyningen and demonstrated to be free of any B subunits (17). Using purified subunit A, van Heyningen and King have shown that this fragment is capable of stimulating adenylate cyclase activity in intact and in broken-cell preparations of pigeon erythrocytes, albeit at concentrations of subunit A considerably greater than that of whole toxin (18). It became of interest, therefore, to determine if subunit A could be shown to have effects in other model systems as well; the adrenal cell model seemed
1479
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DONTA, KREITER, AND WENDELSCHAFER-CRABB
especially useful in this regard, not only because of its exquisite sensitivity to the effects of the enterotoxin, but also because a biological end product, i.e., steroidogenesis, would be measured. In addition, one would predict that, because of the failure of choleragenoid to inhibit the morphological and steroidogenic effects of the whole enterotoxin on adrenal cells, the binding portion of the toxin molecule would not be an absolute prerequisite for toxininduced activity; hence, the active subunit should be capable of inducing morphological and steroidogenic changes similar to those induced by the native toxin. MATERIALS AND METHODS Toxins, toxoid, gangliosides, and antisera. Purified preparations of cholera enterotoxin and choleragenoid were obtained from R. A. Finkelstein (10). Purified subunit A of cholera enterotoxin was provided by S. van Heyningen (17). Ganglioside GGnSLC (GM,) was obtained from W. E. van Heyningen (13). Antisera directed against cholera enterotoxin and against choleragenoid used in experiments reported here were the same as those used and reported previously (3). Dilutions of the toxins and toxoid were in a 0.05 M Tris(hydroxymethyl)aminomethane-0.001 M ethylenediaminetetraacetic acid buffer system at pH 7.5; dilutions of ganglioside and antisera were in a phosphate-buffered saline solution, pH 7.2. Tissue culture methods. Monolayer cultures of Y1 adrenal cells were propagated and maintained in minimal essential medium (MEM) supplemented with 10% fetal calf serum at 37 C in a humidified atmosphere of 95% air and 5% CO2. More specific details of the tissue culture experimental methods, as well as those of the spectrophotometric assay for A4,3-ketosteroids extractable from the tissue culture medium, have been previously described (5). Steroid production is expressed as nanomoles of steroid per plate of cells per incubation time period. For experiments in which the effects of ganglioside or antiserum on toxin-induced steroidogenesis were studied, the toxin was preincubated with ganglioside or antiserum for 30 min at 37 C before the addition of an 0.2-ml portion of the preincubation mixture to the Y1 cells. RESULTS AND DISCUSSION
When purified preparations of subunit A were tested on Y1 adrenal cells, the cells' morphology was affected and their output of ketosteroids increased. An analysis of the dose-response curves of subunit A and the whole toxin revealed that approximately 1,000 times more subunit A, on a weight basis, was necessary for maximal steroidogenic rates, that amount of subunit A being 3 to 10 ,ug/ml. No morphological changes were observed with. concentrations of subunit A less than 0.1 ,ug/ml, concentrations of subunit that are 10,000 to 20,000 times
INFECT. IMMUN.
greater than that amount of whole toxin required to observe minimal morphological and steroidogenic effects (4). Using maximal steroidogenic concentrations (5 ,ug/ml) of subunit A, cell rounding was not observed until 2.5 h had elapsed, with maximal effects requiring an additional 3 h of incubation. In contrast, 5 ng of whole cholera enterotoxin per ml, tested in parallel with subunit A, effected structural alterations beginning 1.5 h postexposure of Y1 cells to toxin, with maximal rounding observed within an additional hour of incubation. Analysis of the nature of the morphological changes induced by the intact toxin and those induced by subunit A by scanning electron microscopy showed that the changes effected by the subunit were essentially identical to those induced by whole enterotoxin, with no obvious differences (Fig. 1). As with whole toxin, both morphological and steroidogenic activities of subunit A could be eliminated by prior heating at 56 C for 30 min. Prior incubation of GN, ganglioside or antibodies to either the whole enterotoxin or to choleragenoid with the whole toxin will prevent the morphological and steroidogenic activities of the toxin (3, 3a, 6). In contrast, preincubation of subunit A with either ganglioside or antibodies directed against choleragenoid was without effect on the activities of the subunit on Y1 adrenal cells (Table 1). Some minimal inhibition of the effects of fragment A could be observed if the subunit was preincubated with an antibody preparation directed against the native enterotoxin (Table 2). Although more inhibition of the effects of the subunit might have been elicited with the use of larger amounts of antitoxin, the results obtained suggest that the active subunit is not an important immunochemical determinant of the intact toxin molecule and is probably buried to some extent within the molecule. Attempts to alter the effects of subunit A on Y1 cells by co-incubation or preincubation of the subunit with varying concentrations of choleragenoid were not successful. Similarly, attempts to increase the effects of submaximal steroidogenic concentrations of fragment A by possibly facilitating its entry into cells with the use of amphotericin B, at concentrations of amphotericin (5 ,g/ml) known to facilitate the entry of a substance such as cyclic AMP, and which concentration is not apparently toxic to the cell, did not result in any increased steroidogenic activities. The results of these experiments demonstrate that the proposed active fragment of cholera enterotoxin is in fact capable of inducing morphological changes and steroidogenesis
VOL. 13, 1976
1481
SUBUNIT A OF CHOLERA TOXIN AND ADRENAL CELLS
TABLE 1. Effect of Gml ganglioside and anticholeragenoid (anti-CG) on morphological and steroidogenic activities of fragment A and cholera enterotoxin (CT) nmol/plate per 22 h 22.4 ± 3.8
Prepn
Control GM a (0.2 jig/ml)
Anti-CG (1:8,000)
26.2 ± 4.1 21.4 ± 0.4
Fragment A (5 ,ug/ml) A + GM, A + GM,
38.0 ± 1.6 36.6 ± 3.8 40.0 ± 0.4
Rounding Ronng
+ + +
CT (2 ng/ml) 64.2 ± 1.0 + CT + GM, 25.6 ± 1.8 CT + anti-CG 22.2 ± 0.2 a Concentrations shown are final concentrations in MEM. b +, Rounding; -, no rounding at end of incubation period.
TABLE 2. Effect of anticholeragenoid (anti-CG) and antitoxin (anti-CT) on the steroidogenic activities of fragment A and cholera toxin (CT) Prepn
FIG. 1. Photomicrographs of Yl adrenal cells in MEM supplemented with 10% fetal calf serum as seen with the scanning electron microscope. Top, Untreated cells; middle, cells treated with cholera enterotoxin (5 nglml) for 18 h; bottom, cells treated with subunit A (5 jig/ml) for 18 h. Magnification bars represent 10 ,um.
grown
in Y1 adrenal cells and that these changes cannot be accounted for by the presence of any contaminating quantities of intact toxin. Furthermore, the changes effected by subunit A
nmol/plate per 22 h %
Itnhibi-
Control Anti-CGa (1:8,000) Anti-CT (1 Lg/ml)
16.0 ± 2.6 14.4 ± 0.6
Fragment A (4.2 ,ug/ml) A + anti-CG A + anti-CT
35.8 ± 0.8 34.4 ± 4.8 30.6 ± 2.4
CT (2 ng/ml) CT + anti-CG CT + anti-CT a Concentrations shown in MEM.
43.2 ± 2.4 16.0 ± 0.2 100 15.2 ± 4.2 100 are final concentrations
13.4 ± 1.4 7 .3 27.1
were done so without the necessity of any prior binding assistance or fixation to the cell surface effected by fragment B. This does not exclude the possibility, however, that fragment B significantly contributes to either the binding of subunit A to any specific receptors or to its localization near membrane-bound adenylate cyclase. Whether the receptor sites for fragment B and for any existing sites for fragment A on adrenal cells are different from those present in other extraintestinal and intestinal cell systems remains to be delineated. Although the results of experiments with amphotericin B did not demonstrate an increase in the steroidogenic effects of subunit A, these results do not preclude the possibility that the active subunit exerts its effects after gaining access to the intracellular compartment; support for this hypothesis would have
1482
DONTA, KREITER, AND WENDELSCHAFER-CRABB
been obtained had there been increased effects in the presence of amphotericin. The differences observed in the amounts of subunit and whole toxin required to effect minimal and maximal morphological and steroidogenic effects suggest that either the subunit was denatured in some fashion during its purification or that the intrinsic activity of the subunit is less than that of the parent toxin. Distinguishing between these two possibilities is not currently possible. ACKNOWLEDGMENTS These studies were supported by grants from the Veterans Administration Research and Education Service and Public Health Service grant AI 11416 from the National Institute of Allergy and Infectious Diseases. S. T. Donta is a Clinical Investigator of the Veterans Administration. 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. 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. 3. Donta, S. T. 1974. Neutralization of cholera enterotoxin-induced steroidogenesis by specific antibody. J. Infect. Dis. 129:284-288. 3a. Donta, S. T. 1976. Interactions of choleragenoid and GMI ganglioside with cholera and E. coli enterotoxins in adrenal tissue cultured cells. J. Infect. Dis. 133: S11iS-SI19. 4. Donta, S. T., M. King, and K. Sloper. 1973. Induction of steroidogenesis in tissue culture by cholera enterotoxin. Nature (London) New Biol. 243:246-247. 5. Donta, S. T., and D. M. Smith. 1974. Stimulation of steroidogenesis in tissue culture by enterotoxigenic Escherichia coli and its neutralization by specific antiserum. Infect. Immun. 9:500-505. 6. Donta, S. T., and J. P. Viner. 1975. Inhibition of the
7.
8. 9.
10. 11.
12.
13. 14. 15.
16.
17. 18. 19.
INFECT. IMMUN.
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. Field, M. 1974. Mode of action of cholera toxin: stabilization of catecholamine-sensitive adenylate cyclase in turkey erythrocytes. Proc. Natl. Acad. Sci. U.S.A. 71:3299-3303. Finkelstein, R. A. 1973. Cholera. Crit. Rev. Microbiol. 2:553-623. Finkelstein, R. A., M. K. LaRue, and J. J. LoSpalluto. 1972. Properties of the cholera exo-enterotoxin: effects of dispersing agents and reducing agents in gel filtration and electrophoresis. Infect. Immun. 6:934944. Finkelstein, R. A., and J. J. LoSpalluto. 1972. Crystalline cholera toxin and toxoid. Science 175:529-530. Holmgren, J. 1973. Comparison of the tissue receptors for Vibrio cholerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid. Infect. Immun. 8:851-859. Holmgren, J., I. Lonnroth, and L. Svennerholm. 1973. Tissue receptor for cholera exotoxin: postulated structure fro6n studies with GM, ganglioside and related glycolipids. Infect. Immun. 8:208-214. King, C. A., and W. E. van Heyningen. 1973. Deactivation of cholera toxin by a sialidase-resistant monosialosylganglioside. J. Infect. Dis. 127:639-647. Kwan, C. N., and R. M. Wishnow. 1974. Escherichia coli enterotoxin-induced steroidogenesis in cultured adrenal tumor cells. Infect. Immun. 10:146-151. LoSpalluto, J. J., and R. A. Finkelstein. 1972. Chemical and physical properties of cholera exo-enterotoxin (choleragen) and its spontaneously formed toxoid (choleragenoid). Biochim. Biophys. Acta 257:158-166. Pierce, N. F. 1973. Differential inhibitory effects of cholera toxoids and ganglioside on the enterotoxins of Vibrio chokerae and Escherichia coli. J. Exp. Med. 137:1009-1023. van Heyningen, S. 1974. Cholera toxin: interaction of subunits with ganglioside GMI. Science 183:656-657. 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. van Heyningen, W. E. 1974. Gangliosides as membrane receptors for tetanus toxin, cholera toxin and serotonin. Nature (London) 249:415-417.
Vol. 13, No. 5
Printed in U.SA.
Morphological and Steroidogenic Changes in Cultured Adrenal Tumor Cells Induced by a Subunit of Cholera Enterotoxin SAM T. DONTA,* SHELLEY R. KREITER,
AND
GWEN WENDELSCHAFER-CRABB
Department of Internal Medicine, University of Iowa,* and Veterans Administration Hospitals, Iowa City, Iowa 52242
Received for publication 23 October 1975
A purified subunit of the cholera enterotoxin molecule was found to have morphological and steroidogenic inducing effects similar to those induced by the native enterotoxin on monolayer tissue cultures of Y1 adrenal tumor cells, although 1,000 times more subunit than toxin (weight basis) was required for maximal effects. In contrast to the whole toxin, the effects of the active subunit could not be prevented by prior incubation with either G.,, ganglioside or with antibodies directed against choleragenoid (the binding subunit). These results suggest that different receptor sites may exist on cells for the binding and for the active subunits of cholera enterotoxin and/or that the active toxin fragment may exert its effects after gaining access to the intracellular compartment.
INTRODUCTION
Cholera enterotoxin induces adenylate cyclase-mediated events in a number of intestinal and extraintestinal model systems by mechanisms that remain to be defined (8). In all intact systems studied, there is a lag period of at least 20 to 30 min after the binding of toxin to cell membranes before the membrane-bound adenylate cyclase is activated. Initially, the toxin rapidly binds to a receptor site that is or resembles the monosialosyl, sialidase-resistant ganglioside GGnSLC (Gm,,) (12, 19). It has been suggested that subsequently the toxin, or a portion thereof, is translocated within the membrane to adenylate cyclase sites (1), with resultant activation of the enzyme and formation of intracellular cyclic adenosine 5'-monophosphate (AMP) from adenosine 5'-triphosphate. The enterotoxin molecule has been isolated and found to be a protein weighing 84,000 daltons (15); like diphtheria toxin, it is thought to be composed of an active fragment, A, and a binding portion, B. The B fragment has a molecular weight of 56,000 and consists of five or six subunits; the A fragment appears to consist of a subunit of approximately 23,000 daltons linked to a smaller 5,000-molecular-weight subunit through a sulfhydryl bridge, through which smaller subunit the B fragment may be associated by noncovalent interactions (9). A naturally occurring fermentation product of certain toxinogenic strains of Vibrio cholerae that is devoid of toxin activity in most systems
studied and thought to be functionally the B fragment has also been isolated and named choleragenoid (11). In several systems studied, choleragenoid has been found to inhibit binding of the whole toxin to G,Nu ganglioside and to inhibit the stimulation by the toxin of adenylate cyclase and cyclic AMP-associated events (7, 11, 16). One model system in which it has not been possible to demonstrate any inhibition of the effects of cholera enterotoxin by choleragenoid is that comprised of certain clonal lines of adrenal tumor cells in monolayer tissue culture (2, 3a, 14). These cells respond to picogram quantities of the enterotoxin by undergoing morphological changes (rounding) and increasing their production of M4,3-ketosteroids (4). The lack of inhibition of the effects of the toxin on the adrenal cells by choleragenoid might mean that there are different cellular receptor sites for choleragenoid (fragment B) and for the toxin, or for its active portion (fragment A). Recently, fragment (subunit) A has been isolated from highly purified enterotoxin by van Heyningen and demonstrated to be free of any B subunits (17). Using purified subunit A, van Heyningen and King have shown that this fragment is capable of stimulating adenylate cyclase activity in intact and in broken-cell preparations of pigeon erythrocytes, albeit at concentrations of subunit A considerably greater than that of whole toxin (18). It became of interest, therefore, to determine if subunit A could be shown to have effects in other model systems as well; the adrenal cell model seemed
1479
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DONTA, KREITER, AND WENDELSCHAFER-CRABB
especially useful in this regard, not only because of its exquisite sensitivity to the effects of the enterotoxin, but also because a biological end product, i.e., steroidogenesis, would be measured. In addition, one would predict that, because of the failure of choleragenoid to inhibit the morphological and steroidogenic effects of the whole enterotoxin on adrenal cells, the binding portion of the toxin molecule would not be an absolute prerequisite for toxininduced activity; hence, the active subunit should be capable of inducing morphological and steroidogenic changes similar to those induced by the native toxin. MATERIALS AND METHODS Toxins, toxoid, gangliosides, and antisera. Purified preparations of cholera enterotoxin and choleragenoid were obtained from R. A. Finkelstein (10). Purified subunit A of cholera enterotoxin was provided by S. van Heyningen (17). Ganglioside GGnSLC (GM,) was obtained from W. E. van Heyningen (13). Antisera directed against cholera enterotoxin and against choleragenoid used in experiments reported here were the same as those used and reported previously (3). Dilutions of the toxins and toxoid were in a 0.05 M Tris(hydroxymethyl)aminomethane-0.001 M ethylenediaminetetraacetic acid buffer system at pH 7.5; dilutions of ganglioside and antisera were in a phosphate-buffered saline solution, pH 7.2. Tissue culture methods. Monolayer cultures of Y1 adrenal cells were propagated and maintained in minimal essential medium (MEM) supplemented with 10% fetal calf serum at 37 C in a humidified atmosphere of 95% air and 5% CO2. More specific details of the tissue culture experimental methods, as well as those of the spectrophotometric assay for A4,3-ketosteroids extractable from the tissue culture medium, have been previously described (5). Steroid production is expressed as nanomoles of steroid per plate of cells per incubation time period. For experiments in which the effects of ganglioside or antiserum on toxin-induced steroidogenesis were studied, the toxin was preincubated with ganglioside or antiserum for 30 min at 37 C before the addition of an 0.2-ml portion of the preincubation mixture to the Y1 cells. RESULTS AND DISCUSSION
When purified preparations of subunit A were tested on Y1 adrenal cells, the cells' morphology was affected and their output of ketosteroids increased. An analysis of the dose-response curves of subunit A and the whole toxin revealed that approximately 1,000 times more subunit A, on a weight basis, was necessary for maximal steroidogenic rates, that amount of subunit A being 3 to 10 ,ug/ml. No morphological changes were observed with. concentrations of subunit A less than 0.1 ,ug/ml, concentrations of subunit that are 10,000 to 20,000 times
INFECT. IMMUN.
greater than that amount of whole toxin required to observe minimal morphological and steroidogenic effects (4). Using maximal steroidogenic concentrations (5 ,ug/ml) of subunit A, cell rounding was not observed until 2.5 h had elapsed, with maximal effects requiring an additional 3 h of incubation. In contrast, 5 ng of whole cholera enterotoxin per ml, tested in parallel with subunit A, effected structural alterations beginning 1.5 h postexposure of Y1 cells to toxin, with maximal rounding observed within an additional hour of incubation. Analysis of the nature of the morphological changes induced by the intact toxin and those induced by subunit A by scanning electron microscopy showed that the changes effected by the subunit were essentially identical to those induced by whole enterotoxin, with no obvious differences (Fig. 1). As with whole toxin, both morphological and steroidogenic activities of subunit A could be eliminated by prior heating at 56 C for 30 min. Prior incubation of GN, ganglioside or antibodies to either the whole enterotoxin or to choleragenoid with the whole toxin will prevent the morphological and steroidogenic activities of the toxin (3, 3a, 6). In contrast, preincubation of subunit A with either ganglioside or antibodies directed against choleragenoid was without effect on the activities of the subunit on Y1 adrenal cells (Table 1). Some minimal inhibition of the effects of fragment A could be observed if the subunit was preincubated with an antibody preparation directed against the native enterotoxin (Table 2). Although more inhibition of the effects of the subunit might have been elicited with the use of larger amounts of antitoxin, the results obtained suggest that the active subunit is not an important immunochemical determinant of the intact toxin molecule and is probably buried to some extent within the molecule. Attempts to alter the effects of subunit A on Y1 cells by co-incubation or preincubation of the subunit with varying concentrations of choleragenoid were not successful. Similarly, attempts to increase the effects of submaximal steroidogenic concentrations of fragment A by possibly facilitating its entry into cells with the use of amphotericin B, at concentrations of amphotericin (5 ,g/ml) known to facilitate the entry of a substance such as cyclic AMP, and which concentration is not apparently toxic to the cell, did not result in any increased steroidogenic activities. The results of these experiments demonstrate that the proposed active fragment of cholera enterotoxin is in fact capable of inducing morphological changes and steroidogenesis
VOL. 13, 1976
1481
SUBUNIT A OF CHOLERA TOXIN AND ADRENAL CELLS
TABLE 1. Effect of Gml ganglioside and anticholeragenoid (anti-CG) on morphological and steroidogenic activities of fragment A and cholera enterotoxin (CT) nmol/plate per 22 h 22.4 ± 3.8
Prepn
Control GM a (0.2 jig/ml)
Anti-CG (1:8,000)
26.2 ± 4.1 21.4 ± 0.4
Fragment A (5 ,ug/ml) A + GM, A + GM,
38.0 ± 1.6 36.6 ± 3.8 40.0 ± 0.4
Rounding Ronng
+ + +
CT (2 ng/ml) 64.2 ± 1.0 + CT + GM, 25.6 ± 1.8 CT + anti-CG 22.2 ± 0.2 a Concentrations shown are final concentrations in MEM. b +, Rounding; -, no rounding at end of incubation period.
TABLE 2. Effect of anticholeragenoid (anti-CG) and antitoxin (anti-CT) on the steroidogenic activities of fragment A and cholera toxin (CT) Prepn
FIG. 1. Photomicrographs of Yl adrenal cells in MEM supplemented with 10% fetal calf serum as seen with the scanning electron microscope. Top, Untreated cells; middle, cells treated with cholera enterotoxin (5 nglml) for 18 h; bottom, cells treated with subunit A (5 jig/ml) for 18 h. Magnification bars represent 10 ,um.
grown
in Y1 adrenal cells and that these changes cannot be accounted for by the presence of any contaminating quantities of intact toxin. Furthermore, the changes effected by subunit A
nmol/plate per 22 h %
Itnhibi-
Control Anti-CGa (1:8,000) Anti-CT (1 Lg/ml)
16.0 ± 2.6 14.4 ± 0.6
Fragment A (4.2 ,ug/ml) A + anti-CG A + anti-CT
35.8 ± 0.8 34.4 ± 4.8 30.6 ± 2.4
CT (2 ng/ml) CT + anti-CG CT + anti-CT a Concentrations shown in MEM.
43.2 ± 2.4 16.0 ± 0.2 100 15.2 ± 4.2 100 are final concentrations
13.4 ± 1.4 7 .3 27.1
were done so without the necessity of any prior binding assistance or fixation to the cell surface effected by fragment B. This does not exclude the possibility, however, that fragment B significantly contributes to either the binding of subunit A to any specific receptors or to its localization near membrane-bound adenylate cyclase. Whether the receptor sites for fragment B and for any existing sites for fragment A on adrenal cells are different from those present in other extraintestinal and intestinal cell systems remains to be delineated. Although the results of experiments with amphotericin B did not demonstrate an increase in the steroidogenic effects of subunit A, these results do not preclude the possibility that the active subunit exerts its effects after gaining access to the intracellular compartment; support for this hypothesis would have
1482
DONTA, KREITER, AND WENDELSCHAFER-CRABB
been obtained had there been increased effects in the presence of amphotericin. The differences observed in the amounts of subunit and whole toxin required to effect minimal and maximal morphological and steroidogenic effects suggest that either the subunit was denatured in some fashion during its purification or that the intrinsic activity of the subunit is less than that of the parent toxin. Distinguishing between these two possibilities is not currently possible. ACKNOWLEDGMENTS These studies were supported by grants from the Veterans Administration Research and Education Service and Public Health Service grant AI 11416 from the National Institute of Allergy and Infectious Diseases. S. T. Donta is a Clinical Investigator of the Veterans Administration. 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. 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. 3. Donta, S. T. 1974. Neutralization of cholera enterotoxin-induced steroidogenesis by specific antibody. J. Infect. Dis. 129:284-288. 3a. Donta, S. T. 1976. Interactions of choleragenoid and GMI ganglioside with cholera and E. coli enterotoxins in adrenal tissue cultured cells. J. Infect. Dis. 133: S11iS-SI19. 4. Donta, S. T., M. King, and K. Sloper. 1973. Induction of steroidogenesis in tissue culture by cholera enterotoxin. Nature (London) New Biol. 243:246-247. 5. Donta, S. T., and D. M. Smith. 1974. Stimulation of steroidogenesis in tissue culture by enterotoxigenic Escherichia coli and its neutralization by specific antiserum. Infect. Immun. 9:500-505. 6. Donta, S. T., and J. P. Viner. 1975. Inhibition of the
7.
8. 9.
10. 11.
12.
13. 14. 15.
16.
17. 18. 19.
INFECT. IMMUN.
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. Field, M. 1974. Mode of action of cholera toxin: stabilization of catecholamine-sensitive adenylate cyclase in turkey erythrocytes. Proc. Natl. Acad. Sci. U.S.A. 71:3299-3303. Finkelstein, R. A. 1973. Cholera. Crit. Rev. Microbiol. 2:553-623. Finkelstein, R. A., M. K. LaRue, and J. J. LoSpalluto. 1972. Properties of the cholera exo-enterotoxin: effects of dispersing agents and reducing agents in gel filtration and electrophoresis. Infect. Immun. 6:934944. Finkelstein, R. A., and J. J. LoSpalluto. 1972. Crystalline cholera toxin and toxoid. Science 175:529-530. Holmgren, J. 1973. Comparison of the tissue receptors for Vibrio cholerae and Escherichia coli enterotoxins by means of gangliosides and natural cholera toxoid. Infect. Immun. 8:851-859. Holmgren, J., I. Lonnroth, and L. Svennerholm. 1973. Tissue receptor for cholera exotoxin: postulated structure fro6n studies with GM, ganglioside and related glycolipids. Infect. Immun. 8:208-214. King, C. A., and W. E. van Heyningen. 1973. Deactivation of cholera toxin by a sialidase-resistant monosialosylganglioside. J. Infect. Dis. 127:639-647. Kwan, C. N., and R. M. Wishnow. 1974. Escherichia coli enterotoxin-induced steroidogenesis in cultured adrenal tumor cells. Infect. Immun. 10:146-151. LoSpalluto, J. J., and R. A. Finkelstein. 1972. Chemical and physical properties of cholera exo-enterotoxin (choleragen) and its spontaneously formed toxoid (choleragenoid). Biochim. Biophys. Acta 257:158-166. Pierce, N. F. 1973. Differential inhibitory effects of cholera toxoids and ganglioside on the enterotoxins of Vibrio chokerae and Escherichia coli. J. Exp. Med. 137:1009-1023. van Heyningen, S. 1974. Cholera toxin: interaction of subunits with ganglioside GMI. Science 183:656-657. 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. van Heyningen, W. E. 1974. Gangliosides as membrane receptors for tetanus toxin, cholera toxin and serotonin. Nature (London) 249:415-417.
