308
Biochimica et Biophysica Acta, 381 (1975) 308--323 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27593
STIMULATION O F C Y C L I C ADENOSINE 3':5'-MONOPHOSPHATE AND CORTICOSTERONE FORMATION IN ISOLATED RAT A D R E N A L CELLS BY CHOLERA ENTEROTOXIN COMPARISON WITH THE EFFECTS OF ACTH
AJAI HAKSAR, DAVID V. MAUDSLEY and FERNAND G. PI~RON
Worcester Foundation for Experimental Biology, Shrewsbury, Mass. 01545 (U.S.A.) (Received July 22nd, 1974)
Summary 1. The production of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and corticosterone in isolated rat adrenal cells was increased by cholera enterotoxin. Both responses were accompanied by a lag period which is characteristic of other known actions of enterotoxin. The duration of the lag period in the production of corticosterone depended on the concentration of enterotoxin; with the maximally stimulating amounts it was ~30--45 min. 2. Maximum rates of cyclic AMP and corticosterone synthesis, after the lag period, were constant for at least 1 h. Although the m a x i m u m rate of corticosterone formation was the same as that obtained with adrenocorticotropic hormone, the m a x i m u m rate of cyclic AMP formation was only 8--10% of that with adrenocorticotropic hormone. 3. Pretreatment of the cells with enterotoxin had no effect on their subsequent steroidogenic response to maximally stimulating amounts of adrenocorticotropic hormone. 4. Cycloheximide inhibited the effect of both enterotoxin and adrenocorticotropic h o r m o n e on corticosterone production. 5. Enterotoxin stimulation of both cyclic AMP and corticosterone formation was dependent on the presence of Ca 2÷ in the medium although the Ca 2÷ requirement was not same as that for adrenocorticotropic hormone. Thus, EGTA at concentrations which completely abolished the effect of adrenocorticotropic h o r m o n e caused only a partial reduction in the effects of enterotoxin. 6. Exogenously added choleragenoid and gangliosides abolished the effects of enterotoxin w i t h o u t having any significant effect on the response of the cells to adrenocorticotropic hormone. 7. After treatment with neuraminidase, the adrenal cells showed an increased response to enterotoxin in terms of both cyclic AMP and cortico-
309
sterone formation which was due to a combination of two effects: (a) increased rate of synthesis of both compounds and (b) shortening of the characteristic lag period. This is in sharp contrast to the results obtained with adrenocorticotropic hormone where neuraminidase-treatment made the cells less sensitive to adrenocorticotropic hormone.
Introduction The active principle of Vibrio cholerae, which produces diarrhea in clinical cholera, has been identified as a specific exotoxin [ 1--4]. The exotoxin, also called enterotoxin and choleragen, has been purified and chemically characterized [1,2,5,6]. Much evidence has accumulated indicating that most, if not all, of the effects of the enterotoxin on the intestinal cells are mediated via stimulation of intestinal adenyl cyclase [7--12]. In addition to the effects on the intestinal cells, enterotoxin has also been shown to increase adenyl cyclase activity, and consequently the biochemical processes dependent on cyclic AMP, in a number of other tissues such as fat cells [13--17], platelets [18], liver [19,20], leukocytes [21--24], thyroid [25] and cultured adrenal tumor cells [26,27]. In this respect, the actions of enterotoxin are similar to those hormones which exert their effects through regulation of intracellular cyclic AMP production. Enterotoxin, therefore, is potentially useful in elaborating the role of various membrane components involved in the regulation of cyclic AMP levels in the tissues. In the present study we have compared the effects of enterotoxin and ACTH on cyclic AMP and corticosterone production in isolated rat adrenal cells to determine the points of congruence between the two stimuli. Materials Trypsin (TRSF-IGA 150 units/mg) and lima bean trypsin inhibitor were obtained from Worthington Biochemical Corporation; collagenase (Serva, 387 Mandl units/mg) from Gallard Schlesinger Chemical Manufacturing Corp.; Pentex Bovine Serum albumin, fraction V powder, fatty acid-poor, from Miles Laboratories; neuraminidase of Clostridium perfringens, type VI chromatographically purified (0.78 units/mg protein), cyclic AMP, dibutyryl cyclic AMP, bovine brain gangliosides, and sialic acid from Sigma Chemical Co.; cyclic [3 HI AMP (spec. act. 27.1 Ci/mmol) from New England Nuclear Corp. Purified cholera enterotoxin was obtained from Dr Richard A. Finkelstein and also from Dr Carl E. Miller, Geographic Medicine Branch, NIAID. The material supplied by the latter was prepared under contract for NIAID by R.A. Finkelstein, Ph.D., The University of Texas, South Western Medical School, Dallas, Texas. Both materials were prepared essentially according to procedure described in ref. 5. Choleragenoid was also obtained from Dr R.A. Finkelstein. Purified ~s-adrenocorticotropic hormone (ACTH) was obtained from Dr C.H. Li, Hormone Research Laboratory, University of California, Berkeley.
310 Methods
Cell suspensions. Suspensions of isolated rat adrenal cells were prepared by collagenase-trypsin treatment of adrenal sections in Krebs-Ringer bicarbonate buffer containing 0.2% glucose (Buffer A) by the m e t h o d described previously [ 28,29]. Cell incubations. All incubations were carried out in duplicate or triplicate in 10 ml beakers in a D u b n o f f metabolic shaker at 37°C in an atmosphere of 95% 0 2 : 5 % CO2. Each beaker contained 1 ml of the cell suspension, test substances dissolved in Buffer A and an appropriate volume of the buffer to make the final volume 1.5 ml. Neuraminidase treatment. The cells were suspended at a concentration of 0 . 6 - - 0 . 8 . 1 0 6 cells/ml in 0.01 M phosphate buffer and neuraminidase treatm e n t carried out as described before [29,30]. Measurement of cyclic AMP. Extraction and quantitative measurement of cyclic AMP was carried out according to previously published methods [31--33] and has been described by these authors before [30]. All cyclic AMP experiments were carried out in triplicate and three measurements were made on each replicate. Each value reported in this paper is the mean of 9 observations. The standard deviations were always less than 10% of the respective means. Measurement of corticosterone. Corticosterone was measured by the sulfuric acid fluorescence m e t h o d described by Silber et al. [34]. Each corticosterone experiment was carried out in duplicate and the average values are reported in the paper. The duplicate determinations were always found to be within + 5% of the respective means. Results
Effects o f enterotoxin and ACTH on cyclic AMP and corticosterone production Fig. 1 shows the time course of corticosterone formation from endogenous precursors in the presence of different concentrations of enterotoxin; in the absence of enterotoxin, no corticosterone synthesis is detectable (< 0.05 nmol/10 s cells) even at 5 h. It is clear that there is a delay or lag period before corticosterone production is observed. The duration of this lag period, however, is dependent upon the concentration of enterotoxin used. With maximally stimulating doses of enterotoxin, i.e. 1 pg/ml, an increase in corticosterone production is evident at 1 h whereas with 1 ng/ml a significant elevation in corticosterone is n o t observed until 3 h. Fig. 2(A) shows the time course of corticosterone formation in the presence of maximally stimulating concentrations of either enterotoxin or ACTH. With ACTH, the m a x i m u m rate of corticosterone production is established within the first 15 min of incubation, a finding in agreement with earlier published reports [35,36]. On the other hand, no corticosterone synthesis is detectable for the first 30 min when enterotoxin is added and maximum rates of production are not established until 45--60 min of incubation. It is to be noted, however, that once established, the m a x i m u m rate of corticosterone production with enterotoxin is similar to that with ACTH.
311
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Fig. 1. T i m e c o u r s e o f e n t e r o t o x i n - s t i m u l a t e d s y n t h e s i s of c o r t i c o s t e r o n e b y t h e isolated a d r e n a l cells, In the a b s e n c e o f e n t e r o t o x i n t h e r e was negligible ( < 0 . 0 5 n m o l / 1 0 s cells) s y n t h e s i s of c o r t i c o s t e r o n e e v e n ~t5h. 450
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Fig. 2, C o m p a r i s o n o f c o r t i c o s t e r o n e a n d cyclic AMP s y n t h e s i s in t h e p r e s e n c e of m a x i m a l l y s t i m u l a t i n g a m o u n t s of A C T H ( e ) a n d e n t e r o t o x i n (A). In Fig. 2 ( A ) t h e c o n c e n t r a t i o n s of A C T H a n d e n t e r o t o x i n w e r e 5 nM a n d 1 ~ g / m l r e s p e c t i v e l y w h e r e a s in 2(B) t h e y w e r e 50 nM a n d 3 ~ g / m l r e s p e c t i v e l y .
312
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ENTEROTOXIN F i g . 3. L o g d o s e - r e s p o n s e
curves of enterotoxin
100
4OO
1600
i
3200
NANOGRAMS/ML for corticosterone
and cyclic AMP synthesis. Incubations
w e r e c a r r i e d o u t :for 2 h.
Fig. 2(B) shows the time course of cyclic AMP form at i on in the presence of supra-maximally stimulating levels of ACTH (50 nM) or e n t e r o t o x i n (3 pg/ml). The increase in cyclic AMP f or m at i on in response to ACTH is immediate whereas the response to e n t e r o t o x i n is observed only after 30 min. Maxim u m rates o f p r o d u c t i o n with the latter are not established until after 60 min of incubation. In contrast to the corticosterone results, the m a x i m u m rate of cyclic AMP f o r mat i on with e n t e r o t o x i n is only about 8--10% of that observed with ACTH. This difference in m a x i m u m rates was observed even when the experiments were carried in the presence o f 8 mM theophylline. Fig. 3 shows the dose-response curves of e n t e r o t o x i n for corticosterone and cyclic AMP production. Maximum p r o d u c t i o n of both cyclic AMP and corticosterone is obtained at a c o n c e n t r a t i o n of a b o u t 1 #g/ml. This is in contrast with ACTH, where m a x i m u m p r o d u c t i o n of corticosterone is observed at much smaller concentrations than those producing m a x i m u m amounts of cyclic AMP [ 3 5 , 3 6 ] . Experiments were carried out to see if the effects of ACTH and enterotoxin were additive. Table I shows that in the presence of maximally stimulating levels o f ACTH, addition of e n t e r o t o x i n did n o t produce any further increase in co r tico s t er one production. In the presence of submaximally stimulating co n cen tr ati ons of ACTH, e n t e r o t o x i n produced a further increase in corticosterone synthesis, bringing it to the levels obtained with maximally stimulating c o n c e n t r a t i o n s of ACTH.
313 TABLE I EFFECT OF ACTH AND ENTEROTOXIN
ON CORTICOSTERONE
Additions
ACTH ACTH ACTH Toxin Toxin
PRODUCTION
C o r t i c o s t e r o n e f o r m e d ( n m o l / 1 0 5 cells)
(2 n M ) (2 n M ) + t o x i n ( 1 0 0 n g / m l ) (2 n M ) + t o x i n (1 p g / m l ) (100 ng/ml) (1 p g / m l )
A C T H (0.5 nM) A C T H (0.5 n M ) + t o x i n (1 p g / m l )
lh
2h
4h
2.0 1.9 2.1 0.5 0.7
4.2 4.2 4.3 1.4 2.9
6.8 6.9 6.7 5.2 7.1
1.6 1.9
3.2 4.1
4.4 6.9
Fig. 4 shows the steriodogenic effect of ACTH in cells pretreated with enterotoxin. Pretreatment of the cells with enterotoxin has essentially no effect on their subsequent response to maximally stimulating concentrations of ACTH.
Effect of cycloheximide Table II shows that cycloheximide inhibits the corticosterone synthesis stimulated by both ACTH and enterotoxin. The extent of inhibition is dependent on the concentration of cycloheximide and at each tested concentration of the inhibitor it is similar for the two stimulants.
0 ~UO 1.6
1.2
0.8 0 ~ 0.4 0 U
ACTH
./
/
30
45
60
I,-
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0
,
TOXIN
TIME
90
,
120
MINUTES
Fig. 4. E f f e c t o f p r e t r e a t r a e n t o f a d r e n a l cells w i t h e n t e r o t o x i n T h e cells w e r e i n c u b a t e d w i t h or w i t h o u t 1 /~g/ml e n t e r o t o x i n c o r t i c o s t e r o n c s y n t h e s i s f o l l o w e d f o r t h e n e x t 9 0 rain. • at 3 0 r a i n ; o o, e n t e r o t o x i n a d d e d at 0 t i m e , A C T H at 3 0 t i m e , A C T H a t 3 0 rain.
o n t h e i r s u b s e q u e n t r e s p o n s e to A C T H . f o r 3 0 rain, 5 n M A C T H w a s a d d e d a n d "-, e n t e r o t o x i n a d d e d at 0 t i m e , n o A C T H rain~ • • , no e n t e r o t o x i n a d d e d a t 0
314 T A B L E II INHIBITION HEXIMIDE
OF
ACTH
AND
ENTEROTOXIN-STIMULATED
Additions
Corticosterone formed (nmol/105 c e l l s p e r 2 h)
A C T H 5 nM + Cycloheximide ( 1 pM) + Cycloheximide ( 3 #M) + Cycloheximide (10 pM)
3.0 2.0 1.2 0.5
33.3 60.0 83.3
Toxin 1 #g/ml + C y c l o h e x i m i d e ( 1 pM) + Cycloheximide ( 3 pM) + C y c l o h e x i m i d e (10 pM)
2.1 1.5 0.8 0.3
28.6 61.9 85.7
STEROIDOGENESIS
BY
CYCLO-
% Inhibition d u e to cycloheximide
Ca ~÷ d e p e n d e n c e
Since the role of Ca 2÷ in the steroidogenic effect of ACTH is well known [37,38], the importance of this ion in enterotoxin-stimulated steroidogenesis was also investigated. Fig. 5 shows that at all time intervals studied, Ca 2÷ increased and EGTA decreased the a m o u n t of corticosterone synthesized by the cells in response to enterotoxin. Table III shows that although the steroidogenic response to ACTH is completely inhibited by 100 pM EGTA, the effects of enterotoxin and dibutyryl cyclic AMP are only partially inhibited (67% and 51% respectively) with as much as 1 mM EGTA. Addition of Ca 2÷ produces a concentration-dependent
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Fig. 5. T h e e f f e c t o f Ca 2+ a n d E G T A on t h e c o r t i c o s t e r o n e r e s p o n s e o f t h e a d r e n a l cells to e n t e r o t o x i n . Cells w e r e p r e p a r e d in r e g u l a r B u f f e r A, c e n t r i f u g e d a n d t h e p e l l e t s s u s p e n d e d in B u f f e r A c o n t a i n i n g t h e u s u a l a m o u n t s o f b o v i n e s e r u m a l b u m i n a n d t r y p s i n i n h i b i t o r b u t w i t h o u t Ca 2+. I n c u b a t i o n s w e r e t h e n c a r r i e d o u t in t h e p r e s e n c e or a b s e n c e o f 2.5 m M Ca 2+ or a f t e r t h e a d d i t i o n o f 1 m M E G T A .
315 TABLE III C O M P A R I S O N O F T H E Ca 2+ R E Q U I R E M E N T F O R T H E S T I M U L A T I O N O F C O R T I C O S T E R O N E F O R M A T I O N BY E N T E R O T O X I N , A C T H A N D D I B U T Y R Y L C Y C L I C AMP Cells w e r e p r e p a r e d in B u f f e r A as usual. A f t e r c e n t r i f u g a t i o n t h e y w e r e r e s u s p e n d e d in B u f f e r A w i t h o u t Ca 2+ a n d i n c u b a t e d w i t h v a r y i n g a m o u n t s of Ca 2+ or E G T A , as s h o w n in t h e table, for 2 h at 37 ° C. C o r t i c o s t e r o n e f o r m e d ( n m o l / 1 0 5 cells p e r 2 h)
Additions (uM)
N o n e (no Ca 2+) EGTA 10 30 100 300 1000
Toxin (1 ~ g / m l )
ACTH (5 nM)
Dibutyryl cyclic AMP (0.5 m M )
1.5
1.8
2.3
1.5 1.2 0.5 0.5 0.5
1.3 0.6 < 0.05 < 0.05 < 0.05
2.2 1.8 1.1 1.1 1.1
Ca 2+ 100 300 1000 3000
2.2 2.5 2.8 2.9
2.7 3.3 3.7 3.7
2.5 3.0 3.4 3.2
increase in the effects of all three stimulants with m a x i m u m increase being observed with about 1 mM concentration of the cation. At this concentration of Ca 2÷ the increases in the effects of enterotoxin, ACTH and dibutyryl cyclic AMP were 90, 106 and 48 percent respectively. Table IV shows that omission o f Ca 2÷ from the incubation medium has a more pronounced effect on cyclic AMP formation in response to ACTH than that in response to enterotoxin; the former is reduced by approx. 90% whereas the latter by only about 50%. Furthermore, 1 mM EGTA almost completely abolishes the effect of ACTH although it produces only a partial reduction in the a m o u n t of cyclic AMP produced in response to enterotoxin.
Effect of choleragenoid Table V shows that choleragenoid effectively inhibited the steroidogenic
TABLE IV Ca 2+ D E P E N D E N C E O F E N T E R O T O X I N - A N D A C T H - S T I M U L A T E D C Y C L I C AMP S Y N T H E S I S I n c u b a t i o n c o n d i t i o n s as in T a b l e I I I Additions
2.5 m M Ca 2+ N o Ca 2+ 1 mM EGTA
Cyclic AMP f o r m e d ( p m o l / 1 0 s cells p e r 2 h ) Control
ACTH (50 n M )
Toxin (1 p g / m l )
Toxin (5 p g / m l )
3.9 3.5 3.9
206.7 24.2 4.9
27.7 13.4 9.8
27.6 16.5 12.1
316
TABLE V EFFECT OF CHOLERAGENOID ON THE STIMULATION E N T E R O T O X I N A N D DIBUTYI=tYL C Y C L I C AMP
OF CORTICOSTERONE
S Y N T H E S I S BY
T h e cells w e r e i n c u b a t e d w i t h c h o l e r a g e n o i d f o r 30 m i n at 37°C. D i b u t y r y l c y c l i c A M P or e n t e r o t o x i n w e r e t h e n a d d e d a n d t h e i n c u b a t i o n c o n t i n u e d f o r 2 h. T h e v a l u e s r e p o r t e d in the table s h o w the a m o u n t o f c o r t i c o s t e r o n e f o r m e d d u r i n g the 2 h i n c u b a t i o n a f t e r the a d d i t i o n o f the s t i m u l a t i n g a g e n t s . Cholerag e n o i d b y i t s e l f h a d n o e f f e c t on t h e n e g l i g i b l e c o r t i c o s t e r o n e s y n t h e s i s ( < 0 . 0 5 n m o l / 1 0 5 cells) in the absence of the s t i m u l a t i n g agents. Choleragenoid added (ng/ml)
C o r t i c o s t e r o n e f o r m e d ( n m o l / 1 0 S cells p e r 2 h) Enterotoxin (1 p g / m l )
Dibutyryl cyclic A M P (1 p m o l / m l )
0 100 300 1000 3000
1.2 1.1 1.0 0.5 0.2
3.0 2.8 2.9 2.7 2.8
reponse to enterotoxin. The inhibition by choleragenoid does not appear to be due to effect(s) on the intracellular reactions involved in steroidogenesis because it has no effect on the dibutyryl cyclic AMP-stimulated corticosterone synthesis. Table VI shows that the concentration of choleragenoid which almost completely inhibits the effects of l p g / m l enterotoxin has virtually no effect on the stimulation of corticosterone synthesis observed with various concentrations of ACTH.
Effect of gangliosides Recently it was shown that incubation of enterotoxin with gangliosides resulted in a loss of activity of the toxin [15,40,41] which was attributed to its incapacity to bind to tissues [42]. Fig. 6 shows the effects of exogenously
T A B L E VI EFFECT OF CHOLERAGENOID ACTH
ON T H E S T I M U L A T I O N
OF CORTICOSTERONE
S Y N T H E S I S BY
Cells w e r e i n c u b a t e d w i t h 3 ~tg/ml c h o l e r a g e n o i d at 3 7 ° C f o r 30 rain, A C T H w a s a d d e d as i n d i c a t e d in t h e t a b l e a n d i n c u b a t i o n c o n t i n u e d f o r 1 2 0 rain. T h e v a l u e s s h o w t h e a m o u n t s o f c o r t i c o s t e r o n e f o r m e d a f t e r t h e a d d i t i o n o f A C T H . C h o l e r a g e n o i d h a d n o e f f e c t on the n e g l i g i b l e c o r t i c o s t e r o n e s y n t h e s i s ( ~ 0 . 0 5 n m o l / 1 0 5 cells) in t h e a b s e n c e o f A C T H . ACTH added (nM)
- Choleragenoid
+ Choleragenoid
0.05 0.1 0.2 0.4 0.8 1.6
0.8 1.6 1.8 2.5 2.8 2.7
0.8 1.5 1.6 2.7 2.9 2.6
C o r t i c o s t e r o n e f o r m e d ( n m o l / 1 0 5 cells p e r 2 h)
317
!
8o
60
o 4O
a ~ o I
3 GANGLIOSlDE$
10
30
10~
MICROGRAMS/ML
Fig. 6. E f f e c t of e x o g e n o u s l y a d d e d gangliosides o n the s t i m u l a t i o n o f c o r t i c o s t e r o n e s y n t h e s i s b y A C T H a n d e n t e r o t o x i n . G a n g l i o s i d e s w e r e i n c u b a t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s of e n t e r o t o x i n or A C T H at 4°C for 20 rain in a 0,5 m l v o l u m e , I m l o f t h e cell s u s p e n s i o n was a d d e d a n d the i n c u b a t i o n c a r r i e d o u t for 2 h at 3 7 ° C . T h e final c o n c e n t r a t i o n s of e n t e r o t o x i n w e r e 1, 3 a n d 10 ~zg/ml ( o ~ ~) a n d A C T H 0.1, 0.4 a n d 1.6 nM ( e A e). T h e c o n c e n t r a t i o n s of gangliosides given in t h e figure are t h e final c o n c e n t r a tions. Values o b t a i n e d in t h e a b s e n c e of gangliosides are e x p r e s s e d as c o n t r o l ( 1 0 0 % ) a n d t h o s e o b t a i n e d in t h e p r e s e n c e o f gangliosides are e x p r e s s e d as p e r c e n t of t h e i r r e s p e c t i v e c o n t r o l s .
added gangliosides on the stimulation of corticosterone synthesis by enterotoxin and ACTH. At all levels of enterotoxin tested, the stimulatory effect was completely abolished when the gangliosides were present in a 3-fold excess on a weight basis. On the other hand gangliosides, even at high concentrations, had only a marginal effect on the ACTH-stimulated corticosterone production. Table VII shows the concentration-dependent inhibition of enterotoxinstimulated cyclic AMP formation by gangliosides. Again, the effect of ACTH is only marginally inhibited by 100 pg/ml gangliosides while lower concentrations show almost no effect.
TABLE VII EFFECT OF GANGLIOSIDES FORMATION
ON T H E E N T E R O T O X I N
A N D A C T H - S T I M U L A T E D C Y C L I C AMP
T r e a t m e n t w i t h gangliosides a n d t h e i n c u b a t i o n c o n d i t i o n s w e r e the s a m e as t h o s e d e s c r i b e d in Fig. 6. Gangliosides (ug/ml)
0 3 1O 30 100
Cyclic AMP f o r m e d ( p m o l / 1 0 s cells/2 h ) Control
Enterotoxin (3 ~zg/ml)
ACTH (5 n M )
1.7 -1.6 -1.8
25.'/ 17.4 8.6 5.0 3.1
62.2 56.8 57.9 60.8 51.8
318
Effect o f neuraminidase-treatment o f adrenal cells on their response to enterotoxin Recently it was shown that after treatment with neuraminidase, adrenal cells became less sensitive to ACTH both in terms of cyclic AMP and corticosterone production [29,30]. Fig. 7(A) shows that unlike the results obtained with ACTH the neuraminidase-treated cells produce more corticosterone than untreated cells in response to enterotoxin. The maximum rates in this experiment, obtained between 60--120 rain, are 2.12 and 1.75 nmol/105 cells/h for the treated and untreated cells respectively. Extrapolation of the curves shows that increase in the a m o u n t of corticosterone produced by the treated cells may have also been due to shortening of the lag period by 6--7 min. Fig. 7(B) shows that neuraminidase-treated cells also produce more cyclic AMP than the untreated cells. There is an increase of approx. 20% in the maximum rate (calculated between 60--120 min) as well as a shortening of the lag period by about 10 min in the neuraminidase-treated cells. Fig. 8(A) shows that after neuraminidase treatment the cells become more sensitive to enterotoxin. Thus, in a 2 h incubation reported here, no corticosterone synthesis was detected in the control cells with 1 or 3 ng/ml entero-
(A)
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F i g . 7. T h e e f f e c t o f n e u r a m i n i d a s e t r e a t m e n t o f a d r e n a l cells o n t h e i r c o r t i s t e r o n e ( A ) a n d c y c l i c A M P (B) response to enterotoxin. The ceils were suspended in 0.01 M phosphate buffer (pH 6.5) containing 0.85% NaC1, 0.5% bovine serum albumin and 0.1% trypsin inhibitor and divided into two groups. Both groups were incubated at 37°C for 15 rain, neuraminidase (20 munits/ml) was added to one group and the incubation continued for 30 rain. After centrifugation, the cells in both groups were washed with 0.01 M phosphate b u f f e r c o n t a i n i n g 0 . 8 5 % NaC1 ( 5 t i m e s o r i g i n a l i n c u b a t i o n v o l u m e ) , c e n t r i f u g e d a n d t h e n s u s p e n d e d in B u f f e r A c o n t a i n i n g b o v i n e s e r u m a l b u m i n a n d t r y p s i n i n h i b i t o r f o r t h e f i n a l i n c u b a t i o n . The concentration of enterotoxin in both experiments was 3 ~g/ml. Control cells (e ~-); n e u r a m i n i d a s e - t r e a t e d cells (A A).
319
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toxin although neuraminidase-treated cells produced significant amounts of the steroid (0.16 and 0.31 nmol/10 s cells) in response to these two concentrations of enterotoxin. The concentration of enterotoxin required to give halfm a x i m u m amounts of corticosterone in the neuraminidase-treated cells was about 1/3 of that required in the control cells. Fig. 8(B) shows that the m a x i m u m a m o u n t of cyclic AMP formed by neuraminidase-treated cells in response to enterotoxin is about twice that formed by control cells and the concentration of enterotoxin required to produce half-maximum amounts of cyclic AMP is reduced considerably in the neuraminidase-treated cells. Discussion Recent reports from a number of laboratories have indicated that most, if not all, of the biological effects of cholera enterotoxin in the cells of the intestine as well as other organs are mediated via cyclic AMP (See 'Introduction' for references). Cholera enterotoxin, therefore, is potentially useful for investigating the regulation of adenyl cyclase in cell membranes and for clarifying the mechanism of action of a variety of hormones which exert their effects through manipulation of cyclic AMP levels. In the present study we have compared the characteristics of ACTH-stimulated cyclic AMP and corticosterone production with those of enterotoxin to determine if there are any c o m m o n components or "overlapping receptor d o m a i n s " [27] for the two stimuli. A major similarity between the effects of ACTH and enterotoxin is that the m a x i m u m rates of corticosterone production are the same for the two
320 stimuli. F u r t h e r m o r e , m a x i m u m doses of ACTH and e n t e r o t o x i n were not additive suggesting t hat the steroidogenic pathway stimulated by the two agents was the same. The response of adrenal cells to ent erot oxi n, however, exhibits certain characteristics which are different from those observed with ACTH. These centered initially on the differences in the temporal response to stimulation and in the discrepancies in the m a x i m u m rates of cyclic AMP production. F u r ther differences between e n t e r o t o x i n and ACTH were subsequently elaborated by manipulation of cell m em brane sialic acid with neuraminidase and by changing the ionic envi r onm ent by means of EGTA. A delayed response or lag period is a characteristic of the e n t e r o t o x i n response in a n u m b e r of systems [4,7,9,10,14] and was also observed in the adrenal cells. With maximally stimulating levels of the toxin, no cyclic AMP or corticosterone synthesis was detected up to 30--45 min and m axi m um rates were established only after 60 min of incubation. It should be not ed that although the m a x i m u m rates of corticosterone product i on were similar for ACTH and e n t e r o t o x i n , those of cyclic AMP p r o d u c t i o n were not. Thus, the m a x i m u m rate o f cyclic AMP p r o d u c t i o n with e n t e r o t o x i n was only about 8--10% o f that with ACTH. These results support the suggestion of others [43,44] that all of the cyclic AMP pr oduced in the adrenal cells in response to ACTH is n o t required for obtaining maximal rates of corticosterone production. The Ca 2÷ r equi r e m ent for the steroidogenic response of ACTH has been known for man y years [ 3 7 , 3 8 ] . Recently, it was shown that this requi rem ent is greater for some event(s) occurring at the plasma m em brane leading to increased cyclic AMP p r o d u c t i o n than for the reactions involved in the stimulation o f steroidogenesis by cyclic AMP [ 4 5 - - 4 7 ] . A Ca 2+ requi rem ent for the effect of e n t e r o t o x i n on fat cells was shown by Vaughan et al. [ 1 4 ] . In this report we have shown that although Ca 2÷ is also required for the effect of e n t e r o t o x i n on adrenal cells, this r e qui r em ent is not the same as that for ACTH. Thus, EGTA abolished the effect of ACTH on corticosterone synthesis completely but caused only a partial reduction in the effect of ent erot oxi n. Omission o f Ca 2÷ from the incubation medium reduced the a m o u n t of cyclic AMP p r o d u ced in response to ACTH to a b o u t 10% of that produced in the presence of 2.5 mM Ca 2÷. On the other hand omission of Ca 2÷ produced only about 40% decrease in the cyclic AMP response of ent erot oxi n. EGTA, which almost co mp letel y abolished ACTH-stimulated cyclic AMP p r o d u c t i o n again had only a partial effect on the e n t e r o t o x i n response. Two possibilities may be considered to explain these differences. First, e n t e r o t o x i n may stimulate adenyl cyclase by the two distinct mechanisms; one d e p e n d e n t on Ca 2÷, the o t h er not. If this is the case then there is a possibility that some c o m p o n e n t of the f o r m e r mechanism may overlap with c o m p o n e n t s involved in ACTH stimulation of adenyl cyclase. The second possibility is that EGTA does not remove all m e m b r a n e - b o u n d Ca 2÷. If this is true then it follows that the Ca ~÷ involved in the effect o f ACTH is in a separate c o m p a r t m e n t from that involved in the effect o f e n t e r o t o x i n . We have not a t t e m p t e d to distinguish between the two possibilities at this time. Cuatrecasas [39] has shown recently that choleragenoid, a protein immunologically identical with the cholera e n t e r o t o x i n but lacking in biologi-
321 cal activity [1,5,14], can inhibit the lipolytic effect of the latter in fat cells by competing for the receptor. Our results with adrenal cells support their findings. The localization of choleragenoid action at the plasma membrane is supported by the finding that it did not alter the corticosterone response to dibutyryl cyclic AMP. The choleragenoid was also w i t h o u t effect on the doseresponse curves of ACTH which makes it unlikely that inhibition of the toxin response was due to a non-specific interaction with the cell membrane. Several investigators have shown that the effects of enterotoxin can be abolished by incubating the toxin with gangliosides [15,40,41]. The loss in the activity of enterotoxin is attributed to the inability of the toxin/ganglioside complex to bind to receptors in the target tissue [42]. The present report demonstrates that exposure of enterotoxin to gangliosides prevents the effects of the former on cyclic AMP and corticosterone synthesis in normal adrenal cells, results which are similar to those obtained by Wolff et al. [27] using cultured mouse adrenal t u m o r cells. Further, we have shown that gangliosides, at concentrations that abolished the effects of enterotoxin, did not prevent the stimulation of cyclic AMP or corticosterone synthesis by ACTH. At high concentrations (around 100 pg/ml) the gangliosides did have a marginal inhibitory effect on these parameters; however, this may be a non-specific effect of these charged molecules on the cell because at such concentrations the stimulation of corticosterone synthesis by dibutyryl cyclic AMP was also inhibited by about 15--20%. The most significant difference between ACTH- and enterotoxin stimulation of adrenal cells was observed after treatment of the cells with neuraminidase. In earlier reports we showed that neuraminidase treatment of the cells reduced the sensitivity to ACTH in terms of both cyclic AMP [ 30] and corticosterone production [29]. Since the steroidogenic response of the cells to dibutyryl cyclic AMP was not affected by neuraminidase treatment [29] it was concluded that removal of sialic acid by neuraminidase resulted in an alteration of some membrane component(s) involved in the stimulation of adenyl cyclase by ACTH. In contrast to the results obtained with ACTH, adrenal cells became more sensitive to enterotoxin after treatment with neuraminidase under conditions identical to those used in the ACTH work [29,30]. Thus, neuraminidasetreated cells produced more cyclic AMP and corticosterone than untreated cells in response to enterotoxin. The increased response of the neuraminidasetreated cells at a given time interval seems mainly to be due to an increased rate of synthesis of cyclic AMP (or corticosterone) although shortening of the lag period may also be involved. A comparison of the dose-response curves for control and treated cells showed that the concentrations of enterotoxin required to give half-maximum production of cyclic AMP (or corticosterone) were substantially reduced after neuraminidase treatment. The reason for increased sensitivity of the cells after neuraminidase treatment is not clear at this time. It is possible that removal of membrane sialic acid results in exposure of more enterotoxin receptors on the cell surface or in a reduction of non-specific binding of enterotoxin to sialic acid-containing glycoproteins. Indeed, several glycoproteins have been shown to be capable of binding enterotoxin [48]. Alternatively, neuraminidase treatment may have increased the binding capacity of an otherwise less active or inactive molecule,
322
This last alternative is supported, in part, by recent studies which indicate that the biological receptor for enterotoxin may be a ganglioside [15,40,41,42,48]. King and van Heyningen [41] showed that in vitro stimulation of intestinal adenyl cyclase by enterotoxin was abolished if the toxin was added after pretreatment with neuraminidase-resistant monosialosylganglioside. The neuraminidase-sensitive di- and trisialosylgangliosides were not effective in their system although treatment of these gangliosides with neuraminidase did render them capable of neutralizing the toxin, presumably by converting them to neuraminidase-resistant monosialosylganglioside. Among the gangliosides that have been shown by Cuatrecasas [48] to inhibit the binding of enterotoxin to liver membranes, neuraminidase-resistant monosialosylganglioside was found to be the most effective. In the same study it was also found that liver membranes after treatment with neuraminidase exhibited a slight enhancement in the binding of enterotoxin. Therefore, it is possible that the complex membrane gangliosides in the intact adrenal cells were converted by neuraminidase into neuraminidase-resistant monosialosylganglioside and as a result of this change the cells became more sensitive to enterotoxin [49]. Acknowledgments The authors would like to express their gratitude to: Dr. R.A. Finkelstein, South Western Medical School, University of Texas, for kindly supplying the enterotoxin and choleragenoid; Dr Carl E. Miller, Geographic Medicine Branch, NIAID for additional supplies of enterotoxin, and Dr C.H. Li, University of California for ~s-ACTH. We would also like to acknowledge the excellent technical assistance provided by William F. Robidoux, Jr, Susan R. Levy and George Gagnon. This research was supported by grant No. AM 04899 from the National Institutes of Arthritis, Metabolism and Digestive Diseases. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
Finkelstein, R.A. and LoSpalluto, J.J. (1969) J. Exp. Med. 130, 185--202 LoSpalluto, J.J. and Finkelstein, R.A. (1972) Biochim. Biophys. Acta 257, 158--166 Burrows, W, (1968) Annu. Rev. Microbiol. 22, 245--268 Pierce, N.F. Greenough III, W.B. and Carpenter, C.C. (1971) Bacteriol. Rev. 35, 1--13 Finkelstein, R.A. and LoSpoUuto, J.J. (1970) J. Infect. Dis. 121, $63--$72 Richardson, S.H. and Noftle, K,A. (1970) J. Infect. Dis. 121, $73--$79 Field, M., Plotkin, G.R. and Silen, W. (1968) Nature 2 1 7 , 4 6 9 - - 4 7 1 Schafer, D.E., Lust, W.D., Sircar, B. and Goldberg, N.D. (1970) Proc. Natl. Acad. Sci. U.S. 67, 851--856 Sharp, G.W.G. and Hynie, S. (1971) Nature 229, 266--269 Kimberg, D.B., Field, M., Johnson, J., Henderson, A. and Gershon, E. (1971) J, Clin. Invest. 50, 1218--1231 Pierce, N.F., Carpenter, Jr, C.C., Elliot, H.L. and Greenough, W.B. (1971) Gastroenterology 60, 22--32 Gurrant, R.L., Chen, L.C. and Sharp, G.W.G. (1972) J. Infect. Dis. 125, 377--381 Greenough, III, W.B., Pierce, N.F. and Vaughan, M. (1970) J. Infect. Dis. 121, S l 1 1 - - $ 1 1 3 Vaughan, M., Pierce, N.F. and Greenough, III, W.B. (1970) Nature 226, 658--659 van Heyningen, W.E., Carpenter, W.B., Pierce, N.F. and Greenough, III, W.B., (1971) J. Infect. Dis. 124, 415--418 Evans, Jr, D.J., Chen, L.C., Curlin, G.T. and Evans, D.G. (1972) Nat. New Biol. 236, 137--138 Cuatrecasas, P. (1973) Biochemistry 12, 3567--3577 Zieve, P.D., Pierce, N.F. and Greenough, W.B. (1971) Joh n H opki ns Med. J. 129, 299--303
323
19 Gorman, R.E. and Bitensky, M.W. (1972) Nature 235, 4 3 9 - - 4 4 0 20 Baker, A.L., Kaplan, M.D., Kimberg, D.V. and Pierce, N.F. (1971) Gastroenterology 60, 739 21 Strnm, T.B., Deisseroth, A., Morganroth, J., C~Lrpenter, C.B. and Merrill, J.B. (1972) Proe. Natl. Acad. Sci. U.S. 69, 2 995--2999 22 Bourne, H.R. (1973) Nature 241, 399 23 Lichtenstein, L.M., Henney, C.S., Bourne, H.R. and Greenough, W.B. (1973) J. Clin. Invest. 52, 691--697 24 Bourne, H.R., Lehrer, R.I., Lichtenstein, L.M., Weissrnann, G. and Zurier, R. (1973) J. Clin. Invest. 52, 6 98--708 25 Mashiter, K., Mashiter, G.D., Hauger, R.L. and Field, J.B. (1973) Endoc ri nol ogy 92, 541--549 26 Donta, S.T., King, M. and Sloper, K. (1973) Nat. New Biol. 243, 246--247 27 Wolff, J., Temple, R. and Cook, G.H. (1973) Proc. Natl. Acad. Sci. U.S. 70, 2741--2744 28 Haksar, A. and P~ron, F.G. (1972) J. Steroid Biochem. 3, 847--857 29 Haksar, A., Baniukiewicz, S. and P~ron, F.G. (197"3) Biochem. Biophys. Res. Commun. 52, 959--966 30 Haksar, A., Maudsley, D.V., Kimmel, G.L. and P~ron, F.G. (1974) Biochim. Biophys. Acta 362, 356--365 31 Brown, B.L., Ekins, R.P. and Albano, J.D.M. (1972) Adv. Cyclic Nueleotide Res. 2, 25--40 32 Albano, J.D.M., Barnes, G.D., Maudsley, D.V., Brown, B.L. and Ekins, R.P. (1974) Anal. Biochern. 60, 130--141 33 Brown, B.L., Albano, J.D.M., Ekins, R.P., Sgherzi, A.M. and Tampion, W. (1971) Biochem. J. 121, 561--562 34 Silber, R.H., Busch, R.D. and Oslapas, R. (1958) Clin. Chem. 4, 278--285 35 Mackie, C., Richardson, M.C. and Sehulster, D. (1972) FEBS Lett. 23, 345--348 36 Beall, R.J. and Sayers, G. (1972) Arch. Biochern. Biophys. 148, 70--76 37 Birmingham, M.K., Elliot, F.H. and Valero, H.L.P. (1953) Endocrinology 53, 687--689 38 Peron, F.G. and Koritz, S.B. (1958) J. Biol. Chem. 233, 256--259 39 Cuatrecasas, P. (1973) Biochemistry 12, 3577--3581 40 Holrngren, J., L6nnroth, J. and Svennerholrn, L. (1973) Infect. lmrnun. 8, 208--214 41 King, C.A. and van Heyningen, W.E. (1973) J. Infect. Dis. 127. 639--647 42 Cuatrecasas, P. (1973) Biochemistry 12, 3558--3566 43 Seelig, S. and Sayers, G. (1973) Arch. Biochem. Biophys. 154, 230--239 44 Moyle, W.R., Kong, Y.C. and Ramachandran, J. (1973) J. Biol. Chem. 248, 2409--2417 45 Sayers, G., BeaD, R.J. and Seelig, S. (1972) Science 175, 1131--1133 46 Haksar, A. and P~ron, F.G. (1972) Biochem. Biophys. Res. Commun. 47, 445--450 47 Haksar, A. and P~ron, F.G. (1973) Biochim. Biophys. Acta 313, 363--371 48 Cuatrecasas, P. (1973) Biochemistry 12, 3547--3557 49 Haksar, A., Maudsley, D.V. and P~ron, F.G. (1974) Nature 251, 514--515
Biochimica et Biophysica Acta, 381 (1975) 308--323 © Elsevier Scientific Publishing Company, Amsterdam -- Printed in The Netherlands
BBA 27593
STIMULATION O F C Y C L I C ADENOSINE 3':5'-MONOPHOSPHATE AND CORTICOSTERONE FORMATION IN ISOLATED RAT A D R E N A L CELLS BY CHOLERA ENTEROTOXIN COMPARISON WITH THE EFFECTS OF ACTH
AJAI HAKSAR, DAVID V. MAUDSLEY and FERNAND G. PI~RON
Worcester Foundation for Experimental Biology, Shrewsbury, Mass. 01545 (U.S.A.) (Received July 22nd, 1974)
Summary 1. The production of cyclic adenosine 3':5'-monophosphate (cyclic AMP) and corticosterone in isolated rat adrenal cells was increased by cholera enterotoxin. Both responses were accompanied by a lag period which is characteristic of other known actions of enterotoxin. The duration of the lag period in the production of corticosterone depended on the concentration of enterotoxin; with the maximally stimulating amounts it was ~30--45 min. 2. Maximum rates of cyclic AMP and corticosterone synthesis, after the lag period, were constant for at least 1 h. Although the m a x i m u m rate of corticosterone formation was the same as that obtained with adrenocorticotropic hormone, the m a x i m u m rate of cyclic AMP formation was only 8--10% of that with adrenocorticotropic hormone. 3. Pretreatment of the cells with enterotoxin had no effect on their subsequent steroidogenic response to maximally stimulating amounts of adrenocorticotropic hormone. 4. Cycloheximide inhibited the effect of both enterotoxin and adrenocorticotropic h o r m o n e on corticosterone production. 5. Enterotoxin stimulation of both cyclic AMP and corticosterone formation was dependent on the presence of Ca 2÷ in the medium although the Ca 2÷ requirement was not same as that for adrenocorticotropic hormone. Thus, EGTA at concentrations which completely abolished the effect of adrenocorticotropic h o r m o n e caused only a partial reduction in the effects of enterotoxin. 6. Exogenously added choleragenoid and gangliosides abolished the effects of enterotoxin w i t h o u t having any significant effect on the response of the cells to adrenocorticotropic hormone. 7. After treatment with neuraminidase, the adrenal cells showed an increased response to enterotoxin in terms of both cyclic AMP and cortico-
309
sterone formation which was due to a combination of two effects: (a) increased rate of synthesis of both compounds and (b) shortening of the characteristic lag period. This is in sharp contrast to the results obtained with adrenocorticotropic hormone where neuraminidase-treatment made the cells less sensitive to adrenocorticotropic hormone.
Introduction The active principle of Vibrio cholerae, which produces diarrhea in clinical cholera, has been identified as a specific exotoxin [ 1--4]. The exotoxin, also called enterotoxin and choleragen, has been purified and chemically characterized [1,2,5,6]. Much evidence has accumulated indicating that most, if not all, of the effects of the enterotoxin on the intestinal cells are mediated via stimulation of intestinal adenyl cyclase [7--12]. In addition to the effects on the intestinal cells, enterotoxin has also been shown to increase adenyl cyclase activity, and consequently the biochemical processes dependent on cyclic AMP, in a number of other tissues such as fat cells [13--17], platelets [18], liver [19,20], leukocytes [21--24], thyroid [25] and cultured adrenal tumor cells [26,27]. In this respect, the actions of enterotoxin are similar to those hormones which exert their effects through regulation of intracellular cyclic AMP production. Enterotoxin, therefore, is potentially useful in elaborating the role of various membrane components involved in the regulation of cyclic AMP levels in the tissues. In the present study we have compared the effects of enterotoxin and ACTH on cyclic AMP and corticosterone production in isolated rat adrenal cells to determine the points of congruence between the two stimuli. Materials Trypsin (TRSF-IGA 150 units/mg) and lima bean trypsin inhibitor were obtained from Worthington Biochemical Corporation; collagenase (Serva, 387 Mandl units/mg) from Gallard Schlesinger Chemical Manufacturing Corp.; Pentex Bovine Serum albumin, fraction V powder, fatty acid-poor, from Miles Laboratories; neuraminidase of Clostridium perfringens, type VI chromatographically purified (0.78 units/mg protein), cyclic AMP, dibutyryl cyclic AMP, bovine brain gangliosides, and sialic acid from Sigma Chemical Co.; cyclic [3 HI AMP (spec. act. 27.1 Ci/mmol) from New England Nuclear Corp. Purified cholera enterotoxin was obtained from Dr Richard A. Finkelstein and also from Dr Carl E. Miller, Geographic Medicine Branch, NIAID. The material supplied by the latter was prepared under contract for NIAID by R.A. Finkelstein, Ph.D., The University of Texas, South Western Medical School, Dallas, Texas. Both materials were prepared essentially according to procedure described in ref. 5. Choleragenoid was also obtained from Dr R.A. Finkelstein. Purified ~s-adrenocorticotropic hormone (ACTH) was obtained from Dr C.H. Li, Hormone Research Laboratory, University of California, Berkeley.
310 Methods
Cell suspensions. Suspensions of isolated rat adrenal cells were prepared by collagenase-trypsin treatment of adrenal sections in Krebs-Ringer bicarbonate buffer containing 0.2% glucose (Buffer A) by the m e t h o d described previously [ 28,29]. Cell incubations. All incubations were carried out in duplicate or triplicate in 10 ml beakers in a D u b n o f f metabolic shaker at 37°C in an atmosphere of 95% 0 2 : 5 % CO2. Each beaker contained 1 ml of the cell suspension, test substances dissolved in Buffer A and an appropriate volume of the buffer to make the final volume 1.5 ml. Neuraminidase treatment. The cells were suspended at a concentration of 0 . 6 - - 0 . 8 . 1 0 6 cells/ml in 0.01 M phosphate buffer and neuraminidase treatm e n t carried out as described before [29,30]. Measurement of cyclic AMP. Extraction and quantitative measurement of cyclic AMP was carried out according to previously published methods [31--33] and has been described by these authors before [30]. All cyclic AMP experiments were carried out in triplicate and three measurements were made on each replicate. Each value reported in this paper is the mean of 9 observations. The standard deviations were always less than 10% of the respective means. Measurement of corticosterone. Corticosterone was measured by the sulfuric acid fluorescence m e t h o d described by Silber et al. [34]. Each corticosterone experiment was carried out in duplicate and the average values are reported in the paper. The duplicate determinations were always found to be within + 5% of the respective means. Results
Effects o f enterotoxin and ACTH on cyclic AMP and corticosterone production Fig. 1 shows the time course of corticosterone formation from endogenous precursors in the presence of different concentrations of enterotoxin; in the absence of enterotoxin, no corticosterone synthesis is detectable (< 0.05 nmol/10 s cells) even at 5 h. It is clear that there is a delay or lag period before corticosterone production is observed. The duration of this lag period, however, is dependent upon the concentration of enterotoxin used. With maximally stimulating doses of enterotoxin, i.e. 1 pg/ml, an increase in corticosterone production is evident at 1 h whereas with 1 ng/ml a significant elevation in corticosterone is n o t observed until 3 h. Fig. 2(A) shows the time course of corticosterone formation in the presence of maximally stimulating concentrations of either enterotoxin or ACTH. With ACTH, the m a x i m u m rate of corticosterone production is established within the first 15 min of incubation, a finding in agreement with earlier published reports [35,36]. On the other hand, no corticosterone synthesis is detectable for the first 30 min when enterotoxin is added and maximum rates of production are not established until 45--60 min of incubation. It is to be noted, however, that once established, the m a x i m u m rate of corticosterone production with enterotoxin is similar to that with ACTH.
311
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Fig. 2(B) shows the time course of cyclic AMP form at i on in the presence of supra-maximally stimulating levels of ACTH (50 nM) or e n t e r o t o x i n (3 pg/ml). The increase in cyclic AMP f or m at i on in response to ACTH is immediate whereas the response to e n t e r o t o x i n is observed only after 30 min. Maxim u m rates o f p r o d u c t i o n with the latter are not established until after 60 min of incubation. In contrast to the corticosterone results, the m a x i m u m rate of cyclic AMP f o r mat i on with e n t e r o t o x i n is only about 8--10% of that observed with ACTH. This difference in m a x i m u m rates was observed even when the experiments were carried in the presence o f 8 mM theophylline. Fig. 3 shows the dose-response curves of e n t e r o t o x i n for corticosterone and cyclic AMP production. Maximum p r o d u c t i o n of both cyclic AMP and corticosterone is obtained at a c o n c e n t r a t i o n of a b o u t 1 #g/ml. This is in contrast with ACTH, where m a x i m u m p r o d u c t i o n of corticosterone is observed at much smaller concentrations than those producing m a x i m u m amounts of cyclic AMP [ 3 5 , 3 6 ] . Experiments were carried out to see if the effects of ACTH and enterotoxin were additive. Table I shows that in the presence of maximally stimulating levels o f ACTH, addition of e n t e r o t o x i n did n o t produce any further increase in co r tico s t er one production. In the presence of submaximally stimulating co n cen tr ati ons of ACTH, e n t e r o t o x i n produced a further increase in corticosterone synthesis, bringing it to the levels obtained with maximally stimulating c o n c e n t r a t i o n s of ACTH.
313 TABLE I EFFECT OF ACTH AND ENTEROTOXIN
ON CORTICOSTERONE
Additions
ACTH ACTH ACTH Toxin Toxin
PRODUCTION
C o r t i c o s t e r o n e f o r m e d ( n m o l / 1 0 5 cells)
(2 n M ) (2 n M ) + t o x i n ( 1 0 0 n g / m l ) (2 n M ) + t o x i n (1 p g / m l ) (100 ng/ml) (1 p g / m l )
A C T H (0.5 nM) A C T H (0.5 n M ) + t o x i n (1 p g / m l )
lh
2h
4h
2.0 1.9 2.1 0.5 0.7
4.2 4.2 4.3 1.4 2.9
6.8 6.9 6.7 5.2 7.1
1.6 1.9
3.2 4.1
4.4 6.9
Fig. 4 shows the steriodogenic effect of ACTH in cells pretreated with enterotoxin. Pretreatment of the cells with enterotoxin has essentially no effect on their subsequent response to maximally stimulating concentrations of ACTH.
Effect of cycloheximide Table II shows that cycloheximide inhibits the corticosterone synthesis stimulated by both ACTH and enterotoxin. The extent of inhibition is dependent on the concentration of cycloheximide and at each tested concentration of the inhibitor it is similar for the two stimulants.
0 ~UO 1.6
1.2
0.8 0 ~ 0.4 0 U
ACTH
./
/
30
45
60
I,-
U
0
,
TOXIN
TIME
90
,
120
MINUTES
Fig. 4. E f f e c t o f p r e t r e a t r a e n t o f a d r e n a l cells w i t h e n t e r o t o x i n T h e cells w e r e i n c u b a t e d w i t h or w i t h o u t 1 /~g/ml e n t e r o t o x i n c o r t i c o s t e r o n c s y n t h e s i s f o l l o w e d f o r t h e n e x t 9 0 rain. • at 3 0 r a i n ; o o, e n t e r o t o x i n a d d e d at 0 t i m e , A C T H at 3 0 t i m e , A C T H a t 3 0 rain.
o n t h e i r s u b s e q u e n t r e s p o n s e to A C T H . f o r 3 0 rain, 5 n M A C T H w a s a d d e d a n d "-, e n t e r o t o x i n a d d e d at 0 t i m e , n o A C T H rain~ • • , no e n t e r o t o x i n a d d e d a t 0
314 T A B L E II INHIBITION HEXIMIDE
OF
ACTH
AND
ENTEROTOXIN-STIMULATED
Additions
Corticosterone formed (nmol/105 c e l l s p e r 2 h)
A C T H 5 nM + Cycloheximide ( 1 pM) + Cycloheximide ( 3 #M) + Cycloheximide (10 pM)
3.0 2.0 1.2 0.5
33.3 60.0 83.3
Toxin 1 #g/ml + C y c l o h e x i m i d e ( 1 pM) + Cycloheximide ( 3 pM) + C y c l o h e x i m i d e (10 pM)
2.1 1.5 0.8 0.3
28.6 61.9 85.7
STEROIDOGENESIS
BY
CYCLO-
% Inhibition d u e to cycloheximide
Ca ~÷ d e p e n d e n c e
Since the role of Ca 2÷ in the steroidogenic effect of ACTH is well known [37,38], the importance of this ion in enterotoxin-stimulated steroidogenesis was also investigated. Fig. 5 shows that at all time intervals studied, Ca 2÷ increased and EGTA decreased the a m o u n t of corticosterone synthesized by the cells in response to enterotoxin. Table III shows that although the steroidogenic response to ACTH is completely inhibited by 100 pM EGTA, the effects of enterotoxin and dibutyryl cyclic AMP are only partially inhibited (67% and 51% respectively) with as much as 1 mM EGTA. Addition of Ca 2÷ produces a concentration-dependent
~o
4
2.5 mM Co**
3
NO
0
~E c
Co * ÷
i.u
2
0
m: m |
1.0 mM EGTA
0
u 0 u
1
2 TIME
3
4
HOURS
Fig. 5. T h e e f f e c t o f Ca 2+ a n d E G T A on t h e c o r t i c o s t e r o n e r e s p o n s e o f t h e a d r e n a l cells to e n t e r o t o x i n . Cells w e r e p r e p a r e d in r e g u l a r B u f f e r A, c e n t r i f u g e d a n d t h e p e l l e t s s u s p e n d e d in B u f f e r A c o n t a i n i n g t h e u s u a l a m o u n t s o f b o v i n e s e r u m a l b u m i n a n d t r y p s i n i n h i b i t o r b u t w i t h o u t Ca 2+. I n c u b a t i o n s w e r e t h e n c a r r i e d o u t in t h e p r e s e n c e or a b s e n c e o f 2.5 m M Ca 2+ or a f t e r t h e a d d i t i o n o f 1 m M E G T A .
315 TABLE III C O M P A R I S O N O F T H E Ca 2+ R E Q U I R E M E N T F O R T H E S T I M U L A T I O N O F C O R T I C O S T E R O N E F O R M A T I O N BY E N T E R O T O X I N , A C T H A N D D I B U T Y R Y L C Y C L I C AMP Cells w e r e p r e p a r e d in B u f f e r A as usual. A f t e r c e n t r i f u g a t i o n t h e y w e r e r e s u s p e n d e d in B u f f e r A w i t h o u t Ca 2+ a n d i n c u b a t e d w i t h v a r y i n g a m o u n t s of Ca 2+ or E G T A , as s h o w n in t h e table, for 2 h at 37 ° C. C o r t i c o s t e r o n e f o r m e d ( n m o l / 1 0 5 cells p e r 2 h)
Additions (uM)
N o n e (no Ca 2+) EGTA 10 30 100 300 1000
Toxin (1 ~ g / m l )
ACTH (5 nM)
Dibutyryl cyclic AMP (0.5 m M )
1.5
1.8
2.3
1.5 1.2 0.5 0.5 0.5
1.3 0.6 < 0.05 < 0.05 < 0.05
2.2 1.8 1.1 1.1 1.1
Ca 2+ 100 300 1000 3000
2.2 2.5 2.8 2.9
2.7 3.3 3.7 3.7
2.5 3.0 3.4 3.2
increase in the effects of all three stimulants with m a x i m u m increase being observed with about 1 mM concentration of the cation. At this concentration of Ca 2÷ the increases in the effects of enterotoxin, ACTH and dibutyryl cyclic AMP were 90, 106 and 48 percent respectively. Table IV shows that omission o f Ca 2÷ from the incubation medium has a more pronounced effect on cyclic AMP formation in response to ACTH than that in response to enterotoxin; the former is reduced by approx. 90% whereas the latter by only about 50%. Furthermore, 1 mM EGTA almost completely abolishes the effect of ACTH although it produces only a partial reduction in the a m o u n t of cyclic AMP produced in response to enterotoxin.
Effect of choleragenoid Table V shows that choleragenoid effectively inhibited the steroidogenic
TABLE IV Ca 2+ D E P E N D E N C E O F E N T E R O T O X I N - A N D A C T H - S T I M U L A T E D C Y C L I C AMP S Y N T H E S I S I n c u b a t i o n c o n d i t i o n s as in T a b l e I I I Additions
2.5 m M Ca 2+ N o Ca 2+ 1 mM EGTA
Cyclic AMP f o r m e d ( p m o l / 1 0 s cells p e r 2 h ) Control
ACTH (50 n M )
Toxin (1 p g / m l )
Toxin (5 p g / m l )
3.9 3.5 3.9
206.7 24.2 4.9
27.7 13.4 9.8
27.6 16.5 12.1
316
TABLE V EFFECT OF CHOLERAGENOID ON THE STIMULATION E N T E R O T O X I N A N D DIBUTYI=tYL C Y C L I C AMP
OF CORTICOSTERONE
S Y N T H E S I S BY
T h e cells w e r e i n c u b a t e d w i t h c h o l e r a g e n o i d f o r 30 m i n at 37°C. D i b u t y r y l c y c l i c A M P or e n t e r o t o x i n w e r e t h e n a d d e d a n d t h e i n c u b a t i o n c o n t i n u e d f o r 2 h. T h e v a l u e s r e p o r t e d in the table s h o w the a m o u n t o f c o r t i c o s t e r o n e f o r m e d d u r i n g the 2 h i n c u b a t i o n a f t e r the a d d i t i o n o f the s t i m u l a t i n g a g e n t s . Cholerag e n o i d b y i t s e l f h a d n o e f f e c t on t h e n e g l i g i b l e c o r t i c o s t e r o n e s y n t h e s i s ( < 0 . 0 5 n m o l / 1 0 5 cells) in the absence of the s t i m u l a t i n g agents. Choleragenoid added (ng/ml)
C o r t i c o s t e r o n e f o r m e d ( n m o l / 1 0 S cells p e r 2 h) Enterotoxin (1 p g / m l )
Dibutyryl cyclic A M P (1 p m o l / m l )
0 100 300 1000 3000
1.2 1.1 1.0 0.5 0.2
3.0 2.8 2.9 2.7 2.8
reponse to enterotoxin. The inhibition by choleragenoid does not appear to be due to effect(s) on the intracellular reactions involved in steroidogenesis because it has no effect on the dibutyryl cyclic AMP-stimulated corticosterone synthesis. Table VI shows that the concentration of choleragenoid which almost completely inhibits the effects of l p g / m l enterotoxin has virtually no effect on the stimulation of corticosterone synthesis observed with various concentrations of ACTH.
Effect of gangliosides Recently it was shown that incubation of enterotoxin with gangliosides resulted in a loss of activity of the toxin [15,40,41] which was attributed to its incapacity to bind to tissues [42]. Fig. 6 shows the effects of exogenously
T A B L E VI EFFECT OF CHOLERAGENOID ACTH
ON T H E S T I M U L A T I O N
OF CORTICOSTERONE
S Y N T H E S I S BY
Cells w e r e i n c u b a t e d w i t h 3 ~tg/ml c h o l e r a g e n o i d at 3 7 ° C f o r 30 rain, A C T H w a s a d d e d as i n d i c a t e d in t h e t a b l e a n d i n c u b a t i o n c o n t i n u e d f o r 1 2 0 rain. T h e v a l u e s s h o w t h e a m o u n t s o f c o r t i c o s t e r o n e f o r m e d a f t e r t h e a d d i t i o n o f A C T H . C h o l e r a g e n o i d h a d n o e f f e c t on the n e g l i g i b l e c o r t i c o s t e r o n e s y n t h e s i s ( ~ 0 . 0 5 n m o l / 1 0 5 cells) in t h e a b s e n c e o f A C T H . ACTH added (nM)
- Choleragenoid
+ Choleragenoid
0.05 0.1 0.2 0.4 0.8 1.6
0.8 1.6 1.8 2.5 2.8 2.7
0.8 1.5 1.6 2.7 2.9 2.6
C o r t i c o s t e r o n e f o r m e d ( n m o l / 1 0 5 cells p e r 2 h)
317
!
8o
60
o 4O
a ~ o I
3 GANGLIOSlDE$
10
30
10~
MICROGRAMS/ML
Fig. 6. E f f e c t of e x o g e n o u s l y a d d e d gangliosides o n the s t i m u l a t i o n o f c o r t i c o s t e r o n e s y n t h e s i s b y A C T H a n d e n t e r o t o x i n . G a n g l i o s i d e s w e r e i n c u b a t e d w i t h d i f f e r e n t c o n c e n t r a t i o n s of e n t e r o t o x i n or A C T H at 4°C for 20 rain in a 0,5 m l v o l u m e , I m l o f t h e cell s u s p e n s i o n was a d d e d a n d the i n c u b a t i o n c a r r i e d o u t for 2 h at 3 7 ° C . T h e final c o n c e n t r a t i o n s of e n t e r o t o x i n w e r e 1, 3 a n d 10 ~zg/ml ( o ~ ~) a n d A C T H 0.1, 0.4 a n d 1.6 nM ( e A e). T h e c o n c e n t r a t i o n s of gangliosides given in t h e figure are t h e final c o n c e n t r a tions. Values o b t a i n e d in t h e a b s e n c e of gangliosides are e x p r e s s e d as c o n t r o l ( 1 0 0 % ) a n d t h o s e o b t a i n e d in t h e p r e s e n c e o f gangliosides are e x p r e s s e d as p e r c e n t of t h e i r r e s p e c t i v e c o n t r o l s .
added gangliosides on the stimulation of corticosterone synthesis by enterotoxin and ACTH. At all levels of enterotoxin tested, the stimulatory effect was completely abolished when the gangliosides were present in a 3-fold excess on a weight basis. On the other hand gangliosides, even at high concentrations, had only a marginal effect on the ACTH-stimulated corticosterone production. Table VII shows the concentration-dependent inhibition of enterotoxinstimulated cyclic AMP formation by gangliosides. Again, the effect of ACTH is only marginally inhibited by 100 pg/ml gangliosides while lower concentrations show almost no effect.
TABLE VII EFFECT OF GANGLIOSIDES FORMATION
ON T H E E N T E R O T O X I N
A N D A C T H - S T I M U L A T E D C Y C L I C AMP
T r e a t m e n t w i t h gangliosides a n d t h e i n c u b a t i o n c o n d i t i o n s w e r e the s a m e as t h o s e d e s c r i b e d in Fig. 6. Gangliosides (ug/ml)
0 3 1O 30 100
Cyclic AMP f o r m e d ( p m o l / 1 0 s cells/2 h ) Control
Enterotoxin (3 ~zg/ml)
ACTH (5 n M )
1.7 -1.6 -1.8
25.'/ 17.4 8.6 5.0 3.1
62.2 56.8 57.9 60.8 51.8
318
Effect o f neuraminidase-treatment o f adrenal cells on their response to enterotoxin Recently it was shown that after treatment with neuraminidase, adrenal cells became less sensitive to ACTH both in terms of cyclic AMP and corticosterone production [29,30]. Fig. 7(A) shows that unlike the results obtained with ACTH the neuraminidase-treated cells produce more corticosterone than untreated cells in response to enterotoxin. The maximum rates in this experiment, obtained between 60--120 rain, are 2.12 and 1.75 nmol/105 cells/h for the treated and untreated cells respectively. Extrapolation of the curves shows that increase in the a m o u n t of corticosterone produced by the treated cells may have also been due to shortening of the lag period by 6--7 min. Fig. 7(B) shows that neuraminidase-treated cells also produce more cyclic AMP than the untreated cells. There is an increase of approx. 20% in the maximum rate (calculated between 60--120 min) as well as a shortening of the lag period by about 10 min in the neuraminidase-treated cells. Fig. 8(A) shows that after neuraminidase treatment the cells become more sensitive to enterotoxin. Thus, in a 2 h incubation reported here, no corticosterone synthesis was detected in the control cells with 1 or 3 ng/ml entero-
(A)
50
2,5 LM
U
"~ F~ rt~
LM
40
~,,u
1.5
ao
:ll •
1.G
20
O r-
•..m 2.0 O
O maa
O IN
°c..~
O u i,- 0.5
10
m
u
0
30
60
90
120
TIME
0
30
60
90
}20
MINUTES
F i g . 7. T h e e f f e c t o f n e u r a m i n i d a s e t r e a t m e n t o f a d r e n a l cells o n t h e i r c o r t i s t e r o n e ( A ) a n d c y c l i c A M P (B) response to enterotoxin. The ceils were suspended in 0.01 M phosphate buffer (pH 6.5) containing 0.85% NaC1, 0.5% bovine serum albumin and 0.1% trypsin inhibitor and divided into two groups. Both groups were incubated at 37°C for 15 rain, neuraminidase (20 munits/ml) was added to one group and the incubation continued for 30 rain. After centrifugation, the cells in both groups were washed with 0.01 M phosphate b u f f e r c o n t a i n i n g 0 . 8 5 % NaC1 ( 5 t i m e s o r i g i n a l i n c u b a t i o n v o l u m e ) , c e n t r i f u g e d a n d t h e n s u s p e n d e d in B u f f e r A c o n t a i n i n g b o v i n e s e r u m a l b u m i n a n d t r y p s i n i n h i b i t o r f o r t h e f i n a l i n c u b a t i o n . The concentration of enterotoxin in both experiments was 3 ~g/ml. Control cells (e ~-); n e u r a m i n i d a s e - t r e a t e d cells (A A).
319
0 Z
(A)
2.0
50
(B)
r-
t~
~t ..z ,.a
•" ~
-~ t~
4O
-g
30
m
20
0pm
10
~
1.5
i
I
~ ,.01 u.
0 tM
0.5
0 U
0 C 0 U
I
10
100
ENTEROTOXI N
i
i
i
i
i
1000
3OOO
100
3OO
1000
Fig. 8. L o g d o s e r e s p o n s e
curves for the stimulation
by
(o) and neuraminidase-treated
enterotoxin
tion conditions
in control
3OOO
NANOGRAMS/ML of corticosterone
(A) and cyclic AMP (B) synthesis
(A) c e l l s . N e u r a m i n i d a s e
treatment
and incuba-
w e r e t h e s a m e as i n F i g . 7.
toxin although neuraminidase-treated cells produced significant amounts of the steroid (0.16 and 0.31 nmol/10 s cells) in response to these two concentrations of enterotoxin. The concentration of enterotoxin required to give halfm a x i m u m amounts of corticosterone in the neuraminidase-treated cells was about 1/3 of that required in the control cells. Fig. 8(B) shows that the m a x i m u m a m o u n t of cyclic AMP formed by neuraminidase-treated cells in response to enterotoxin is about twice that formed by control cells and the concentration of enterotoxin required to produce half-maximum amounts of cyclic AMP is reduced considerably in the neuraminidase-treated cells. Discussion Recent reports from a number of laboratories have indicated that most, if not all, of the biological effects of cholera enterotoxin in the cells of the intestine as well as other organs are mediated via cyclic AMP (See 'Introduction' for references). Cholera enterotoxin, therefore, is potentially useful for investigating the regulation of adenyl cyclase in cell membranes and for clarifying the mechanism of action of a variety of hormones which exert their effects through manipulation of cyclic AMP levels. In the present study we have compared the characteristics of ACTH-stimulated cyclic AMP and corticosterone production with those of enterotoxin to determine if there are any c o m m o n components or "overlapping receptor d o m a i n s " [27] for the two stimuli. A major similarity between the effects of ACTH and enterotoxin is that the m a x i m u m rates of corticosterone production are the same for the two
320 stimuli. F u r t h e r m o r e , m a x i m u m doses of ACTH and e n t e r o t o x i n were not additive suggesting t hat the steroidogenic pathway stimulated by the two agents was the same. The response of adrenal cells to ent erot oxi n, however, exhibits certain characteristics which are different from those observed with ACTH. These centered initially on the differences in the temporal response to stimulation and in the discrepancies in the m a x i m u m rates of cyclic AMP production. F u r ther differences between e n t e r o t o x i n and ACTH were subsequently elaborated by manipulation of cell m em brane sialic acid with neuraminidase and by changing the ionic envi r onm ent by means of EGTA. A delayed response or lag period is a characteristic of the e n t e r o t o x i n response in a n u m b e r of systems [4,7,9,10,14] and was also observed in the adrenal cells. With maximally stimulating levels of the toxin, no cyclic AMP or corticosterone synthesis was detected up to 30--45 min and m axi m um rates were established only after 60 min of incubation. It should be not ed that although the m a x i m u m rates of corticosterone product i on were similar for ACTH and e n t e r o t o x i n , those of cyclic AMP p r o d u c t i o n were not. Thus, the m a x i m u m rate o f cyclic AMP p r o d u c t i o n with e n t e r o t o x i n was only about 8--10% o f that with ACTH. These results support the suggestion of others [43,44] that all of the cyclic AMP pr oduced in the adrenal cells in response to ACTH is n o t required for obtaining maximal rates of corticosterone production. The Ca 2÷ r equi r e m ent for the steroidogenic response of ACTH has been known for man y years [ 3 7 , 3 8 ] . Recently, it was shown that this requi rem ent is greater for some event(s) occurring at the plasma m em brane leading to increased cyclic AMP p r o d u c t i o n than for the reactions involved in the stimulation o f steroidogenesis by cyclic AMP [ 4 5 - - 4 7 ] . A Ca 2+ requi rem ent for the effect of e n t e r o t o x i n on fat cells was shown by Vaughan et al. [ 1 4 ] . In this report we have shown that although Ca 2÷ is also required for the effect of e n t e r o t o x i n on adrenal cells, this r e qui r em ent is not the same as that for ACTH. Thus, EGTA abolished the effect of ACTH on corticosterone synthesis completely but caused only a partial reduction in the effect of ent erot oxi n. Omission o f Ca 2÷ from the incubation medium reduced the a m o u n t of cyclic AMP p r o d u ced in response to ACTH to a b o u t 10% of that produced in the presence of 2.5 mM Ca 2÷. On the other hand omission of Ca 2÷ produced only about 40% decrease in the cyclic AMP response of ent erot oxi n. EGTA, which almost co mp letel y abolished ACTH-stimulated cyclic AMP p r o d u c t i o n again had only a partial effect on the e n t e r o t o x i n response. Two possibilities may be considered to explain these differences. First, e n t e r o t o x i n may stimulate adenyl cyclase by the two distinct mechanisms; one d e p e n d e n t on Ca 2÷, the o t h er not. If this is the case then there is a possibility that some c o m p o n e n t of the f o r m e r mechanism may overlap with c o m p o n e n t s involved in ACTH stimulation of adenyl cyclase. The second possibility is that EGTA does not remove all m e m b r a n e - b o u n d Ca 2÷. If this is true then it follows that the Ca ~÷ involved in the effect o f ACTH is in a separate c o m p a r t m e n t from that involved in the effect o f e n t e r o t o x i n . We have not a t t e m p t e d to distinguish between the two possibilities at this time. Cuatrecasas [39] has shown recently that choleragenoid, a protein immunologically identical with the cholera e n t e r o t o x i n but lacking in biologi-
321 cal activity [1,5,14], can inhibit the lipolytic effect of the latter in fat cells by competing for the receptor. Our results with adrenal cells support their findings. The localization of choleragenoid action at the plasma membrane is supported by the finding that it did not alter the corticosterone response to dibutyryl cyclic AMP. The choleragenoid was also w i t h o u t effect on the doseresponse curves of ACTH which makes it unlikely that inhibition of the toxin response was due to a non-specific interaction with the cell membrane. Several investigators have shown that the effects of enterotoxin can be abolished by incubating the toxin with gangliosides [15,40,41]. The loss in the activity of enterotoxin is attributed to the inability of the toxin/ganglioside complex to bind to receptors in the target tissue [42]. The present report demonstrates that exposure of enterotoxin to gangliosides prevents the effects of the former on cyclic AMP and corticosterone synthesis in normal adrenal cells, results which are similar to those obtained by Wolff et al. [27] using cultured mouse adrenal t u m o r cells. Further, we have shown that gangliosides, at concentrations that abolished the effects of enterotoxin, did not prevent the stimulation of cyclic AMP or corticosterone synthesis by ACTH. At high concentrations (around 100 pg/ml) the gangliosides did have a marginal inhibitory effect on these parameters; however, this may be a non-specific effect of these charged molecules on the cell because at such concentrations the stimulation of corticosterone synthesis by dibutyryl cyclic AMP was also inhibited by about 15--20%. The most significant difference between ACTH- and enterotoxin stimulation of adrenal cells was observed after treatment of the cells with neuraminidase. In earlier reports we showed that neuraminidase treatment of the cells reduced the sensitivity to ACTH in terms of both cyclic AMP [ 30] and corticosterone production [29]. Since the steroidogenic response of the cells to dibutyryl cyclic AMP was not affected by neuraminidase treatment [29] it was concluded that removal of sialic acid by neuraminidase resulted in an alteration of some membrane component(s) involved in the stimulation of adenyl cyclase by ACTH. In contrast to the results obtained with ACTH, adrenal cells became more sensitive to enterotoxin after treatment with neuraminidase under conditions identical to those used in the ACTH work [29,30]. Thus, neuraminidasetreated cells produced more cyclic AMP and corticosterone than untreated cells in response to enterotoxin. The increased response of the neuraminidasetreated cells at a given time interval seems mainly to be due to an increased rate of synthesis of cyclic AMP (or corticosterone) although shortening of the lag period may also be involved. A comparison of the dose-response curves for control and treated cells showed that the concentrations of enterotoxin required to give half-maximum production of cyclic AMP (or corticosterone) were substantially reduced after neuraminidase treatment. The reason for increased sensitivity of the cells after neuraminidase treatment is not clear at this time. It is possible that removal of membrane sialic acid results in exposure of more enterotoxin receptors on the cell surface or in a reduction of non-specific binding of enterotoxin to sialic acid-containing glycoproteins. Indeed, several glycoproteins have been shown to be capable of binding enterotoxin [48]. Alternatively, neuraminidase treatment may have increased the binding capacity of an otherwise less active or inactive molecule,
322
This last alternative is supported, in part, by recent studies which indicate that the biological receptor for enterotoxin may be a ganglioside [15,40,41,42,48]. King and van Heyningen [41] showed that in vitro stimulation of intestinal adenyl cyclase by enterotoxin was abolished if the toxin was added after pretreatment with neuraminidase-resistant monosialosylganglioside. The neuraminidase-sensitive di- and trisialosylgangliosides were not effective in their system although treatment of these gangliosides with neuraminidase did render them capable of neutralizing the toxin, presumably by converting them to neuraminidase-resistant monosialosylganglioside. Among the gangliosides that have been shown by Cuatrecasas [48] to inhibit the binding of enterotoxin to liver membranes, neuraminidase-resistant monosialosylganglioside was found to be the most effective. In the same study it was also found that liver membranes after treatment with neuraminidase exhibited a slight enhancement in the binding of enterotoxin. Therefore, it is possible that the complex membrane gangliosides in the intact adrenal cells were converted by neuraminidase into neuraminidase-resistant monosialosylganglioside and as a result of this change the cells became more sensitive to enterotoxin [49]. Acknowledgments The authors would like to express their gratitude to: Dr. R.A. Finkelstein, South Western Medical School, University of Texas, for kindly supplying the enterotoxin and choleragenoid; Dr Carl E. Miller, Geographic Medicine Branch, NIAID for additional supplies of enterotoxin, and Dr C.H. Li, University of California for ~s-ACTH. We would also like to acknowledge the excellent technical assistance provided by William F. Robidoux, Jr, Susan R. Levy and George Gagnon. This research was supported by grant No. AM 04899 from the National Institutes of Arthritis, Metabolism and Digestive Diseases. References 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18
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