Prostaglandin E In Cholera Toxin-Induced Intestinal Secretion

Lack Of An Intermediary Role

Norman Hudson, MD, Suleiman El Hindi, MD, Donald E. Wilson, MD, and Larissa Poppe

Prostaglandin E1 (PGE1) and cholera enterotoxin stimulate small-intestine mucosal adenylate cyclase and intestinal secretion of water and electrolytes. The previous suggestion that PGE may mediate cholera-toxin effects was explored in these studies. Closed rabbit jejunal loops were injected in vivo with cholera toxin and compared to similar loops in the same animal injected with buffer. Loop mucosal homogenates and intestinal secretions were ana- lyzed by radioimmunoassay for cAMP and PGE concentrations. Cholera tox- in produced significant increases in mucosal and intestinal fluid cAMP; how- ever, there were no significant increases in PGE in the toxin-treated loops when compared to the control loops. In addition, there was no correlation between cAMP and PGE in the same samples. These studies indicate that cholera toxin stimulates intestinal cAMP and secretion independent of PGE synthesis and provide evidence against a specific role for PGE in mediating cholera-toxin effects.

The massive secretion of fluid and elec- trolytes from the small intestine caused by infec- tion with Vibrio ckolerae in humans is known to be caused by an enterotoxin produced by the organism (1-4). While the exact mechanisms of toxin-induced secretion are not dear , it has been recently shown that cholera toxin stimu- lates adenylate cyclase, resulting in the intra- cellular accumulation of cAMP ( 5 - 7 ) The ef- fects of cholera toxin on intestinal secretion can be mimicked by admistering compounds which either st imulate c A M P production or interfere

From the Department of Medicine, Section of Gastroen- terology: University of Illinois, Abraham Lincoln School of Medicine, Chicago, Illinois.

Dr. Hudson's present address is Department of Medi- cine, Duke University, Medical Center, Durham, North Carolina

Address for reprint requests: Dr. D. E. Wilson, Depart- ment of Medicine, Section of Gastroenterology, University of Illinois. Abraham Lincoln School of Medicine, P.O. Box 6998, Chicago, Illinois 60680.

with c A M P degradation (8, 9). A secretory diarrhea in some respects similar to that caused by cholera has been reported in several patients

who were found to have tumors that were se- creting large amounts of prostaglandins (10, 11 ).

Prostaglandin E1 (PGE1) like cho]era toxin increases mucosat cAMP and stimulates in- testinal secretion (9, 12). Several studies in ani- mals reported an inhibitory effect on cholera

toxin-induced intestinal secretion when the animals were pretreated with aspirin or indo- methacin (13-15). Since aspirin and indometh- acin are known inhibitors of prostaglandin synthesis (16-19), it was suggested that prosta- glandins may mediate the cholera toxin effect and further that aspirin may be efficacious in the t reatment of cholera (14).

The present study was undertaken to mea- sure directly the cholera-toxin effect on intes- tine mucosal c A M P and PGE and on the intes- tinal secretion of c A M P and PGE in order to

Digestive Diseases, Vol. 20, No. 11 (November 1975) 1035

Table 1. Effect of Cholera Toxin on Intestinal-Loop Secretory Volume (ml/cm loop) (Mean 4- SE )

Time (hr) Control Toxin

(N = 44) 2 0.24 4- 0.05 0.48 + 0.07* (N = 36) 4 0.16 + 0.06 0.89 + 0.131"

*P < 0.05. I P < 0.001.

evaluate any possible role for pros taglandins in

media t ing cholera toxin- induced s t imulat ion of

c A M P and intestinal secretion.

M A T E R I A L S A N D M E T H O D S New Zealand white rabbits of either sex, weighing be-

tween 4 and 5 kg were anesthetized with intravenous pen- mbarbital following an overnight fast. The proximal jeju- num was exposed through an abdominal incision and flushed with 0.9% saline. Following the distal run-offof in- trahlminal fluid, 5 closed jejunal loops, each measured to be 10 cm tong, were prepared by ligation. 2 adjacent loops were each injected intraluminally with 2 cc of isotonic phos- phate buffer to serve as control loops. The other 2 adjacent loops separated from the control loops by an inactive loop were injected with 2 cc of isotonic phosphate buffer contain- ing 10 #g/co of purified cholera toxin (Choleragen*). The bowel was then replaced into the abdominal cavity and the abdomen closed. At time periods of ~, 1, 2, and 4 hr, indi- vidual animals were sacrificed, the loops removed, and ac- cumulated fluid drained and measured. The loops were rinsed in iced saline and everted over a glass rod. The mu- cosal surface was removed by scraping as described by Kim- berg et al (6). The mucosal samples and secretions were rapidly frozen in liquid nitrogen and stored until prepared for extraction.

Cyclic AMP was measured by radioimmunoassay using a commercially available kit (Schwarz-Mann, Orangeburg, New York). Cyclic AMP was extracted from mucosal sam- pies according to the kit protocol and from intraluminai se- cretions as previously described (20). Prostaglandins were extracted from tissue and secretions and the PGE fraction separated and measured by radioimmunoassay as pre- viously described (20). All samples were assayed in dupli- cate with duplicate variability being 15% or less. Tissue protein content was determined by the method of Lowry et al (21). A total of 120 loops were analyzed. The data are presented as the mean • 1 SE. Statistical analyses were per- formed using Student's t test.

*Chuleragen, Lot #0172, was supplied by Dr. Carl T. Miller, NIH, Bethesda, Maryland.

HUDSON ET AL

R E S U L T S

Pre l imina ry studies (not included) indicated

that 20 #g of Cho le ragen produced the greatest

vo lume of fluid secretion at 4 hr; therefore, this

dosage was used th roughou t the study.

T h e loop lengths were 10.4 4- 0.54 and 10.2

4- 0.52 cm for the control and toxin groups, re-

spectively. Cho le ragen significantly increased

the vo lume of intestinal secretion at 2 and 4 hr

(Table 1). Mucosa l c A M P was s imi lar in both

groups at 30 min and 1 hr, but increased signifi-

cantly in the toxin- t rea ted group at 2 and 4 hr

(Figure 1). T h e concentra t ion of c A M P in in-

test inal secretions was significantly h igher in

the toxin- t rea ted group at 4 hr (Figure 1). Pros-

taglandin concentra t ions increased in both mu-

cosa and intestinal secretions over the 4-hr per i -

od; however , there were no significant

differences between the control and toxin-

t reated loops (Figure 2). At 30 min and 1 hr,

the mucosal concentra t ion of P G E in the toxin-

t reated loops was in fact slightly lower than in

the control loops. To t a l P G E output in loop se-

cretions was s imilar in both groups th roughout

the study even though volume output was sig-

nificantly greater in the toxin- t rea ted group at 2

and 4 hr. In addit ion, there was no l inear corre-

lat ion between c A M P levels and P G E levels in

the same mucosal sample.

D I S C U S S I O N

Whi le both PGE1 and cholera toxin s t imu-

late intestinal adenylate cyclase and fluid secre-

tion, certain differences exist between the ac-

tions of these two compounds. A lag per iod

general ly occurs be tween the admin is t ra t ion of

cholera toxin and s t imulat ion of adenylate cy-

clase, whi le the effects of P G E 1 are rapid (6). In

addition, the effects of max ima l concentra t ions

of the individual compounds on adenyla te cy-

clase s t imulat ion are addit ive when the com-

pounds are adminis tered together suggest ing

the involvement of different receptor

sites (6, 12). In contrast to animal studies link-

ing pros tag landin synthesis to cholera- toxin

s t imulat ion of intestinal secretion (13-15) ,

1036 Digestive Diseases, Vol. 20, No. 11 (November 1975)

PGE IN CHOLERA TOXIN- INDUCED SECRETION

35

30 o E ,, 25

20

< 15 r ..a 10 r

O 5

. . . . . . . . . . . . . .

i i i i i

t -

2 e~

E e~

e~

,<

(J , M

20

15

10

~ J ~ _ v C . 4 ~

. . . . . _L /

] I I I I

1/2 1 2 3 4 hours

Fig 1. Changes in cAMP concentrat ion in intestinal secretion (top panel) and in mucosa (bottom panel) for control and toxin-treated loops. Vertical lines indicate 1 SEM (N = 20, 20, 44, and 36 at 1/2, 1, 2, and 4 hr, respectively), a, P < 0.02; b, P < 0.01; c, P < 0.001. Unbroken line signifies control; broken line signifies toxin.

Bourne (22) failed to find that aspirin or in- domethacin (inhibitors of prostaglandin syn- thesis) prevented the cholera toxin-induced ac- cumulation of c A M P in leukocytes. More recently, Kimberg et al (12) could not demon- strate any effect for indomethacin or aspirin on cholera toxin-induced increases in intestinal c A M P or in levels of prostaglandin B (aggre- gate of E's and A's) or PGF2~.

The present study did not reveal a relation- ship between c A M P and P G E content in in-

testinal mucosa or intestinal secretions in re- sponse to cholera toxin. As expected, cholera toxin produced significant increases in in- testinal fluid secretion and in cAMP. However, although there was a general trend of increasing prostaglandin concentrations in the intestinal mucosa, no difference existed between the con- trol and toxin-treated groups. These studies

s u p p o r t previous conclusions that prostaglan- dins and cholera toxin act by different mecha- nisms to stimulate small intestine cAMP. In

Digestive Diseases, Vol. 20, No. 11 (November 1975) 1037

H U D S O N ET AL

I -

LU

(5 n

1.0

.9

.8

.7

.6

.5

. 4 -

. 3 -

.2 I I T I ', I

' t T c-

O .5 / / -

/

E . 4 -

lal . 2 - O a _1_

.1 1' ' ' ~ '

0 Y2 1 2 3 4 hours

Fig 2. Changes in prostaglandin E concentration in intestinal secretion (top panel) and in mucosa (bottom panel) for control and toxin-treated loops. Verti- cal lines indicate 1 SEM (N = 20, 20, 44, and 36 at V2, 1,2, and 4 hr, respective- ly). Unbroken line signifies control; broken line signifies toxin.

addition, they refute the concept of an inter- mediary role for prostaglandins in the patho- genesis of cholera toxin-induced diarrhea.

R E F E R E N C E S

1. Dutta NK, Habbu MK: Experimental cholera in infant rabbits: A method for chem- otherapeutic investigation. Br J Pharmacol 10:153-159, 1955

2. De SN, Chatterje DN: An experimental study of the mechanism of action of vibrio cholerae on the

4 .

5.

intestinal mucous membrane. J Pathol Bacteriol 66:559-562, 1953 Sack RB, Carpenter CCJ, Steenburg RW, Pierce NF: Experimental cholera: A canine model. Lancet 2:206-207, 1966 Benyajati C: Experimental cholera in humans. Br Med J 1:140-142, 1966 Schafer DE, Lust WD, Sircar B, Goldberg ND: Elevated concentration of adenosine 3':5'-cyclic monophosphate in intestinal mucosa after treat- ment with cholera toxin. Proc Natl Acad Sci U.S.A. 67:851-856, 1970

1038 Digestive Diseases, Voi. 20, No. 11 (November 1975)

PGE IN CHOLERA TOXIN-INDUCED SECRETION

6. Kimberg DV, Field M, Johnson J, Henderson A, Gershon E: Stimulation of intestinal mucosal adenyl cyclase by cholera enterotoxin and pros- taglandins. J Clin Invest 50:1218-1230, 1971

7. Sharp GWG, Hynie S: Stimulation of intestinal adenyl cyclase by cholera toxin. Nature (Lon- don) 229:266-269, 1971

8. Field M, Fromm D, Wallace CK, Greenough III WB: Stimulation of active chloride secretion in small intestine by cholera exotoxin. J Clin In- vest 48:24a, 1969

9. Pierce NF, Carpenter CCJ, Elliott HL, Gree- nough III WB: Effects of prostaglandins, theo- phylline and cholera exotoxin upon trans- mucosal water and electrolyte movement in the canine jejunum. Gastroenterology 60:22-32, 1971

10. Sandler M, Karim SMM, Williams ED: Pros- taglandins in amine-peptide-secreting turnouts. Lancet 11:1053-1055, 1968

11. Williams ED, Karim SMM, Sandler M: Pros- taglandin secretion by medullary carcinoma of the thyroid. Lancet 1:22-23, 1968

12. Kimberg DV, Field M, Gershon E, Henderson A: Effects of prostaglandins and cholera entero- toxin on intestinal mucosal cyclic AMP accumu- lation. Evidence against an essential role for prostaglandins in the action of toxin. J Clin In- vest 53:941-949, 1974

13. Jacoby HI, Marshall CH: Antagonism of chol- era enterotoxin by anti-inflammatory agents in the rat. Nature (London) 235:163-165, 1972

14. Finch A, Katz R: Prevention of cholera-induced intestinal secretion in the cat by aspirin. Nature (London) 238:273-274, 1972

15. Gots RE, Formal SB, Giannella RA: In- domethaein inhibition of salmonella typhimu- rium, shigella flexneri, and cholera-mediated rabbit ileal secretion. J Infect Dis 130:280-284, 1974

16. Fer re i ra SH, Moncada S, Vane JR : In- domethacin and aspirin abolished prostaglandin release from the spleen. Nature (London), New Biol 231:237-239, 1971

17. Vane JR: Inhibition of prostaglandin synthesis as a mechanism of action for aspirin-like drugs. Nature (London), New Biol 231:232-235, 1971

18. Smith AP: Response of aspirin-allergic patients to challenge by some analgesics in common use. Br Med J 2:494-496, 1971

19. Smith ID, Temple DM: The influence of anal- gesic drugs on the actions of prostagtandin F2a on the human uterus in vivo and human and rabbit myometrial strips in vitro. Prostaglandins 4:469-477, 1973

20. Hinsdale JG, Engel J j , Wilson DE: Prosta- glandin E in peptic ulcer disease. Prostaglandins 6:495-500, 1974

21. Lowry OH, Rosebrough N J, Farr AL, Randall R J: Protein measurement with the folin phenol reagent. J Biol Chem 193:265-275, 1951

22. Bourne HR: Cholera enterotoxin: Failure of anti-inflammatory agents to prevent cyclic AMP accumulation. Nature (London) 241:399, 1973

Digestive Diseases, Vol. 20, No. 11 (November 1975) 1039