Prostaglandin E2: A factor in the pathogenesis of cholera

PROSTAGLANDINS

PROSTAGLANDINE2:

A FAcMlR IN THE PA!iROGERESISOFCROLEXA

Anne Tothill

Department of Pharmacology and Therapeutics The Middlesex Hospital Medical School Windeyer Building Cleveland Street London WlP ?PN

ABSTRACT Injection of cholera toxin -w in vivo into loops of intestine in rats caused the production of an exudate.

This was found to contain

prostaglandinE2 by assay on the rat stomach strip and by thin-layer chromatography. The amounts found ranged from 20 to 40 ng per loop of intestine.

Introductionof 30 ng of prostaglandinE2 into

intestinal loops caused the production of an exudate similar in volume to that found after the introductionof cholera toxin.

These

results indicate that the exudate in cholera is caused by the action of prostaglandinliberatedby the enterotoxin.

It is suggested that

an inhibitor of prostaglamdinrelease could be added to the solutions ueed in treatment for the re+oration of fluids and electrolytes,with the object of blocking the-.action of toxin still present in the intestinal lumen, thereby achieving a.more rapid therapeuticresult.

A&NOWLEDGR4nVT

I am grateful to Mre

A

Gallart for her technical

assistance,and to Upjohn for the gifts of samples of prostaglandin.

JUNE

1976

VOL. 11 NO. 6

925

PROSTAGLANDINS

There is little

i n f o r m a t i o n a b o u t t h e e x a c t œ e c h a n i s m b y which

the outpouring of fluid cholera.

into the intestinal

L o r e e t a l . 1 s t u d i e d sodium f l u x e s i n t h e i n t e s t i n e

concluded that electrolytes

absorption

normally there is s large transport

larger

of fluid

occurs.

net

I n c h o l e r a t h e r e c o u l d be an i n c r e a œ e d

or a decreased lulen to plasma flux or a combination

of t h e two, which r e s u l t s

in anet

loas of fluid

i n t o t h e lumen.

Neogy

2

cholera toxin induces the formation of emcoprotein and/or

œ u c o p o l y s a c c h a r i d e which b i n d s e l e c t r o l y t e s

and s o i n t e r f e r e s

with

exchanges. The l o s s o f f l u i d

and e l e c t r o l y t e s

c o u l d be due t o t h e a c t i o n o f

an e n d o g e n o u s s u b s t a n c e s u c h a s p r o s t a g l a n d i n , precursor

in large asounta in the cells

cholera toxin is not absorbed, its membrane.

indicating

that

have s i m i l a r

effects,

experimental results

to support thia

review of the evidence,

prostaglandin

Since

prostaglandin

and h a v e t a k e n t h i s

and

as

B e n n e t t , 8 however~ s u g g e s t e d t h a t c h o l e r a

prostaglandin,

Nevertheless

wall.

a c t i o n must take place a t t h e c e l l

toxin œight liberate

the previously

b u t h e d i d n o t p r o d u c e any hypothesis.

concluded that

quoted authors all

Sharp, this

9

in a

is unlikely.

atteœpted to isolate

but were not s u c c e s a f u l .

In view of the theoretical directly

of the intestinal

t h e s e two s u b s t a n c e s h a v e a common pathway t h r o u g h t h e

c y c l i c ~qP s y s t e m . 3 ' 4 ' 5 ' 6 ' 7

critical

which occura a s a

S e v e r a l a u t h o r s have p o i n t e d o u t t h a t

cholera enterotoxin

possibility

that prostaglandins

involved in the pathogenesis of chol e ra it

to endeavour to establiah

926

and a

f l u x from t h e lumen t o t h e p l a s m a , s o t h a t

p l a s m a t o lumen f l u x ,

suggested that

and

o f w a t e r and

f r o n t h e p l a s m a i n t o t h e lumen o f t h e i n t e s t t n e

very slightly

fluid

lumen t a k e a p l a c e i n

this

possibility

are

seemed o f i n t e r e s t

or definitely

JUNE 1976

exclude it.

VOL. 11 NO. 6

PROSTAGLANDINS

MATERIALS AND METHODS

The t o x i n u s e d was c h o l e r a f i l t r a t e extract

of a culture

was s e p a r a t e d addition

f r o œ t h e a g a r and b a c t e r i a

to toxins

destroying

it

enzyme).

reconstituted

contains

grown on a g a r .

The t o x i n

and t h e n f r e e z e - d r i e d .

In

t h e enzyme n e u r a œ i n i d a s e ( r e c e p t o r

The ampoule o f f r e e z e - d r i e d

by t h e a d d i t i o n

by t h e a d d i t i o n solution

of cholera vibrios

(Duphar) which i s a crude

material

o f 2 ml o f 0 . 9 ~ s a l i n e ,

o f 0 . 1 ml o f O. IM c a l c i u œ c h l o r i d e

t h u s p r e p a r e d was u a e d i m m e d i a t e l y .

was

and was a c t i v a t e d

solution.

The

Samplee o f p r o s t a g l a n d i n

and F2« were p r o v i d e d by U p j o h n .

The m a t e r i a l s

distilled

o f 1 mg/ml and s t o r e d a t 4°C.

water at a concentration

Working d i l u t i o n s

f o r 72 h o u r s ,

by a m o d i f i c a t i o n

Sprague-Dawley female rats

t h e n r e d 5~ g l u c o s e i n 0 . 9 ~ s a l i n e .

was made i n t o t h e a n t e r i o r

drawn t o t h e s u r f a c e .

instances

At t h e end o f t h i s

with ether.

The i n t e s t i n e

a b d o m i n a l w a l l and t h e i l e u m was

a t each end,

l e a v i n g a s p a c e o f 2 cm

was t h e n c u t b e t w e e n t h e l o o p s .

t h e b l o o d s u p p l y was a U o w e d t o r e m a i n i n t a c t .

2 l o o p s w e r e made i n each a n i m a l .

Each l o o p was i n j e c t e d

0 . 4 ml c h o l e r a t o x i n w i t h 0 . 1 ml o f 0 . 1 M c a l c i u œ c h l o r i d e and 0 . 1 ml c a l c i u m c h l o r i d e was t h e n r e p l a c e d ,

solution

for control

Ninety minutes later

Any e x u d a t e p r e s e n t

in the test

a s y r i n g e and t h e volume was r e c o r d e d .

J U N E 1976

purposes.

In all

Not more t h a n with either o r 0 . 4 ml s a l i n e The i n t e s t i n e

t h e abdomen was s u t u r e d and t h e a n i œ a l s were a l l o w e d t o

recover consciousness. ether.

A email

Loops o f i l e u m a b o u t lO cm i n l e n g t h were t i e d

o f f f r o œ each o t h e r w i t h l i g a t u r e s between loops.

o f t h e œethod o f

w e i g h i n g 250-300 g w e r e s t a r v e d

p e r i o d t h e a n i œ a l s were 1Lghtly a n a e s t h e t i z e d incision

were d i a a o l v e d i n

were p r e p a r e d as r e q u i r e d .

E x u d a t e s were p r o d u c e d i n r a t e A z i z e t a l . 10

E2

VOL. 11 NO. 6

t h e animals were k i l l e d

or control

with

l o o p s was removed w i t h

The e x u d a t e s w e r e t h e n t r e a t e d

927

PROSTAGLANDINS

11 by the method of Green and Samuelson for the extractionof prostaglandin. They were adjusted to pH 3 with 0.1 N hydrochloricacid and extracted into ethyl acetate. evaporator.

This was then evaporated to dryness in a rotary

Some of the dry residues were fe-dissolvedin 0.1 ml of

Ringer solution and examined for the presence of prostaglandinby the 12 rat stomach strip method.

Other residues were dissolved in 0.02 ml

of ethyl acetate and examined for the presence of prostaglandinby 11 thin layer chromatographywith the Al solvent system. Reference samples of authentic prostaglandinE2 and Fzo,were run at the same time using the same volume of 0.02 ml for spotting. separationswere carried out in duplicate. developed with ammonium molybdate.

The

One plate of each pair was

The intensityof the spots which

appeared was compared with that of known amounts of authentic prostaglandin in order to fom an approximateestimate of the amount present.

On the

other plates the area correspondingto the position of the prostaglandin spot was scraped off and extracted with 3 separate washings of 5 ml of methanol, which were combined and reduced to dryness in a rotary evaporator.

The dry residue was dissolved in 0.1 ml of Ringer solution

and assayed for prostaglandincontent on the rat stomach strip.

Volumes

of 2 ml of the toxin solution were extracted in the came way and the redissolved residue was also examined by chromatography. In further experiments in rats, various amounts of an authentic solution of prostaglandinE2 were injected into loops of intestine, which were examined 90 minutes later for the presence of exudate.

RESULTS In preliminaryexperimentsthe animals were kept under continuous anaesthesiawith pentobarbitoneor urethane.

928

In no instance was an

JUNE 1976

VOL. 11 NO. 6

PROSTAGLANDINS

exudate obtained.

The a n a e s t h e t i c was t h e r e f o r e c h a n g e d t o e t h e r and

t h e a n i s m l s were l i g h t l y after

a n a e s t h e t i z e d and a l l o w e d t o r e c o v e r i m n e d i a t e l y

t h e o p e r a t i o n and r e m a i n e d c o n ~ i o u s

I t was o n l y t h e n t h a t p o s i t i v e aince it

until

the time of sacrifice.

r e s u l t 8 were o b t a i n e d o

E t h e r was c h o s e n

i s r a p l d l y e x c r e t e d v i a t h e l u n g 8 t h u s m i n i m i z i n g t h e œeœbrane

stabilizing

effect

e x e r t e d by a l l

anaenthetics.

I n a d d i t i o n a t t e m p t n were made t o t e s t i n i a o l a t e d p r e p a r a t i o n 8 i n an o r g a n b a t h .

t h e a c t i o n of c h o l e r a t o x i n E n t e r o t o x i n was i n t r o d u c e d

t o t h e b a t h and t h e ~ a r r o u n d i n g f l u i d e v e n t u a l l y removed t o be a s s a y e d f o r p r o s t a g l a n d i n a s had b e e n p o n s i b l e i n t h e c a s e o f t h e r a t u t e r u s . 1 2 ' 1 3 Only n e g a t i v e r e s u l t 8 were o b t a i n e d . Cholera flltrate o u t i n 19 a n i m a l s . 0 . 8 t o 1.4 ml.

was i n j e c t e d

i n t o 19 l o o p s i n e x p e r i m e n t 8 c a r r i e d

F~mdate a p p e a r e d i n 18 l o o p s i n amounts v a r y i n g from

I n t v o i n n t a n c e s i t c o n t a i n e d b l o o d , and was t h e r e f o r e

discarded.

Detection of prostaKlandin in the exudate B i o l o ~ i c a I assa~r:

The d r i e d s a m p l e was r e c o n n t i t u t e d

i n 0 . 1 œl R i n g e r

s o l u t i o n and added t o an o r g a n b a t h c o n t a i n i n g a r a t 8tomach 8 t r i p . all

In

c a s e s a c o n t r a c t i o n o c c u r r e d which r e n e m b l e d t h a t p r o d u c e d by

pro•taglandin.

In eachrun

t h e stomach s t r l p

was a l s o t r e a t e d w i t h

a o l u t i o n s o f p r o n t a g l a n d i n E2 o f known c o n c e n t r a t i o n s ,

and c a l c u l a t i o n s

b a s e d on t h e h e i g h t s o f c o n t r a c t i o n o b t a i n e d i n d i c a t e d t h a t t h e s a m p l e s of i n t e s t i n a l

e x u d a t e c o n t a i n e d p r o n t a g l a n d i n i n a m o u n t s v a r y f n g froœ

20 t o 40 n g p e r e a m p l e . Thin la~er chrosmtography: of t h e t r e a t e d

asmaya o f e t h y l a c e t a t e s o l u t i o n s

e x t r a c t 8 and r e f e r e n c e s a m p l e s o f known c o n c e n t r a t i o n o f

p r o s t a g l a n d i n s E2 and F ~ ,

J U N E 1976

In parallel

all the samples gare spots corresponding in

VOL. 11 NO. 6

929

PROSTAGLANDINS

position

to that

of the reference

sample of prostaglandin

E2 .

Meaaurements

o f t h e v a l u e g a r e a mean R . F . v a l u e o f 0 . 6 4 ~ 0.0fi (12 o b s e r v a t i o n s ) l close

agreement with the aecepted value of 0.62.11

duplieate

plates

were r u n a n d t h e c o a t i n g

to the stomach etrip. presence

In all

of prostaglandin

c o n f i r m e d by b i o l o g i c a l

Production

by c h o l e r a role,

extracted

i n t h e aame way no p r o s t a g l a n d i n

prostaglandin

can be detected

does not neceeaarily

of the toxin.

of proata~landi n

mean t h a t

of prostaglandin

This is unlikely,

it

cauaes the exudate it

prostaglandin

into

intestinal

is neceasary

in the exudate produeed it

plays an aetiological

œight be a side

since

e x u d a t e s e a n b e p r o d u c e d by p r o s t a g l a n d i n , that

it

were therefore

0 . 3 œl o f 0 . 9 ~ s a l i n e solution.

9 but before

that

t o show t h a t

Control

930

injected

with the addition

as8uming

will

found.

of the

intestinal

injection

w i t h 30 n g o f p r o s t a g l a n d i n

of produce Loops of

E2 i n

o f 0 . 1 ml o f M c a l c i u m c h l o r i d e loops contained

exudatea

in

f r o œ 0 . 8 t o 1 . 5 ml (mean 1 . 1 5 ~ 0 . 2 1 ml) ( 1 0 o b s e r v a t i o n a ) ,

i 8 , v o l u m e s o f t h e same o r d e r a s t h o s e o b t a i n e d

of toxin

finally

loops in the amounte detected

On e x a m i n a t i o n 90 m i n u t e a l a t e r

volumes ranging

effect

i s known t h a t

e x u d a t e i n v o l u m e s o f t h e aame o r d e r a s t h o e e a c t u a l l y intestine

was t h u s

œeans.

aince the liberation

action

and t h e

by c h r o m a t o g r a p h y .

that

toxin

and a p p l i e d

was o b t a i n e d ,

i n t h e e h r o m a t o g r a p h and m a t e r i a l

o f e x u d a t e by t h e i n ~ e c t i o n

The f a c t

correaponding

as described

caaes a contraction

In samples of enterotoxin could be detected

With 5 a a m p l e s

in the position

to the developed apota were acraped off~ treated

in

in amounts aufficient loops contained

to liberate

after

the injection

20-40 ng of prostaglandin.

no f l u i d .

J U N E 1976

VOL. 11 NO. 6

PROSTAGLANDINS

DISCUSSION

The r e s u l t s

show t h a t

contains prostaglandin m i g h t be i n c i d e n t a l , latter

of the E serieso

and t h i s

since it

It

i s known t h a t

A possible prostaglandin

liberated explanation

is that

that

of the failure

o f some a u t h o r s 4 ' 7 t o f i n d

they used anaesthetized

since the production of prostaglandin

phenomenon and a n a e s t h e t i c s or release

stabilize

animals.

are rapidly tissues

from t h e t i s s u e .

no e x u d a t e c o u l d be

unless they are present

It

destroyed during the extraction

used purified with it.

JUNE 1976

difficult

to isolate

in very large quantities. p r e s e n t work i t

t o x i n and h a v e drawn t h e i r

prostaglandin,

VOL. 11 NO. 6

from

In view of the is possible

that

e x p e r i m e n t s were

from t h e t i s s u e s .

t h e a u t h o r s who a t t e m p t e d t o i s o l a t e

and i s o l a t e d

to isolate

i s w e i l known t h a t p r o s t a g l a n d i n s

Hudson e t a l . 4 d i d p r e l i m i n a r y

crude extract

and f a i l e d

appearing during the course of their

all

was r e a s o n e d

i s t h o u g h t t o be a m e i b r a n e

preparations

small amounts found in exudates in this

In addition

It

membrane, t h e y would p r e v e n t any

d e s t r o y e d and a r e n o t o r i o u s l y

any p r o s t a g l a n d i n s

Preliminary

from t a k i n g p l a c e .

Kimberg et a l . 5 used i s o l a t e d prostaglandin

actually

by t h e a c t i o n o f e n t e r o t o x i n .

o b t a i n e d when t h e a n i m a l s were k e p t u n d e r a n a e s t h e s i a .

synthesis

produce

the exudate in c h o I e r a i s caused

e x p e r i m e n t s i n t h e p r e s e n t work had shown t h a t

that

will

of t h e amounts of p r o s t a g l a n d i n

is proposed therefore

by p r o s t a g I a n d i n

prostaglandin

The

was c o n f i r m e d by t h e p r o d u c t i o n o f e x u d a t e i n t h e

e x p e c t e d v o l u m e s by i n j e c t i o n found.

The p r e s e n c e o f p r o s t a g l a n d i n

o r i t c o u l d be t h e c a u s e o f t h e ex~~date.

i s more l i k e l y ,

an e x u d a t e ,

t h e e x u d a t e c a u s e d by c h o l e r a e n t e r o t o x i n

prostaglandin

c o n c l u s i o n s from r e s u l t s experiments in rabbits

obtained with a

hut discounted these results

931

PROSTAGLANDINS

when i t was found t h a t p r o s t a g l a n d i n was n o t 1 L b e r a t e d by t h e p u r L f i e d toxin.

The c h o l e r a v i b r L o p r o d u c e s t h r e e t o x L n s , an e n d o t o x i n whLch i s

1 L b e r a t e d from c e l l s

whLch h a v e u n d e r g o n e l y s i s ,

and a h e a t - l a b L l e e x o t o x i n . 15

cholera filtrate

sLnce m e t h o d s f o r p u r i f y i n g i t have

The l o g i c a l i n t e r p r e t a t i o n liberates

prostaglandin,

i s n o t t h e f a c t o r Lnvolvedo

of theLr results

is that

but that the heat-labile

exotoxin

F u r t h e r work s h o u l d t h e r e f o r e be d L r e c t e d

t o s t u d y i n g t h e e f f e c t o f t h e o t h e r two t o x i n s o p r o d u c e s t h e enzyme n e u r a m i n L d a s e . 16 a c i d froœ s L a l o p r o t e i n s ,

exotoxtn,

The p u r i f L e d t o x i n which t h e s e w o r k e r s

have u s e d i s p r o b a b l y t h e l a t t e r , been publLshed.

a heat-stable

The c h o l e r a v i b r i o a l s o

This splits

off N-acetylneuraminic

and t h u s p r o f o u n d l y m o d i f i e s t h e s t r u c t u r e

membranes, a p r o c e s s which c o u l d w e l l l e a d t o t h e l i b e r a t i o n

of

of prostaglandins.

The c o n c l u s i o n t o be drawn from t h e p r e s e n t work i s t h a t d L a r r h o e a i n c h o l e r a i s c a u s e d by p r o s t a g l a n d i n l i b e r a t e d intestinal

from t h e c e l l s

of the

mucosa by a p r o d u c t o f t h e m e t a b o l i s m o f t h e c h o l e r a v i b r i o ,

and t h a t t h e f a c t o r r e s p o n s i b l e h a s n o t y e t b e e n L d e n t L f i e d . S a l i n e i s o r t e n gLven by mouth i n t h e t r e a t m e n t o f c h o l e r a i n t h e field,

and i t would be r e a s o n a b l e t o add an i n h i b i t o r

release as a rational

of prostaglandin

method o f p r e v e n t i n g f u r t h e r p r o d u c t i o n o f e x u d a t e

by t h e a c t L o n o f t o x i n s t i l l

present in the intestinal

lumen, t h u s

a d d i n g a t h e r a p e u t i c coœponent t o t h e a c c e p t e d method o f s y m p t o m a t i c treatment.

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VOL. 11 NO. 6

PROSTAGLANDINS

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933