THE JOURNAL OF INFECTIOUS DISEASES
•
VOL. 125, NO.5'
MAY 1972
© 1972 by the University of Chicago. All rights reserved.
Effect of Cholera Toxin on Jejunal Osmoregulation
of Mannitol Solutions in Dogs
From the Cholera Research Laboratory,
Dacca, Bangladesh, (formerly East Pakistan)
D. R. Noon, K. Ally, R. Hare, and K. Hare
It is well known that the small intestine can
rapidly adjust the osmolarity of its contents [1, 2].
The mechanisms involved are chiefly addition of
sodium and removal of water in the case of solutions with osmolarity lower than that of plasma or
addition of water and removal of solute in the
case of hypertonic solutions [3, 4]. The present
investigation was undertaken to examine the way
in which osmoregulation was affected by cholera
toxin, which causes net movement of water and
sodium into the intestinal lumen [5].
Received for publication December 17, 1971.
The Pakistan-SEATO Cholera Research Laboratory is
a part of the SEATO Cholera Research Program in
Dacca. It was formerly the Pakistan-SEATO Cholera Research Laboratory and was supported by the U.S. Agency
for International Development, Department of State; the
National Institutes of Health and the National Communicable Disease Center of the Department of Health,
Education and Welfare; and the governments of Pakistan, the United Kingdom, and other SEATO nations.
The N.I.H. Cholera Advisory Committee coordinates the
research program.
These studies were supported in part by research
agreement no. 196802 between the National Institutes of
Health and the Pakistan-SEATO Cholera Research Laboratory, as well as by research agreement no. RO 7
A110048 11 between the National Institutes of Health
and the Johns Hopkins University Center for Medical
Research and Training, Baltimore, Maryland.
Please address requests for reprints to Dr. D. R. Nalin,
CMRT, 550 N. Broadway, Baltimore, Maryland 21205.
528
Methods
Adult mongrel dogs weighing 10-15 kg were
anesthetized with iv sodium pentobarbitol (20
mg/kg) . Small additional doses were given as
needed to maintain light anesthesia. Body temperature of the experimental animals was maintained
at 37-38 C. At laparotomy two adjacent 20em jejunal loops were identified, beginning 15
ern from the ligament of Treitz. The loops were
intubated with multipore plastic sampling tubes
via a 1-3-mm puncture in an adjacent segment
(figure 1). This method is a modification of that
used by Ingraham and Code [6, 7]. After intubation the loops were tied off at both ends, replaced
into the peritoneal cavity, and rinsed with normal
saline at 37 C. Then hypotonic, isotonic, or hypertonic mannitol solutions containing phenolsulfophthalein (PSP) or polyethylene glycol (PEG)
as a nonabsorbable marker were injected into the
loops through the sampling tube.
Samples of venous blood for determination of
sodium and osmolarity were obtained immediately
after induction of anesthesia and at regular intervals during the studies. The values obtained
remained constant and normal during the studies.
Both long-term and short-term studies were
carried out before and 3 hr after treatment with
10 ml of N.I.H. lot 001 cholera toxin (Wyeth)
(table 1). This material is a lyophilized, crude
culture filtrate, which was reconstituted with distilled water and deep frozen at -19 C for three
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This study was done to determine how cholera toxin affects osmoregulation. Mannitol solutions of various concentrations were instilled into jejunal loops of anesthetized dogs, before and after treatment with toxin. Solutions of high osmolarity
within the loops approach the osmolarity of plasma more slowly than do dilute
solutions. After treatment with toxin adjustment is slower, and the concentration
of Na + is higher in all cases. Net movement of water and solute was out of the
loop, or zero, in control studies, but always into the loop after treatment with
toxin. The mechanism of adjustment in the control loop seems to be primarily
absorption, while after administration of toxin it is primarily secretion. It is concluded that the relative inefficiency of the latter process accounts for the slower
regulation.
Cholera Toxin and Osmoregulation
529
to five weeks. Within this interval, the ability of
the toxin to produce a typical diarrheal response
in the jejunal loops did not change.
The culture filtrate was diluted at the time of
use by addition to the freshly thawed material of
10 ml of normal saline at room temperature, making a total volume of 20 ml. This volume was
then injected into each loop and left in for 10
min. After 10 min the residue was withdrawn, and
Table 1. Design of experiments for study of effect
of cholera toxin on solutions within jejunal loops of
dogs.
Solution in loop
Experiment
Long-term
No. dogs
No. loops
No. studiest
Short-term
No. dogs
No. loops
No. studies
Hypotonic
Isotonic
Hypertonic
3
4*
8
2
3*
6
3
3
6
5:1:
3
3
6
3
3
6
7
14
NOTE. Studies with He-mannitol were carried out in
two additional animals.
* There were two loops in one of the dogs.
t Half of the studies represent the control periods,
the other half the periods during which toxin was present.
:/: The fluid in the loops of two of the dogs was distilled water.
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Figure 1. Method of injection into canine jejunal
loops. Solutions were injected into adjacent loops
and withdrawn at specified intervals.
the loop was rinsed with 20 ml of 0.9% NaCl.
The loop contents were thereafter aspirated every
hr for 3 hr to ascertain the presence of typical
rice-water fluid and to prevent excessive distention. Fluid was generally aspirated by the second
hour and increased further by the third hour when
studies began. The volume aspirated was usually
under 15 ml at 2 hr and 15-30 ml at 3 hr.
In the long-term studies, 20 ml of test solution
was injected into the loop, and the luminal contents were sampled after 5 min and then every
10 min for measurement of changes in concentration. To ensure mixing, a small volume of air
equivalent to the volume of the samples withdrawn was injected before each sample was taken.
In the short-term studies, 20 ml of test solution
was injected into the loop; immediate mixing was
carried out by withdrawal and rapid reinjection
of 15 ml; 2 ml was then withdrawn as a zero-time
specimen. The loop was emptied after 10 or 15
min to measure terminal concentrations and net
fluxes of sodium and water. Before the next test
solution was injected, the loops were rinsed with
10 ml of 0.9% NaCI (37 C). Total recovery of
this lO-ml rinse helped to rule out the presence
of residual solution and assured complete drainage before injection of another test solution into
the loop.
The range of osmolarities of solutions in the
long-term studies was 115 to 575 milliosmols/
liter; in the short-term studies it was 0 to 430
milliosmols/liter. Osmolarity was measured in a
vapor-pressure osmometer (Hewlitt-Packard) calibrated with NaCI solutions.
At regular intervals samples were removed for
determination of osmolarity, sodium, and PSP or
PEG. Each concentration of each solution was
tested in at least three jejunal loops. Mannitol labeled with HC was counted on a Beckman liquidscintillation counter. Sodium was measured on a
Baird Atomic flame photometer. PSP was measured colorimetrically after alkalinization, and
PEG by a modification method of Hyden [8].
Changes in volume were calculated from concentrations of the markers.
Exploratory studies, not presented here, showed
that loops adjacent to toxin-treated loops were not
suitable as controls due to variation in response
from one loop to another. In contrast, the variation in response of any individual loop to osmotic
challenge over 4 hr was insignificant. Therefore,
Nalin et al,
530
each loop was used as its own control. The results
used for comparison and presented here were
those obtained in the same loop before and 3 hr
after treatment with toxin.
Results
ter and solutes in the long-term studies of hyperand hypotonic solutions that resulted in the osmoVOLUME
CHANGe
30
mL
20
&00
LOOP FLUID
~x
~x
~
~~_o
10
l'nOs/uter
x
INCREAs£t
0
500
D£CJtEASG~
...-----x
__ -)(--
~~
"'-0- __
-0--_-0
-10
SOLUTE
CHANGE
400
P<O·OOl
mO,
6
x
x/
5
___ ~_IIlI--~--~
x.....----..
8
4
.PLASMA OSMOLAIUTY
/
.
/
/
3
2
///1
/
/
.>
.r >:
/
300
x
x
~--6---~~o ~o
200
~o
INCA'EASl-t
0
15
100 ...........---...---........- - r - - - r - - - ,
5
15
25
35
45
55
MINUTES
Figure 2. Change in osmolarity of fluid in canine
jejunal loops with time, both before and after toxin.
0= control; X = 3 hr after toxin.
25
minutes
55
45
55
&5
Figure 3. Cumulative net changes in solutes and
volumes within canine jejunal loops in long-term
studies. (- - -) =Hypotonic solution 110 milliosmols/liter; (--) = hypertonic solution 575 milliosmoleslliter; 0 = control; X = 3 hr after toxin.
Downloaded from http://jid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 17, 2016
When nonisotonic solutions were in the loops, the
adjustment of osmolarity within the loop to that
of plasma was slower after treatment with toxin
(figure 2). The difference between periods before
and after administration of toxin was greater in
the case of hypertonic solutions. When isotonic
solutions were in the loops, osmolarity increased
to the same extent both before and after toxin.
Both before and after exposure to cholera toxin,
adjustment of osmolarity was faster with hypotonic than with hypertonic solutions.
Concentration of sodium was always higher
after toxin than in control periods (table 2), especially in the case of hypertonic solutions, in which
the difference in concentration of sodium accounted for the difference in osmolarity.
Figure 3 shows cumulative net balances of wa-
Table 2. Concentration of sodium in canine jejunal
loops (mEq/liter) before and after treatment with
toxin.
Time (min)
Solution
in loop
25
35
45
55
5
15
Hypotonic
27
56
81
89
QNS*
control
Hypotonic
toxin
33
65
87
101
Isotonic
53
control
16
36
63
76
84
Isotonic
toxin
27
50
68
83
95
111
Hypertonic
32
40
control
18
48
59
72
Hypertonic
44
toxin
33
63
84
93
72
* QNS = quantity not sufficient.
Cholera Toxin and Osmoregulation
531
LOOP
SOLUTE
CHANGE
0·30
0.25
LOOP
VOLUME
CHANGE
0·2
ml/mil1.
0.1
/ir'CitEASE
lJEC~EA%
t
t
O·j
0·2
0·+
0·5
0·6
P<O·Ol
0·7
Figure 5. Change in volume of solution in canine
jejunal loops in short-term studies before and after
administration of cholera toxin.
0 = control,
mm = 3 hr after toxin.
absorbable. As higher luminal-fluid osmolarities
were infused, the change in rate of net water
movement was much greater during control periods than after toxin.
Discussion
mOs/"l-"Wil'V
020
0·15
0·10
0·05
INCUASEt
- - - - 0 +-..JJJ.J..lL-.L-J.J.W.1.-=LllUIL......,,--j'W.ll--,--I-Wll
lIECA'EAS£~
0·05
r <0·01
110
~
OSMOLARITY
LJiQJ LillJ
mOs/titre.
Figure 4. Change in concentration of solutes in canine jejunal loops in short-term studies, before and
after cholera toxin.
0
control; [JJI]
3 hr
after toxin.
=
=
Both before and after administration of toxin, net
movement of solute varies directly with the concentration of solute in the loop. The mechanism
of the response differs in that, when initial osmolarity of luminal fluid is more hypertonic, control
loops adjust by increasing solute absorption, while
after treatment with toxin the same loops adjust
by decreasing net secretion of solutes. Toxintreated loops can decrease net secretion of solutes
as initial luminal fluid osmolarity rises, but net
secretion is always more than during comparable
control periods.
Under the conditions of the study, net movements of solute chiefly reflect movement of sodium into and solutes out of the loops. In control
periods higher concentrations were reduced more
slowly than lower concentrations, and the osmolarity of isotonic solutions actually increased. This
Downloaded from http://jid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 17, 2016
larities shown in figure 2. During control periods,
water entered the hypertonic solution but was absorbed from the hypotonic solution. In the period
after toxin was given, water always entered the
loop regardless of initial osmolarity. Solute entered both hypotonic and hypertonic mannitol solutions, but much more entered after exposure to
toxin.
Figure 4 shows net change of solutes in the
short-term studies. During the control period, net
movement of solute was into hypotonic solutions
but out of hypertonic solutions. After treatment
with toxin, net movement of solute was always
into the loop.
Solute entering loops during control or toxintreatment periods was chiefly sodium salts. Solute
leaving the loops was chiefly mannitol, as suggested by net balance of solutes in the short-term
studies and proven by 97% recovery of the 14C_
label in the urine of two other dogs within 3 hr
of injection into the lumen.
Figure 5 shows net balance of water in relation
to initial osmolarity in the same studies as figure
4. At all concentrations of mannitol tested in
these studies net movement of water was out of
the loops in control periods, and into the loops
after toxin. Distilled water was rapidly absorbed
before toxin, but after toxin it was essentially non-
532
change in net movement of water after toxin may
be due to a relatively lower efficiency of secretion
than absorption as a mechanism of osmoregulation.
References
1. Visscher, M. B., Roepke, R. R, Lifson, N. Osmotic
and electrolyte concentration relationships during
the absorption of autogenous serum from ileal
segments. Am. J. Physiol. 144:457-463, 1945.
2. Grim, E. Water and electrolyte flux rates in the duodenum, jejunum, ileum and colon, and effects of
osmolarity. Am. J. Dig. Dis. 7: 17-27, 1962.
3. Kalser, M. H., Williams, R M., Peterson, A. R,
Smitherman, B. Relation of osmolality to jejunal
sorption of water, cations and glucose in humans.
Gastroenterology 46:260-266, 1964.
4. Vogel, G., Stoeckert, I. Functional differences between the small intestine and the colon-the
different reactions of the intestinal mucosa to solutions of nonabsorbable solutes of increasing osmolarity. [in German] Pfluegers Arch. 303: 262273, 1968.
5. Swallow, J. H., Code, C. F. The effect of cholera
toxin on water and ion fluxes in the canine bowel.
In Proceedings of the cholera research symposium. Honolulu. USPHS publication no. 1328,
Washington, 1965, p. 283-285.
6. Ingraham, R. C., Visscher, M. B. The production
of chloride-free solutions by the action of the intestinal epithelium. Am. J. Physiol, 114:676-680
1936.
7. Code, C. F., Bass, P., McClary, G. V., Jr., Newnum,
R L., Orvis, A. L. Absorption of water, sodium
and potassium in small intestine of dogs. Am. J.
Physiol. 199:281-288, 1960.
8. Hyden, S. The recovery of polyethylene glycol after
passage through the digestive tract. Kgl, Lantbrucks-Hogskol, Ann. 22:411-424, 1956.
Downloaded from http://jid.oxfordjournals.org/ at Penn State University (Paterno Lib) on May 17, 2016
must mean that sodium enters more rapidly than
mannitol can leave.
In the case of hypotonic solutions, there is net
absorption of water from control loops but net
entry into loops after toxin. Increase in osmolarity
is slower after toxin exerts its effect, even though
the rate of sodium movement into the loop is
higher. This must be due to the decrease of net
water absorption after toxin takes effect.
Isotonic solutions are handled in very much
the same way as hypertonic solutions except that
water balance in the control loops is nearly zero.
It is interesting that both before and after administration of cholera toxin, the osmolarity of isotonic luminal solutions rises above that of plasma
and remains elevated for as long as 55 min.
The mechanism of reduction in osmolarity of
hypertonic solutions is qualitatively the same before and after toxin, but the rate of change is
slower after toxin. This is chiefly due to the fact
that the concentration of sodium is maintained at
a higher level in the toxin-treated loop, either because it enters the loop more rapidly than water
or because it leaves the loop more slowly than
water. Slower change in osmolarity after toxin
may also be due to depressed absorption of solute
other than sodium, such as mannitol.
In control periods, absorption of water falls as
the activity of water in the loop falls (i.e., as
osmolarity rises). After treatment with toxin, net
water flux is less dependent on water activity:
water always enters the loop, and only slightly less
water enters the hypotonic solution as compared
to the hypertonic solution. As initial osmolarity
within the lumen rises, the relatively smaller
Nolin et al,