Altered regulation of intestinal ion transport

by enteric nerves in diabetic rats

MARY H. PERDUE AND JOSEPH S. DAVISON

Intestinal Disease Research Unit, Department of Pathology, Faculty of Health Sciences, MeMaster
University, Hamilton, Ontario L8N 325; and Gastrointestinal Research Group, Department of Me&al
Physiology, Faculty of Medicine, University of Calgary, Calgary, Alberta T2N 4N1, Canada

PERDUE, MARY H., AND JOSEPH S. DAVISON. AZtered regu- use this technique to identify abnormalities of ion trans-
lation of intestinal ion transport by enteric nerves in diabetic port regulation by enteric nerves in diabetes.
rats. Am. J. Physiol. 254 (Gastrointest. Liver Physiol. 17):
G444-G449, 1988.-We compared ion transport parameters in METHODS
isolated ileal mucosa from diabetic rats (8 wk after streptozo-
tocin injection) and littermate controls under basal conditions Animals. Diabetes was induced in male Wistar rats
and in response to electrical transmural stimulation (TS). (Charles River, St. Constant, Canada) by intraperitoneal
Stripped ileal mucosa (submucosal plexus intact) was mounted injection of 65 mg/kg body wt streptozotocin freshly
in Ussing flux chambers modified to include stimulating elec- prepared in buffered saline. Controls were sham-treated
trodes on opposite sides of the tissue. Under basal conditions
unidirectional fluxes of Na’ and Cl- were decreased across (injected with saline) littermates of the same age and
mucosa from diabetic rats compared with controls, whereas net initial weight. Animals were housed in metabolic cages
fluxes were not significantly different. TS caused a tetrodotoxin that enabled us to determine food and water intake and
(TTX)-sensitive transient increase in short-circuit current (I=) collect urine. The rats were weighed weekly. Diabetes
that was significantly less in tissue from diabetic than control was confirmed by the presence of polydipsia, hyper-
rats. The muscarinic cholinergic receptor antagonist, atropine, phagia, polyuria, and glucosuria. Rats were studied at 8
significantly reduced the I, response to TS in ileum from wk postinjection because histochemical studies had iden-
control but not diabetic rats. In addition, the noncholinergic tified abnormalities of enteric nerves at this time (15).
component of the response was smaller. The muscarinic ago- Animals were anesthetized with intramuscular urethan.
nist, Urecholine chloride (bethanechol chloride), caused an
Ten-centimeter segments of jejunum, beginning 5 cm
increase in I, that was unaffected by pretreatment with TTX
and was the same in tissue from control and diabetic rats. Our distal to the ligament of Treitz, and of ileum, ending 5
results suggest that the intestinal abnormalities that occur in cm proximal to the ileocecal valve, were measured as
diabetes may include a defect in the regulation of ion transport accurately as possible using a standard length of suture.
by enteric nerves resulting in an abnormal ability to respond (Under these conditions the intestine is in a relaxed,
to luminal and other stimuli. noncontracted state.) The segments of intestine were
removed, flushed with cold buffer, blotted, and weighed.
intestinal epithelium; intestinal transport; enteric nervous sys- Mucosa was removed by scraping firmly with a glass
tem; neurotransmitters slide. Sections were prepared for histology from adjacent
regions of gut and from the mid small intestine. Segments
of ileum were used for ion transport studies. The external
muscularis was stripped by slipping the segment over a
RECENT REPORTS have suggested that diabetic rats de- plastic rod, making a light incision along the mesenteric
velop a neuropathy that involves intestinal nerves (2, 3, border with a dull scalpel blade, and gently peeling the
15-17). In addition, these animals demonstrate abnor- muscle off with fine forceps as described (19). Segments
malities of intestinal function. Changes in motility and were then opened and cut into sheets -2 cm in length.
fluid transport (8,10,23) have been described in diabetes, Histology of these segments demonstrated that the sub-
including an alteration in adrenergic regulation of ab- mucosal plexus was intact.
sorption (5, 6). Evidence for abnormal cholinergic neu- Flux chambers. Four adjacent sheets of ileum from
romuscular transmission has also been presented (18). each animal were mounted in flux chambers that con-
We previously showed that neurotransmitters released tained agar-salt bridges to monitor potential difference
from submucosal plexus neurons by electrical transmural (PD) and inject I,, (20). In addition, Ag-AgCl stimulating
stimulation (TS) of guinea pig small intestine caused an electrodes were incorporated at opposite ends of each
increase in short-circuit current &) and net Cl- ion half-chamber. (A threaded hole was drilled through the
secretion (19). In the present study we examined ion Leucite wall, and a plug attached to a 0.5 cm length of
transport parameters under basal conditions and in re- thick silver wire was constructed to fit the opening. The
sponse to nerve stimulation in preparations of ileal mu- electrodes, which were aligned parallel to the tissue on
cosa from diabetic rats and compared results with those opposite sides, could be removed for cleaning when nec-
from controls. Our objective was to determine if we could essary by unscrewing the plug.) The chamber opening
G444 0193-1857/M $1.50 Copyright 0 1988 the American Physiological Society

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Copyright © 1988 American Physiological Society. All rights reserved.
 ALTERED TRANSPORT REGULATION G445

was rectangular in shape exposing 0.6 cm2 of mucosal Materials. Streptozotocin, urethan, Urecholine chlo-
and serosal surfaces to 10 ml of buffer that contained (in ride (bethanechol chloride), and tetrodotoxin were ob-
mM) 115 NaCl, 8.0 KCl, 1.25 CaC12, 1.2 MgC12, 2.0 tained from Sigma Chemical, St. Louis, MO. Atropine
KH2P04, and 25 NaHC04, pH 7.35, at 37°C. The serosal was from Glaxo Laboratories, Toronto, Canada. Radio-
buffer also contained 10 mM glucose and the mucosal isotopes were purchased from Amersham, Oakville, Can-
buffer contained 10 mM mannitol. The tissue was ada.
clamped at zero volts using a WPI automatic voltage Statistics. Statistical analyses were performed using
clamp (Narco Scientific, Downsview, Ontario, Canada), Student’s paired or unpaired t test when appropriate.
which monitored spontaneous PD and injected an appro-
priate Isc. The I,, was recorded continuously. Determi- RESULTS
nations were begun after an equilibrium time of -15 min
by which time the I,, was stable. In experiments in which Animals. Although diabetic rats consumed more food
ion fluxes were to be measured, radioisotopes, 10 &i of they lost weight (Fig. 1). Initially, weight fell off rapidly
22Na+ or 36C1-, were added to either the mucosal or serosal and then stabilized at a new lower level. At 8 wk the
reservoir, the “hot side”. After 30 min to reach isotopic diabetic rats did not have diarrhea but had large pasty
steady state, four l.O-ml samples were withdrawn from stools. At laparotomy the upper small intestine appeared
the “cold side” at lo-min intervals, and the volume was relatively normal but the ileum, cecum, and colon were
corrected with the appropriate buffer. Two duplicate O.l- distended and atonic. Intestinal weights are shown in
ml samples were removed from the hot side. Unidirec- Table 1. Weights of lo-cm segments (flushed of contents
tional and net ion fluxes were calculated from standard and blotted) of jejunum and ileum and mucosa scraped
equations as described (9). Fluxes were determined dur- from these segments from diabetic rats were significantly
ing three lo-min intervals and reported as overall flux
during the 30-min period. For net flux calculations tissue
pairs were used in which conductance did not differ by
>25%. Na’ and Cl- fluxes are expressed as micro- 450
equivalents per square centimeter per hour, PD as mil-
livolts, and conductance (G) as millisiemens per square
centimeter. To maintain consistency with on-line record-
ings, I,, in tables is expressed as microamps rather than
microamps per square centimeter. G and PD were re-
corded after the equilibration period and at lo-min in- 400
tervals thereafter during the course of the experiment. 0 control
3l
V
Stimulation. Electrical TS was achieved by passing q diabetic
rectangular current pulses from one side of the tissue to z
c3
the other via a stimulator connected to the stimulating w
3
electrodes via the plug (19). The parameters for the
stimulus were 10 mA, 10 Hz, 0.5 ms pulse width, delivered 350
in trains for a total time of 5 s. (During the 5-s stimula-
tion period a stimulus artifact was produced; I,, changes
in response to TS were recorded after the stimulating
current was turned off.) Preliminary experiments indi-
cated that these parameters resulted in consistent re-
sponses that could be reproduced several times over the 300
course of a 60-min experiment. In some experiments 1 I 1 I 1 I 1 I

after the initial I,, response to TS was determined, either 0 2 4 6 8

the neurotoxin, tetrodotoxin (TTX, 5 x 10B6 M), or the TIME (weeks)

muscarinic antagonist, atropine (2 x low6 M), was added 1. Weights

FIG. of control rats and diabetic rats after administra-
to the serosal reservoir. After a 15- to 20-min incubation tion of streptozotocin (65 mg/kg). Values represent means t SE;
number of controls = 11, number of diabetics = 9.
the response to TS was again determined and compared
with the original in the same tissue and to responses in
control tissues from the same animal at the same stage
TABLE 1. Intestinal weights in control and diabetic rats
of the experiment.
Morphometric measurements. Diameter of the small Weight, g
intestine was determined by measuring the width of P
Controls Diabetic
intestinal sheets that had been flattened and stapled (11) (9)
onto cardboard. These segments of intestine were then
Jejunum (10 cm) l.OtO.l 1.4kO.l <O.OOl
fixed in formalin and processed for histology. Mucosal 0.46t0.05 0.70~0.04 <O.OOl
Mucosa
thickness (villus tip to crypt bottom) was measured on Ileum (10 cm) 1.0tO.l 1.5kO.l <O.OOl
at least three representative villus-crypt units per sec- Mucosa 0.46t0.03 0.78~0.09 co.01
tion; only well-oriented sections were used. All measure- Values are means t SE; number of animals is in parentheses.
ments were made by the same investigator on coded Segments of jejunum and ileum were flushed and blotted before being
sections.
1 l

weighed. Mucosa was removed by scraping with a glass slide.

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Copyright © 1988 American Physiological Society. All rights reserved.
G446 ALTERED TRANSPORT REGULATION

MUCOSAL THICKNESS DIAMETER

cl control

800

Je junum Mid-gut Ileum Jejunum Ileum

FIG. 2. Mucosal thickness (A) and diameter of segments of small intestine (B) from control and diabetic rats.
Mucosal thickness was measured from villus tip to crypt bottom of at least 3 representative well-oriented villus-crypt
units per section. Diameter was measured on segments of intestine that had been opened and stapled to cardboard.
Values represent means * SE; * P c 0.01, ** P c 0.001 compared with controls; n = 5-7 for controls, 6 for diabetic
rat tissues.

increased above control values. In addition, mucosal are shown in Table 3. Mucosal-to-serosal flux (Jm+J and
thickness (measured in histological sections from villus serosal-to-mucosal flux (&-& for both Na’ and Cl- were
tip to crypt bottom) and intestinal diameters were greater significantly less across tissue from diabetic rats com-
in intestine from the experimental animals (Fig. 2). pared with controls. Values for net fluxes were not sig-
Zon transport. Values (means t SE) for ZsoPD, and G nificantly different.
in ileum from control and diabetic animals are shown in Responseto 5%. Figure 3 demonstrates the I,, response
Table 2. I,, and PD were not significantly different in to TS in control tissues in the presence and absence of
tissue from diabetic rats, but G was reduced by a small the neurotoxin, TTX. In the absence of TTX after the
but significant amount. In preparations from both 5-s stimulation, the I,, increased rapidly to a peak by 30
groups, G remained stable over the course of the experi- s and then returned to base line within -2-3 min. This
ment; values for I,, and PD decreased very gradually with response could be reproduced several times with only
time. Unidirectional and net fluxes of Na’ and Cl- ions small variation during the course of a 60-min experiment
and, therefore, was used as an index of tissue viability.
The addition of TTX caused the I,, base line to fall in
TABLE 2. Electrical parameters in preparations of deal some preparations; however, when the data from all
mucosafrom control and diabetic rats preparations were analyzed the change was not signifi-
cantly different. In the presence of TTX the I,, response
Control Diabetic P to TS was abolished. Addition of the muscarinic cholin-
ergic agonist, Urecholine at 5 x 10D6M (dose for maxi-
Lc,PA 13.5kO.7 11.3-eO.8 NS
mum response), also caused an increase in Zsc.However,
PD, mV 0.99t0.06 0.93t0.08 NS
G, mS/cm2 34.7k1.7 29.2k2.0 co.05 this response was similar in the presence and absence of
Values are means k SE for short-circuit current (I,), potential TTX (maximum increase in I,, was 18.3 t 2.1 vs. 16.6 t
difference (PD), and conductance (G); tissue segments from 11 rats in 2.1 PA, mean t SE, n = 7). The rapidly changing I,,
each group were studied. following TS made it impossible to measure changes in

TABLE 3. Fluxes of Na’ and Cl- across ileal mucosa from control and diabetic rats

Na’ Fluxes, peg - cmm2 - h-’ Cl- Fluxes, peq - cmm2 ah-’

J- J n-m J net J m-0 J a+m J net

Control 13.99kO.76 13.2720.56 0.91kO.54 16.69t1.41 16.04kO.92 0.71kO.32

Diabetic 9.49k0.98 9.30t1.27 0.62t0.35 10.66k1.12 10.47U.19 0.50t0.44
P co.01 co.05 NS CO.01 CO.01 NS
Values are means t SE. J,,, flux in mucosal-to-serosal direction; J,,, flux in serosal-to-mucosal direction; Jnet, net flux obtained by
subtraction of numerical values. P compares fluxes in tissue from diabetic rats with those from controls; preparations from 11 rats in each group
were studied.

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 ALTERED TRANSPORT REGULATION G447

showing short-circuit current responses

lb& to electrical transmural stimulation (TS,
A urecholine A) and Urecholine in presence (A) and
b absence (B) of TTX. Stripped ileal mu-
TTX
cosa was used. Parameters for TS are as
in text. Concentration of TTX was 5 x
B
10m6M; concentration of Urecholine was
1O-5 M.

b
A A urecholine

ion fluxes under steady-state conditions. However, in As in the study by Lincoln et al. (l5), the rats in our
tissues from control rats, Jmrn for Cl- significantly in- study did not have diarrhea but had abnormal stools that
creased (P < 0.05) from 16.7 t 1.5 to 20.4 t 1.6 peg* were large and bulky. In another experimental investi-
crnD2*h-’ (mean t SE, n = 6) during the 2-min period gation of chronic diabetes, administration of streptozo-
immediately following TS; Jm- for Cl- was unaffected tocin resulted in rats with diarrhea when studied after 6
(17.3 t 2.3 vs. 16.3 t 1.6). mo (5). It is difficult to speculate on the reasons for the
Total changes in Isc in response to TS as well as cho- different symptoms produced. In the latter studies the
linergic and noncholinergic components of the response drug was administered intravenously, whereas in our
are shown in Fig. 4. The noncholinergic component was studies the drug was administered intraperitoneally. In
determined in tissues pretreated with 2 x 10B6M atro- addition, their rats (younger and a different strain) were
pine (dose that eliminated the I,, increase to Urecholine) studied much later after streptozotocin administration.
for at least 15 min and was compared with the original In our studies the base-line I,, of ileal mucosa from
(total) response. In control tissues, atropine significantly diabetic rats was not significantly different from that of
reduced (P < 0.01) the I,, response from 24.2 t 1.7 to controls; net fluxes of Na+ and Cl- ions under basal
17.6 t 1.9 PA (mean t SE, n = 18). (Similar results were conditions were also similar. However, conductance and
found in tissues paired with respect to conductance and unidirectional fluxes of Na’ and Cl- were reduced in
I,, at the beginning of the experiment in which one of diabetic tissue, suggesting some inherent difference in
each pair was treated with atropine.) In tissues from the epithelium. As is the custom in Ussing chamber
diabetic rats, atropine had no significant effect. Com- experiments, data were normalized to serosal surface
pared with responses in control tissues, total, noncholi- area, and no correction was made for the increase in
nergic, and choline@ responses were significantly less mucosal mass in the intestine of diabetic animals. Such
(P < 0.005) in tissues from diabetic rats. To determine a correction would be difficult because we have no infor-
whether the abnormality was in the nerves or in the mation on whether the hypertrophy involved both ab-
epithelium, we examined the response to Urecholine. I,, sorptive and secretory cells or affected one population
increases after addition of 10B6M Urecholine were sim- preferentially.
ilar in ileum from control and diabetic animals. As in our previous studies using guinea pig small
intestinal mucosa (19) electrical TS for 5 s of normal rat
DISCUSSION ileal mucosa caused a transient increase in 1%that ap-
Rats treated with streptozotocin and studied after 8 peared to be due, at least in part, to luminally directed
wk demonstrated weight loss but selective preservation Cl- ion transport. This response was abolished by the
of intestinal tissue as demonstrated by mucosal hyper- neurotoxin, TTX, suggesting that it was mediated by
trophy. Weight and thickness of the mucosa were signif- neurotransmitters released from submucosal plexus neu-
icantly increased. This finding has been reported previ- rons. TTX did not alter the base line Isc, and in the
ously (14, 15, 21). The reason for the hypertrophy is presence of TTX the I, response to Urecholine was
unknown but may be related to decreased glucose utili- unaltered. These results imply that TTX itself does not
zation and hyperphagia (10,14,24). Increased absorptive affect ion transport in this preparation and doesnot have
capacity of the small intestine for hexoses and amino a toxic effect on the epithelium. Studies of the effects of
acids has been described in experimental diabetes (4). TTX in rat colon have demonstrated that, after removal

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G448 ALTERED TRANSPORT REGULATION

total non-cholinergic cholinergic

v TRANSMURAL STIMULATION + URECHOLINE

FIG, 4. Total, noncholinergic, and cholinergic changes in short-circuit current (I,) in response to electrical
transmural stimulation (TS) and Urecholine in ileal mucosa from control and diabetic rats. Values represent differences
between base-line I,, and peak increases, means t SE; n = 18 for responses to TS, 12 for responses to Urecholine; * P
< 0.005 compared with controls. Noncholinergic component was measured after pretreatment of tissues with 2 x 10s6
M atropine for at least 15 min; cholinergic component was numerical difference between total response and
noncholinergic component. Parameters for TS are as in text. Concentration of Urecholine was 10B5 M.

of the submucosal plexus, the neurotoxin had no effect abnormality of cholinergic nerves was found. However,
on ion transport (1). that study examined mainly the myenteric plexus and
The I,, response to electrical nerve stimulation was muscle, and as a histochemical study, only small seg-
reduced in mucosa from diabetic rats. This result may ments of intestine were studied in detail. Functional
have been due to neural or nonneural abnormalities that studies have recently provided evidence for abnormal
cause altered ion transport. If the change was due to a cholinergic neuromuscular transmission in diabetic rat
difference in epithelial mass one would have expected small intestine (18).
the response to be increased in diabetic tissue. In fact, Subtraction of the Isc response to TS in the presence
the overall ability of the epithelium to respond to secre- of atropine from the total response indicated that the
tory stimuli did not appear to be affected, since I,, noncholinergic component was reduced in diabetic rat
incre ases to Urecholine were similar in control and ex- intestine. The nature of the neurotransmitter responsible
perimental rat ileum. Choline@ agonists are believed for this component has not been identified but may
to produce net fluid and Cl- secretion by a direct action possibly be vasoactive intestinal peptide (VIP) or an-
on muscarinic receptors on enterocytes (7, 12, 13, 25). other peptide. Belai et al. (3) recently reported the pres-
Therefore, in diabetes the reduced ion transport re- ence of enlarged VIP-like immunoreactive varicosities in
sponses to nerve stimu latio n were more likely to have the ileum of diabetic rats, changes that they felt m.aY
been due to neural abnormalities. This is not surprising, have been indicative of the early stages of disruption of
since autonomic neuropathy has been described during VIP innervation of the gut. In addition, changes in
diabetes in both experimental animals and in humans calcitonin gene-related peptide-containi .ng neurons have
(11, 15-17, 22). been reported (2). Interestingly, in our studies diabetes
In tissue from control rats the addition of atropine 9at affected the cholinergic and noncholinergic components
a dose that completely prevented the I,, response to of the response to TS unequally. In ileum from diabetic
Urecholine, reduced the 1% response to TS by ~25%. rats the cholinergic component was virtually absent,
This indicates that in normal rats enteric cholinergic thereby increasing the relative contribution of the non-
neurons contribute to the response. We (19) and others cholinergic component. In addition, in other studies of
(7) have demonstrated the presence of a similar cholin- ileal mucosa from rats with chronic diabetes 6 mo after
ergic component in the response to enteric nerve stimu- streptozotocin treatment the I,, response to endogenous
lation in normal guinea pig small intestine. In contrast, adrenergic transmitter released by tyramine treatment
in tissue from diabetic rats atropine had no significant was reduced (5). Perhaps during diabetes destruction of
effect on the response to TS. These results suggest that different types of nerves occurs at different rates under
there is an abnormality of enteric cholinergic neurons in different circumstances with the overall consequence,
this model of diabetes. In the neurochemical and histo- diarrhea or constipation, being dependent on which
chemical study reported by Lincoln et al. (15) no specific nerves have been affected to the greatest degree.

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Copyright © 1988 American Physiological Society. All rights reserved.
 ALTERED TRANSPORT REGULATION G449

Our results provide further evidence that the intestinal 10. GRANNEMAN, J. G., AND E. M. STRICKER. Food intake and gastric
emptying in rats with streptozotocin-induced diabetes. Am. J.
abnormalities that occur in diabetes include a defect in
Physiol. 247 (Regulatory Integrative Comp. Physiol. 16): R1054-
the regulation of ion transport by enteric nerves resulting RlO61,1984.
in an abnormal ability to respond to luminal and other 11. HOSKING, D. J., T. BENNETT, AND J. R. HAMPTON. Diabetic
stimuli. autonomic neuropathy. Diabetes 27: 1043-1054,1978.
12. HUBEL, K. A. Intestinal ion transport: effect of norepinephrine,
pilocarpine, and atropine. Am. J. Physiol. 231: 252-257, 1976.
The authors thank Roger Galbraith and Nancy Dyck for excellent 13. ISAACS, P. E. T., C. L. CORBEIT, A. K. RILEY, P. C. HAWKER,
technical assistance. AND L. A. TURNBERG. In vitro behavior of human intestinal
This research was supported by a grant from the Canadian Foun- mucosa. The influence of acetylcholine on ion transport. J. Clin.
dation for Ileitis and Colitis. These studies were presented in part at Invest. 58: 535-542, 1976.
the Canadian Society for Clinical Investigation Annual Meeting and 14. JERVIS, E. L., AND R. J. LEVIN. Anatomic adaptation of the
were published in abstract form (Clin. Inuest. Med. 9: A56, 1986). alimentary tract of the rat to the hyperphagia of chronic alloxan-
diabetes. Nature Lord 210: 391-393,1966.
Received 20 January 1987; accepted in final form 15 October 1987. 15. LINCOLN, J., J. T. BOKOR, R. CROWE, S. G. GRIFFITH, A. J.
HAVEN, AND G. BURNSTOCK. Myenteric plexus in streptozotocin-
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