22

Indian
Tripathi,
J Physiol
Gangopadhyay,
Pharmacol Sharma,
2016; 60(1)
Kar and
: 2229
Mandal

Indian J Physiol Pharmacol 2016; 60(1)

Original Article

In Vitro Evaluation of Carbachol and Endothelin on Contractility of

Colonic Smooth Muscle in Hirschsprungs Disease
Brijesh K. Tripathi 1, A. N. Gangopadhyay 1, S. P. Sharma 1,
Amrita G. Kar 2 and Maloy B. Mandal 3*
Departments of Paediatric Surgery 1 , Pathology 2 and Physiology 3 ,
Institute of Medical Sciences,
Banaras Hindu University, Varanasi, U.P.

Abstract
Background: The hypomotility of colon observed in Hirschsprungs disease (HD) has been attributed to
congenital aganglionosis only. So far, it is not clear whether the contractility of colonic smooth muscle in
this condition is altered or not. Therefore, the present study attempted to understand the contractile status
of colonic segments of HD patients by examining carbachol and endothelin (ET-1) evoked colonic smooth
muscle contractions in vitro .
Methods: Contractile responses were recorded from strips of colonic segments obtained from HD patients,
using organ bath preparations. Cholinergic agonist carbachol and ET-1 along with their antagonists were
used to evoke contractile responses. Thereafter, the samples were histopathologically confirmed for HD.
Results: Colonic strips of HD did not show any spontaneous contractions but responded to carbachol and
ET-1 to a lesser extent. In HD, response of carbachol was blocked by atropine and hexamethonium by
nearly 73% and 50% respectively. ET-1 induced contractile responses were blocked by ET-A and ET-B
antagonist up to 40%, signifying the possible role of ET-A and ET-B receptors in HD colon contractility.
Conclusion: As evidenced by lack of spontaneous contractions and impaired carbachol and ET-1-induced
contractile responses, it is concluded that, in addition to aganglionosis, decreased contractility of colonic
smooth muscle may contribute to hypomotility observed in patients with HD.

Introduction
Hirschsprungs disease is characterised by intestinal
obstruction due to hypomotility of colon and
*Corresponding author :
Maloy B. Mandal, Department of Physiology, Institute of
Medical Sciences, Banaras Hindu University, Varanasi,
U.P.; Email address [email protected]
(Received on April 12, 2015)

congenital absence of ganglion cells in the enteric

nervous system. The aganglionosis in HD is caused
by the failure of the migration of neural crest cells
during foetal development (1). Further, the
pathogenesis of aganglionosis has been attributed
to the genetically lack of the endothelin receptors
(ET-A and ET-B) in some cases (2-4). So far, the
colonic hypomotility observed in HD has been
assigned to only aganglionosis or hypoganglionosis.
However, even after complete excision of aganglionic

Indian J Physiol Pharmacol 2016; 60(1)

segment of bowel, continued dysmotility of remaining

bowel have been reported and patients present as
post-operative recurrent enterocolitis and persistent
constipation (5). On the other hand, there is lack of
studies on the contractile status of colonic smooth
muscles in HD patients. Therefore, the possibility of
impaired contraction of colonic smooth muscle per
se cannot be ruled out. Further, in studies with animal
(rat) model of HD, it was shown that application of
cholinergic agonist, carbachol caused enhanced
contractile response in HD as compared to control
(6). These experiments failed to provide concluding
evidence about the contractile status of colonic
smooth muscle in HD for two reasons. Firstly, the
experiments were carried out in rat model and
secondly, the HD model was produced by genetically
knocking out endothelin ET-B receptors. Thus, the
animal model seemed to be inadequate to represent
the human HD, where there may not be total absence
of ET-B receptors in various tissues. Consequently,
it was felt that direct examination of the colonic
tissue from HD patients is required to resolve the
issue.
Therefore, the present study aimed for in vitro
assessment of the contractile status of colonic
smooth muscle in HD patients, by recording the
contractions induced by cholinergic agonist carbachol
and ET-1, with the help of organ bath preparations.

Colonic Contractility in Hirschsprungs Disease

23

areas (e.g. atretic part of colonic atresia) were not

considered for contractility studies. In case of HD,
central areas were used for the study. Immediately
after excision in the operation theatre, the specimens
were collected in a wide mouth bottle containing
ice-cold Krebs-Ringer solution bubbled with 100%
oxygen. They were quickly transferred to the
laboratory in the Department of Physiology for
contractile studies. All the experiments were
conducted as per the guidelines laid down by the
ethical committee of the institute for handling human
tissues.
Preparation of muscle strips

The excised specimens of HD and non-HD were

transferred to a petri dish containing oxygenated icecold (4C to 6C) Krebs Ringer solution having the
composition (in mM): NaCl, 119; KCl, 4.7;
CaCl 2 .2H 2O, 2.5; KH 2PO 4 , 1.2; MgSO 4.7H 2 O, 1.2;
NaHCO 3, 5; and glucose, 11. Each specimen was
cleaned with freshly prepared cold Krebs-Ringer
solution to remove the faecal matter adhered to the
tissue. Thereafter, the adventitious layer was removed
and 2 to 3 mm wide and 15 to 20 mm long, rectangular
colonic strips (1-2 strips) oriented along the
longitudinal layer of smooth muscle were prepared
from the HD and non-HD cases.
Groups, treatment and recording of contractile responses

Methods
Specimens

The present study was carried out on the excised

specimens (total 30 cases) of colon obtained from
19 patients of HD, 6 cases of ano-rectal malformation
(ARM) and 5 cases of colonic atresia. These
specimens were collected from the operation theatre
in the Department of Paediatric Surgery, Institute of
Medical Sciences, Banaras Hindu University,
Varanasi, India. In absence of availability of normal
colonic tissue, ARM and colonic atresia (i.e. 11 nonHD cases) were considered as working control to
HD. In both of these cases, colonic strips were
prepared from relatively healthy looking areas towards
the end of dissected specimens. Grossly abnormal

Specimens were divided into two groups, HD and

non-HD groups. HD group was further subjected to
treatment with a) Carbachol, in presence or absence
of atropine and hexamethonium and b) ET-1, with or
without ET-A / ET-B antagonist. The non-HD group
had same treatment excepting the use of ET-A / ETB antagonists.
The procedure for recording of contractile response
has been described earlier (7). Briefly, the prepared
strips were mounted in Krebs-Ringer filled organ bath
(12 mL) maintained at 37C1C and continuously
bubbled with 100% oxygen. One end of the muscle
strip was fastened to a glass tube support, and the
other end was fixed to an isometric force transducer
(MLT 0210). The strip was placed under an initial

24

Tripathi, Gangopadhyay, Sharma, Kar and Mandal

tension of 0.5 g and then left to equilibrate for 30

minutes, with replacement of Krebs-Ringer solution
every 15 minutes. The output signals from the
transducer were amplified by bridge amplifier and
digitized by A/D converter (Power Lab 4/ST system)
and the recording of isometric contractions was
stored in a personal computer. The recording was
displayed and analysed with the help of software
Chart-5 for windows. The transducer, amplifier,
digitizer system, and software were procured from
AD Instruments, Sydney, Australia. Before, as well
as after recording the contractile responses,
calibration for the tension (0-10 g) was performed.
After stabilization, the initial recording was made
for 30 minutes without any external chemical
interventions, so as to assess the presence of
any spontaneous contraction. Subsequently, the
tissue was exposed to different concentrations of
carbachol (0.1, 1, 10, and 100 M) and endithelin-1
(1, 10 and 100 nM). The contractions were recorded
for a minimum period of 15 minutes for each
concentration. In one set of experiments, carbachol
(100 M)-induced contractions were recorded in
presence of atropine (100 M), and in another set
same was recorded after pre-treatment with
hexamethonium (100 M). After the recording of
contractions, the strips were removed from the bath
and placed on blotting paper for lightly soaking the
extra water from the tissue. Two ends of the strip
beyond their attachments were cut and discarded
because these parts of tissue did not participate
in the recorded contractions. The wet tissue was
then weighed in a fine balance to express the
contractile response per unit weight of tissue (g/g
wet tissue).
Drugs and solutions

Aqueous solutions of carbachol, ET-1, hexamethonium,

(Sigma Aldrich, New Delhi, India) and atropine
sulphate (Sd-Fine Chemicals, Mumbai, India),
were used in this study. The stock solutions of
these chemicals were prepared with distilled
water and refrigerated. Required dilutions were
made in Krebs-Ringer solution just before the
experimentation.

Indian J Physiol Pharmacol 2016; 60(1)

Histopathological examination

Fresh tissue from spastic site of all clinically

suspected cases of HD were processed for
preparing frozen section and subsequently stained
with haematoxylin and eosin (H&E) and
acetylcholinesterase (AChE) stains for confirmation
of HD.
Statistical analysis

The amplitude of contractions was noted as tension

(g weight) after the calibration procedure. The
tensions were then expressed per unit mass of
colonic tissue (g/g of wet tissue). The initial tension
was normalised as 100% and the change in tension
following chemical intervention was expressed as %
of initial. The values were pooled to calculate the
meanSEM. The statistical significance of differences
in mean values was examined with the help of
Students t - test and 2-way analysis of variance
(ANOVA) as and when applicable. P value of less
than 0.05 was considered as significant. The software
GraphPad Prism, version 5.0 was used for statistical
analysis.

Results
Contractile study was carried out with a total of 29
colonic strips obtained from 19 cases of HD and 18
strips obtained from 11 cases of non-HD to evaluate
spontaneous as well as chemically (ET-1 and
carbachol) evoked contractions. Most of the strips
responded well to carbachol and ET-1.
Absence of spontaneous contractions in Hirschsprungs
disease

Only two (6.9%) of 29 HD strips demonstrated

spontaneous contractions and another two strips from
two cases of HD did not respond to any concentration
of carbachol and ET-1. In contrast, most of the strips
from the non-HD cases (78%) i.e. 14 out of 18
showed spontaneous contractions. The spontaneous
contractions observed in non-HD specimens were
characterized with tonic contractions and

Indian J Physiol Pharmacol 2016; 60(1)

Colonic Contractility in Hirschsprungs Disease

superimposed by phasic rise in tension. Frequency

and strength of tonic and phasic contractions varied
from strip to strip (Figure 1 B-D).
Characteristics
contractions

of

carbachol

and

ET-1-induced

25

contractions as compared to non-HD samples. ET-1

produced quicker response with lower duration of
contraction in HD as compared to non-HD. The data
for carbachol and ET-1 responses in HD and non-HD
samples are presented in the Table I.
Dose-responses to carbachol

In HD specimens, carbachol produced less quick

response (increased latent period) and more prolonged

A concentration dependent increase in the amplitude

of contractions was observed with 4 different
concentrations (0.1-100 M) of carbachol in both HD
and the non-HD specimens. There was a significant
(P<0.05, 2-way ANOVA) lower response evoked by
carbachol on HD as compared to non-HD specimens
(Figure 2 upper right panel). At 100 M bath
concentration of carbachol, the response in HD was
increased by 5.5 times (554.52104.58% of initial;
n=5) as compared to nearly 8 (795.63291.71% of
initial; n=5) times in non-HD samples. EC-50 of
carbachol for non-HD was approximately 2 M and
that in HD was 7 M.
Carbachol-induced
atropine

contractions

were

blocked

by

In HD, after treatment with atropine (100 M),

carbachol (100 M) produced approximately 27%
(n=7) of its initial contraction. Thus, there was nearly
73% blockade of carbachol-induced response after
atropine pre-treatment. Whereas, in non-HD samples
the blockade was 53%, i.e. after atropinisation
carbachol produced 47% (n=6) of initial response
(Table II).
Fig. 1 : A c t u a l c o n t r a c t i o n s r e c o r d e d f r o m c o l o n i c s t r i p s
obtained from HD (A), colonic atresia (B) and ARM
(C, D). Please note the absence of spontaneous
contractions in HD and presence of same in other
cases. Vertical and horizontal calibration represents
tension (g) and time (minute) respectively.

Carbachol-induced
hexamethonium

contractions

were

blocked

by

In HD as well as in non-HD cases, after pre-

TABLE I : Showing meanSEM values of latent period, contraction period/duration in minutes

from various experimental groups after carbachol and ET-1 treatments.

Treatments

Carbachol (100 M)
ET-1 (100 nM)

Experimental
group
HD
Non-HD
HD
Non-HD

Latent period
(minutes)
0.390.01*
0.290.01
0.180.003*
0.390.03

Contraction durations
(minutes)
7.890.07*
5.840.13
2.540.231*
8.120.29

Time to reach peak

(minutes)
1.840.03*
1.120.03
0.990.1*
4.720.29

*p<0.05 (Students t -test, paired) as compared to Non-HD group. M=Micromole, nM=Nano-mole, HD=Hirschsprungs disease,
Non-HD=Non-Hirschsprungs disease.

26

Tripathi, Gangopadhyay, Sharma, Kar and Mandal

administration of hexamethonium (100 M), carbachol

(100 M) produced nearly 50% (HD, n=17; Non-HD,
n=7),) of its initial contraction, indicating
50% blockade in both HD and non-HD cases (Table
II).

Indian J Physiol Pharmacol 2016; 60(1)

TABLE II : Showing meanSEM values of contractile response
to carbachol after pre-treatment with atropine and
hexamethonium in HD and Non-HD group of samples.
The values are expressed as % of carbachol or
ET-1 alone response, i.e. % of initial response.

Drugs used in
pre-treatment

% of initial
response in
HD samples

% of initial
response in
Non-HD samples

Atropine (100 M)
Hexamethonium (100 M)
ET-A antagonists (100 nM)
ET-B antagonists (100 nM)

26.599.93*
50.636.57*
61.721.59
60.3413.53

46.749.51*
50.745.93*

Dose response to ET-1

A concentration dependent increase in the amplitude

of contractions was observed with 3 different
concentrations of ET-1 in both HD and the non-HD
specimens. There was significantly reduced response
in HD as compared to non-HD group. At 100 nM
bath concentration of ET-1, the response of HD

*p<0.05 (Students t - test, paired) as compared to the

response without antagonist. Data for ET-A/ET-B
antagonists on Non-HD group was not available.

Fig. 2 : Left panel: Showing actual recordings of contractions induced by carbachol in non-HD (A) and HD (B) and ET-1 in
HD (C) non-HD (D) samples. Vertical and horizontal bars represent contractile tension (g) and time (min) respectively.
Arrows indicate point of application of drugs. Right panel: The upper and lower right panel shows dose-response curve
for carbachol and ET-1 respectively. Data points indicate meanSEM. An asterisk indicates P<0.05 (Two-way ANOVA).

Indian J Physiol Pharmacol 2016; 60(1)

Colonic Contractility in Hirschsprungs Disease

27

(352.7699.72 % of initial tension, n=5) was nearly

3 times lesser than that of non-HD (866.78318.30%
of initial tension, n=4) (Figure 2, lower right panel).

ET-1 induced response (Table II).

ET-1-induced contractions were blocked by ET-A and

ET-B antagonists

All the sections from the spastic segment of clinically

suspected cases of HD showed absence of ganglion
cells. In addition, some of the cases also showed
presence of hypertrophied nerve bundle. These cases
also demonstrated positive staining for AChE, and
therfore confirming the diagnosis of HD. Section from
the Non-HD cases showed presence of ganglion cells.

On pre-treatment with either ET-A or ET-B antagonists

(100 nM each), ET-1 produced nearly 60% of initial
contractile tension in both cases. In other words,
these antagonists caused around 40% blockade of

Confirmation of HD by histopathological examination

Fig. 3 : A . HD samples showing positive AChE stained thin nerve fibres (arrow) in between the crypts in lamina propria and
muscularis mucosa in a case of HD, (100X).
B. Negative stain with AChE in the lamina propria, in a case of non-HD (100X).
C. Non-HD sample stained with H&E stain, showing several neural units (arrow) and presence of ganglion cells (200X).
D. No ganglion cell was seen with H&E stain. Arrow shows hypertrophied nerve bundles in myenteric plexus in a case
of HD (100X).

28

Tripathi, Gangopadhyay, Sharma, Kar and Mandal

Discussion
The present study was carried out to understand the
functional status of colonic smooth muscles in HD,
by studying in vitro contractility of colonic strips. In
this study, except two strips, none of the HD
specimens showed spontaneous contractions. In
contrast, in earlier in vitro studies with normal colon,
spontaneous contractions from almost all of the
colonic strips were observed (8-9). Similar
observations in non-HD colon were also made in our
study. The origin of spontaneous contraction is
related to the activity of interstitial cells of Cajal
(10). These contractions have been found reduced in
other pathologic conditions of colon including
ulcerative colitis (9, 11). The nonappearance of
spontaneous contractions in HD samples could be
correlated to absence or very sparse ganglion cells
as observed in histopathological examinations.
However, the present functional study demonstrated
that the colonic strips from HD responded to both
ET-1 and carbachol in vitro .
Carbachol dose-response curve indicated that the
colonic smooth muscle responded feebly in HD as
compared to non-HD cases (EC-50 for HD was around
7 M, against 2 M in non-HD samples). Thus it
may be presumed that the cholinergic contractions
are preserved in HD, although to a lesser extent.
Further, it was seen that pre-administration of
atropine could abolish 73% of carbachol response in
HD. This observation indicated that the carbacholinduced response was largely mediated through
muscarinic receptors. Interestingly, carbachol-induced
response was also reduced by 50% after pretreatment with ganglion blocker hexamethonium. At
present, it is difficult to explain, how hexamethonium
could interfere with muscarinic action of carbachol
in colonic muscle. However, there is evidence that
hexamethonium can antagonise the carbachol
induced contractions in canine vascular smooth
muscle (12). The same may be true here also, since
hexamethonium produced 50% blockade of carbacholinduced contractile responses in both HD and nonHD cases.
The HD specimens also responded to application of
ET-1. However, the contractile response to ET-1 was

Indian J Physiol Pharmacol 2016; 60(1)

lesser as compared to non-HD samples, as evidenced

by EC-50, which was 5 times more in HD (EC-50,
HD=10 nM; non-HD=2 nM). In mouse colon, it was
shown that ET-1-induced contractions are mediated
via ET-A and ET-B receptors (13). On the other hand,
in rat model of HD (produced by knocking out ET-B
receptors), demonstrated that the ET-1-induced
contractions were not mediated through ET-B
receptors (14). In our observations with human HD,
it was revealed that, at least 40% of ET-1-induced
contraction was mediated through ET-B receptors.
This difference in responses for ET-B receptors may
be due to variations in species. Therefore, it is
suggested that the colonic contractions in human
HD samples were partially mediated via ET-A and
ET-B receptors.
The ET-B receptors are implicated in the pathogenesis
of some but not all cases of short-segment HD. The
expression of this receptor is required for neural crest
cell migration and development of ganglion in enteric
nervous system. Failure or absence of these
receptors may lead to development of HD (15-17).
However, our results on contractions with ET-1 and
its blockers indicated the presence of ET-B receptors
in colonic smooth muscles of HD.
It may be noted that we, for the first time, used
human colonic tissue from HD patients and attempted
to resolve the contractility issues with the help of in
vitro techniques. In earlier experiments in rat model
(6), it was shown that carbachol increased the
contractile response in colonic smooth muscle. On
the other hand, we did not observe any substantial
histological change in muscle layer. Further, the ETB receptors were present in colonic tissue, as
evidenced by ET-1-induced contractile response and
its antagonism by ET-B antagonist. Therefore, it is
reasonable to say that, the rat model of HD does
not represent a perfect model of human HD.
Histologically, though there was absence of ganglion
cells in myenteric plexus and submucosa and also
presence of hypertrophied nerve fibre in the
submucosa but no gross abnormality in muscle layer,
attributable to the altered contractile responses, was
seen.
The major limitation of this study was non-availability

Indian J Physiol Pharmacol 2016; 60(1)

Colonic Contractility in Hirschsprungs Disease

of age matched normal human colonic tissue.

Difficulty in obtaining the normal colon was obviously
due to ethical reasons. In the present study, the
non-HD specimens obtained from the paediatric
patients with ARM and colonic atresia were
considered as working control. These non-HD
specimens were histologically normal but may not
be functionally healthy tissue. Although, at this stage
of the study it is hard to clearly correlate the
histological findings with contractile responses of
colonic smooth muscle, nevertheless, this study

29

provides evidence for altered contractile function of

the colonic muscle in HD. Thus, the observations of
this study may help in the formulation of better
clinical management strategies in future.
In conclusion, it may be said that, in addition to
aganglionosis, impaired contractility of colonic
smooth muscle may be responsible for colonic
hypomotility in Hirschsprungs disease as evidenced
by lack of spontaneous contractions and reduced
carbachol/ET-1 induced contractions.

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