Scorpion venom stimulates biliary/duodenal motility

and pancreatic exocrine secretion

J. W. C. CHEN, C. X. SHI, M. J. TENG, A. C. SCHLOITHE, J. TOOULI & G. T. P. SACCONE
Department of General and Digestive Surgery, Pancreatobiliary Research Group, Flinders Medical Centre, Flinders University,
SA, Australia
Abstract Scorpion envenomation causes severe upper
abdominal pain associated with nausea and vomiting.
Although scorpion venom (SV) stimulates pancreatic
and gastric secretion in animal models, its effects on
duodenal and biliary motility have not been reported.
The aim of this study was to determine the effects of
SV on sphincter of Oddi (SO), duodenal and gall
bladder motility and pancreatic amylase output.
Anaesthetized Australian possums (n 21) were
infused with SV via intravenous or closed intra-arter-
ial routes. Blood pressure, SO, duodenal and gall
bladder motility were continuously monitored for 4 h.
Trans-sphincteric ow (TSF), an indicator of bile duct
resistance, was measured concurrently. The amylase
output in pancreatic juice was also measured. SV
infusion resulted in profound transient increase in
blood pressure, SO motility and a signicant decrease
in TSF. No signicant differences were noted in SO
basal pressure changes. A transient increase in gall
bladder tone, duodenal contraction amplitude and
frequency, and amylase output were noted. Following
the peak in blood pressure, amylase output, SO, gall
bladder and duodenal motility were depressed. SV
induces a rapid but transient increase in biliary and
duodenal motility that is associated with stimulation
of pancreatic amylase output. These changes may
contribute to gastrointestinal symptoms associated
with early phases of envenomation.
Keywords duodenum, envenomation, gall bladder,
Oddis sphincter, pancreatic secretion, scorpion
venom.
INTRODUCTION
Scorpion envenomation causes profound systemic
changes occasionally resulting in deaths. Clinical
manifestations of scorpion evenomation include vom-
iting, abdominal pain, salivation, hypertension, tachy-
cardia, muscle rigidity, seizure and other symptoms.
13
Most of these effects can be explained by direct
neurotoxicity scorpion venom (SV) has on cholinergic
and sympathetic nerves resulting in release of neuro-
transmitters causing intense stimulation of target
organs. The actions of SV on various muscles of the
body have been studied. They cause intense stimula-
tion of smooth muscles like in the ileum,
4
uterus,
5
arterial smooth muscle,
6
vas deferens
7
and cardiac
muscle.
8,9
SV from Brazilian scorpion (Tityus serrula-
tus) has shown to cause vomiting, abdominal pain and
acute pancreatitis. Tityus serrulatus venom induces
prolonged delayed gastric emptying for up to 48 h in
experimental animals
10
and stimulates gastric circular
smooth muscle.
11,12
These effects combined with
stimulation of gastric secretion
13
could explain vom-
iting and abdominal pain associated with acute scor-
pion envenomation. However, there is no direct
evidence that the effects of delayed gastric emptying
were due to intense contraction of gastric muscle or
pyloric sphincter. SV has shown to cause ileal smooth
muscle contraction
4
and in contrast lower oesophageal
sphincter smooth muscle relaxation.
14,15
Scorpion envenomation can result in acute pancre-
atitis.
16,17
This was postulated to be due to stimulatory
effects of SV on sphincter of Oddi (SO) causing
obstruction coupled with stimulation of pancreatic
exocrine secretion.
18
The effects of SV on duodenal,
gall bladder and SO motility in vivo have not been
studied.
The aims of this study were to characterize the
effects of T. serrulatus venom, on biliary gall bladder,
SO and duodenal motility as well as its effects on
pancreatic exocrine secretion.
Address for correspondence
Dr John W C Chen, Department of General and Digestive
Surgery, Flinders Medical Centre, Bedford Park, SA 5042,
Australia.
Tel: 618-8204-5511; fax: 618-8204-5843;
e-mail: john.chen@inders.edu.au
Received: 12 August 2003
Accepted for publication: 27 January 2004
Neurogastroenterol Motil (2004) 16, 447454 doi: 10.1111/j.1365-2982.2004.00566.x
2004 Blackwell Publishing Ltd 447
METHODS
Animal preparation
Twenty-one Australian Brush-tailed possums (Tri-
chosurus vulpecular) of either sex (1.53.5 kg) were
prepared as previously described.
1921
Briey, anaes-
thesia was induced with a combination of intramus-
cular ketamine (20 mg kg
)1
Ketalar; Park Davis,
Caringbah, NSW, Australia) and xylazine (10 mg kg
)1
Rompun; Bayer, Botany, NSW, Australia) IM. The left
and right femoral veins were cannulated and anaesthe-
sia maintained for the duration of the experiment with
a constant infusion of sodium pentobarbitone (nembu-
tal 1520 mg kg
)1
h
)1
; Boehringer Ingelheim Pty. Ltd,
Artarmon, NSW, Australia) into the left vein. The right
femoral vein was used for infusion of SV or vehicle.
The left femoral artery was cannulated and connected
to a pressure transducer (Transpac

IV; Abbot Ireland,

Silgo, Ireland) for blood pressure monitoring. A trach-
eostomy was performed and the animal ventilated with
a small animal ventilator (Phillips and Bird Inc.,
Richmond, VA, USA). In experimental groups subjec-
ted to closed intra-arterial infusion to the biliary tree,
pancreas and duodenum, the splenic artery was can-
nulated retrogradely and the tip of the catheter placed
at the origin of the hepatic artery.
Sphincter of Oddi, duodenal and gall bladder
motility and trans-sphincteric ow measurement
At laparotomy, a small choledochotomy was made in
the common bile duct distal to the entry of the cystic
duct. A single lumen side-hole polyethylene manome-
try catheter (OD 1 mm, ID 0.6 mm) and a polyethylene
bile diversion tube (OD 1.2 mm, ID 0.8 mm) were
inserted (Fig. 1).
19
The distal end of this catheter was
placed in the distal duodenum to divert bile from
proximal bile duct. The manometry catheter, to meas-
ure SO motility, was connected to a minimally com-
pliant pneumohydraulic pump (Arndorfer Medical
Specialties Inc., Greendale, WI, USA) and a pressure
transducer. To collect pancreatic exocrine secretion, the
pancreatic duct proximal to the SO was incised and one
end of a polyethylene catheter (OD1.2 mm, ID0.8 mm)
was inserted and secured with a 6/0 silk suture. An
inowcatheter was inserted into the common bile duct
with the manometry catheter to measure trans-sphinc-
teric ow (TSF), as previously reported.
1921
Briey, a
polyethylene inow catheter (OD 1.2 mm, ID 0.8 mm)
was positioned in the common bile duct proximal to the
manometry catheter, secured with a ligature and con-
nected to a saline-lled reservoir suspended froma force
transducer (FT03; Grass Instruments, Quincy, MA,
USA) acting as an electromagnetic balance. As TSF
proceeds, the weight of the reservoir decreases and the
rate of change (slope) represents TSF. Aliquots of saline
(1 mL) were delivered into the reservoir at regular
intervals to maintain a relatively constant inow
pressure. Cessation of TSF was indicated an upward
slope as complete obstruction at the SO resulted in
transfer of the manometry catheter perfusate into the
saline reservoir via the inow catheter.
Gall bladder motility
The gall bladder was aspirated to empty the bile. The
fundus of the gall bladder was incised and a saline-
lled balloon connected to a pressure transducer is
then inserted as shown in Fig. 1 and secured with a
4/0 silk purse string.
Duodenal activity
A strain gauge is then attached to the serosal surface of
the duodenum 2 cm distal to the SO junction for
continuous monitoring of duodenal activity. All trans-
ducers were connected to a MacLab

(AO Instruments,
Castle Hill, NSW, Australia) recording system.
Dose of scorpion (T. serrulatus) venom
Previous study had demonstrated that each manual
extraction of SVfromthis species yield between 100 and
1000 mcg of venom. Hence, doses of 100500 mcg kg
)1
were used in this study. This is equivalent to each
manual extraction of venom from this species. The
serum concentration following scorpion sting varies
between 1 and 40 mcg L
)1
in one study.
22
However, the
concentration could be higher than measured as the
duration between inoculation of the venom and samp-
ling of sera was not documented in this particular study.
Experimental design
The possums were divided into four groups (Table 1).
These included the control group (n 6), which
received vehicle (0.01% bovine serum albumin; Sigma
Chemical Co., St Louis, MO, USA) infusion into the
hepatic artery, a second group (n 5) with close intra-
arterial infusion received SV (T. serrulatus, Sigma
Chemical Co.) at 500 mcg kg
)1
over 1 h using a syringe
pump. The intra-arterial route was used to achieve a
higher concentration of the SV in the region of the
pancreas and biliary tree. The third (n 5) and fourth
(n 5) groups received intravenous infusion of SV at
448 2004 Blackwell Publishing Ltd
J. W. C. Chen et al. Neurogastroenterology and Motility
100 and 500 mcg kg
)1
, respectively. All parameters
measured were monitored for 4 h after initial period of
stabilization (30 min), after which, the animals were
killed by sodium pentobarbitone (Lethabarb

, Virbac
Pty. Ltd., NSW, Australia).
Data analysis
The effects of SV on SO basal pressure and phasic
amplitude (mmHg) and frequency of contraction (con-
tractions per minute) were evaluated. Integrated SO
motility as represented by mean amplitude under the
curve (mmHg) was measured for 2 min every half
hourly.
The change in TSF (lL min
)1
) was determined by the
difference between the mean ow rate (over 2 min)
every half and the ow rate before SV infusion
(baseline).
Duodenal motility was measured as contractions
frequency and amplitude (mV). The magnitude of
strain registered is proportionate to voltage output.
Motion artefacts from ventilation (when present) rep-
resent changes of less than 0.15 mV and were not
included in the analysis. The duodenal frequencies
were measured as mean contraction per minute over a
period of 2 min. As SV causes a burst of duodenal
activity about 3060 min after commencing infusion,
the period of maximal activity was evaluated. Duode-
nal motility was expressed as change from baseline in
frequency and amplitude. Gall bladder motility was
manifested as tonic contraction and is expressed as
change from baseline pressure.
Figure 1 Schematic representation of the animal preparation used in this study showing bile diversion tube, sphincter of Oddi (SO)
manometry catheter, duodenal strain gauge (for duodenal motility), duodenal decompression tube, and saline-lled balloon for
measurement of gall bladder motility. A catheter was also inserted into the pancreatic duct with the outlet set at 1 cm below the
duodenum for pancreatic juice collection. An inow catheter was also inserted into common bile duct to measure trans-sphincteric
ow. All parameters were recorded using MacLab

computer recording system.

Table 1 Experimental groups
Groups Route of infusion n
Control (vehicle) Intra-arterial 6
Scorpion venom
500 mcg kg
)1
Intra-arterial 5
100 mcg kg
)1
Intravenous 5
500 mcg kg
)1
Intravenous 5
2004 Blackwell Publishing Ltd 449
Volume 16, Number 4, August 2004 Pancreatobiliary effects of scorpion venom
Pancreatic juice amylase output
After a 30-min stabilization period, pancreatic juice
was collected half hourly, the secretion rate deter-
mined gravimetrically. The pancreatic juice amylase
activity was measured using enzymatic colorimetric
spectroanalysis (Boehringer Mannheim, Meylan,
France and Sigma) in a centrifugal analyser (Cobas

Bios; F.Hoffmann-La Roche, Basle, Switzerland). Plas-

ma amylase and lipase levels were expressed as a
percentage of the baseline levels.
Statistical analysis
The statistical analysis of the data utilized SSPS
software. Changes in blood pressure, motility param-
eters, TSF and pancreatic amylase output were ana-
lysed using MannWhitney test. A P value of <0.05 was
regarded as signicant. All data are expressed as
mean SEM unless otherwise stated. This study was
approved by the Animal Welfare Committee of the
Flinders University.
RESULTS
Low- or high-dose SV infusion by either intravenous or
intra-arterial routes cause a profound increase in blood
pressure, gall bladder, duodenal and SO motility
(Fig. 2). Pancreatic exocrine amylase secretion was also
increased. Although salivation was noted in all ani-
mals subjected to SV infusion, this was not quantied.
Blood pressure
Scorpion venom causes a dose-dependent increase in
blood pressure (Fig. 3A). Intravenous SV causes a
greater increase than both intra-arterial and lower
intravenous dose (P < 0.01). The blood pressure peaked
D
U
O
-
S
G
(
m
v
)
G
B
(
m
m
H
g
)
F
L
O
W
(
U
L
)
B
D
-
(
m
m
H
g
)
B
P
(
m
m
H
g
)
1 h
Figure 2 A typical multi channel recording showing blood pressure (BP), sphincter of Oddi (SO) pressure, trans-sphincteric ow
(FLOW), gall bladder pressure (GB) and duodenal tension (DUO-SG) response to intravenous infusion of scorpion venom. The 1-h
infusion period is indicated by horizontal bar and the commencement of the infusion by the vertical arrow. The peak activity
occurs at 3060 min during the infusion. Note the slowing of trans-sphincteric ow with peak SO activity.
450 2004 Blackwell Publishing Ltd
J. W. C. Chen et al. Neurogastroenterology and Motility
between 30 and 60 min of the onset of infusion then
declined to baseline levels by about 2 h after cessation
of the infusion.
Sphincter of Oddi motility and trans-sphincteric
ow
Scorpion venom infusion caused a profound increase in
SO phasic contraction amplitude and frequency, with
maximal effects noted between 30 and 60 min follow-
ing the onset of SV infusion. (Fig. 3B) The SO ampli-
tude declined after 2 h and remained low for the
duration of the experiment. However, the frequency of
contractions increased as shown in Fig. 4. There was,
however, no signicant change in basal pressures. The
integrated SO motility was also increased with SV
infusion (P < 0.01, Fig. 3C). This change in integrated
motility corresponds to a signicant slowing of TSF
rate (Fig. 3D) with subsequent recovery and increase in
ow rate as the phasic amplitude declines for the
remainder of the experiment. The high dose intra-
arterial SV infusion produced the greatest increase in
SO motility and the most signicant decrease in TSF
(Fig. 3E).
**
**
**
*
*
+ #
40
20
0
20
40
60
80
100
120
0 30 60 90 120 150 180 210 240
Time (min)
B
P

C
h
a
n
g
e

f
r
o
m

B
a
s
e
l
i
n
e

(
m
m
H
g
)
Control
SV500 IA
SV100 IV
SV500 IV
SO amplitude change
20
10
0
10
20
30
40
50
0 60 120 180 240
Time (min)
C
h
a
n
g
e

i
n

A
m
p
l
i
t
u
d
e

f
r
o
m

B
a
s
e
l
i
n
e

(
m
m
H
g
)
*
SO frequency
0
2
4
6
8
10
12
0 60 120 180 240
Time (min)
C
o
n
t
r
a
c
t
i
o
n
s
/
m
i
n
*
4
2
0
2
4
6
8
10
0 60 120 180 240
Time (min)
I
n
t
e
g
a
t
e
d

S
O

m
o
t
i
l
i
t
y

c
h
a
n
g
e

(
m
m
H
g
)
*
*
*
Flow change from baseline
10
8
6
4
2
0
2
4
6
0 60 120 180 240
Time (min)
T
r
a
n
s
-
s
p
h
i
n
c
t
e
r
i
c

F
l
o
w

C
h
a
n
g
e
(
u
l
/
m
i
n
)

*
*
*
A
B
C
D E
Figure 3 Scorpion venom induces a dra-
matic increase in systemic blood pressure
(*P < 0.02, **P < 0.01). (A) Intravenous
(i.v.) high-dose scorpion venom resulted in
the highest increase in blood pressure
which was signicantly higher than intra-
arterial (i.a.) (+P < 0.05) or intravenous low
dose (P < 0.01). (B) Signicant increase in
sphincter of Oddi (SO) amplitude is noted
with all doses of scorpion venom irre-
spective of route of administration. The
amplitude declined following cessation of
the infusion (*P < 0.05). (C) Signicant
increase in sphincter of Oddi (SO) fre-
quency is noted with all doses of scorpion
venom irrespective of route of adminis-
tration. The frequency continues to
increase subsequent to initial rise
following cessation of the infusion
(*P < 0.05). (D) Scorpion venom infusion
produces dramatic increase in sphincter of
Oddi (SO) motility from baseline
(*P < 0.01). A dose-related increase in SO
motility was noted with the doses of
scorpion venom and routes of administra-
tion tested. (E) Scorpion venom infusion
decreased trans-sphincteric ow. High-
dose scorpion infusion results in maximal
decrease of trans-sphincteric ow
(*P < 0.01). In control group, there is a
minor increase in ow with time.
2004 Blackwell Publishing Ltd 451
Volume 16, Number 4, August 2004 Pancreatobiliary effects of scorpion venom
Gall bladder and duodenal motility
Scorpion venom infusion caused a dramatic increase in
gall bladder tone (Fig. 4A). This effect is dose-related
lasting for about 60 min. Intra-arterial infusion caused a
greater increase in peak gall bladder tone than intraven-
ous infusion (P < 0.02). Under baseline conditions,
duodenal motility was usually quiescent; however, SV
infusion induced duodenal contraction, with bursts of
activity evident at 3060 min after onset of infusion.
These bursts of activity consisted of increased contrac-
tion amplitude and frequency (Fig. 4B,C). As seen with
gall bladder motility, greater responses were observed
when SV was infused via the intra-arterial compared
with the intravenous route (P < 0.05).
Pancreatic amylase output
Scorpion venom stimulated pancreatic amylase output
(Fig. 5) and this was dose dependent (P < 0.01). Amy-
lase secretion was signicantly elevated even at a
lower dose of SV when compared with control
(P < 0.05). The rate of amylase secretion declined soon
after cessation of SV infusion as seen with several other
parameters measured.
DISCUSSION
Scorpion envenomation is serious and often associated
with life-threatening complications such as hyperten-
sive crisis, cardiac dysrhythmia
23
or acute pancreati-
tis.
16,17
This study evaluated direct effects of SV
through intravenous and intra-arterial route demon-
strating that the effects of scorpion envenoma-
tion occur very early (within 1530 min) and cause
profound transient hyperstimulation followed by a
prolonged period of depression of motility, secretion
as well as blood pressure. Although the hyperten-
sive effects of SV is well documented,
23
its stimulatory
effects on SO, gall bladder and duodenal motility in
vivo have not been demonstrated previously.
Several subtypes of SV have been identied and
some have been used in the experimental setting to
deplete neurotransmitter and hence eliminate neur-
0
1
2
3
4
5
6
7
8
9
10
D
u
o
d
e
n
a
l

f
r
e
q
u
e
n
c
y

(
c
o
n
t
r
a
c
t
i
o
n
s

p
e
r

m
i
n
u
t
e
)
Control
SV100D IV
0
5
10
15
20
25
30
35
D
u
o
d
e
n
a
l

p
h
a
s
i
c

a
m
p
l
i
t
u
d
e

(
m
m
H
g
)
SV500D IV
SV500D IA
3
2
1
0
1
2
3
4
5
6
Groups
G
B

p
r
e
s
s
u
r
e

(
m
m
H
g
)
**
**
*
*
*
*
*
*
#
##
A
B C
Figure 4 Scorpion venom infusion produ-
ces dramatic increase in gall bladder
motility. (A) Gall bladder motility, as
reected by gall bladder pressure,
increased following scorpion venom infu-
sion, reaching a maximum 3060 min
after commencement of the infusion. The
high dose of scorpion venom administered
by the intra-arterial route produced the
greatest increase in gall bladder pressure
(***P < 0.02), followed by the intravenous
infusion of high and low dose (**P < 0.02,
*P < 0.01 respectively). In the control
group, gall bladder pressure declined over
the same period. Scorpion venom stimu-
lates duodenal contraction frequency and
amplitude. (B) The maximal increase in
duodenal motility occurred 3060 min
after the commencement of the scorpion
venom infusion (*P < 0.01). (C) Intra-
arterial administration produced the
greatest increase in amplitude, followed
by the high and low doses administered
intravenously (P < 0.01, ##P < 0.05,
respectively). In control groups, the
duodenum is inactive prior to the
administration of scorpion venom.
452 2004 Blackwell Publishing Ltd
J. W. C. Chen et al. Neurogastroenterology and Motility
onal inuence on parameters studied.
14
SVs action
appears to cause depletion of neurotransmitter from
nerve endings resulting in profound stimulation of
target organs. This stimulatory response is then fol-
lowed by a period of exhaustion of the organ con-
cerned.
The mechanisms of action of SV are complex and
incompletely understood. The current data suggest
that the toxins (and other components) in the venom
depolarize nerve endings to release neurotransmitters
and also have prejunctional effects.
24,25
As the regula-
tion of biliary and duodenal motility and also pancre-
atic exocrine secretion involves muscarinic receptors,
we attempted to determine whether muscarinic recep-
tors are involved in the observed responses. However,
we found that treatment with atropine, at concentra-
tions which denitely blocked muscarinic receptors
based on functional tests, signicantly increase blood
pressure. Furthermore, the combination of atropine
treatment with SV infusion resulted in cardiovascular
collapse. Therefore, we have not been able to pursue
this question.
The action of SV on SO function and acute pancre-
atitis has received little attention. The single published
study that investigated the effects of SV on SO activity
noted that scorpion toxin stimulated SO contractile
activity resulting in a decrease in TSF.
26
The authors
also noted a stimulatory effect on pancreatic exocrine
secretion.
They postulated that acute pancreatitis associated
with scorpion envenomation could be caused by
stimulation of SO activity resulting in pancreatic duct
obstruction in conjunction with a stimulated exocrine
pancreas. The results of the current in vivo study
indicate that SV is associated with a transient stimu-
lation of SO motility and an increase in bile duct
resistance. Although SO dysfunction may play a role in
causation of various forms of acute pancreatitis,
27
this
mechanism has not been shown to be the basis of
SV-induced acute pancreatitis. We have previously
demonstrated in an in vivo model that stimulation of
SO motility with a cholinergic agonist, carbachol, can
induce not only sphincteric obstruction but can also
result in acute pancreatitis.
19
However, complete bile
duct obstruction was achieved and this lasted for a
period of 5 h. In contrast, during the current study,
there was only a transient decrease in TSF. This
difference in the magnitude and duration of these
effects may be related to the concentration of circula-
ting agonists. In this study, we demonstrated that
following the initial period of stimulated activity, there
was exhaustion or depression of SO activity with an
associated increase in TSF. These responses are not
consistent with pancreatic duct obstruction as the
major mechanism responsible for the induction of
acute pancreatitis.
In addition to biliary and pancreatic responses, SV
also stimulated salivary secretion in this model,
although this parameter was not quantied. The
increased biliary and duodenal motility coupled with
increased stimulation of the exocrine pancreas may
explain why upper abdominal pain with nausea and
vomiting are frequent and early symptoms of scorpion
envenomation in the presence or absence of acute
pancreatitis.
1,23
From this study, it is evident that
profound physiological changes accompany scorpion
envenomation including an early period of intense
stimulation (hypertensive crisis) followed by a long
period of depressed motility activity and blood pres-
sure. This response prole has prompted some authors
to suggest that treatment should include vasodilator
support early followed by inotropic support in the later
period of envenomation.
2
In a previous study, the concentration of venom in
victims sera is directly proportional to the severity of
clinical manifestations. This observation is similar to
the dose-related increase in SO and GB motility as well
as pancreatic exocrine secretion prior to the period of
exhaustion.
22
This study of SV levels in human
victims sera and our ndings of dose-related effects
on the parameters measured suggest that the degree of
toxicity related to scorpion envenomation is dose
related. Larger scorpion (hence the size of inoculation)
envenomation in children has been shown to correlate
Pancreatic amylase secretion
1
10
100
1000
10 000
100 000
0 1 2 3 4 5 6 7 8
Time (h)
A
m
y
l
a
s
e

(
%

o
f

c
o
n
t
r
o
l
)
Control
SV500 IA
SV100 IV
SV500 IV
Figure 5 Scorpion venom increased pancreatic amylase
secretion. Administration of scorpion venom at low and high
doses, via either the intravenous or intra-arterial routes, sig-
nicantly increased pancreatic amylase secretion compared
with control (*P < 0.05, **P < 0.01). Infusion of the high dose
of scorpion venom by either route then produced the greatest
effects (
+
P < 0.05).
2004 Blackwell Publishing Ltd 453
Volume 16, Number 4, August 2004 Pancreatobiliary effects of scorpion venom
with increased clinical severity of envenomation, indi-
cating a dose-related effect of scorpion envenomation.
1
In conclusion, we have demonstrated for the rst
time that intravenous or close intra-arterial infusion of
SV results in transient profound effects on biliary and
duodenal motility. These effects occur early following
infusion and may explain the upper gastrointestinal
symptoms that accompany scorpion envenomation.
ACKNOWLEDGMENTS
The study was supported by the Royal Australasian
College of Surgeons and the National Health and
Research Council of Australia. Permission for the use
of the Australian Brush tailed possum in this study was
granted by the South Australian National Parks and
Wildlife Service. The technical assistance of Mr Aaron
Citti is acknowledged.
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2 Amitai Y. Clinical manifestations and management of
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3 Bucaretchi F, Baracat EC, Nogueira RJ et al. A comparative
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