Investigation of the distribution and function of alpha-adrenoceptors in the sheep isolated internal anal sphincter.
ABSTRACT We have investigated the distribution of alpha-adrenoceptors in sheep internal anal sphincter (IAS), as a model for the human tissue, and evaluated various imidazoline derivatives for potential treatment of faecal incontinence.
Saturation and competition binding with (3)H-prazosin and (3)H-RX821002 were used to confirm the presence and density of alpha-adrenoceptors in sheep IAS, and the affinity of imidazoline compounds at these receptors. A combination of in vitro receptor autoradiography and immunohistochemistry was used to investigate the regional distribution of binding sites. Contractile activity of imidazoline-based compounds on sheep IAS was assessed by isometric tension recording.
Saturation binding confirmed the presence of both alpha(1)- and alpha(2)-adrenoceptors, and subsequent characterization with sub-type-selective agents, identified them as alpha(1A)- and alpha(2D)-adrenoceptor sub-types. Autoradiographic studies with (3)H-prazosin showed a positive association of alpha(1)-adrenoceptors with immunohistochemically identified smooth muscle fibres. Anti-alpha(1)-adrenoceptor immunohistochemistry revealed similar distributions of the receptor in sheep and human IAS. The imidazoline compounds caused concentration-dependent contractions of the anal sphincter, but the maximum responses were less than those elicited by l-erythro-methoxamine, a standard non-imidazoline alpha(1)-adrenoceptor agonist. Prazosin (selective alpha(1)-adrenoceptor antagonist) significantly reduced the magnitude of contraction to l-erythro-methoxamine at the highest concentration used. Both prazosin and RX811059 (a selective alpha(2)-adrenoceptor antagonist) reduced the potency (pEC(50)) of clonidine.
This study shows that both alpha(1)- and alpha(2)-adrenoceptors are expressed in the sheep IAS, and contribute (perhaps synergistically) to contractions elicited by various imidazoline derivatives. These agents may prove useful in the treatment of faecal incontinence.
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ABSTRACT: The focus of the British Society for Research on Ageing (BSRA) annual scientific meeting 2012 was aging mechanisms and mitigants. The themes covered included epigenetics, stem cells and regeneration, aging pathways and molecules, the aging bladder and bowel, as well as updates from the New Dynamics of Ageing (NDA) programme. The topics incorporated new directions for staple aging research in caloric restriction (CR), inflammation, immunesenescence, neurodegeneration, homeostasis and stress resistance, as well as newer research areas such as bioengineering of tissues, including the internal anal sphincter and thymus.British Society for Research on Ageing (BSRA) annual scientific meeting 2012; 07/2012
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ABSTRACT: The role of α-adrenoceptors in promoting continence through modulation of sphincter tone has focused primarily on the effects of α1 -adrenoceptors. We have used three clinically available agents, which are selective for α2 -adrenoceptors, to investigate their role in contractile and neurogenic responses on the internal anal sphincter (IAS).Neurogastroenterology and Motility 06/2014; · 2.94 Impact Factor
Investigation of the
distribution and function of
a-adrenoceptors in the
sheep isolated internal anal
SJ Rayment1, T Eames1, JAD Simpson1, MR Dashwood2, Y Henry3,
H Gruss3, AG Acheson1, JH Scholefield1and VG Wilson4
1Centre for Integrated Systems Biology and Medicine, Department of Surgery and4School of
Biomedical Sciences, The University of Nottingham Medical School, Queen’s Medical Centre,
Clifton Boulevard, Nottingham, UK,2Clinical Biochemistry, Royal Free and University College
Medical School, Royal Free Campus, London, UK, and3Clinical Development, Norgine Ltd, R&D
Division, Harefield, UK
Dr VG Wilson, School of
Biomedical Sciences, The
University of Nottingham
Medical School, Queen’s Medical
Centre, Clifton Boulevard,
Nottingham NG7 2UH, UK.
imidazoline; faecal incontinence;
7 January 2010
16 March 2010
22 March 2010
BACKGROUND AND PURPOSE
We have investigated the distribution of a-adrenoceptors in sheep internal anal sphincter (IAS), as a model for the human
tissue, and evaluated various imidazoline derivatives for potential treatment of faecal incontinence.
Saturation and competition binding with3H-prazosin and3H-RX821002 were used to confirm the presence and density of
a-adrenoceptors in sheep IAS, and the affinity of imidazoline compounds at these receptors. A combination of in vitro
receptor autoradiography and immunohistochemistry was used to investigate the regional distribution of binding sites.
Contractile activity of imidazoline-based compounds on sheep IAS was assessed by isometric tension recording.
Saturation binding confirmed the presence of both a1- and a2-adrenoceptors, and subsequent characterization with
sub-type-selective agents, identified them as a1A- and a2D-adrenoceptor sub-types. Autoradiographic studies with3H-prazosin
showed a positive association of a1-adrenoceptors with immunohistochemically identified smooth muscle fibres.
Anti-a1-adrenoceptor immunohistochemistry revealed similar distributions of the receptor in sheep and human IAS. The
imidazoline compounds caused concentration-dependent contractions of the anal sphincter, but the maximum responses
were less than those elicited by L-erythro-methoxamine, a standard non-imidazoline a1-adrenoceptor agonist. Prazosin
(selective a1-adrenoceptor antagonist) significantly reduced the magnitude of contraction to L-erythro-methoxamine at the
highest concentration used. Both prazosin and RX811059 (a selective a2-adrenoceptor antagonist) reduced the potency
(pEC50) of clonidine.
CONCLUSIONS AND IMPLICATIONS
This study shows that both a1- and a2-adrenoceptors are expressed in the sheep IAS, and contribute (perhaps synergistically)
to contractions elicited by various imidazoline derivatives. These agents may prove useful in the treatment of faecal
IAS, internal anal sphincter
British Journal of
British Journal of Pharmacology (2010) 160 1727–17401727
© 2010 The Authors
Journal compilation © 2010 The British Pharmacological Society
Faecal incontinence is a common condition which,
depending on criteria applied, affects up to 2% of
the population (Perry et al., 2002) and can have a
devastating effect on quality of life. Clinical man-
agement of the condition has historically relied on
non-pharmacological approaches, although there is
increasing interest in modulating the contractile
state of the anal sphincter smooth muscle to allevi-
ate symptoms (Tuteja and Rao, 2004).
It has long been recognized that sympathetic
activation of a-adrenoceptors contributes to conti-
nence in man (Parks et al., 1969; Frenckner and
Ihre, 1976). In 1975, Gutierrez and Shah reported
that intravenous infusion or oral administration of
a-adrenoceptor antagonists caused a reduction in
anal sphincter pressure (Gutierrez and Shah, 1975).
Similarly, the selective a1-adrenoceptor antagonist
indoramin caused a reduction in anal sphincter
pressure in patients suffering from anal fissures
(Pitt et al., 2001). These observations are consistent
with reports on isolated segments of the human
internal sphincter that a1-adrenoceptors mediate
constriction (Speakman et al., 1990; Glavind et al.,
1997). Several recent clinical trials have been con-
ducted to examine the effect of a-adrenoceptor
agonists on anal sphincter pressure in both healthy
volunteers and patients (Carapeti et al., 1999;
2000; Cheetham et al., 2001; Badvie and Andreyev,
2005; Park et al., 2007). Anal (topical) application of
the selective a1-adrenoceptor agonist phenyleph-
rine was found to cause a significant increase in
resting anal pressure in healthy volunteers (Cara-
peti et al., 1999). Subsequent use of this agonist in
patients with faecal incontinence noted a signifi-
cant increase in maximum resting pressures, but
only when phenylephrine was employed at higher
concentrations (10% w/w) than in volunteers
and which may account for the incidence of local
side effects (Carapeti et al., 2000). Later trials with
patients suffering with passive faecal incontinence
(Cheetham et al., 2001), radiation-induced faecal
incontinence (Badvie and Andreyev, 2005) and
incontinence resulting from low anterior resection
(Park et al., 2007) produced mixed results. Based
on incontinence scoring, radiation-induced faecal
incontinence was improved by 10–20% (w/w)
while a 30% (w/w) gel was insufficient to improve
the continence scores or resting pressure of low
anterior resection patients (Park et al., 2007). Con-
versely, in patients suffering with passive faecal
incontinence, 30% (w/w) phenylephrine gel was
sufficient to increase resting pressure (Cheetham
et al., 2001).
Mortensen and colleagues reported that an
isomer of methoxamine, L-erythro-methoxamine,
was four times more potent than phenylephrine as a
constrictor agent in the porcine isolated internal
anal sphincter (IAS) in vitro (Jones et al., 2003). Based
on these findings, trials have been conducted in
human volunteers and patients. Intra-anal and
rectal application of L-erythro-methoxamine gel in
healthy volunteers demonstrated rapid and sus-
tained increase in resting pressure from a concen-
tration as low as 1% w/w (Nisar et al., 2005).
applied to patients suffering with faecal inconti-
nence, resting pressures similarly increased rapidly
and gave a sustained response (Nisar et al., 2007).
However, application of 3% L-erythro-methoxamine
in an immediate release formulation was associated
with an increased systemic absorption and side
effects of hypertension and bradycardia in some
The potential role of a2-adrenoceptors in faecal
continence has not been explored in man, but work
in the anaesthetized opossum suggests that the imi-
dazoline derivative clonidine can activate the myen-
teric neurones responsible for inhibition of the
recto-anal inhibitory reflex (Yamato and Rattan,
1990). Although there are no comparable studies in
man, a2-adrenoceptors are known to be present
in the duodenum and proximal colon (Zhang
et al., 1992; Valet et al., 1993), and administration of
selective a2-adrenoceptor agonists (e.g. clonidine) is
associated with an increase in colonic and rectal
compliance (Viramontes et al., 2001; Camilleri et al.,
2003). Interestingly, a2-adrenoceptors have been
reported to play a role in noradrenaline-mediated
constriction of the pig isolated IAS, but only at high
concentrations, suggesting that a1-adrenoceptors
are predominant in contractile responses to a-
adrenoceptor agonists (Mills et al., 2008).
As changes in modern surgical practice have led
to a significant increase in sphincter-preserving
surgery which allows the post-operative patient to
maintain voluntary continence, access to human
tissue is limited. With this in mind, the current
study has utilized a sheep model of the IAS to
characterize the a-adrenoceptors in sheep IAS, and
used this model to investigate potential pharmaco-
logical tools for the therapeutic treatment of faecal
incontinence. The aim of the current study was
twofold. First, to establish the presence and where
possible the distribution of a-adrenoceptors in
sheep and human IAS; and second, to investigate
whether a variety of imidazoline compounds com-
pared to known a1-adrenoceptor agonists exhibit
functional effects at a-adrenoceptors expressed in
SJ Rayment et al.
1728British Journal of Pharmacology (2010) 160 1727–1740
Human IAS tissue was collected in accordance with
ethical approval obtained from the University of
from patients undergoing a proctectomy either as
part of an abdominoperineal resection or comple-
tion procedure as a consequence of suffering from
ulcerative colitis. Care was taken to ensure that
none of the patients had active disease affecting the
anal canal at the time of operation.
Sheep anus, containing IAS and rectum, were
obtained from a local abattoir and transported to
the lab on ice within 120 min and stored at 4°C.
Within a further 2 h, the anal canal was divided
along its ventral longitudinal axis and pinned to a
corkboard with the mucosa uppermost. The mucosa
was reflected at the dentate line to expose the under-
lying white circular muscle fibres of the IAS. Further
dissection from the underlying external sphincter
enabled a thin strip of circular internal sphincter to
be prepared. The IAS was dissected and used for the
Radioligand binding experiments
Sheep IAS was dissected from anal tissue on the day
of collection and frozen at -20°C until required.
When required, the defrosted tissue was crudely
chopped and homogenized using an Omni macro
homogenizer (Omni International, Kennesaw, GA,
USA) in a minimal volume of ice-cold buffer
(50 mM, Tris–HCl pH 7.6). Tissue typically required
three 30 s homogenizations at maximum speed to
achieve complete tissue disruption. Ice-cold Tris
buffer was used to increase the homogenate volume
to 10 times original tissue wet weight before cen-
trifugation at 1500¥ g for 10 min at 4°C. The super-
natant was passed through a gauze filter then
centrifuged at 30 000¥ g for 30 min at 4°C. The
resulting pellet was then washed and resuspended in
2 volumes tissue wet weight of ice-cold Tris buffer.
Following radioligand binding, fractions of mem-
brane preparations were retained and stored at
-20°C for estimation of protein concentration by
Lowry assay (Lowry et al., 1951). In all cases, mem-
brane preparations were made from frozen tissue
and used for experiments on the same day, because
preliminary experiments had previously revealed
that if frozen membrane preparations were used,
they failed to reveal significant binding of the
Saturation binding with [3H]-prazosin (selective
antagonist for a1-adrenoceptors) was performed in a
500 mL reaction volume consisting of 200 mL mem-
brane preparation; 200 mL 50 mM Tris–HCl, pH 7.6;
50 mL of 50 mM TE buffer (50 mM Tris, 1 mM EDTA,
pH 7.4) or 100 mM noradrenaline (used to define
non-specific binding) and 50 mL [3H]-prazosin using
eight doubling concentrations in a range of 0.04–
5 nM (final concentration). Reactions were initiated
by the addition of membrane preparation, and
after thorough mixing, were incubated at 25°C for
30 min. Reactions were terminated by filtration
under vacuum using a Brandel cell harvester (Semat,
Herts, UK). Filters were washed three times with
ice-cold TE buffer to remove unbound radioligand,
and remaining bound ligand was evaluated by
performed in triplicate. Saturation studies with
a2-adrenoceptors) were performed as described
above, but rauwolscine (100 mM final concentra-
tion) was used instead of noradrenaline, to define
incubated for 60 min rather than 30 min, and the
concentration range for [3H]-RX821002 was 0.08–
10 nM. These conditions have been used in previous
studies using these radioligands (Wright et al.,
1995). Competition studies using [3H]-prazosin and
[3H]-RX821002 were performed in a 500 mL volume
using 200 mL membrane preparation, 200 mL 50 mM
Tris–HCl, 50 mL [3H]-antagonist at a fixed concentra-
tion and 50 mL of competitor at increasing concen-
trations. The relative affinities of seven different
imidazoline derivatives at a1- and a2-adrenoceptors
were compared to L-erythro-methoxamine and nor-
drenaline using competition binding.
Strips of sheep and human circular IAS were embed-
ded in OCT (BDH, Poole, Dorset, UK), and sections
(10 mm thick) were cut in a cryostat at approxi-
mately -20°C and thaw-mounted on poly-lysine-
coated slides. Immunohistochemical staining of
sections proceeded according to instructions for
Vectastain ABC-AP kit (Vector laboratories Inc., Bur-
lingame, CA, USA). Briefly, sections were allowed to
equilibrate to room temperature prior to fixation in
cold acetone at -20°C for 20 min. After residual
fixative had evaporated, slides were incubated in
phosphate-buffered saline (PBS) containing 10%
normal horse serum and incubated at room tem-
perature for 10 min in humid conditions to block
non-specific binding. All subsequent incubations
were also carried out at room temperature in humid
conditions. After removing the blocking solution,
the slides were incubated in PBS (negative control)
or PBS containing one of the following antibodies
for 30 min: anti-a-smooth muscle actin (1:200 dilu-
tion; to identify smooth muscle cells), anti-CD31
(1:100; endothelial cell marker), anti-NF200 (1:200;
foridentification of neurones), anti-tyrosine
a-Adrenoceptor function in sheep anal sphincter
British Journal of Pharmacology (2010) 160 1727–17401729
hydroxylase (1:250; adrenergic neurone marker) or
anti-a1A-adrenoceptor (1:400; Genway Biotech Inc.,
San Diego, CA, USA). The slides were washed three
times in PBS before incubating with biotinylated
universal antibody in blocking solution (as per
washing three times in PBS again and incubating
with the ABC-AP reagent for 5 min. The slides were
once again washed three times in PBS before devel-
opment with alkaline phosphatase substrate solu-
tion (in 200 mM Tris buffer, pH 8.2). Reactions were
terminated by immersion and rinsing in water.
The slides were subsequently counterstained
with haematoxylin for 15 s, rinsed in running tap
water, dehydrated in increasing concentrations of
methanol, clearing in Histoclear (National Diagnos-
tics, Hull, UK) and coverslipped using DPX moun-
tant (BDH). A minimum of three tissues (two
non-consecutive sections from each tissue) were
evaluated for each antibody used.
for 10 minbefore
Sections of sheep IAS (10 mm) were thaw-mounted
on poly-lysine-coated slides as described for the
50 mM Tris–HCl, pH 7.6, for 10 min at 4°C to
remove endogenous ligand. The sections were then
transferred to 50 mM Tris–HCl, pH 7.6, containing
Non-specific binding was evaluated by incubating
adjacent sections in the presence of 100 mM norad-
renaline. The binding reaction was terminated by
washing the slides twice in TE buffer (4°C) before
rinsing in distilled water (4°C). The sections were
dried in a continuous stream of cold air, followed
(~22°C). A parallel series of experiments were con-
ducted using [3H]-RX821002 (2–25nM) where non-
specific binding was evaluated in the presence of
rauwolscine (100–250 mM).
mounted sections were exposed to
(Amersham Biosciences, Little Chalfont, UK) in
X-ray cassettes for up to 9 weeks at 4°C. Films were
developed using Kodak D19 and fixed using Hypam
(Ilford, Moberly, Cheshire, UK). For high-resolution
autoradiography, sections were dipped in molten
nuclear emulsion (K2, Ilford) following air-drying.
The slides were stored in light-proof boxes con-
taining dessicantat 4°C
evidence of binding to tissue sections. Following
development, the sections were counterstained with
Olympus BX50 microscope (Olympus Corporation,
Tokyo, Japan) and photographed where appropriate
3H-prazosin, then incubated for 1 h at 4°C.
at room temperature
on an AxioCam digital camera (Carl Zeiss MicroIm-
aging GmBH, Göttingen, Germany) with images
stored on a KS300 imaging system (Imaging Associ-
ates, Bicester, UK).
Following collections of the sheep anal tissue (as
described above), a thin strip of circular internal
sphincter measuring approximately 50 mM long,
5 mM wide and 2 mM thick was removed and stored
overnight in Krebs–Henseleit solution at 4°C. The
following day, tissue strips were allowed to warm up
to room temperature. Additional connective tissue
was removed by sharp dissection from the speci-
men, and up to a maximum of four strips of internal
sphincter tissue (approximately 10 ¥ 2 ¥ 2 mM) con-
taining visible parallel muscle fibres were prepared
and used for experiments. Silk ligatures were tied to
each end of the strips, and the lower end was
attached to a perspex holder between two parallel
platinum wire electrodes (although electrical field
stimulation was not performed in this series of
experiments). The upper end was secured to a Grass
FT-03 isometric force transducer (Grass Technolo-
gies, West Warwick, RI, USA) ensuring some laxity in
the suspended tissue which was bathed for approxi-
mately 30 min in 20 mL organ baths containing
Krebs–Henseleit solution (pH 7.4), gassed (95%
O2/5% CO2) at 37°C. The transducer was connected
to a MacLab Bridge amplifier (AD Instruments,
Chalgrove, UK) and linked via a four-channel
MacLab unit to a Macintosh LC II computer
running Chart 3.5.
The strips were initially placed under 19.62 mN
tension, which has previously been identified as
an optimal stretch tension necessary for the devel-
opment of spontaneous myogenic tone in sheep
isolated IAS (Mundey et al., 2000). Once stable myo-
genic tone had been reached, cumulative concentra-
tion curves were constructed by half-logarithm
increments using a total of six imidazoline com-
pounds, which were compared to responses to the
and noradrenaline. Only one agonist was used per
strip. A minimum of 5 min was allowed between the
addition of individual concentrations, and subse-
quent additions were made only after the previous
response had reached equilibrium. In addition, the
responses of L-erythro-methoxamine and clonidine
were examined in the presence or absence of
100 nM prazosin and 100 nM RX-811059, antago-
nists selective for a1- and a2-adrenoceptors, respec-
tively (McGrath et al., 1989; Mallard et al., 1992).
The antagonists were allowed to equilibrate for
30 min prior to construction of the concentration
SJ Rayment et al.
1730 British Journal of Pharmacology (2010) 160 1727–1740
For saturation binding experiments, Kd and Bmax
(GraphPad, San Diego, CA, USA) and represented
as the mean ? SEM for triplicate experiments.
Competition binding curves were similarly con-
structed, and the mean pKi values ? SEM for trip-
licate experiments calculated.
studies, changes in tension with the addition of
drugs were determined as the maximal increase or
decrease in tone, and are expressed as a percentage
of the initial stable myogenic tone. Individual
concentration–response curves were constructed
for each tissue, and the negative logarithm of the
concentration required to achieve 50% of the
maximum response (pEC50) and the maximum
response relative to myogenic tone (Rmax) were
between mean pEC50 and Rmax values for L-erythro-
methoxamine and clonidine in the presence or
absence of antagonists were compared using a one-
way ANOVA with Dunnett’s post hoc test and con-
sidered significant if P < 0.05.
The composition of the modified Krebs–Henseleit
saline was (in mM): NaCl 118.4, KCl 4.7, CaCl2
1.25, MgSO41.2, NaHCO324.9, KH2PO41.2, glucose
11.1. TE buffer was (in mM): Tris 50, EDTA 1, pH 7.4.
The drugs used were obtained from Sigma-Aldrich
(Poole, UK) with the exception of: prazosin hydro-
methoxamine (Norgine, Hengoed, UK), rauwolscine
(Carl Roth GmbH & Co. KG, Karlsruhe, Germany),
moxonidine (Tocris Bioscience, Bristol, UK), xylome-
tazoline (Chemos GmbH, Regenstauf, Germany),
hydrochloride (RS100329, Tocris Bioscience), 8-[2-
o[4.5]decane-7,9-dione dihydrochloride (BMY7378,
Tocris Bioscience), 2-[(4,5-dihydro-1H-imidazol-2-
eate (BRL44408, Tocris Bioscience).
[3H]-prazosin were obtained from GE Healthcare Ltd
(Little Chalfont, UK). Prazosin (1 mM) was dissolved
in 0.1 M lactic acid. All further dilutions were
made in distilled water. The concentration of the
vehicle never exceeded 0.3% v/v. All drug and
molecular target nomenclature follows Alexander
et al. (2009).
prazosin and [3H]-RX-821002 were able to bind to
membranes of the sheep IAS in a concentration-
dependent manner that was inhibited by 100 mM
noradrenaline and 100 mM rauwolscine respectively.
At the half-maximally effective concentration (Kd),
approximately 87 and 90% of the binding of
[3H]-prazosin and [3H]-RX-821002, respectively, was
inhibited. [3H]-prazosin was calculated to have a Kd
value of 0.58 ? 0.07 nM (n = 3) and identified sites
at density of 222 ? 4 fmol·mg-1protein (n = 3),
while [3H]-RX821002 was slightly less potent (Kd =
0.7 ? 0.14 nM, n = 3) and labelled approximately
one-third the number of sites (72 ? 7 fmol·mg-1
protein, n = 3). These findings indicate that the
sheep isolated IAS possesses both a1- and a2-
adrenoceptor binding sites.
Figure 1shows that[3H]-
Discrimination of a-adrenoceptor sub-type using radio-
Competition binding analysis was
subsequently used to investigate which sub-type of
a-adrenoceptor was identified by each ligand in this
tissue. The pKivalues for the competing agents gen-
erated from these experiments are summarized in
Table 1. With the exception of RS100329, all of the
Hill slope values were close to unity (>0.8). In each
instance, the competing agents displaced >90% of
the specific binding of the ligands (data not shown).
RS100329 (an a1A-adrenoceptor-selective antagonist;
Williams et al., 1999), 5-methyl-urapidil (discrimi-
nates a1A from a1B/a1D-adrenoceptor sub-types; Ford
et al., 1997) and BMY7378 (a1D-adrenoceptor sub-
type-selective antagonist; Piascik et al., 1995) were
used to investigate the sub-type of a1-adrenoceptor
present in sheep IAS. The data summarized in
Table 1 indicate that the rank order of affinity for
these agents is 5-methylurapidil ?RS100329 >>
BMY7378, suggesting the presence of the a1A-
Four agents were used to provide information on
the a2-adrenoceptor sub-type present. BRL44408
was used as a selective a2A/a2D-adrenoceptor anta-
gonist (Wikberg-Matsson et al., 1995); prazosin,
although non-selective at a1-adrenoceptor sub-
types, can be employed to discriminate between a2C-
adrenoceptors (for which it has high affinity) and
a2A/a2D-adrenoceptors (McGrath et al., 1989); phen-
tolamine and rauwolscine were also used, as the
relative affinities of these two agents can be used to
discriminate between a2A- and a2D-adrenoceptor
sub-types. If the affinity of rauwolscine is greater
than phentolamine, it is indicative of the presence
a-Adrenoceptor function in sheep anal sphincter
British Journal of Pharmacology (2010) 160 1727–17401731
of an a2A-adrenoceptor; if the affinity of phentola-
mine is greater than rauwolscine, then an a2D-
adrenoceptor is implied (Trendelenburg et al., 1996;
Naselsky et al., 2001). The data summarized in
Table 1 indicate that the rank order of affinity for
these agents is BRL44408 > phentolamine > rauwols-
cine > prazosin, which is consistent with the pres-
ence of a2D-adrenoceptors.
Saturation binding data for [3H]-prazosin (A,B) and [3H]-RX-821002 (C,D). Representative data showing total and non-specific binding from a
single experiment are shown in (A) for [3H]-prazosin and (C) for [3H]-RX821002. (B) and (D) show representative specific binding curves from a
single experiment using [3H]-prazosin and [3H]-RX821002 respectively.
Mean pKi values (?SEM of three experiments) for sub-type-selective inhibitors at a1- ([3H]-prazosin) or a2-adrenoceptors ([3H]-RX821002) on
pKi(Hill slope) [3H]-RX-821002 pKi(Hill slope) [3H]-prazosin
8.85 ? 0.13 (-0.81 ? 0.06)
5.32 ? 0.13 (-1.07 ? 0.09)
7.91 ? 0.12 (-0.83 ? 0.08)
8.66 ? 0.08 (-0.84 ? 0.07)
9.14 ? 0.11 (-0.68 ? 0.06)
9.52 ? 0.03 (-1.27 ? 0.08)
6.61 ? 0.04 (-0.98 ? 0.09)
n/d, the experiment was not done.
SJ Rayment et al.
1732 British Journal of Pharmacology (2010) 160 1727–1740
Competition binding with agonists.
tive agonists examined using competition binding
showed concentration-dependent displacement of
both radioligands and, with the exception of tizani-
dine ([3H]-prazosin) and moxonidine ([3H]-RX-
821002), the Hill slope values were close to unity
(>0.8). The pKi values for these compounds are
included in Table 2. The affinity of the imidazoline
compounds against [3H]-prazosin varied a 1000-
fold, with oxymetazoline and moxonidine the
most andleast potent
[3H]-RX821002 binding sites, a 100-fold range in
potency was observed between the imidazoline
derivatives (Table 2).
The ratio of mean [3H]-RX821002 and [3H]-
prazosin pKivalues (a2:a1adrenoceptor binding) was
used to establish receptor selectivity of agonists.
Based on this analysis, a ratio with a value higher
than 1 indicates an a2-selective agonist, whereas a
value of less than 1 suggests an a1-adrenoceptor
selective agent. The majority of the agonists exhib-
ited 3- to 30-fold selectivity for a2-adrenoceptor
binding sites over a1-adrenoceptor binding sites,
with moxonidine exhibiting the greatest selectivity.
binding sites (Table 2). Noradrenaline, xylometazo-
line and oxymetazoline did not distinguish between
the a1- and a2-adrenoceptor binding sites.
All of the puta-
Sections of sheep IAS that had been exposed to the
anti-a-smooth muscle actin antibody and counter-
stained with haematoxylin showed a pronounced
positive staining (red colouration) throughout the
tissue (Figure 2A,B). The staining was most intense
in bundles surrounded by a loose network of pale
Anti-CD31 antibodies were used as a marker for
the presence of endothelial tissue, and therefore
indicative of the vasculature associated with the
tissue sections of sheep IAS (Figure 2C,D). Unlike
the smooth muscle actin, this antibody stained dis-
crete areas of the tissue sections that appear to cor-
respond to the areas of pale pink immunostaining
surrounding the bundles of a-smooth muscle actin-
Two antibodies were used to identify neuronal-
derived tissue. As shown in Figure 2E,F, anti-NF200
was found to have a distribution of staining largely
similar to anti-CD31 antibodies in the areas of weak
a-smooth muscle actin staining. However, closer
examination of the tissue also revealed a weaker
distribution of staining with anti-NF200 that also
corresponds to the a-smooth muscle actin-rich
bundles of tissue. In addition to NF200, an anti-
tyrosine hydroxylase antibody was also used to
identify adrenergic nerves. The profile of intense
staining was similar to that observed using the
observed in both the a-smooth muscle actin-rich
tissue and surrounding tissue which was weakly
stained by the a-smooth muscle actin antibody
(Figure 2G,H). In the absence of primary antibody,
the slides revealed a low level of staining (pale pink)
that corresponded to the a-smooth muscle actin-
poor regions of the tissue (Figure 2I,J).
Based on the results of a1-adrenoceptor sub-type
competition binding, sections of sheep and human
IAS were also incubated with an a1A-adrenoceptor
antibody. Results using this antibody can be seen in
Figure 3 with their corresponding negative controls.
Overall, both tissues showed a similar distribution
of positive staining: with a diffuse pink positive
staining over the smooth muscle bundles, as well as
discrete areas of dense positive staining which could
Mean pKivalues (?SEM of three experiments) for imidazoline compounds at both a1- ([3H]-prazosin) and a2-adrenoceptor ([3H]-RX-821002) on
the sheep IAS
pKi(Hill slope) [3H]-RX-821002 pKi(Hill Slope) [3H]-prazosin
7.99 ? 0.01 (-0.79 ? 0.01)
7.55 ? 0.09 (-0.91 ? 0.07)
7.23 ? 0.09 (-0.94 ? 0.02)
6.92 ? 0.05 (-1.07 ? 0.10)
6.75 ? 0.03 (-0.82 ? 0.04)
6.45 ? 0.04 (-0.87 ? 0.05)
5.98 ? 0.06 (-0.75 ? 0.05)
5.62 ? 0.08 (-0.90 ? 0.14)
4.00 ? 0.03 (-0.99 ? 0.08)
6.84 ? 0.09 (-1.04 ? 0.08)
6.39 ? 0.10 (-0.95 ? 0.02)
7.67 ? 0.13 (-0.87 ? 0.05)
7.10 ? 0.15 (-0.82 ? 0.04)
5.57 ? 0.05 (-0.76 ? 0.03)
5.33 ? 0.04 (-0.89 ? 0.05)
4.62 ? 0.02 (-0.88 ? 0.02)
5.47 ? 0.06 (-0.83 ? 0.02)
5.40 ? 0.07 (0.86 ? 0.04)
a-Adrenoceptor function in sheep anal sphincter
British Journal of Pharmacology (2010) 160 1727–17401733
be found either in or between the muscle bundles.
This discrete dense staining was particularly appar-
ent in the human specimens.
Using 5 nM [3H]-prazosin, significant binding was
observed in sheep IAS after 8 weeks exposure (a
typical example is shown in Figure 4). Specific
binding occurs in discrete areas of smooth muscle
bundles, with no specific binding observed in con-
nective tissue between smooth muscle bundles or
adventitia (Figure 4A,B). Attempts to generate com-
parable images with 2–25 nM [3H]-RX-821002 were
unsuccessful. Even after 12 weeks exposure, no
evidence of 25 mM [3H]-RX821002 rauwolscine-
sensitive ‘hotspots’ were observed (n = 3), although
specific binding could be observed using sections of
rat brain as positive controls incubated under the
same conditions (data not shown).
The sheep IAS responded to the initial stretch with
the generation of sustained increase tension above
baseline (35.61 ? 2.84 mN, n = 28). All of the com-
dependent contractions that took 5–10 min to
attain a peak response that declined slowly there-
after. Rilmenidine was not investigated in detail
maximum response was not appreciably different
from the other imidazoline derivatives (data not
shown). Moreover, the sub-type selectively dis-
played in the radioligand binding experiments was
Table 2). Based on the pEC50 values (Table 3), there
was less than a 10-fold difference in potency
between all six of the imidazoline compounds
tested; in the case of moxonidine, many prepara-
tions failed to produce a true maximum response so
the pEC50values shown is an estimate. Xylometazo-
line was the most potent imidazoline agonist, which
was between 10- and 100-fold more potent than
L-erythro-methoxamine and noradrenaline, respec-
tively (Table 3). Although L-erythro-methoxamine
and noradrenaline were less potent than the imida-
zoline derivatives, the maximum responses were
significantly larger (see Table 3); based on the
increase in tone calculated relative to the baseline
tone (% of resting tone), the response to L-erythro-
methoxamine was approximately twofold greater
than that elicited by clonidine (Student’s unpaired
t-test, P < 0.01).
Figure 5A showscumulative
curves for L-erythro-methoxamine in the presence
or absenceof 100 nM
a1-adrenoceptor antagonist) or 100 nM RX-811059
and tizanidine (see
Anti-a-smooth muscle actin antibody at low (A) and high magnifi-
cation (B); CD31 antibody at low (C) and higher magnification (D);
NF200 antibody at low (E) and high magnification (F); tyrosine
hydroxylase antibody at low (G) and high magnification (H); nega-
tive control (no primary antibody) at low (I) and high magnification
(J). Arrows are used to indicate dense areas of positive staining in
Figure 2 (G). Positive immunostaining is red and sections counter-
stained with haematoxylin (blue).
SJ Rayment et al.
1734British Journal of Pharmacology (2010) 160 1727–1740
(a selective a2-adrenoceptor antagonist). Contractile
responses observed in the presence of 100 nM
RX-811059 did not differ significantly in terms of
pEC50 (control 5.63 ? 0.09, n = 14; RX-811059 5.43
? 0.10, n = 9) or maximum contraction (control
50.72 ? 5.59 mN, n = 14; RX811059 56.31 ?
3.83 mN, n = 9) from those observed in control
experiments. In contrast, 100 nM prazosin caused a
30-fold rightward displacement of the concentra-
tion response curve to L-erythro-methoxamine; at
the highest concentration of the agonist employed
(100 mM), there was a significant reduction in the
response (control 136 ? 18%, n = 14; prazosin 59 ?
15%, n = 12; Student’s paired t-test P < 0.005).
Comparison of staining using anti-a1A-adrenoceptor antibody in sheep (A) and human (B) IAS at high magnification. Negative controls (no primary
antibody) are also shown for sheep (C) and human (D) tissues. Positive immunostaining is pink or red, with haematoxylin counterstaining (blue).
Film images showing the localization of3H-prazosin binding in sections of sheep IAS: (A) Total binding of 5 nM3H-prazosin to sheep IAS with some
areas of dense binding. (B) Tissue underlying total binding, counterstained with haematoxylin and eosin. (C) Non-specific binding of 5 nM
3H-prazosin in the presence of 100 mM noradrenaline. These images are representative of sections taken from three individual sheep IASs.
a-Adrenoceptor function in sheep anal sphincter
British Journal of Pharmacology (2010) 160 1727–1740 1735
Cumulative concentration curves for clonidine
in the presence or absence of 100 nM prazosin and
100 nM RX811059 are shown in Figure 5B. Both
antagonists significantly affected the pEC50 values
calculated compared to control (control 6.55 ?
0.10, n = 8; prazosin 4.52 ? 0.52, n = 9; RX811059
6.03 ? 0.16, n = 8) based on analysis by one-way
ANOVA with Dunnett’s post hoc test (prazosin
P < 0.01; RX811059 P < 0.05). Neither antagonist
affected the Rmax values observed (control 37.57 ?
5.40 mN, n = 8; prazosin 43.65 ? 9.03 mN, n = 9;
RX811059 34.43 ? 4.91 mN, n = 8).
Presence and distribution of a-adrenoceptors
in the sheep IAS
Saturation binding analysis of membrane pre-
parations of the sheep IAS with [3H]-prazosin and
[3H]-RX821002 revealed the presence of two dis-
crete populations of a-adrenoceptor binding sites.
The density of a1-adrenoceptor binding sites was
approximately threefold greater than that of a2-
adrenoceptor binding sites.
Using sub-type-selective a1-adrenoceptor antago-
nists, the rank order of affinity against [3H]-prazosin
bindingin the sheep
5-methylurapidil >> BMY7378. Based on the pub-
lished data for recombinant human receptors
(McGrath et al., 1989; Mallard et al., 1992), the rank
order of affinity is consistent with that described for
the a1A-adrenoceptor sub-type. This conclusion is in
agreement with recent observations in the pig iso-
lated IAS where RS100329 was reported to competi-
tively inhibit phenylephrine-induced contractions
with a pA2 value of 9.0 (Mills et al., 2008).
For the population of a2-adrenoceptor binding
sites, sub-type-selective antagonists suggested the
presence of a2D-adrenoceptors. This conclusion is
based on the finding that BRL4408 showed high
affinity (pKi 8.85) against [3H]-RX821002 binding,
while prazosin was only weakly active (see Table 1),
adrenoceptor. These species orthologues of a2-
adrenoceptors can be distinguished on the basis of
the relative potency of phentolamine and rauwols-
cine (Trendelenburg et al., 1996; Naselsky et al.,
2001). As phentolamine was approximately fivefold
more potent than rauwolscine, the sub-type present
in sheep is similar to that found in the rat and
cow (a2D-adrenoceptor) rather than that previously
described in man and pig (a2A-adrenoceptor). As far
as we are aware, the available data suggest that
the two species orthologues respond in a similar
manner to known a2-adrenoceptor agonists, so the
functional significance of this difference is unclear.
Immunohistochemical analysis revealed that
the sheep IAS is composed of bundles of smooth
muscle fibres. Interestingly, the smooth muscle-poor
regions of the tissue were highly stained by antibod-
ies to CD31 and NF200, which suggests that vascular
endothelium and enteric nerves are closely associ-
ated with connective tissue that links the bundles of
smooth muscles. The finding that positive staining
for adrenergic nerves (using a tyrosine hydroxylase
antibody) can be identified in close association
with the smooth muscle bundles is consistent with
adrenergic innervation of the IAS as observed in
studies with [3H]-prazosin revealed the presence of
a1-adrenoceptor binding sites on the smooth muscle
bundles that were largely absent from the smooth
muscle-poor regions of the tissue. Positive staining
The potency of imidazoline agonists, noradrenaline and L-erythro-methoxamine as contractile agents in sheep IAS
Maximal responses (%
of resting tone)n
6.89 ? 0.06
6.78 ? 0.36
6.46 ? 0.20
6.44 ? 0.07
6.01 ? 0.17
5.22 ? 0.11
5.60 ? 0.08
4.68 ? 0.29
21.88 ? 5.49
19.13 ? 2.84
28.15 ? 7.26
30.51 ? 4.22
27.37 ? 5.79
19.91 ? 4.02
42.58 ? 4.91
34.14 ? 5.20
63 ? 23
58 ? 13
57 ? 13
62 ? 7
102 ? 37
67 ? 10
133 ? 17
214 ? 57
Values shown are the mean ? SEM.
SJ Rayment et al.
1736British Journal of Pharmacology (2010) 160 1727–1740
in smooth muscle bundles of human and sheep IAS
was further confirmed using an antibody specifically
targeting the a1-adrenoceptor sub-type indicated
adrenoceptors). In light of the pattern of adrenergic
neurones revealed by the immunohistochemistry,
confirmation of the presence of a1-adrenoceptors in
sheep and human tissue lends further weight to the
interpretation of adrenergic innervation.
Surprisingly, the distribution of a2-adrenoceptor
binding sites could not be revealed by this method.
Even with several modifications to the protocol,
including elevation of the concentration of [3H]-
RX821002 to 10-fold greater than the Kd value,
elevation of the concentration of rauwolscine (the
displacing agent) to 100 mM and increased duration
of exposure of the tissue segments to the film, no
specific binding was detected. In contrast, ‘hot
spots’ of [3H]-RX821002 binding sites was observed
in slicesoftherat cerebral
250 fmol·mg-1protein) when run in parallel experi-
ments (unpublished observations). Thus, it seems
likely that the low density of a2-adrenoceptor
binding sites in the sheep IAS precludes autoradio-
The activity of imidazoline derivatives
compounds at a-adrenoceptors
Three observations prompted a detailed study
of biological activity of imidazoline derivatives in
the sheep IAS. First, clinical evidence that the
greater potency of L-erythro-methoxamine at a1-
allowed for low concentrations to be used to raise
anal sphincter pressure in volunteers and patients
(Carapeti et al., 1999; 2000; Cheetham et al., 2001;
Badvie and Andreyev, 2005; Nisar et al., 2007; Park
et al., 2007). Second, imidazoline derivatives (e.g.
xylometazoline, and clonidine) are generally known
as agonists at a-adrenoceptors with greater potency
than either noradrenaline or adrenaline, but possess
varying selectivity for the sub-types (McGrath et al.,
1989). Compounds in this class are used clinically as
nasal decongestants, anti-hypertensive agents and
anti-spasticity agents, and to reduce symptoms of
opiate withdrawal. Third is the finding that anal
sphincter smooth muscle possesses both a1- and
a2-adrenoceptor binding sites (see above). Thus,
seven different imidazoline derivatives were exam-
ined in competition binding studies to determine
their relative affinity at a1- and a2-adrenoceptors in
the anal sphincter.
With the exception of xylometazoline and
oxymetazoline, all of the imidazoline derivatives
exhibited a greater selectivity (>3-fold) for a2-
adrenoceptor binding sites over a1-adrenoceptor
binding sites.In general,
possessed a higher affinity (3- to 300-fold) at a2-
adrenoceptor binding sites than either L-erythro-
methoxamine or noradrenaline. For the majority
adrenoceptor binding sites in the sheep IAS agrees
with the known activity at recombinant human a2A/
a2D-adrenoceptors expressed in HEK cells (Jasper
et al., 1998). In contrast, at a1-adrenoceptor binding
sites, the imidazoline derivatives can be divided into
two groups: those with an affinity comparable to
L-erythro-methoxamine and noradrenaline (tizani-
dine, rilmenidine and moxonidine) and those that
Comparison of the effect of (A) L-erythro-methoxamine and (B)
clonidine on the sheep isolated IAS in the absence (control) and the
presence of 0.1 mM prazosin and 1 mM RX-811059. The responses
shown are the mean ? SEM of 9–14 observations.
a-Adrenoceptor function in sheep anal sphincter
British Journal of Pharmacology (2010) 160 1727–17401737
possessed a 10-fold higher affinity (naphazoline,
clonidine, oxymetazoline and xylometazoline).
Contraction-based experiments revealed that
all the agents examined elicited concentration-
dependent responses. A characteristic feature of the
results is that the maximum responses for the imi-
dazoline compounds were approximately 30% of
that elicited by noradrenaline, an effect similar to
that observed in other smooth muscle preparations
that possess a1-adrenoceptors, such as rat aorta
(Ruffolo and Waddell, 1982), porcine splenic artery
(Barbieri et al., 1998) and human nasal mucosa
(Johannssen et al., 1997). Also, with the exception
of moxonidine, L-erythro-methoxamine and norad-
renaline, the potency of the compounds was more
closely related to the affinity detected against the
a1-adrenoceptor binding sites. Confirmation of the
predominant role of a1-adrenoceptors was obtained
when examining the effect of sub-type-selective
contractions. In each case, prazosin (100 nM)
caused an approximate 30-fold rightward dis-
placement of the concentration–response curve.
However,while the selective
tions, it caused a threefold rightward displacement
of clonidine-induced contractions. The latter obser-
vation provides unambiguous evidence for the pre-
sence of a2-adrenoceptors on the smooth muscle
of the sheep isolated internal sphincter and raises
the possibility of a synergistic interaction with
A functional role for a2-adrenoceptors in the
control of defaecation has largely been confined to
the enteric nervous system; by reducing the rate
of gastrointestinal transit (Scheibner et al., 2002),
interference in the ano-rectal inhibitory response in
the opossum (Yamato and Rattan, 1990) and modu-
lation of colonic and rectal compliance in man
(Viramontes et al., 2001; Camilleri et al., 2003). The
finding that a2-adrenoceptors are also present on
the sphincter muscle and have the potential to
enhance a1-adrenoceptor responses, may explain
the observation in the anaesthetized opossum that
naphazoline causes both increased IAS pressure
and inhibited the recto-anal inhibitory response
(Yamato and Rattan, 1990).
The original basis for examining the potential of
L-erythro-methoxamine for treating faecal inconti-
nence was the finding that it possessed approxi-
mately fourfold greater potency than phenylephrine
(Jones et al., 2003). With the exception of moxoni-
dine, all of the imidazoline derivatives examined
in this study were more potent than L-erytho-
methoxamine, but significantly less ‘efficacious’.
The impact of the latter property on their therapeu-
tic potential is unclear, but it is noteworthy that
many imidazoline derivatives have been success-
fully used as potent decongestants in man despite
possessing significantly low constrictor ‘efficacy’ at
a-adrenoceptors in nasal blood vessels (Johannssen
et al., 1997; Corboz et al., 2003).
In conclusion, we have demonstrated the pres-
ence of a1A- and a2D-adrenoceptors on the sheep IAS
muscle. Although a1A-adrenoceptors appear to make
the major contribution to the constrictor effect of
known a-adrenoceptor agonists, in the case of selec-
tive a2-adrenoceptor imidazoline derivatives, there
is the possibility of synergistic interaction between
derivatives (e.g. clonidine) have greater potency at
both a-adrenoceptor sub-types compared to existing
selective a1-adrenoceptor agonists, these agents
could be effective at lower doses, thereby reducing
the propensity for local or systemic side effects. In
light of this, some imidazoline derivatives would be
worthy candidates for investigating in the treatment
of faecal incontinence.
We thank Woods Ltd, Clipstone, Nottinghamshire
and M Najib and sons, Foston, Derbyshire for the
supply of tissue.
Conflict of interest
None to declare.
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