shortening of gastrointestinal transit time (Figure2).

DA-9701 increased cAMP concentration

in electrical field stimulation (Figure 3). Conclusion: DA-9701 increased the gastric contraction by anti-dopaminergic and cholinergic effects. DA-9701 increased gastrointestinal motility
by 5-HT4, anti-dopaminergic, and cholinergic effects.

AGA Abstracts

of DA9701 for expression level of BDNF and ASIC subunits. Methods: Colonic hypersensitivity was induced in twenty adult male Sprague-Dawley rats by butyrate enemas in the distal
colon, and 10 control rats served as vehicle only. After butyrate enema, the rats were divided
into 2 groups and were given respectively DA 9701 3 mg/kg or vehicle. The rats were sacrificed
at 1 and 2 days after treatment, and colonic damage score and tissue myeloperoxidase
activity were evaluated. In lumbosacral L1 and L2 DRG, expression level of BDNF, ASIC
1a, ASIC 2b, IL-1, and TNF- were assessed using quantitative real-time PCR, ELISA, and
western blot. Results: In L1 DRG, the levels of BDNF protein were increased at both 1 and
2 days of colitis (2.2- to 3.2-fold, p < 0.05). There were no significant changes in the BDNF
protein level in L2 DRG at both days of colitis. In addition, level of ASIC 1a (2.5- to 4.3fold, p < 0.05) and 2b (2.7- to 3.6-fold, p < 0.05) protein expression in L2 DRG significantly
higher after administration. RT PCR results showed significant increases in the level of BDNF
mRNA in L1 DRG (2.1- to 2.3-fold, p < 0.05) at 1 and 2 days of colitis. ASIC 1a and 2b
mRNA expression was significantly increased in L1 level. There was no change in IL-1
and TNF- expression in colitis group. In butyrate-treated colitis model, administration of
DA 9701 led to significantly decreased level of BDNF, ASIC 1a, and ASIC 2b protein and
mRNA expression in both L1 and L2 DRG levels compared with control groups. Conclusion:
Our data suggest that peripheral BDNF and ASIC1a/2b concomitantly contribute to the
development of butyrate-induced colon hypersensitivity. In addition, DA 9701 can negatively
modulate the activity of ASICs and BDNF in lumbosacral DRG.
Su1870
Entacapone, A Peripherally Restricted COMT Inhibitor, Regulates
Gastrointestinal Motility and Pain Sensitivity in Mice
Xiaoqing Li, Qian Li, Liansheng Liu, Pankaj J. Pasricha
Introduction: The enzyme catechol-O-methyl transferase (COMT) is important in the regulation of signaling by catecholamines, including dopamine. However, its role in regulating
gastrointestinal motility and visceral pain is not known. We tested this hypothesis by studying
the pharmacological effects of entacapone, a peripherally restricted COMT inhibitor currently
being used as an adjunct to levodopa for patients with Parkinson's disease. Methods: We
tested the effects of entacapone on the dose response of whole gut transit time (WGTT),
gastric emptying and distal colon transit time (DCTT) in healthy C57/BL6 mice. We also
tested the acute effects of entacapone on response to colorectal distention (CRD) in a mouse
model of irritable bowel syndrome (IBS) induced by neonatal colonic irritation by acetic
acid. Colonic pain sensitivity to graded colorectal balloon distension (CRD, 15, 30, 50 and
70mmHg) was determined by visceral motor response (VMR) measured by electromyography
of external oblique muscle. Results: Acute administration of entacapone (100 mg/kg, i.p.)
significantly attenuated the hyperalgesic visceral motor response to CRD in "IBS" mice (Figure
1). At this dose, it also significantly increased the DCTT with a peak at 75 min after injection
(vehicle: 271.271.89 sec, entacapone: 1560.67 518.52 sec, P<0.01). An additional group
of healthy mice were administrated entacapone orally (100mg/kg, 5 ml/kg) or vehicle 3
times a day for 5 days. On day 7, liquid gastric emptying was measured using phenol red
and found to be increased in the entacapone group as compared with control associated
with acceleration of small bowel transit; however, no effect was seen on WGTT. Conclusions:
Entacapone appears to have multiple region-specific effects on gastrointestinal motility and
sensation. It accelerates upper gastrointestinal transit while delaying distal colon transit.
Further, entacapone is also capable of attenuating the hyperalgesia in an IBS model. These
results indicate an important role for COMT in gastrointestinal motility and suggest new
therapeutic targets for disorders of the same.

Su1872

Effects of DA-9701, a Prokinetic Agent on Gastric Motor Function in Guinea

Pig; In Vivo and In Vitro Study
Hyun Chul Lim, Hyojin Park, Young Ju Lee

High Resolution Neuronal Imaging Reveals a Novel Rhythmic Firing Pattern

Across Multiple Myenteric Ganglia Which Underlies Rhythmic Cholinergic
Depolarisations in Smooth Muscle During the Colonic Migrating Motor
Complex
Nicholas J. Spencer, Lee Travis, Timothy J. Hibberd, Marcello Costa, Simon J. Brookes,
Philip G. Dinning, Lukasz Wiklendt

Introduction: DA-9701 is a newly developed drug made from the vegetal extracts of Pharbitidis semen and Corydalis tuber for treatment of functional dyspepsia. This study aimed to
clarify the pharmacological mechanisms of DA-9701 on gastrointestinal motility via in vitro
and in vivo study. Methods: To investigate the effects DA-9701 on gastrointestinal tract in
vivo, amplitudes from transducers on stomach and duodenum in conscious guinea pig were
checked and to investigate the effects of DA-9701 on stomach muscle contraction in vitro,
the amplitudes on antral circular muscle in tissue bath after electrical field stimulation (15 Hz) were checked. To evaluate the pharmacological mechanism, amplitude of contraction
was checked after pre-treatment with atropine, dopamine, and GR113808 (5-HT4 antagonist).
To investigate the gastrointestinal transit of DA-9701, the distance of charcoal migration
from pylorus to distal intestine was measured after administration of DA-9701, and additional
experiments with infusion of atropine, dopamine, and GR113808 were performed. To
investigate the gastrointestinal transit of DA-9701, the distance of charcoal migration from
pylorus to distal intestine was measured after administration of DA-9701, and additional
experiments with infusion of atropine, dopamine, and GR113808 were performed. To clarify
the 5-HT4 effect of DA-9701, cAMP from gastric antral circular muscle after electrical field
stimulation (1, 5 Hz) was measured by ELISA method. Results: In vivo study, DA-9701
DA-9701 improved contraction amplitude in 3mg/kg and in vitro study, DA-9701 improved
contraction amplitude of gastric muscle in dose-dependent manner. Atropine, dopamine
suppressed DA-9701 induced contraction, but GR113808 did not suppress DA-9701 induced
contraction(Figure 1). DA-9701 shortened the gastrointestinal transit time in dose-dependent
manner and atropine, dopamine, and GR113808 significantly suppressed DA-9701 induced

Colonic migrating motor complexes (CMMCs) are cyclical neurogenic contractions that
propagate along the colon of mammals and play a major role in the propulsion of colonic
content. Whilst it is clear that CMMCs require the enteric nervous system (ENS), it is not
clear how the activation of the different classes of myenteric neurons is coordinated over
large regions colon to generate an orderly contraction of smooth muscle. In this study, we
used the latest high resolution, EMCCD camera (Evolve Delta; Photometrics) to visualize
the temporal activation properties of different morphological classes of myenteric neurons
in multiple myenteric ganglia along the colon. To do this, the entire colon was removed
from mice, euthanized in accordance with the animal welfare committee of Flinders University
and mounted serosal side uppermost in an oxygenated Krebs solution at 35oC. Intracellular
electrophysiological recordings from the circular muscle confirmed the presence of CMMCs
every 2-4 minutes, that propagated along the colon. Each CMMC consisted of repetitive
discharge of hexamethonium-sensitive cholinergic EJPs producing rapid oscillations in membrane potential (at 2Hz) in the circular muscle, lasting ~20s (N=5). To characterize the
temporal firing properties of myenteric neurons during these rapid oscillations in the muscle,
the calcium indicator, Fluo-4 was loaded into multiple neighbouring myenteric ganglia.
Simultaneous mechanical recordings along the colon, combined with calcium imaging at
35oC revealed that each CMMC was associated with a rhythmic discharge of synchronized
calcium transients in large populations of Dogiel Type II and Type I neurons across multiple
rows of neighbouring myenteric ganglia and internodal strands (N=6). The frequency of the
rhythmic calcium transients in Dogiel type I and Type II neurons (2.0 0.1Hz) and internodal
strands (2.1 0.1Hz) was the same as the cholinergic EJPs recorded at the same time in

Su1871

S-539

AGA Abstracts

AGA Abstracts

the smooth muscle. Rhythmic calcium transients occuring simultaneously in many Dogiel
Type I and II neurons, across many rows of myenteric ganglia, were found to cause timelocked calcium action potentials arising from EJPs in neigbouring smooth muscle layers,
that lead to muscle contraction underlying each CMMC. All synchronized calcium transients
across multiple ganglia were abolished by hexamethonium (300 micromolar; N=6) or tetrodotoxin (1 micromolar; N=4). This is the first demonstration of a rhythmic neuronal firing
pattern in mammalian ENS that is responsible for rhythmic cholinergic EJPs in the smooth
muscle during a complex intestinal motor pattern. Of particular interest was the discovery
that both Dogiel Type II (intrinsic sensory neurons) and Type I neurons (interneurons and
motor neurons) generated simultaneous and rhythmic calcium transients during each CMMC,
over large spatial fields of ENS.

toxicity in the gut need further investigation. In the mean time, caution is warranted when
using high protein diets to obtain weight loss. 1Attene-Ramos MS, et al. Mol Cancer Res
2007;5:455-9 2Windey K, et al. PLoS One 2012;7:e52387 3Russell WR, et al. Am J Clin
Nutr 2011;93:1062-72
Su1875
High Levels of Sulfate-Reducing Bacteria Predispose to Protein-Induced Fecal
Water Genotoxicity
Karen Windey, Vicky De Preter, Lise Deroover, Geert Huys, Greet Vansant, Kristin
Verbeke
Background: In healthy subjects, increased fecal levels of sulfides were associated to higher
fecal water genotoxicity1 confirming in vitro experiments that demonstrated the genotoxic
potential of hydrogen sulfide (H2S) in colonocytes2. In the colon, H2S is produced by the
sulfate reducing bacteria (SRB) from sulfur-containing amino acids or inorganic sulfate and
sulfite, both frequently used additives in preserved foods. Objective: We investigated whether
levels of SRB at the start of a high protein diet (either isocaloric or caloric restricted)
predispose to increases in fecal water genotoxicity. Methods: Fecal samples were obtained
from 20 normal-weight subjects before and after 2-weeks of an isocaloric high protein (HP;
27% protein) diet and from 30 overweight subjects before and after 12 weeks of a high
protein weight loss diet (HP-WL; 30% protein, 25% energy restriction). The numbers of
SRB were quantified in the fecal samples using RT-PCR. Fecal water genotoxicity was assessed
using the Comet Assay and expressed as m tail length (TL). Relative concentrations of
sulfides were determined using GC-MS. Results: Absolute protein intake amounted to 2.0g/
kg BW/d during the isocaloric HP diet and to 1.7g /kg BW/d during the HP-WL diet. Fecal
numbers of SRB were significantly higher after the isocaloric HP diet compared to the LP
diet (p=0.03). The HP-WL diet tended to increase numbers of SRB compared to baseline
(p=0.10). After the isocaloric HP diet, baseline numbers of SRB correlated positively with
an increase in fecal water genotoxicity (p=0,04; Spearman's rho=0,46). In subject with low
numbers of SRB, the effect of the HP diet on fecal water genotoxicity [-13.2 m TL (-43.7
- 21.2)] was significantly different from the effect in the group with high SRB numbers
[37.0 mTL (-2.4 - 55.2); p=0.04]. Relative concentrations of sulfides were higher in the
group with high numbers of SRB compared to the group with low numbers. After the HPWL diet baseline numbers of SRB were not correlated to changes in fecal water genotoxicity
(p=0.72; Spearman's rho=0.071). The changes in fecal water genotoxicity after the diet were
not different between the group with high SRB [-13.9 m TL (-52.3 - 14.6)] and low
numbers of SBR [0.7 m TL (-46.2 - 5.6); p=0.57]. Conclusion: High protein intake
stimulates the number of SRB in the colon. The baseline number of SRB predisposes to
increased fecal water toxicity only after an isocaloric high protein diet, but not after a high
protein calorierestricted diet suggesting that a minimal absolute protein intake is required.
The increased fecal water genotoxicity after increased protein intake is associated with an
increased production of sulfides which are known genotoxic agents. 1Windey K, et al. PLoS
One 2012;7:e52387 2Attene-Ramos MS, et al. Mol Cancer Res 2007;5:455-9

Su1873
Dopamine D1 and D2 Receptors Expressed by Musculomotor and
Secretomotor Neurons in the Enteric Nervous System (ENS) and the Dorsal
Vagal Motor Nucleus for Guinea Pig and Mouse
Jackie D. Wood, Guo-Du Wang, Xi-Yu Wang, Yun Xia
Background and Aims: Dopamine is a neurotransmitter in the brain and ENS. Dopaminergic
transmission is a key in the neural control of motility and secretion in the gut. Importance
in the ENS overlaps the CNS in Parkinson's disease and psychiatric disorders. We expanded
our earlier work on ENS dopaminergic D1 and D2 receptor subtypes in guinea pig gut to
mice and compared results with dopaminergic neuropharmacology in the dorsal motor
nucleus of the vagus (DMV). Methods: Patch clamp recording methods were used in the
DMV and intracellular recording with "sharp" microelectrodes in the ENS. Organ bath
motility and Ussing chamber methods were used for study of muscular contractile behavior
and mucosal secretion. RT-PCR and Western blotting assessed genotypes and receptor protein
expression by ENS neurons in D1 and D2 knockout and wild type mice (C57Bl/6J). Results:
Dopamine depolarized the membrane potential in 12/15 neurons in the DMV of D2R
knockout (D2R-KO) mice. The D1 receptor agonist, SKF38393, but not the D2 agonist
quinpirole, mimicked the action of dopamine. Dopamine application hyperpolarized the
membrane potential in 11/11 DMV neurons of D1 knockout (D1R-KO) mice. The D2 agonist,
quinpirole, but not the D1 agonist SKF38393, mimicked the action of dopamine. Quinpirole
application depolarized the membrane potential in 6/9 neurons in the ileal myenteric plexus
of D1R-KO mice, but not in 7/7 myenteric neurons in D2R-KO mice. SKF38393 suppressed
the force of spontaneously occurring contractions and neurogenic twitch-like contractions
evoked by electrical field stimulation in the colon (n=28) and ileum (n=24) of wild-type
mice, but not in D1R-KO mice (n=6). The inhibitory effects of SKF38393 were blocked by
tetrodotoxin or the D1 antagonist, SCH23390. Quinpirole enhanced the force and frequency
of spontaneously-occurring contractions in 5/8 wild-type and 8/8 D1R-KO mice. Application
of quinpirole in Ussing chambers enhanced neurogenic secretory responses (i.e., shortcircuit current) in 5/8 ileal and 6/8 colonic preparations from wild-type mice (n=4) and 7/
8 ileal and 8/8 colonic preparations from D1R-KO (n=4). Quinpirole did not stimulate
neurogenic secretion in preparations from 8/8 ileal or 8/8 colonic preparations from D2RKO mice. Action of quinpirole on neurogenic secretion was suppressed by the selective D2R
antagonist, L741626, or tetrodotoxin in all preparations. Conclusion: Neuronal responses
to stimulation of D1 and D2 receptors in the ENS are reversed in relation to the DMV.
Stimulation of D1R depolarizes the membrane potential and potentiates neuronal excitability
in the DMV while stimulation of D2R evokes membrane hyperpolarization and suppresses
neuronal excitability. Stimulation of D2R in ENS neurons elevates excitability while stimulation of D1R suppresses excitability of ENS motor neurons. Acknowledgment: NIH
2R01DK037238

Su1876
Replacement of Insoluble Fiber (IF) With Soluble Fiber (SF) in a PurifiedIngredient Openstandard Diet Increases Cecal and Colonic Weights Similar to
Grain-Based Chows (Chows) and Distinctly Alters Microbiota in Weanling
Male C57BL/6N Mice
Michael A. Pellizzon, Matthew R. Ricci, Douglas S. Compton, Erik C. Rocheford,
Dionysios A. Antonopoulos, Jason C. Koval, Edward Ulman
Grain-based (GB) chows have been used for decades to maintain the health of lab animals.
However, research on how the gut microbiome affects the biology of rodents is confounded
by the use of chows. GB chows can contain numerous phytochemicals and provide 20-25%
total fiber (TF) as both undefined IF and SF. Unlike IF, SF provides a fermentable substrate
for cecal and colonic bacteria, which in turn can provide energy substrates for surrounding
tissues. In contrast to chow, purified diets (PDs) contain refined ingredients with one main
nutrient per ingredient and have historically used 5% IF as cellulose (CEL). The current
work assessed the effect of replacing CEL with 2 doses of 2 SF sources (inulin [IN] and
fructooligosaccharides [FOS]) on cecal and colonic microbiota and weight (wt). Weanling
4 week old male C57Bl/6N mice were split into 8 dietary groups (n=6/grp): 6 PDs included
IN, FOS, or CEL at 100 g/4084 kcals (100IN, 100FOS, 100CEL) or 200 g/4084 kcals
(200IN, 200FOS, 200CEL), or 2 chows (5001 or 5002) for 14 days ad-lib. Body wt increased
similarly on all diets except for 200FOS, which was lower compared to chows. 100CEL
and 100FOS reduced colon length relative to 5002 but all other PDs were similar to chows.
Combined cecum and colon wt with contents were similar for 100IN (939mg35) and
100FOS (75237), both of which were higher than 100CEL (49825); 100IN was similar
to 5001 (83926) and 5002 (96526) while 100 FOS was lower than 5002; 200IN
(1.3g0.04) or 200FOS (1.10.1) increased wt further, while 200CEL (460mg12) did not;
200IN was higher than both 5001 (83926) and 5002 (96526). These results were mirrored
by changes in both cecal and colon wall wt and contents. Within both cecum and colon
contents, Firmicutes (35.3%) and Bacteroidetes (53.2%) made up the greatest proportion
of phyla among all groups. Cecal Firmicutes (mainly Clostridiales) were reduced by 100IN
(27%2), 200IN (213), 100FOS (395), and 200FOS (204) relative to 100CEL (502),
200CEL (493), 5001 (644) and 5002 (595). Cecum Bacteroidetes (mainly Bacteroides)
were higher for 100IN (60%3) and 200IN (624) relative to 5001 (334) and 5002
(376), while 100FOS (444), 200FOS (546), 100CEL (442) and 200CEL (473) were
intermediate. Cecal Verrucomicrobia were elevated in 100IN (112) and 100FOS (132)
relative to 5001 (11), 5002 (21) and 100CEL (11); 200FOS (191) was higher than
200IN (102) or 200CEL (11). Cecum Actinobacteria (mainly Bifidobacterium) were detected
in groups fed 100FOS (3%1), 200FOS (52), 200IN (32) and to a lesser proportion in
100IN (0.30.1) while being virtually absent from other groups. Similar dietary changes in
these aforementioned cecal phyla were also found in the colon. These data show that
replacement of IF with SF in a PD changes microbiota content and improves cecal and
colonic tissue wt similar to chow in male C57BL/6N mice.

Su1874
High and Standard Protein Weight Loss Diets Modulate Colonic Fermentation
but Do Not Affect Fecal Water Toxicity
Karen Windey, Evelien Backx, Vicky De Preter, Lisette De Groot, Kristin Verbeke
Background: High protein diets are increasingly popular as they result, at least on the short
term, in increased weight loss. However, concerns are raised considering the increase in
protein fermentation induced by high protein intake. In vitro and animal studies suggest
protein fermentation to be detrimental to gut health, although evidence in humans is mostly
lacking. Aim: The impact of weight loss diets differing in amount of protein on colonic
fermentation and on fecal water genotoxicity and cytotoxicity was investigated in overweight
subjects. Methods: Sixty subjects (age: 62.7 5, BMI = 31.2 3.0 kg/m2) performed a
double-blind, parallel RCT comparing a standard protein weight loss diet (SP, 15% protein)
to a high protein weight loss diet (HP, 30% protein). Both diets were caloric restricted with
25%. Fecal samples were collected before the start of the diet and after 12 weeks. Colonic
fermentation was characterized through an untargeted metabolomics approach using GCMS. Fecal water genotoxicity and cytotoxicity were analyzed as parameters of gut health
using the Comet Assay and WST-1 assay, respectively. Clustering techniques were applied
to detect fermentation metabolites associated with cytotoxicity or genotoxicity. Results:
Weight loss after 12 weeks amounted to 9.1 3.4 kg after the SP diet (p<0.001) and 8.9
2.9 kg after the HP diet (p<0.001). Absolute protein intake during the HP diet was 116.7g/
d and 83.5g/d during the SP diet. Both weight loss diets modified colonic fermentation.
Short chain fatty acids (FA) and medium chain FA were reduced after both diets, while
aldehydes were more prevalent in the samples collected at the end of both diets. Samples
collected after the HP diet are associated with higher levels of sulfides. Fecal branched chain
FA-concentrations were reduced after the SP-diet (p=0.05) and remained unaffected after
the HP-diet (p=0.21). Parameters of gut health were not affected by any diet (genotoxicity:
p=0.25 for SP and p=0.22 for HP; cytotoxicity: p=0.36 for SP and p=0.32 for HP). No
correlation was found between body weight loss and fecal water cytotoxicity (p=0.23) nor
genotoxicity (p=0.69). However, higher levels of acids and sulfides were associated with
higher cytotoxicity and genotoxicity. Conclusion: Parameters of gut health were not affected
by weight loss per se nor by diet composition (protein intake). However sulfides, previously
suggested as genotoxic agents1,2, were more prevalent after the HP diet and were associated
with fecal water genotoxicity and cytotoxicity. Therefore, the mechanisms of sulfide-induced

AGA Abstracts

S-540