Tu1996

Glycogen Metabolism Defects Produce Gastrointestinal Myopathy WithPolyglucosan BodiesYasotha Duraisamy, Milly West, Charles H. Knowles, Hasan O. Akman, SalvatoreDiMauro, Mohammed M. Rahman, Berge Minassian, Jo Martin

INTRODUCTION The intestine is a significant site of gluconeogenesis, with high expressionof glucose 6 phosphatase. Abnormal forms of glycogen, including polyglucosan bodies,occur in the muscularis propria in a subset of patients with gastrointestinal motility disorders.Little is known, however, of the distribution of glycogen metabolic enzymes and relatedproteins in normal gastrointestinal tract, or effects of deficiencies of these proteins on thebowel. METHODS Key enzymes in glycogen metabolism, including glycogen synthase (GS),glycogen branching enzyme (GBE), malin and laforin were examined in stomach, ileum andcolon of two mouse strains and in normal human bowel using immunohistochemistry withsemiquantitiative analysis of epithelium, muscle, submucosal and myenteric plexus, validatedby western blotting and positive and negative controls. The pathological effects of GBE,laforin and malin gene knockout on mouse gastrointestinal tract were also studied. RESULTSGS was present at high levels in the smooth muscle of muscularis propria, GBE in themajority of large ganglion cells of the submucosal and myenteric plexuses, and laforin andmalin in submucosal ganglia. Analysis of human bowel showed no significant regionaldifferences in the distribution of GBE, laforin and malin, GS was more prominent in themuscularis propria of the small intestine, compared with stomach and colon. GBE knockoutproduced a progressive severe myopathy with numerous polyglucosan bodies and vacuola-tion. Polyglucosan bodies were also present in both laforin and malin knockout models,but with a less severe phenotype than that seen in the GBE knockout, and were not seenin controls. CONCLUSION The distribution of glycogen metabolic proteins in the bowelwall of mouse and man shows a specific neuromuscular distribution for each pathwaycomponent. Defects in glycogen metabolism in mouse knockout models reproduce histolog-ical features of polyglucosan body myopathy in human digestive motility disorders.

Tu1997

A Novel In Vivo Method to Measure Gastrointestinal Transit in MiceDavid E. Reed, Jun Lu, Stephen M. Collins, Premysl Bercik

Introduction: Altered gastrointestinal transit is a feature of many gastrointestinal and systemicdisorders (e.g. GI infections, diabetes mellitus). To date, analysis of intestinal transit inanimal models has been hampered by a need to euthanize the animal at a given time pointto remove the intestine and localize the tracer substance. This study explores the developmentof a novel In Vivo method to determine gastrointestinal transit in mice. Methods: Steel beads(n=5, diameter=0.79mm) were used as a marker for intestinal transit. BALB/c mice (n=17)were gavaged with beads 3 hours before fluoroscopic imaging. Barium (40% dilution, 100-200 μl) was gavaged 3 hours and 10 min prior to imaging. Some mice were also gavagedwith either milk of magnesia or loperamide 2 hours prior to gavage of beads to accelerateor slow down the transit. Each mouse was fluoroscoped for 20-30 seconds and imagesrecorded for off-line analysis. Immediately after completing the fluoroscopy exam, micewere euthanized. The GI tract, from the stomach to the rectum, was removed and oneinvestigator localized each bead within the GI tract. A second blinded investigator localizedthe beads by analyzing the fluoroscopic images. A bead was given a numeric score dependingon its location within the GI tract (range 0: stomach to 5: expelled from GI tract) and atotal score was calculated by adding the individual bead scores. The total scores obtainedfrom the 2 investigators were compared. Results: Steel beads were easily identified in eachanimal. Barium was consistently seen outlining the stomach, proximal small bowel andcecum, identifying position of beads localized in these gut segments. Beads seen in fecalpellets indicated their localization in mid to distal colon. Total scores ranged from 7 to 26.There was a good correlation between fluoroscopic and anatomical (post-mortem) localizationof beads (Pearson correlation, r= 0.87, p<0.0001). Differences between investigators' scoresfor individual beads occurred in differentiating a bead in distal small bowel versus proximalcolon. Conclusion: Fluoroscopic localization of small metallic beads shows good correlationwith their anatomical position within the GI tract. Longer imaging protocols, that allowdetection of high frequency slow wave-driven contractions within the small bowel, mayfurther improve discrimination of whether a bead is located in the distal small bowel orcolon. This novel imaging method enables repetitive measurements of GI transit In Vivo,and will be useful to investigate dysmotility in animal models of disease. Supported by grantfrom CIHR.

Tu1998

Third Instar Drosophila Larvae Expressing RNAI Against CA-Channel Proteinα1 Subunit D in the Visceral Muscle Serve as a Model for Decreased IntestinalMotilityKate Buzzi, Benjamin Ohlstein

Peristalsis propels nutrients in a wave-like fashion anterograde through the intestine providingnourishment necessary for proper growth and survival of an organism. The alimentary canalof Drosophila has a significant number of similarities to mammals making it an ideal modelto study intestinal motility. The Drosophilamidgut is a composed of a monolayer of epithelialcells surrounded by an inner circular layer and an outer longitudinal layer of visceral muscles.The muscle is striated, yet it ultrastructurally resembles vertebrate smooth muscle. Muscularcontraction is initiated by an increase in intracellular calcium. To generate an optimal modelfor evaluating intestinal transit in Drosophila, we tested the hypothesis that L-type calciumchannels in the midgut of Drosophila are required for peristalsis. To investigate the role ofL-type calcium channels in muscle contraction, we decreased the expression of a gene forDrosophila L-type calcium channels, Ca-channel proteinα1 subunit D, using RNA interference(RNAi) in visceral muscle using the muscle-specific driver how-Gal4. For the larval clearanceassay, larvae were reared on corn molasses food with 4% blue dye which turns their intestinescompletely blue. Individual third instar larvae were assessed in standard food without dyeand the clearance time was calculated from the time the larvae were removed from the blue

S-897 AGA Abstracts

food and the time when the intestine was no longer blue. 33 larvae containing the RNAitransgene, but lacking the Gal4 muscle driver, and 30 larvae containing both the muscledriver and RNAi transgene were tested. Larvae expressing RNAi against the Ca-channelprotein α1 subunit D expressed in the visceral muscle cleared the dye in an average of 291min versus 137.3 min for larvae lacking the muscle driver (p<0.05, Figure 1), demonstratinga role for visceral muscle L-type Ca-channels in food clearance. In addition, our data validatesthe larval clearance assay as a powerful method to swiftly identify genes required for functionof the visceral muscle. This model will be valuable in future studies investigating Drosophilawith altered expression of G-proteins and innexins, two potential gene targets for regulat-ing peristalsis.

Figure 1: Clearance of intestinal blue dye in larvae expressing RNAi for Ca-channel proteinα1 subunit D in visceral muscle versus controls

Tu1999

Gender and Estradiol as Major Factors in the Expression and Dimerization ofnNOSα in Rats With Experimental Diabetic GastroparesisShowkat Ali, Iliana Tiscareno-Grejada, Siviu Locovei, Rebecca Smiley, Todd Colins, IreneSarosiek, Jerzy Sarosiek, Richard McCallum

The molecular mechanisms of cellular changes responsible for diabetic gastroparesis, primar-ily seen in middle-aged women, still remain incompletely defined. Nitric oxide is a majorneurotransmitter in the gut and a key physiological mediator of the non-adrenergic, non-cholinergic relaxation of gastrointestinal smooth muscle. Nitric oxide is synthesized by thenitric oxide synthase, nNOSα, which is predominantly expressed in the enteric nerve andplays a pivotal role in various parts of the GI tract. Because of the strong evidence fornNOSα involvement and a gender preference in gastroparetic dysfunction, estrogen may beinvolved in normal gastric emptying via a nitric oxide regulatory role. We hypothesizedthat a decrease in the estrogen-mediated expression, dimerization, and post-translationalmodification of nNOSα is responsible for the gender-specific prevalence of this malady. Theapproach used in this study was to induce diabetes by injecting male and female rats withstreptozotocin. Male diabetic rats without gastroparesis were then injected with estrogen for3 weeks and evaluated for gastroparesis development. Gastric tissues were collected andanalyzed for the elucidation of the biochemical events associated with diabetes and gastropar-etic dysfunction. Although male diabetic and gastroparetic rats exhibited similarity in diseasepathology to that of females, the molecular mechanism of development was different. Ourresults indicated that slow gastric emptying in male diabetic, gastroparetic rats was notassociated with the level of expression of nNOSα in gastric tissue. However, nNOSα dimeriz-ation declined with borderline significance in diabetic male rats with estrogen-inducedgastroparesis, suggesting possible inactivation of nNOSα. Females with diabetic gastroparesis,in contrast, demonstrated significantly impaired levels and dimerization of nNOSα in atissue-specific fashion, with maximum effect in the antrum and pylorus. Although the precisemechanism remains unknown, the observed impairment of nNOSα dimerization in theantrum of these female rats is apparently associated with reduced phosphorylation ofSer(1416) in the activation loop of nNOSα. Taken together, these results demonstrate thatgender and estrogens may be leading factors, through molecular changes involved in nitricoxide synthesis down-regulation, responsible for gastroparetic dysfunction manifestationswithin the antrum and pylorus.

Tu2000

High Resolution Manometry in Healthy Women: Normal Values and Effects ofAgeAdil E. Bharucha, Jessica Noelting, Shiva K. Ratuapli, Jessica Edge, Karthik Ravi, DorisHarvey, Alan R. Zinsmeister

BACKGROUND. High resolution anorectal manometry (HRM) is increasingly used in clinicalpractice. However, normal values for HRM are not available, limiting its utility. While theeffects of age on anal pressures and rectal sensation have been extensively evaluated bytraditional manometry, effects on anorectal pressures during simulated evacuation and rectalballoon expulsion have not. AIMS. To evaluate normal values and the effects of age onanorectal pressures and rectal sensation in healthy women using HRM. METHODS. Usinghigh resolution manometry (HRM, Sierra Scientific Instruments), anorectal pressures wereassessed at rest, during voluntary contraction (squeeze), and simulated evacuation (Simulatedevacuation) in 62 healthy women (age 44 ± 17 years (mean ± SEM), BMI 26 ± 4 kg/m2 ).Rectal sensation was evaluated by balloon distention (20 ml increments up to 120 mL, 30ml increments thereafter) until patients recorded maximum discomfort. RESULTS (Table).Compared to younger women, anal pressures at rest and during Simulated evacuation andthe rectal BET were lower in older than younger women. However, length of the high

AG

AA

bst

ract

s

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AA

bst

ract

spressure zone (HPZ), anal squeeze pressures and rectal sensory thresholds were not signific-antly correlated with age. The 90th percentile value for anal resting pressures in youngerwomen was 112 mmHg. Since anal but not rectal pressures declined with age, the rectoanalgradient during Simulated evacuation was more negative in younger than older women.CONCLUSIONS. In asymptomatic women, increasing age was associated with anal restingpressures and a shorter BET. Contrary to current concepts, a majority of healthy womenhad a negative rectoanal gradient during Simulated evacuation. The notable differencesversus traditional manometry are: (i) a higher 90th percentile value for anal resting pressurein younger women (i.e., 112 versus 90 mmHg); (ii) negative rectoanal gradient duringSimulated evacuation even in asymptomatic women; and (iii) lower rectal sensory volumethresholds than historical data with a latex balloon. Confirming barostat studies, age didnot affect rectal sensory thresholds. Also, the 90th percentile value for BET in women ≥50y was only 15s, which is lower than the cutoff (60s) currently used in clinical practice.A lower cutoff may improve identification of evacuation disorders in older women, whohave lower weak resting pressures. Based on these data, the utility of the anorectal gradientduring Simulated evacuation needs to be reassessed.

Values are mm Hg unless stated otherwise. *Spearman correlation coefficient,†p < 0.01

Tu2001

Development of Myogenic IAS Reconstructs From Human Internal AnalSphincter (IAS) Smooth Muscle Cells (Smcs) With Functional and MolecularProperties Similar to Intact Human IASSatish C. Rattan, Jagmohan Singh

Background & Aims: Rectoanal incontinence and a number of other rectoanal motilitydisorders have been associated with the IAS smooth muscle (SM) dysfunction, for whichthere is no satisfactory treatment. Present studies were performed to explore the possibilityof IAS smooth muscle reconstructs from the human IAS SMCs and compare their functionaland molecular attributes with the intact human IAS. Methods: The IAS reconstructs in theform of rings were developed from the SMC isolated from the circular smooth muscle layerof the human IAS. These SMCs were grown in DMEM containing 10% serum and 50 μg/ml of sodium ascorbate. SMCs (107) in 1 ml of collagen I (4 mg/ml) were transferred to aSylgard coated sterile tissue culture plates (35 mm OD) containing a central Sylgard post(4 mm OD), and left undisturbed at 37oC for 1 h. Once the collagen gelated, DMEMcontaining 15% FBS and 50 μg/ml sodium ascorbate were added to the plates. After 24 hnormal media was replaced by SM differentiation media. Plates were incubated for 72 to96 h at 37oC. The reconstructs were transferred to 2ml organ bath, and following 0.2 g oftension were allowed to equilibrate for 1 h. Effects of 0Ca2+, KCl, bethanechol, isoproterenol,PKC activator PDBu, permeant RhoA, ROCK and PKC inhibitors C3 exoenzyme, Y 27632,and Gö 6850, respectively, were determined. Western blot (WB), immunofluorescence (IF),and immunocytochemical (IC) analyses were also performed. Results: The reconstructsdeveloped spontaneous tone (592.0 ± 35.9 μN; n = 24). Bethanechol (a muscarinic agonist)and KCl depolarization produced contraction, while isoproterenol (β-adrenoceptor agonist),C3 exoenzyme, and Y 27632 produced conc.-dependent relaxation. Maximal decrease inbasal tone in the constructs with Y 27632 and Gö 6850 (each 10-5 M) was 80.45 ± 2.56%and 12.24 ± 2.45%, respectively. WB data with the constructs revealed higher levels ofRhoA/ROCK, CPI-17, phospho- CPI-17, MYPT1 and MLC20. WB, IF and IC studies oforiginal SMCs as well as re-dispersed from the reconstructs for the relative distributionand location of RhoA/ROCK and other signal transduction proteins further confirmed theconstitutive role of this pathway rather than PKC, similar to the intact IAS. These data suggestthe preservation of the original phenotypic SMCs throughout the reconstructs transformation.Conclusion: It is feasible to tissue engineer IAS constructs using human IAS SMC thatmaintain phenotypic characteristics of the tonic smooth muscle of the intact IAS. Thestudies suggest potential use of such reconstructs for the replacement of dysfunctional IASsmooth muscle.

S-898AGA Abstracts

Tu2002

Effect of Pooled Human Intravenous Globulin (IVIG) on the Reversal ofCholinergic Inhibition of Smooth Muscle by IgG Immunoglobulins FromPatients With Scleroderma (Scl)Jagmohan Singh, Vaibhav Mehendiratta, Sergio A. Jimenez, Sidney Cohen, Anthony J.DiMarino, Satish C. Rattan

Background & Aims: The marked changes in gastrointestinal motility in Patients withScleroderma (Scl) have been shown to be mediated through cholinergic blockade of themuscarinic (Type M3R) smooth muscle receptor by circulating IgG antibodies (AJP, G1206;2009). The purpose of this study is to determine if commercial pooled human intravenousglobulin (IVIG) can alter this inhibition through binding of the antibody in a validated InVitromodel.Methods:We determined the effect of Scl IgGs onM3R activation by bethanechol(methyl derivate of carbachol) (BeCh; 10-7 to 10-4 M) in human and rat IAS smooth musclecells (SMC) and rat smooth muscle strips, in comparison with normal IgGs and IVIG. TheIgGs from six GI symptomatic Scl patients (SclIgGs) and two normal volunteers (NIgGs)were purified using protein G-sepharose columns. To determine M3R occupancy, we quanti-fied SMC membrane M3R immunofluorescence (IF) intensities before and after SclIgG vs.other IgGs. Functional displacement of M3R occupancy was determined by a decrease inthe BeCh-induced sustained IAS contraction induced by different concentrations of theSclIgGs vs. NIgGS and IVIG.Results: SclIgGs caused significant (p < 0.05) and concentration-dependent (0.3, 0.6 and 1 mg/ml) rightward shifts in the BeCh concentration responsecurves (p < 0.05) in contrast with NIgGs or IVIG. However, SclIgGs had no significant effect(p > 0.05) on the IAS SMC contraction by phenylephrine and K+-depolarization. Thisinhibitory effect of SclIgGs on the IAS SMCs was significantly (p < 0.05) blocked by theIVIG (1 to 10 mg/ml). Similar data were obtained in the rat smooth muscle strips: SclIgGs-attenuated BeCh responses were significantly (p < 0.05) reversed by the IVIG. IF andfunctional studies revealed that SclIgG examined from all patients specifically decreased thebasal M3R IF, and caused a concentration-dependent decrease in the BeCh-induced IAScontraction. Collectively, these data suggest that SclIgG specifically binds to the M3R andcauses displacement of the M3R occupancy. Secondly, the IVIG blocks M3R-inactivation bySclIgG. This effect most likely occurs speculatively via neutralization of the SclIgG in circula-tion rather than at the receptor site. Conclusion: Cholinergic blockage of the M3R muscarinicreceptor by immunoglobulins from Scl patients can be reversed In Vitro by commercialhuman pooled immunoglobulins. This is the first evidence for a potential new approach totreating the gastrointestinal changes of scleroderma.

Tu2003

Rho Kinase is the Major Molecular Pathway for the Basal Tone in IntactHuman Internal Anal Sphincter (IAS) Smooth MuscleSatish C. Rattan, Jagmohan Singh

Background & Aims: Because of the lack of the knowledge of molecular control mechanismsunderlying the basal tone in the intact human IAS tone, presently there is no satisfactorytreatment for a number of debilitating rectoanal motility disorders associated with the IASdysfunction. Therefore, present investigation determined the role of RhoA/ROCK and PKCpathways, known to play a significant role in the agonist-induced sustained contraction ofsmooth muscles via Ca2+-sensitization and inhibition of myosin light chain phosphatase(MLCP). The latter is primarily responsible for the dephosphorylation of p-MLC20 and thusrelaxation of the smooth muscle. Methods: We compared the basal tone in the human IASwith the rectal smooth muscle (RSM), and determined responses to ROCK and PKC-selectiveinhibitors Y 27632 and Gö 6850, respectively. Western blot studies determined the levelsof RhoA/ROCKII, protein kinase Cα (PKCα), myosin-binding subunit of MLCP (MYPT1),protein kinase C-potentiated inhibitor (CPI-17), and MLC20 in the unphosphorylated andphosphorylated forms, in the particulate and cytosolic fractions in the IAS vs. RSM. Phospho-levels of most signal transduction proteins represent their activated forms. Phospho-MYPT1leads to the inhibition of MLCP. The particulate levels of RhoA/ROCK and PKCα provideadditional data on the status of their activation. Confocal microscopy validated the membranedistribution of ROCKII before, and redistribution after the ROCK inhibitor Y-27632. Finally,in order to confirm a direct relationship, we examined the correlation between the enzymaticactivities and changes in the basal IAS tone and p-MYPT1, p-CPI-17, and p-MLC20, beforeand after Y 27632 and Gö 6850 (10-8 to 10-4 M). Results: Data show significantly higherlevels of RhoA as well as ROCK II in the human IAS vs. RSM tissues and the SMCs. Inaddition, data show a significant correlation between the active RhoA/ROCK levels, ROCKenzymatic activity, and the basal IAS tone, before and after ROCK vs. the PKC inhibitor.Additionally, data with the downstream signal transduction cascade proteins show that thebasal tone correlates with the constitutively active RhoA/ROCK pathway and not the PKC.Higher levels of phospho- and non-phospho- RhoA/ROCK in the particulate fractions ofthe IAS vs. RSM provide further evidence in support of this hypothesis. Conclusion: 1).RhoA/ROCK pathway is the critical determinant of the basal tone in intact human IAS; and2). RhoA/ROCK provide novel therapeutic targets for several rectoanal motility disorders inhumans for which currently there is no satisfactory treatment.

Tu2004

Rectoanal Function in Patients With Large Rectal Adenomas Before and AfterTreatmentRenée M. Barendse, Jac Oors, Eelco J. de Graaf, Willem A. Bemelman, Paul Fockens,Evelien Dekker, Andreas J. Smout

Introduction. Patients with large rectal adenomas often present with fecal urge, incontinenceand soiling. The underlying rectoanal dysfunction remains to be quantified. Standard treat-ments of these patients include endoscopicmucosal resection (EMR) and transanal endoscopicmicrosurgery (TEM). We aimed to determine rectoanal function in these patients beforeand after treatment. Methods. Patients with large (≥ 3 cm) rectal adenomas, eligible foran ongoing prospective randomized trial comparing EMR and TEM (TREND-study), were