These data indicate that in the early postnatal period SERT expression and function is lowbut DAT and NET contribute to 5-HT clearance. As the animals mature beyond 3 weeks,SERT function increases while DAT function declines. NET and SERT both contribute to5-HT clearance in the mucosa of 6 week old guinea pigs. These data indicate that there areredundant transport mechanisms for 5-HT clearance in the gut mucosa. Monoamine transportinhibitors used to treat behavioral problems in pediatric patients could have unanticipatedeffects on gut function. (Supported by DK57039, DK082967, HD05197)

Su1963

Excitation of Enteric Neurons by Supernatants of Colonic Biopsies FromIrritable Bowel Syndrome Patients (IBS) is Linked to Visceral SensitivitySabine Buhner, Qin Li, Breg Braak, Tamira K. Klooker, Sheila Vignali, Michael Schemann,Guy E. Boeckxstaens

We have previously shown that incubation supernatants of colonic mucosal biopsies fromIBS patients activate neurons of human and guinea pig submucous plexus. Interestingly,supernatants from C-IBS (constipation predominant) and D-IBS patients (diarrhea predomin-ant) evoked similar effects. We hypothesized that neural activation may be linked to thevisceral sensitivity of the patients rather than to their altered bowel habits. Methods: Rectaldistension with a barostat was used to classify IBS patients according to their visceralsensitivity into normosensitive (discomfort threshold > 23 mmHg; 8 female; age: 37 yrs(21-46 yrs); 1 C-IBS, 3 D-IBS, 4 A-IBS)and hypersensitive (discomfort threshold < 23 mmHg;6 female, 2 male; age: 34 yrs (20-50 yrs); 1 C-IBS, 4 D-IBS, 3 A-IBS). Fast neuroimagingtechniques with a voltage sensitive dye (Di-8-ANEPPS) was used to record form single guineapig submucous neurons after local spritz-application of IBS supernatants for 200 ms. Results:Supernatants from hypersensitive IBS patients evoked a higher rate of action potentialdischarge compared to normosensitive patients (3.7 Hz (2.2/5.3) vs. 2.4 Hz (1.2/4.2);P<0.001, Mann-Whitney Rank Sum Test, median (IQR)). Also the number of activatedneurons was higher after application of supernatants from hypersensitive IBS patients (49%(27/67) vs. 25% (9/36); P=0.002). Stronger nerve activation was highly correlated withlower barostat distending pressure values for discomfort (correlation coefficient: -0.624, P=0.0096; Spearmann rank order correlation). Conclusion: Supernatants from normo- andhypersensitive IBS patients both activate enteric neurons. The excitatory action is significantlymore pronounced for supernatants from hypersensitive patients and the degree of activationcorrelates with discomfort thresholds. Thus sensitization of enteric neurons and visceralsensitivity are linked phenomena. (Supported by DFG Sche 267/7-2 and 7th EU researchframework IPODD)

Su1964

Calcium Sensing Receptor in Rat Myenteric Plexus of ColonJialie Luo, Haitang Li, Dongsheng Shu, Hongzhen Hu

Neuronal development, signaling and plasticity are largely controlled by cellular calciumsignaling. In mammals, voltage-gated calcium channels, calcium transporters, calcium per-meable cation channels and calcium-sensitive G-protein coupled receptors sense and translatechanges of extracellular calcium into signaling events. Calcium sensing receptor (CASR) isa calcium-sensitive Gq-coupled receptor and abundantly distributed in the gastrointestinaltract. CASR is a polymodal receptor activated by metal ions (calcium, Gadolinium, magnesiumetc), polyamines as well as amino acids in the food. It has been reported that CASR in thecolon epithelium is involved in electrolytes absorption. Although CASR is also expressed inthe neural tissues in the gut, the neurogenic effect of CASR in the enteric nervous systemremains unknown. By using indirect immunofluoresence and chemical coding we studiedthe distribution pattern of CASR in the myenteric plexus of rat colon. There is no co-localization of CASR- immunoreactivity (IR) with choline acetyltransferase (ChAT)-IR, sug-gesting that CASR is not a calcium sensor for cholinergic neurons in the myenteric plexusof rat colon. Accordingly, CASR-IR was not co-localized with calbindin- or calretinin-IR.On the other hand, 99.7% of CASR-immunoractive neurons had nitric oxide synthase (NOS)-IR. About 73% of the NOS-immunoreactive neurons had CASR-IR. Calcium imaging assayrevealed that the CASR agonist gadolinium (0.5 mM) evoked calcium increase in about 10%of the myenteric neurons isolated from the myenteric plexus of rat colon. The gadoliniumaction was not affected by removing extracellular calcium in the extracellular buffer, sug-gesting the calcium increase stems from calcium mobilization from the intracellular calciumstores. The data suggest that there exist functional CASR in the descending inhibitory motorneurons in the myenteric plexus of rat colon. Activation of CASR by chemical cues in thegut will likely excite the inhibitory motor neurons and cause smooth muscle relaxation byreleasing nitric oxide.

Su1965

Antibodies to Campylobacter Jejuni Cytolethal Distending Toxin Subunit B(CDT-B) Bind Enteric Neural Elements in Uninfected Mice SuggestingMolecular MimicryVenkata B. Pokkunuri, Winnie M. Ho, Walter Morales, Gene Kim, Stacy Weitsman, KeithA. Sharkey, Patricia Guerry, Christopher Chang, Mark Pimentel

We have recently developed a rat model of post-infectious IBS that exhibits chronicallyaltered bowel habits, reduced DMP-ICC and bacterial overgrowth several months afterresolution of acute Campylobacter jejuni infection. A cytolethal distending toxin B (CDT-B) mutant of C. jejuni in the same model has no effect on DMP-ICC and mitigates bowelchanges. This implies a role for CDT-B in the pathogenesis of PI-IBS. Two different antibodiesraised against near full-length CDT-B (the main toxin subunit) and another against an 18amino acid CDT-B peptide fragment, stain cross sections of rat intestinal neuromuscularelements in Campylobacter-infected animals. Here we examine whether anti-CDT antibodiesbind neural elements of C. jejuni naïve rats and similar structures in the mouse. Methods:Sprague-Dawley rats never exposed to C. jejuni were euthanized and the ileum was resectedand fixed in formalin. Cross-sections were incubated with antibody raised in rabbits to the

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whole CDT protein and an 18 residue sequence of the CDT-B peptide. This was then washedand visualized with a goat anti-rabbit secondary antibody. In another preparation, CD1mouse colon was opened along the mesenteric border and fixed in Zamboni's fixative. Wholemount preparations of submucosal and myenteric plexuses were incubated with the sameanti-CDT antibodies for 48 hours at 4 degrees celcius. They were visualized with donkeyanti-rabbit secondary antibody conjugated to CY3. (1:100). The tissue was then washed inPBS, mounted in bicarbonate-buffered glycerol, and examined. Results: In Sprague-Dawleyrat ileum, immunohistochemistry with both anti-CDT-B antibodies stained DMP ICC, myent-eric and submucosal ganglia. However, due to cross-sectioning, we could not delineatewhether there was staining of enteric neurons. Using immunofluorescence staining, theantibody to whole CDT specifically stained colonic myenteric ganglia and enteric neuronsin CD1 mouse colon that was not previously infected with C. jejuni. Exposure to rabbitpre-immune sera failed to stain any region of the rat ileum or mouse colon under theseconditions. Conclusions: In this study, antibodies raised against the toxin subunit (CDT-B)of C. jejuni cytolethal distending toxin specifically stained the enteric nervous sytem andICCs in rat ileal cross-sections and enteric neurons in mouse colon. This finding supportsthe hypothesis that cytolethal toxin is important in the pathophysiology of post-infectiousIBS, possibly through an immune mechanism directed some component of the entericnervous system.

Su1966

Rat Embryo Enteric Nervous System Primary Cultures and Jug2 Enteric GlialCells Respond to Bacterial Cell Components Through Up-Regulation of TLR2Joan F. Burgueño, Elena Eyre, Carolina Romero, Michel Neunlist, Ester Fernández

Background: Toll-like receptors (TLR) recognize microorganism associated molecular pat-terns (MAMPs), and have been involved in the pathogenesis of several gastrointestinaldisorders. Previous data from our lab and others describe their expression in the submucousand myenteric plexuses of the adult enteric nervous system (ENS). Aim: To determinewhether ENS components are capable of sensing and responding to MAMPs through TLR.Methods: An ENS primary culture from Sprague-Dawley rat embryos and the enteric glialcell line JUG2 were stimulated with two logarithmic doses of PAM2CSK4, LPS and CpGODN 1826, and NF-κB activation and TLR2, TLR4, TLR9 and glial cell-line derived neuro-trophic factor (GDNF) expression were monitored. Expression and distribution of TLR werestudied by quantitative PCR (qPCR) and immunofluorescence, while activation of the NF-κB pathway was semi-quantitatively evaluated by western blot, through determination ofphospho-IκB alpha (P-IκBα) levels in cell lysates. GDNF levels were also measured by qPCR.Results: TLR2, TLR4 and TLR9 were either expressed in ENS primary cultures and JUG2cells. mRNA levels of these three receptors were similar in primary cultures, while TLR4was markedly the most expressed in JUG2 cells (in fold number, TLR2: 1±0; TLR4: 69.2±12.8;TLR9: 25.1±7.2; n=7 for each; arbitrary units; P<0.001 vs. TLR2 and P<0.01 vs. TLR9).Distribution of TLR immunoreactivity (IR) was similar in both types of cultures, TLR2-IRbeing found in the cell cytoplasm and perinuclear areas, TLR4-IR localization close to thenucleus and TLR9-IR present in the cytoplasm and its prolongations with a fibrous pattern.Treatment with the above mentioned TLR ligands led to differential activation of the NF-κB pathway. ENS primary cultures responded to all MAMPs, as P-IκBα levels were increasedfrom 1 to 48 hours, and reached their peak activation at 8 hours (table). Conversely, JUG2cells responded strongly to LPS, but did not to CpG ODN 1826, and only mildly toPAM2CKS4. Activation kinetics in JUG2 cells was also faster, beginning at 10 minutes,peaking at 1 h and lasting until 8 h for LPS. Both cultures reacted to the assayed TLR ligandsby up-regulating TLR2, without affecting TLR4, TLR9 or GDNF expression. In ENS primarycultures, up-regulation began around 8 hours and remained high at 24 hours, while JUG2cells exhibited again faster responses, starting around 2 hours and ending before 8 hours(table). Conclusions: ENS components express functional TLR and are thus responsive toMAMPs. The assayed ligands do not elicit down-regulation of their respective receptors, butincrease TLR2 expression, suggesting a major role for this receptor in the modulation ofthe gut immune response.Responses elicited by MAMPs in ENS and enteric glial cell cultures

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