April 1995 Motility and Nerve-Gut Interactions A697

• A N O V E L AND S E L E C T I V E MOTILIN R E C E P T O R ANTAGONIST, GM-109, INHIBITS CONTRACTILE RESPONSE TO GM-611, AN ACID-STABLE E R Y T H R O M Y C I N A DERIVATIVE, IN THE RABBIT SMALL INTESTINE. H. Takanashi, K. Yogo, K, Ozaki, M. Akima, H. Koga, H. Nabata. Fuji- Gotemba Research Laboratories, Chugai Pharmaceutical Co., Ltd., Shizuoka 412, Japan.

In order to assess functional and physiological roles of the motilin receptor activation, we have synthesized a novel motilin receptor antagonist, Phe-cyclo[Lys-Tyr(3-tBu)-13Ala-] • trifluoroacetate (GM-109), which was developed by optimization studies of synthetic fragments of porcine motilin (pMTL). In the present study, therefore, pharmacological properties of GM-109 were investigated in rabbit small intestine. GM- 109 as well as pMTL inhibited t25I-pMTL binding to motilin receptors in the homogenate of rabbit small intestinal smooth muscle tissue in a concentration-dependent manner. The pKi value of GM-109 and pKd value of unlabelled pMTL were 7.99 + 0.04 and 9.25 + 0.06, respectively. To determine whether GM-109 interacts competitively with motilin receptor, Scatchard analysis of motilin binding was carried out in the presence or absence of GM-109. The presence of GM-109 (10 nM) decreased the pKd value of 125I-pMTL binding (control, 9.25 _+ 0.06; presence of GM-109, 8.84 + 0.05; P<0.01) with no significant effect on the Bmax (control, 22.5 -+ 1.9 fmol/mg protein; presence of GM-109, 23.1 + 2.0 fmol/mg protein). This result indicates that motilin binding to the motilin receptor is competitively inhibited by GM 109. Secondary, the in vitro functional activity of GM-109 was examined in longitudinal muscle strips of rabbit duodenum. GM-109, up to concentration of 100 gM, did not affect the baseline tonus and did not induce contraction in rabbit duodenal strips. However, GM-109 (0.1 - 3 gM) competitively inhibited contractions induced by pMTL and GM-611 (an acid-stable erythromycin A derivative which induces contractions in the rabbit small intestine in a similar manner as motilin). The pA2 value of GM- 109 for motilin and GM-611 were 7.37 -+ 0.24 and 7.42 _+ 0.21, respectively. Furthermore, the contractile response to acetylcholine, substance P, prostaglandin F2c, and KCI was unaffected by the same preparation of GM-109 at a concentration of 10 gM. Therefore, these results indicate that GM-109 is a selective and competitive motilin receptor antagonist which lacks agonistic activity in rabbit small intestine. Thus, this compound is a useful pharmacological tool to examine the functional role(s) of motilin and prokinetic effects of motitin agonists.

• MAST CELL INFILTRATION AND DEGRANULATION IN COLONIC MUCOSA IN IRRITABLE BOWEL SYNDROME. N.J. Talley t, J.H. Butterfield 2, and E. Peterson 2. Department of Medicine, University of Sydney, Nepean Hospital, NSW, Australia I and Division of Allergic Diseases, Mayo Clinic, Rochester, Minnesota, USA :[ .

Intact mucosal mast cells have been reported to be increased in patients with irritable bowel syndrome (IBS) compared with controls (Dig Dis Sci 1993;38:1590). Mast cells contain mediators (e.g. histamine, prostaglandins) that on cell degranulation could directly alter smooth muscle function. No studies have evaluated whether mast cell degranulation occurs in IBS. We aimed to test the hypothesis that increased mast cell infiltration and degranulation occurs in the colon in IBS. METHODS: Recruited were patients with well-documented diarrhea-predominant IBS based on the Rome criteria (n=7) and healthy controls (n=5). After colonic lavage, patients and controls underwent colonoscopy and biopsy (e2) from the ascending or mid- transverse colon. Tissue sections were stained for tryptase by an indirect immunofiuorescence procedure. Three 6 ,urn serial sections from each paraffin block were processed; they were incubated with either rabbit antihuman tryptase antiserum or normal rabbit serum and subsequently incubated with fluorescein isothiocyanate-labelled goat antirabbit IgG. Antitryptase is highly specific for mast cells and does not stain eosinophils, neutrophils, basophils or other cells. All sections were blindly scored for mast cell infiltration and degranulation from 0-3. RESULTS: Intact mast cells in the colonic mucosa were observed in both patients and controls. Mast cell degranulation was present in 5 of 7 IBS patients and 4 of 5 controls (table).

Group Median age % female Median Median Infiltration Degranulation

Patients 22 86 1.0 0.5 Controls 21 80 2.0 0.5

One 1BS patient and one control had moderate degranulation; none had marked degranulation. In patients versus controls, mast cell infiltration (p=0.38) and degranulation scores (p=0.89) were not significantly different. CONCLUSION: Mast cell infiltration and degranulation in the colon do not occur to a greater extent in diarrhea-predominant IBS than in health, suggesting that mast cells are not important in modulating colonic motor function in IBS.

• PSYCHOLOGICAL DISTRESS AND SEASONAL SYMPTOM CHANGES IN COMMUNITY SUBJECTS WITH IRRITABLE BOWEL SYNDROME ~ , P. Boyce, B. Owen and K. Paterson. Departments of

icine and Psychiatry, University of Sydney, Nepean Hospital, N.S.W., Australia

The role of psychosocial factors in irritable bowel syndrome (IBS) in the corm'unity continues to be controversial. It is also unknown whether IBS is a seasonal disorder. We aimed to determine whether psychosocial and seasonal factors play a role in community subjects with IBS.

: A valid 83 item questionnaire that measured gastrointestinal (GI) GI symptoms, and psychosocial factors including neuroticisrn

(EPQ), psychological morbidity (GHQ), lifetime depression and seasonality (seasonal pattern assessment scale) was sent to a random sanlole of a Sydney population selected from the electoral roles. The response rate was 69% (n=99). Presence of IBS was assessed from the responses based on abdominal pain plus >_ 2 of the Manning criteria.

Results: The prevalence of IBS was estimated at 17% (mean age 43 years; 59% female). Univariate analysis showed that IBS was associated with neuroticism (mean EPQ score in IBS 3.6 vs 5;5 in non-IBS, sd=2.8, 2.2 respectively; p<0.05) but not psychological morbidity scores. Subjects with IBS (71%)were six times more likely than non-IBS (29%) to have a history of lifetime depression (OR 6.1, 95% CI 1.9-19.1). A significant correlation was detected between the EPQ score and the global seasonality score (r=0.2, p=0.02)i An association between IBS and seaSonality was detected (global seasonality score in IBS 9.0, sd=6.4 vs 4.8, sd=4.5 in non-IBS, p=0.01). IBS subjects were mare likely to report that a change in bowel habit (OR 5.8, 95% CI 1.7-20.6), change in mood (OR 3.4, 95'% CI 1.02-11.34) and change in level of social activity (OR 3.4, 95% CI 1.1-10.5) were affected bY the seasons. Neuroticism was not a significant predictor of the perception of a seasonal change in bowel habit.

Conclusion: IBS in the corrrnunity is associated with greater trait and depression but not state psychological disturbances. IBS

is also associated with a high seasonality score indicating that GI syrrlotorns are, to an extent, seasonally determined and suggesting that these subjects are sensitive to environmental factors.

CALBINDIN IMMUNOREACTIVITY OF IDENTIFIED MYENTERIC NEURONS IN THE GUINEA PIG DISTAL COLON. K. Tamura, H. Itoh and P.R. Wade. Dept. Physiology I, Tokai University, School of Medicine, Bohseidai, Isehara 259-11, JAPAN and Dept. Anatomy & Cell Biology, Columbia University College of Physicians & Surgeons, New York, NY 10032 USA

Calbindin, a calcium-binding protein, has been reported m be a marker of myenteric neurons having AH/Type 2 elecu-ophysiological and/or Dogiel Type 2 morphological characteristics in the guinea pig small intestine. It has also been proposed that calbindin might be a marker for intrinsic sensory neurons of the gut. Myenteric neurons in different regions of the g.i. tract express different eleetrophysiological and synaptic properties. The aim of this study was to test the hypothesis that the expression of calbindin immunoreactivity (IR) in distal colonic myenteric neurons can be correlated with their elec~ophysiological and morphological characteristics. Intracellular recordings from neurons in myenteric ganglia adherant to flat sheet preparations of colonic longitudinal muscle were made with microelectrodes containing 2% Neurobiotin® (NB) in IM KCI. The electrophysiological properties of the neurons were determined before the injection of NB. Tissues were fixed and processed for calbindin IR using a monoclonal primary antibody against calbindin-D-28K and fluoroscein (FITC)- or rhodamine (TRITC)-labeled secondary antisera. NB-labeled neurons were revealed by incubation with avidin-TRITC or -FITC. After analysis of calbindin-IR, the precise morphology of NB-labeled neurons was revealed by immunoperoxidase histochemistry, in the NB/FITC labeled neurons with an anti-FITC antibody followed by a peroxidase coupled secondary antibody and in the NB/TRITC-labeled neurons with peroxidase-DAPA-biotin. The neurons in this ~study (n=68) were classified morphologically as having a single long process (SLP, 81%), bipolar (BP, 3%), or multiple long processes (MLP, 16%). All of the MLP neurons and 13% of SLP neurons were caIbindin-IR. AI-I./Type 2 physiological properties were recorded from 3 SLP neurons not expressing calbindin-IR. Six of the SLP neurons were calbindin-IR, 4 had S/Type 1 and 2 had neither S/Type 1 nor AH/Type 2 electrical properties. These results suggest that the distribution of calbindin-IR in distal colonic myenteric neurons is different from that in the small intestine in the guinea pig. We conclude that calbindin-IR can be used as an indicator or predictor of neither the morphology nor the electrophysiology of myenteric neurons in the guinea pig distal colon. (Supported by Grant 02044134 MESC of Japan, NIH NS 12969 and funded in part by Glaxo Research Inst., Research Triangle Park, NC).