appetite before and 1 rain after the infusion (VAS). Repeated measurements ANOVA was used for final analysis of spatial and infusion-induced differences. Results: Lipid showed a specific duodenal motor response in all volunteers (data:Ameans-+SEM vs. 5ml saline: number of contractions/5min 80.7-+ 19.3 p=O.O03 vs saline 0.8 -+2.6, motility index 3.7-+0.7 p=O.001 vs saline -0.56-+0.4), acid in 6 of 8 volunteers (contracrions/5min 15.4-+6.4, p=0.021 vs saline, MI 1.7-+0.8 p= 0.02 vs saline). The motor response to lipid but not to acid was abolished by gramsetron (lipid/granisetron: contraction~5min 0.46-+ 5.2 p = 0.002, MI -0.08 + 0.8 p = 0.001 vs lipid/saline; acid: contractions/5min - 0.63 -+ 6.1, p = 0.129, M1-0.17 -+ 0.9, p = O.108). Perception was unaffected by all infusions. Basic motility was unchanged by granisetron. Conclusions: Lipid and acid elicit specific duodenal motility patterns in terms of the magnitude and timing of this response. The lipid induced duodenal motility is abolished by the 5-HT3 receptor antagonist granisetron indicating the involvement of the enterochromaffine cells in the sensing of lipids and the 5-HT3 receptor in mediating the motor response. The rapid local motor response to acid infusion is 5-HT3 receptor independent. S1019 Ryanodine-Sensitive Stores in Gallbladder Smooth Muscle Maria J. Pozo, Sara Morales, Gary M. Mawe, Pedro J. Cameno Ca2§ release from intracellular stores is a ubiquitous signal in many types of cells. In smooth muscle, two main receptors or channels mediate this release: IP3 and ryanodine receptors. The exact location of these two receptors and their contributions in the regulation of smooth muscle physiology greatly varies upon the type of smooth muscle considered. To date, ryanodine Ca2+ stores in the gallbladder smooth muscle have not been characterized. There- fore, we sought to examine these Ca2+ stores in the gallbladder by measuring [Ca2+]t in fura-2 loaded isolated myocytes, and by recording isometric contractions in whole mounted preparations. Application of caffeine (200 tim - 10 mM) to single myocytes induced a rapid spike-like elevation in [Ca2+]~that reached a peak within 6-9 s after the onset of elevation and had a concentration dependent amplitude with a maximum increase reached at 10 mM of 0.60 +- 0.09 (expressed as a ratio of the fluorescence at 380 and 340 nM, F3so/F34o, n = 10). When cholecystokinin (CCK, 10 nM) was used as an agent to induce IP3-dependent Ca2+ release, the mean amplitude of the peak was 3 fold smaller than that caused by caffeine (n = 14). These data support the concept that ryanodine receptors (RyRs) could release a substantial amount of Ca2+ in these cells. The caffeine-sensitive Ca 2+ stores have a leakage, as there was an inverse relationship between time in Ca:+ free medium and amplitude of the peak, being totally depleted after the cells were for 20 rain in Ca2+ free medium. The refilling of caffeine-sensitive stores involves SERCA activation, as it was inhibited in the presence of 1 tiM thapslgargine. The moderate Ca2+ elevation caused by CCK induced a contraction of 13.1 -+ 1.3 mN (n = 8), but caffeine-induced Ca2+ elevation was unable to induce gallbladder contraction. Thus, addition of caffeine (100 tiM - 10 mM) to the organ bath did not alter resting tone. Similarly, no changes were recorded in response to ryanodine application. The inability of caffeine to trigger contraction could be related to the hyperpolar- ization (6. 6 -+ 0.9 mV, n = 5) that this drug caused in the tissue or to the activation of spontaneous outward transitory currents that we have previously reported (Pozo et al, Am J Physiol Gastr L, 2002). Taken together, our results suggest that gallbladder smooth muscle cells have functional ryanodine receptors, and Ca2+ release through these channels may not have a direct role in inducing contraction, but rather, may play an important role in controlling excitability of gallbladder smooth muscle. Supported by SAF-2001-0295, NIH-NS26995 S1020 Cnnfocal Visualization of Nitric Oxide in the Myenteric Plexus of the Gastric Fundns Obtained from Endotoxin-Treated Rats Elsa Qnintana, Alberto M. Alvarez, Angeles Alvarez, Juan V. Esplugues, Maria D. Barrachina Sr. Nitric oxide (NO) has been shown as a NANC neurotrasmitter in the gastric fundus. However no direct anatomical evidence exist about the NO synthesis in this tissue. Endotoxin has been shown to decrease fundns gastric tone through a non-transcriptional regulation of peripheral NO (Quintana eta[. 2002). AIM: To visualize, by a bio-imaging system, the anatomical pattern of NO synthesis in the gastric fundus of endotoxin-treated rats and to correlate results obtained with changes in fundns contractility. METHODS: Fasted rats received endotoxin (40 p-g/kg, i.p.) or saline (lml/kg, i.p.) and 30 mm later the stomach was removed and four circular fundns strips from each rat were cut. a) Imaging of nitric oxide synthesis using NO specific fluorescent dye DAF-FM: The fundus strips were pre- incubated (25 rain) with a tyrode solution containing vehicle, L-NAME (1 raM), tetrodotoxin (I~M, TTX) or hexamethonium (100 p,M, HEX). Afterwards, strips were changed to an identical medium than before but containing in all cases: DAF-FM diacetate (1p-M), CMxROS (1 p-g/m]) and Hoestch (i ttg/ml) and incubated for 60 min. Basal fluorescence in the enteric plexus and smooth muscle layer was visualized and quantified with a confocal laser micro- scope, b) Registration of isometric contraction in organ bath chambers: TRIM (lO0p-M, a nNOS selective inhibitor), TTX (II~M), HEX (100tiM) or vehicle were added to the organ bath and the contractile response to carhachol was analyzed. RESULTS: a) Data (n>3) express intensity of fluorescence (a.u.). In the enteric plexus, pre-treatmem with endotoxin induced a significant increase in DAF basal fluorescence (9.2 _+ 0.7 a.u.) compared with that observed in control animals (3.9-+0.6 a.u.). This effect was significantly reversed by L- NAME (68 - 16%), TTX (92 + 8%) or HEX (58 -+ 23%). In smooth muscle layer, endotoxin (4.4-+0.6 a.u.) failed to modify basal fluorescence compared with vehicle (3.2 -+ 1 a.u.), b) The maximal contractile response to carbachol (6.3-+0.4g) was significantly reduced in strips from endotoxin-treated rats (2.6 + 0.6g). This effect was reversed by TRIM (98-+ 11%), TTX (72 -+ 3%) or HEX (88 + 10%)(n>5). CONCLUSION: Synthesis of NO in post-gangli- onic enteric neurones of the gastric fundus is increased by in vivo pre-treatment with endotoxin. Once released, NO is involved in the fundus hypotonicity associated with endotox- emia. This fluorescence-based technique constitutes a highly sensitive method to analyze the role of the nitrergic neurotransmission on GI function. S1021 Group 1 Metabotropic Glutamate Receptor Antagonism Inhibits Propulsion in Guinea Pig Colon Bridget R. Southwell, Madeleine Stephens, Vllija G. Vilija G. Jokubaitis, Joel C. Bomatein Background: Glutamate, a major neurotransmitter in the brain, is present in vagal fibres and in the intestine, but its rote as a transmitter in the colon is not clear. In guinea pig distal colon, glutamate enhances acetylcholine (ACh) and noradrenaline (NA) overflow. The specific agoinsts, N-methyl-D-aspartate (NMDA) inhibits colonic propulsion via NA release ~, while alpha-amino-3-hydroxy-5-methyl-4-isoxazole propionic acid (AMPA) enhances colonic peristalsis (while inhibiting ACh overflow and increasing electrically evoked NA overflow) 2. Glutamate evokes slow depolarizing responses via group 1 metabotropic glutamate receptors (mGfuR) in myenteric3 and submucosal neurons4 in the guinea pig ileum, but the role of group I mGluR in intestinal propulsion is not known. Aims: Determine if group I mGluR have a role in control of motility in the guinea pig distal colon. Methods: Colonic propulsion was measured as the movement of artificial fecal pellets through 5-7 cm segments of intact distal colon (in Krebs, 37~ taken from guinea pigs killed by being stunned and having their spinal cords cut. Speed of transit was measured 6 times (4 rain rest between), drug added for 30 rain, speed of transit measured 6 times, the colon was washed for 30 ram and transit measured another 6 times. Six animals were used for each treatment. Group 1 antagonists N-phenyl-7-(hydroxyimino) cyclopropa[b]chromen-la-carboxamide (PHCCC) and 1-aminoindan-l,5-dicarboxylic acid (AIDA), NMDA antagomat D-2-amino-5-phospho- nopemanoate (APS)and non-NMDA antagonist 6,7-Dinitroquinoxaline-2,3-dione (DNQX) were tested. Results: In the absence of antagonist, pellets moved through the colon at 1.3- 1.8 mm/s. PHCCC and AIDA dose-dependently depressed colonic transit with 10 I~M PHCCC reducing transit by 50% (P < 0.001) and 3 p-M PHCCC having no significant effect, while 10 p-M AIDA depressed transit by about 30% (P < 0.05). Neither DNQX nor AP5 altered transit. Conclusion: Blockade of group 1 mGluR inhibits colonic propulsion in isolated colon. This suggests that group i mGfuR play a role in the control of colonic motility and this effect does not require intact extrinsic pathways. This effect is not mediated via ionotropic glutamate receptors which have previously been shown to affect colonic propulsion ~. Cosentino et al Neurosci Lett 1995;183:139-42. Giaroni et al. Life Sci 2000;67:1747- 57. Ren et al Neuroreport 1999;10:3045-8. Hu et al Br J Pharmacol 1999;128:1631-5. $1022 ATP-sensitive K+ Channel Demonstrates Enhanced Bursting Activity in a Murine Experimental Colitis Model Hamid I. Akbarali, X/aochun Jin Inflammation of the colon results in decreased smooth muscle contractility which may be associated with altered ion channel function. We have previously demonstrated that in the murine model of experimental colitis induced with dextran sulphate (DSS), L-type Ca ++ channel activity is markedly reduced without changes in the amplitude or kinetics of the transient outward current (Akbarali et al, 1999). In the present study, we further examined the effect of DSS-induced colitis on the ATP-sensitive K + channel function. Inflammation was induced in Balb/C mice by 5% dextran sulphate in drinking water provided ad libitum for 4-6 days. The extent of inflammation was evident from H & E stained sections of the distal colon. Single colonic smooth muscle cells were enzymatically dispersed and ionic currents measured using the patch clamp technique. Activation of IKCAW)was examined in whole cell recordings with cells bathed in high K + solution. From a Vh -70 mV, levcromakalim (20 p,M) induced an inward current of 7.6 -+ 1.1 pA/pF in control cells and 26.3 +_ 7 pA/ pF in DSS treated cells. The currents were inhibited by glibenclamide (10 ~M). In cell attached patches with symmetrical K+ concentrations, IK~Tp~ in the presence of 20 tiM levcromakalim, demonstrated bursting activity at negative potentials (-20 to -100 mV) which was markedly increased in DSS colonic cells. Burst durations were increased from 38 -+ 8 ms in control cells (n = 10), to 632 + 100 ms in DSS mice (n = 10). DSS cells were significantly more sensitive to levcromakalim. At lit M the open probability (NPo) of I~A~p) was <0.05 while in DSS-treated colonic cells, NPo was 0.25 (n=4). The interburst interval but not the time constants for openings within bursts was significantly reduced in cells from inflamed colon. The single channel conductance remained unchanged between control and DSS mice (43_+ 2 pS in control and 44 _+ 8 pS in DSS colon). These data suggest that decreased excitability of smooth muscle may he partly mediated by up-regulation of 1KIArP) and changes in the sensitivity of the sulphonyfurea receptor following inflammation. $1023 Identification of Galanin Receptor 1 on Ascending Excitatory Pathways: Immunohistochemical and Functional Evidence Marcello Tonini, Fabrizio De Ponti, Roberto De Giorgio, Anthony Ho, Laura Anselmi, Elisabetta Cervio, Stelania Guerrini, Catia Sternini Galainn is a brain-gut neuropeptide exerting a variety of cellular functions. In the gastrointesti- nal tract, galanin is localized to myenteric and submucosal neurons and to fibers projecting to the gut wall. This peptide affects motility and secretion mainly acting as an inhibitory modulator. Indeed, in the guinea pig ileum gahnin inhibits the excitatory neuro-neuronal and neuro-muscular transmission to the longitudinal muscle layer. Among the three G protein-coupled galanin receptors so far identified, the Gi-coupled GAL-R1 is likely to be mostly involved in the modulation of neurotransmitter release. Aims: To identify the distribu- tion of GAL-R1 on ascending excitatory pathways subserving the distension-evoked ascending excitatory reflex in isolated segments of guinea pig ileum. Methods: The study was conducted using both immunohistochemical and functional approaches. Myenteric neurons were identi- fied using specific antibodies raised against GAL-R1 and calbindin (a general marker for intrinsic sensory neurons) or CHAT, VIP and NOS with double labehng immunofluorescence and confocal microscopy. The ascending excitatory reflex was studied using a three-compart- ment bath allowing to establish the site of action of drugs on the afferent, intermediate and efferent components of the reflex pathways. Results: Double labeling studies showed that numerous GAL-R1 munnnoreactive neurons contained ChAT immunoreactivity, some were AGA Abstracts A- 138