Al102 AGA ABSTRACTS GASTROENTEROLOGY Vol. 118, No.4 5101 ENTERAL EPIDERMAL GROWTH FACTOR REDUCES INSU- LIN-LIKE GROWTH FACTOR-I MRNA IN DUODENUM OF DE- VELOPING RATS. Bohuslav Dvorak, Hwal Chun, Jessica A. Dominguez, Catherine S. Wil- liams, Debra L. McWilliam, Anthony F. Philipps, Univ of Arizona, Tuc- son, AZ; Univ of CA, Davis, Davis, CA. Epidermal growth factor (EGF) is known to be present in milk, but its physiologic effect in the neonatal digestive tract remains unclear especially 5100 SOMATOSTATIN RELEASED FROM STC-l CELLS IS NOT IN- VOLVED IN AUTOCRINE REGULATION OF SECRETIN AND CCK RELEASE. Cecilia H. Chang, William Y. Chey, Francis L. Roth, Ta-min Chang, Univ of Rochester Med Ctr, Rochester, NY. STC-I, a murine intestinal neuroendocrine tumor cell line, contains several regulatory peptides including secretin, CCK and somatostatin. It has been widely used as a model to study the cellular mechanism of secretin and CCK secretion. Exogenous somatostatin has been shown to inhibit CCKI secretin secretion in vivo. The purpose of present study was to examine whether or not somatostatin released from STC-1 cells would act as an autocrine factor to regulate CCKlsecretin release. Methods: Monolayer cultures of STC-1 cell were preincubated with or without an anti-soma- tostatin antibody and then incubated with various stimulants at 37°C for various periods of time. Somatostatin, secretin and CCK released to the medium and their cellular contents were determined by respective specific RIA. Results STC-I cells had a higher content of somatostatin (60.3 ± 7 pmollmg cell protein) than secretin (11.8 ± 0.6 pmollmg) and CCK (\2.4 ± 1.4 pmollmg). Gastrin-releasing peptide (GRP), sodium oleate and L-phenylalanine stimulated the release of all three peptides as listed in the Table below(mean ± SE, n = 4). GRP and sodium oleate also time- dependently stimulated the release of SS and CCKlsecretin. Incubation of STC-I cells with an anti-somatostatin antibody (anti-SS) or unimmuized IgG did not affect significantly the release of CCK and secretin stimulated by all three secretagogues. Prolonged incubation with the anti-SS to elim- inate a possible receptor desensitization due to continuous exposure to the secreted somatostatin also did not affect the three secretagogues-stimulated secretin and CCK release, despite of the fact that no free somatostatin was found in the medium. Conclusion: Although co-secreted with CCK and secretin, endogenously released somatostatin does not act as an autocrine inhibitory factor in STC-I cells to regulate CCK and secretin release. 5099 THROMBOXANE A z -INDUCED CONTRACTION OF CAT ESOPHAGEAL AND LOWER ESOPHAGEAL SPHINCTER CIR- CULAR SMOOTH MUSCLE. Weibiao Cao, Karen M. Harnett, Jose Behar, Piero Biancani, RI Hosp and Brown Univ, Providence, RL We have previously shown that spontaneous LES tone depends on arachi- donic acid metabolism and formation of prostaglandin F Za , and of throm- boxane A z . These arachidonic acid metabolites act on receptors linked to G i3 and Gq-proteins to activate phospholipases, produce second messen- gers, and maintain lower esophageal sphincter tone(LES). In the present investigation we examined the signal transduction pathway activated by the thromboxane A z analog U46619 to contract circular smooth muscle cells isolated by enzymatic digestion from cat esophagus (ESO) and LES. U46619 (\0- 1 3_10-7 M) caused dose-dependent contraction of ESO and LES. In ESO U46619-induced contraction was inhibited by antibodies against G i3-type G proteins, and a 35S_GTPyS binding assay confirmed that G 9 and G i3 G proteins were activated by U46619. LES contraction was innibited by G q antibodies and the 35S_GTPyS binding assay confirmed Gq-proteins were activated by U46619. In ESO U46619-induced contrac- tion was reduced by the selective phosphatidylcholine-specific phospho- lipase C (PC-PLC) inhibitor D609, and by propranolol, which inhibits the phospholipase D (PLD)-dependent pathway, but was not affected by the phosphatidylinositol-specific phospholipase C (PI-PLC) inhibitor U73122. The contraction was inhibited by the PKC inhibitor chelerythrine, and not affected by the calmodulin inhibitor CGS9343B. LES contraction was inhibited by U73122 and not affected by D609 and propranolol. The contraction induced by a low dose of U466 19 was blocked by cheleryth- rine, but not by CGS9343B. In contrast, the contraction induced by a maximal dose of U46619 was blocked by CGS9343B, but not by chel- erythrine. These data suggest that ESO thromboxane receptors are linked to Gi3 to activate a PC-PLC-and PKC-dependent pathway. In LES U46619 at high concentration activates thromboxane receptors linked to Gq to acti- vate a PI-PLC and calmodulin-dependent pathway. At low doses, however U46619 activates a PKC-dependent pathway. Supported by NIH ROI-DK- 28614 Peptide released in pmol/mg cellprotein (%of control) Somatostatin Secretin CCK 4 Day·l Group Food intake (g/100 9 body weighUday) before (day -1) and after (days 1-5) induction of colitis at day 0 Food intake in9-41 CRF/colitic significantly greater (P < 0.05) than control colilic 1l-41CRF 86 ± 22 9.2 ± 23 11 ± 06 23 ± 18 43 ± 30 65 ± 4.2 55 ± 3.1 Controls 7.7+ 37 9.9 + 1.7 10 + 0.8 0.9+ 0.7 19 + 0.6 2.7+ 08 2.1 + 0.8 when administered enterally. Insulin-like growth factors (lGF-I and IGF-II) are postulated to be important regulators of somatic growth during the perinatal period. The aim of this study was to evaluate the effect of milk-borne EGF on the endogenous production of IGF-I and IGF-II mRNA in the small intestine of suckling rats. METHODS: Eight-day old suckling rats underwent intragastric cannulation and then were artificially fed either growth factor-free rat milk substitute (RMS) or RMS supplemented with EGF (500 ng/ml) for 4 days. Artificially reared rats were compared with their dam-fed littermates (Control). The rats were sacrificed and the small intestine was collected. The expression of rat IGF-I and IGF-II mRNAs was determined using reverse transcription (RT) competitive-polymerase chain reaction in all intestinal segments. RESULTS: There were no sig- nificant differences in body weight gain between the three groups of rats. In the duodenum, IGF-I mRNA levels were significantly lower in the RMS+EGF group compared to those in the RMS group or controls. Additionally, immunohistochemistry showed that IGF-I was present in enterocytes of the duodenal villi in the dam fed and RMS group, but the signal was weak in the RMS+EGF group. Jejunum and ileum !GF-I mRNA levels, and intestinal IGF-II mRNA levels did not differ between groups. CONCLUSION: These results indicate that enteral administration of EGF can regulate endogenous production of IGF-I in the duodenum of developing rats. Supported by the NIH Grant HD-26013. 5103 THE EFFECT OF CHOLINERGENIC STIMULATION ON CA H EVENTS AND STOCS IN MURINE COLONIC MYOCYTES. Brian Hagen, Orline P. Bayguinov, Kenton M. Sanders, Unr, Reno, NV. Confocal microscopy and whole cell patch clamp recording using the perforated-patch technique were used to investigate the effects of acetyl- choline (ACh) on isolated colonic myocytes. Smooth muscle cells, isolated from murine colon and loaded with Fluo-3, exhibited spontaneous, local- ized Ca z + transients and Ca?" waves. These events corresponded to spontaneous transient outward currents (STOCs). In a previous study we showed that Ca z + transients and STOCs depend upon activity of the phospholipase C-catalyzed formation of inositol I (IP3)' ACh caused an increase in basal cytosolic Ca + and reduced localized Ca + transients and STOCs. Atropine (lO/-LM) abolished the effects of ACh. Pretreatment of cells with nicardipine (\/-LM), Cd z + or NiH (200/-LM) did not prevent the effects of ACh. An inhibitor of phos- pholipase C, 1-[6-[[17 13)-3-methoxyestra-I,3,5(lO)-trien-17-yl] amin- 0]hexyl]-IH-pyrrole-2,5-dione (U-73122), blocked Ca z + transients and STOCs but did not affect the increase of basal Ca z + in response to ACh. A blocker of nonselective cation channels, Gd3+ (lO/-LM), prevented the increase in basal Ca2+ due to ACh and increased the frequency and amplitude of Ca z + transients and waves. These results suggest that occu- pation of muscarinic receptors increases Ca z + influx through nonselective 5102 INHIBITION OF CORTICOTROPHIN RELEASING FACTOR (CRF) INCREASES FOOD INTAKE BUT NOT GASTRIC EMPTY- ING IN ANOREXIA ASSOCIATED WITH INTESTINAL INFLAM- MATION. Tarek T. Elhaj, Anne B. Ballinger, Michael Jg Farthing, St Bartholomew's and The Royal London Sch of Medicine, London, United Kingdom. Inflammatory bowel disease is often associated with loss of appetite, which, in children, contributes to linear growth retardation and delayed puberty. In health, hypothalamic CRF is a potent inhibitor of feeding and may act in part by delaying gastric emptying. CRF is released by interleu- kin-I, a key cytokine implicated in anorexia associated with inflammation. The aim of this study was two-fold: (i) to determine if inhibition of CRF increases food intake in anorexia induced by intestinal inflammation; and (ii) to determine if increased food intake is associated with accelerated gastric emptying. Methods: Colitis was induced in Wistar rats by intrarectal administration of trinitrobenzene (TNBS) in ethanol. lZ- 4I CRF a potent antagonist of CRF, or vehicle (control) was administered by intracerebro- ventricular infusion via osmotic minipumps implanted subcutaneously. Gastric emptying was assessed in colitic rats, free-feeding healthy controls and animals pair-fed to the colitic group to control for the effects of weight loss on gastric emptying. Intestinal inflammation was assessed macroscop- ically and by tissue myeloperoxidase (MPO). Results: Inhibition of CRF increased food intake in TNBS-colitis (Table). Severity of inflammation was similar in lZ- 4I CRF (macroscopic score, 9.0+ 1.4; MPO, 320+80 tissue) and controllcolitic ./iroups 1.9, 270.2+ 181). 90 min gastnc emptying was similar In 9- CRF/cohtiC (64+5.2%), control/colitic (65+16%), pair-fed (60+21%) and healthy controls (71+9%). Conclu- sion: CRF mediates, in part, the loss of appetite associated with intestinal inflammation. However, its mechanism of action is independent of any alteration in gastric emptying. 0.12±0.01 o 27±0.04 (226) 0.36±0.06 (304) 0.20±0.01 (165) 0.09±0.01 o 24±0.01 (287) 0.42±0.09 (503) 0.16±0.02 (189) 2.92±0.18 5.86±0.09 (200) 1053±195(361) 476±026 (163) Secretagogue added None GRP,5nM Na oleate, 0.2mM l·Phe, 20 mM