A7J.4 AGA ABSTRACTS GASTROENTEROLOGY, Vol. J.08, No. 4

Q IDENTIFICATION OF THE GABA A RECEPTOR atl SUBUNIT GENE IN RAT INTESTINE. D. K. Zeiter, X. Li, D. L. Broussard. Div. of GI and Nutrition, Children's Hospital of Phila. and Univ. of Penna. School of Med., Phila., PA.

There is convincing evidence that GABA is a neurotransmitter in the enteric nervous system (ENS) via the GABA A receptor. The central nervous system (CNS) GABAA receptor consists of multiple subunits which form a ligand-gated ion channel. Alterations in the tz subunit isoforms result in changes in benzodiazepine receptor binding affinities and contribute to the heterogeneity of the GABAA receptor.in the CNS. GABA^ receptor ct subuuit subtypes differ in regional distribution throughout the brain. The ENS GABAA receptor has similar pharmacologic and electrophysiologic properties to those previously described in the CNS, including potentiation by benzodiazepines. However, the subunit composition of the ENS GABA A receptor has not been described. We investigated the molecular expression of the GABA A receptor o~1 subunit (the most abundant c~ subtype in the brain) in the rat proximal small intestine.

Total RNA was isolated from rat proximal duodenum and brain. Following reverse transcription , polymerase chain reaction (PCR), using primers derived from the GABA A receptor czl eDNA, amplified a 307 nueleotide sequence in rat brain and intestine. An oligonucleotide probe homologous to a sequence between the primers in the rat brain hybridized to PCR products from rat brain and intestine on Southern blot analysis. Sequencing of the rat intestine PCR product revealed 98% homology from position g82 to 1168 of the rat brain tzl subunit eDNA. Nonisotopic in situ hybridization, using digoxigenin-labeled antisense GABA A tzl riboprobe, localized the GABA~ txl mRNA to many myeuterie neurons in frozen sections of rat duodenum.

The gene for the GABA^ receptor subunit tzl is expressed in myenterio neurons of the rat proximal small intestine. Determination of the subunit composition of enterie GABA A receptors may permit a better Understanding of the functional properties of this receptor. Demonstration of genetic expression of the GABA^ receptor subunit supports a role for GABA in the modulation of intestinal motility. Supported by the Robert Wood Johnson Foundation and NIH grant 5T32DK07066-19.

OSIMULTANEOUS POST-PRANDIAL ASSESSMENT OF LOWER ESOPHAGEAL SPHINCTER TONE, PROXIMAL GASTRIC TONE AND PLASMA CHOLECYSTOKININ IN NORMAL MAN. F Zerbiba, S Bruley des Varannes a, N Bentouimou a, V Leray a, C Cherbut b, C Roz6 c, JP Galmiche a. aFonctions Digestives et Nutrition, blNRA, 44035 Nantes; ClNSERM U410, 75870- Paris - FRANCE.

The proximal stomach may participate tO the control of the lower esophageal sphincter (LES). Gastric distension induces transient LES relaxations in dogs and humans. In dogs such relaxations are mediated by CCK (Gastroenterology 1994;107:1059). Our aim was to simultaneously assess proximal gastric and LES tone and plasma CCK in normal humans receiving a fat meal. Methods. Esophageal motility and LES tone (Dent sleeve) were measured on two separate days in 7 healthy volunteers (mean age 23 years). During one of these sessions, proximal gastric tone was simultaneously assessed with a high compliance balloon (750 ml) placed in the proximal stomach and connected to an electronic barostat (mean pressure: 7.3 mm Hg). Motility was monitored lh before and 4h after a liquid fat meal (400ml, 600kcal, 75% lipids). Plasma CCK was determined by bio-assay (J Clin Invest 1985;75:1144) before and 5 to 240 min after the meal. Results. Simultaneous use of gastric barostat and esophageal motility device was well tolerated in 6/7 volunteers. The presence of the barostat bag did not significantly affect LES resting tone nor the rate of transient LES relaxations. Immediately after the meal, LES tone decreased to a minimum observed within the first 15rain (from 13.6 + 4.1 to 5.1 + 3.2 mm Hg, m + SD, P<0.01) while plasma CCK peaked at 10 min (from 0.7 + 0.3 to 7.2 + 2.9pM), and gastric relaxation proceeded within 15 rain to 50°/, (92 + 89 ml) of its peak value (which occurred between 60 and 75 rain): Mean LES tone then slowly increased to reach fasting values 210 min after the meal. The mean LES tone was correlated to the mean gastric tone over the whole time of the study (r = 0.86, P<0.001). Plasma CCK decreased slowly from 30 rain after the meal, to reach a plateau (2.0 + 1.0 pM at 240 min). Plasma CCK was correlated neither with oesogastric tone nor to transient LES relaxations. Conclusion; 1. Simultaneous assessment of LES and gastric tone is feasible and well tolerated. 2. Post-prandial LES and gastric tone are closely correlated. 3: Post-prandial changes of LES and gastric tone occur early after a fat meal and are not correlated with plasma CCK, suggesting other, likely neural, regulatory mechanisms. Nevertheless, using CCK receptors antagonists remains necessary to fully assess the.exact rote of CCK.

O F A T IN DISTAL GUT INHIBITS INTESTINAL TRANSIT MORE POTENTLY THAN FAT IN PROXIMAL GUT. XT Zhou, LJ Wang, JD Elashoff, HC Lin. Department of Medicine, Cedars-Sinai Medical Center and UCLA, Los Angeles, CA

Fat in the distal (I1¢al Brake, Gut 1988,29:1042) and proximal (Jejunal Brake, Gastro 1994,106:A531) gut inhibits intestinal transit. It is unknown whether the feedback response to proximal vs. distal fat is different. Since. surgical removal of the distal small intestine induces faster transit and greater steatorrhea than resection of the proximal gut (Ann Surg 1954,140:439), we hypothesize that ileal brake inhibits intestinal transit more potently that jejunal brake. In 4 dogs equipped with duodenal (10 cm from pylorus) and midgut (160 cm from pyloms) fistulas, we compared intestinal transit across an isolated 150 cm test segment (between fistulas) while 0, 15, 30 or 60 mM oleate was delivered into either the proximal 1/2 of gut (between fistulas) or distal 1/2 'of gut (beyond the midgut fistula). Oleate was delivered as solutions of mixed micelles in pH 7.0 phosphate buffer at 2 ml/min for 90 rain. The 1 / 2 0 f gut not receiving oleate was perfused with buffer at 2 ml/min. 60 rain after the start of the perfusion, ~ 20 gCi of 99m-Tc-DTPA was delivered as a bolus into the test segment. Intestinal transit was then measmed by counting the radioactivity of 1 ml samples collected every 5 min from tli¢ diverted output of the midgut fistula. Intestinal transit was calculated by determining the area under the curve (AUC) of the cumulative percent recovery of the radioactive marker. The square root values of the AUC (sqrt AUC), where 0 = no recovery by 30 min and 47.4 = theoretical, instantaneous complete recovery by time 0 were compared across region O f fat exposure and oleate dose using 2-way repeated measures ANOVA.

Oleate dose (mM) (mean + SE) R e , o n of fat exoosul~ 15 30 (~0 Proximal l /2 of gut 4 1 . 6 + i 1 4 40.6+_ 10.2 34 .4+ 3.0 Dis ta i l /2ofgu t 25.6+- 1 . 4 18.9+ 1.5 7.0+- 3.8 Control: buffer into both proximal and distal 1/2 of gut ---41.1 + 4.6. 1. Intestinal transit was slower with fat in the distal 1/2 of gut (region effect p<.01), 2. Oleate inhibited intestinal transit in a dose-dependent fashion (dose effect, p<.05), and 3. Dose dependent inhibitirn of intestinal transit by oleate depended on the region of exposure (interaction.between region and dose, p<.01). Fat in the distal gut (Ileal Brake) inhibits intestinal transit more potgntly than fat in the proximal gut (Jejunal Brake). Supported by AHA-GLAA.