Antioxidants in patients with acquired megacolon

AU54 AGA ABSTRACTS • G4722 INTRACISTERNAL INJECTION OF LOW-DOSES OF SOMATOSTATIN ANALOGUE, ODT8-SS, INCREASES THE GASTRIC RESISTANCE TO ETHANOL THROUGH VAGAL, NON-MUSCARINIC PATHWAY IN RATS. K. Kawakubo and Y. Tach4. CUREYDigestive Diseases Res. Center, VA Medical Center, Dept. of Medicine, UCLA, Los Angeles, CA 90073. BACKGROUND: Intracisternal (ic) injection of the octapeptide analogs of somatostatin such as ODT8-SS or sandostatin produces vagal-dependent stimulation of gastric acid output (Regul. Pept., 1:307, 1981; Brit J.Pharmacol. 116:2303, 1995). Low levels central stimulation induced by ic TRH prevents the gastric mucosa against ethanol injury (AIP 260:R1610, 1994). AIM: To investigate 1) whether low-doses of ODT8-SS injected ic induces a vagal dependent protective effect on ethanol-induced gastric damage, and if so, 2) the peripheral mechanisms involved. METHODS: Under urethane anesthesia (1.25 g/kg, ip), fasted SD rats (250 - 300 g) were injected ic with saline (10 lal) or ODT8-SS 30 min before intragastric administration of 45% ethanol (5 ml/kg). Percentage of corpus mucosa containing lesions was determined by computerized image analyzer 1 hr after ethanol injection. The influence of bilateral truncal vagotomy (-2h), atropine sulfate (1 mg/kg, sc, -30 rain), indomethacin (5 mg/kg, ip, -60 min), CGRP antagonist, hCGRP8.37 (100 lag/kg, iv, -2 min), nitric oxide synthase inhibitor, L-NG-Nitro arginine methyl ester (L-NAME, 3 mg/kg, iv, -1 min) and a VIP antagonist, [4C1-D-Phe6,LeulT]VIP (75 lag, iv, -5 min) on cytoprotection were tested. L- or D-arginine (300 mg/kg, iv) was injected immediately before LNAME (3 mg/kg, iv). RESULTS: Ethanol induced gastric lesion in 15 ± 4% of the glandular area in vehicle-treated rats (n--4). ODT8-SS (30 and 100 ng, ic) decreased gastric injury to 9 ± 3% (p>0.05, n--4) and 2 ± 1% (p<0.05, n=5), respectively. Higher doses (200, 500 and 1000 ng/, ic, n=5-6/group) tend to reverse the protective effect (9±2%, 1 6 ± 2 % and 23±4%, respectively, p>0.05). The cytoprotection induced by ODT8-SS (I00 ng, ic) was completely blocked by vagotomy, L-NAME and [4CI-D-Phe6,LeulT]VIP (15 ± 3%, 16 ± 5% and 14 ± 4%, respectively, p<0.05, nf4-5/group), but not by atropine, indomethacin and hCGRPs.37. L-arginine, but not D-arginine, reversed the effects of L-NAME. Vagotomy, L-NAME and [4CI-DPhe6,LeulT]VIP themselves did not alter the ethanol-induced gastric damage. Combined ic injections of the TRH analog, RX 77368, (1 ng) and ODT8-SS (30 ng), at doses subthreshold given singly, reduced by 52% (P<0.05) ethanol-induced gastric lesion. CONCLUSIONS: These data indicate that ic injection of ODT8-SS at low-doses increases the resistance of the gastric mucosa to ethanol injury through vagal non-muscarinic pathways involving nitric oxide and VIP. • G4723 DETECTION OF CCK-8 INDUCED CEREBRAL NEURONAL ACTIVITIES BY FUNCTIONAL MAGNETIC RESONANCE IMAGING (FMRI) IN HUMANS. M. Kern, K. Saeian, R, Arndorfer, L. Eskowski, E. Stein, A. Bloom, J. Hyde, R. Shaker. MCW Dysphagia Institute, MCW Biophysics Institute, Departments of Medicine, Radiology, Psychiatry, and Pharmacology, Medical College of Wisconsin, and VAMC, Milwaukee, Wl. Cholecystokinin (CCK) is present abundantly within the central nervous system. It is involved in central control of gastrointestinal motility, satiety, and also induces nausea. Aims: To detect and characterize the cerebral cortical effect of clinical doses of intravenous CCK-8 using FMRI. Methods: Whole brain imaging of 10 normal subjects (ages: 19-33 yrs) was performed using 1.5T General Electric Signa and 3T Bruker scanners. The scanners were fit with a three axis gradient head coil which was used with a gradient echo, echo planar image pulse sequence ( ~ 0 msec, TR=6000 msec, FOV=24cm) to acquire 64x64 pixel images of 13 contiguous 10.4mm sagittal slices. For each CCK injection scan, 200 images/slice were acquired during a 1-min. baseline, 30 sec. of drug administration and 18.5 min. of post-CCK injection period. Two doses of CCK (20ng/kg and 40ng/kg) were tested in the same session but in two separate scanning intervals. After each scan, subjects were asked to rate the level of nausea on a scale of 0 (no nausea) to 5 (severe nausea). In 5 subjects, an additional scan was performed injecting an equivalent volume of normal saline. A waveform recognition algorithm was used to detect FMRI signal changes. Results: An FMRI response was detectable in all volunteers following IV injection of 40ng/kg CCK and in 4 subjects with injections of 20ng/kg (Table). Common areas of activation corresponded to anterior cingulate, the parietal/occipital region, prefrontal regions and the amygdala on super-imposed anatomical images. Characteristics of these responses were different from historical values (t) for a simple motor task and sensory stimuli originating from the esophagus.

20 ng/kg CCK i40 ng/kg CCK saline motor taskt esoph distendt esoph acidt

# Subjects Median Activation Activation Peak to to Peak Base with FMRI Nausea Latency (minutes) (minutes) (minutes) Response Score 4/10 2 0.55±0.70 2.3±0.12 4.79±0.24 4 0.45±0.05 2.1±0.11 6.01±0.29 10/10" 0/5 0 . . . . . . -0.04 + 0.02 0.08 ± 0.02 -10/10 -0.08 ± 0.02 0.14 ± 0.02 -8/10 -4.70 ± 1.20 9.80 ± 1.40 -5/5

GASTROENTEROLOGY Vol. 114, No. 4

After each CCK injection scan, all subjects reported some degree of nausea following injection that subsided shortly thereafter. Conclusions: Exogenous CCK elicits a detectable cerebral FMRI response that appears to be different from historical values of GI tract related FMRI responses. The detected CCK response may be due to primary CCK effect or cortical processes associated with the perception of nausea. G4724 1,25(OH)2 VITAMIN D 3 AND TPA STIMULATE PHOSPHATIDYLCHOLINE-PHOSPHOLIPASE D (PC-PLD) IN CACO-2 CELLS: ROLE OF PKC, RHO, AND C-SRC IN PLD STIMULATION. S. Khare, R. K. Wall, M. Bissonnette, B. Scaglione-Sewell, M.D. Sitrin and T.A. Brasitus. Dept. of Medicine, University of Chicago, Chicago, IL. PLD has been implicated in the regulation of mitogenesis, as well as a number of other important cellular processes. Our laboratory has previously reported that both 1,25(OH)2D 3 and TPA stimulated PC-PLD in a time- and concentration-dependent manner via a PKC-dependent mechanism in CaCo-2 cells, a cell line derived from a human colon carcinoma. The alms of the present study were to: 1) investigate the specific PKC isoforms involved in PLD stimulation by these agonists; and 2) determine whether heteromeric and/or monomeric G-proteins and/or c-Src are involved in the regulation of PLD activity. Methods: PLD activity was assayed by measuring phosphatidylbutanol [Pbt], the specific transphosphatidylation product of the PLD reaction by HP-TLC. PKC isoforms were detected by Western blotting using isoform-specific polyclonal antibodies. CaCo-2 ceils were transfected with human protein kinase C-~t in sense or antisense orientation, c-Src was immunoprecipitated with anti-Src monoclonal antibodies and kinase activity was assayed by 32p-incorporation into a peptide substrate derived from p34 coo-2. Results: 1,25(OH)zD 3 (100 nM) rapidly, but transiently activated PKC-o~, but not PKC-13I, II, 6 or e, as assessed by increased membrane association. TPA (200 nM), however, not only stimulated PKC-cx, but also 13I and & Preincubation of ceils with 2 taM indocarbazole G~6976, a specific inhibitor of Ca2*-dependent isoforms of PKC, caused an 85% and 95% inhibition of PLD stimulation by 1,25(OH)2D 3 or TPA, respectively. Furthermore, overexpression of PKC-tx enhanced PLD stimulation by 1,25(OH)2D 3 or TPA. Pretreatment of ceils with pertussis toxin (100 ng/ml, 18 h), but not cholera toxin (100 ng/ml, 2h), completely blocked the ability of 1,25(OH)2D 3 or TPA to stimulate PLD. PLD activation by 1,25(OH)2D 3, but not TPA, was significantly blocked after pretreatment of cells with C3-transferase, an inhibitor of Rho (monomeric G-protein)-dependent processes. PP1, a specific Src family protein tyrosine kinase inhibitor, also blocked PLD stimulation by 1,25(OH)2D 3, but not TPA. In summary, 1,25(OH)2D 3 stimulation of PC-PLD in Caco-2 cells appears to involve PKC-ct alone, while both PKC-et and/or 13I may mediate the effects of TPA on PLD. Furthermore, a pertussis toxinsensitive heterotrimeric G protein is involved in stimulation of PLD by both these agonists. PLD stimulation by 1,25(OH)2D3, but not TPA, however, requires c-Src and Rho signaling. G4725

ANTIOXIDANTS IN PATIENTS WITH ACQUIRED MEGACOLON. TR Koch. L Yuan, West Virginia University, Morgantown, WV; GL Telford, Medical College of Wisconsin; SJ Stryker, Northwestern University Medical Center; E Abdel-Rahman, EC Opara, Duke University Med Center, Durham, NC. In a rodent model, chronic depletion of the antioxidant, glutathione, alters expression of enteric inhibitory neurochemicals, VIP and NO synthase. We have also reported reduced glutathione levels associated with abnormal levels of VIP and NO synthase in muscularis externa (ME) layer from acquired megacolon. Since it is not clear whether glutathione depletion is related to decreased bioavailability of antioxidants or to increased utilization, this study was designed to examine the total antioxidant capacity in colon and colonic levels of lipid peroxides. METHODS: Colon from non-obstructed neoplasia (grossly normal; n=8; 5 males & 3 females; ages 64-88 years) and acquired megacolon (n=4; 2 males & 2 females; ages 45-51 years) were obtained at surgery and quick frozen. Each sample was microdissected into mucosalsubmucosal (MS) and ME layers. All layers were extracted prior to determination of lipid peroxide (LP) levels (mainly 4-hydroxyalkenals; pmoles/g tissue) and total antioxidant capacity (TAC; absorbance of ABTS radical cation using Trolox as standard; nmoles/g tissue). RESULTS:

Tissue Normals: Megacolon:

Layer MS ME MS ME

Mean _+SEM LP 162 ± 31 130 ± 28 200 ± 35 137 ± 24

*t-test: p = .01 TAC 383 ± 74* 661 ± 103" 541 ± 34 578 ± 58

In the ME layer, TAC in 4 patients <72 years was 769+/-137 and in 4 patients >76 years it was 553+#151 (p>.05). SUMMARY: Total antioxidant capacity is higher in the muscularis externa and appears to decline with aging. The presence of similar lipid peroxide levels supports: 1) decreased bioavailability of antioxidants rather than increased consumption in acquired megacolon, 2) the altered expression of VIP and NO synthase in megacolon may not be the result of oxidative damage. The similarity in total antioxidant capacity raises the question of the relative contributions of individual free radical scavengers. Supported in part by West Virginia University