April 1 9 9 5 Intest inal Disorders A 2 6 9

• IN VITRO INTESTINAL EPITHELIAL RESTITUTION IS CHARACTERIZED BY ALTERATIONS IN THE ENTEROCYTE CYTOSKELETON AND IS INHIBITED BY CYTOCHALASIN D. T. Albers and R, Moore, Dept. of Pathology, School of Veterinary Medicine and Medicine, Tufts University, Boston, MA 02111 Intestinal epithelial injury occurs in many disease states, including IBD and viral enteritits. Expedient repair of such injuries is crucial for maintenance of critical intestinal barrier function. Cell migration in response to intestinal injury is poorly understood, however phenotypic alterations in migrating cells are known to occur. To study potential cytoskeletal alterations we utilized immunohistochemistry using a monoclonal anti-villin antibody and phalloidin, ultrastructure(US), and Ussing chamber(UC) techniques with restituting native intestine in vitro. Guinea pig ileal mueosa was injured briefly in Triton X-100 and restituted for 60 minutes(Lab Invest 60:237). Spatial distribution of F-actin and villin was graded on a scale from 0-3 in epithelial cells bordering the injury and compared to n0n-injured(NI) villus tip enterocytes. Subtle changes in F-actin distribution in the leading edge of migrating cells that were not evident by conventional US techniques were detectable by phalloidin histochemistry. The advancing cytoplasmic process of migra t ing cel ls did not exhibi t F-actin in 95% of the cel ls examined(n=37). Basolateral and brush border F-actin was decreased in restituting and fully restituted villi in 70-100% of cells examined (n=l 11). Villin in the brush border of migrating epithelial cells was slightly decreased in staining intensity, 64% strongly positive(n--41) as compared to 71% in NI epithelial cells(n=366). 56% of cells Shouldering the injury(n=41) exhibited villin staining in the advancing cytoplasmic process. Basal staining of villin was increased in injured villi(33-38%, n=123) but returned to pre-injury state after restitution in 100% of ceils examined(n=24). US studies of migrating cells revealed a previously unreported phenotypic alteration, namely diminution and loss of microvilli adjacent to the advancing cytoplasmic process. In UC studies transepithelial electric resistance dropped by 30% following injury and returned to pre-injury levels within 60 minutes. Parallel morphological studies revealed that following injury the epithelial monolayer was reestablished at 60 minutes in 100% of villi(n=36). In the presence of cytochalasin D, however, resistance continued to drop after injury, to 64% of pre-injury levels at 60 minutes. Correspondingly, by morphological assessment a defect remained at the villus tip on 64% of the vil l i examined(n=33) and the appearance of cells bordering the injury remained columnar. These data suggest that significant cytoskeletal alterations, perhaps including microvill i loss and changes in actin polymerization, are necessary for intestinal epithelial restitution.

INTESTINAL ABSORPTION OF NEWLY SYNTHESIZED BILE ACIDS: A STRUCTURE-ACTIVITY STUDY. Aldini R. 1, Roda A. 2, Montagnani M. 3, Polimeni C. 3, Cerre C. 2, Roda E.\ llstituto di Scienze Chimiche, 2Dipartimento di Scienze Farmaceutiche, 3Cattedra di Gastroenterologia, Universita di Bologna, Bologna, Italy.

New synthetic unconjugated, conjugated and "pseudoconjugated" BA absorption was compared with the corresponding physiological unconjugated and conjugated forms. The absorption of ursodeoxycholic (UDCA) and ursocholic (UCA) acid with a methyl group in 6 position (6MUDCA and 6MUCA) and a fiuoro group in 6 position (6FUDCA and 6FUCA) was studied in rabbit ileum and compared with UDCA, UCA hyodeoxycholic (HDCA), hyocholic (HCA), deoxycbolic (DCA), cholic (CA) and ehenodeoxycholic (CDCA) acid. Taurine conjugated UDCA and UCA with a methyl group in 23 position (T23MUDCA and T23MUCA) were studied and compared with tauroursodeoxycholic (TUDCA), tauroursocholic (TUCA), taurocholic (TCA), taurochenodeoxycholic (TCDCA), teurodeoxyeholic (TDCA) acid, and a bioisoster of glycoursodeoxycholic acid, in which the C24 amide bond (-CO-NH-) was replaced with a (-CO-CH2) (24PUDCA). The net absorption was evaluated from the mesenteric vein concentratio.p in steady-state conditions, BA lipophilicity by reverse-phase HPLC (rK '). Unconjugated BA showed passive diffusion, which was in the following order: DCA>CDCA>UDCA>HDCA>CA>HCA>UCA; 6MUDCA absorption was higher than UDCA. 6FUDCA absorption was only slightly lower than UDCA. 6MUCA and 6FUCA diffusion rates were both higher than UCA. T23MUDCA and T23MUCA were actively transported with a Vmax slightly lower than TUDCA and TUCA. 24PUDCA showed also an active saturable transport with a Vmax lower than the natural bioisoster GUDCA. The order of the Vmax was: TCA>TUCA>TUDCA>T23MUCA> T23MUDCA>24PUDCA>TCDCA>TDCA. Structure, number, topographical distribution of the steroid substituents, lipophilicity are the main determinants for BA passive diffusion. A 23 methyl group in the side chain still affords active transport of TUDCA and TUCA. Replacement of an amide bond with a (-CO-CH2) bond does not compromise the active transport. This suggests that the structural modification is bioisosteric and that active transport is still possible without the C-24 amide bond.

• EFFECT OF PROSTANOIDS ON JEJUNAL GLUCOSE TRANSPORT AND OXYGEN CONSUMPTION IN VITRO. A. Alemayehu and C.C. Chou, Depts. of Physiology & Medicine, Michigan State University, E. Lansing, MI 48824-1101.

Endogenous prostanoids inhibit the increases in jejunal blood flow and oxygen uptake occurring during nutrient absorption (AJP 257:G798- G808, 1989). In order to assess whether the vascular action of the prostanoids is independent of their actions on jejunal mucosal oxygen consumption (QO2) and glucose transport (GT), the effects of PGE2, CPGI 2 (carbaprostacyelin, a PGI 2 analog), U-44069 (FxA 2 analog), and PGF~ on jejunal QO 2 and GT were determined in vitro using constant volume manometry (Warburg Apparatus) (Series I) and everted gut sac technique (Series II), respectively. Mucosal QO 2 (pl/hr/mg dry tissue) and GT (mmole/hr/mg wet tissue) were measured for 1 hour. In series I, glucose (5 mM) significantly increased the rate of canine jejunal mucosal QO2 (n=26) from 5.126 -+ 0.059 to 8.258 -+ 0.083 I.d/hr/mg. Addition of PGF-, z , CPGI 2 , U-44069, or PGF2~ , (3 and 30 nM) to glucose (G) in the media (n = 8-10) resulted in the following percent changes in the mucosal QO2: * = p<0.05 relative to G alone.

G + PGE2 G + CPGI 2 G ÷ PGF2~ 3nM - 7 - + 2 % * - 5 - + 1 % + 3 ± 1 % 30nM -16 -+ 1%* -11 ± 1%* +7 -+ 1%*

Thus, PGE 2 and CPGI 2 inhibit whereas PGF2~ enhances jejunal mucosal QO 2 . In Series II, rat intestinal sacs were incubated in 5 mM glucose. The rates of mucosal GT in the absence (0 nM) and presence of PGE 2 , CPGI 2 , or U-44069 (3 and 30 nM), (n = 6-7) are as follows:

PGE2 CPGI 2 U-44069 0 nM 9.5 -+ 0.3 8.2 -+ 0 3 9.5 -+ 0.3 3 nM 9.7 -+ 0.4 7.0 -+ 0.7 8.9 - 0.4 30 nM 10.2 _+ 0.4 6.2 -+ 1.2 11.1 -+ 0.4

Ouabain (1 mM, n= 7) significantly (p<0.05) attenuated the GT from 9.5 + 0.3 to 5,9 -+ 0.2 mmole/hr/mg. Thus, PGE2, CPGI2, and U-44069 did not significantly alter intestinal GT. Conclusion: PGE~ and PGI 2 inhibit the glucose-induced increase in mucosal oxygen consumption without altering the rate of intestinal glucose transport. The vascular action of prostanoids during nutrient absorption may be in part due to their inhibitory action on mucosal oxygen consumption. (support: NIH #HL- 15231)

• SQUID MAGNETOMETER DIAGNOSIS OF MESENTERIC VEIN THROMBOSIS SH Alins, M Son'ell, S Slater, D Staton, JP Wikswo Jr., and WO Richards. Depts of Surgery and Pathology, VA Medical Center and Dept of Surgery, Living State Physics Group, and Physics, Vanderhilt University, Nashville, TN. Supported by Department of Veterans Affairs, NIH 1RO124751

Background: Mesenteric Venous Thrombosis (MVT) carries a poor prognosis because of the lack of noninvasive diagnostic tests to detect compromised bowel. Previous studies have demonstrated that SQUID magnetometers can nonlnvasively measure electrical activity of small bowel and identify arterial ischemia prior to pathologic changes. This study was conducted to test the possibility of utilizing the Superconducting QUantum Interference Device (SQUID) for early noninvasive detection of MVT.

Methods: Six New Zealand rabbits were anesthetized using IM ketamine (30mg/kg) and acepromazine (0.75mg/kg). A laparotomy was performed and silver electrodes were attached to a loop of ileum. The laparotomy incision was closed and simultaneous recordings of small intestinal electrical activity was obtained by the SQUID through the abdominal wall and with the serosal electrodes. Baseline recordings were taken for 15 minutes following which the superior mesenteric vein was ligated, and 1000 units of thrombin injected distally. Recordings were resumed 10-15 minutes later after closure of the abdomen and continued for 90 minutes.

Results: The dominant BER frequency was determined for 5 minute segments of data using Fast Fourier Transformation (Acknowledge Software, Biopac, Goleta, CA). The Basic Electrical Rhythm (BER) of the small bowel dropped from 14.93-+0.36 (mean -4- SEM) to 8.58-+0.36 at thirty minutes and to 6.82-+0.722 al~er 90 minutes ofischemia (P v a l u ~ < 0.0001, paired T test). The correlation coefficient between the SQUID and electrical recordings was O.91. Histopathologic examination of the ileum after 90 minutes showed evidence of sloughing and necrosis of mucosa only.

Conclusion: These data suggest that the iscbemia caused by mesenteric venous thrombosis results in slowing of electrical activity which can be detected noninvasively using a SQUID. This technique has many advantages over other tests for intestestinal ischemia - it is noninvasive, fast, detects problems prior to pathologic change, is sensitive, and specific for the determination of smooth muscle viability. This may be helpful in early noninvasive diagnosis of MVT.