April 1995 intestinal Disorders A281

• CALCIUM AS A MARKER FOR SMALL BOWEL CELL INJURY. U. Dan'. R. Abraham, J. Geibel. Depts. of Surgery and C&M Physiology. Yale Univ. Seh. of Med. New Haven, CT 06520

Investigators involved in research dealing with prevention of cell injury often face the hardship of the lack of a reproducible cell model. The goal of our study was to develop a reliable assay for determining reversible and irreversible cell damage for live analysis. To address this question we chose the rabbit small bowel model. Numerous studies have documented the small bowel to be particularly sensitive to isehemic injury, never-the- less, the mechanism of this injury as well as methods for its prevention and treatment remain poorly understood. Ileal villi from New Zealand White rabbits were isolated by microdissection at 4°C in standard HEPES solution (pH adjusted to 7.4 at 4°C). Isolated villi are fixed to cover slips pre-treated with Cell-Tak and mounted onto the microscope perfusion chamber. We chose eonfocal microscope since it allows continuos optical sections to be taken during biochemical manipulation of live tissue and simultaneously generating state-of-the-art image analysis. Tissue response to i schemia was assessed by monitoring: i) apoptosis (loss of morphological integrity) and ii) changes in intracellular calcium concentration using Calcium Orange, a calcium sensitive fluorescent indicator. Live micro-dissected villi were exposed to a superfusion of anoxic (0% 021100% N2) solution at 37°C to mimic ischemia. Morphological assessment of the tissue prior to exposure to the anoxic medium was performed to document viabi l i ty . Simul taneous measurements ofintracellular calcium and cell morphometry demonstrated that exposure to HEPES buffer bubbled with 100% nitrogen caused a rapid uncontrolled rise in intracellular calcium and the development of apoptotic lesions along the apical membrane of the villus. Both of these effects were evident within 2-3 minutes. The blebs are an attempt by the cell to volume regulate, and to compensate for the cytosolic swelling that occurs secondary to the uncoupling of the Na-K ATPase pump. The elevation in intracellular calcium levels closely parallels this event. We conclude that measurements of intracellular calcium in isolated villi simulate in-vivo conditions and present an effective alternative to whole organ studies.

O INHIBITION OF Na+-H + EXCHANGE PREVENTS ANOXIC DAMAGE IN ISOLATED SMALL BOWEL VILLI. U.M. Darr, R. Abraham, J. Geibel. Depts. of Surgery and C&M Physiology. Yale Univ. Seh. of Med. New Haven, CT 06520.

The small bowel mucosa is particularly sensitive to ischemia and reperfusion injury. The goal of our study was to enhance tissue resistance to ischemic injury and to develop useful pharmacotherapeutic tools for these purposes. Ileal villi from New Zealand White rabbits were isolated by microdissection. Using laser confocal microscopy isolated villi were examined for two parameters of cell injury: (i) apoptosis (loss of morphological integrity) and, (ii) rise in intracellular calcium concentration [Ca2*]i.Ischemic injury simulated by treatment of villi with nitrogenated HEPES (0% 02/100% N2) solution, led to an uncontrolled rise in [Ca2÷]~ (monitored by Calcium Orange 101.tM, a fluorescent marker for Ca2÷), resulting in apoptosis and irreversible cell damage. This phenomena was delayed significantly in the presence of butyrate (20mM), an already established therapeutic agent in the colon. To corroborate the hypothesis that butyrate exerted its protective effect by converting to butyric acid inside the cell, a second set of experiments was performed with amiloride ( lmM) a Na÷/I-I ÷ exchange inhibitor. Intracellular pH measurements done with SNARF (a pH sensitive fluorescent dye) 101.tM, show that amiloride locks the cell in a steady state of relatively low pH. Pre-treatment with amiloride when compared with controls delayed anoxic damage significantly (10 min. vs. 2-3 rain.) p<0.05; i.e., amiloride delayed the appearance of apical membrane blebs and prevented the rise in [Ca2÷]~. In concordance with our hypothesis that intraeellular acidification is protective, tissue when subjected to an alkaline medium (HEPES titrated to pH 8.0 with N-methyl-D-Glucamine) blebbed instantly along with a rapid rise in [Ca2+]~. We conclude that: (i) butyrate and amiloride increase tissue resistance to ischemia by inducing intracellular acidification, and, (ii) amiloride is an effective reversible pharmacologic agent that causes a five-fold increase in the duration of cell viability under anoxic conditions.

O EGF, a physiological regulator of barrier function and actin in epithelial cells.

Shehan B. Dissanayake, Robert Bacallao* and Eugene B. Chang Dept of Physiology and Pharmacology, Dept. of Medicine, The University of Chicago, Chicago Illinois, and *Dept. of Nephrology, Northwestern University, Chicago, Illinois.

Introduction: Previous studies demonstrate that actin is important in maintaining epithelial barrier function. Several pathogens and toxins such as C. difficile and ZOT induce changes in barrier function that are coincident with gross alterations of the actin cytoskeleton. Although the kinetics and the mechanisms for pathogen induced changes in barrier function have been characterized, little is known about the potential of physiological agents such as EGF to regulate barrier function and actin in epithelial cells. Preliminary experiments conducted in our laboratory on CaCo-2 cells suggests that EGF induces a transient decrease in barrier function and a coincident reorganization of actin. To better define the physiological significance of this effect, we have chosen to study EGF induced changes in barrier function and actin in a nontransformed high resistance MDCK cell line.

Methods: Barrier function of MDCK's were assessed on monolayers grown on type collagen I coated transwells 5 days post plating. Barrier function was assayed on monolayers treated with EGF on the apical, basal and apical+basal surfaces. Changes in barrier function in control and EGF treated monolayers were measured using transepithelial resistance and confirmed by following 3H- mannitol flux. The reorganization of actin in control and EGF treated monolayers was studied by 1) fixing and staining cells with FITC-phalloidin and 2) visualizing by laser scanning confocal microscopy. Two dimensional and three dimensional analysis of monolayers was performed using a Silicon Graphics workstation and Noran Instruments InterVision software.

Results: The addition of EGF to either the basal or apical+basal sides caused a transient 15% decrease in transepithelial resistance relative to control. Monolayers treated with EGF apically displayed a diminished response with similar kinetics. The EGF effect of MDCK monolayers is dose dependent with an EDs0 of 10 ng/ml. Two and three dimensional visualization of actin in EGF treated monolayers indicated a reorganization of basal and cortical actin. Actin at the perijucntional ring and microvillus membrane remained relatively unchanged.

Conclusion: EGF caused a dose dependent decrease in barrier function of MDCK's which is coincident with changes in actin cytoskeleton. Moreover, this study suggests a potential means by which barrier function in the kidney and intestine may be dynamically regulated by physiological agents such as EGF.

E F F E C T OF I S C H E M I A ON I N T E S T I N A L P E R M E A B I L I T Y OF L I P O P O L Y S A C C H A R I D E S J. Drewe , U. Behrens and G. Fricker, Depts. of Anesthesia and Research, Univ. Hospital, CH-4031 Basel, Switzerland and Albert-Ludwigs-Univ. , D-79104 Freiburg, Germany

I N T R O D U C T I O N : Enteral absorption of luminal lipopoly- saccharides (LPS) may play an important role in the release of various cytokines and the devolpment of multiorgan system failure after temporal gut iscbemia. Therefore, mechanisms of transepithelial transport of LPS were investigated using different in vitro and in situ methods. METHODS: In vitro transepithelial permeation was studied using bmsh- border membrane vesicles (BBMV) from the jejunal mucose of rabbits and fluorescence (FITC) labelled LPS derived from Salmonella typhimurium. Uptake of FITC-LPS into BBMV was investigated by rapid filtration technique and fluorescence detection. Adhesion of FITC-LPS was assessed by determination of uptake at different osmolarity of incubation medium and extrapolation to infinite osmolarity. In addition, transepithelial transport through Caco-2 cell monolayer was investigated in the presence and absence of oxigenization. Visualization of absorption pathways was assessed by fluorescence microscopy after in situ administration of FITC-LPS into isolated jejunal loops of rats with and without prior gut ischemia (occlusion of A. mesenterica) over 1 hour. RESULTS: FITC-LPS is taken up into BBMV in a dose-dependent manner. Uptake was not pH and sodium dependent and could not be inhibited by addition of unlabeUed LPS. 74.5 % of vesicle associated fluorescence were due to adhesion of FITC-LPS to the vesicle membranes, 25.5 % due to uptake. Transporth across Caco-2 celt monolayers was dose-dependent and linear over 120 rain. Transport increased under ischemic conditions with apparent increase in paracellular permeability. CONCLUSION: The results of the present study indicate, that LPS is taken up by intestinal mucosa predominately by passive transcellular diffusion through enterocytes. Under ischemic conditions, the permeability of LPS is increased mainly by an enhanced paracellular permeability.