Abstracts
1999 Digestive Disease Week

# 2163 HUMAN DUODENAL VESICLES: NEW ADVANCES IN NON INVASIVE INTRACELLULAR PH MEASUREMENTS
Michael J Sessler, Michael Weinlich, Christina Baumstark, Engin Usta, Horst D Becker, Univ of Tuebingen, Tuebingen Germany

To enlighten the pathogen mechanisms of intestinal diseases in vitro we badly need a suitable cell culture model. Aim of our study was to cultivate a new three-dimensional and polar cell culture from human duodenum biopsies (so called vesicles) and to develop a perfusion chamber for intracellular measurement. Furthermore we investigated the intracellular regulation mechanisms exemplary by measuring non invasive the pHi- and Ca2+i-regulation. Methods: Intact vesicles were mounted in a special micro perfusion chamber. The vesicles were physically fixed with a nylon net. The pHi was measured by ratio imaging of BCECF fluorescence and Ca2+i with Fura-2. Sodium free solution, amiloride and H2DIDS were used to specify proton exchanger and Verapamil to specify Ca-channels. Results: Viability throughout the experiment was always greater than 90%. Resting pHi of the vesicles was 7.31. After intracellular acidification sodium free buffer led to a complete and Amiloride exposure led to a partial inhibition of pHi recovery. The combination of H2DIDS and Amiloride led to a complete inhibition of pHi recovery. Intracellular Ca2+ concentration was about 190mM; Ca2+ backregulation after Ca2+ free perfusion was nearly complete inhibited with Verapamil. Conclusion: Because human duodenal cells in form of tissue like vesicles consist of highly polarized cells with normal cytoarchitecture comparable to those known from native tissue, they are adequate for non invasive pHi and Ca2+i measurements using a special micro perfusion chamber. The vesicles exhibited a Na+/H+ exchanger and a Na+/HCO-3 exchanger to compensate acidification of the cells. They also exhibit Ca2+ channel. This experimental setup represent a suitable model for further investigations of regulatory mechanisms of human duodenal mucosa.

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