Duodenal mucosal defense was assessed by measuring blood flow and epithelial intracellular pH (pH_i) of rat proximal duodenum in vivo. Fluorescence microscopy was used to measure epithelial pH_i using the trapped, pH_i-indicating dye 2',7'-bis(2-carboxyethyl)-5(6)-carboxyfluorescein-AM. Blood flow was measured with laser-Doppler flowmetry. The mucosa was briefly superfused with NH₄Cl, pH 2.2 buffer, the potent Na⁺/H⁺ exchange inhibitor 5-(N,N-dimethyl)-amiloride (DMA), or the anion exchange and Na⁺-HCO₃ cotransport inhibitor DIDS. Cryostat sections localized dye fluorescence to the villus tip. Steady-state pH_i was 7.02 ± 0.01, which remained stable for 60 min. Interventions that load the cells with protons without affecting superfusate pH (NH₄Cl prepulse, nigericin with low superfusate K⁺ concentration, DMA, and DIDS) all decreased pH_i, supporting our contention that the dye was faithfully measuring pH_i. An acid pulse decreased pH_i, followed by a DIDS-inhibitable overshoot over baseline. Intracellular acidification increased duodenal blood flow independent of superfusate pH, which was inhibited by DMA, but not by DIDS. We conclude that we have established a novel in vivo microscopy system enabling simultaneous measurements of pH_i and blood flow of duodenal epithelium. Na⁺/H⁺ exchange and Na⁺-HCO₃ cotransport regulate baseline duodenal epithelial pH_i. Intracellular acidification enhances duodenal blood flow by a unique, amiloride-inhibitable, superfusate pH-independent mechanism.

epithelial cells; in vivo microscopy; fluorescent dyes; amiloride analogs; laser-Doppler flowmetry

This article has been cited by other articles:

				M. Mizumori, Y. Choi, P. H. Guth, E. Engel, J. D. Kaunitz, and Y. Akiba CFTR inhibition augments NHE3 activity during luminal high CO2 exposure in rat duodenal mucosa Am J Physiol Gastrointest Liver Physiol, June 1, 2008; 294(6): G1318 - G1327. [Abstract] [Full Text] [PDF]

				Y. Akiba, M. Mizumori, P. H. Guth, E. Engel, and J. D. Kaunitz Duodenal brush border intestinal alkaline phosphatase activity affects bicarbonate secretion in rats Am J Physiol Gastrointest Liver Physiol, December 1, 2007; 293(6): G1223 - G1233. [Abstract] [Full Text] [PDF]

				O. Furukawa, M. Hirokawa, L. Zhang, T. Takeuchi, L. C. Bi, P. H. Guth, E. Engel, Y. Akiba, and J. D. Kaunitz Mechanism of augmented duodenal HCO3- secretion after elevation of luminal CO2 Am J Physiol Gastrointest Liver Physiol, March 1, 2005; 288(3): G557 - G563. [Abstract] [Full Text] [PDF]

				O. Furukawa, L. C. Bi, P. H. Guth, E. Engel, M. Hirokawa, and J. D. Kaunitz NHE3 inhibition activates duodenal bicarbonate secretion in the rat Am J Physiol Gastrointest Liver Physiol, January 1, 2004; 286(1): G102 - G109. [Abstract] [Full Text] [PDF]

				S Tanaka, S Chu, M Hirokawa, M H Montrose, and J D Kaunitz Direct measurement of acid permeation into rat oesophagus Gut, June 1, 2003; 52(6): 775 - 783. [Abstract] [Full Text]

				Y. Akiba, O. Furukawa, P. H. Guth, E. Engel, I. Nastaskin, and J. D. Kaunitz Acute adaptive cellular base uptake in rat duodenal epithelium Am J Physiol Gastrointest Liver Physiol, June 1, 2001; 280(6): G1083 - G1092. [Abstract] [Full Text] [PDF]

				F. Schweda, H. Seebauer, B. K. Kramer, and A. Kurtz Functional role of sodium-calcium exchange in the regulation of renal vascular resistance Am J Physiol Renal Physiol, January 1, 2001; 280(1): F155 - F161. [Abstract] [Full Text] [PDF]

				Y. Akiba, P. H. Guth, E. Engel, I. Nastaskin, and J. D. Kaunitz Dynamic regulation of mucus gel thickness in rat duodenum Am J Physiol Gastrointest Liver Physiol, August 1, 2000; 279(2): G437 - G447. [Abstract] [Full Text] [PDF]

				Y. Akiba, P. H. Guth, E. Engel, I. Nastaskin, and J. D. Kaunitz Acid-sensing pathways of rat duodenum Am J Physiol Gastrointest Liver Physiol, August 1, 1999; 277(2): G268 - G274. [Abstract] [Full Text] [PDF]