Regulation of intracellular pH and blood flow in rat duodenal epithelium in vivo

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⁺-${HCO}_3^{-}$ 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⁺-${HCO}_3^{-}$ cotransport regulate baseline duodenal epithelial pH_i. Intracellular acidification enhances duodenal blood flow by a unique, amiloride-inhibitable, superfusate pH-independent mechanism.