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+-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 pHi and blood flow of duodenal epithelium. Na+/H+ exchange and Na+-HCO3- cotransport regulate baseline duodenal epithelial pHi. Intracellular acidification enhances duodenal blood flow by a unique, amiloride-inhibitable, superfusate pH-independent mechanism.