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

Duodenal mucosal defense was assessed by measuring blood flow and epithelia l intracellular pH (pH(i)) of rat proximal duodenum in vivo. Fluorescence m icroscopy was used to measure epithelial pH(i) using the trapped, pH(i)-ind icating dye 2',7'-bis(2-carboxyethyl)-5(6)carboxyfluorescein-AM. Blood flow was measured with laser-Doppler flowmetry. The mucosa was briefly superfus ed with NH4Cl, pH 2.2 buffer, the potent Na+/H+ exchange inhibitor 5-(N,N-d imethyl)-amiloride (DMA), or the anion exchange and Na+-HCO3- cotransport i nhibitor DIDS. Cryostat sections localized dye fluorescence to the villus t ip. Steady-state pH(i) was 7.02 +/- 0.01, which remained stable for 60 min. Interventions that load the cells with protons without affecting superfusa te 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 D IDS-inhibitable overshoot over baseline. Intracellular acidification increa sed 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 vi vo microscopy system enabling simultaneous measurements of pHi and blood fl ow of duodenal epithelium. Na+/H+ exchange and Na+-HCO3- cotransport regula te baseline duodenal epithelial pHi. Intracellular acidification enhances d uodenal blood flow by a unique, amiloride-inhibitable, superfusate pH-indep endent mechanism.