No abstract

We hypothesized that duodenal HCO(3)(-) secretion alkalinizes the microclimate surrounding intestinal alkaline phosphatase (IAP), increasing its activity. We measured AP activity in rat duodenum in situ in frozen sections with the fluorogenic substrate ELF-97 phosphate and measured duodenal HCO(3)(-) secretion with a pH-stat in perfused duodenal loops. We examined the effects of the IAP inhibitors L-cysteine or L-phenylalanine (0.1-10 mM) or the tissue nonspecific AP inhibitor levamisole (0.1-10 mM) on AP activity in vitro and on acid-induced duodenal HCO(3)(-) secretion in vivo. AP activity was the highest in the duodenal brush border, decreasing longitudinally to the large intestine with no activity in stomach. Villous surface AP activity measured in vivo was enhanced by PGE(2) intravenously and inhibited by luminal L-cysteine. Furthermore, incubation with a pH 2.2 solution reduced AP activity in vivo, whereas pretreatment with the cystic fibrosis transmembrane regulator (CFTR) inhibitor CFTR(inh)-172 abolished AP activity at pH 2.2. L-Cysteine and L-phenylalanine enhanced acid-augmented duodenal HCO(3)(-) secretion. The nonselective P2 receptor antagonist suramin (1 mM) reduced acid-induced HCO(3)(-) secretion. Moreover, L-cysteine or the competitive AP inhibitor glycerol phosphate (10 mM) increased HCO(3)(-) secretion, inhibited by suramin. In conclusion, enhancement of the duodenal HCO(3)(-) secretory rate increased AP activity, whereas inhibition of AP activity increased the HCO(3)(-) secretory rate. These data support our hypothesis that HCO(3)(-) secretion increases AP activity by increasing local pH at its catalytic site and that AP hydrolyzes endogenous luminal phosphates, presumably ATP, which increases HCO(3)(-) secretion via activation of P2 receptors.

Regulation of localized extracellular pH (pH o ) maintains normal organ function. An alkaline microclimate overlying the duodenal enterocyte brush border protects the mucosa from luminal acid. We hypothesized that intestinal alkaline phosphatase (IAP) regulates pH o due to pH-sensitive ATP hydrolysis as part of an ecto-purinergic pH regulatory system, comprised of cell-surface P2Y receptors and ATP-stimulated duodenal bicarbonate secretion (DBS). To test this hypothesis, we measured DBS in a perfused rat duodenal loop, examining the effect of the competitive alkaline phosphatase inhibitor glycerol phosphate (GP), the ecto-nucleoside triphosphate diphosphohydrolase inhibitor ARL67156, and exogenous nucleotides or P2 receptor agonists on DBS. Furthermore, we measured perfusate ATP concentration with a luciferin-luciferase bioassay. IAP inhibition increased DBS and luminal ATP output. Increased luminal ATP output was partially CFTR dependent, but was not due to cellular injury. Immunofluorescence localized the P2Y 1 receptor to the brush border membrane of duodenal villi. The P2Y 1 agonist 2-methylthio-ADP increased DBS, whereas the P2Y 1 antagonist MRS2179 reduced ATP-or GP-induced DBS. Acid perfusion augmented DBS and ATP release, further enhanced by the IAP inhibitor l-cysteine, and reduced by the exogenous ATPase apyrase. Furthermore, MRS2179 or the highly selective P2Y 1 antagonist MRS2500 co-perfused with acid induced epithelial injury, suggesting that IAP/ATP/P2Y signalling protects the mucosa from acid injury. Increased DBS augments IAP activity presumably by raising pH o , increasing the rate of ATP degradation, decreasing ATP-mediated DBS, forming a negative feedback loop. The duodenal epithelial brush border IAP-P2Y-HCO 3 − surface microclimate pH regulatory system effectively protects the mucosa from acid injury.

Presence of taste receptor families in the gastrointestinal mucosa suggests a physiological basis for local and early detection of a meal. We hypothesized that luminal L-glutamate, which is the primary nutrient conferring fundamental umami or proteinaceous taste, influences mucosal defense mechanisms in rat duodenum. We perfused the duodenal mucosa of anesthetized rats with L-glutamate (0.1-10 mM). Intracellular pH (pH(i)) of the epithelial cells, blood flow, and mucus gel thickness (MGT) were simultaneously and continuously measured in vivo. Some rats were pretreated with indomethacin or capsaicin. Duodenal bicarbonate secretion (DBS) was measured with flow-through pH and CO(2) electrodes. We tested the effects of agonists or antagonists for metabotropic glutamate receptor (mGluR) 1 or 4 or calcium-sensing receptor (CaSR) on defense factors. Luminal L-glutamate dose dependently increased pH(i) and MGT but had no effect on blood flow in the duodenum. L-glutamate (10 mM)-induced cellular alkalinization and mucus secretion were inhibited by pretreatment with indomethacin or capsaicin. L-glutamate effects on pH(i) and MGT were mimicked by mGluR4 agonists and inhibited by an mGluR4 antagonist. CaSR agonists acidified cells with increased MGT and DBS, unlike L-glutamate. Perfusion of L-glutamate with inosinate (inosine 5'-monophosphate, 0.1 mM) enhanced DBS only in combination, suggesting synergistic activation of the L-glutamate receptor, typical of taste receptor type 1. L-leucine or L-aspartate had similar effects on DBS without any effect on pH(i) and MGT. Preperfusion of L-glutamate prevented acid-induced cellular injury, suggesting that L-glutamate protects the mucosa by enhancing mucosal defenses. Luminal L-glutamate may activate multiple receptors and afferent nerves and locally enhance mucosal defenses to prevent subsequent injury attributable to acid exposure in the duodenum.