We hypothesized that duodenal HCO3− 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 HCO3− 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 HCO3− 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 PGE2 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 CFTRinh-172 abolished AP activity at pH 2.2. l-Cysteine and l-phenylalanine enhanced acid-augmented duodenal HCO3− secretion. The nonselective P2 receptor antagonist suramin (1 mM) reduced acid-induced HCO3− secretion. Moreover, l-cysteine or the competitive AP inhibitor glycerol phosphate (10 mM) increased HCO3− secretion, inhibited by suramin. In conclusion, enhancement of the duodenal HCO3− secretory rate increased AP activity, whereas inhibition of AP activity increased the HCO3− secretory rate. These data support our hypothesis that HCO3− secretion increases AP activity by increasing local pH at its catalytic site and that AP hydrolyzes endogenous luminal phosphates, presumably ATP, which increases HCO3− secretion via activation of P2 receptors.
Intestinal alkaline phosphatase regulates protective surface microclimate pH in rat duodenum
