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.