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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.

Controversy exists as to the role of oxygen-derived free radicals in tissue injury and the no-reflow phenomenon in reperfusion injury after ischemia. In this study using an experimental rat model, left hepatic lobar ischemia followed by reperfusion resulted in an increase of serum glutamic pyruvic transaminase at 30 min with concomitant histological evidence of hepatocellular necrosis at 24 hr. In the in vivo liver microcirculation, reperfusion after ischemia resulted in an initial transient return of blood flow, but stasis of blood flow later developed in the liver sinusoids. Thus a no-reflow phenomenon in the microcirculation was demonstrated. Intravenous administration of a long-acting form of superoxide dismutase (half-life 6 hr, dose 4 or 8 mg/kg) significantly decreased the hepatocellular necrosis and reduced the microcirculatory stasis in the liver sinusoids. These studies established the important contribution of the no-reflow phenomenon in ischemia-reperfusion injury to the liver and the participation of superoxide anions in mediating the no-reflow phenomenon.