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The aim of this study was the identification of the mechanism of oxidant-induced intestinal secretion.The action of H2O2 on ion transport across rat distal colon was evaluated in Ussing chambers. Changes in cytosolic Ca2+ concentration were measured using fura-2.H2O2 concentration-dependently induced an increase in short-circuit current (Isc), which was due to a stimulation of Cl− secretion. The effect of H2O2 was dependent on the presence of serosal Ca2+. It was inhibited after emptying of intracellular Ca2+ stores by cyclopiazonic acid or blockade of ryanodine receptors by ruthenium red, whereas a blocker of inositol 1,4,5-trisphosphate receptors was less effective. Fura 2-experiments confirmed an increase in the cytosolic Ca2+ concentration in the presence of H2O2. Measurements of Cl− currents across the apical membrane at basolaterally depolarized epithelia revealed the activation of a glibenclamide-sensitive, SITS-resistant Cl− conductance by the oxidant. The activation of this conductance was inhibited after blockade of protein kinases with staurosporine. When the apical membrane was permeabilized with nystatin, two sites of action of H2O2 were identified at the basolateral membrane. The oxidant stimulated a basolateral tetrapentylammonium-sensitive K+ conductance and increased the current generated by the Na+–K+ pump. Pretreatment of the tissues with H2O2 reduced the action of subsequently administered Ca2+-, cAMP- and cGMP-dependent secretagogues demonstrating a long-term downregulation after the initial secretory response evoked by the oxidant.H2O2 affects colonic anion secretion by action sites at both the apical, as well as the basolateral membrane.