Brush border phosphatidylinositol 3-kinase mediates epidermal growth factor stimulation of intestinal NaCl absorption and Na+/H+ exchange.

In terminally differentiated ileal villus Na+-absorptive cells, epidermal growth factor (EGF) stimulates NaCl absorption and its component brush border Na+/H+ exchanger, acting via basolateral membrane receptors, and as we confirm here, a brush border tyrosine kinase. In the present study we show that brush border phosphatidylinositol 3-kinase (PI 3-kinase) is involved in EGF stimulation of NaCl absorption and brush border Na+/H+ exchange. In rabbit ileum studied with the Ussing chamber-voltage clamp technique, EGF stimulation of active NaCl absorption is inhibited by the selective PI 3-kinase inhibitor wortmannin. PI 3-kinase, a largely cytosolic enzyme, translocates specifically to the brush border of ileal absorptive cells following EGF treatment. This translocation occurs as early as 1 min after EGF treatment and remains increased at the brush border for at least 15 min. EGF also causes a rapid (1 min) and large (4-5-fold) increase in brush border PI 3-kinase activity. Involvement of PI 3-kinase activity in intestinal Na+ absorption is established further by studies done in the human colon cancer cell line, Caco-2, stably transfected with the intestinal brush border isoform of the Na+/H+ exchanger, NHE3 (Caco-2/NHE3 cells). Brush border Na+/H+ exchange activity was measured using the pH-sensitive fluorescent dye 2'7'-bis(carboxyethyl)5-(6)-carboxyfluorescein. EGF added to the basolateral surface but not apical surface of Caco-2/NHE3 cells increased brush border Na+/H+ exchange activity. The EGF-induced increase in brush border Na+/H+ exchange activity was completely abolished in cells pretreated with wortmannin. EGF treatment caused increased tyrosine phosphorylation of PI 3-kinase in both ileal brush border membranes and Caco-2/NHE3 cells, suggesting that a tyrosine kinase upstream of the PI 3-kinase is involved in the EGF effects on Na+ absorption. In conclusion, the present study provides evidence in two separate intestinal models, the ileum and a human colon cancer cell line, that PI 3-kinase is an intermediate in EGF stimulation of intestinal Na+ absorption.