Abstract
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.
Highlights
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
We reported previously that in terminally differentiated rabbit ileal villus cells, epidermal growth factor (EGF)1 increases NaCl absorption acting via basolateral membrane (BLM) receptors [1]
In terminally differentiated intestinal epithelial cells, EGF does not act as a mitogen but, as shown previously [1] and confirmed here, stimulates active NaCl absorption
Summary
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. PI 3-kinase, a largely cytosolic enzyme, translocates to the brush border of ileal absorptive cells following EGF treatment. 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. We reported previously that in terminally differentiated rabbit ileal villus cells, epidermal growth factor (EGF) increases NaCl absorption acting via basolateral membrane (BLM) receptors [1]. Whether PI 3-kinase is involved in regulation of transport processes in epithelial cells is unknown
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