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.
Khurana
S
S
Department of Medicine, Gastrointestinal Division, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
Nath
S K
SK
Levine
S A
SA
Bowser
J M
JM
Tse
C M
CM
Cohen
M E
ME
Donowitz
M
M
D000730:Androstadienes / Q000494:pharmacology; D000818:Animals; D002460:Cell Line; D016764:Cell Polarity; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000502:physiology*; D066246:ErbB Receptors / Q000502:physiology*; D019833:Genistein; D005947:Glucose / Q000378:metabolism; D006801:Humans; D007408:Intestinal Absorption; D007413:Intestinal Mucosa / Q000378:metabolism*; D007529:Isoflavones / Q000494:pharmacology; D008871:Microvilli / Q000502:physiology*; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor) / Q000037:antagonists & inhibitors / Q000378:metabolism*; D019000:Phosphotyrosine / Q000378:metabolism; D011505:Protein-Tyrosine Kinases / Q000378:metabolism; D011817:Rabbits; D015398:Signal Transduction; D012965:Sodium Chloride / Q000378:metabolism*; D017923:Sodium-Hydrogen Exchangers / Q000378:metabolism*; D014162:Transfection; D000077191:Wortmannin
D000730:Androstadienes; D004791:Enzyme Inhibitors; D007529:Isoflavones; D017923:Sodium-Hydrogen Exchangers; D019000:Phosphotyrosine; D012965:Sodium Chloride; D004815:Epidermal Growth Factor; D019833:Genistein; D019869:Phosphatidylinositol 3-Kinases; D017853:Phosphotransferases (Alcohol Group Acceptor); D066246:ErbB Receptors; D011505:Protein-Tyrosine Kinases; D005947:Glucose; D000077191:Wortmannin