Khurana S, Nath S K, Levine S A, Bowser J M, Tse C M, Cohen M E, Donowitz M
Department of Medicine, Gastrointestinal Division, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205, USA.
J Biol Chem. 1996 Apr 26;271(17):9919-27. doi: 10.1074/jbc.271.17.9919.
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.
在终末分化的回肠绒毛钠吸收细胞中，表皮生长因子（EGF）通过基底外侧膜受体刺激氯化钠吸收及其组成部分刷状缘钠/氢交换体，正如我们在此所证实的，还有一种刷状缘酪氨酸激酶。在本研究中，我们表明刷状缘磷脂酰肌醇3激酶（PI 3激酶）参与了EGF对氯化钠吸收和刷状缘钠/氢交换的刺激作用。在用尤斯灌流室-电压钳技术研究的兔回肠中，选择性PI 3激酶抑制剂渥曼青霉素可抑制EGF对活性氯化钠吸收的刺激。PI 3激酶是一种主要存在于胞质中的酶，在EGF处理后特异性地转位至回肠吸收细胞的刷状缘。这种转位在EGF处理后1分钟就开始出现，并在刷状缘持续增加至少15分钟。EGF还会使刷状缘PI 3激酶活性迅速（1分钟）且大幅（4 - 5倍）增加。在稳定转染了钠/氢交换体NHE3（Caco - 2/NHE3细胞）的人结肠癌细胞系Caco - 2中进行的研究进一步证实了PI 3激酶活性参与肠道钠吸收。使用对pH敏感的荧光染料2'7'-双（羧乙基）5 -（6）-羧基荧光素测量刷状缘钠/氢交换活性。添加到Caco - 2/NHE3细胞基底外侧而非顶端表面的EGF增加了刷状缘钠/氢交换活性。在用渥曼青霉素预处理的细胞中，EGF诱导的刷状缘钠/氢交换活性增加被完全消除。EGF处理导致回肠刷状缘膜和Caco - 2/NHE3细胞中PI 3激酶的酪氨酸磷酸化增加，这表明PI 3激酶上游的酪氨酸激酶参与了EGF对钠吸收的作用。总之，本研究在两个不同的肠道模型，即回肠和人结肠癌细胞系中提供了证据，表明PI 3激酶是EGF刺激肠道钠吸收过程中的一个中间环节。
