Cell signaling pathways mediating epidermal growth factor stimulation of Na:K:2Cl cotransport activity in rabbit corneal epithelial cells

被引:26
作者
Yang, H
Wang, Z
Miyamoto, Y
Reinach, PS
机构
[1] SUNY Coll Optometry, Dept Biol Sci, New York, NY 10036 USA
[2] Univ Tokyo, Grad Sch Frontier Sci, Dept Integrat Biosci, Bunkyo Ku, Tokyo 1138657, Japan
关键词
cornea; epidermal growth factor; Na : K : 2Cl; cAMP; proliferation; epithelium;
D O I
10.1007/s00232-001-0057-6
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
We characterized the signaling and ion transport pathways that mediate epidermal growth factor receptor physiological control in SV40-immortalized rabbit corneal epithelial cells (tRCEC). Our evaluation employed single-cell fluorescence imaging to measure the intracellular [Na+](i) in these cells loaded with the Na+ sensitive dye, SBFI. EGF (I to 5 ng/ml) transiently increased [Na+](i) from 10 nim to as much as 35 mm after 25 min, which was followed by a decline towards its control value. These increases waned at higher EGF concentrations up to 50 ng/ml. Both inhibition of EGF receptor-linked tyrosine kinase activity (50 mum RG-13022) and cPLA(2) activity (10 mum AACOCF(3)) obviated EGF-induced increases in [Na+](i). In contrast, PGE(2) (10 mug/ml) and cAMP (2 mM) increased [Na+](i) by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 mum furosemide in NaCl Ringers eliminated EGF-induced increases in [Na+](i). Similarly, EGF failed to increase [Na+](i) following inhibition of. 1) PKA activity (10 muM H-89); 2) Erk1/2 (15 mum PD98059) or 3) p38 (15 mum SB203580) activity. Stimulation protein kinase C activity (0.1 mum PMA) transiently increased [Na+], followed by a decline towards its baseline value. EGF-induced increases in [Na+](i) were unaltered by inhibition of K+ conductance (100 mum 4-AP). Taken together, EGF stimulates Erk1/2; P38 and cPLA(2) activity. Their stimulation increases PGE(2) and cAMP levels resulting in PKA and NKCC activation.
引用
收藏
页码:93 / 101
页数:9
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