Cell signaling pathways mediating epidermal growth factor stimulation of Na:K:2Cl cotransport activity in rabbit corneal epithelial cells.
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 (1 to 5 ng/ml) transiently increased [Na+]i from 10 mm 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 microm RG-13022) and cPLA2 activity (10 microm AACOCF3) obviated EGF-induced increases in [Na+]i. In contrast, PGE2 (10 microg/ml) and cAMP (2 mm) increased [Na+]i by 25 mm. Inhibition of NKCC activity through exposure to either Cl-free Ringers or 300 microm 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 microm H-89); 2) Erk1/2 (15 microm PD98059) or 3) p38 (15 microm SB203580) activity. Stimulation protein kinase C activity (0.1 microm PMA) transiently increased [Na+]i followed by a decline towards its baseline value. EGF-induced increases in [Na+]i were unaltered by inhibition of K+ conductance (100 microm 4-AP). Taken together, EGF stimulates Erk1/2; p38 and cPLA2 activity. Their stimulation increases PGE2 and cAMP levels resulting in PKA and NKCC activation.
Yang
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College of Optometry, Department of Biological Sciences, State University of New York, 33 W. 42nd Street, New York, NY 10036, USA.
Wang
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Z
Miyamoto
Y
Y
Reinach
P S
PS
D000818:Animals; D001572:Benzofurans / Q000378:metabolism; D002455:Cell Division / Q000187:drug effects; D002461:Cell Line, Transformed; D002478:Cells, Cultured; D000242:Cyclic AMP / Q000378:metabolism; D017868:Cyclic AMP-Dependent Protein Kinases / Q000037:antagonists & inhibitors / Q000378:metabolism; D015232:Dinoprostone / Q000494:pharmacology; D004305:Dose-Response Relationship, Drug; D004791:Enzyme Inhibitors / Q000494:pharmacology; D004815:Epidermal Growth Factor / Q000037:antagonists & inhibitors / Q000494:pharmacology*; D019573:Epithelium, Corneal / Q000378:metabolism*; D066246:ErbB Receptors / Q000378:metabolism; D004988:Ethers, Cyclic / Q000378:metabolism; D005453:Fluorescence; D017136:Ion Transport; D007700:Kinetics; D020928:Mitogen-Activated Protein Kinases / Q000378:metabolism; D011493:Protein Kinase C / Q000037:antagonists & inhibitors / Q000502:physiology; D011817:Rabbits; D015398:Signal Transduction; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters / Q000378:metabolism / Q000502:physiology*; D064506:Solute Carrier Family 12, Member 2; D013755:Tetradecanoylphorbol Acetate / Q000494:pharmacology; D013997:Time Factors; D048051:p38 Mitogen-Activated Protein Kinases
D001572:Benzofurans; D004791:Enzyme Inhibitors; D004988:Ethers, Cyclic; D049994:Sodium Potassium Chloride Symporter Inhibitors; D028021:Sodium-Potassium-Chloride Symporters; D064506:Solute Carrier Family 12, Member 2; C061665:sodium-binding benzofuran isophthalate; D004815:Epidermal Growth Factor; D000242:Cyclic AMP; D066246:ErbB Receptors; D017868:Cyclic AMP-Dependent Protein Kinases; D011493:Protein Kinase C; D020928:Mitogen-Activated Protein Kinases; D048051:p38 Mitogen-Activated Protein Kinases; D015232:Dinoprostone; D013755:Tetradecanoylphorbol Acetate