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P Iserovich, J Fischbarg, V Bildin, Z Wang, PS Reinach; EGF Receptor Linked Signaling and Water Secretion in SV40-Immortalized Rabbit Lacrimal Gland Cells . Invest. Ophthalmol. Vis. Sci. 2002;43(13):3272.
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Purpose: We recently reported that in SV40-immortalized rabbit lacrimal gland cells (RLGC) a physiological concentration of epidermal growth factor (EGF) (i.e. 20 ng/ml) stimulates water secretion, produces cell volume increase, accelerates regulatory volume decrease during a hypotonic challenge. To explore the mechanisms for these phenomena, we analyzed the relationship between EGF-induced changes in water secretion and EGF receptor linked signaling. Methods: RLGC initially grown to confluence in flasks coated with Matrigel were seeded at a density of ∼105 cells /cm2 on Transwell® Costar permeable supports and grown in DMEM medium containing high-glucose and 6% FBS, EGF 5 ng/ml and Insulin-Transferin-Sodium Selenite Media Supplement. Experiments were done after 4 days of culture. Cells were preincubated at 37 C for 1 h in the medium containing high glucose DMEM/25 mM HEPES and EGF in concentrations 2, 20, or 40 ng/ml was added. Levels of Erk1/2 activation were determined by Western blotting/ECL assay. Fluid transport activity was directly measured with an automatic volume clamp system. Results: Stimulation of RLGC fluid secretion occurred in less than 15 min after EGF application and it was sustained for 1 h. The EGF dose dependence response was biphasic with maximal increase for 20 ng/ml EGF. On the other hand, EGF-induced Erk1/2 activation in first five min after its application was observed. At EGF concentrations of 2, 20, or 40 ng/ml, the levels of increase in Erk1/2 activity were 2.7, 3.25 and 4.5-fold, respectively. After 1 h of EGF exposure those levels were moderately declined to 1.5, 3.25, and 3.25, respectively. Conclusion: In RGLC EGF-induced Erk1/2 activation and stimulation of fluid secretion have a similar pattern. In addition, the time course of these processes suggested that their activation are dependent on cytosolic events (i.e. protein phosphorylation cascades) rather than on nuclear ones. These associations suggest that EGF-induced stimulation of fluid transport is related with its stimulation of Erk1/2 activity.
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