EGF regulates amiloride-sensitive Na absorption across glandular endometrium

The objective of this study was to investigate the effects of epidermal growth factor (EGF) on insulin-stimulated Na absorption in cultured endometrial epithelial cells. Glandular epithlelial cells, isolated from porcine uterus, were cultured on permeable filter supports (4.5 cm²) in DMEM supplemented with 10 % FBS and 10 μg/ml insulin (standard media) for one week and then replaced with serum-free media (SFM) or SFM supplemented with insulin and/or EGF. Addition of benzamll (5 μM) to the apical solution inhibited 24 ± 6 % of the basal lsc in standard media and 85 ± 2 % in SFM with 10 μg/ml insulin. The IC₅₀ values for benzamil and amiloride were 24 nM and 240 nM respectively. Addition of 5 μg/ml insulin to the basolateral membrane of cell monolayers grown in SFM increased amiloride-sensitive Na absorption but had no effect on cells grown in SFM with EGF for 4 days Addition of EGF (10 ng/ml) to the basolateral solution, not apical solution, inhibited both basal and insulin-stimulated Na absorption after 30-45 min. In addition, incubation with EGF for 4 days inhibited insulin-stimulated Na absorption in a dose-dependent fashion with an IC₅₀ value of 2 ng/ml. Experiments using amphotericin B-permeabilized monolayers demonstrated that the reversal potential of the benzamil-sensitive current in cells grown in SFM with insulin was 65 ± 4 mV and the Na to K selectivity ratio was calculated to be 11.6:1. Addition of EGF in this media did not significantly change the reversal potential however the benzamil-sensitive Na conductance was reduced compared to cells grown in the absence of EGF These results indicate that EGF regulates the Na conductance properties to the apical membrane and reduces the effects of insulin on Na absorption.