Proceedings of The Physiological Society
University of Oxford (2011) Proc Physiol Soc 23, C35
17β-Estradiol increases amiloride-sensitive Na+ current and PKCδ activity in renal collecting duct M1 cells
M. Y. Yusef Robles1, B. J. Harvey1, W. Thomas1
1. Department of Molecular Medicine, Royal College of Surgeons in Ireland, Education and Research Centre, Beaumont Hospital, Dublin 9, Ireland.
17β-estradiol (E2), the most abundant oestrogen occurring naturally in the female body, regulates chloride secretion in distal colon in a gender-specific manner via inhibition of K+ recycling through basolateral K+ channels (1). The anti-secretory effect of E2 could provide an explanation for the fluid retention observed in females during periods of high circulating plasma oestrogen. The effect of E2 on ion transport in other organs contributing to electrolyte and fluid homeostasis in females has not been characterised. For example, oestrogen modulation of Na+ re-absorption in the renal cortical collecting duct could contribute to fluid conservation observed during periods of high circulating plasma oestrogen. The aim of this study was to determine whether E2 had an effect on Na+ re-absorption in the renal cortical collecting duct using the well-characterised M1-CCD cell line and to reveal the molecular mechanisms underlying effects of E2 on the amiloride-sensitive epithelial Na+ channel ENaC. We found that treatment with E2 (25nM) for 15 min increased the amplitude of the amiloride-sensitive current in M1-CCD cells grown as polarized monolayers in Ussing chambers (Control 1.5±0.4 μA/cm2, E2 2.8±0.4 μA/cm2; n=6). Experiments in amphotericin-B perforated epithelia using ouabain demonstrated that this response was not attributable to changes in Na+/K+ ATPase pump current (Control 2.9±0.2 μA/cm2, E2 3.3±0.8 μA/cm2; n=4). Previous work from our laboratory has shown that the anti-secretory effect of E2 in distal colon is PKCδ-dependent (1). To test the role of PKCδ in the E2 activation of ENaC we measured the phosphorylation state of the PKCδ autophosphorylation residue Ser643 following E2 treatment. E2 stimulated PKCδ activity in a concentration-dependent manner and within a similar time frame as the E2 stimulatory effect on the amiloride-sensitive current. We found that PKCδ autophosphorylation was also stimulated by the ERα selective agonist PPT (1nM) but not by the ERβ agonist DPN (5nM). The stimulatory effect of E2 on PKCδ autophosphorylation was blocked by pre-treatment with the matrix metalloproteinase (MMP) inhibitor GM6001 (1μM) and by the EGFR inhibitor AG1478 (10μM). Moreover, E2 was unable to increase the amplitude of the amiloride-sensitive current when M1-CCD cells were pre-treated with the PKCδ inhibitor rottlerin (5μM) (E2 2.4±0.4 μA/cm2, E2 + rottlerin 0.6±0.3 μA/cm2; n=6). In conclusion, E2 treatment rapidly and concurrently increased the amplitude of the ENaC current and stimulated PKCδ activity in M1-CCD cells. These rapid responses to oestrogen were transduced via ERα and required the MMP-mediated transactivation of EGFR.
Where applicable, experiments conform with Society ethical requirements