Mineralocorticoid receptor antagonists do not block rapid ERK activation by aldosterone

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Abstract

Aldosterone can elicit rapid nongenomic effects both in vivo and in vitro, often mediated by signal transduction cascades. However, it is not understood how these rapid effects are initiated. In this study we show that aldosterone leads to rapid activation of mitogen activated protein kinases ERK1/2 in the cortical collecting duct cell line M-1. Inhibitors of transcription and translation could not block this activation, which suggests an extranuclear (nongenomic) mechanism. Although it is known that M-1 cells do not contain a transcriptionally functional MR, it is not known whether a closely related protein still could mediate the effects, or an unrelated nonclassic receptor. To test this hypothesis, the effects of four classical mineralocorticoid receptor antagonists were studied. None of the compounds could block the response to aldosterone. Altogether, the data suggest that rapid aldosterone effects in M-1 cells are initiated by a receptor different from the classical mineralocorticoid receptor.

Section snippets

Materials and methods

Materials. Aldosterone was purchased from Fluka (Buchs, Switzerland), ethanol was obtained from Merck (Darmstadt, Germany), and actinomycin, cycloheximide, canreonate, spironolactone, and RU 38486 (mifepristone) were from Sigma (Taufkirchen, Germany). MEK inhibitors PD98059 and U0126 were from Cell Signaling Technology (Frankfurt, Germany). Cell culture media and reagents were from Invitrogen (Karlsruhe, Germany). RU 26752 and RU 28318 were kindly provided by Hoechst (now Aventis,

ERK1/2 phosphorylation

By using a specific antibody which recognizes the active, phosphorylated forms of MAPK ERK1/2, we analyzed the activation of ERK1/2 in M-1 cells after treatment with aldosterone by Western immunoblot. To ensure equal loading of samples, an antibody that recognizes both the phosphorylated and non-phosphorylated forms of ERK1/2 was used.

Preliminary time course experiments revealed a maximum effect of aldosterone after 5 min which decreased after 10 min; no effect at all was seen in the time

Discussion

In this study we have demonstrated that the rapid activation of the ERK1/2 MAPK cascade in M-1 cells is not significantly inhibited by a structurally diverse set of antagonists at the classic mineralocorticoid receptor. The cell line used in these studies is derived from murine kidney cortical collecting duct, resembling principal cells which belong to the main targets for aldosterone [19]. In this cell line other rapid aldosterone effects (e.g., increases in intracellular Ca2+ levels, pH, and

Acknowledgements

This work was supported by a grant from the Deutsche Forschungsgemeinschaft (DFG We 1184/9-1). Parts of this work were done in the course of MD thesis preparation by Q.Z. The expert technical skills of Martina Seyfert are gratefully acknowledged.

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