Aldosterone Rapidly Activates Na ⁺/H ⁺ Exchange in M-1 Cortical Collecting Duct Cells via a PKC-MAPK Pathway

Abstract

Background: In this study, the mechanism of the rapid non-genomic effect of aldosterone on Na⁺/H⁺ exchanger (NHE)-mediated intracellular pH (pH_i) recovery from an acid load in murine M-1 cortical collecting duct cells was assessed. Methods: Spectrofluorescence microscopy and Western blot analysis was carried out and NH₄Cl was used to induce the acid load. Results: Aldosterone (10 n M) induced a rapid (<5 min) concentration-dependent increase in pH_i recovery in M-1 cells, an effect mimicked by its precursor deoxycorticosterone (1 n M). This response was unaffected by the mineralocorticoid receptor (MR) antagonist spironolactone (10 µ M) but was significantly reduced by the NHE antagonists 5′-(N-ethyl- N-isopropyl)amiloride (EIPA) (20 µ M) and cariporide (1 µ M). The PKC inhibitor chelerythrine chloride (1 µ M) significantly attenuated the aldosterone-induced increase in NHE1 activity. HBDDE (80 µ M), a PKC_α inhibitor, inhibited the rapid aldosterone effect whereas rottlerin (15 µ M), a PKC_δ antagonist, did not. The glucocorticoid receptor agonists hydrocortisone (1 µ M) and dexamethasone (100 n M) decreased NHE activity, whereas the synthetic mineralocorticoid fludrocortisone (1 n M) had no significant effect. MAPK inhibition using PD98059 (25 µ M) significantly attenuated the rapid aldosterone effect; Western blot analysis showed that aldosterone activation of ERK 1/2 was unaffected by pretreatment with spironolactone but was inhibited following chelerythrine chloride. Conclusion: Aldosterone causes a rapid non-genomic increase in NHE1 activity in M-1 cells via a PKC_α/MAPK pathway independent of the classical MR.