Fig. 2.
Aldosterone increases NHE3 mRNA and NHE3 gene promoter activity. A: RNA was harvested from cells at varying times after aldosterone (1 nM) and analyzed by real-time PCR for human NHE3 and GAPDH. Ct values were calculated, and ΔΔCt was derived from these values and the value at 0 time set to 1. Data are means ± SE for 3 separate experiments. *P < 0.05, +P < 0.01 compared with 0 time by analysis of variance using a Bonferroni correction. B: aldosterone-stimulated NHE3 promoter-luciferase reporter activity, an effect inhibited by the receptor antagonist, spironolactone (Sp) (10 μM). Interestingly, the Na+-K+-ATPase inhibitor, ouabain (Ouab) (100 μM), significantly inhibited aldosterone-induced reporter activity as well (third bar from far right), an effect that was not additive to the inhibition caused by spironolactone. Neither agent alone nor a combination of ouabain and spironolactone affected baseline reporter activity. Some cells were treated with the PI3K kinase inhibitor (L294002, LY, 30 μM) or MEK-1 inhibitor (PD98059, PD, 30 μM), the p38 MAP kinase inhibitor (SB203580, SB, 30 μM) or the SAPK/JNK MAP kinase inhibitor (SP600125, SP, 30 μM), all for 2 h before aldosterone treatment (1 nM) of NHE3 promoter-luciferase reporter-transfected cells. Cells were also harvested after 6-h aldosterone stimulation. Cell monolayers were grown for 14 days, transfected with rat NHE3 promoter firefly luciferase and Rous sarcoma virus promoter Renilla luciferase constructs. Twenty-four hours later, cells were treated with aldosterone (1 nM) and harvested for luciferase activities 6 h later. Values are means ± SE for 3 separate determinations. *P < 0.05, +P < 0.01 compared with untreated control by analysis of variance with a Bonferroni correction.