Glucocorticoids acutely increase cell surface Na+/H+ exchanger-3 (NHE3) by activation of NHE3 exocytosis

Abstract

Glucocorticoids have important effects on renal function, including the modulation of renal acidification by the major proximal tubular Na(+)/H(+) exchanger, NHE3. While the chronic effect of glucocorticoids is considered to be primarily at the transcriptional level, with increases in NHE3 mRNA and protein expression driving increased transport activity, the mechanisms by which glucocorticoids activate NHE3 in an acute setting have not been investigated. Previous studies have shown that a glucocorticoid-stimulated increase in NHE3 activity can occur before any detectable change in NHE3 mRNA. The present study examines the acute effects of glucocorticoids on NHE3 using opossum kidney (OKP) cells as a cell model. In OKP cells, total NHE3 protein abundance was not changed by 3 h of treatment with dexamethasone (10(-6) M). However, the biotin-accessible fraction representing NHE3 at the apical membrane as well as Na(+)/H(+) exchange activity measured fluorimetrically using the pH-sensitive dye BCECF-AM were significantly increased. These effects were not prevented by the protein synthesis inhibitor cycloheximide. NHE3 insertion (biotinylatable NHE3 after sulfo-NHS-acetate blockade) was stimulated by dexamethasone incubation, with or without cycloheximide. The rate of NHE3 endocytic retrieval, assessed either by the avidin protection assay (early endocytosis) or by the sodium 2-mercaptoethane sulfonate (MesNa) cleavage assay (early and late endocytosis), was not affected by dexamethasone. These findings suggest that trafficking plays a key role in the acute stimulation of NHE3 by glucocorticoids, with exocytosis being the major contributor to the glucocorticoid-induced rapid increase in cell surface NHE3 protein abundance and Na(+)/H(+) exchange activity.

Fig. 1

Dexamethasone (Dex) acutely increases Na⁺/H⁺ exchanger-3 (NHE3) activity. Confluent quiescent opossum kidney (OKP) cells were treated with vehicle (Con), dexamethasone (10⁻⁶ M × 3 h), cycloheximide (10⁻⁵ M × 4 h), or combined dexamethasone and cycloheximide. NHE3 activity was measured under V_max conditions as Na⁺-dependent cell pH recovery (dpH_i/dt) after acidification, where pH_i is intracellular pH. Bars and error bars denote means and SE, respectively. n, No. of independent measurements; vehicle, n = 8; dexamethasone, n = 7; cycloheximide, n = 10; cycloheximide + dexamethasone, n = 11. *P < 0.05 compared with vehicle by ANOVA.

Fig. 2

Acute treatment with dexamethasone does not alter NHE3 mRNA and total protein abundance. A: confluent quiescent OKP cells were treated with vehicle or dexamethasone (10⁻⁶ M) for 1, 3, or 12 h, and NHE3 mRNA was measured by RNA blotting. For each experiment, the densitometric values of NHE3 mRNA were normalized to 18S rRNA. B: confluent quiescent OKP cells were treated with vehicle, dexamethasone (10⁻⁶ M × 3 h), cycloheximide (10⁻⁵ M × 4 h), or combined dexamethasone and cycloheximide. NHE3 antigen in whole cell lysates was measured by immunoblot, and β-actin was used as a loading control (Con, vehicle). In A and B, representative RNA and protein blots (top) and the summaries of results from 3 independent experiments (bottom) are shown as bars (means) and error bars (SD). *P < 0.001 by Student’s t-test.

Fig. 3

Dexamethasone increases cell surface NHE3. Confluent quiescent OKP cells were treated with vehicle, dexamethasone (10⁻⁶ M × 3 h), cycloheximide (10⁻⁵ M × 4 h), or combined dexamethasone and cycloheximide. Surface NHE3 antigen was quantified as biotin-accessible and streptavidin-precipitated NHE3 from whole cell lysates measured by immunoblot. Top: representative immunoblot. Bottom: summary of results from 5 different experiments is shown as bars (means) and error bars (SD). *P < 0.05 by unpaired Student’s t-test.

Fig. 4

Dexamethasone increases exocytic insertion of NHE3 independently of de novo protein synthesis. A: typical immunoblot. B: summary of results from 6 independent experiments shown as bars (means) and error bars (SD). Cells were pretreated with or without 10⁻⁵ M cycloheximide, labeled with sulfo-NHS-acetate, and then incubated with 10⁻⁶ M dexamethasone or vehicle for 3 h at 37°C. Cells kept at 4°C or not labeled with sulfo-NHS-acetate were used as additional controls. Cell surface biotinylation was then performed as described in EXPERIMENTAL PROCEDURES. For each experiment, the densitometric values were normalized to the control that had not been labeled with sulfo-NHS-acetate. *P < 0.05 by unpaired Student’s t-test.

Fig. 5

Dexamethasone does not alter NHE3 endocytosis. A: typical immunoblots. B: summary of results from 3 independent experiments (bars are means, error bars are SD). After surface labeling with sulfo-NHS-SS-biotin, confluent quiescent OKP cells were warmed to 37°C in the presence of dexamethasone (10⁻⁶ M × 3 h) or vehicle. NHE3 endocytosis was measured as the fraction of biotinylated NHE3 protected from either reductive cleavage by sodium 2-mercaptoethane sulfonate (MesNa) or saturation by avidin, as described in EXPERIMENTAL PROCEDURES. For each experiment, the densitometric values were normalized to the control that had not been treated with either MesNa or avidin. *P < 0.05 by unpaired Student’s t-test.