Regulation of sodium-proton exchanger isoform 3 (NHE3) by PKA and exchange protein directly activated by cAMP (EPAC)

Abstract

The Na(+)/H(+) exchanger 3 (NHE3) is expressed in the brush border membrane (BBM) of proximal tubules (PT). Its activity is down-regulated on increases in intracellular cAMP levels. The aim of this study was to investigate the contribution of the protein kinase A (PKA) and the exchange protein directly activated by cAMP (EPAC) dependent pathways in the regulation of NHE3 by adenosine 3',5'-cyclic monophosphate (cAMP). Opossum kidney cells and murine kidney slices were treated with cAMP analogs, which selectively activate either PKA or EPAC. Activation of either pathway resulted in an inhibition of NHE3 activity. The EPAC-induced effect was independent of PKA as indicated by the lack of activation of the kinase and the insensitivity to the PKA inhibitor H89. Both PKA and EPAC inhibited NHE3 activity without inducing changes in the expression of the transporter in BBM. Activation of PKA, but not of EPAC, led to an increase of NHE3 phosphorylation. In contrast, activation of PKA, but not of EPAC, inhibited renal type IIa Na(+)-coupled inorganic phosphate cotransporter (NaPi-IIa), another Na-dependent transporter expressed in proximal BBM. PKA, but not EPAC, induced the retrieval of NaPi-IIa from BBM. Our results suggest that EPAC activation may represent a previously unrecognized mechanism involved in the cAMP regulation of NHE3, whereas regulation of NaPi-IIa is mediated by PKA but not by EPAC.

Fig. 1.

EPAC1 localization in mouse kidney. (A) EPAC1 staining was attenuated after preincubation of the affinity purified antibody with the antigenic peptide indicating specificity of the applied antiserum. (B) Consecutive cryosections were stained for NaPi-IIa, EPAC, and NHE3. All three proteins were localized in the BBM of proximal tubular cells.

Fig. 2.

NHE3 activity and expression in OK cells. (A) Example of a typical intracellular pH trace. Cells were preincubated with BCECF, followed by a 15-min treatment in the absence or presence of 100 μM 8-Br-cAMP; ≈20 OK cells were monitored per experiment. (B) Effect of the different cAMP analogs on the Na-dependent intracellular pH recovery (NHE activity). Data are presented as mean ± SEM (n = 4). (C) Total expression of NHE3. Cells were incubated in duplicate in the absence or presence of 50 μM 8-Br-cAMP, EPAC activator, or PKA activator for 15 min. Cell lysates were processed for Western blot with an anti-NHE3 antibody (n = 3). (D) Surface expression of NHE3. Cells were incubated for 15 min or 4 h in the absence or presence of 50 μM 8-Br-cAMP, EPAC activator, or PKA activator as well as with 10 nM PTH; PTH was always applied for 4 h. Upon biotinylation and streptavidine precipitation, samples were subjected to SDS/PAGE and incubated with an anti-NHE3 antibody (n = 4 and 3, respectively).

Fig. 3.

NHE3 and PKA activities in mouse kidney samples. Kidney slices were incubated for 30 min with the indicated concentrations of EPAC and PKA activators. Homogenates and BBMV were then prepared and the NHE and PKA activities were measured as described in Materials and Methods. (A) Reproduction showing a superposition of original acridine orange fluorescence traces obtained with BBMV isolated from kidney slices incubated in the absence or presence of PKA activator. (B) Concentration response of both activators over a range of 10–100 μM. Data are presented as mean ± SEM of (n = 4). (C) Determination of PKA activity in homogenates isolated from kidney cortex slices incubated in the absence or presence of 50 μM of the PKA and EPAC activators (n = 3). (D) Western blot with an anti-PKA-substrate antibody of BBMV incubated in the absence or presence of 50 μM PKA or EPAC activator. Substrates phosphorylated on PKA treatment are indicated by arrows. (E) Effect of H89 on the PKA- and EPAC-dependent NHE inhibition. Kidney slices were incubated in the absence or presence of 50 μM H89 for 5 min, before addition of 50 μM PKA or EPAC activators (n = 4). (F) Effect of incubation with 50 μM EPAC and/or PKA activator. (G) Effect of PD098059 on the PKA- and EPAC-dependent NHE inhibition. Kidney slices were incubated in the absence or presence of 20 μM PD098059 for 5 min, before addition of 50 μM PKA or EPAC activators (n = 3).

Fig. 4.

Expression of NHE3 in mouse BBMV. Kidney slices were incubated for 30 min with the indicated analogs (50 μM). (A) Western blot of BBMV with an anti-NHE3 antibody. BBMV isolated from four independent experiments were processed in parallel. (B) Immunofluorescence of kidney slices with anti-NHE3 antibodies (green) as well as with phalloidin (red).

Fig. 5.

Phosphorylation of NHE3 in OK cells. Confluent cultures were phosphorylated and NHE3 was immunoprecipitated as described. (A) Typical autoradiography. (B) Quantification of seven independent experiments. The signal detected in untreated samples was taken as 100% for each experiment.

Fig. 6.

NaPi-IIa activity and expression. (A) Na-dependent ³²P-uptake. Cells were incubated for 4 h in the absence or presence of 10 nM PTH (–34) or the indicated concentrations of the cAMP analogs. (B) Kidney slices were incubated in the absence or presence of 50 μM indicated analogs and processed for immunofluorescence with anti-NaPi-IIa antibodies (green) and phalloidin (red). Rectangles show an overview of NaPi-IIa signal; squares are magnified images.