Glucocorticoids enhance acid activation of the Na+/H+ exchanger 3 (NHE3)

Abstract

In the absence of exogenous glucocorticoids, decreasing media pH (from 7.4 to 6.8) for 24 hours increased the Na+/H+ exchanger 3 (NHE3) activity in opossum kidney (OKP) cells. 10(-7) M and 10(-8) M hydrocortisone increased NHE3 activity, and in their presence, acid incubation further increased NHE3 activity. Hydrocortisone (10(-9) M) had no effect on NHE3 activity, but in its presence, the effect of acid incubation on NHE3 activity increased twofold. Aldosterone (10(-8) M) had no effect. In the absence of hydrocortisone, acid incubation increased NHE3 protein abundance by 47%; in the presence of 10(-9) M hydrocortisone, acid incubation increased NHE3 protein abundance by 132%. The increase in NHE3 protein abundance was dependent on protein synthesis. However, 10(-9) M hydrocortisone did not modify the effect of acid incubation to cause a twofold increase in NHE3 mRNA abundance. In the absence of protein synthesis, 10(-9) M hydrocortisone did potentiate an effect of acid on NHE3 activity, which was due to trafficking of NHE3 to the apical membrane. These results suggest that glucocorticoids and acid interact synergistically at the level of NHE3 translation and trafficking.

Figure 1

Effect of 10^–7 M hydrocortisone and media pH on Na/H antiporter activity. Cells were incubated in the presence or absence of 10^–7 M hydrocortisone for 48 h, and at pH 7.4 or 6.8 for the last 24 h. Na/H antiporter activity was then measured at pH 7.4 and is reported as dpH_i/dt. *P < 0.03 vs. pH 7.4 (–HC); **P < 0.005 vs. pH 7.4 (–HC); ***P < 0.01 vs. pH 6.8 (–HC), P < 0.05 vs. pH 7.4 (+HC), P < 0.02 vs. pH 7.4 (–HC). n = 7 (–HC/7.4, +HC/6.8), n = 8 (–HC/6.8, +HC/7.4). HC, hydrocortisone.

Figure 2

Effect of 10^–8 M hydrocortisone and media pH on Na/H antiporter activity. Cells were incubated in the presence or absence of 10^–8 M hydrocortisone for 48 h, and at pH 7.4 or 6.8 for the last 24 h. Na/H antiporter activity was then measured at pH 7.4 and is reported as dpH_i/dt. *P < 0.03 vs. pH 7.4 (–HC); **P < 0.02 vs. pH 7.4 (–HC); ***P < 0.005 vs. pH 6.8 (–HC), P < 0.005 vs. pH 7.4 (+HC), P < 0.001 vs. pH 7.4 (–HC). n = 7 (–HC/7.4), n = 8 (–HC/6.8, +HC/6.8, +HC/7.4).

Figure 3

Effect of 10^–9 M hydrocortisone and media pH on Na/H antiporter activity. Cells were incubated in the presence or absence of 10^–9 M hydrocortisone for 48 h, and at pH 7.4 or 6.8 for the last 24 h. Na/H antiporter activity was then measured at pH 7.4 and is reported as dpH_i/dt. *P < 0.01 vs. pH 7.4 (–HC); **P < 0.02 vs. pH 6.8 (–HC), P < 0.003 vs. pH 7.4 (+HC), P < 0.002 vs. pH 7.4 (–HC). n = 9.

Figure 4

Effect of 10^–8 M aldosterone and media pH on Na/H antiporter activity. Cells were incubated in the presence or absence of 10^–8 M aldosterone for 48 h, and at pH 7.4 or 6.8 for the last 24 h. Na/H antiporter activity was then measured at pH 7.4 and is reported as dpH_i/dt. *P < 0.03, 6.8 vs. 7.4; **P < 0.02, 6.8 vs. 7.4. n = 8.

Figure 5

HOE694 does not inhibit the effect of 10^–9 M hydrocortisone and media pH on Na/H antiporter activity. Cells were incubated in the presence of 10^–9 M hydrocortisone for 48 h, and at pH 7.4 or 6.8 for the last 24 h. Na/H antiporter activity was then measured at pH 7.4 in the absence or presence of 100 μM HOE694 and is reported as dpH_i/dt. *P < 0.01 vs. pH 7.4. n = 8.

Figure 6

Effect of 10^–7 M hydrocortisone on NHE3 protein abundance. Cells were incubated in the presence or absence of 10^–7 M hydrocortisone for 48 h at pH 7.4. NHE3 protein abundance was measured by Western blot. (a) Typical blot. (b) Summary of data. *P < 0.05. n = 4. NHE, Na⁺/H⁺ exchanger.

Figure 7

Effect of 10^–9 M hydrocortisone and media pH on NHE3 protein abundance. Cells were incubated in the presence or absence of 10^–9 M hydrocortisone for 48 h, and at pH 7.4 or 6.8 for the last 24 h. NHE3 protein abundance was measured by Western blot. All groups were paired to cells studied in the absence of hydrocortisone at pH 7.4, and NHE3 abundance is expressed as the percent increase over this condition. (a) Typical blot. (b) Summary of data. *P < 0.003, n = 18; **P = 0.05, n = 11; ***P < 0.001, n = 6.

Figure 8

Effect of media pH on NHE3 protein abundance in the presence of 10^–9 M hydrocortisone and 10^–4 M cycloheximide. Cells were incubated in the presence of 10^–9 M hydrocortisone for 48 h, and at pH 7.4 or 6.8 for the last 24 h. 10^–4 M cycloheximide was added for the last 24 h. NHE3 protein abundance was measured by Western blot. (a) Typical blot. (b) Summary of data. *P < 0.05. n = 6.

Figure 9

Effect of 10^–9 M hydrocortisone and media pH on NHE3 mRNA abundance. Cells were incubated in the presence or absence of 10^–9 M hydrocortisone for 48 h, and at pH 7.4 or 6.8 for the last 24 h. NHE3 mRNA abundance was measured by Northern blot. (a) Typical blot. (b) Summary of data. *P < 0.05. n = 5 (–HC/6.8), n = 6 (–HC/7.4, +HC/6.8, +HC/7.4).

Figure 10

Effect of 10^–9 M hydrocortisone and media pH on Na/H antiporter activity: cycloheximide. Cells were incubated in the presence or absence of 10^–9 M hydrocortisone for 48 h, and at pH 7.4 or 6.8 for the last 24 h. 10^–4 M cycloheximide was added for the last 24 h. Na/H antiporter activity was then measured at pH 7.4 and is reported as dpH_i/dt. *P < 0.005 vs. 7.4 (–HC and +HC). n = 9 (–HC/7.4), n = 10 (–HC/6.8, +HC/6.8, +HC/7.4).

Figure 11

Effect of media pH on apical membrane NHE3 protein abundance in the presence of 10^–9 M hydrocortisone and 10^–4 M cycloheximide. Cells were incubated in the presence of 10^–9 M hydrocortisone for 48 h, and at pH 7.4 or 6.8 for the last 24 h. 10^–4 M cycloheximide was added for the last 24 h. Apical membrane NHE3 protein abundance was measured by biotinylation followed by precipitation with streptavidin and Western blotting with anti-NHE3 antisera. (a) Typical blot. (b) Summary of data. *P < 0.05. n = 6.