Small-Conductance Ca2+-Activated Potassium Channels Negatively Regulate Aldosterone Secretion in Human Adrenocortical Cells

Abstract

Aldosterone, which plays a key role in maintaining water and electrolyte balance, is produced by zona glomerulosa cells of the adrenal cortex. Autonomous overproduction of aldosterone from zona glomerulosa cells causes primary hyperaldosteronism. Recent clinical studies have highlighted the pathological role of the KCNJ5 potassium channel in primary hyperaldosteronism. Our objective was to determine whether small-conductance Ca(2+)-activated potassium (SK) channels may also regulate aldosterone secretion in human adrenocortical cells. We found that apamin, the prototypic inhibitor of SK channels, decreased membrane voltage, raised intracellular Ca(2+) and dose dependently increased aldosterone secretion from human adrenocortical H295R cells. By contrast, 1-Ethyl-2-benzimidazolinone, an agonist of SK channels, antagonized apamin's action and decreased aldosterone secretion. Commensurate with an increase in aldosterone production, apamin increased mRNA expression of steroidogenic acute regulatory protein and aldosterone synthase that control the early and late rate-limiting steps in aldosterone biosynthesis, respectively. In addition, apamin increased angiotensin II-stimulated aldosterone secretion, whereas 1-Ethyl-2-benzimidazolinone suppressed both angiotensin II- and high K(+)-stimulated production of aldosterone in H295R cells. These findings were supported by apamin-modulation of basal and angiotensin II-stimulated aldosterone secretion from acutely prepared slices of human adrenals. We conclude that SK channel activity negatively regulates aldosterone secretion in human adrenocortical cells. Genetic association studies are necessary to determine whether mutations in SK channel subtype 2 genes may also drive aldosterone excess in primary hyperaldosteronism.

Keywords: adrenal cortex aldosterone secretion; angiotensin II; apamin; hyperaldosteronism; small-conductance Ca2+-activated potassium channels.

Figure 1. H295R cells express functional SK channels

(A-C) representative K⁺ current traces in the absence and presence of SK channel activator 1-EBIO, or inhibitor apamin. (D) Apamin-sensitive SK channel currents obtained by subtracting C from B. (E) Current-voltage relationship (I–V curves) generated from peak current density at each test voltage. Data are presented as means ± S.E.M. from 10 cells. (F) SK1, SK2 and SK3 channel mRNA expression were detected in H295R cells by RT-PCR. NTC: no template control.

Figure 2. Apamin increases aldosterone secretion mediating Ca²⁺ entry through T-type Ca²⁺ channels in H295R cells

(A) Apamin increased aldosterone secretion dose-dependently. 12 h serum starved H295R cells were incubated in 0.1% FBS DMEM / F12 for 24 h with/without apamin at various concentrations. Data were expressed as fold- increase over control. P < 0.05 compared to control using a one-way ANOVA test. (n = 3∼12). (B) 24 h treatment with 1nmol/L apamin increased basal intracellular calcium concentration. Results are presented as mean ± S.E.M. of fluorescence excitation ratios. *, P < 0.05 compared to control using Students' t-test. (C, D) Ca²⁺ currents were evoked by 80-ms steps ( –60 to +35 mV in 5-mV increments) applied every 6 s from a V_H of –90 or –50 mV. Shown are representative currents evoked by steps to –40, –10, or +10 mV from a V_H of –90, or V_H of –50 mV to reduce Cav3.x channel availability. Current-voltage relationship was constructed from peak currents (n = 7). (E) Effects of L-type Ca²⁺ channel inhibitor nifedipine (3 μmol/L) and T-type Ca²⁺ channel inhibitor TTA-P2(2 μmol/L) on Ca²⁺ currents. (F) TTA-P2 but not nifedipine precluded apamin-induced aldosterone production (n = 4, *, P < 0.05 compared to control by ANOVA).

Figure 3. Effect of apamin on mRNA expression of the key proteins that control steroid biosynthesis: STAR, CYP11A1, HSD3B2, CYP21A2, CYP17A1, CYP11B1 and CYP11B2

(A) Apamin increased HSD3B2, CYP11B1 and CYP11B2 but decreased CYP21A2 mRNA expression. 12 h serum starved H295R cells were incubated in DMEM/F12 with 0.1% FBS for 24 h with / without 1nmol/L apamin. Data are expressed as fold- change over control. *, P < 0.05 compared with control (n = 4∼7); (B) Apamin upregulated the expression of STAR at early time points. *, P < 0.05 compared with control (n = 3).

Figure 4. Effect of knockdown of SK2 channels on aldosterone secretion in H295R cells

(A) The mRNA expression of SK2 channels was reduced by 70% in H295R cells transduced with shRNA-SK2 lentiviruses. (B) knockdown of SK2 channels increased basal aldosterone secretion, and abrogated apamin-induced increase. *, P < 0.05 compared with scramble control (n = 6). (C) knockdown of SK2 channels increased mRNA expression of CYP11B1 , CYP11B2 and HSD3B2, but decreased that of CYP21A2. *, P < 0.05 compared with scramble control (n = 4∼5).

Figure 5. Effects of apamin and 1-EBIO on Ang II-stimulated aldosterone secretion in H295R cells

(A) Apamin increased both basal and Ang II-stimulated aldosterone secretion, whereas 1-EBIO, an activator of SK channels, suppressed secretion. Data are presented as fold- change over control. *, P < 0.05 compared with control (n = 3). #, P < 0.05 compared with Ang II alone (n = 3). (B) 24h incubation with apamin increased Ang II-induced elevation of intracellular calcium (left). Baseline Ca²⁺ fluorescent signal was recorded for 30 sec; the Ang II -induced signal for 120 sec. Statistical analysis of Ang II-induced Ca²⁺ increase measured in control cells or apamin treated cells (right). Data expressed as fold increase over control cells. *, P < 0.05 compared with control (n = 4).

Figure 6. Effects of apamin and 1-EBIO on high K⁺-stimulated aldosterone secretion in H295R cells

(A) Apamin did not alter K⁺ -stimulated aldosterone secretion, whereas 1-EBIO suppressed it. *, P < 0.05 compared with control. #, P < 0.05 compared with high K⁺ alone. (B) 24h incubation with apamin did not alter high K⁺ -induced elevation of intracellular calcium (left). Baseline Ca²⁺ fluorescent signal was recorded for 30 s, the high K⁺-induced signal for 120 s. Statistical analysis of high K⁺ -induced intracellular Ca²⁺ level in control or apamin treated cells (right). Data expressed as fold- increase over control cells. P > 0.05 compared with control cells (n = 4).

Figure 7. Changes in membrane voltage (Vm) of H295R cells elicited by Ang II or K⁺ with/without Apamin

Membrane voltage was determined in current-clamp. Plotted values are differences in Vm induced by 1 nmol/L apamin. Depolarization by apamin was additive to that of 10 nmol/L Ang II, but was subsumed by the voltage change induced by 22 mmol/L K⁺ (n = 5-7). *, P < 0.05 compared with Ang II alone.

Figure 8. SK2 channel expression in the zona glomerulosa of human adrenals and SK channel regulation of aldosterone

(A) Representative examples of immunofluorescence images showing high expression of SK2 channels in zona glomerulosa of the human adrenal cortex. (B-E) 1 nmol/L apamin increased basal and 10 nmol/L Ang II induced aldosterone production, but failed to increase 15 mmol/L K⁺-stimulated aldosterone secretion from human adrenal slices. (S1-11 =samples from 11 individual patients)