Zona glomerulosa cells of the mouse adrenal cortex are intrinsic electrical oscillators

Abstract

Aldosterone, which plays a central role in the regulation of blood pressure, is produced by zona glomerulosa (ZG) cells of the adrenal gland. When dysregulated, aldosterone is pathogenic and contributes to the development and progression of cardiovascular and renal disease. Although sustained production of aldosterone requires persistent Ca2+ entry through low-voltage activated Ca2+ channels, isolated ZG cells are considered nonexcitable, with recorded membrane voltages that are too hyperpolarized to permit Ca2+ entry. Here, we show that mouse ZG cells within adrenal slices spontaneously generate membrane potential oscillations of low periodicity. This innate electrical excitability of ZG cells provides a platform for the production of a recurrent Ca2+ signal that can be controlled by Ang II and extracellular potassium, the 2 major regulators of aldosterone production. We conclude that native ZG cells are electrical oscillators, and that this behavior provides what we believe to be a new molecular explanation for the control of Ca2+ entry in these steroidogenic cells.

Figure 1. ZG cells are electrically excitable.

(A) Representative current clamp recording of a ZG cell within an adrenal slice generating spontaneous Vm oscillations of low periodicity, and the calculated mean cycle waveform (see Methods) at 22°C and at 35°C (inset). (B) Average oscillation frequency versus baseline Vm, both determined per cell (see Methods) over recording of 3–10 minutes duration (n = 85). (C) Average peak amplitude per cell (determined from the same oscillation analysis as above) versus baseline Vm (n = 73). (D) Average oscillation frequency per cell was binned to calculate probability density distribution of oscillation frequency versus baseline Vm. The 2D contour plot shows behavior of an electrical oscillator with a voltage threshold for activity and a voltage dependence to oscillatory activity.

Figure 2. ZG cell oscillations depend on a Ni²⁺-sensitive current.

Perfusion application of TTX to target Nav1.x, nifedipine to target Cav1.x, and Ni²⁺ to target Cav3.x. (A) Representative current clamp recording of a ZG cell stably oscillating 1 min before and during bath exposure to 60 μM TTX (rendering block of TTX-sensitive and insensitive Nav1 channels). Mean oscillation frequency of each of 9 cells before and after exposure to TTX at 100 nM (n = 5) or 60 μM (n = 4) is also shown. (B) Vm oscillations recorded before and during bath exposure to 1 μM nifedipine (NIF). Mean oscillation frequency of each recorded cell calculated before and after exposure to nifedepine at 100 nM (n = 4) or 1–3 μm (n = 3) is also shown. (C) Example of a cell with periods of rhythmic oscillations interrupted by intermittent periods of silence, before and after bath exposure to 50 μM Ni²⁺. Mean oscillation frequency was silenced by Ni²⁺ in all recorded cells (n = 6).

Figure 3. ZG Ca²⁺ currents are robust and Cav3.2-like.

(A) Ca²⁺ currents evoked by 100-ms steps from –70 to +40 mV in 10-mV increments applied every 6 s from a V_H of –90 or –50 mV. Shown are representative currents evoked by steps to –60, –40, or +10 mV from a V_H of –90 or –50 mV to reduce Cav3.x channel availability. Current-voltage relationship was constructed from peak current densities (n = 16). (B) Representative currents as in A evoked in the presence of 1 μM (S)-(–)BAYK8644 or 1 μM nifedipine from a V_H of –90 mV. Current-voltage relationships were constructed from peak current densities (n = 8 per group). (C) Ca²⁺ currents evoked by step (100-ms to –40 mV) or tail current (35-ms repolarization to –70 mV following 9-ms step to –40 mV) voltage protocols from a V_H of –90 mV applied every 6 s, in the absence, then presence, of Ni²⁺ in the bath solution from 1 to 100 μM (mean ± SEM). (D) Ni²⁺ inhibition curves of Ca²⁺ channel currents (ICa). Percent inhibition was calculated as measured current relative to maximal current recorded in the absence of Ni²⁺. Datasets were fitted with a logistic equation yielding IC₅₀ 22.5 μM (n = 6) for Ni²⁺ block of Ca²⁺ currents expressed in ZG cells and 9.2 μM (n = 6) for block of recombinant Cav3.2 currents expressed in HEK293 cells.

Figure 4. External K⁺ and Ang II change ZG cell oscillatory behavior.

Representative current clamp recordings of ZG cells oscillating in standard buffer (3 mM K⁺) and after K⁺ substitution with 2 mM (A) or 5 mM (B) K⁺. Expanded traces are calculated average cycles from same voltage recordings. (C and D) Average oscillation frequency per cell (C) and average baseline Vm (D), calculated before and after hyperpolarization with 2 mM K⁺ (n = 5) or depolarization with 5 mM K⁺ (n = 5). (E) Representative current clamp recording of a cell spontaneously oscillating before and during exposure to 100 nM Ang II. Expanded traces are calculated average cycles from the same voltage recording. (F) Average oscillation frequency (calculated before and after exposure to a single concentration of Ang II), expressed as fold increase over the pre–Ang II frequency. Data points represent mean ± SEM and were fitted with a logistic equation. EC₅₀ = 85.2 nM (n = 5 per concentration). *P < 0.05.

Figure 5. Vm oscillations increase Ca²⁺ entry carried by Cav3.2 channels.

(A) Vm oscillatory voltage commands generated from voltage recordings to construct average cycles (see Methods) that represent control and Ang II oscillatory activity. 4 (control; 2.14 s) or 8 (Ang II; 1.24 s) Vm cycles were delivered in 10 s (top traces). Mean Ca²⁺ currents evoked sequentially by control and Ang II voltage commands (n = 16) are shown below. Also displayed are magnified representations of the mean current elicited during first and last cycles. (B) Cycle comparison of averaged peak Ca²⁺ currents and averaged Ca²⁺ current areas. Peak current and current area per cycle per cell (n = 16) were calculated and averaged. Average Ca²⁺ current area summed across cycle number (i.e., total current) evoked by Ang II command was 240% that of control command. (C) Ni²⁺-sensitive Cav3.2 Ca²⁺ current. Mean Ca²⁺ currents recorded in the absence or presence of 100 μM Ni²⁺ evoked by the first or last Vm cycle. The blue difference current defines the Ni²⁺-sensitive Cav3.2 current, which, because of incomplete block by Ni²⁺, was an underestimate. (D) Average peak Ca²⁺ current and average Ca²⁺ current area compared between first and last cycle, showing persistent Ni²⁺ block (n = 6). Average Ni²⁺-sensitive component (Cav3.2) of Ca²⁺ current area summed across cycle number (i.e., total Ni²⁺-sensitive current) evoked by Ang II command was 220% that of control. (E) Mean Ni²⁺-sensitive current averaged during interspike intervals elicited by Ang II command (n = 6). ⁺P < 0.05 vs. first cycle; *P < 0.05 vs. control. Bars represent mean ± SEM.