Effects of K+ Channel Blockers on K+ Channels, Membrane Potential, and Aldosterone Secretion in Rat Adrenal Zona Glomerulosa Cells* (2024)

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Abstract

THE K⁺ channel classes controlling the plasma mem-brane potential and, consequently, Ca²⁺ influx through voltage-dependent Ca²⁺ channels in adrenal zona glomerulosa cells have been hypothesized to play an important role in the stimulation of aldosterone secretion by the two primary physiological stimuli: elevated extracellular K⁺ and angiotensin II (ANG II) (1; for review, see Refs. 2–4). K⁺-induced membrane depolarization is directly attributed to the depolarizing shift in the K⁺ equilibrium potential (E_K) caused by elevation of the extracellular K⁺ concentration (5, 6). ANG II-induced membrane depolarization has been attributed to AT₁ receptor-mediated inhibition of K⁺ channels controlling the resting membrane potential (1, 7–10), although the specific K⁺ channel classes involved and their properties have remained a controversial topic (10). ANG II-induced membrane depolarization may also involve contributions from T-type voltage-dependent Ca²⁺ channels and nonselective cation channels (11).

The relative contributions of K⁺ channel inhibition to ANG II-induced membrane depolarization and stimulation of aldosterone secretion have not been elucidated. Previous studies examining the effects of K⁺ channel blockers on aldosterone secretion reported little effect on basal secretion for most of the blockers examined (12–14). The nonselective blocker tetraethylammonium was reported to induce a small stimulation of aldosterone secretion and potentiate ACTH-induced secretion (12). Tetraethylammonium was also reported to weakly inhibit ANG II-induced aldosterone secretion (13). Most blockers were found to inhibit, rather than potentiate, stimulus-induced aldosterone secretion. Furthermore, the K⁺ channel opener, pinacidil, was reported to inhibit both ANG II- and K⁺-stimulated aldosterone secretion (13, 14). Pinacidil inhibition of K⁺-stimulated aldosterone secretion suggested that pinacidil acted by a mechanism other than opening plasma membrane K⁺ channels. Such results suggest that ANG II inhibition of K⁺ channels does not significantly contribute to the stimulation of aldosterone secretion. However, these studies did not examine blocker/opener effects on either membrane potential or K⁺ permeability in glomerulosa cells.

Whole cell, perforated patch, and single channel patch-clamp studies of glomerulosa cell K⁺ channels have been carried out in several laboratories (8, 10, 15–18). There has been little consensus among these studies regarding the K⁺ channel classes present or their pharmacological properties. In our study of rat glomerulosa cells maintained in primary cell culture (<48 h), we observed two electrophysiologically distinct cell types (10). The most prevalent cell type (type 1; 80% of cells) expressed a rapidly activating, noninactivating, weakly voltage-dependent K⁺ channel class (leak K⁺ channels) that appeared to mediate most of the K⁺ permeability at membrane potentials below− 40 mV (the resting membrane potential is approximately −85 mV under our re-cording conditions). In the second cell type (type 2), a charybdotoxin-sensitive, voltage-dependent K⁺ current attributable to Ca²⁺-dependent maxi-K⁺ channels accounted for most of the K⁺ current activated by membrane depolarization above −50 mV; this current was absent from the first cell type. This second cell type was similar to that described by Payet et al. (18) for rat glomerulosa cells maintained in cell culture.

In the present study we reexamined the effects of K⁺ channel blockers, focusing on blockers previously reported to inhibit some aspect of glomerulosa cell K⁺ permeability. The effects of several K⁺ channel blockers were measured on basal and stimulated aldosterone secretion, macroscopic and single K⁺ channel currents, and membrane potential in rat adrenal glomerulosa cells. The results demonstrated that inhibition of resting K⁺ conductance depolarized the membrane potential and was a strong stimulus for aldosterone secretion. Blockers that did not inhibit the resting K⁺ conductance did not depolarize the cells or stimulate aldosterone secretion.

Materials and Methods

Animals were humanely cared for by professional staff before experimentation. All animals were anesthetized before use, and all protocols were approved by the institutional animal care committee at Northern Illinois University.

Aldosterone secretion

Aldosterone secretion was measured in acutely dissociated glomerulosa cell suspensions under static incubation conditions at 37 C in a shaking water bath as previously described (19). Isolated cell suspensions were prepared from female Sprague-Dawley rats (Harlan, Indianapolis, IN), weighing 125–175 g, using a collagenase dispersion method as previously described (19). After isolation, glomerulosa cells were equilibrated for periods of 1–2 h in medium 199 adjusted to contain 4 mm K⁺, 1.25 mm Ca²⁺, 1.2 mm Mg²⁺, 4.2 mm NaHCO₃, 0.1% fatty acid-free BSA, 50 U/ml penicillin, 50 μg/ml streptomycin, and 10 mm HEPES (pH adjusted to 7.4 with NaOH). After the equilibration period, cells were pelleted by centrifugation, resuspended, and aliquoted into a 1.0-ml final volume containing fresh medium 199 plus the various treatments to a final cell density of approximately 25,000 cells/ml. Cells were incubated for 1 h, and incubation was terminated by transfer of tubes to an ice water bath. Cells were separated from the medium by centrifugation, and the medium was saved and stored at −20 C until assayed for aldosterone content. Each treatment, including the controls, was performed in triplicate, and treatment effects were examined in at least three separate experiments. In experiments using elevated K⁺ or Cs⁺, the NaCl concentration was reduced by an equimolar amount to maintain the osmolarity of the solution. Quinidine was dissolved in dimethylsulfoxide and diluted to the final concentration; the final di-methylsulfoxide concentration was always equal to or less than 0.1%.

Secreted aldosterone was measured in medium 199 without extraction using a commercial aldosterone RIA kit (Diagnostic Products Corp., Los Angeles, CA). The inter- and intraassay coefficients of variation were 6.9% and 5.4%, respectively.

Cell culture

Electrophysiological measurements used rat glomerulosa cells maintained in primary cell culture as previously described (11). Glomerulosa cells were isolated using a collagenase dispersion method; isolated cells were plated at low density (<10⁵ cells/ml) on fibronectin-treated glass coverslip chips. Cultures were maintained at 37 C in a humidified atmosphere of 5% CO₂-95% air. Culture medium consisted of a mixture of Ham’s F-12 and DMEM (1:1) supplemented with 2% FBS, 8% horse serum, 0.1 mm ascorbic acid, 1 μm vitamin E, 1 μg/ml insulin, 50 U/ml penicillin G, and 50 μg/ml streptomycin.

Sera were obtained from Life Technologies (Grand Island, NY), and collagenase was obtained from Worthington Biochemical Corp. (Freehold, NJ); all other reagents were obtained from Sigma Chemical Co. (St. Louis, MO).

Electrophysiology

Patch-clamp recordings (20) were performed on cells maintained in culture between 12–48 h. Glass coverslip chips containing adherent glomerulosa cells were transferred to a small volume (0.5 ml) recording chamber mounted on an inverted microscope equipped with phase contrast optics.

Glomerulosa cells were identified by visual appearance. Adherent cells identified as glomerulosa cells initially retained their characteristic spherical shape and granular cytoplasm. Over 1–2 days in culture, many cells flattened and extended short processes along the substrate as previously described (21). After 2 days in culture, cells retained their ability to secrete aldosterone in response to hormone stimulation; 1-h stimulation with 1 nm ANG II or 100 pm ACTH increased aldosterone 5- and 35-fold over basal secretion, respectively.

The chamber was continuously perfused at a rate of 1 ml/min with modified Hanks’ saline equilibrated with 100% O₂: 140 mm NaCl, 4 mm KCl, 1.25 mm CaCl₂, 1.2 mm MgCl₂, 4.2 mm NaHCO₃, 10 mm HEPES, and 5.5 mm glucose (pH adjusted to 7.4 with NaOH). For experiments in which the KCl concentration of the saline was increased or CsCl was added, the NaCl concentration was reduced by an equimolar amount. All recordings were performed at room temperature.

Macroscopic membrane currents and membrane potential were measured using the perforated patch variation of the whole cell patch clamp (22), employing nystatin as the pore-forming agent. A stock solution of nystatin (50 mg/ml) in dimethylsulfoxide was freshly prepared each day and diluted in pipette solution to a concentration of 200 μg/ml. The patch pipette solution contained 55 mm KCl, 70 mm K₂SO₄, 8 mm MgCl₂, and 10 mm HEPES (pH adjusted to 7.3 with KOH). Patch pipettes were fabricated from Corning 7052 capillary glass to give a pipette resistance of 3–4 megaohms when filled with pipette solution. Pipettes were coated with sylgard (Dow Corning, Midland, MI) to within 100 μm of the tip. Membrane potentials were corrected for liquid junction potentials as described previously (23).

Single channel recordings were made using inside-out patches. Pipettes for inside-out patches were constructed using borosilicate TW-150 glass capillaries (World Precision Instruments, Sarasota, FL) to give pipette resistances of 6–8 megohms when filled with pipette solution. The pipette solution was formulated to distinguish weakly voltage-dependent K⁺ channels from nonselective cation channels (10) and contained 20 mm KCl, 125 mm NaCl, 2.4 mm MgCl₂, 0.1 mm CaCl₂, and 10 mm HEPES (pH adjusted to 7.4 with NaOH). The saline on the cytosolic membrane face (bath) contained 145 mm KCl, 2.5 mm MgCl₂, and 10 mm HEPES (pH adjusted to 7.4 with KOH).

Membrane current or membrane potential (in the current clamp mode) was measured with an Axopatch 200 amplifier (Axon Instruments, Foster City, CA). Macroscopic currents were low pass filtered at 10 kHz (−3 decibels) with a four-pole Bessel filter. Currents were sampled at 10 kHz (TL-1–125 analog to digital converter, Axon Instruments) and stored on computer for subsequent analysis using PCLAMP software (Axon Instruments). Single channel currents were low pass filtered at 2 kHz and stored on computer or on digital audiotape (Sony, Tokyo, Japan) for subsequent analysis. In experiments measuring membrane potential, potentials were recorded on digital audiotape. For computer analysis of recorded data, current records were sampled at 10 kHz (or 200 Hz for membrane potential records) during playback; single channel events were selected for analysis by the 50% current amplitude threshold criteria and analyzed for mean current amplitude and open times using PCLAMP software.

Results

Electrophysiological results are reported for cells expressing the type 1 current-voltage (IV) relationship described previously (10). This cell type exhibited a rapidly activating, noninactivating, voltage-dependent outward current pattern, shown as the control pattern in Fig. 1. The steady state macroscopic current (at the end of the voltage command) was previously shown to be carried by K⁺ and predominantly attributable to a single class of weakly voltage-dependent “leak” K⁺ channels at membrane potentials below approximately −40 mV. At membrane potentials above −40 mV, a second class of voltage-dependent K⁺ channels blocked by quinidine may also contribute to the steady state macroscopic current. However, this second voltage-dependent K⁺ channel class has not been well separated from the weakly voltage-dependent K⁺ channels at either the macroscopic or the single channel current level (10).

Inhibition of K⁺channels

Cs⁺ is an effective nonselective blocker of K⁺ channels and intracellular Cs⁺ has previously been shown to effectively block K⁺ currents in the glomerulosa cells (8, 10, 17). The effects of 5 and 20 mm extracellular Cs⁺ on macroscopic membrane current responses to hyperpolarizing and depolarizing voltage commands are shown in Fig. 1. Leak subtraction was not used in these experiments, because leak current could not be cleanly separated from K⁺ current. The kinetics and magnitude of Cs⁺-blocked current evoked by hyperpolarizing and depolarizing voltage commands (to −125 and −65 mV) are shown in Fig. 1B. The Cs⁺-sensitive current component was obtained by subtracting the current remaining in the presence of Cs⁺ from the control current traces. Both 5 and 20 mm Cs⁺ caused an instantaneous and stable blockade of inward current evoked by a hyperpolarizing command to −125 mV, but only 20 mm Cs⁺ blocked the outward current response at− 65 mV. The Cs⁺ blockade of outward current was rapid in onset and stable for the duration of the voltage command. In response to larger depolarizing commands, the kinetics of the Cs⁺ blockade became more complex, possibly due to the kinetics of K⁺ channel activation and the unblocking reaction as well as to activation of an additional K⁺ channel class that may differ in sensitivity to the Cs⁺ blockade.

The effects of 5 and 20 mm Cs⁺ on the steady state membrane current-voltage (IV) relationship are shown in Fig. 1C. These data demonstrated that Cs⁺ blockade of membrane K⁺ current was dependent on both membrane potential and Cs⁺ concentration; the Cs⁺ blockade of outward current was diminished by membrane depolarization, and this effect was opposed by increasing the Cs⁺ concentration. The blockade by 5 mm Cs⁺ was nearly abolished at membrane potentials above −65 mV, whereas blockade by 20 mm Cs⁺ persisted to much more depolarized membrane potentials. The intersection of the control and Cs⁺ blockade curves (the reversal potential) should equal the K⁺ equilibrium potential (E_K) if the only effect of Cs⁺ on membrane current was K⁺ channel blockade and if the effectiveness of the K⁺ channel block did not change over the voltage range used to determine the intersection of the curves. The mean (±sd) reversal potential measured using 15 and 20 mm Cs⁺ was −93.6 ± 3.0 mV (n = 5), which is near the expected E_K (5, 10), indicating that effects of Cs⁺ were largely mediated through blockade of K⁺ channels.

The voltage dependence of K⁺ conductance blockade by 5 and 20 mm Cs⁺ is presented in Fig. 1D, in which the steady state IV relationship from Fig. 1C is plotted as the slope conductance. Increasing the Cs⁺ concentration extended the voltage range over which K⁺ channels were blocked.

The concentration dependence of Cs⁺ blockade was estimated from inhibition of steady state slope conductance measured between −85 and −105 mV of the IV curve (Fig. 1E), a membrane potential range that included the reversal potential and, therefore, Cs⁺ blockade of both inward and outward K⁺ currents. Again, membrane current values were not leak subtracted before calculation of slope conductance. Assuming a single Cs⁺-binding site, the data were fit to the Hill equation: G = [1 + (K_d/[Cs⁺])⁻ⁿ]⁻¹, where G is the slope conductance, K_d is the apparent dissociation constant, and n is the Hill coefficient. The best fit to the data yielded a Hill coefficient of 1 and an apparent K_d of 2.4 ± 0.32 mm (±se; giving a 95% confidence interval for K_d of 1.52–3.28).

In type 1 cells, resting K⁺ conductance appeared to be attributable to a single class of weakly voltage-dependent K⁺ channels (10). Thus, if Cs⁺ blockade of macroscopic current was due to blockade of this channel class, then extracellular Cs⁺ would be predicted to block inward single channel K⁺ currents much more strongly than outward current through these channels. The effect of 15 mm Cs⁺ on single K⁺ channel currents measured from inside-out patches is shown in Fig. 2. In these experiments the pipette solution contained 20 mm K⁺ to facilitate measurement of inward single channel currents and readily separate unitary K⁺ currents from those carried through nonselective cation channels (10). Control current traces (Fig. 2A) from a patch containing multiple active channels illustrated the weakly voltage-dependent K⁺ channel characteristics: a voltage-dependent low open probability that increased with membrane depolarization and a brief mean open time of approximately 2 msec. The channels exhibited an outward single channel conductance of 12.2 picosiemens and an inward conductance of 7.8 picosiemens measured from the slope of the IV relationship (Fig. 2B). The asymmetric slope conductance was due to the asymmetric K⁺ distribution across the plasma membrane. Inclusion of 15 mm Cs⁺ in the patch pipette solution (reducing Na⁺ by an equimolar amount) blocked inward unitary current events at membrane potentials below E_K (approximately −50 mV under these recording conditions). Outward unitary current amplitude appeared to be slightly decreased by Cs⁺, but neither the extrapolated reversal potential, the mean open time, nor the open probability of the outward currents was significantly affected by 15 mm Cs⁺ under these recording conditions (data not shown). These results were consistent with the hypothesis that Cs⁺ blockade of the resting macroscopic K⁺ conductance is primarily attributable to blockade of the weakly voltage-dependent K⁺ channels. The relief of Cs⁺ blockade at membrane potentials positive to E_K in both the macroscopic and single channel currents is consistent with a multi-ion occupancy model for these K⁺ channels. In such a model, Cs⁺ would block K⁺ conduction by entering the channel pore from the extracellular side and binding to a site in the pore. At membrane potentials positive to E_K, K⁺ entry into the channel pore from the cytosolic side would electrostatically repel Cs⁺ from the pore, thus opposing Cs⁺ blockade (24).

Other K⁺ channel blockers examined did not consistently inhibit the resting macroscopic K⁺ conductance in the type 1 glomerulosa cells. We have previously shown that quinidine strongly blocked depolarization-activated K⁺ current, but this effect occurred at membrane potentials above −50 mV (10). In this regard, quinidine decreased the mean open time of the weakly voltage-dependent K⁺ channel, but this effect was apparent only at more positive membrane potentials (data not shown). Charybdotoxin had no significant effect on resting or depolarization-activated K⁺ conductance in the type 1 cells (10). Apamin was previously reported to inhibit a transient ANG II-stimulated ⁸⁶Rb flux in bovine adrenal glomerulosa cells, but did not affect ANG II inhibition of ⁸⁶Rb flux (7). In the rat type 1 cells, 0.1 μm apamin was observed to inhibit resting and depolarization-activated K⁺ conductance by as much as 20% in approximately 25% of the cells examined (n = 20; data not shown); this effect of apamin was not highly reproducible in these cells.

Membrane depolarization

Cs⁺ inhibition of resting K⁺ conductance suggested that extracellular Cs⁺ should depolarize the resting membrane potential. As shown in Fig. 3, bath perfusion of 15 mm Cs⁺ rapidly depolarized the resting membrane potential. The time course of depolarization may be largely attributed to the time needed for Cs⁺ equilibration in the recording chamber. Membrane depolarization was caused by blockade of resting K⁺ conductance, as indicated by the simultaneous increase in membrane input resistance. Input resistance was monitored throughout the experiments by measuring the membrane potential response to repeated hyperpolarizing constant current pulses applied through the patch pipette. After the initial slow depolarization, the membrane potential remained relatively stable throughout the remainder of the treatment period (10–15 min) and recovered to its control value upon washout of Cs⁺.

Cs⁺ stimulation also increased the amplitude of short-lived transient depolarizing events that could usually be detected before Cs⁺ treatment (Fig. 3). These events may have been caused by nonselective cation channel gating and/or activation of T-type voltage-dependent Ca²⁺ channels. Before Cs⁺, these events were usually between 1–2 mV in amplitude and increased in amplitude during the Cs⁺-induced depolarization, although they usually remained less than 5 mV in amplitude. The increased amplitude was probably attributable to the increased input resistance of the membrane. In the record shown (Fig. 3), the amplitude of these events was blunted by the low sampling frequency used to display the records (200 Hz).

The concentration dependence of Cs⁺-induced membrane depolarization is shown in Fig. 4A. The threshold for depolarization was approximately 2 mm Cs⁺; increasing the Cs⁺ concentration above 2 mm increased the amplitude of the stable minimum depolarization attained. These results were in close agreement with the effects of Cs⁺ on the macroscopic IV relationship (Fig. 1C), in which elevated Cs⁺ levels progressively reduced outward K⁺ current, shifting the zero current potential toward more positive membrane potentials.

Other K⁺ channel blockers examined (50 μm quinidine, 50 nm charybdotoxin, and 0.1 μm apamin) did not depolarize the resting membrane potential, in agreement with their lack of significant inhibition of resting K⁺ conductance.

Aldosterone secretion

Cs⁺ caused a concentration-dependent stimulation of aldosterone secretion from the acutely dissociated glomerulosa cell preparation (Fig. 4B). Over the Cs⁺ concentration range examined (1–20 mm), maximal stimulation occurred at a concentration of 10 mm, with half-maximal stimulation at approximately 3.5 mm. Cs⁺ stimulation of aldosterone secretion was completely blocked by omission of Ca²⁺ from the medium (nominally Ca²⁺-free medium), as expected if Cs⁺-induced stimulation was mediated by membrane depolarization and activation of Ca²⁺ influx through voltage-dependent Ca²⁺ channels (data not shown). The maximal secretory response to Cs⁺ stimulation was similar to that produced by 1.0 nm ANG II (Fig. 5A) or 8 mm K⁺ (Table 1), both of which are near maximally effective stimuli for aldosterone secretion.

If both Cs⁺ and ANG II stimulated aldosterone secretion through inhibition of K⁺ conductance, then their stimulatory effects would not be expected to be additive. Cs⁺ (10 mm) significantly increased aldosterone secretion (Fig. 5B) induced by a submaximal concentration of ANG II (0.1 nm; Fig. 5A), although their combined effects were not fully additive. However, Cs⁺ (10 mm) only marginally increased the secretory response to a supramaximal concentration of ANG II (10 nm; Fig. 5, A and B).

Other K⁺ channel blockers that did not significantly inhibit resting K⁺ conductance either did not stimulate aldosterone secretion or inhibited secretion (Table 1). Charybdotoxin neither stimulated basal aldosterone secretion nor affected ANG II- or K⁺-induced aldosterone secretion when tested at a concentration (50 nm) that completely blocked Ca²⁺-dependent maxi-K⁺ channels in type 2 cells (10). Apamin did not significantly affect basal aldosterone secretion in the acutely dissociated cell preparation or consistently affect ANG II- or K⁺-stimulated aldosterone secretion. Quinidine strongly inhibited basal and ANG II- or K⁺-stimulated aldosterone secretion. Quinidine-induced inhibition became apparent at 10 μm quinidine (data not shown), which corresponded to its concentration dependence for inhibition of depolarization-activated K⁺ conductance (10). However, quinidine inhibition of basal aldosterone secretion in the absence of effects on membrane potential suggested that quinidine inhibited secretion by a mechanism other than inhibition of plasma membrane K⁺ channels.

Discussion

The results of the present study demonstrated that inhibition of the resting K⁺ conductance can be an effective stimulus for aldosterone secretion. Although extracellular Cs⁺ treatment did not mimic the effects of ANG II on glomerulosa cell K⁺ conductance, it provided an effective means for examining the relationships among inhibition of resting K⁺ conductance, membrane depolarization, and aldosterone secretion. The magnitude of Cs⁺-induced depolarization was dictated by the Cs⁺ concentration and E_K, which imparted voltage dependence to the Cs⁺ blockade. Outward K⁺ current relieved the Cs⁺ blockade in proportion to the electrochemical gradient for K⁺ (the difference between membrane potential and E_K). This effect was opposed by increasing the Cs⁺ concentration, which extended the voltage range for effective Cs⁺ blockade and caused larger depolarizations. The concentration dependencies of Cs⁺-induced inhibition of resting K⁺ conductance and membrane depolarization suggested that resting K⁺ conductance must be inhibited by approximately 50% to initiate membrane depolarization through this mechanism. The threshold for Cs⁺-induced aldosterone secretion was similar to that for membrane depolarization (∼2 mm Cs⁺). Maximal aldosterone secretion occurred at approximately 10 mm Cs⁺, a concentration that produced an average minimum depolarization of approximately 7 mV, excluding the numerous small spike-like depolarizing events.

Stimulation of aldosterone secretion by membrane depolarization will be affected by the mechanism of depolarization as well as the absolute magnitude of the depolarization. This can be seen by comparing the effects of elevated extracellular K⁺ and Cs⁺. Increasing K⁺ shifts E_K in a depolarizing direction, causing membrane depolarization and stimulation of aldosterone secretion through activation of voltage-dependent T-type Ca²⁺ channels (25–27). The maximally effective K⁺ concentration for stimulation of aldosterone secretion is reported to be approximately 10 mm K⁺ (27, 28); increasing K⁺ from 4 to 10 mm will induce a stable depolarization of approximately 20 mV (5, 6, 11). This depolarization is thought to induce maximal steady state Ca²⁺ influx through T-type Ca²⁺ channels (25, 27–30). Increasing extracellular K⁺ will also increase K⁺ conductance (10); this effect stabilizes the membrane potential near the new E_K and blunts depolarizing responses to inward current (11). These effects offer an explanation for the apparently greater aldosterone response to Cs⁺- vs. K⁺-induced depolarization based on the voltage dependence of T-type Ca²⁺ channel activation, deactivation, and inactivation. Cs⁺ (10 mm) and 8 mm K⁺ induced nearly equivalent aldosterone secretory responses, yet the K⁺-induced depolarization is expected to be nearly twice as great as that induced by Cs⁺. K⁺ (8 mm) will cause a stable membrane depolarization of 15–16 mV based on the observation that K⁺-induced depolarization is closely predicted by the Nernst equation for E_K at extracellular K⁺ concentrations above 4 mm (5, 6, 11). The minimum depolarization induced by 10 mm Cs⁺ was approximately 7 mV, although this minimum depolarization was superimposed with small amplitude, transient, depolarizing spikes. This suggests that voltage-dependent fluctuations in T-type Ca²⁺ channel gating during Cs⁺-induced depolarization more effectively mediate Ca²⁺ influx and aldosterone secretion than the steady state Ca²⁺ influx response to larger, more stable depolarizations produced by elevated K⁺.

It is also of interest to compare the effects of ANG II with those of Cs⁺, as maximal Cs⁺-induced aldosterone secretion was as great as maximal ANG II-induced secretion. ANG II has been hypothesized to stimulate aldosterone secretion at least partially through membrane depolarization-induced activation of voltage-dependent Ca²⁺ channels (1–4). ANG II-induced depolarization has been attributed to AT₁ receptor-mediated inhibition of K⁺ conductance (1, 8–10, 18), but ANG II activation of nonselective cation channels (11) and modulation of T-type Ca²⁺ channels (31) may also contribute to the depolarization. Cs⁺- and ANG II-induced aldosterone secretion were nonadditive, suggesting a common mechanism of stimulation through inhibition of K⁺ conductance. ANG II probably inhibits at least two K⁺ channel classes in the type 1 rat adrenal glomerulosa cells (10): a weakly voltage-dependent (leak) K⁺ channel, which appeared to dominate the resting K⁺ conductance, and a more strongly voltage-dependent K⁺ channel activated by membrane depolarization, although this second K⁺ channel has not been identified at the single channel level or fully isolated as a component of macroscopic current. ANG II inhibition of leak K⁺ channels would be predicted to initiate membrane depolarization and enhance depolarizing responses to inward currents, whereas inhibition of the voltage-dependent K⁺ channel class would be predicted to enhance depolarizing responses initiated by other mechanisms. Compared with Cs⁺-induced depolarization, ANG II-induced depolarization appeared highly variable from one cell to the next (11, 18). ANG II-induced depolarizations usually consisted of a slow, maintained depolarization superimposed by both large (10–40 mV) and small (2–8 mV) transient depolarizing events (18) (Lotshaw, D. P., unpublished observations). The magnitude of the ANG II-induced slow minimum depolarization appears similar to that induced by Cs⁺ (10 mm), averaging 9.5 ± 3.2 mV (mean ± sd; n = 6) (Lotshaw, D. P., unpublished observations) when measured during the initial 15 min of ANG II stimulation. The slow, maintained phase of ANG II-induced depolarization coincided with an increase in membrane input resistance (11) and, therefore, may be largely attributed to inhibition of resting K⁺ conductance. The large amplitude transient depolarizations, which are not observed in response to Cs⁺, are hypothesized to represent concerted activation of several nonselective cation channels. Overall, differences between the membrane potential responses to Cs⁺ and ANG II may be attributed to several possible mechanisms: the steep voltage dependence of Cs⁺ blockade, ANG II inhibition of both leak and depolarization-activated K⁺ conductances, ANG II activation of nonselective cation channels (11), and ANG II stimulation of T-type voltage-dependent Ca²⁺ current (31). The possible contribution of T-type Ca²⁺ channels to membrane depolarization is unclear; ANG II was reported to stimulate T channels in calf glomerulosa cells (31) and inhibit T channels in adult bovine glomerulosa cells (32).

K⁺channel classes controlling the membrane potential

The results of the present study provide further support for the hypothesis that the weakly voltage-dependent leak K⁺ channels are the primary determinant of the resting membrane potential in the type 1 rat glomerulosa cell. Although Cs⁺ is a nonselective K⁺ channel blocker, the effects of Cs⁺ on single leak K⁺ channels were similar to those on the macroscopic K⁺ current, blocking inward, but not outward, K⁺ current. K⁺ channel blockers that did not inhibit leak K⁺ channels at the resting membrane potential did not induce membrane depolarization or stimulate aldosterone secretion.

The contribution of the postulated quinidine-sensitive voltage-dependent K⁺ channels to control of the resting membrane potential is unclear. These channels appeared to be active at membrane potentials more positive than approximately −50 mV, suggesting a primary role in limiting membrane depolarization and driving membrane repolarization in response to activation of large inward currents (10). Quinidine strongly inhibited both basal and stimulated aldosterone secretion. Inhibition of basal aldosterone secretion occurred without membrane depolarization, suggesting that this effect was mediated through a mechanism other than inhibition of plasma membrane K⁺ channels. The inhibitory effects of quinidine on aldosterone secretion precluded attempts to mimic the inhibitory effects of ANG II on K⁺ conductance using combinations of Cs⁺ and quinidine. The optical isomer of quinidine, quinine, was also reported to inhibit, rather than stimulate, basal and stimulated aldosterone secretion in bovine cells (13, 33).

Charybdotoxin-sensitive Ca²⁺-dependent maxi-K⁺ channels have been suggested to play an important role in control of the membrane potential (18). ANG II inhibition of these channels was postulated to increase membrane depolarization and Ca²⁺ influx during stimulation of aldosterone secretion in rat glomerulosa cells. However, in the present study and in a previous study using bovine glomerulosa cells (13), charybdotoxin did not affect either basal or stimulated aldosterone secretion, suggesting that Ca²⁺-dependent maxi-K⁺ channels did not significantly contribute to control of the membrane potential in the acutely dissociated cell preparation. In this regard, the type 1 glomerulosa cells that predominated in our primary cell cultures during the first 48 h after preparation did not express a significant charybdotoxin-sensitive membrane current (10). On the other hand, the type 2 cells that were more commonly encountered in older cultures exhibited a large charybdotoxin-sensitive Ca²⁺-dependent maxi-K⁺ current. These observations suggest that type 2 cells represented either a small proportion of the acutely dissociated cell preparation that was selected for under our culture conditions or a phenotype induced by culture conditions.

Apamin was previously reported to inhibit a transient phase of ANG II-stimulated ⁸⁶Rb efflux, but not a maintained phase of ANG II-inhibited ⁸⁶Rb efflux in bovine glomerulosa cells (7, 33), suggesting a role for an apamin-sensitive K⁺ channel. However, in rat cells, apamin did not significantly affect either basal or stimulated aldosterone secretion. In bovine cells, one study reported that apamin caused a small inhibition of ANG II-induced aldosterone secretion (33); in another study, apamin caused no measurable effect on aldosterone secretion (13). In the present study, apamin did not consistently affect cellular K⁺ currents, and these results indicated that apamin-sensitive K⁺ channels do not make a major contribution to the control of membrane potential and aldosterone secretion under basal conditions or during stimulation.

^*

This work was supported by grants-in-aid from the American Heart Association, Illinois Affiliate.

References

Copyright © 1997 by The Endocrine Society

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