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Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60115
Address all correspondence and requests for reprints to: Dr. David P. Lotshaw, Department of Biological Sciences, Northern Illinois University, DeKalb, Illinois 60112.
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Abstract |
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2 mM). Depolarization consisted
of a slow, maintained phase proportional to Cs+
concentration superimposed with 2- to 5-mV transient depolarizing
events. Cs+ induced a Ca2+-dependent
stimulation of aldosterone secretion in acutely dissociated cells,
exhibiting an EC50 of approximately 3 mM.
Maximal Cs+-induced secretion was quantitatively
similar to 1 nM ANG II- or 8 mM
K+-induced secretion. Cs+-induced secretion was
not additive with that of ANG II. K+ channel blockers that
did not inhibit weakly voltage-dependent K+ channels at
rest (quinidine, apamin, and charybdotoxin) did not cause
depolarization or stimulate aldosterone secretion. Furthermore,
charybdotoxin did not significantly affect ANG II-induced aldosterone
secretion, indicating that Ca2+-dependent
maxi-K+ channels did not contribute to the control of
aldosterone secretion in acutely dissociated cells. These data strongly
support involvement of weakly voltage-dependent K+ channels
in ANG II-induced aldosterone secretion, but also implicate roles
for other channel classes in controlling membrane potential during ANG
II-induced aldosterone secretion. |
Introduction |
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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, 13, 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, 16, 17, 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.
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Materials and Methods |
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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.
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Results |
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. 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).
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. 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 (EK) 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 EK (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 +
(Kd/[Cs+])-n]-1,
where G is the slope conductance, Kd 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 Kd
of 2.4 ± 0.32 mM (±SE; giving a 95%
confidence interval for Kd 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 EK (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
EK 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
EK, K+ entry into the channel pore from the
cytosolic side would electrostatically repel Cs+ from the
pore, thus opposing Cs+ blockade (24).
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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+.
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). 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.
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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.
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) 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.
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Discussion |
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TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
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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 EK in a depolarizing direction, causing membrane depolarization and stimulation of aldosterone secretion through activation of voltage-dependent T-type Ca2+ channels (25, 26, 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, 28, 29, 30). Increasing extracellular K+ will also increase K+ conductance (10); this effect stabilizes the membrane potential near the new EK 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 EK 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, 2, 3, 4). ANG II-induced depolarization has been attributed to AT1 receptor-mediated inhibition of K+ conductance (1, 8, 9, 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.
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Footnotes |
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Received February 21, 1997.
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I. Arrighi, M. Bloch-Faure, F. Grahammer, M. Bleich, R. Warth, R. Mengual, M.-D. Drici, J. Barhanin, and P. Meneton Altered potassium balance and aldosterone secretion in a mouse model of human congenital long QT syndrome PNAS, July 17, 2001; 98(15): 8792 - 8797. [Abstract] [Full Text] [PDF]
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