From the
In adrenal glomerulosa cells, low threshold voltage-activated
(T-type) calcium channels play a crucial role in coupling physiological
variations of extracellular potassium to aldosterone biosynthesis.
Angiotensin II markedly reduced the activity of these channels by
shifting their activation curve toward positive voltage values. This
inhibition of the channels resulted in a marked decrease of the
cytosolic free calcium concentration maintained by potassium. This
effect was abolished by losartan, a specific antagonist of the
angiotensin II AT
The regulation of voltage-operated calcium channels by
intracellular messengers has been recognized as a way for hormones and
neurotransmitters to exert their effect on cell function. The
modulation of L-type (high threshold and dihydropyridine-sensitive)
calcium channel activity by various protein kinases, including the
cyclic AMP-dependent kinase and protein kinase C, as well as by
GTP-binding proteins has been extensively
documented(1, 2) . In contrast, the regulation of low
threshold, T-type calcium channels is still poorly characterized.
In
adrenal glomerulosa cells, both low and high threshold Ca
In the present study, we
used the patch-clamp technique to demonstrate that AngII inhibits
T-type Ca
Percoll was obtained from Pharmacia Biotech Inc..
Tetrodotoxin, sodium ATP, sodium GTP, and nicardipine were purchased
from Sigma, and Cs
In adrenal glomerulosa cells, the low threshold, slowly
deactivating (T-type) Ca
Protein kinase C activation by DiC
The action of AngII on
voltage-operated Ca
It
might seem paradoxical that AngII, a potent agonist of aldosterone
secretion, inhibits T-type Ca
The inhibition of glomerulosa
cell T-type Ca
In conclusion, the modulation of T-type Ca
The recording conditions were the same as those described in the
legend of Fig. 2, but the shifts in the activation and inactivation
curves (
We are grateful to Drs. P. Q. Barrett and U. Lang for
helpful discussions, to Drs. W. Schlegel and S. R. Rawlings for their
comments concerning this manuscript, and to Dr. R. D. Smith (Du Pont
Merck) for providing us with losartan (DuP753). We have also benefited
from the excellent technical assistance of L. Bockhorn, G. Dorenter,
and M. Lopez.
ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS AND DISCUSSION
FOOTNOTES
ACKNOWLEDGEMENTS
REFERENCES
receptor. Hormone action on T-type
channels appeared to be mediated by protein kinase C because 1) it was
mimicked by phorbol ester and diacylglycerol, and 2) it was
significantly reduced by decreasing protein kinase C activity with
specific inhibitors such as chelerythrine chloride or a pseudosubstrate
of the enzyme, as well as by protein kinase C down-regulation.
Similarly, protein kinase C activation reduced the cytosolic calcium
response to potassium and the steroidogenic action of this agonist. Low
threshold T-type calcium channels therefore appear as potential sites
for the modulation of steroidogenesis by protein kinase C in adrenal
glomerulosa cells.
channels have been described(3, 4) . The exquisite
sensitivity of these cells to small variations of extracellular
K
concentrations, as well as their very negative
membrane potential(5) , strongly suggest a role for T-type
calcium channels in the steroidogenic response to this
agonist(6) . The involvement of these channels in the sustained
cytosolic free calcium ([Ca
]
)
response to AngII
(
)is more debated. Indeed, by
closing some K
conductances, AngII is expected to
depolarize glomerulosa cells (5, 7, 8, 9) and, therefore, to activate
T-type channels. However, the poor effect of nicardipine, a
dihydropyridine blocking both L- and T-type channels, on the
[Ca
]
response to AngII suggests
that other Ca
influx pathways are stimulated by the
hormone(10) . In addition, AngII has been shown to antagonize
the sustained [Ca
]
response
induced by K
(11, 12), suggesting a negative
modulation of T channels by the hormone.
channels in bovine adrenal glomerulosa
cells by shifting the channel activation curve toward more positive
potential values. This inhibition of the channels is mediated by
protein kinase C (PKC) and results in a marked reduction of the
[Ca
]
response and aldosterone
secretion induced by K
.
BAPTA and fura-2 acetoxymethyl ester from
Molecular Probes (Eugene, OR). 1-oleoyl-2-acetyl-sn-glycerol
(OAG), sn-1,2-dioctanoylglycerol (DiC
), phorbol
12-myristate 13-acetate (PMA), chelerythrine chloride, and thapsigargin
were from LC Laboratories (Woburn, MA), AngII was from Bachem AG
(Bubendorf, Switzerland), and protein kinase C pseudosubstrate peptide
inhibitor was obtained from Peninsula Laboratories Inc. (Belmont, CA).
Losartan (DuP753) was a generous gift from Dr. R. D. Smith, DuPont
Merck Pharmaceuticals (Wilmington, DE).
Adrenal Glomerulosa Cell Isolation and Culture
Bovine
adrenal glands were obtained from a local slaughterhouse, and
glomerulosa cells were prepared by enzymatic dispersion, purified on a
Percoll density gradient, and maintained in culture for 2-4 days,
as described in detail elsewhere(4) .
Patch-Clamp Measurements
The activity of slowly
deactivating (T-type) Ca channels was recorded under
voltage clamp in the whole cell configuration of the patch-clamp
technique, as described previously(4) . The bath solution
contained (in mM): 117 tetraethylammonium chloride, 20
BaCl
, 0.5 MgCl
, 5 D-glucose, 32
sucrose, and 200 nM tetrodotoxin, and was buffered to pH 7.5
with 10 mM Hepes/CsOH. The patch pipette (3-6 megohm,
Clark 150T, Reading, United Kingdom) contained (in mM): 85
CsCl, 10 tetrabutylammonium chloride, 6 MgCl
, 5 sodium ATP,
and 0.04 sodium GTP, and pH was buffered to 7.2 with 20 mM Hepes/CsOH. The pipette solution also contained 0.9 mM
CaCl
and 11 mM Cs
BAPTA in order to
buffer free calcium below 50 nM. Agents were directly added to
the bath or introduced in the patch pipette, as indicated in the legend
of . Diacylglycerols (OAG and DiC
) were stored
in small aliquots at -20 °C, in chloroform and under N
atmosphere to prevent oxidation by air. Before use, aliquots were
dried under N
, resuspended in experiment buffer and
sonicated. The reference electrode was placed in a KCl solution linked
to the bath with an agar bridge; the resulting liquid junction
potential was smaller than 2 mV and has been neglected. The cell was
voltage-clamped (Axopatch 1D, Axon Instruments Inc., Foster City, CA)
at a holding potential of -90 mV and depolarized as indicated.
Fairly round and small cells, with a diameter of approximately 20
µm and a membrane capacitance of 15.8 ± 5.6 picofarads
(S.D., n = 50), were chosen in order to optimize the
spatial voltage clamp. The Ba
currents were filtered
at 1 kHz and sampled at 6.2 kHz. Leak was subtracted either digitally
after the experiment or automatically by a P/4 protocol (pclamp 5.5,
Axon Instrument Inc.). In a few experiments, pipettes coated with
Sylgard (Dow Corning, Seneffe, Belgium) instead of plain pipettes have
been used for recording T-type currents and similar characteristics
(kinetics of activation and deactivation, V
of
activation and inactivation) have been observed under both conditions.
Cytosolic Free Calcium Measurements
For
[Ca]
determinations, freshly
isolated glomerulosa cells were purified on a Percoll density gradient
and resuspended in a Krebs-Ringer medium (4) at a concentration
of 5
10
cell/ml. Cells were then incubated for 30
min at 37 °C in the presence of 2 µM fura-2
acetoxymethyl ester, washed, and immediately used for determination of
the fura-2 fluorescence (excitation at 340/380 nm and emission at 500
nm) in a Jasco CAF-110 fluorometer (Hachioji City, Japan). The
fluorescence signal was digitized (DaQSys 2.0, Sicmu, University of
Geneva), and [Ca
]
was
calibrated using the ratio values of emitted fluorescence (340 nm/380
nm) as described in Ref. 13.
Determination of Aldosterone Formation
Measurement
of aldosterone production was performed as described
elsewhere(10) . Glomerulosa cells, cultured for three days, were
incubated at 37 °C in multiwell plates containing a Krebs-Ringer
medium and various concentrations of potassium and PMA or
DiC. At the end of the incubation period, the aldosterone
content of the medium was determined by direct radioimmunoassay, using
a commercially available kit (Diagnostic Products Corp., Los Angeles,
CA). Cellular proteins were measured using the Coomassie Blue method of
Bradford(14) .
channels appear perfectly
suited for controlling Ca
influx, a signal sufficient
for the activation of steroidogenesis(15, 16) , in
response to small depolarizations of the membrane. The analysis of
slowly deactivating Ba
tail currents, elicited upon
cell repolarization after a short (20 ms) depolarizing pulse (Fig. 1, A and B), allowed us to discriminate
between T- and L-type channels in these cells. Indeed, L channels have
been shown to rapidly deactivate (in a few milliseconds) at -65
mV(6) , and T channels can be considered as exclusively
responsible for the slowly decaying current(4) . The time
constant of T channel deactivation at -65 mV, as assessed by
single exponential fitting, starting 5 ms after cell repolarization,
was 6.6 ± 0.3 ms (n = 61). The analysis of
activation (voltage-dependent opening) and steady-state inactivation
(lack of opening upon strong depolarization) characteristics of T
channels (Fig. 1C) revealed the presence of a permissive
``window'' of voltage, in which activation and inactivation
curves overlap. As a consequence, at these voltages, the channels are
already partially activated but not yet completely inactivated. This
window therefore determines the range of voltage over which a
steady-state current can flow through T channels, and the relative
amplitude of this current can be calculated (6) as a function of
voltage using Ohm's law (Fig. 1D). The resting
potential of glomerulosa cells has been estimated to be -79
mV(5) , a value that is close to the threshold of this current.
Figure 1:
Electrophysiological properties of low
threshold (T-type) calcium channels in bovine adrenal glomerulosa
cells. A, slowly deactivating Ba currents,
recorded in the whole cell configuration of the patch-clamp technique
(see ``Materials and Methods''), were elicited upon
repolarization after a short period of activation (20 ms) at various
depolarizing potentials. Example of six superimposed tail currents
evoked in a representative glomerulosa cell at -65 mV, after
membrane depolarization to various voltages (-45 to +5 mV,
steps of 10 mV) from a holding potential of -90 mV. The time
constant of the slowly decaying current in this cell was 6.9 ±
0.1 ms. B, slowly deactivating Ba
currents
elicitable after steady-state inactivation at various potentials. Tail
currents (time constant = 7.5 ± 0.2 ms) were similarly
elicited (at -65 mV) in the same cell, but after steady-state
inactivation of T channels for 10 s at various holding potentials (from
-80 to -30 mV) and 20 ms of activation at +20 mV. C, activation and inactivation curves of the T-type channels.
Voltage-dependent activation (
) and inactivation (
) of
slowly deactivating currents were measured from traces similar to those
presented in panelsA and B, respectively,
and as described elsewhere (19). The maximal current, elicited at the
time of cell repolarization, was determined by extrapolating the tail
currents fitted to a single exponential. Current amplitudes were
plotted as a function of test voltage, after fitting to
Boltzman's equation and normalization to the maximum of the
function (I
). The potentials at which the ratio of
currents I/I
is 0.5 (V
) were -23.5 and -50.3 mV for
activation and inactivation, respectively. D, steady-state
current flowing through T-type channels. The theoretical steady-state
current (I
) was determined as a function of
voltage from the Ohm's equation (6): I
= g
mh(V
- V), where g
is the maximal
barium conductance through T channels (when all channels are open) and
arbitrarily chosen equal to 1.0, m and h are the
fractions of open channels (I/I
),
calculated from Boltzman's equation in activation and
inactivation experiments, respectively, and V
is
the reversal potential measured to be = +50 mV in this
cell.
AngII, a physiological secretagogue of aldosterone, significantly
shifted the activation curve of T channel toward positive voltage
values, without affecting the inactivation curve (Fig. 2A and ). This resulted in a marked reduction of the size
of the permissive voltage window and therefore of the amplitude of the
steady-state current (Fig. 2B).
Figure 2:
Inhibition of T-type channels by
angiotensin II. A, angiotensin II effect on the T channel
activation curve. Cultured glomerulosa cells were voltage-clamped as
indicated in the legend of Fig. 1, and Ba currents
were recorded before and 3 min after addition of 50 nM AngII
to the bath. Each cell was independently analyzed; the parameters of
Boltzman's function were determined, and the currents were
normalized before being averaged. A, activation (
,
) and inactivation (
,
) curves were established, as
described in the legend of Fig. 1, before (
,
) or 3 min
after exposure of the cell to 50 nM AngII (
,
).
Data are the mean ± S.E. from 13 cells obtained from 9
independent preparations. The mean V
for
inactivation was -53.5 mV before and -53.8 mV after hormone
application, whereas the V
for activation was
shifted from -29.3 to -20.9 mV by the same treatment. B, inhibition of the steady-state current by angiotensin II.
The theoretical steady-state currents, before and after hormone
treatment, were determined as described in the legend of Fig.
1D.
This inhibitory
action of AngII on T channels could also be demonstrated by measuring
the decrease of the cytosolic free calcium concentration after
stimulation of fura-2-loaded glomerulosa cells with K (Fig. 3A). After depletion of intracellular
Ca
pools and activation of the capacitative
Ca
influx with 400 nM thapsigargin (10), the
addition of 12 mM KCl induced a marked and sustained rise in
[Ca
]
. This maximal response to
K
was rapidly reduced upon stimulation with AngII (50
nM), and nicardipine (2 µM) completely blocked
the residual T channels (10), which had remained unaffected by the
hormone. The fact that [Ca
]
decreased below the basal level after nicardipine treatment
suggests the presence of a basal T channel activity in some resting
cells, a fact reported previously(10) . We have estimated that,
at this concentration, AngII inhibited by 70% the nicardipine-sensitive
[Ca
]
response to K
(n = 7), a value in agreement with the inhibition
predicted by the shift of the current activation curve ().
The action of AngII was prevented by the presence of 10 µM losartan (DuP753), a specific antagonist of the AT
receptor subtype (Fig. 3B).
Figure 3:
Inhibition of the potassium-induced
cytosolic calcium response by angiotensin II. A, fura-2-loaded
cells were sequentially exposed to 400 nM thapsigargin, 12
mM KCl, 50 nM AngII, and 2 µM
nicardipine. In traceB, 10 µM losartan
was added 100 s before AngII. [Ca]
was determined as described under ``Materials and
Methods.'' The traces are representative of two independent
experiments.
Since
hormone-induced inhibition of Ca influx through T
channels required activation of AT
receptors but was not
dependent upon Ca
release from intracellular stores
or activation of the capacitative influx, a possible role for PKC was
investigated. Indeed, three exogenous activators of PKC, PMA (1
µM), OAG (50 µM), and, to a smaller extent,
DiC
(100 µM) mimicked hormone action by
significantly shifting the activation curve (). Moreover,
the effect of AngII was markedly decreased when PKC activity was
partially reduced by exposure to chelerythrine chloride (1
µM), or when protein kinase C pseudosubstrate peptide
inhibitor (17) was present in the patch pipette at a
concentration of 10-100 µM. Protein kinase C
down-regulation by a 24-h treatment with PMA (100 nM), which
reduced enzyme activity by approximately 85%(18) , also
partially prevented AngII action (). No shift of T channel
activation curve larger than 2 mV was observed in control
(unstimulated) cells or in cells exposed to 5 µM thapsigargin (19) or 25 µM forskolin (not
shown), and no significant effect of the various treatments was
observed on the inactivation characteristics of the channel (). The inhibition of the maximal steady-state current
through T channels due to the shift of their activation curve was also
calculated (). This inhibition, greater than 60% in the
presence of AngII, OAG, or PMA, was significantly (p <
0.05) reduced to less than 40% after decreasing PKC activity.
(100 µM)
resulted in a marked reduction of the
[Ca
]
maintained by potassium (Fig. 4B) with a kinetics similar to the inhibition
induced by AngII (Fig. 4A). When added after
diacylglycerol, AngII only reduced
[Ca
]
minimally, a result
suggesting that both agents act through the same pathway. The effect of
DiC
was concentration-dependent, 50% inhibition of the
response to K
being observed at approximately 100
µM (not shown). The low solubility of this agent in
aqueous solutions prevented a use at concentrations above 300
µM.
Figure 4:
Involvement of protein kinase C in
angiotensin II-induced inhibition of the calcium response to potassium. A, fura-2-loaded cells were exposed to 200 nM thapsigargin, 9 mM KCl, 10 nM AngII, and 2
µM nicardipine. In B, addition of KCl was
followed by addition of 100 µM DiC. The same
experiment was repeated in three independent cell preparations, giving
similar results.
The inhibition of the
[Ca]
response to K
by DiC
was not due to a ``desensitization''
of the cell to extracellular K
. Fig. 5A shows the response to 3 mM step increases in
[K
] in control (untreated) cells and in
cells pretreated for 5 min with 100 µM DiC
.
The response in treated cells was reduced at each K
concentration (Fig. 5B), and the inhibition (44%)
could not be overcome by increasing K
. The EC
for the [Ca
]
response was
5.6 ± 0.2 mM added K
in control cells
and 5.7 ± 0.2 mM in DiC
-treated cells. This
is in agreement with the predicted inhibition of the relative size of
the steady-state current, without noticeable shift in the sensitivity
to potential (Fig. 2B). Interestingly, other activators
of PKC, such as OAG or PMA, were much less efficient in reducing the
K
-induced [Ca
]
response. This difference could possibly be explained by a less
efficient activation of PKC in adrenal glomerulosa cells by these
agents, as already suggested by others(20) .
Figure 5:
Inhibition of the calcium response to
potassium by dioctanoyl-glycerol. A, fura-2-loaded cells,
either untreated (C) or exposed for 5 min to 100 µM DiC, were stimulated by stepwise increases in
extracellular K
concentration; basal
[K
]: 3 mM, step increase: 3
mM. B, mean increase in
[Ca
]
induced by various
increases in KCl concentrations, calculated from four independent
experiments performed as in panelA. Data were fitted
to a four-parameter logistic function.
As expected, PMA
and DiC also markedly reduced aldosterone secretion
activated by extracellular potassium (Fig. 6). PMA inhibited up
to 80% of the steroidogenic response to 12 mM potassium in a
concentration-dependent manner, with an IC
of 42 nM (Fig. 6A). At 100 nM (Fig. 6B), this agent similarly reduced aldosterone
production in response to increasing concentrations of potassium,
without significantly affecting the sensitivity of steroidogenesis to
the agonist. Although we cannot completely exclude a direct effect of
PMA (and PKC) on the various steps of steroidogenesis, our data suggest
that the main inhibitory action of PMA on aldosterone secretion is
secondary to its effect on the Ca
channel. Indeed, in
a separate study, we have observed that when aldosterone production is
activated by a rise of [Ca
]
induced by ionomycin, a selective ionophore, and not through
opening of T channels, PMA does not affect steroid output(16) .
Figure 6:
Protein kinase C-mediated inhibition of
the steroidogenesis induced by potassium. A and C,
concentration-dependent inhibition by PMA (A) and DiC (C). Aldosterone secretion induced by 12 mM KCl
was inhibited by increasing concentrations of PMA or DiC
;
data of panelA (mean values ± S.E. from 3
independent experiments) were fitted to a sigmoidal four-parameter
logistic function to determine an IC
value of 42
nM. A maximal inhibition of 80% was achieved at 1 µM PMA. In C, the effect of DiC
on unstimulated
cells is also shown (n = 4). B and D,
effect of 100 nM PMA (B) or 100 µM DiC
(D) on the steroidogenic response to
extracellular potassium. Data represent the mean values ± S.E.
from 11 and 6 independent cell preparations for B and D, respectively. EC
values were estimated after
fitting the data to logistic functions and were not significantly
different in treated and control cells.
DiC action on aldosterone secretion appeared somewhat
more complex. Although a marked inhibition of aldosterone secretion was
observed at 100 µM, a slight potentiation of the response
to potassium was obtained at concentrations ranging between 1 and 30
µM (Fig. 6C). The relevance of this
potentiation is currently under investigation in our laboratory, but it
does not appear to be dependent upon T channel activity, as suggested
by the lack of a positive effect of DiC
on the
[Ca
]
response (not shown). At
100 µM, DiC
reduced steroidogenesis at each
KCl concentration by approximately 40%. Like PMA, this agent did not
affect the cell sensitivity to potassium.
channels in adrenal glomerulosa
cells is quite controversial. A hormone-induced, PMA-insensitive,
activation of L-type, but not of T-type, currents has been reported in
Y1 cells, an adrenal cortical cell line(21) . However, an
involvement of high threshold (L-type) channels in normal glomerulosa
cells, whose resting potential is much more negative than that of Y1
cells, is questionable. Recently, McCarthy et al.(22) described a GTP-dependent activation of T-channels by
AngII in bovine adrenal glomerulosa cells. In this study, AngII
appeared to shift the channel activation curve toward more negative
values without affecting the inactivation curve. Another group (23)
showed evidence for a role for calmodulin-dependent protein kinase II
in this effect of AngII. Although we have at the present time no
explanation for the discrepancy between these results and our data, an
inhibitory action of AngII on T channel is more in agreement with the
well documented reduction by AngII of the
[Ca
]
response to
potassium(11, 12) . In non-differentiated NG
108-15 cells, AngII has been shown to reduce the T-type
Ca
current through the angiotensin AT
receptor subtype, by a mechanism presumably involving a
phosphotyrosine phosphatase(24) . In bovine adrenal glomerulosa
cells, which almost exclusively express the AT
subtype(25) , AngII appears to exert its action through
the latter subtype (Fig. 3B) and PKC activation.
channels, whose
activity is indispensable for the steroidogenic response to potassium.
However, we recently demonstrated that the majority of the
Ca
entering the cell in response to AngII uses a
different mechanism, namely the capacitative Ca
influx pathway, which is regulated by intracellular
Ca
pools and independent of voltage-operated
Ca
channels(10) . In addition, the inhibitory
effect of AngII on T channels, highlighted under voltage-clamp
conditions, is partially balanced by its depolarizing action on
glomerulosa cells(7) . Interestingly, as a consequence, the net
hormonal effect on T channels appears to be different in resting or in
potassium-stimulated cells,(
)
but the
physiological relevance of this observation remains to be determined.
Finally, the steroidogenic action of AngII, in contrast to that of
K
, also involves Ca
-independent
mechanisms(11, 16) .
channels through a shift of their
activation curve toward positive potentials is not the only modulatory
mechanism of the activity of these channels. Indeed, atrial natriuretic
peptide (ANP), an antagonist of aldosterone secretion, has been shown
to reduce T-type current by selectively shifting the inactivation curve
of the channel toward negative voltages (6). It is noteworthy that two
different hormones inhibit the same channel by affecting distinct
properties of this channel. Because ANP induces cGMP formation and
therefore activates a cGMP-dependent kinase, one could speculate that
the latter kinase is involved in the modulation of the channel by ANP
and that various regulatory sites are present on the same channel.
channels by AngII provides the hormone with a mechanism to finely
control Ca
entry and, therefore, steroidogenesis in
adrenal glomerulosa cells. The demonstration of a complex hormonal
regulation of this channel, by protein kinases, phosphatases, or
G-proteins, should trigger further studies leading to the purification,
cloning, and molecular characterization of such an important effector
in glomerulosa cell function.
Table: Role of protein kinase C in the
angiotensin II-induced shift of T-type calcium channel activation curve
V
) induced by AngII, PMA, OAG, or
DiC
were determined for each cell independently. These
agents were directly added to the bath. The 5th group of cells was
pretreated for 1 h at 37 °C in the presence of 1 µM chelerythrine chloride before being exposed to AngII (50
nM) in the continuous presence of chelerythrine. PKCI, the
enzyme pseudo substrate peptide inhibitor (17), was introduced into the
pipette solution at a concentration of 10-100 µM.
The last group of cells was incubated for 24-32 h in the presence
of 100 nM PMA before current recording. The steady-state
current inhibition due to the shift of the activation curve observed in
each experimental condition was also determined as in Fig. 2B,
and expressed as percent inhibition of maximal current. Results are the
mean values ± S.E., and the number of cells tested (n)
is indicated. The statistical significance of the difference between V
values of activation before and after
treatment with AngII, PMA, OAG, or DiC
was assessed by the
Student's t test; NS, not significantly different from
untreated cells.
, sn-1,2-dioctanoylglycerol; PMA, phorbol 12-myristate
13-acetate; ANP, atrial natriuretic peptide.
©1995 by The American Society for Biochemistry and Molecular Biology, Inc.