(Received for publication, March 13, 1995; and in revised form, June 8, 1995)
From the
In bovine adrenal zona fasciculata (AZF) cells, angiotensin II
(AII) may stimulate depolarization-dependent Ca entry
and cortisol secretion through inhibition of a novel potassium channel
(I
), which appears to set the resting potential of these
cells. Aspects of the signaling pathway, which couples AII receptors to
membrane depolarization and secretion, were characterized in patch
clamp and membrane potential recordings and in secretion studies.
AII-mediated inhibition of I
, membrane depolarization, and
cortisol secretion were all blocked by the AII type I (AT
)
receptor antagonist losartan. These responses were unaffected by the
AT
antagonist PD123319. Inhibition of I
by AII
was prevented by intracellular application of guanosine
5`-O-2-(thio)diphosphate but was not affected by
pre-incubation of cells with pertussis toxin. Although mediated through
an AT
receptor, several lines of evidence indicated that
AII inhibition of I
occurred through an unusual
phospholipase C (PLC)-independent pathway. Acetylcholine, which
activates PLC in AZF cells, did not inhibit I
. Neither the
PLC antagonist neomycin nor PLC-generated second messengers prevented
I
expression or mimicked the inhibition of this current by
AII. I
expression and inhibition by AII were insensitive
to variations in intracellular or extracellular Ca
concentration. AII-mediated inhibition of I
was
markedly reduced by the non-hydrolyzable ATP analog adenosine
5`-(
,
-imino)triphosphate and by the non-selective protein
kinase inhibitor staurosporine. The protein phosphatase antagonist
okadaic acid reversibly inhibited I
in whole cell
recordings. These findings indicate that AII-stimulated effects on
I
current, membrane voltage, and cortisol secretion are
linked through a common AT
receptor. Inhibition of I
in AZF cells appears to occur through a novel signaling pathway,
which may include a losartan-sensitive AT
receptor coupled
through a pertussis-insensitive G protein to a staurosporine-sensitive
protein kinase. Apparently, the mechanism linking AT
receptors to I
inhibition and Ca
influx in adrenocortical cells is separate from that involving
inositol trisphosphate-stimulated Ca
release from
intracellular stores. AII-stimulated cortisol secretion may occur
through distinct parallel signaling pathways.
AII ()is a peptide that physiologically regulates the
secretion of corticosteroid hormones. In most mammals, AII stimulates
the secretion of the mineralocorticoid aldosterone from the AZG, while
in other species the peptide also enhances cortisol production by the
AZF cell. AII regulates corticosteroid production by
Ca
-dependent mechanisms, which involve both
Ca
release from an intracellular pool and
Ca
influx across the plasma
membrane(1, 2) . The cellular mechanisms that underlie
AII-stimulated increases in Ca
are only partially
understood and may involve multiple receptors and signaling pathways.
Two pharmacologically distinct types of AII receptors have been
identified in various cells, including those of the adrenal cortex (3, 4, 5) . Losartan-sensitive AT receptors are coupled through separate G proteins to at least two
signaling pathways. Most physiological responses mediated through
AT
receptors involve activation of PLC, which catalyzes the
synthesis of inositol trisphosphate (IP
) and diacylglycerol
from phosphotidylinositol
4,5-bisphosphate(4, 6, 7) . IP
triggers the release of Ca
from intracellular
stores. Other AT
receptors are coupled to adenylate cyclase
inhibition through a PTx-sensitive G protein(8) . AT
receptors are losartan-insensitive but are blocked by
PD123319(3, 4) . Although AT
receptors
comprise 20% of AII receptors in rat adrenocortical cells, the
signaling pathway and function of these receptors in corticosteroid
secretion is unknown(3) .
In adrenal cortical cells,
AT receptor activation leads to IP
synthesis
and Ca
release from intracellular stores(9) .
The signaling pathway that mediates AII-stimulated Ca
influx remains to be identified. In many secretory cells,
Ca
enters through specific voltage-gated channels
activated in response to membrane depolarization. Recently, we
identified a novel K
permeable channel expressed in
bovine AZF cells that appears to set the membrane
potential(10) . AII inhibits this K
current
(I
) and depolarizes AZF cells at concentrations that
stimulate cortisol secretion. Further, AII-stimulated secretion is
blocked by selective antagonists of T-type Ca
channels, the major Ca
channel expressed in
these cells(11, 12) . These findings appear to
identify a specific cellular mechanism, whereby the binding of AII to a
specific receptor on AZF membranes is coupled to
depolarization-dependent Ca
entry and cortisol
secretion. The present study was done to characterize components of
this signal transduction pathway in bovine adrenal cortical cells.
Tissue culture media, antibiotics, fibronectin, and fetal
calf serum were obtained from Life Technologies, Inc. Culture dishes
were purchased from Corning. Coverslips were from Bellco (Vineland,
NJ). Enzymes, AII, GTP, MgATP, AMP-PNP, GDPS, GTP
S, pertussis
toxin, IP
, neomycin, arachidonic acid, acetylcholine,
phenidone, phorbol 12-myristate 13-acetate (PMA), and indomethacin were
obtained from Sigma. AII receptor antagonists losartan and Du 532 were
kindly provided by Dr. Ronald Smith (Dupont-Merck Pharmaceutical Co.).
PD123319 (4) was kindly provided by Dr. Joan Keiser
(Parke-Davis, Ann Arbor, MI). Staurosporine was purchased from Biomol
(Plymouth Meeting, PA). Okadaic acid was obtained from Calbiochem.
AZF cells were used for patch clamp experiments 2-12
h after plating. Typically, cells with diameters of 15-25 µm
and capacitances of 15-30 picofarads were selected. Coverslips
were transferred from 35-mm culture dishes to the recording chamber
(volume, 1.5 ml), which was continuously perfused by gravity at a rate
of 3-5 ml/min. Patch electrodes with resistances of 1.0-2.0
megohms for whole cell or 10-20 megaohms for single channel
recording were fabricated from Corning 0010 glass (Garner Glass Co.,
Claremont, CA). K currents were recorded at room
temperature (22-24 °C) following the procedure of Hamill et al.(15) using a List EPC-7 patch clamp amplifier.
Pulse generation and data acquisition were done using an IBM-AT computer and PCLAMP software with an Axolab interface (Axon Instruments, Inc., Burlingame, CA). Currents were digitized at 1-50 kHz after filtering with an 8-pole Bessel filter (Frequency Devices, Haverhill, MA). Linear leak and capacity currents were subtracted from current records using scaled hyperpolarizing steps of to amplitude. Data were analyzed and plotted using PCLAMP (CLAMPAN and CLAMPFIT) and GraphPAD InPLOT. Drugs were applied by bath perfusion, controlled manually by a six-way rotary valve.
Previously, it was shown that AII inhibits I by a
maximum of approximately 77% with an IC
of 145
pM(10) . We have explored the mechanism of
AII-mediated inhibition of I
in adrenocortical cells.
Cells were clamped in the whole cell configuration, and K
currents were recorded at 30-s intervals until I
grew to a stable amplitude. In voltage protocols designed to
activate both I
and the transient A-type K
current, I
was measured as the non-inactivating
K
current present at the end of a 300-ms test pulse to
+20 mV, applied from a holding potential of -80 mV. A second
voltage protocol was designed to isolate I
by elimination
of the rapidly inactivating A-type K
current (Fig. 1B), which inactivated completely at -20 mV (16) .
Figure 1:
Effect of losartan on AII inhibition of
I. Whole cell I
current was recorded from an
AZF cell at 30-s intervals. I
was allowed to grow to a
stable amplitude before superfusing drugs. Voltage clamp protocol is
shown in B. The 10-s prepulse to -20 mV served to
inactivate the transient A-type current. I
was then
measured as the non-inactivating current present during the subsequent
300-ms test pulse to +20 mV. A, I
amplitude
plotted against time. Losartan (500 nM) and/or AII (2
nM) were superfused for the indicated times. B,
voltage protocol. C, I
current records from the
same cell as in A in control saline, in the presence of both
losartan and AII, and after exposure to AII
alone.
The selective AT receptor antagonist
losartan and the AT
receptor antagonist PD123319 (4, 5) were used to identify the AII receptor subtype
mediating inhibition of I
. In the experiment illustrated
in Fig. 1, the cell was pre-exposed to losartan (500
nM) for 5 min before superfusing saline containing both this
antagonist and AII (2 nM). In the presence of losartan, AII
had no effect on I
(Fig. 1, A and C). When the same cell was re-exposed to AII after losartan
was removed by a 5-min wash in control saline, I
was
inhibited by 70%. Similar results were obtained in each of three
experiments (Fig. 2B). AII-mediated inhibition of
I
was not easily reversed, even after prolonged washing
with saline containing losartan.
Figure 2:
Effect of PD123319 and losartan on AII
inhibition of I. Whole cell I
current was
recorded from an AZF cell at 30-s intervals as described in the legend
to Fig. 1. Cell was perfused with solutions containing PD123319
(500 nM) or PD123319 and AII (2 nM). A,
I
amplitude plotted against time before and during
superfusion of drugs as indicated. B, summary of data from
experiments similar to those described in Fig. 1and Fig. 2A. Bars indicate fraction of I
inhibited by 1 nM AII (n = 7), 5 nM AII (n = 5), 2 nM AII + 500 nM losartan (n = 3), and 2 nM AII + 500
nM PD123319 (n =
3).
The AT-antagonist
PD123319 did not blunt or prevent AII-mediated inhibition of I
(Fig. 2A). In the presence of 500 nM PD123319, 2 nM AII inhibited I
by 83.5
± 12.5% (n = 3). By comparison, AII alone at
concentrations of 1 and 5 nM inhibited I
by 65.7
± 4.9% (n = 7) and 71.6 ± 5.1% (n = 5) (Fig. 2B). These results show clearly
that AII-mediated inhibition of I
in AZF cells occurs
exclusively through activation of a losartan-sensitive receptor.
Figure 3: Effect of AII antagonists on AII-stimulated depolarization of AZF cells. Membrane potential was recorded continuously with KCl-filled electrodes as described under ``Materials and Methods.'' After impaling the cells and obtaining a stable resting potential, cells were superfused with AII (2 nM) and/or losartan (500 nM) or PD 123319 (500 nM).
Figure 4: Effect of AII antagonists on AII-stimulated cortisol production. Cultured AZF cells were incubated for 24 h in serum-free control defined media (DMEM/F12 with 50 µg/ml bovine serum albumin, 100 µM ascorbic acid, 1 µM tocopherol, 100 nM insulin, and 10 µg/ml transferrin) or the same media containing AII and the antagonists losartan, PD 123319, or Dup 532 at the indicated concentrations. Media samples were collected at 6 and 24 h for cortisol measurement. Values are mean ± S.E. of triplicate determinations. Results are representative of three similar experiments.
Complete inhibition of cortisol secretion by the competitive
antagonist losartan could be overcome by increasing the AII
concentration to 200 nM (Fig. 4B). In
contrast, the non-competitive AT antagonist, Du 532, at an
equivalent concentration (relative to reported K
values) completely inhibited cortisol secretion stimulated
by 200 nM AII (Fig. 4B).
Figure 5:
Voltage independence and specificity of
AII inhibition of I. After obtaining whole cell clamp,
K
currents were recorded at 30-s intervals in response
to voltage steps to +20 mV from a holding potential of -80
mV (inset). When I
had grown to a stable value,
voltage ramps of 100 mV/s were applied from a holding potential of 0 mV
to voltages between -140 and +60 mV. A second voltage ramp
was recorded after steady state block with 10 nM AII. Pseudo
steady state current-voltage relationships were obtained for I
currents activated by ramps.
Figure 6:
Effect of guanine nucleotides and PTx on
AII-mediated inhibition of I. The amplitude of I
was measured at 30-s intervals as described in Fig. 1with
pipette solutions containing 200 µM GTP (control), no
added GTP (0 GTP), or no added GTP with 1 mM GDP
S
(GTP
S). Alternatively, cells were pretreated for 6-12 h with
200 ng/ml PTx before recording currents using control pipette solution. A, effect of GDP
S on AII block of I
.
I
was continuously recorded at 30-s intervals over a 1-h
period using a pipette containing 1 mM GDP
S. Cell was
superfused at indicated AII concentrations for durations shown.
I
amplitude is plotted against time. B, effect of
PTx. PTx-treated cell was superfused with 1 and 10 nM AII as
indicated. C, summary of results from experiments similar to
those in A and B. Bars indicate fraction of
I
inhibited by 10 nM AII with control pipette
solution, 76.5 ± 4.6% (n = 6); with pipette
solution containing no GTP, 79 ± 8.0% (n = 2);
with 1 mM GDP
S in the pipette, 12.8 ± 10.4% (n = 7); or with control pipette solution using cells
pretreated with PTx, 73 ± 10.6% (n = 4).
Experiments with GDPS indicated that
AII-mediated inhibition of I
occurred through a G protein
intermediate. To determine if either G
or G
mediated this inhibition, adrenal cortical cells were
pre-incubated prior to patch clamping with PTx, which suppresses
activation of G
and G
. PTx had no significant
effect on the time-dependent growth of I
or its inhibition
by AII (Fig. 6, B and C). In cells pretreated
for 6-12 h with PTx (200 ng/ml) (Fig. 6C), AII (2
nM) inhibited I
by 73 ± 10.6% (n = 4). Results with guanine nucleotides and PTx indicated
that AII-mediated inhibition of I
requires a G protein
other than G
or G
.
Figure 7:
Inhibition
of unitary K currents by AII. Unitary K
currents were recorded from cell-attached patches of bovine AZF
cells. Unitary K
channel activity was continuously
recorded at a potential of +100 mV positive to the resting
potential. Pipette contained standard external solution. AII was
applied by bath perfusion. Trace shows continuous recording of unitary
K
current activity before and following superfusion of
cell with 2 nM AII.
Acetylcholine (Ach) binds to muscarinic
receptors on bovine AZF cells, activating PLC and triggering cortisol
secretion(6, 17) . However, Ach at concentrations that
produce maximal stimulation of PLC and cortisol secretion produced no
inhibition of I in whole cell patch clamp recordings. In
the experiment illustrated in Fig. 8A, a cell was
sequentially superfused with Ach (10 µM) and AII (10
nM) for 10 min each. At the end of the exposure to Ach,
I
(measured as the non-inactivating component of current
present at the end of the 300-ms voltage step) had not changed. AII
selectively reduced I
by 60% from 110 to 44 pA. Similar
results were obtained in each of six separate cells.
Figure 8:
Effect of Ach, IP, and PMA on
I
. A, Ach, mixed A-type and I
.
K
current was activated in response to depolarizing
steps to +20 mV applied at 30-s intervals from a holding potential
of -80 mV. After recording control current, cell was sequentially
superfused with 10 µM Ach and 10 nM AII. Current
records were obtained after a 10-min exposure to either agent. B, IP
. I
was recorded at 30-s
intervals using the voltage protocol shown in Fig. 1B and pipette solution containing 5 µM IP
.
AII (10 nM) was superfused at the indicated time. C,
PMA. I
current was recorded at 30-s intervals for 10 min
before superfusing 200 nM PMA. Current records show
K
current 1 min (left) and 10 min (middle) after beginning whole cell recording and after a 5
min exposure to PMA (right). Lefttrace shows combined A-type and I
K
currents at +20 mV, activated from a holding potential of
-80 mV. I
amplitude is plotted against time at bottom.
PLC-generated
second messengers also failed to mimic AII-mediated inhibition of
I. The membrane-permeable phorbol ester PMA (200
nM), which mimics diacylglycerol in activating kinase C, when
superfused for times ranging from 5 to 9 min, did not reduce I
in each of four experiments similar to the one illustrated in Fig. 8C. When the membrane-impermeant phospholipid
IP
(5 µM) was included in the patch electrode,
the time-dependent expression of I
was not affected, and
I
was inhibited to the same extent by AII as under control
conditions (Fig. 8B, Fig. 9, A and B).
Figure 9:
Effect of Ca and
phospholipases on I
expression and inhibition by AII.
Whole cell I
currents were recorded using the voltage
protocol shown in Fig. 1B and a variety of pipette
solutions. I
was allowed to reach a stable maximum value
before superfusing the cell with 10 nM AII. A,
maximum I
current density (pA/pF) observed with pipette
solutions where free Ca
was buffered to nominally 0
(Ca = 0, n = 5), 22 nM (control, n = 6), or 220 nM (Ca*10, n = 2)
using the Bound and Determined program(14) . Current densities
are also shown for standard pipette solutions supplemented with 5
mM IP
(n = 2) or 10 µM AA (n = 3). B, the maximum inhibition of
I
by AII (10 nM) was measured using pipette
solutions containing 0 (Ca = 0, n = 5), 22
nM (control, n = 6), or 220 nM (Ca*10, n = 2) free
[Ca
] as described above. I
block by AII was also measured when standard pipette solution was
supplemented with 5 µM IP
(n =
2), neomycin (200 or 500 µM, n = 6), 10
µM AA (n = 3), 1 mM phenidone (n = 2) and 100 µM indomethacin (n = 2). Results are mean ± S.E.
As in most cells, PLC-generated IP in
adrenal cortical cells acts to release Ca
from
intracellular stores(2) . However, changes in
[Ca
]
were not involved
in the modulation of I
in our studies. In unstimulated
cells, I
expression was not affected by pipette solutions
in which Ca
was buffered over a range of nominally
0-230 nM (Fig. 9A). Further, AII
inhibited I
to approximately the same extent with each of
these pipette solutions (Fig. 9B). In particular, AII
effectively inhibited I
in Ca
-free
pipette solutions where Ca
was strongly buffered with
20 mM BAPTA (Fig. 9B). AII also effectively
reduced I
in Ca
-free external solutions
containing 1 mM EGTA (data not shown).
The antibiotic
neomycin prevents activation of PLC in a variety of cell
types(18) . The inclusion of neomycin in the recording pipette
failed to significantly alter AII-mediated inhibition of I (Fig. 9B). In a total of six experiments with
either 200 or 500 µM neomycin in the pipette, AII (10
nM) inhibited I
by 61.4 ± 10.6%.
The
phospholipase A pathway is another source of second
messengers that have been shown to behave as ion channel modulators.
Arachidonic acid (AA) liberated from cell membranes following
activation of phospholipases as well as biologically active arachidonic
acid metabolites modulate K
channels in some
cells(19, 20) . Neither AA nor its metabolites were
responsible for AII-mediated inhibition of I
. The
inclusion of AA (10 µM) in the pipette solution did not
prevent the typical growth of I
in whole cell recordings
or inhibition of this current by AII (Fig. 9, A and B). Also, including the lipo-oxygenase inhibitor phenidone (1
mM) or the cyclo-oxygenase inhibitor indomethacin (100
µM) in the pipette to block, respectively, the formation
of leukotrienes and prostaglandins from AA did not alter AII-mediated
inhibition of I
(Fig. 9B). Manoalide (10
µM in the recording pipette), which inhibits both
phospholipase A
and phospholipase
C(21, 22) , was also ineffective in preventing AII
inhibition of I
(data not shown).
Figure 10:
I block and kinase inhibition by staurosporine and AMP-PNP. A, staurosporine. K
current was activated by
voltage steps to +20 mV from a holding potential of -80 mV.
Cell was superfused with 100 nM staurosporine for 10 min
before switching to solutions containing this antagonist as well as AII
(10 nM) and then ACTH (100 pM) as indicated. I
amplitude (measured at the end of 300-ms voltage step) is plotted
against time. B, AMP-PNP and staurosporine. The inhibition of
I
by 10 nM AII was studied in experiments similar
to those described in A. Staurosporine (100 nM) was
applied by bath perfusion, and AMP-PNP (1 mM) was substituted
for ATP in the pipette solution. Fraction of I
inhibited
expressed as mean ± S.E. for AII, AII + staurosporine, and
AII + AMP-PNP as indicated.
MgATP is
the phosphate donor for most protein kinases. To determine whether ATP
hydrolysis is necessary for AII-mediated inhibition of I,
the non-hydrolyzable ATP analog AMP-PNP (1 mM) was substituted
for ATP in the recording pipette. With this ATP analog in the pipette,
AII (10 nM) inhibited I
by only 18.1 ±
8.4% (n = 6) (Fig. 10B). The relative
ineffectiveness of AII in the presence of staurosporine and AMP-PNP
suggests that a protein kinase mediates inhibition of I
channels.
The activity of many ion channels appears to be
regulated by opposing phosphorylation/dephosphorylation mediated by
kinase and phosphatases. If AII inhibits K current
through a kinase-stimulated phosphorylation, the potent phosphatase
inhibitor okadaic acid (26) might be expected to produce a
similar inhibition of I
by enhancing steady state
phosphorylation of the channel. At a concentration of 200 nM,
okadaic acid reversibly inhibited I
in AZF cells by a
maximum of 84% (Fig. 11). Similar results were obtained in three
of four cells exposed to okadaic acid at this concentration.
Figure 11:
Inhibition by okadaic acid (OKAA). I was activated in an AZF cell by voltage
steps to +20 mV as described in the legend of Fig. 1. The
cell was then superfused with 100 nM okadaic acid for the
indicated durations. I
amplitude is plotted against time.
Although our results indicate that AII-mediated inhibition of
I is mediated by a protein kinase, it does not appear to
be one typically activated through AT
receptor stimulation
of PLC. Specifically, the inability of PMA to mimic AII inhibition of
I
indicates that protein kinase C does not mediate the
response. The Ca
/calmodulin kinase inhibitor KN-62 (5
µM) failed to prevent AII-mediated inhibition of I
in three cells where I
was reduced by 68 ±
12%.
We have demonstrated that in bovine AZF cells, AII inhibits
I K
current, depolarizes cells, and
stimulates cortisol secretion through a losartan-sensitive AT
receptor. I
inhibition appears to occur through a
novel PLC-independent pathway, which includes a PTx-insensitive G
protein and a staurosporine-sensitive protein kinase. The results
indicate that in AZF cells, AT
receptors may regulate
cortisol secretion by two parallel, Ca
-dependent
pathways: a PLC-dependent release of Ca
from
intracellular stores and a PLC-independent influx of Ca
secondary to membrane depolarization.
Several distinct
AT receptor cDNAs, each coding for losartan-sensitive
receptors have been cloned including two from rat adrenal
cortex(7, 27, 28, 29) . Possibly,
one of these AT
receptor subtypes is specifically coupled
to I
inhibition through a mechanism that does not involve
PLC. However, both adrenal AT
receptor subtypes cloned thus
far are coupled to PLC-mediated Ca
mobilization.
Perhaps identical AT
receptors are linked to several
signaling pathways through one or more G proteins.
In addition to
activating PLC, losartan-sensitive AT receptors in some
cells are also coupled to adenylate cyclase inhibition through a
PTx-sensitive G protein(8) . This second AT
-coupled
pathway appears to be unrelated to I
inhibition and
cortisol secretion in AZF cells since the associated G protein is PTx
insensitive. Inhibition of cAMP synthesis would suppress rather than
stimulate cortisol secretion.
Second messengers generated
in response to PLC activation (or the biochemical equivalent) were
ineffective in suppressing the expression of I or
mimicking the action of AII on this current. PMA was applied after
recording I
in the whole cell mode for a period of
10-35 min. Failure of this phorbol ester to suppress I
under these conditions might be due to ``washout'' of
the target enzyme, protein kinase C. Even large macromolecules such as
enzymes may reach diffusional equilibrium within minutes after
initiating whole cell recording(32) . However, since AII
effectively inhibits I
under identical conditions, it
remains unlikely that diacylglycerol is the second messenger mediating
inhibition of I
by AII.
The failure of IP to prevent the time-dependent expression of I
in
whole cell recordings or to alter the inhibition of this current by AII
demonstrates that AII-stimulated IP
synthesis is not
necessary for I
inhibition by this peptide and that this
phospholipid does not directly inhibit I
channels.
However, in most of our experiments, intracellular Ca
was strongly buffered by pipette solutions containing 11 mM BAPTA. Under these conditions, IP
-stimulated increases
in intracellular Ca
would be eliminated or strongly
suppressed(33, 34) . Our findings then do not exclude
the possibility that under physiological conditions, AII might also
inhibit I
through IP
-stimulated release of
Ca
where [Ca
]
may reach micromolar levels. Several types of
Ca
-inhibited K
channels have been
identified(33, 35) . However, our results do not
indicate a major role for Ca
in I
expression and modulation, since varying pipette Ca
concentration from 0 to 220 nM did not alter I
expression or inhibition by AII. The observation that buffering
[Ca
]
to nominally zero
with 20 mM BAPTA failed to prevent I
inhibition
by AII argues strongly against the involvement of this cation in the
response. At this concentration, BAPTA has been shown to produce near
complete elimination of agonist-induced Ca
transients
in cells(33, 34) . Accordingly, in rat glomerulosa
cells, AII inhibits K
permeability measured by
Rb efflux, an effect not mimicked by the Ca
ionophore ionomycin(36) .
We conclude that
AII-mediated inhibition of I occurs through a
phospholipase C-independent pathway. A similar phospholipase
C-independent parallel pathway appears to function in muscarinic
inhibition of K
current in neuronal
cells(37) .
Staurosporine (100 nM) reduced
AII-mediated inhibition of I by approximately 64%. Since
this drug inhibits the identified serine-threonine kinases with
IC
values ranging from <1 to 20
nM(25) , the incomplete block of AII effects on
I
may suggest that a different protein kinase is involved.
Staurosporine also inhibits tyrosine kinases, although at somewhat
higher concentrations(25) . In this regard, AII has been shown
to induce losartan-sensitive tyrosine phosphorylation in rat aortic
smooth muscle cells. This phosphorylation is inhibited by
staurosporine(38) . Perhaps a similar tyrosine kinase is
involved in AII-mediated inhibition of I
.
The ability
of okadaic acid to reversibly inhibit I raises the
possibility that AII might act through the inhibition of a protein
phosphatase. Our results with staurosporine and AMP-PNP suggest that
this phosphatase inhibition might require prior activation of a protein
kinase. The delay of up to several minutes that precedes AII's
effect on I
and membrane potential is consistent with such
a multi-step process. Complex pathways requiring both kinases and
phosphatases in peptide-mediated ion channel modulation have been
described(39, 40) .
Major components of the pathway
that links AT receptors to I
are yet to be
specifically identified. The delay of 1 to several minutes that
precedes AII-mediated inhibition of the current is inconsistent with a
response that involves a tight coupling between AII receptor, G
protein, and I
channels. A delay of this duration is
excessive, even for responses that require the synthesis of a
cytoplasmic diffusible second messenger. Further, since AII-mediated
inhibition was observed in cells that had been held in whole cell patch
clamp for periods in excess of 30 min, it seems doubtful that a freely
diffusible cytoplasmic messenger is involved. Protein kinases tightly
associated with rat brain K
channels have been shown
to modulate these channels after reconstitution into lipid
bilayers(24) . Our results suggest that a similar
membrane-associated kinase may couple AT
receptors in AZF
cells to phosphorylation and inhibition of I
channels. The
successful inhibition of unitary I
currents in
cell-attached patches upon superfusion of the cell membrane outside the
pipette with AII is consistent with the suggestion that diffusible,
lipid-soluble intramembranous second messengers may be functioning.
Secretion studies showed that AII-stimulated cortisol production,
like membrane depolarization and I inhibition, occurs
through losartan-sensitive AT
receptors. Apparently,
AT
receptors are coupled through PTx-insensitive G proteins
to cortisol secretion by two parallel Ca
-dependent
signaling pathways. In addition to the previously described PLC- and
IP
-mediated release of Ca
, we have
identified a novel PLC-independent path that couples losartan-sensitive
receptors to membrane depolarization and Ca
entry
through voltage-gated T-type Ca
channels(10, 12) . Because components of this
signaling mechanism appear to be identical in AZF and AZG cells,
AII-mediated inhibition of I
should function as a stimulus
for both cortisol and aldosterone secretion.