| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Protein Subunit and Cyclic Adenosine 3',5'-Monophosphate
Department of Physiology and Biophysics (P.D., L.B., A.C., M.D.P.) and Department of Medicine Endocrine Service (P.D., L.L., N.G-P.), Faculty of Medicine, University of Sherbrooke Sherbrooke, Quebec, Canada J1H 5N4
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
s-GDP, the inhibitory
effect is reduced and cannot be recovered by the addition of GTP
S,
indicating that the s is not involved, but
rather the ß-subunit. Indeed, DA-induced inhibition was mimicked
by Gß in the pipette and 8-Br-cAMP in the bath. Similarly, Gß
release from the activation of the AT1 receptor
of angiotensin II did affect the current amplitude only in the presence
of 8-Br-cAMP in the bath. The mitogen-activated protein kinase cascade,
which can be activated by receptors coupled to
Gs, was not involved as shown by the lack of
activation of p42mapk by DA and the absence of
effect of the mitogen-activated protein kinase inhibitor, PD 098059, on
the DA-induced inhibition. Because the binding of Gß-subunits to
various effectors involves the motif QXXER, we therefore tested the
effect of the QEHA peptide on the inhibition of the T-type
Ca2+ current induced by DA. The peptide, added
to the medium pipette (200 µM), abolished the
effect of DA. We conclude that the presence of the Gß and an
increase in cAMP concentration are both required to inhibit the T-type
Ca2+ current in rat adrenal glomerulosa cells. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Both dopamine (DA) receptors, D1 and D2, have been described in rat and
bovine glomerulosa cells (11, 12). Binding of DA or specific agonists
to D1 receptors activates adenylyl cyclase through a Gs
protein, which results mainly in an increase in cytosolic cAMP. In
contrast, one of the best known effects of D2 receptors is the
inhibition of adenylyl cyclase through the pertussis toxin
(PTX)-sensitive Gi/Go family of G proteins
(13). Modulation of ionic channels is also an important component of
the physiological effects of DA. In rat neostriatal neurons, D1
receptors inhibit high voltage-activated (HVA) currents (N- and
P-types) through the activation of a Ser/Thr phosphatase (PP1-type)
(14) and depress the amplitude of the Na+ current through a
direct cAMP-protein kinase A (PKA)-dependent phosphorylation of the
channels (15). The role of D2 receptors in the regulation of
K+ and Ca2+ currents has been reported in many
cell types. The signaling pathway was described in rat pituitary cells,
where Go and Gi3 proteins are involved
(16).
Little is known about the modulation of ionic currents by DA in adrenal
glomerulosa cells. A recent report showed that the T-type
Ca2+ current is blocked by activation of the D2 receptor
(17). In the present study, we also report that DA inhibits the T-type
Ca2+ current in glomerulosa cells. However, using agonists
and antagonists of DA receptors, we demonstrate that the type of DA
receptor involved is the D1 receptor. We also show that the DA-induced
inhibition is mediated by the Gs-coupling G protein,
through its ß- subunit. Because current inhibition was prevented
by the QEHA peptide, we propose that the ß-subunit must bind to a
specific site on the T-channel. Moreover, at odds with previous works
on Gß-subunit-modulated ionic channels, we demonstrate that the
inhibitory effect of DA also requires cAMP-dependent
phosphorylation.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
shows the
current traces obtained in control conditions and after addition of 0.3
mM DA to the bath. DA caused the current amplitude to
decrease rapidly, reaching a plateau after 70 sec, with a level of
inhibition of 33.0 ± 1.8% (n = 75). The current-voltage
relationships (I/V curve) are illustrated in Fig. 1B. The current
threshold was close to -60 mV, the peak current voltage near -30 mV,
and the zero current voltage around +50 mV. DA reduced the current
amplitude at each potential without any change in the voltage threshold
or peak current voltage. Figure 1C illustrates the dose-dependent
blockage effect of DA. A cumulative dose-response curve was constructed
with five DA concentrations (0.1 to 0.5 mM). Current traces
are presented in the inset. Percent inhibition of the
current amplitude was plotted as a function of DA concentration (Fig. 1D); 50% inhibition (IC50) was obtained with 0.25
mM DA. Total inhibition was never observed, even with DA
concentrations as high as 1 mM. Partial recovery of the
current amplitude was obtained after washing (data not shown).
|
). In addition, preincubation of the cells with the D1
antagonist SCH 23390 (5 µM) markedly reduced their
response to DA (0.8 mM) (Fig. 2B). We also found that 5 or
10 µM (n = 5) SCH 23390 has by itself a blocking
effect on the T-type current. In contrast, when the D2 antagonist
spiperone (10 µM) was added to the bath before DA (0.3
mM), the current amplitude was still reduced with similar
time course and potency as observed in the absence of the antagonist
(Fig. 2C, n = 5). These results suggest that the inhibitory effect
of DA we have recorded on the T-type Ca2+ current is
produced through the activation of the D1 receptor subtype.
|
confirms and extends
these observations. The maximun increase in cAMP production measured
with DA was 11.6-fold, with a concentration of 0.3 mM
(n = 3), compared with a 7.2-fold increase with the D1 agonist
Cl-APB (1 µM) (n = 3). The use of specific D1 and D2
antagonists indicates that the increase in cAMP production is linked
only to D1 receptor activation, since SCH 23390 blocked the DA
stimulation, while sulpiride was ineffective.
|
shows that addition of 1 mM 8-Br-cAMP to the bath
did not affect the current amplitude, which indicates that cAMP does
not modulate directly the T-current in adrenal glomerulosa cells.
However, pretreatment of the cells with 8-Br-cAMP drastically decreased
DA potency (9.1 ± 1.9% inhibition; n = 7). Moreover, when
the cells were preincubated 5 min in the presence of the specific
adenylyl cyclase inhibitor 2',3'-dideoxyadenosine (DDA, 100
µM) (20, 21, 22), DDA had no effect on the T-type
Ca2+ current, but prevented current inhibition by DA (0.3
mM) (5.2 ± 3.6%, n = 5) (Fig. 3C). Thus,
although cAMP does not directly affect the channel activity, adenylyl
cyclase activation is part of the D1-receptor-signaling pathway.
Activation of the cAMP-dependent PKA, known to phosphorylate L-type
Ca2+ channels in numerous cell types, seems to be involved
in the regulation of the T-type Ca2+ channel, because
preincubation of the cells with the PKA inhibitor H-89 (10
µM) reduced the impact of DA on the current amplitude
(16.4 ± 3.1% inhibition; n = 11); the IC50 of
the dose-effect curve was 8.1 µM (data not shown). It has
been reported that D1 receptor can signal through the phospholipase C
pathway (23, 24, 25). However, application of Ang II (100 nM)
had no effect on the T-type Ca2+ current and did not
modulate the DA-induced inhibition of the current (data not shown).
Several ionic channels are modulated by the p21ras
protein (26) or by the Ras/Raf transduction cascade (27). This
possibility was tested in glomerulosa cells. The T-type
Ca2+ current was not affected by an antibody raised against
the p21ras protein added to the pipette medium (35.2
± 1.2% inhibition; n = 4; data not shown), or when the Ras/Raf
cascade was blocked at a more distal stage by preincubation of the
cells with the MAP kinase inhibitor PD 098059 (1 h, 30
µM) (Fig. 4A; 30.3 ± 5.7%
inhibition; n = 7). Furthermore, MAP kinase activity was assessed
by in-gel kinase analysis after cell stimulation by Ang II or DA. As
shown in Fig. 4B, treatment of glomerulosa cells with Ang II for 5 min
activates the p42mapk (ERK2) MAP kinase (see also Ref.28),
while DA treatment was ineffective. A PP1-type phosphatase, activated
by PKA-dependent phosphorylation, was shown to be involved in the
D1-like receptor-induced inhibition of HVA Ca2+ currents
(14). This possibility was also ruled out, because okadaic acid (1
mM), a blocker of Ser/Thr phosphatase, did not impair
DA-induced current inhibition (38.8 ± 3.2%; n = 8; data not
shown).
|
), which confirms the involvement of a G
protein in the signaling pathway between the D1 receptor and the T-type
Ca2+ channel. To identify the type of G protein, the cells
were incubated 18 h with 100 ng/ml PTX. In glomerulosa cells, this
PTX concentration was shown to inactivate the Gi protein
through ADP ribosylation (30). Figure 5B shows that DA was still able
to reduce the T-type current amplitude (35. 6 ± 7.2% inhibition;
n = 5). Similar results were found when an antibody raised against
the -subunit of the Gi protein was added to the pipette
medium (data not shown, n = 4). The absence of effect of Ang II,
as mentionned earlier, confirms that neither Gi nor
Gq coupling proteins were involved. However, if Ang II (100
nM) and 8-Br-cAMP (1 mM) were added together, a
slow inhibition took place, reaching 25.3 ± 3.9% (Fig. 5C;
n = 4).
|
-subunit of
the G protein by Clapham’s group (31, 32). This modulation would
result from a direct binding of the Gß-subunit to the
IKAch channel (33). Here, when Gß concentration was
reduced by cell dialysis with Gs-GDP (300
nM), placed in the pipette medium, the inhibitory effect of
DA was considerably decreased (Fig. 6A, 16.9 ±
2.3% inhibition; n = 4). When GTPS (1 µM) was
added to the Gs-GDP medium pipette, DA did not recover
its inhibitory effect (10.2 ± 3.2% inhibition; n = 4; data
not shown), suggesting that the G-subunit was not involved in
DA-induced inhibition of the T-type Ca2+ current. In
contrast, although Gß-subunits (100 nM), added to the
pipette medium, did not affect the current under control conditions, DA
inhibition was impaired (Fig. 6B; 14.8 ± 3.9% inhibition; n
= 6). Denaturated Gß was inactive (data not shown). We previously
showed that DA-induced inhibition of the T current was considerably
reduced by cAMP (Fig. 3B). However, when 8-Br-cAMP (1 mM)
was added to the bath medium and Gß-subunit to the pipette medium,
current inhibition increased slowly (Fig. 6C) to reach a level similar
to the one measured with DA (Fig. 6D; 31.2 ± 4.9% inhibition;
n = 4).
|
-subunits to various effectors involves the
motif QXXER (34). The effects of Gß have been reported to be
specifically blocked by a peptide containing the sequence corresponding
to residues 956 to 982 of adenylyl cyclase 2, the peptide called QEHA
(34). We therefore tested the effect of the QEHA peptide on the
DA-induced inhibition of the T-type Ca2+ current. The
peptide, added to the medium pipette (200 µM), suppressed
the effect of DA (Fig. 7A; 10.3 ± 3.7%
inhibition; n = 4); QEHA peptide blunted the DA inhibition in a
dose-dependent manner (Fig. 7B).
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-subunit of the Gs-coupling protein is
involved in the inhibition of the current. However, our data clearly
demonstrated that cAMP plays a pivotal role in the inhibition of the
current.
Dopamine Receptors in Adrenal Glomerulosa Cells
Several subtypes of DA receptors have been described and
classified in two families: the D1-like (D1 and D5) and the D2-like
(D2, D3, D4) receptors (for reviews, see Refs. 19 and 29). This
classification is based mainly on the modulation of adenylyl cyclase.
The D1-like receptors stimulate adenylyl cyclase through a
Gs protein (35), whereas the D2-like receptors inhibit the
enzyme activity through a Gi protein (36). D1-receptor
activation was also associated with stimulation of phosphoinositide
hydrolysis (23, 24, 25) and Ca2+ mobilization (37, 38) whereas
D2 receptors were reported to stimulate ATP-mediated arachidonic acid
release (39).
Conflicting results were reported concerning the role of DA in the
control of steroid secretion from isolated glomerulosa cells (40, 41).
They were shown to originate from experimental conditions. Freshly
isolated cells express DA receptors from both D1- and D2-subclasses,
while 3-day cultured cells express only the D1 subclass (5, 6). In this
study, all experiments were performed on glomerulosa cells after 1 or 2
days of culture, when the majority of DA receptors are expected to
belong to the D1-like subclass. Expression of the D1-like receptors was
confirmed by an increase in cAMP production measured after the addition
of DA or the D1 agonist Cl-APB (see Fig. 3A). Moreover, preincubation
of the cells with the D1 antagonist SCH 23390 prevented DA-mediated
increase in cAMP production. The fact that cells preincubated with the
D2 antagonist sulpiride did not respond to DA by a faster rate of cAMP
production indicates that the DA receptors expressed were mainly of the
D1 type.
DA Receptors Modulate Ionic Currents
In the majority of the cells we studied (95%), DA reduced the
current amplitude of the T-type Ca2+ channel in a
dose-dependent manner, whereas transient inhibition were occasionally
observed (data not shown) as previously reported for K+
current enhancement by PACAP 38 (42). Several experiments were designed
to confirm that DA inhibition of the T-type current was mediated
through the activation of a D1 receptor type. First, the inhibitory
potency of DA was not modified by the presence of spiperone, a specific
D2 antagonist (Fig. 2C). Second, the D1 agonist Cl-APB was also able to
reduce the T-type Ca2+ current amplitude. Finally, the
DA-inhibitory effect was greatly attenuated when cells were pretreated
5 min with the D1 antagonist SCH 23390. As shown in Fig. 2B, this D1
antagonist SCH 23390 does have an inhibitory effect per se
on the Ca2+ channel activity. Such an effect was recently
reported on calcium currents in rat retinal ganglion cells (43).
Most cases of ionic current modulation by DA reported in the literature
were believed to be due to the activation of D2-like receptors. D2
receptor activation was reported to modulate Ca2+ currents.
It was found that LVA and/or HVA Ca2+ currents are
inhibited by DA or D2-agonists (17, 44, 45) and that this inhibition is
suppressed by PTX (44). DA also induces an hyperpolarization of the
cell membrane, which was attributed to an activation of K+
channels (44, 46, 47, 48). This hyperpolarization is believed to play a key
role in the inhibition of cell secretion by DA. PTX treatment of the
cells blocks both the DA-induced membrane hyperpolarization and
activation of the K+ channels, indicating that a
Gi/o protein is involved (44, 47, 48). Modulation of
K+ and Ca2+ currents by D2 type receptors
occurs through different G protein coupling: Gi3 for
IK and IA channels, and Go for
the T- and L-types of Ca2+ channels (16).
Modulation of ionic currents by D1 receptor activation has been less frequently reported. However, cAMP-dependent PKA phosphorylation was always involved. For instance, in rat striatal neurons, D1 receptor activation reduces the amplitude of the fast Na+ current. This effect is mediated by a GTP-binding protein that induces an increase in cAMP level, which then activates the cAMP-dependent PKA (15). Phosphorylation of the Na+ channel by PKA results in a reduction in current amplitude (49). D1 receptor activation also inhibits HVA Ca2+ currents (N- and P-types) of neostriatal neurons (14). In this case, the cAMP-dependent PKA activates a protein phosphatase (PP1), which dephosphorylates the Ca2+ channels. In some neurons, D1 receptor activation leads to an increase in the L-type Ca2+ current (14). An increase in the cytosolic Ca2+ concentration in rat pituitary GH4C1 cells transfected with the human D1 receptor was also explained by an L-type channel activation (38).
Coupling between the D1 Receptor and the T-Type
Ca2+ Channel
Our results show that the T-type current inhibition by DA is
mediated by the cAMP-dependent PKA. The PKA inhibitor H-89 (50, 51)
blocks the DA-inhibitory effect. However, the fact that 8-Br-cAMP did
not inhibit the T current provides strong evidence that in addition to
a PKA-dependent phosphorylation, a parallel signal must be delivered.
The involvement of a PKA/phosphatase cascade, as described for the
inhibition of the HVA Ca2+ channels in neurons (14), was
ruled out using a Ser/Thr phosphatase inhibitor, okadaic acid. Some
reports suggest that phospholipase C activation could also be part of
the D1 receptor-signaling pathway (23, 24), through a
Gq-coupling protein (25). However, previous results from
our group showed that DA was not able to increase inositol phosphate
production in rat glomerulosa cells (6), which, together with the lack
of effect of Ang II application on the T-type Ca2+ current,
ruled out a possible involvement of the phospholipase C pathway.
A G protein coupling is part of the D1 receptor-inhibitory process.
Blockage of G protein activity with GDPßS completely prevented
current inhibition by DA. Pretreatment of the cells with PTX or the use
of an antibody raised against the i-subunit did not
impair the inhibitory action of DA. These results, along with those
showing that Ang II application has no effect on the T current
amplitude (data not shown), indicate that the inhibition of the T
current we recorded is not transduced by the coupling protein
Gi and thus strengthen our findings that D2-like receptors
are not involved.
Whether G protein modulation of ionic channels is mediated by the G-
or Gß-subunit is a matter of debate (52, 53, 54). Here, the fact that
Gß-subunits from bovine brain, added to the internal solution, did
not have any effect on the T-type currents (Fig. 6B) raised several
possibilities. The effectiveness of the Gß-subunits we used in our
experiments could be questioned. However, DA-induced inhibition of the
T current was greatly reduced by Gß dialysis of the cell whereas
temperature-inactivated Gß was ineffective. On the basis of the
existence of several Gß- combinations (53), the bovine brain
Gß-subunit may not efficiently interfere with the inhibitory
pathway of the T-type Ca2+ channels. However, it has been
recently shown that various Gß-forms, including bovine brain
Gß, activate the IKAch channel (32) by direct binding
(33). Consequently, this absence of effect did not exclude the
participation of Gß in the DA-signaling pathway, but raises the
possibility of a second factor that should act together with Gß to
inhibit the T-type Ca 2+ current.
The involvement of the Gß-subunit in the inhibition of the T-type
Ca2+ current by the D1 receptor was further assessed by
decreasing the Gß concentration in the cell using specific
Gß-subunit scavengers. First, we added the -subunit of the
Gs protein under its -GDP form to the pipette medium.
Under these conditions, DA could no longer inhibit the T-type current.
Second, it has been shown that a 27-amino acid peptide, the QEHA
peptide, was able to block the Gß-induced regulation of adenylyl
cyclase (AC), phospholipase C-ß3, the ß-adrenergic receptor kinase
(ß-ARK), and the G protein-coupled inward rectifer K +
channel (GIRK-1) (34). The inhibitory effect of DA was also abrogated
by the peptide placed in the pipette medium. Moreover, the
IC50 was found near 100 µM, a concentration
similar to that reported by Chen et al. (34) for various
effectors of the Gß-subunit. These results suggest that binding of
Gß on a site that could be located on the T channel is involved in
current inhibition, as demonstrated for the IKAch channel
(32, 33). Because several effectors of the Gß-subunit (AC2, AC4,
ß-ARK-1 and 2, and GIRK-1) display a common amino acid sequence (see
Ref.34), we looked for this sequence in the T-type channel. A
Ca2+ channel has been cloned from rat brain cells (55).
When expressed in oocytes, the 1-subunit (rbE-II) shows
voltage-dependent and pharmacological properties similar to the LVA
Ca2+ channel (T-type) found in neuronal cells (56) or
glomerulosa cells (18). A unique consensus sequence (QQIER) was found
at position 325 (Fig. 7C). Cloned Ca2+ channels can be
classified into two subfamilies: the dihydropyridine (DHP)-insensitive
(T-, N-, P-, and Q-type) and the DHP-sensitive (L-type) families. The
two-Ca2+ channel subfamilies display the QXXER motif, which
may confer the modulating properties of the Gß-subunit. Indeed, it
has been recently reported that N-type (57) and P/Q-type (58)
Ca2+ channels are modulated by various forms of the
Gß-subunit. However, evidence has not yet been obtained in the
case of the DHP-sensitive channel.
As shown in Fig. 3B, superfusion of the cells with 8-Br-cAMP (1
mM) completely abolished the inhibition of the T current
induced by DA. Nevertheless, in order to trigger the DA effect, it
appears that cAMP is required at a level higher than the basal level.
Indeed, we show that DA inhibition of the Ca2+ current will
not occur if the adenylyl cyclase is blocked by the DDA (100
µM) (Fig. 3C). Requirement of cAMP for the development of
the T-type inhibition was further strengthened by results showing that
bovine Gß-subunit, placed in the medium pipette or disengaged from
Gq and/or Gi by Ang II binding on the
AT1 receptor, only reduces Ca2+ current in the
presence of 8-Br-cAMP.
This kind of double regulation by a Gs-coupled membrane
receptor has been recently reported. It has been shown that the
ß-adrenergic receptor is coupled to a Gs protein that
activates adenylyl cyclase and potently activates the MAP kinase
cascade through Gß and that application of 8-Br-cAMP inhibits this
effect in a PKA-dependent way (59). Similarly, the pituitary adenylyl
cyclase-activating polypeptide (PACAP 38) increases the amplitude of
the K+ current both by the Gß-Ras/Raf and the
Gs-adenylyl cyclase cAMP pathways; nevertheless, this
effect is blocked by preincubation with cAMP (42). Using a specific
inhibitor of the MAP kinase cascade, the PD 098059 (60), and the MAP
kinase assay (70), we ruled out the MAP kinase pathway in favor of a
direct interaction of the Gß-subunit with the T channel.
Conclusion
Our data support the fact that the T-type Ca2+ channel
is inhibited upon binding of the ß-subunit of the G-coupling
protein. Various forms of Gß-subunits could be involved, since
they can originate from the Gs protein (D1-like receptor),
the Gq and/or Gi protein(s) (AT1
receptor of Ang II), or from bovine brain
(ß12). Since the first work of Clapham and
co-workers (31), showing that the Gß-subunit was able to activate
the IKAch channel, numerous effectors of Gß-subunits
have been described (34, 42, 54, 57, 58). One important observation is
that several Gß combinations are effective, which implies that the
same effector can be modulated by different G protein-coupled
receptors. Pathways specificity for an effector can be obtained in
different ways: binding of G or Gß alone or a synergistic or
antagonistic binding of G and Gß (53). Yet another factor could
be the relative proximity between the effectors and the receptors
releasing Gß-subunits (62). Our results show that, for the T-type
channel, cAMP is a strong modulator of the Gß effect. The question
can be raised whether the T channel and/or the ß-subunit should be
phosphorylated by PKA. The 1 (rbE-II)-subunit displays the concensus
sequence RRXS for a putative phosphorylation by PKA. However,
experimental evidence for PKA-dependent phosphorylation of the T-type
channel has not yet been provided. On the other hand, PKC-dependent
phosphorylation of the ß-subunit has been reported (63, 64). As
stated by Neer (62), the role of ß-subunit phosphorylation, if
any, is not known. In any case, PKA is not able to phosphorylate the
ß-subunit (64) and, more important, unphosphorylated forms of
ß-subunit are active on several effectors (31, 32, 33, 34). In the
framework of our results we can postulate that the ß-subunits bind
to the T channel and that activation of PKA, by an increase in cAMP,
phosphorylates the channel. The sequence of the events and the
mechanism involved are not yet resolved. This could constitute an
example in which cAMP acts as a gating factor (62). Indeed, we show
that the Ang II-signaling pathway, which was shown to increase T-type
current amplitude (65, 66, 67), could be modulated by a cAMP-dependent
process in such a way that T-type current is blocked. This raises the
possibility of interactions between hormones and neurotransmitters
coupled to G protein receptors in the control of aldosterone secretion
by adrenal glomerulosa cells.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
S and GDPßS from Boeringher
(Indianapolis, IN); the -subunit of the Gs coupling
protein and the bovine brain Gß-subunit from Calbiochem (San
Diego, CA); H-89
(N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinoline-sulfonanide)
from Seikagaku America (St. Petersburg, FL); angiotensin II from Bachem
(Marina Delphen, CA); dopamine, 6-chloro-APB Hbr (SKF 82958), SCH
23390, and spiperone from RBI (Natich, MA); collagenase, MEM (Eagle
medium) and OPTI-MEM from GIBCO (Burlington, Ontario, Canada). The
specific inhibitor of the MAP kinase cascade, PD 098059, was a gift
from Dr. D. T. Dudley (Parke-Davis Pharmaceuticals Research Division,
Warner-Lambett Co, Ann Harbor, MI 48105). The QEHA peptide was
synthesized by "Service de Séquence de Peptides de l’Est du
Québec" (Le Centre Hospitalier de l’Université Laval,
Qué, Canada). The peptide was purified by HPLC (> 90%) and its
identity verified by mass spectrometry. All other chemicals were of
A-grade purity.
Preparation of Glomerulosa Cells
The zonae glomerulosa were obtained from adrenal glands of
female Long Evans rats weighting 200–250 g. Rats were killed according
to a protocol approved by the Local Ethics Animal Care Committee, and
adrenals were isolated according to the method described in detail
elsewhere (6). The successive steps of zona glomerulosa isolation and
cell dissociation were performed in MEM Eagle medium (supplemented with
100 U/ml penicillin and 100 mg/ml streptomycin). After a 20-min
incubation at 37 C in collagenase (2 mg/ml, 4 capsules/ml) and DNAse
(25 µg/ml), the cells were disrupted by gentle aspiration with a
sterile 10 ml pipette, filtered, and centrifuged for 10 min at 100
x g. They were then resuspended in OPTI-MEM supplemented
with 2% FBS, 100 U/ml penicillin, and 100 µg/ml streptomycin and
plated in 35-mm tissue culture dishes at a density of approximately
5 x 104 cells per dish. The cells were cultured at 37
C in a humidified atmosphere of 95% air-5% CO2. The
culture medium was changed every day, and the cells were used after 1
or 2 days of culture.
Solutions and Recording Conditions
Solutions
The physiological solutions used for the patch clamp experiments had
the following compositions. The basic extracellular solution contained
(mM): NaCl, 100; CaCl2, 10; tetraethylammonium,
35; MgCl2, 1; CsCl, 5.4; HEPES, 5; and glucose 2 g/liter at
pH 7.4. The pipette solution contained (mM): CsCl, 126;
NaCl, 18; CaCl2, 1; EGTA, 11; MgCl2, 2; HEPES,
5; ATP, 3; and GTP, 0.4 at pH 7.2. The external solution was
supplemented with glutathione to avoid DA oxydation. Solutions
containing hormones, antibodies, or drugs were freshly prepared before
each experiment.
Electrophysiology
Experiments were performed at room temperature and in the dark. The
Petri dish (1 ml volume solution) was mounted on the stage of an
inverted microscope, and the cells were observed at a magnification of
300x. Ionic currents were recorded using the whole-cell configuration
of the patch-clamp method (68). Patch electrodes with a resistance of 3
to 5 megohms (M
) were pulled from Pyrex Glass capillaries (Corning
7740, Corning Glass Works, Corning, NY). Ionic currents were recorded
with an axopatch 1B (Axon Instruments, Burlingame, CA), whereas pulse
stimulation and data acquisition were performed with an A/D interface
DAS 16F (Metrabyte Taunton, MA) and an IBM-compatible computer under
the control of a custom-built program. Linear leak and capacitative
currents were subtracted. Currents were filtered at 2 kHz and sampled
at 5 kHz. Analysis was performed with a custom-made program. Each
figure is representative of several experiments conducted on the number
of cells (n) indicated in the text.
Cyclic AMP Determination
Intracellular cAMP production was determined by measuring the
conversion of [3H]ATP into [3H]cAMP, as
described previously (6). Briefly, cultured cells were incubated at 37
C in the same MEM Eagle culture medium containing 2 µCi/ml
[3H]adenine. After 1 h, the cells were washed and
then incubated in Hanks buffer saline glucose containing 1
mM isobutyl methylxanthine for 15 min at 37 C. Hormones or
drugs were then added to the medium for an additional 15-min incubation
period at 37 C. The reaction was stopped by aspiration of the medium
and addition of 1 ml TCA 5%. The cells were scraped with a rubber
policeman and transfered to 100 µl of cold 5 mM ATP-cAMP.
Cell membranes were pelleted at 5,000 x g for 15 min,
and the supernatant was sequentially chromatographed on Dowex and
alumina columns, according to the method of Salomon (69), allowing the
separation of [3H]ATP from [3H]cAMP. Cyclic
AMP accumulation was calculated from the equation: % conversion =
([3H]cAMP/[3H]cAMP + [3H]ATP)
x 100.
Analysis of MAP Kinase Activity
Cells are washed and stimulated for 5 min at 37 C with or
without Ang II (100 nM) or DA (0.3 mM). Cells
are then washed and solubilized in 300 ml of cold lysis buffer (50
mM HEPES, pH 7.8, 1% Triton X-100, 2.5 mM
EDTA, 100 mM sodium fluoride, 10 mM sodium PPi,
2 mM sodium orthovanadate, 0.5 U aprotinin, 1
mM phenylmethylsulfonylfluoride, 1 mM
benzamidin) and centrifuged at 10,000 x g for 15 min
at 4 C. Equal amounts of protein (10 µg) were electrophoresed on a
12% SDS-polyacrylamide gel containing 0.5 mg/ml myelin basic protein
(MBP, Sigma, Mississauga, Ontario, Canada). Kinase assays in
MBP-containing gel were performed at room temperature as described by
Holt et al. (70). After further denaturation and
renaturation of the proteins, the kinase reaction was initiated by
placing the gel in fresh kinase buffer containing 30 µM
[32P]ATP (9 µCi/ml) and incubated for 1 h at
room temperature with gentle agitation. The gel was washed extensively
with repeated changes of wash buffer (5% trichloroacetic acid, 1%
sodium PPi), dried, and subjected to autoradiography.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
Nicole Gallo-Payet is a recipient of a Scholarship from "Le Fonds de La Recherche en Santé du Québec." This work was supported by grants (MT-10998 and MA-6813) from the Medical Research Council of Canada and the "Fondation des Maladies du Coeur du Québec" to Marcel Daniel Payet and Nicole Gallo-Payet.
Received for publication October 18, 1996. Revision received January 13, 1997. Accepted for publication January 17, 1997.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
-subunits activate the muscarinic-gated atrial
potassium channel. Nature 368:255–257[CrossRef][Medline]
binds directly to the G protein-gated K+ channel,
IKAch. J Biol Chem 270:29059–29062 subunits. Science 268:1166–1169-dimers in transmembrane signalling. Nature 365:403–406[CrossRef][Medline]
subunits. Nature 380:255–258[CrossRef][Medline]
subunits. Nature 380:258–262[CrossRef][Medline]
-dependent activation and a Ras-cAMP-mediated inhibition.
J Biol Chem 270:25259–25265 subunit of G protein, 12, and
its phosphorylation by protein kinase C. J Biol Chem 270:29469–39475 subunits complexes of brain GTP-binding proteins composed of
distoncts subunits. FEBS Lett 266:41–44[CrossRef][Medline]
This article has been cited by other articles:
 |
|
 |
|
  E. Perez-Reyes G Protein-Mediated Inhibition of Cav3.2 T-Type Channels Revisited Mol. Pharmacol., February 1, 2010; 77(2): 136 - 138. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. Hu, S. D. DePuy, J. Yao, W. E. McIntire, and P. Q. Barrett Protein Kinase A Activity Controls the Regulation of T-type CaV3.2 Channels by G{beta}{gamma} Dimers J. Biol. Chem., March 20, 2009; 284(12): 7465 - 7473. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
J. Tao, M. E. Hildebrand, P. Liao, M. C. Liang, G. Tan, S. Li, T. P. Snutch, and T. W. Soong Activation of Corticotropin-Releasing Factor Receptor 1 Selectively Inhibits CaV3.2 T-Type Calcium Channels Mol. Pharmacol., June 1, 2008; 73(6): 1596 - 1609. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. R. Costa and W. A. Varanda Intracellular calcium changes in mice Leydig cells are dependent on calcium entry through T-type calcium channels J. Physiol., December 1, 2007; 585(2): 339 - 349. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. SPAT and L. HUNYADY Control of Aldosterone Secretion: A Model for Convergence in Cellular Signaling Pathways Physiol Rev, April 1, 2004; 84(2): 489 - 539. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. Barbas, L. DesGroseillers, V. F. Castellucci, T. J. Carew, and S. Marinesco Multiple Serotonergic Mechanisms Contributing to Sensitization in Aplysia: Evidence of Diverse Serotonin Receptor Subtypes Learn. Mem., September 1, 2003; 10(5): 373 - 386. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
E. Perez-Reyes Molecular Physiology of Low-Voltage-Activated T-type Calcium Channels Physiol Rev, January 1, 2003; 83(1): 117 - 161. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 M. Cote, G. Guillon, M. D. Payet, and N. Gallo-Payet Expression and Regulation of Adenylyl Cyclase Isoforms in the Human Adrenal Gland J. Clin. Endocrinol. Metab., September 1, 2001; 86(9): 4495 - 4503. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 C. K. Naber, J. Husing, U. Wolfhard, R. Erbel, and W. Siffert Interaction of the ACE D Allele and the GNB3 825T Allele in Myocardial Infarction Hypertension, December 1, 2000; 36(6): 986 - 989. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 A. Chorvatova, L. Gendron, L. Bilodeau, N. Gallo-Payet, and M. D. Payet A Ras-Dependent Chloride Current Activated by Adrenocorticotropin in Rat Adrenal Zona Glomerulosa Cells Endocrinology, February 1, 2000; 141(2): 684 - 692. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
, control; 
, DA) shows
current traces generated by a depolarizing pulse from a holding
potential of -80 mV to -20 mV. Scale: horizontal, 30 msec; vertical,
220 pA. B, Current-voltage relationships of the T-current in control
conditions (
) and with 0.3 mM DA (
