Interaction of ␣1-Adrenoceptor Subtypes With Different G
Proteins Induces Opposite Effects on Cardiac L-type
Ca2ⴙ Channel
Jin O-Uchi, Hiroyuki Sasaki, Satoshi Morimoto, Yoichiro Kusakari, Hitomi Shinji, Toru Obata,
Kenichi Hongo, Kimiaki Komukai, Satoshi Kurihara
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Abstract—We examined the effect of ␣1-adrenoceptor subtype-specific stimulation on L-type Ca2⫹ current (ICa) and
elucidated the subtype-specific intracellular mechanisms for the regulation of L-type Ca2⫹ channels in isolated rat
ventricular myocytes. We confirmed the protein expression of ␣1A- and ␣1B-adrenoceptor subtypes at the transverse
tubules (T-tubules) and found that simultaneous stimulation of these 2 receptor subtypes by nonsubtype selective
agonist, phenylephrine, showed 2 opposite effects on ICa (transient decrease followed by sustained increase). However,
selective ␣1A-adrenoceptor stimulation (ⱖ0.1 ␮mol/L A61603) only potentiated ICa, and selective ␣1B-adrenoceptor
stimulation (10 ␮mol/L phenylephrine with 2 ␮ mol/L WB4101) only deceased ICa. The positive effect by
␣1A-adrenoceptor stimulation was blocked by the inhibition of phospholipase C (PLC), protein kinase C (PKC), or
Ca2⫹/calmodulin-dependent protein kinase II (CaMKII). The negative effect by ␣1B-adrenoceptor stimulation disappeared after the treatment of pertussis toxin or by the prepulse depolarization, but was not attriburable to the inhibition
of cAMP-dependent pathway. The translocation of PKC␦ and ␧ to the T-tubules was observed only after ␣1Aadrenoceptor stimulation, but not after ␣1B-adrenoceptor stimulation. Immunoprecipitaion analysis revealed that
␣1A-adrenoceptor was associated with Gq/11, but ␣1B-adrenoceptor interacted with one of the pertussis toxin-sensitive G
proteins, Go. These findings demonstrated that the interactions of ␣1-adrenoceptor subtypes with different G proteins
elicit the formation of separate signaling cascades, which produce the opposite effects on ICa. The coupling of
␣1A-adrenoceptor with Gq/11-PLC-PKC-CaMKII pathway potentiates ICa. In contrast, ␣1B-adrenoceptor interacts with Go,
of which the ␤␥-complex might directly inhibit the channel activityat T-tubules. (Circ Res. 2008;102:0-0.)
Key Words: ␣1-adrenoceptor 䡲 L-type Ca2⫹ channel 䡲 G protein 䡲 PKC
he ␣1-adrenoceptor (AR) stimulation has an important
role for the regulation of mammalian cardiac muscle
contraction.1– 4 We have previously shown that ␣1-AR stimulation modulates the function of voltage-gated L-type Ca2⫹
channels (VLCC) which is one of the important regulatory
factors in cardiac excitation-contraction coupling.5 The effects of ␣1-AR stimulation on cardiac Ca2⫹ current through
VLCC (ICa) can be classified into 2 opposite effects (negative
and positive effects): the positive effect is dependent on
protein kinase C (PKC) and Ca2⫹/calmodulin-dependent protein kinase II (CaMKII) activity, but the negative effect is
not.5 Although we have proposed this novel model for
understanding the molecular mechanisms underlying the
modulation of VLCC by ␣1-AR stimulation, 2 important
questions remain to be solved: (1) What is the molecular
mechanism which simultaneously induces two opposite effects during ␣1-AR stimulation?; (2) What are the molecular
components for evoking the negative effect on ICa by␣1-AR
T
stimulation? We postulated that these 2 opposite effects
simultaneously occur via (1) different ␣1-AR subtypes, ␣1A
and ␣1, which are the dominant receptor subtypes in mammalian heart1,4 and (2) subtype-specific intracellular signal
transduction pathways. The aims of this study are to characterize the effects of ␣1-AR subtype-selective stimulation on
ICa and to clarify the ␣1-AR subtype-specific signaling pathway for the regulation of ICa. Here, we show the direct
evidence that cardiac ␣1-AR signaling diverges at the level of
the ␣1-AR subtype and G protein, which produce the opposite
effects on I Ca in rat ventricular myocyes. Alpha 1A adrenoceptor coupled with Gq/11 and activated phospholipase
C (PLC)-PKC-CaMKII pathway, which evoked the potentiation of ICa. In contrast, ␣1B-adrenoceptor interacted with Go,
of which the ␤␥-complex could directly inhibit ICa. These
results represent the whole picture of intracellular mechanism
for the unique regulation of VLCC by cardiac ␣1-AR signaling and also provide the significant insight into the regulation
Original received November 13, 2007; revision received April 16, 2008; accepted April 30, 2008.
From the Department of Cell Physiology (J.O.-U., S.M., Y.K., S.K.), the Division of Molecular Cell Biology (H.S., T.O.), the Division of Cardiology
(S.M., K.H., K.K.), and the Department of Bacteriology (H.S.), The Jikei University School of Medicine, Tokyo, Japan.
Correspondence to Jin O-Uchi, Department of Cell Physiology, The Jikei University School of Medicine, 3-25-8 Nishi-Shimbashi, Minato-ku, Tokyo,
105-8461 Japan. E-mail [email protected]
© 2008 American Heart Association, Inc.
Circulation Research is available at http://circres.ahajournals.org
DOI: 10.1161/CIRCRESAHA.107.167734
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Figure 1. Detection and cellular localization of ␣1-AR subtypes in rat ventricle. A,
Detection of ␣1-AR subtypes in rat ventricle by Western immunoblot (IB) using
specific antibodies against ␣1A- (left),
␣1B- (middle) or ␣1D-AR (right). Each well
contained 50-␮g membrane protein from
rat ventricular myocytes (heart), urinary
bladder (bladder), brain, or liver. B, Confocal images of isolated ventricular myocytes labeled with ␣1-AR subtypespecific antibody (␣1A- or ␣1B-AR) (red,
left) and the plasma membrane marker
Wheat Germ Agglutinin-FITC (WGA)
(green, middle). The overlay images are
also shown (right). Bars⫽10 ␮m. C,
Immunoelectron microscopic images of
ventricular tissue labeled with 15-nm
gold-␣1A-AR (left) or ␣1B-AR (right). A
high intensity of gold labeling was
observed directly under T-tubule membranes (indicated by arrows). Mt indicates mitochondrion; Z, Z-line; Bar⫽
500 nm.
of cardiac excitation-contraction coupling by ␣1-AR subtypespecific signaling.
Materials and Methods
For details, please see the Data Supplement (available online at
http://circres.ahajournals.org). Single ventricular myocytes and papillary muscles were prepared from adult male Wistar rats (300 to
400 g).5 The measurement of ICa using a perforated patch clamp,5
Western immunoblot,5 immunoprecipitation,6 cAMP determination
using an enzyme immunoassay,7 and immunofluorescence microscopy5 were performed on freshly isolated ventricular myocytes.
Papillary muscles were used for immunoelectron microscopy.5 All
results are shown as mean⫾SD. Bars in the graphs indicate SD.
Paired data were evaluated by Student t test. For multiple comparisons, 1-way or 1-way repeated ANOVA followed by Bonferroni post
hoc test with the significance level set at P⬍0.05.
Results
Detection and Cellular Localization of ␣1-AR
Subtypes in Cardiomyocytes
The protein expression of ␣1-AR subtypes in isolated adult rat
ventricular myocytes was confirmed by Western immunoblot
with the commercially available antibodies against ␣1A-, ␣1B-,
and ␣1D-AR (Figure 1A). In the membrane proteins from
cardiomyocytes and urinary bladder, single bands were detected with the expected molecular size for glycosylated
␣1A-AR (68 kDa)8 using specific antibody against human
␣1A-AR (Figure 1A, left). However, in the parallel measurement with membrane proteins from rat brain, no positive band
was observed. The specific antibody against human ␣1B-AR
showed a major band with the expected molecular size for
glycosylated ␣1B-AR (⬇80 kDa)9 in rat cardiomyocytes, liver,
and brain (Figure 1B, middle). The ␣1D-AR (60 kDa) was
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Figure 2. Alpha1A-AR stimulation shows only positive effect in ICa, but ␣1B-AR stimulation shows only negative effect in ICa. A, A representative record of the time-dependent change in ICa during the application of the nonsubtype selective ␣1-AR agonist, 10 ␮mol/L Phe.
B, A representative record of time-dependent change in ICa during application of the selective ␣1A-AR agonist, 0.1 ␮mol/L A61603. C,
Concentration-dependent effect of A61603 on ICa. Time course of ICa in the absence of A61603 is also shown (red diamond). The amplitudes of currents at each period were normalized by the current before the application of A61603. The number of the cells tested is
shown in parentheses. *P⬍0.05, **P⬍0.01 compared to the normalized current in the absence of A61603 (red diamond) at each time.
D, Effect of 0.1 ␮mol/L A61603 on ICa in the presence of PLC inhibitor (1 ␮mol/L U73122), PKC inhibitor (10 ␮mol/L chelerythrine), or
CaMKII inhibitors (0.5 ␮mol/L KN-93 or 10 ␮mol/L AIP). One ␮mol/L U73343 and 0.5 ␮mol/L KN-92 were also applied as the inactive
analogues of U73122 and KN-93, respectively. Each inhibitor or inactive analogue was applied 10 minutes before the agonist application. Graphs show the ratios of ICa 15 minutes after application of A61603 to that before the application of A61603. The number of the
cells tested is shown in parentheses. *P⬍0.05, compared to the control (0.1 ␮mol/L A61603). †P⬍0.05, compared to the current in the
presence of inactive form analogues (KN-92 or U73343). E, A representative time course of ICa during application of 10 ␮mol/L Phe in
the presence of selective ␣1A-AR antagonist, 2 ␮mol/L WB4101. F, Concentration-dependent effect of Phe in the presence of 2 ␮mol/L
WB4101 on ICa,. WB4101 was applied 10 minutes before agonist application. The amplitudes of currents at each period were normalized by the current before the application of Phe. Time-dependent changes in ICa in the absence of Phe is also shown (red diamond).
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only found in rat brain; no significant bands were observed in
cardiomyocytes and liver cells using the specific antibody
against rat ␣1D-AR (Figure 1A, right). These results show that
␣1A- and ␣1B-AR (but not ␣1D-AR) were detectable at the
protein level in our preparation of cardiomyocytes. Thus, we
focused on the role of these 2 subtypes of ␣1-AR (␣1A and ␣1B)
in native cardiomyocytes in the following experiments.
We determined the cellular localization of ␣1A-AR and
␣1B-AR in cardiac cells using an immunofluorescence microscope (Figure 1B). In ventricular myocytes, ␣1A-AR was
detectable at the plasmalemma and along the Z-lines, which
coincides with the sarcolemmal invaginations termed transverse tubules (T-tubules) where the majority of VLCC are
located.10 On the other hand, ␣1B-AR was not detectable at the
plasmalemma; rather it was localized at T-tubules and intercalated disks. The light microscopic images obtained from
papillary muscle cells also showed a similar tendency of the
localization of ␣1A-AR and ␣1B-AR as observed in the isolated
cells (supplemental Figure I).
To confirm the detailed subcellular localization of ␣1A-AR
and ␣1B-AR, ultrathin cryosections of the left ventricular
papillary muscles were incubated with these receptor
subtype-specific antibodies (Figure 1C). The membranes of
T-tubules were specifically labeled with the antibodies
against ␣1A-AR and ␣1B-AR. These results suggest that 2
␣1-AR subtypes (␣1A and ␣1B) are detectable at the protein
level in cardiac membrane, and they are preferentially localized at the T-tubules.
Alpha1A-AR Stimulation Showed Only a Positive
Effect on ICa Without a Negative Effect
Alpha1-AR stimulation by the nonsubtype selective agonist,
10 ␮mol/L phenylephrine (Phe), showed a biphasic change in
ICa measured using the perforated patch clamp (a transient
decrease followed by a sustained increase) in the presence of
␤-AR antagonist, 1 ␮mol/L bupranolol, which we used
previously5 (Figure 2A). Similar results were obtained when
we used another ␤-AR antagonist, 1 ␮mol/L propranolol, as
shown in supplemental Figure II. Following experiments
were all performed in the presence of 1 ␮mol/L bupranolol.
Next we observed the effect of selective ␣1A-AR stimulation on ICa by using the selective ␣1A-AR agonist A61603.
Fifteen-minute treatment with A61603 (0.1 ␮mol/L) evoked
only potentiation of ICa (Figure 2B) without changing the
current-voltage relationship (supplemental Figure III), and
there was no negative effect in the initial period, which was
observed in the presence of nonsubtype selective ␣1-AR
stimulation by Phe (see Figure 2A). This positive effect after
15-minute treatment with A61603 was saturated at
Figure 2 (Continued). The number of the cells tested is shown
in parentheses. *P⬍0.05 compared to the normalized current in
the absence of Phe (red diamond) at each time. G, Concentrationdependent effect of the selective ␣1B-AR antagonist, L765,314 on a
transient decrease in ICa 2 minutes after application of 10 ␮mol/L
Phe. L765,314 was applied 10 minutes before agonist application.
Graphs show the percent inhibition of ICa 2 minutes after application of 10 ␮mol/L Phe in the presence (10⬇100 nmol/L, n⫽4) or in
the absence of L765,314 (0 nmol/L, n⫽12). *P⬍0.05, compared to
the control (0 nmol/L L765,314).
1 ␮mol/L A61603 (0.1 ␮mol/L, 35.36⫾14.37%, n⫽6;
1 ␮mol/L, 42.64⫾27.49%, n⫽8; P⫽1.00) and was blocked
by the selective ␣1A-AR antagonist, 2 ␮mol/L WB4101 (n⫽5,
data not shown). All concentrations of A61603 (0.1⬇
1␮mol/L) used showed only a positive without a negative
effect (Figure 2C).
As we previously reported that the positive effect of ␣1-AR
stimulation on ICa is evoked through a PKC- and CaMKIIdependent mechanism,5 next we investigated the involvement
of PKC and CaMKII in the signaling pathways which evoke
the potentiation of ICa during ␣1A-AR stimulation. In the
presence of a PKC inhibitor chelerythrine, the positive effect
of A61603 was not observed. CaMKII inhibition by KN-93 or
autocamtide-2 inhibitory peptide (AIP; a membranepermeable and a highly specific peptide type inhibitor of
CaMKII) also abolished the potentiation of ICa by A61603
(Figure 2D). Moreover, in the presence of a PLC inhibitor,
U73122, the positive effect of A61603 completely disappeared (Figure 2D). These results suggest that ␣1A-AR stimulation shows only a positive effect on ICa and this effect is
mediated through the PLC-PKC-CaMKII pathway.
Alpha1B-AR Stimulation Showed Only a Negative
Effect on ICa Without a Positive Effect
We investigated the effect of ␣1B-AR stimulation on ICa by the
application of a nonsubtype selective ␣1-AR agonist (Phe)
with a selective ␣1A-AR antagonist (WB4101), because no
selective ␣1B-AR agonist is available at present.4 Ten-minute
exposure to 2 ␮mol/L WB4101 significantly decreased ICa
without changing the shape of the current-voltage relationship (supplemental Figure III) and reached another steady
state (Figure 2F, red diamonds). In the continuous presence of
WB4101, 10 ␮mol/L Phe only decreased ICa (Figure 2E and
2F) without changing the shape of the current-voltage relationship (supplemental Figure III). In contrast, 1 ␮mol/L Phe
(no negative effect on ICa was observed at this concentration5), in the presence of WB4101, showed no significant
positive or negative effects (Figure 2F). Thus, ␣1B-AR stimulation produced only a negative effect without potentiation
of ICa, which was opposite to the effect of ␣1A-AR stimulation.
To further confirm the negative effect by ␣1B-AR stimulation on ICa, we also investigated the effect of 10 ␮mol/L Phe
in the presence of the selective ␣1B-AR antagonist, L-765,314.
The negative effect of 10 ␮mol/L Phe on ICa was significantly
inhibited by the treatment of L-765,314 in a concentrationdependent manner (Figure 2G and supplemental Figure IV).
PKC Was Activated After ␣1A-AR Stimulation, but
Not After ␣1B-AR Stimulation
We previously showed that the positive effect of ␣1-AR
stimulation on ICa is dependent on PKC activity.5 Therefore,
we examined the involvement of PKC in the signaling
pathway after ␣1A- or ␣1B-AR stimulation. One of the hallmarks of PKC activation is the translocation of soluble
enzymes to particle fractions, presumably near their protein
substrates that include sarcolemmal proteins.11 We determined the isoform-specific PKC translocation to the membrane by calculating the membrane-to-cytosolic (M/C) ratio
before and after selective ␣1-AR-subtype stimulation (Figure
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Figure 3. Effect of subtype-specific ␣1-AR stimulation on PKC translocation. A to C, Immunoblot analysis of PKC␣ (A), PKC␦ (B), and
PKC␧ translocation (C). Blots show the redistribution of each PKC isoform in cytosol (C), membrane (M), and filament (F) fraction of
intact myocytes treated with 10 ␮mol/L phenylephrine (Phe), 1 ␮mol/L A61603 (␣1A), 10 ␮mol/L phenylephrine in the presence of
2 ␮mol/L WB4101 (␣1B) or 1 ␮mol/L PMA (PMA) for 15 minutes (25 ␮g protein/well). Control (CTR) represents untreated cells. Total
PKC␣, ␦, and ␧ in whole cell lysates (W) are also shown (50 ␮g protein/well). D to F, Graphs show M/C ratios for the evaluation of
PKC-isoform-specific translocation (n⫽7). The M/C ratio after each stimulation (Phe, ␣1A, ␣1B, or PMA) was normalized by the M/C ratio
before stimulation (CTR). *P⬍0.05, compared to the control (CTR).
3A to 3F). In our preparation, one of the Ca2⫹-dependent PKC
isoforms, PKC␣ was not significantly translocated by nonsubtype selective ␣1-AR stimulation or by subtype-selective
␣1-AR stimulations, although endogenous PKC activator,
phorbol 12-myristate 13-acetate (PMA) did translocate PKC␣
from cytosol to membrane and filament fractions (Figure 3A
and 3D). On the contrary, significant translocation of the
Ca2⫹-independent PKCs (␦ and ␧) from cytosol to membrane
fraction was found 15 minutes after ␣1A-AR stimulation
(Figure 3B, 3C, 3E, and 3F). However, no remarkable
translocation of PKC after ␣1B-AR stimulation was observed
(Figure 3E and 3F).
Phosphorylation of PKC itself was also measured because
it is another hallmark of PKC activation.11 Immunoreactivity
of PKC␧ phosphorylation at the hydrophobic motif and PKC␦
phosphorylation at the activation loop significantly increased
after ␣1A-AR stimulation or PMA treatment in the membrane
fraction. However, the amount of phosphorylated PKC␣ in
the membrane fraction did not increase after ␣1A- or ␣1B-AR
stimulation (supplemental Figure V).
We identified the subcellular localization of the activated
PKC isozymes to elucidate their roles in the regulation of
VLCC before and after selective ␣1-AR-subtype stimulation
by using an immunofluorescence microscope (Figure 4A to
4C). Significant translocation of PKC␣ was not observed
after the treatment with Phe as shown in Western immunoblot
(Figure 4A). Most of PKC␦ was localized in the nucleus or at
the nuclear membrane, but the remainder was diffusely
distributed in the cytosol at rest (Figure 4B). After Phe
treatment, a striated pattern also became visible, which was in
accordance with the location of T-tubules (Figure 4B). PKC␧
was diffusely distributed in the cytosol before stimulation
(Figure 4C). After Phe treatment, PKC␧ was accumulated at
the T-tubules and intercalated disks (Figure 4C).
These results suggest that Ca2⫹-independent PKCs were
activated and the activated PKCs were redistributed to the
membrane fraction, presumably to the T-tubules after ␣1A-AR
stimulation. However, there was no obvious involvement of
PKC in the ␣1B-AR signaling pathway.
Negative Change in ICa During ␣1-AR Stimulation
Was Mediated via the Pertussis Toxin–Sensitive G
Protein Pathway
We showed that the positive effect on ICa caused by ␣1A-AR
stimulation was dependent on PKC, but the negative effect of
␣1B-AR stimulation was independent of PKC activation (Figures 2 and 3). Several reports demonstrated that ␣1-AR
couples not only with Gq/11 which in turns leads to activation
of PLC and PKC, but also with the pertussis toxin (PTX)sensitive G proteins, and it shows diverse physiological
effects in cardiomyocytes.1,4,12 Therefore, we hypothesized
that ␣1B-AR functionally couples with other G proteins, and
we examined the involvement of PTX-sensitive G protein in
the regulation of ICa by ␣1-AR stimulation.
Inhibition of Gi/o-protein by PTX in our preparations was
confirmed by the ability of PTX to block the muscarinic
inhibition of ICa in the presence of ␤-AR stimulant (supplemental Figure VI). Treatment of PTX significantly inhibited
the negative effect by 10 ␮mol/L Phe at 2 minutes and then
enhanced the positive effect at 15 minutes (Figure 5A).
Moreover, we separately investigated the effects of ␣1-AR
subtype-selective stimulation on ICa in PTX-treated cells. We
confirmed that the negative effect by ␣1B-AR stimulation was
blocked by PTX (Figure 5C), but the magnitude of the
positive effect by ␣1A-AR stimulation did not alter after PTX
treatment (Figure 5B). These results indicate that the negative
phase of ICa during ␣1B-AR stimulation (Figure 2A) was
produced through PTX-sensitive G protein (Gi/o) pathways.
In adult rat cardiomyocytes at least 3 subtypes of PTXsensitive G␣ (G␣i-2, G␣i-3, G␣o) are expressed at the mRNA
level and are detectable at the protein level.13 Therefore, the
possibility that ␣1-AR subtypes directly couple with these
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immunofluorescence images with the specific antibodies
against ␣1B-AR and G␣o showed that G␣o was colocalized with
␣1B-AR at T-tubules (Figure 5E). Thus, these results indicate
that the ␣1A-AR couples with Gq/11-protein in a classical
coupling mode, which activates the PLC-diacylglycerol-PKC
pathway and that ␣1B-AR is linked to Go at the T-tubules and
evokes the negative phase in ICa.
Negative Effect of ␣1-AR Stimulation on ICa Is
Mediated Through ␤␥-Complex of G Protein
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Figure 4. PKC␦ and ␧ (but not ␣) were translocated to the
T-tubules by ␣1-AR stimulation. A to C, Immunofluorescence
localization of PKC in adult rat ventricular myocytes before and
after ␣1-AR stimulation. The immunofluorescence images of ventricular myocytes before stimulation (CTR), 15 minutes after
application of 100 ␮mol/L phenylephrine (Phe), or 15 minutes
after application of 100 ␮mol/L PMA (PMA) are shown by using
specific antibodies against PKC ␣, ␦, and ␧ which were used in
Western immunoblot.
PTX-sensitive G proteins was examined by coimmunoprecipitation of these G␣-subunits with anti–␣1A- or anti–␣1B-AR
antibody. The immunoprecipitants were analyzed by Western
immunoblot probing with the antibodies against G␣-subunits,
of which specificities were checked by using the recombinant
G␣-subunits (see supplemental Figure VII). The ␣1A-AR
antibody coimmunoprecipitated G␣q/11, whereas the ␣1B-AR
antibody coimmunoprecipitated G␣o (Figure 5D). Moreover,
Our biochemical and electrophysiological results indicated
that ␣1B-AR interacted with one of the PTX-sensitive G
proteins, Go. However, the functional roles of Go-protein in
native cardiomyocytes have not been clarified. We postulated
here that ␣1B-AR-Go interaction could inhibit the VLCC
activity through (1) the decrease of basal cAMP level (eg, by
the inhibition of adenylyl-cyclase activity as in the case of
Gi4), or (2) stimulation of protein phosphatase activity,
followed by the reduction of basal phosphorylation level of
the VLCC. However, we found that the basal cAMP level in
our preparations did not significantly change during ␣1-AR
stimulation as described previously14 (Figure 6A), and negative effect of ICa by Phe was clearly observed even in the
presence of cAMP-dependent protein kinase (PKA) inhibitor
(1 ␮mol/L H-89; Figure 6B). Thus, the inhibition of cAMPPKA signaling is not involved in the mechanism for evoking
negative phase of ICa. Moreover, we pretreated the cells with
a protein phosphatase inhibitor, calyculin A in the presence of
H-8915 and then investigated the effects of Phe in the
continuous presence of calyculin A and H-89. Under this
condition,15 we still observed the negative phase of ICa,
suggesting that activation of phosphatases followed by the
reduction of basal VLCC phosphorylation is not involved in
the negative phase (supplemental Figure VIII).
Several reports stated that the ␤␥-complex of heterotrimetric Go-protein directly interacts with N-type or L-type Ca2⫹
channels to inhibit their activity.16,17 Moreover, a depolarization pulse applied to the membrane before channel activation
is known to counteract this inhibition.16 Therefore, we next
observed the effect of a nonsubtype selective ␣1-AR agonist
(10 ␮mol/L Phe) on ICa using this prepulse depolarization
protocol (Figure 7A).
Recording with this prepulse depolarization, there was no
significant transient decrease of ICa for up to 2 minutes after
the application of 10 ␮mol/L Phe (Figure 7A and 7B). Fifteen
minutes after the application of Phe, ICa was significantly
increased (Figure 7A and 7B). Thus, the current inhibition at
the initial stage (⬇2 minutes) induced by ␣1-AR stimulation
was not attributable to the reduction of basal phosphorylation
level of VLCC, but was possibly produced by the direct
interaction of ␤␥-complex of Go with VLCC.
Discussion
In this study, we elucidated the differences between cardiac
␣1A- and ␣1B-AR signaling pathways and provide direct
evidence indicating that different G proteins (and kinases)
are involved in the respective subtype-specific signaling
pathway and induce opposite changes in ICa in native
cardiomyocytes (Figure 8). We showed that ␣1A- and
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Figure 5. Negative effect on ICa during
␣1-AR stimulation is mediated through
␣1B-AR and PTX-sensitive G protein pathway. A to C, Effect of 10 ␮mol/L Phe (A),
1 ␮mol/L A61603 (B), or 10 ␮mol/L Phe with
2 ␮mol/L WB4101 (C) on ICa in PTX-treated
cells (red circles) and in nontreated cells
(black circles). The amplitudes of currents at
each period were normalized by the current
before the application of Phe. *P⬍0.05,
compared to the normalized current in the
PTX-nontreated cells (black circle) at each
time. The number of the cells tested is
shown in parentheses. D, Coimmunoprecipitation of ␣1-ARs with G␣-subunits. Membrane proteins were immunoprecipitated (IP)
with specific ␣1-AR antibodies or control
IgG (IgG). The immunoprecipitates were analyzed by Western immunoblot by probing
with the antibodies against ␣1-ARs and
G␣-subunits. Membrane lysates (ML; 12.5
␮g/lane) are also shown as the positive control. E, Immunofluorescence images of ventricular myocytes costained with antibodies
against anti-G␣o (red, upper panel) and
␣1B-AR (green, middle panel). The overlay
image is also shown (lower panel). Bars,
10 ␮m.
␣1B-AR were functionally expressed at T-tubules where
VLCC is concentrated,10 but ␣1D-AR was not detected at
protein level and was not functionally expressed in our
preparations (supplemental Figure IV). Furthermore, we
clearly separated the effect of ␣1A- or ␣1B-AR stimulation
from that of nonsubtype selective stimulation on ICa by
pharmacological procedure and clarified the detail of each
signaling pathway by biochemical and morphological techniques. Alpha1A-AR and ␣1B-AR signaling pathways couple
with different G proteins, Gq/11 and Go, respectively and
produce different functional outcomes; ␣1A-AR stimulation
activates Gq/11-PLC-diacylglycerol-PKC-CaMKII pathway
and increases ICa. On the contrary, ␣1B-AR interacts with Go
and inhibits the VLCC activity.
Figure 6. Inhibition of cAMP-PKA signaling
was not involved in the mechanism for evoking negative phase of ICa during ␣1-AR stimulation. A, cAMP concentration in isolated rat
ventricular myocytes treated with 10 ␮mol/L
phenylephrine (Phe) for 2 or 15 minutes (n⫽3
or 4). cAMP concentration after treatment of
100 nmol/L isoproterenol (Iso) for 15 minutes
in the absence of ␤-AR antagonist was also
shown as the positive control (n⫽4). *P⬍0.05,
compared to the control (nontreated cells;
CTR). B, The effect of 10 ␮mol/L Phe on ICa in
the presence of the selective PKA inhibitor,
1 ␮mol/L H-89 (n⫽6). The amplitudes of the
currents at each period were normalized by
the current before the application of Phe. *P⬍0.05, compared to the current before stimulation (0 minutes).
8
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June 6, 2008
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In our preparations, we demonstrated that only ␣1A-AR
stimulation induces the activation of novel PKCs and translocates them to the cell membrane structure called T-tubules,
and that ␣1B-AR stimulation did not show any activation of
PKC. This result is consistent with the previous report that
␣1B- or ␣1D-AR signaling pathway does not have any influences on PKC activity.19 We did not detect any significant
activation of PKC␣ after ␣1-AR stimulation, indicating that
PKC␣ was not involved in ␣1-AR signaling19 in our preparations. Moreover, we directly showed the interaction of
␣1A-AR and G␣q/11 which activates PLC.1,6 The ␣1A-AR-Gq/11PLC-diacylglycerol pathway activated novel PKCs and translocated them to T-tubules where VLCC and CaMKII are
prevalent.5,21 The translocated PKC could activate CaMKII at
T-tubules,5 and then the activated CaMKII could directly
potentiate ICa through the phosphorylation of ␣1c and /or ␤
subunits of the channel.21,22 Thus, ␣1A-AR signaling components preferentially localized at the T-tubules and efficiently
regulated cardiac VLCC, as in the case of cardiac endothelinreceptor signaling.23
Alpha1B-AR-Go Interaction Induces the
Inhibition of ICa
Figure 7. Negative effect on ICa during ␣1-AR stimulation is
inhibited by prepulse depolarization protocol. A, Effect of
10 ␮mol/L Phe on ICa recorded by using the prepulse depolarization protocol shown at the top. The horizontal bar indicates
the period of application of Phe. The inset shows the superimposed original current traces at the points indicated. B, Time
courses of the changes in ICa after the application of 10 ␮mol/L
Phe recorded by using the prepulse depolarization protocol
(n⫽11, open circles). Time course of ICa in the absence of Phe is
also shown (n⫽6, closed circles). The amplitudes of currents at
each period were normalized by the current before the application of Phe. *P⬍0.05, ***P⬍0.001 compared to the normalized
current in the absence of Phe (closed circles) at each time.
Alpha1A-AR-Gq-PKC Signaling Pathway Induces
Potentiation of ICa
In this study, we showed that ␣1A-AR was expressed at
T-tubules and also demonstrated that ␣1A-AR stimulation did
affect the VLCC activity which was confirmed by using
␣1A-AR selective agonist, A61603. Alpha1A-AR pathway
potentiated ICa in native cardiomyocytes, which is mediated
through a PKC-dependent mechanism (Figure 8). PKC is a
phospholipid-dependent Ser-Thr kinase, and most isoforms of
the PKC are activated as a result of receptor-dependent
activation of PLC and the hydrolysis of membrane phosphoinositides.1 Although all cloned subtypes of ␣1-AR can
induce PLC activation and inositol phosphate formation,18
receptor subtype-specific activation or downregulation of
PKC has been reported in cultured neonatal cardiomyocytes.19 In cardiac tissue, the isoforms of 1 Ca2⫹-dependent
PKC (PKC␣) and 2 Ca2⫹-independent PKCs (novel PKCs;
PKC␦ and PKC␧) are at least detectable at the protein level.20
We showed that ␣1B-AR was expressed at T-tubules and also
demonstrated that ␣1B-AR stimulation did affect the VLCC
activity confirmed by pharmacological procedures. Alpha1BAR stimulation inhibited ICa, which is mediated through
PKC-independent mechanisms (Figure 8). Our biochemical
studies indicated that ␣1B-AR stimulation shows less influence on the PKC activity than ␣1A-AR stimulation. This result
is compatible with the previous reports that ␣1B-AR subtype
has less potency for the stimulation of phosphoinositide
hydrolysis than ␣1A-AR.24,25 We found that ␣1B-AR coupled
with Go instead of Gq/11 and this pathway brought about
negative modulation of ICa, which was not attributable to the
decline of basal phosphorylation level of VLCC by the
inhibition of cAMP-PKA pathway or activation of protein
phosphatases (Figure 6 and supplemental Figure VIII). Our
result is consistent with the previous results obtained using a
constitutively active mutant of ␣1B-AR or the technique of the
overexpression of wild-type ␣1B-AR, which shows the possibility that only ␣1B-AR (but not ␣1A-AR) couples with the
PTX-sensitive pathway.26,27 ICa inhibition through Go was
reported in secretory cells17 and cardiac cells from genetically
engineered mice,28 but the functional role of Go in native
cardiomyocytes is still poorly understood. Ivanina et al
showed that the direct binding of G␤␥ subunit of Go to VLCC
inhibits the channel activity in heterologous expression systems.29 They also reported that the basal intracellular Ca2⫹
level is essential for this inhibition, which is consistent with
our previous data.5 We also showed that ␣1B-AR and Go
colocalize with VLCC at T-tubules, and we propose the
working model that ␤␥-complex of Go protein could directly
inhibit VLCC.
Conclusion
In conclusion, our results represent the evidence that the
unique combinations of ␣1-AR subtypes and specific G
proteins form subtype-specific signal transduction pathways,
O-Uchi et al
Cardiac ␣1-Adrenoceptor Subtypes and G Proteins
9
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Figure 8. Possible mechanism underlying the opposing modulation of L-type Ca2⫹ channels induced by ␣1-AR subtype-specific signaling.
A, Model of ␣1-AR subtype-specific intracellular signaling pathways in cardiomyocytes. DAG, diacylglycerol. PIP2, Phosphatidylinositol(4,5)bisphosphate. B, Model for the opposing modulation of ICa by ␣1-AR subtype-specific signaling. Alpha1A-AR-Gq/11 pathway potentiates
ICa (showing as the red line) and ␣1B-AR-Go interaction inhibits ICa (showing as the blue line). The sum of these 2 opposite effects could
explain the unique effect (biphasic change) of subtype nonsubtype selective ␣1-AR stimulation by Phe (shown as the black line).
which induce the opposite effects on VLCC in native cardiac
cells. The coupling of specific ␣1-AR subtypes with PTXsensitive G protein could exhibit the negative feedback
response to ␣1-AR stimulation, and this mechanism would
contribute to the protection of the heart from Ca2⫹ overload as
in the relation between ␤1- and ␤2-AR. The approach of
characterizing the receptor subtype-specific interacting G
protein will provide new insight to elucidate the whole
picture of the subtype-specific signaling pathway in native
cardiomyocytes, and further could lead to an understanding of
the functional roles of each ␣1-AR subtype under physiological and pathophysiological condition.
Acknowledgments
The authors thank Prof C. Franzini-Armstrong (University of Pennsylvania School of Medicine) for her helpful comments. The authors
thank N. Tomizawa, M. Murata, H. Arai, E. Kikuchi, M. Nomura, Y.
Natake, H. Saito, and Y. Kimura for their technical assistance.
Sources of Funding
This study was supported by Japan Heart Foundation Young Investigator’s Research Grant (to J.O.-U.), Japan Foundation of Cardiovascular Research (to J.O.-U.), Kato Memorial Bioscience Foundation (to J.O.-U.), The Jikei University Research Fund (to J.O.-U.), a
Grant-in-Aid from the Ministry of Education, Culture, Sports,
Science, and Technology (to K.H., K.K., and S.K.), Uehara Memo-
rial Foundation (to S.K. and Y.K.), Takeda Science Foundation (to
K.H.), Ueda Memorial Foundation (to K.K.), and the Vehicle Racing
Commemorative Foundation (to K.H. and S.K.).
Disclosures
None.
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Interaction of α1-Adrenoceptor Subtypes With Different G Proteins Induces Opposite
Effects on Cardiac L-type Ca 2+ Channel
Jin O-Uchi, Hiroyuki Sasaki, Satoshi Morimoto, Yoichiro Kusakari, Hitomi Shinji, Toru Obata,
Kenichi Hongo, Kimiaki Komukai and Satoshi Kurihara
Circ Res. published online May 8, 2008;
Circulation Research is published by the American Heart Association, 7272 Greenville Avenue, Dallas, TX 75231
Copyright © 2008 American Heart Association, Inc. All rights reserved.
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Expanded Material and Methods
Preparations, solutions, chemicals and antibodies
Single ventricular myocytes and papillary muscles were prepared from adult male Wistar rats
(300-400 g) as previously described1,2 in accordance with the Guiding Principles for the Care
and Use of Animals in the Field of Physiological Sciences published by the Physiological
Society of Japan (December 19, 1988) and suspended in Tyrode’s solution (mM): NaCl,
136.9;
KCl,
5.4;
CaCl2,
1;
MgCl2,
0.5;
NaH2PO4,
0.33;
2-[4-(2-Hydroxyethyl)-1-piperazinyl]ethanesulfoniac acid (HEPES), 5; glucose, 5, pH 7.40
adjusted with NaOH.
One µmol/L bupranolol (donated by Kaken Pharmaceutical Co. Ltd.,
Tokyo Japan) was used in the perfusion solution throughout the experiments to block
β-adrenergic effects (see also Supplementary Figure II).1,2
The pipette solution in
electrophysiological studies was (mmol/L): CsCl, 130; NaCl, 10; MgCl2, 0.5; HEPES, 5;
CaCl2, 1; pH was adjusted to 7.20 with CsOH.1, 3
All reagents were purchased from Sigma-Aldrich Corporation. (St. Louis, MO)
unless otherwise indicated.
Phorbol 12-myristate 13-acetate (PMA) was obtained from
Wako Pure Chemical Industries, Ltd., (Osaka, Japan).
Pertussis toxin (PTX) was purchased
from Seikagaku Corporation (Tokyo, Japan) and used as previously described (0.75 µg/ml, 3
hrs, at 37°C).4
Meeting, PA).
Calyculin A was obtained from Biomol International, L.P. (Plymouth
Full length recombinant proteins of rat Gαq, Gαi-2, Gαi-3 and Gαo were used
1
for the Western immunoblot positive control (Santa Cruz Biotechnology, Santa Cruz, CA).
A61603 (Tocris, Ellisville, MO) was used for selective α1A-AR stimulation because
this agent is 200- to 300-fold more potent against α1A-AR than phenylephrine (Phe).5
WB4101 (Tocris) was used to block α1A-AR at the concentration of 2 µmol/L, because at this
concentration, this compound can completely block the phenylephrine-induced inotropic
effect in rat cardiomyocytes.6
L-765,314 was used to block α1B-AR as it displays a 90- to
130-fold stronger selectivity for binding to rat α1B-AR than to α1A-AR and α1D-AR.7
BMY7378 (Tocris) was used to block α1D-AR at the concentration of 30 nmol/L because its
pKB (the negative log of KB) values against phenylephrine in adult rat right ventricle is
6.87±0.1.8
The synthetic CaMKII inhibitor, KN-93 and its inactive compound, KN-92 were
At a concentration of 0.5 µmol/L, KN-93
obtained from Calbiochem (La Jolla, CA).
selectively
inhibits
CaMKII
without
affecting
other
protein
kinases.9
A
membrane-permeable form of a highly specific peptide inhibitor of CaMKII (AIP) which
corresponds to an auto-inhibitory domain of CaMKII was used at the concentration of 10
µmol/L.1, 10
The PKC inhibitor, chelerythrine was used at the concentration of 10 µmol/L
because this agent selectively inhibits PKC without affecting other protein kinases at this
concentration.11
U73122 was used for the inhibition of PLC at the concentration of 1
µmol/L.12 Control experiments were also done using U73343, the inactive compound of
2
U73122 at the same concentration.
H-89 was used to selectively inhibit cAMP-dependent
protein kinase (PKA) at the concentration of 1 µmol/L.13
The following primary antibodies were used for immunoblot, immunoprecipitation
and immunohistochemical analysis: Anti-α1A-AR antibody (rabbit polyclonal IgG raised
against amino acids 331-466 mapping within a C-terminal cytoplasmic domain of α1A-AR of
human origin) (Santa Cruz Biotechnology), anti-α1B-AR antibody (goat polyclonal IgG raised
against a peptide mapping within an extracellular domain of α1B-AR of human origin) (Santa
Cruz Biotechnology), anti-α1D-adrenergic receptor antibody (goat polyclonal IgG raised
against a peptide mapping at C-terminus of α1D-AR of rat origin)14 (Santa Cruz
Biotechnology), anti-PKCα (mouse monoclonal IgG antibody raised against a peptide
corresponding to an amino acid sequence mapping at carboxy terminus of human PKCα)15
(Santa Cruz Biotechnology), anti-PKCδ (mouse monoclonal IgG raised against a peptide
corresponding to residues 114-289 of human PKCδ)16 (BD Transduction Laboratories,
Franklin Lakes, NJ), anti-PKCε (mouse monoclonal IgG raised against a peptide
corresponding to residues 1-175 of human PKCε)17 (BD Transduction Laboratories),
anti-phospho-PKC (pan) antibody (rabbit polyclonal IgG raised against a synthetic
phospho-peptide corresponding to residues surrounding Ser660 of human PKCβII)18, 19 (Cell
Signaling Technology, Danvers, MA), anti-phospho-PKCδ (Thr505) antibody (rabbit
polyclonal IgG a synthetic phospho-peptide corresponding to residues around Thr505 of
3
human PKCδ)18, 19 (Cell Signaling Technology), anti-Gαq/11 antibody (rabbit polyclonal IgG
raised against a peptide mapping within a domain common to Gαq and Gα11 of mouse
origin)20 (Santa Cruz Biotechnology), anti-Gαi-2 antibody (rabbit polyclonal IgG mapping
within a highly divergent domain of Gαi-2 of human origin)
21, 22
, anti-Gαi-3 antibody (rabbit
polyclonal IgG raised against C terminal region of Gαi-3 the rat origin)22 (Santa Cruz
Biotechnology), anti-Gαo antibody (rabbit immunopurified IgG raised against purified native
bovine Gαo)23 (Upstate, Lake Placid, NY).
The following secondary antibodies were used for immunoblot analysis:
Peroxidase-linked
anti-rabbit
IgG,
anti-mouse
IgG
(GE
Healthcare
Buckinghamshire, England) and anti-goat IgG (Santa Cruz Biotechnology).
UK
Ltd.,
The following
secondary antibodies were used for immunofluorescence microscopy or immunoelectron
microscopy: Alexa-546-conjugated anti-rabbit secondary antibody, Alexa-546-conjugated
anti-goat secondary antibody, Alexa-488-conjugated anti-rabbit secondary antibody
(Molecular Probes, Eugene, OR), 15-nm gold conjugated anti-rabbit IgG (GE Healthcare UK
Ltd.) and 15-nm gold conjugated anti-goat IgG (British Biocell International, Cardiff, United
Kingdom).
Measurement of ICa
A perforated patch clamp was carried out with amphotericin B as a pore forming agent for
measuring the Ca2+ current through L-type channels (ICa) using EPC-8 and EPC-7 PLUS
4
amplifier (HEKA Electronik, Lambrecht/Pfalz, Germany) at room temperature (≈25°C).1, 3
Amphotericin B was dissolved in DMSO (50 µg/µl) and then was diluted with pipette
solution using short vortex and sonication to a final concentration of 400-800 µg/ml.
The
pipette solution also contained 1 mmol/L Ca2+ to ensure that the perforated-patch membrane
was maintained during the recording and accidental rupture of the membrane resulted in cell
death. 1, 3 For measurement of ICa, the holding potential was set at -40 mV to inactivate the
Na+ current and a 200-msec depolarization pulse to 0 mV was applied every 10 sec.
The
amplitude of current was defined as the difference between the peak current and the residual
current at the end of the pulse.
The holding current and remaining current were not altered
throughout the experiments (including the period of α1-adrenoceptor stimulation).
The
application of 10 µmol/L nifedipine in the perfusion solution almost completely blocked this
current, indicating that the measured current was ICa.
Current-voltage relationship was
obtained using a series of test pulses between -30 and +60 mV in 10 mV increments.
Pre-pulse protocol for relieving the effect of direct inhibition of ICa by Gβγ was
designed as previously described.24-26
Depolarization pulse to +80mV from the holding
potential (-40 mV) was applied, followed by a 15-ms refractory period and 200-ms activating
pulse to 0 mV (see Figure 7A).
The amplitude of ICa recording with the pre-pulse
depolarization was stable for up to 15 min (see Figure 7B).
Western immunoblot
5
Whole cell extract of isolated rat cardiomyocytes was prepared as previously described.1
Membrane, cytosolic and filament proteins from cardiomyocytes were prepared using
differential centrifugation.15, 27
Membrane proteins from rat brain, liver and urinary prostate
were obtained using ProteoExtractTM Native Membrane Protein Extraction Kit (Calbiochem).
Each protein was separated on 8 or 10% SDS-PAGE and analyzed by Western immunoblot.1
Membrane proteins from urinary prostate in which 70% of α1-AR mRNA is α1A-subtype28
were used as the positive control to check the reactivity of the antibody against α1A-AR.
Membrane proteins from brain and liver in which α1B-AR mRNA is abundant29 were used as
the positive control for α1B-AR.
Membrane protein from brain was also used as the positive
control for checking the reactivity of the specific α1D-AR antibody because α1D-AR mRNA
was detectable in rat brain at a higher level than in heart and liver.29
Immunoreactive bands were visualized by enhanced chemiluminescence using
ECL-plus detection kit (or otherwise indicated) (GE Healthcare UK Ltd.) and analyzed by the
densitometry (ATTO, Tokyo, Japan).
Isoform-specific PKC activation was evaluated 1) by PKC translocation from the
cytosolic fraction to the membrane fraction30 and 2) by phosphorylation of PKC itself.18
Determination of isoform-specific PKC translocation to the membrane fraction was
performed by the analysis of cytosolic and membrane fractions to calculate the
membrane-to-cytosolic (M/C) ratio as descried previously.30
6
The bands of PKCs in each
fraction were visualized by using ECL-advance detection kit (GE Healthcare UK Ltd.).
For
the detection of PKC phosphorylation, the transferred membranes were blocked by Blocking
One-P solution which is free of phosphate group and endogenous phosphatase (Nakarai
Tesque, INC., Kyoto, Japan) and primary and secondary antibodies were diluted in Can Get
Signal immunoreaction enhancer solution (Toyobo Co., LTD., Osaka, Japan).
Immunoprecipitation
Isolated ventricular myocytes from 6 hearts were suspended in NaH2PO4-Na2HPO4 buffer
(pH 7.6) containing 154 mM NaCl, 0.5 mM phenylmethylsulfonyl fluoride (PMSF), 25
µg/ml aprotinin, 25 µg/ml leupeptin and 25 µg/ml pepstatin A.
Cell suspension was
sonicated and centrifuged at 500 × g for 10min at 4°C. The supernatant was centrifuged at
100,000 × g for 60 min at 4°C.
The resulting pellet was solubilized in PBS containing 1.5%
digitonin, 0.5 mM PMSF, 25 µg/ml aprotinin, 25 µg/ml leupeptin and 25 µg/ml pepstatin A as
described previously.31 The sample was centrifuged again and supernatant was used for the
soluble fraction of the membrane.
At first, solubilized membrane (1000 µg) was incubated
with normal IgG (0.5 µg) for 15 min, followed by a 30-min incubation with 20 µl of 25%
solution of protein G to eliminate the non-specifically bounded proteins (GE Healthcare UK
Ltd.).
After centrifugation, supernatant was incubated with anti-α1A or anti-α1B-AR
antibody, or with control IgGs used at the same concentration as these specific antibodies
(overnight), followed by a 60-min incubation with protein G.
7
The immunoprecipitants were
solubilized in sample preparation buffer and separated on 10 or 14% SDS-PAGE and
analyzed by Western immunoblot by probing with antibodies against α1A-AR, α1B-AR, Gαq/11,
Gαi-2, Gαi-3 and Gαo.
The specificity of the antibodies against Gα-subunits is shown in
Online Supplementary Figure VII.
Immunofluorescence microscopy
Isolated ventricular myocytesor cryosections of papillary muscles were fixed with 4%
paraformaldehyde in distilled water (Cell FixTM, BD Biosciences, San Jose, CA) at 4°C for
10 min, incubated with the primary antibodies and Wheat Germ Agglutinin-FITC
(WGA-FITC) (as a marker of sarcolemma including T-tubules) (Biomeda, Foster City, CA)
(overnight), followed by secondary antibodies for 1 hr.1 Immunostaining was visualized
with a laser scanning confocal microscope LSM510 (Carl Zeiss, Jena, Germany).
Control
experiments were performed using secondary antibodies without primary, which showed no
noticeable labeling.
Immunoelectron microscopy
Papillary muscles from rat heart were fixed in 2% paraformaldehyde in 0.1 mol/L phosphate
buffer (pH 7.4), infused with 1.84 mol/L sucrose containing 20% polyvinylpyrolidone at 4°C,
and frozen in liquid nitrogen.
immunolabeling.1
Ultrathin cryosections were cut and processed for
Samples were examined with a transmission electron microscope H-7500
(Hitachi, Tokyo, Japan) at an accelerating voltage of 100 KV.
8
Measurement of cytosolic cAMP concentration
The cAMP concentration of ventricular myocytes was measured by using the previously
described method with some modifications.32 Isolated cells were suspended in Tyrode’s
solution, followed by phenylephrine stimulation (for 2 or 15 min) or isoproterenol stimulation
(for 15 min).
The reaction was stopped by the addition of ice-cold perchloric acid and by
vortexing. Suspension was centrifuged at 12000 × g for 10 min at 4°C. The pellets were
snap-frozen by liquid nitrogen and stored at -80 °C for subsequent protein determination by
using BCA protein assay kit (Pierce Biotechnology, Inc., Rockford, IL).
The supernatants
were collected and adjusted to pH 4-5 by the addition of 20 % KHCO3. The resulting
precipitate was removed by the centrifugation (12000 × g for 10 min at 4°C).
The cAMP
content of the supernatant was determined by a competitive enzyme immunoassay (EIA) by
cAMP EIA kit (Cayman Chemical Company, Ann Arbor, MI). Each sample was assayed in
duplicate, calibrated against a standard curve and expressed in pmol cAMP/mg protein.
Statistics
All results are shown as mean ± standard deviation (SD).
Paired data were evaluated by Student’s t test.
Bars in the graphs indicate SD.
For multiple comparisons, on e-way or
one-way repeated ANOVA followed by Bonferroni post hoc test was used with the
significance level set at P<0.05.
9
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14
Online Supplementary Figures
Supplementary Figure I. Localization of α1A-AR and α1B-AR in rat papillary muscle
Immunofluorescence images of papillary muscles labeled with α1-AR subtype-specific
antibody (α1A- or α1B-AR) (red, left) and the plasma membrane marker Wheat Germ
Agglutinin-FITC (WGA) (green, middle).
The overlay images are also shown (right).
Bars =10 µm.
15
Supplementary Figure II.
Phenylephrine (Phe) showed a biphasic change in ICa in the
presence of another β-AR antagonist, propranolol. .
Effect of 10 µmol/L Phe on ICa in the absence (n=8, black circles) or in the presence of β-AR
antagonist, 1µmol/L propranolol (Pro) (n=9, red square) measured using the perforated patch
clamp.
The amplitudes of currents at each period were normalized by the current before the
application of Phe. The positive effects by Phe in the absence of Pro (the normalized
current at 15 min: 120.7±19.5%) were slightly (but not significant) larger than that in the
presence of Pro (the normalized current at 15 min: 111.4±14.3%, P=0.273). Vertical bars
indicate the SD.
16
Supplementary Figure III. α1A-AR stimulation or α1B-AR stimulation did not change
the current voltage relationships of ICa
A: Mean current-voltage relationships (n=7) of ICa before (closed circles) and 15 min after
application of 0.1 µmol/L A61603 (closed squares).
*
P<0.05,
**
Vertical bars indicate the standard error.
P<0.01 compared to the current before A61603 at each voltage.
B: Mean
current-voltage relationships (n=5) of ICa before WB4101 (open triangles), 15 min after
application of WB4101 (closed circles) and 15 min after application of 10 µmol/L
phenylephrine (Phe) in the continuous presence of WB4101 (closed squares). Vertical bars
indicate the standard error.
each voltage.
*P<0.05, **P<0.01 compared to the current before WB4101 at
#
P<0.05, # #P<0.01 compared to the current before Phe at each voltage.
17
Supplementary Figure IV. Effect of Phe on ICa in the presence of the selective α1B-AR
antagonist or α1D-AR antagonist.
A: The effect of 10 µmol/L Phe on ICa in the presence of the selective α1B-AR antagonist, 50
nmol/L L765,314 (n=4, open triangles) or in the absence of L765,314 (n=12, closed circles).
The amplitudes of the currents at each period were normalized by the current before the
application of Phe.
The negative phase of ICa caused by 10 µmol/L Phe was abolished by 50
nmol/L L765,314. *P<0.05 compared to the normalized current in the absence of L765,314
(closed circles) at each time.
Vertical bars indicate SD. B: Effect of 10 µmol/L Phe on ICa in
the presence of selective α1D-AR antagonist, 30 nmol/L BMY7378 (n=10, closed circles).
Time course of ICa in the absence of 10 µmol/L Phe is also shown (n=5, open squares).
ICa
showed a significant transient decrease after the 2-min application of Phe and a sustained
increase after the 15-min application in the presence of 30 nmol/L BMY7378.
The
amplitudes of currents at each period were normalized by the current before the application of
Phe.
*P<0.05, **P<0.01 compared to the normalized current in the absence of Phe (open
squares) at each time.
Vertical bars indicate SD.
18
Supplemantary Figure V. Effect of subtype-specific α1-AR stimulation on PKC
phosphorylation in the membrane fraction
Immunoblot analysis of phosphorylated PKC in the cardiac membrane fraction of intact
myocytes treated with 10 µmol/L Phe (Phe), 1 µM A61603 (α1A), and 10 µmol/L Phe in the
presence of 2 µM WB4101 (α1B) or 1 µmol/L phorbol 12-myristate 13-acetate (PMA) for 15
min (50 µg protein/well). A: Western blot showing the phosphorylation levels at conserved
19
hydrophobic motifs of conventional PKC (α) and novel PKCs (δ and ε).
The graphs
(bottom) show the quantification of the band intensities of phospho-PKCε, α and δ. The
band intensities after stimulations were normalized by the control (before stimulation) (n=6).
*P<0.05, compared to CTR. B: Western blot showing the phosphorylation levels at Thr505
in the activation loop of PKCδ.
The graph (bottom) shows the quantification of the band
intensities of phospho-PKCδ (Thr505).
The band intensities after stimulations were
normalized by the control (CTR) (before stimulation) (n=6).
Vertical bars indicate SD.
20
*P<0.05, compared to CTR.
Supplementary Figure VI. Pertussis toxin (PTX) treatment blocked the acetylcholine
(ACh)-induced decrease in isoproterenol (Iso)-stimulated ICa
A and B, effect of muscarinic receptor agonist, 1 µmol/L acetylcholine (ACh) on ICa in the
presence of β-AR agonist, 100 nmol/L isoproterenol (Iso) in PTX-treated cells (n=3) (B) and
in non-treated cells (n=4) (which had been kept at 37°C in the absence of PTX for 3 hrs) (A).
At first, cells were treated with 100 nmol/L Iso for 5 min and significant increase in ICa was
observed and ICa was reached another steady state.
Then, the effect of 1 µmol/L ACh was
investigated in the continuous presence of Iso in PTX-treated cells and in non-treated cells.
*P<0.05.
Vertical bars indicate SD.
21
Supplementary Figure VII. Specificity of the antibodies against Gα-subunits
The specificity of the antibodies against Gα-subunits was tested using full length recombinant
protein of antigens of rat Gαq, Gαi-2, Gαi-3 and Gαo.
recombinant Gα-subunit.
Gαi-3 and Gαo.
Gαo protein.
Each well contained 5 ng of each
There were no cross-reactivity among the antibodies against Gαq,
The antibody against Gαi-2 did detect Gαi-2, but it only weakly reacted with
IB, immunoblot.
22
Supplementary Figure VIII. Activation of phosphatases was not involve in the
mechanism for evoking the negative phase of ICa during α1-AR stimulation
The effect of 10 µmol/L Phe on ICa in the presence of the selective PKA inhibitor, 1 µmol/L
H-89 and a protein phosphatase inhibitor, 30 nmol/L calyculin A (CaA) (n=5). When cells
were pre-treated with a protein phosphatase inhibitor, calyculin A (CaA) for 15 min in the
presence of H-89, significant increase in ICa was observed (from -5.37±1.22 to -13.18±4.33
pA/pF, P<0.01) and ICa was reached another steady state (see also Ref 15).
of Phe was investigated in the continuous presence of CaA and H-89.
Then the effect
The amplitudes of the
currents at each period were normalized by the current before the application of Phe.
*P<0.05, compared to the current before stimulation (0 min).
23
Vertical bars indicate SD.