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Circulation Research. 2008;102:1378-1388
Published online before print May 8, 2008, doi: 10.1161/CIRCRESAHA.107.167734
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(Circulation Research. 2008;102:1378.)
© 2008 American Heart Association, Inc.

Cellular Biology

Interaction of {alpha}1-Adrenoceptor Subtypes With Different G Proteins Induces Opposite Effects on Cardiac L-type Ca2+ Channel

Jin O-Uchi, Hiroyuki Sasaki, Satoshi Morimoto, Yoichiro Kusakari, Hitomi Shinji, Toru Obata, Kenichi Hongo, Kimiaki Komukai, Satoshi Kurihara

From the Department of Cell Physiology (J.O.-U., S.M., Y.K., S.K.), the Division of Molecular Cell Biology (H.Sasaki, T.O.), the Division of Cardiology (S.M., K.H., K.K.), and the Department of Bacteriology (H.Shinji), 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 o-uchi{at}jikei.ac.jp


*    Abstract
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 *Abstract
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We examined the effect of {alpha}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 {alpha}1A- and {alpha}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 {alpha}1A-adrenoceptor stimulation (≥0.1 µmol/L A61603) only potentiated ICa, and selective {alpha}1B-adrenoceptor stimulation (10 µmol/L phenylephrine with 2 µ mol/L WB4101) only decreased ICa. The positive effect by {alpha}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 {alpha}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{delta} and {epsilon} to the T-tubules was observed only after {alpha}1A-adrenoceptor stimulation, but not after {alpha}1B-adrenoceptor stimulation. Immunoprecipitaion analysis revealed that {alpha}1A-adrenoceptor was associated with Gq/11, but {alpha}1B-adrenoceptor interacted with one of the pertussis toxin-sensitive G proteins, Go. These findings demonstrated that the interactions of {alpha}1-adrenoceptor subtypes with different G proteins elicit the formation of separate signaling cascades, which produce the opposite effects on ICa. The coupling of {alpha}1A-adrenoceptor with Gq/11-PLC-PKC-CaMKII pathway potentiates ICa. In contrast, {alpha}1B-adrenoceptor interacts with Go, of which the β{gamma}-complex might directly inhibit the channel activity at T-tubules.


Key Words: {alpha}1-adrenoceptor • L-type Ca2+ channel • G protein • PKC


*    Introduction
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 *Introduction
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The {alpha}1-adrenoceptor (AR) stimulation has an important role for the regulation of mammalian cardiac muscle contraction.1–4 We have previously shown that {alpha}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 {alpha}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 {alpha}1-AR stimulation, 2 important questions remain to be solved: (1) What is the molecular mechanism which simultaneously induces two opposite effects during {alpha}1-AR stimulation?; (2) What are the molecular components for evoking the negative effect on ICa by {alpha}1-AR stimulation? We postulated that these 2 opposite effects simultaneously occur via (1) different {alpha}1-AR subtypes, {alpha}1A and {alpha}1B, 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 {alpha}1-AR subtype-selective stimulation on ICa and to clarify the {alpha}1-AR subtype-specific signaling pathway for the regulation of ICa. Here, we show the direct evidence that cardiac {alpha}1-AR signaling diverges at the level of the {alpha}1-AR subtype and G protein, which produce the opposite effects on ICa in rat ventricular myocyes. Alpha1A-AR coupled with Gq/11 and activated phospholipase C (PLC)-PKC-CaMKII pathway, which evoked the potentiation of ICa. In contrast, {alpha}1B-AR interacted with Go, of which the β{gamma}-complex could directly inhibit ICa. These results represent the whole picture of intracellular mechanism for the unique regulation of VLCC by cardiac {alpha}1-AR signaling and also provide the significant insight into the regulation of cardiac excitation-contraction coupling by {alpha}1-AR subtype-specific signaling.


*    Materials and Methods
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 *Materials and Methods
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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; Sankyo Labo Service, Tokyo, Japan).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
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 *Results
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Detection and Cellular Localization of {alpha}1-AR Subtypes in Cardiomyocytes
The protein expression of {alpha}1-AR subtypes in isolated adult rat ventricular myocytes was confirmed by Western immunoblot with the commercially available antibodies against {alpha}1A-, {alpha}1B-, and {alpha}1D-AR (Figure 1A). In the membrane proteins from cardiomyocytes and urinary bladder, single bands were detected with the expected molecular size for glycosylated {alpha}1A-AR (68 kDa)8 using specific antibody against human {alpha}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 {alpha}1B-AR showed a major band with the expected molecular size for glycosylated {alpha}1B-AR ({approx}80 kDa)9 in rat cardiomyocytes, liver, and brain (Figure 1B, middle). The {alpha}1D-AR (60 kDa) was only found in rat brain; no significant bands were observed in cardiomyocytes and liver cells using the specific antibody against rat {alpha}1D-AR (Figure 1A, right). These results show that {alpha}1A- and {alpha}1B-AR (but not {alpha}1D-AR) were detectable at the protein level in our preparation of cardiomyocytes. Thus, we focused on the role of these 2 subtypes of {alpha}1-AR ({alpha}1A and {alpha}1B) in native cardiomyocytes in the following experiments.


Figure 1
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  		Figure 1. Detection and cellular localization of {alpha}1-AR subtypes in rat ventricle. A, Detection of {alpha}1-AR subtypes in rat ventricle by Western immunoblot (IB) using specific antibodies against {alpha}1A- (left), {alpha}1B- (middle) or {alpha}1D-AR (right). Each well contained 50-µg membrane protein from rat ventricular myocytes (heart), urinary bladder (bladder), brain, or liver. B, Confocal images of isolated ventricular myocytes labeled with {alpha}1-AR subtype-specific antibody ({alpha}1A- or {alpha}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 µm. C, Immunoelectron microscopic images of ventricular tissue labeled with 15-nm gold-{alpha}1A-AR (left) or {alpha}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.

We determined the cellular localization of {alpha}1A-AR and {alpha}1B-AR in cardiac cells using an immunofluorescence microscope (Figure 1B). In ventricular myocytes, {alpha}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, {alpha}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 also showed a similar tendency of the localization of {alpha}1A-AR and {alpha}1B-AR as observed in the isolated cells (supplemental Figure I).

To confirm the detailed subcellular localization of {alpha}1A-AR and {alpha}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 {alpha}1A-AR and {alpha}1B-AR. These results suggest that 2 {alpha}1-AR subtypes ({alpha}1A and {alpha}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 µmol/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 β-AR antagonist, 1 µmol/L bupranolol, which we used previously5 (Figure 2A). Similar results were obtained when we used another β-AR antagonist, 1 µmol/L propranolol, as shown in supplemental Figure II. Following experiments were all performed in the presence of 1 µmol/L bupranolol.


Figure 2
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  		Figure 2. Alpha1A-AR stimulation shows only positive effect in ICa, but {alpha}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 {alpha}1-AR agonist, 10 µmol/L Phe. B, A representative record of time-dependent change in ICa during application of the selective {alpha}1A-AR agonist, 0.1 µmol/L A61603. C, Concentration-dependent effect of A61603 on ICa. Time course of ICa in the absence of A61603 is also shown (red diamonds). 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 diamonds) at each time. D, Effect of 0.1 µmol/L A61603 on ICa in the presence of PLC inhibitor (1 µmol/L U73122), PKC inhibitor (10 µmol/L chelerythrine), or CaMKII inhibitors (0.5 µmol/L KN-93 or 10 µmol/L AIP). One µmol/L U73343 and 0.5 µmol/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 µmol/L A61603). {dagger}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 µmol/L Phe in the presence of selective {alpha}1A-AR antagonist, 2 µmol/L WB4101. F, Concentration-dependent effect of Phe in the presence of 2 µmol/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 diamonds). The number of the cells tested is shown in parentheses. *P<0.05 compared to the normalized current in the absence of Phe (red diamonds) at each time. G, Concentration-dependent effect of the selective {alpha}1B-AR antagonist, L765,314 on a transient decrease in ICa 2 minutes after application of 10 µmol/L Phe. L765,314 was applied 10 minutes before agonist application. Graphs show the percent inhibition of ICa 2 minutes after application of 10 µmol/L Phe in the presence (10 to 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).

Next we observed the effect of selective {alpha}1A-AR stimulation on ICa by using the selective {alpha}1A-AR agonist A61603. Fifteen-minute treatment with A61603 (0.1 µmol/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 {alpha}1-AR stimulation by Phe (see Figure 2A). This positive effect after 15-minute treatment with A61603 was saturated at 1 µmol/L A61603 (0.1 µmol/L, 35.36±14.37%, n=6; 1 µmol/L, 42.64±27.49%, n=8; P=1.00) and was blocked by the selective {alpha}1A-AR antagonist, 2 µmol/L WB4101 (n=5, data not shown). All concentrations of A61603 (0.1 to 1 µmol/L) used showed only a positive without a negative effect (Figure 2C).

As we previously reported that the positive effect of {alpha}1-AR stimulation on ICa is evoked through a PKC- and CaMKII-dependent mechanism,5 next we investigated the involvement of PKC and CaMKII in the signaling pathways which evoke the potentiation of ICa during {alpha}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 membrane-permeable 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 {alpha}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 {alpha}1B-AR stimulation on ICa by the application of a nonsubtype selective {alpha}1-AR agonist (Phe) with a selective {alpha}1A-AR antagonist (WB4101), because no selective {alpha}1B-AR agonist is available at present.4 Ten-minute exposure to 2 µmol/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 µmol/L Phe only decreased ICa (Figure 2E and 2F) without changing the shape of the current-voltage relationship (supplemental Figure III). In contrast, 1 µmol/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, {alpha}1B-AR stimulation produced only a negative effect without potentiation of ICa, which was opposite to the effect of {alpha}1A-AR stimulation.

To further confirm the negative effect by {alpha}1B-AR stimulation on ICa, we also investigated the effect of 10 µmol/L Phe in the presence of the selective {alpha}1B-AR antagonist, L-765,314. The negative effect of 10 µmol/L Phe on ICa was significantly inhibited by the treatment of L-765,314 in a concentration-dependent manner (Figure 2G and supplemental Figure IV).

PKC Was Activated After {alpha}1A-AR Stimulation, but Not After {alpha}1B-AR Stimulation
We previously showed that the positive effect of {alpha}1-AR stimulation on ICa is dependent on PKC activity.5 Therefore, we examined the involvement of PKC in the signaling pathway after {alpha}1A- or {alpha}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 {alpha}1-AR-subtype stimulation (Figure 3A to 3F). In our preparation, one of the Ca2+-dependent PKC isoforms, PKC{alpha} was not significantly translocated by nonsubtype selective {alpha}1-AR stimulation or by subtype-selective {alpha}1-AR stimulations, although endogenous PKC activator, phorbol 12-myristate 13-acetate (PMA) did translocate PKC{alpha} from cytosol to membrane and filament fractions (Figure 3A and 3D). On the contrary, significant translocation of the Ca2+-independent PKCs ({delta} and {epsilon}) from cytosol to membrane fraction was found 15 minutes after {alpha}1A-AR stimulation (Figure 3B, 3C, 3E, and 3F). However, no remarkable translocation of PKC after {alpha}1B-AR stimulation was observed (Figure 3E and 3F).


Figure 3
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  		Figure 3. Effect of subtype-specific {alpha}1-AR stimulation on PKC translocation. A to C, Immunoblot analysis of PKC{alpha} (A), PKC{delta} (B), and PKC{epsilon} 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 µmol/L phenylephrine (Phe), 1 µmol/L A61603 ({alpha}1A), 10 µmol/L Phe in the presence of 2 µmol/L WB4101 ({alpha}1B) or 1 µmol/L PMA (PMA) for 15 minutes (25 µg protein/well). Control (CTR) represents untreated cells. Total PKC{alpha}, {delta}, and {epsilon} in whole cell lysates (W) are also shown (50 µg 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, {alpha}1A, {alpha}1B, or PMA) was normalized by the M/C ratio before stimulation (CTR). *P<0.05, compared to the control (CTR).

Phosphorylation of PKC itself was also measured because it is another hallmark of PKC activation.11 Immunoreactivity of PKC{epsilon} phosphorylation at the hydrophobic motif and PKC{delta} phosphorylation at the activation loop significantly increased after {alpha}1A-AR stimulation or PMA treatment in the membrane fraction. However, the amount of phosphorylated PKC{alpha} in the membrane fraction did not increase after {alpha}1A- or {alpha}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 {alpha}1-AR-subtype stimulation by using an immunofluorescence microscope (Figure 4A to 4C). Significant translocation of PKC{alpha} was not observed after the treatment with Phe as shown in Western immunoblot (Figure 4A). Most of PKC{delta} 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{epsilon} was diffusely distributed in the cytosol before stimulation (Figure 4C). After Phe treatment, PKC{epsilon} was accumulated at the T-tubules and intercalated disks (Figure 4C).


Figure 4
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  		Figure 4. PKC{delta} and {epsilon} (but not {alpha}) were translocated to the T-tubules by {alpha}1-AR stimulation. A to C, Immunofluorescence localization of PKC in adult rat ventricular myocytes before and after {alpha}1-AR stimulation. The immunofluorescence images of ventricular myocytes before stimulation (CTR), 15 minutes after application of 100 µmol/L phenylephrine (Phe), or 15 minutes after application of 100 µmol/L PMA (PMA) are shown by using specific antibodies against PKC {alpha}, {delta}, and {epsilon} which were used in Western immunoblot.

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 {alpha}1A-AR stimulation. However, there was no obvious involvement of PKC in the {alpha}1B-AR signaling pathway.

Negative Change in ICa During {alpha}1-AR Stimulation Was Mediated via the Pertussis Toxin–Sensitive G Protein Pathway
We showed that the positive effect on ICa caused by {alpha}1A-AR stimulation was dependent on PKC, but the negative effect of {alpha}1B-AR stimulation was independent of PKC activation (Figures 2 and 3Up). Several reports demonstrated that {alpha}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 {alpha}1B-AR functionally couples with other G proteins, and we examined the involvement of PTX-sensitive G protein in the regulation of ICa by {alpha}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 β-AR stimulant (supplemental Figure VI). Treatment of PTX significantly inhibited the negative effect by 10 µmol/L Phe at 2 minutes and then enhanced the positive effect at 15 minutes (Figure 5A). Moreover, we separately investigated the effects of {alpha}1-AR subtype-selective stimulation on ICa in PTX-treated cells. We confirmed that the negative effect by {alpha}1B-AR stimulation was blocked by PTX (Figure 5C), but the magnitude of the positive effect by {alpha}1A-AR stimulation did not alter after PTX treatment (Figure 5B). These results indicate that the negative phase of ICa during {alpha}1B-AR stimulation (Figure 2A) was produced through PTX-sensitive G protein (Gi/o) pathways.


Figure 5
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  		Figure 5. Negative effect on ICa during {alpha}1-AR stimulation is mediated through {alpha}1B-AR and PTX-sensitive G protein pathway. A to C, Effect of 10 µmol/L Phe (A), 1 µmol/L A61603 (B), or 10 µmol/L Phe with 2 µmol/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 circles) at each time. The number of the cells tested is shown in parentheses. D, Coimmunoprecipitation of {alpha}1-ARs with G{alpha}-subunits. Membrane proteins were immunoprecipitated (IP) with specific {alpha}1-AR antibodies or control IgG (IgG). The immunoprecipitates were analyzed by Western immunoblot by probing with the antibodies against {alpha}1-ARs and G{alpha}-subunits. Membrane lysates (ML; 12.5 µg/lane) are also shown as the positive control. E, Immunofluorescence images of ventricular myocytes costained with antibodies against anti-G{alpha}o (red, upper panel) and {alpha}1B-AR (green, middle panel). The overlay image is also shown (lower panel). Bars, 10 µm.

In adult rat cardiomyocytes at least 3 subtypes of PTX-sensitive G{alpha} (G{alpha}i-2, G{alpha}i-3, G{alpha}o) are expressed at the mRNA level and are detectable at the protein level.13 Therefore, the possibility that {alpha}1-AR subtypes directly couple with these PTX-sensitive G proteins was examined by coimmunoprecipitation of these G{alpha}-subunits with anti–{alpha}1A- or anti–{alpha}1B-AR antibody. The immunoprecipitants were analyzed by Western immunoblot probing with the antibodies against G{alpha}-subunits, of which specificities were checked by using the recombinant G{alpha}-subunits (see supplemental Figure VII). The {alpha}1A-AR antibody coimmunoprecipitated G{alpha}q/11, whereas the {alpha}1B-AR antibody coimmunoprecipitated G{alpha}o (Figure 5D). Moreover, immunofluorescence images with the specific antibodies against {alpha}1B-AR and G{alpha}o showed that G{alpha}o was colocalized with {alpha}1B-AR at T-tubules (Figure 5E). Thus, these results indicate that the {alpha}1A-AR couples with Gq/11-protein in a classical coupling mode, which activates the PLC-diacylglycerol-PKC pathway and that {alpha}1B-AR is linked to Go at the T-tubules and evokes the negative phase in ICa.

Negative Effect of {alpha}1-AR Stimulation on ICa Is Mediated Through β{gamma}-Complex of G Protein
Our biochemical and electrophysiological results indicated that {alpha}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 {alpha}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 {alpha}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 µmol/L H-89; Figure 6B). Thus, the inhibition of cAMP-PKA 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).


Figure 6
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  		Figure 6. Inhibition of cAMP-PKA signaling was not involved in the mechanism for evoking negative phase of ICa during {alpha}1-AR stimulation. A, cAMP concentration in isolated rat ventricular myocytes treated with 10 µmol/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 β-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 µmol/L Phe on ICa in the presence of the selective PKA inhibitor, 1 µmol/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).

Several reports stated that the β{gamma}-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 {alpha}1-AR agonist (10 µmol/L Phe) on ICa using this prepulse depolarization protocol (Figure 7A).


Figure 7
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  		Figure 7. Negative effect on ICa during {alpha}1-AR stimulation is inhibited by prepulse depolarization protocol. A, Effect of 10 µmol/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 µmol/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.

Recording with this prepulse depolarization, there was no significant transient decrease of ICa for up to 2 minutes after the application of 10 µmol/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 ({approx}2 minutes) induced by {alpha}1-AR stimulation was not attributable to the reduction of basal phosphorylation level of VLCC, but was possibly produced by the direct interaction of β{gamma}-complex of Go with VLCC.


*    Discussion
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 up arrowIntroduction
 up arrowMaterials and Methods
 up arrowResults
 *Discussion
down arrowReferences
 
In this study, we elucidated the differences between cardiac {alpha}1A- and {alpha}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 {alpha}1A- and {alpha}1B-AR were functionally expressed at T-tubules where VLCC is concentrated,10 but {alpha}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 {alpha}1A- or {alpha}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 {alpha}1B-AR signaling pathways couple with different G proteins, Gq/11 and Go, respectively and produce different functional outcomes; {alpha}1A-AR stimulation activates Gq/11-PLC-diacylglycerol-PKC-CaMKII pathway and increases ICa. On the contrary, {alpha}1B-AR interacts with Go and inhibits the VLCC activity.


Figure 8
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  		Figure 8. Possible mechanism underlying the opposing modulation of L-type Ca2+ channels induced by {alpha}1-AR subtype-specific signaling. A, Model of {alpha}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 {alpha}1-AR subtype-specific signaling. Alpha1A-AR-Gq/11 pathway potentiates ICa (showing as the red line) and {alpha}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 nonsubtype selective {alpha}1-AR stimulation by Phe (shown as the black line).

Alpha1A-AR-Gq-PKC Signaling Pathway Induces Potentiation of ICa
In this study, we showed that {alpha}1A-AR was expressed at T-tubules and also demonstrated that {alpha}1A-AR stimulation did affect the VLCC activity which was confirmed by using {alpha}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 {alpha}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{alpha}) and 2 Ca2+-independent PKCs (novel PKCs; PKC{delta} and PKC{epsilon}) are at least detectable at the protein level.20 In our preparations, we demonstrated that only {alpha}1A-AR stimulation induces the activation of novel PKCs and translocates them to the cell membrane structure called T-tubules, and that {alpha}1B-AR stimulation did not show any activation of PKC. This result is consistent with the previous report that {alpha}1B- or {alpha}1D-AR signaling pathway does not have any influences on PKC activity.19 We did not detect any significant activation of PKC{alpha} after {alpha}1-AR stimulation, indicating that PKC{alpha} was not involved in {alpha}1-AR signaling19 in our preparations. Moreover, we directly showed the interaction of {alpha}1A-AR and G{alpha}q/11 which activates PLC.1,6 The {alpha}1A-AR-Gq/11-PLC-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 {alpha}1c and /or β subunits of the channel.21,22 Thus, {alpha}1A-AR signaling components preferentially localized at the T-tubules and efficiently regulated cardiac VLCC, as in the case of cardiac endothelin-receptor signaling.23

Alpha1B-AR-Go Interaction Induces the Inhibition of ICa
We showed that {alpha}1B-AR was expressed at T-tubules and also demonstrated that {alpha}1B-AR stimulation did affect the VLCC activity confirmed by pharmacological procedures. Alpha1B- AR stimulation inhibited ICa, which is mediated through PKC-independent mechanisms (Figure 8). Our biochemical studies indicated that {alpha}1B-AR stimulation shows less influence on the PKC activity than {alpha}1A-AR stimulation. This result is compatible with the previous reports that {alpha}1B-AR subtype has less potency for the stimulation of phosphoinositide hydrolysis than {alpha}1A-AR.24,25 We found that {alpha}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 {alpha}1B-AR or the technique of the overexpression of wild-type {alpha}1B-AR, which shows the possibility that only {alpha}1B-AR (but not {alpha}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 Gβ{gamma} 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 {alpha}1B-AR and Go colocalize with VLCC at T-tubules, and we propose the working model that β{gamma}-complex of Go protein could directly inhibit VLCC.

Conclusion
In conclusion, our results represent the evidence that the unique combinations of {alpha}1-AR subtypes and specific G proteins form subtype-specific signal transduction pathways, which induce the opposite effects on VLCC in native cardiac cells. The coupling of specific {alpha}1-AR subtypes with PTX-sensitive G protein could exhibit the negative feedback response to {alpha}1-AR stimulation, and this mechanism would contribute to the protection of the heart from Ca2+ overload as in the relation between β1- and β2-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 {alpha}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 for Scientific Research (C) from Japan Society for the Promotion of Science (to K.H., K.K., and S.K.), Uehara Memorial 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.


*    Footnotes
 
Original received November 13, 2007; revision received April 16, 2008; accepted April 30, 2008.


*    References
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