Interaction of α1-Adrenoceptor Subtypes With Different G Proteins Induces Opposite Effects on Cardiac L-type Ca2+ Channel
We examined the effect of α1-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 α1A- and α1B-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 α1A-adrenoceptor stimulation (≥0.1 μmol/L A61603) only potentiated ICa, and selective α1B-adrenoceptor stimulation (10 μmol/L phenylephrine with 2 μ mol/L WB4101) only decreased ICa. The positive effect by α1A-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 α1B-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 PKCδ and ε to the T-tubules was observed only after α1A-adrenoceptor stimulation, but not after α1B-adrenoceptor stimulation. Immunoprecipitaion analysis revealed that α1A-adrenoceptor was associated with Gq/11, but α1B-adrenoceptor interacted with one of the pertussis toxin-sensitive G proteins, Go. These findings demonstrated that the interactions of α1-adrenoceptor subtypes with different G proteins elicit the formation of separate signaling cascades, which produce the opposite effects on ICa. The coupling of α1A-adrenoceptor with Gq/11-PLC-PKC-CaMKII pathway potentiates ICa. In contrast, α1B-adrenoceptor interacts with Go, of which the βγ-complex might directly inhibit the channel activity at T-tubules.
The α1-adrenoceptor (AR) stimulation has an important role for the regulation of mammalian cardiac muscle contraction.1–4 We have previously shown that α1-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 α1-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 α1-AR stimulation, 2 important questions remain to be solved: (1) What is the molecular mechanism which simultaneously induces two opposite effects during α1-AR stimulation?; (2) What are the molecular components for evoking the negative effect on ICa by α1-AR stimulation? We postulated that these 2 opposite effects simultaneously occur via (1) different α1-AR subtypes, α1A and α1B, 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 α1-AR subtype-selective stimulation on ICa and to clarify the α1-AR subtype-specific signaling pathway for the regulation of ICa. Here, we show the direct evidence that cardiac α1-AR signaling diverges at the level of the α1-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, α1B-AR interacted with Go, of which the βγ-complex could directly inhibit ICa. These results represent the whole picture of intracellular mechanism for the unique regulation of VLCC by cardiac α1-AR signaling and also provide the significant insight into the regulation of cardiac excitation-contraction coupling by α1-AR subtype-specific 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; 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.
Detection and Cellular Localization of α1-AR Subtypes in Cardiomyocytes
The protein expression of α1-AR subtypes in isolated adult rat ventricular myocytes was confirmed by Western immunoblot with the commercially available antibodies against α1A-, α1B-, and α1D-AR (Figure 1A). In the membrane proteins from cardiomyocytes and urinary bladder, single bands were detected with the expected molecular size for glycosylated α1A-AR (68 kDa)8 using specific antibody against human α1A-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 α1B-AR showed a major band with the expected molecular size for glycosylated α1B-AR (≈80 kDa)9 in rat cardiomyocytes, liver, and brain (Figure 1B, middle). The α1D-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 α1D-AR (Figure 1A, right). These results show that α1A- and α1B-AR (but not α1D-AR) were detectable at the protein level in our preparation of cardiomyocytes. Thus, we focused on the role of these 2 subtypes of α1-AR (α1A and α1B) in native cardiomyocytes in the following experiments.
We determined the cellular localization of α1A-AR and α1B-AR in cardiac cells using an immunofluorescence microscope (Figure 1B). In ventricular myocytes, α1A-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, α1B-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 α1A-AR and α1B-AR as observed in the isolated cells (supplemental Figure I).
To confirm the detailed subcellular localization of α1A-AR and α1B-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 α1A-AR and α1B-AR. These results suggest that 2 α1-AR subtypes (α1A and α1B) 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.
Next we observed the effect of selective α1A-AR stimulation on ICa by using the selective α1A-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 α1-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 α1A-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 α1-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 α1A-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 α1A-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 α1B-AR stimulation on ICa by the application of a nonsubtype selective α1-AR agonist (Phe) with a selective α1A-AR antagonist (WB4101), because no selective α1B-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, α1B-AR stimulation produced only a negative effect without potentiation of ICa, which was opposite to the effect of α1A-AR stimulation.
To further confirm the negative effect by α1B-AR stimulation on ICa, we also investigated the effect of 10 μmol/L Phe in the presence of the selective α1B-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 α1A-AR Stimulation, but Not After α1B-AR Stimulation
We previously showed that the positive effect of α1-AR stimulation on ICa is dependent on PKC activity.5 Therefore, we examined the involvement of PKC in the signaling pathway after α1A- or α1B-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 α1-AR-subtype stimulation (Figure 3A to 3F). In our preparation, one of the Ca2+-dependent PKC isoforms, PKCα was not significantly translocated by nonsubtype selective α1-AR stimulation or by subtype-selective α1-AR stimulations, although endogenous PKC activator, phorbol 12-myristate 13-acetate (PMA) did translocate PKCα from cytosol to membrane and filament fractions (Figure 3A and 3D). On the contrary, significant translocation of the Ca2+-independent PKCs (δ and ε) from cytosol to membrane fraction was found 15 minutes after α1A-AR stimulation (Figure 3B, 3C, 3E, and 3F). However, no remarkable translocation of PKC after α1B-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 PKCε phosphorylation at the hydrophobic motif and PKCδ phosphorylation at the activation loop significantly increased after α1A-AR stimulation or PMA treatment in the membrane fraction. However, the amount of phosphorylated PKCα in the membrane fraction did not increase after α1A- or α1B-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 α1-AR-subtype stimulation by using an immunofluorescence microscope (Figure 4A to 4C). Significant translocation of PKCα was not observed after the treatment with Phe as shown in Western immunoblot (Figure 4A). Most of PKCδ 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). PKCε was diffusely distributed in the cytosol before stimulation (Figure 4C). After Phe treatment, PKCε 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 α1A-AR stimulation. However, there was no obvious involvement of PKC in the α1B-AR signaling pathway.
Negative Change in ICa During α1-AR Stimulation Was Mediated via the Pertussis Toxin–Sensitive G Protein Pathway
We showed that the positive effect on ICa caused by α1A-AR stimulation was dependent on PKC, but the negative effect of α1B-AR stimulation was independent of PKC activation (Figures 2 and 3⇑). Several reports demonstrated that α1-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 α1B-AR functionally couples with other G proteins, and we examined the involvement of PTX-sensitive G protein in the regulation of ICa by α1-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 α1-AR subtype-selective stimulation on ICa in PTX-treated cells. We confirmed that the negative effect by α1B-AR stimulation was blocked by PTX (Figure 5C), but the magnitude of the positive effect by α1A-AR stimulation did not alter after PTX treatment (Figure 5B). These results indicate that the negative phase of ICa during α1B-AR stimulation (Figure 2A) was produced through PTX-sensitive G protein (Gi/o) pathways.
In adult rat cardiomyocytes at least 3 subtypes of PTX-sensitive Gα (Gαi-2, Gαi-3, Gαo) are expressed at the mRNA level and are detectable at the protein level.13 Therefore, the possibility that α1-AR subtypes directly couple with these PTX-sensitive G proteins was examined by coimmunoprecipitation of these Gα-subunits with anti–α1A- or anti–α1B-AR antibody. The immunoprecipitants were analyzed by Western immunoblot probing with the antibodies against Gα-subunits, of which specificities were checked by using the recombinant Gα-subunits (see supplemental Figure VII). The α1A-AR antibody coimmunoprecipitated Gαq/11, whereas the α1B-AR antibody coimmunoprecipitated Gαo (Figure 5D). Moreover, immunofluorescence images with the specific antibodies against α1B-AR and Gαo showed that Gαo was colocalized with α1B-AR at T-tubules (Figure 5E). Thus, these results indicate that the α1A-AR couples with Gq/11-protein in a classical coupling mode, which activates the PLC-diacylglycerol-PKC pathway and that α1B-AR is linked to Go at the T-tubules and evokes the negative phase in ICa.
Negative Effect of α1-AR Stimulation on ICa Is Mediated Through βγ-Complex of G Protein
Our biochemical and electrophysiological results indicated that α1B-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 α1B-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 α1-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).
Several reports stated that the βγ-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 α1-AR agonist (10 μmol/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 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 (≈2 minutes) induced by α1-AR stimulation was not attributable to the reduction of basal phosphorylation level of VLCC, but was possibly produced by the direct interaction of βγ-complex of Go with VLCC.
In this study, we elucidated the differences between cardiac α1A- and α1B-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 α1A- and α1B-AR were functionally expressed at T-tubules where VLCC is concentrated,10 but α1D-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 α1A- or α1B-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 α1B-AR signaling pathways couple with different G proteins, Gq/11 and Go, respectively and produce different functional outcomes; α1A-AR stimulation activates Gq/11-PLC-diacylglycerol-PKC-CaMKII pathway and increases ICa. On the contrary, α1B-AR interacts with Go and inhibits the VLCC activity.
Alpha1A-AR-Gq-PKC Signaling Pathway Induces Potentiation of ICa
In this study, we showed that α1A-AR was expressed at T-tubules and also demonstrated that α1A-AR stimulation did affect the VLCC activity which was confirmed by using α1A-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 α1-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 (PKCα) and 2 Ca2+-independent PKCs (novel PKCs; PKCδ and PKCε) are at least detectable at the protein level.20 In our preparations, we demonstrated that only α1A-AR stimulation induces the activation of novel PKCs and translocates them to the cell membrane structure called T-tubules, and that α1B-AR stimulation did not show any activation of PKC. This result is consistent with the previous report that α1B- or α1D-AR signaling pathway does not have any influences on PKC activity.19 We did not detect any significant activation of PKCα after α1-AR stimulation, indicating that PKCα was not involved in α1-AR signaling19 in our preparations. Moreover, we directly showed the interaction of α1A-AR and Gαq/11 which activates PLC.1,6 The α1A-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 α1c and /or β subunits of the channel.21,22 Thus, α1A-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 α1B-AR was expressed at T-tubules and also demonstrated that α1B-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 α1B-AR stimulation shows less influence on the PKC activity than α1A-AR stimulation. This result is compatible with the previous reports that α1B-AR subtype has less potency for the stimulation of phosphoinositide hydrolysis than α1A-AR.24,25 We found that α1B-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 α1B-AR or the technique of the overexpression of wild-type α1B-AR, which shows the possibility that only α1B-AR (but not α1A-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 Gβγ 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 α1B-AR and Go colocalize with VLCC at T-tubules, and we propose the working model that βγ-complex of Go protein could directly inhibit VLCC.
In conclusion, our results represent the evidence that the unique combinations of α1-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 α1-AR subtypes with PTX-sensitive G protein could exhibit the negative feedback response to α1-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 α1-AR subtype under physiological and pathophysiological condition.
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.).
Original received November 13, 2007; revision received April 16, 2008; accepted April 30, 2008.
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