Published online before print May 8, 2008, doi: 10.1161/CIRCRESAHA.107.167734
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
© 2008 American Heart Association, Inc.
Cellular Biology |
Interaction of 
1-Adrenoceptor Subtypes With Different G Proteins Induces Opposite Effects on Cardiac L-type Ca2+ Channel
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 |
|---|
 Top Abstract IntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 (
0.1 µmol/L A61603) only potentiated ICa, and selective 1B-adrenoceptor stimulation (10 µmol/L phenylephrine with 2 µ mol/L WB4101) only decreased 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 1A-adrenoceptor 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 β
-complex might directly inhibit the channel activity at T-tubules.
Key Words: 1-adrenoceptor • L-type Ca2+ channel • G protein • PKC
|
Introduction |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
The
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 stimulation? We postulated that these 2 opposite effects simultaneously occur via (1) different 1-AR subtypes, 1A and 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 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 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, 1B-AR interacted with Go, of which the β-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 of cardiac excitation-contraction coupling by 1-AR subtype-specific signaling. |
Materials and Methods |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
Detection and Cellular Localization of
1-AR Subtypes in CardiomyocytesThe 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 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 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 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.
|
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
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 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 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 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 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 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 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 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, 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 µmol/L Phe in the presence of the selective 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 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 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 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 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 PTX-sensitive G (Gi-2, Gi-3, Go) 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 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 Gq/11, whereas the 1B-AR antibody coimmunoprecipitated Go (Figure 5D). Moreover, immunofluorescence images with the specific antibodies against 1B-AR and Go showed that Go 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 β-Complex of G Protein
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 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 β-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 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 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-complex of Go with VLCC.
|
Discussion |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
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 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.
|
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 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 Gq/11 which activates PLC.1,6 The 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 1c and /or β 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 endothelin-receptor signaling.23
Alpha1B-AR-Go Interaction Induces the Inhibition of ICa
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. Alpha1B- AR 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 Gβ 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 β-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, which induce the opposite effects on VLCC in native cardiac cells. The coupling of specific 1-AR subtypes with PTX-sensitive 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 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 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 |
|---|
|
TopAbstractIntroductionMaterials and MethodsResultsDiscussionReferences |
|---|
1. Varma DR, Deng XF. Cardiovascular
1-adrenoceptor subtypes: functions and signaling. Can J Physiol Pharmacol. 2000; 78: 267–292.[CrossRef][Medline]
[Order article via Infotrieve]2. Piascik MT, Perez DM. 1-adrenergic receptors: new insights and directions. J Pharmacol Exp Ther. 2001; 298: 403–410.
3. Endoh M, Blinks JR. Actions of sympathomimetic amines on the Ca2+ transients and contractions of rabbit myocardium: reciprocal changes in myofibrillar responsiveness to Ca2+ mediated through - and β-adrenoceptors. Circ Res. 1988; 62: 247–265.
4. Brodde OE, Michel MC. Adrenergic and muscarinic receptors in the human heart. Pharmacol Rev. 1999; 51: 651–690.
5. O-Uchi J, Komukai K, Kusakari Y, Obata T, Hongo K, Sasaki H, Kurihara S. Alpha1-adrenoceptor stimulation potentiates L-type Ca2+ current through Ca2+/calmodulin-dependent PK II (CaMKII) activation in rat ventricular myocytes. Proc Natl Acad Sci U S A. 2005; 102: 9400–9405.
6. Gurdal H, Seasholtz TM, Wang HY, Brown RD, Johnson MD, Friedman E. Role of Gq or Go proteins in 1-adrenoceptor subtype-mediated responses in Fischer 344 rat aorta. Mol Pharmacol. 1997; 52: 1064–1070.
7. Kusakari Y, Hongo K, Kawai M, Konishi M, Kurihara S. Use of Ca-shorting curve to estimate the myofilament responsiveness to Ca2+ in tetanized rat ventricular myocytes. J Physiol Sci. 2006; 56: 219–226.[CrossRef][Medline] [Order article via Infotrieve]
8. Lin F, Owens WA, Chen S, Stevens ME, Kesteven S, Arthur JF, Woodcock EA, Feneley MP, Graham RM. Targeted 1A-adrenergic receptor overexpression induces enhanced cardiac contractility but not hypertrophy. Circ Res. 2001; 89: 343–350.
9. Luther HP, Podlowski S, Schulze W, Morwinski R, Buchwalow I, Baumann G, Wallukat G. Expression of 1-adrenergic receptor subtypes in heart cell culture. Mol Cell Biochem. 2001; 224: 69–79.[CrossRef][Medline]
[Order article via Infotrieve]
10. Brette F, Orchard CH. T-tubule function in mammalian cardiac myocytes. Circ Res. 2003; 92: 1182–1192.
11. Sabri A, Wilson BA, Steinberg SF. Dual actions of the Gq agonist Pasteurella multocida toxin to promote cardiomyocyte hypertrophy and enhance apoptosis susceptibility. Circ Res. 2002; 90: 850–857.
12. Steinberg SF, Drugge ED, Bilezikian JP, Robinson RB. Acquisition by innervated cardiac myocytes of a pertussis toxin-specific regulatory protein linked to the 1-receptor. Science. 1985; 230: 186–188.
13. Kilts JD, Akazawa T, Richardson MD, Kwatra MM. Age increases cardiac Gi2 expression, resulting in enhanced coupling to G protein-coupled receptors. J Biol Chem. 2002; 277: 31257–31262.
14. Schümann HJ, Endoh M, Brodde OE. The time course of the effects of β- and -adrenocepror stimulation by isoprenaline and methoxamine on the contractile force and cAMP level of the isorated rabbit papillary muscle. Naunyn Schmiedebergs Arch Pharmacol. 1975; 289: 291–302.[CrossRef][Medline]
[Order article via Infotrieve]
15. duBell WH, Rogers TB. Protein phosphatase 1 and an opposing protein kinase regulate steady-state L-type Ca2+ current in mouse cardiac myocytes. J Physiol. 2004; 556: 79–93.
16. Mirshahi T, Mittal V, Zhang H, Linder ME, Logothetis DE. Distinct sites on G protein beta gamma subunits regulate different effector functions. J Biol Chem. 2002; 277: 36345–36350.
17. Kleuss C, Hescheler J, Ewel C, Rosenthal W, Schultz G, Wittig B. Assignment of G-protein subtypes to specific receptors inducing inhibition of calcium currents. Nature. 1991; 353: 43–48.[CrossRef][Medline] [Order article via Infotrieve]
18. Taguchi K, Yang M, Goepel M, Michel MC. Comparison of human alpha1-adrenoceptor subtype coupling to protein kinase C activation and related signalling pathways. Naunyn Schmiedebergs Arch Pharmacol. 1998; 357: 100–110.[CrossRef][Medline] [Order article via Infotrieve]
19. Rohde S, Sabri A, Kamasamudran R, Steinberg SF. The 1-adrenoceptor subtype- and protein kinase C isoform-dependence of norepinephrine’s actions in cardiomyocytes. J Mol Cell Cardiol. 2000; 32: 1193–1209.[CrossRef][Medline]
[Order article via Infotrieve]
20. Puceat M, Hilal-Dandan R, Strulovici B, Brunton LL, Brown JH. Differential regulation of protein kinase C isoforms in isolated neonatal and adult rat cardiomyocytes. J Biol Chem. 1994; 269: 16938–16944.
21. Grueter CE, Abiria SA, Dzhura I, Wu Y, Ham AJ, Mohler PJ, Anderson ME, Colbran RJ. L-type Ca2+ channel facilitation mediated by phosphorylation of the beta subunit by CaMKII. Mol Cell. 2006; 23: 641–650.[CrossRef][Medline] [Order article via Infotrieve]
22. Lee TS, Karl R, Moosmang S, Lenhardt P, Klugbauer N, Hofmann F, Kleppisch T, Welling A. Calmodulin kinase II is involved in voltage-dependent facilitation of the L-type Cav1.2 calcium channel: Identification of the phosphorylation sites. J Biol Chem. 2006; 281: 25560–25567.
23. Robu VG, Pfeiffer ES, Robia SL, Balijepalli RC, Pi Y, Kamp TJ, Walker JW. Localization of functional endothelin receptor signaling complexes in cardiac transverse tubules. J Biol Chem. 2003; 278: 48154–48161.
24. Wenham D, Rahmatullah RJ, Rahmatullah M, Hansen CA, Robishaw JD. Differential coupling of 1-adrenoreceptor subtypes to phospholipase C and mitogen activated protein kinase in neonatal rat cardiac myocytes. Eur J Pharmacol. 1997; 339: 77–86.[CrossRef][Medline]
[Order article via Infotrieve]
25. McWhinney C, Wenham D, Kanwal S, Kalman V, Hansen C, Robishaw JD. Constitutively active mutants of the 1a- and the 1b-adrenergic receptor subtypes reveal coupling to different signaling pathways and physiological responses in rat cardiac myocytes. J Biol Chem. 2000; 275: 2087–2097.
26. Rorabaugh BR, Gaivin RJ, Papay RS, Shi T, Simpson PC, Perez DM. Both 1A- and 1B-adrenergic receptors crosstalk to down regulate β1-ARs in mouse heart: coupling to differential PTX-sensitive pathways. J Mol Cell Cardiol. 2005; 39: 777–784.[CrossRef][Medline]
[Order article via Infotrieve]
27. Akhter SA, Milano CA, Shotwell KF, Cho MC, Rockman HA, Lefkowitz RJ, Koch WJ. Transgenic mice with cardiac overexpression of 1B-adrenergic receptors. In vivo 1-adrenergic receptor-mediated regulation of β-adrenergic signaling. J Biol Chem. 1997; 272: 21253–21259.
28. Valenzuela D, Han X, Mende U, Fankhauser C, Mashimo H, Huang P, Pfeffer J, Neer EJ, Fishman MC. Go is necessary for muscarinic regulation of Ca2+ channels in mouse heart. Proc Natl Acad Sci U S A. 1997; 94: 1727–1732.
29. Ivanina T, Blumenstein Y, Shistik E, Barzilai R, Dascal N. Modulation of L-type Ca2+ channels by Gβ and calmodulin via interactions with N and C termini of 1C. J Biol Chem. 2000; 275: 39846–39854.
This article has been cited by other articles:
 |
|
 |
|
  G. S. Pitt Calcineurin Finds a New Partner in the L-Type Ca2+ Channel Circ. Res., July 2, 2009; 105(1): 7 - 8. [Full Text] [PDF]
|
|
|
|
 |
|
 D. Matsuba, T. Terui, J. O-Uchi, H. Tanaka, T. Ojima, I. Ohtsuki, S. Ishiwata, S. Kurihara, and N. Fukuda Protein kinase A-dependent modulation of Ca2+ sensitivity in cardiac and fast skeletal muscles after reconstitution with cardiac troponin J. Gen. Physiol., May 25, 2009; 133(6): 571 - 581. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 E. A. Oestreich, S. Malik, S. A. Goonasekera, B. C. Blaxall, G. G. Kelley, R. T. Dirksen, and A. V. Smrcka Epac and Phospholipase C{epsilon} Regulate Ca2+ Release in the Heart by Activation of Protein Kinase C{epsilon} and Calcium-Calmodulin Kinase II J. Biol. Chem., January 16, 2009; 284(3): 1514 - 1522. [Abstract] [Full Text] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||