Journal of Pharmacology And Experimental Therapeutics Fast Forward
First published on September 29, 2006; DOI: 10.1124/jpet.106.110494
0022-3565/07/3201-14-21$20.00
JPET 320:14-21, 2007
CARDIOVASCULAR
Distinct KATP Channels Mediate the Antihypertrophic Effects of Adenosine Receptor Activation in Neonatal Rat Ventricular Myocytes
Ying Xia,
Sabzali Javadov,
Tracey X. Gan,
Theresa Pang,
Michael A. Cook, and
Morris Karmazyn
Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, University of Western Ontario, London, Ontario, Canada
Received for publication
July 7, 2006
Accepted
September 28, 2006.
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Abstract
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Recent evidence suggests that both adenosine receptor (AR) and KATP channel activation exert antihypertrophic effects in cardiac myocytes. We studied the relative contributions of mitochondrial KATP (mitoKATP) and sarcolemmal KATP (sarcKATP) to the antihypertrophic effects of ARs in primary cultures of neonatal rat ventricular myocytes exposed for 24 h with the 
1 adrenoceptor agonist phenylephrine (PE). The A1R agonist N6-cyclopentyladenosine (CPA), the A2AR agonist CGS21680 [2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine], and the A3R agonist N6-(3-iodobenzyl)adenosine-5'-methyluronamide (IB-MECA) all prevented PE-induced hypertrophy. Glibenclamide, a nonselective KATP channel blocker reversed the antihypertrophic effect of all three AR agonists as determined by cell size and atrial natriuretic peptide expression and early c-fos up-regulation. In contrast, the mitoKATP blocker 5-hydroxydecanoic acid selectively attenuated the effect of CGS21680 and IB-MECA, whereas HMR1098 [1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea, sodium salt], a specific blocker of sarcKATP, only abolished the antihypertrophic effect of CPA. Moreover, both CGS21680 and IB-MECA but not CPA decreased the mitochondrial membrane potential when PE was present, similarly to that seen with diazoxide, and both agents inhibited PE-stimulated elevation in mitochondrial Ca2+. All AR agonists diminished PE-induced phosphoserine/threonine kinase and protein kinase B up-regulation, which was unaffected by any KATP blocker. Our data suggest that AR-mediated antihypertrophic effects are mediated by distinct KATP channels, with sarcKATP mediating the antihypertrophic effects of A1R activation, whereas mitoKATP activation mediates the antihypertrophic effects of both A2AR and A3R agonists.
Both opening of KATP channels and activation of adenosine receptors exert cardioprotective effects against ischemic and reperfusion injury (Cohen et al., 2000
). Early studies on KATP channels suggested that the cardioprotective properties are mediated by sarcolemmal KATP (sarcKATP) activation (Noma, 1983; Auchampach et al., 1992). More recent studies have found that the opening of mitoKATP also plays an important role in cardiac protection such as in ischemic preconditioning (Garlid et al., 1997). Cardiac hypertrophy has traditionally been considered as an adaptive response; however, prolonged hypertrophy is associated with increased risk of sudden death or progression to heart failure (Frey and Olson, 2003). Emerging evidence suggests that, in addition to their cardioprotective effects, both adenosine receptor and KATP activation reduce the remodeling process and inhibit cardiac hypertrophy. For example, the mitoKATP opener nicorandil has been shown to reduce myocardial remodeling in rats treated with the nitric-oxide synthase inhibitor N
-nitro-L-arginine methyl ester (Sanada et al., 2002), whereas the putative mitoKATP opener diazoxide inhibited phenylephrine (PE)-induced cardiac hypertrophy in rat neonatal cardiomyocytes (Xia et al., 2004). Thus, these studies suggest a direct antihypertrophic effect of KATP activation in the heart. Recent studies have also demonstrated an antihypertrophic effect of adenosine receptor activation in both cardiac myocytes as well as in a murine cardiac hypertrophy produced by aortic coarctation (Liao et al., 2003; Gan et al., 2005), although the nature of the receptors involved in this phenomenon is not known with certainty. For example, Liao et al. (2003) showed that only A1 receptor activation could attenuate cardiac hypertrophy, although work from our laboratory has shown that multiple receptor subtypes (A1R, A2AR, and A3R) activation share equal efficacy in their ability to attenuate PE-induced hypertrophy (Gan et al., 2005).
Previous studies have shown that A1 receptor stimulation activates KATP channels in rat cardiac cells (Kirsch et al., 1990). It has also been shown that activation of adenosine A1 receptors induces myocardial preconditioning in the canine heart by opening KATP channels (Auchampach and Gross, 1993). In view of this evidence and the finding that both diazoxide and adenosine receptor agonists attenuate hypertrophy, we hypothesized that the direct antihypertrophic effects of adenosine receptor stimulation could involve KATP activation. Accordingly, the present study was designed to determine whether KATP channels mediate the antihypertrophic effect of adenosine receptors in neonatal rat ventricular myocytes and, if so, to assess and identify the nature of KATP involvement in mediating the antihypertrophic effect of adenosine receptor activation.
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Materials and Methods
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Primary Neonatal Ventricular Myocyte Culture. Myocytes were prepared from the ventricles of 4-day-old Sprague-Dawley rats as described in detail previously (Gan et al., 2003). In brief, the ventricles were excised, washed, and cut into small pieces in 15 ml of Hanks' balanced salt solution (Invitrogen, Burlington, ON, Canada) and then digested in 60 ml of Hanks' balanced salt solution containing 800 U of collagenase (Worthington Biochemical Corporation, Lakewood, NJ)/ventricle. The digestion was performed in a circulating water bath to maintain the reaction temperature at 37°C. The digestion was terminated by adding an identical volume of 20% fetal bovine serum. The cells were sorted by a cell strainer to remove undigested particles and then centrifuged at 600g for 5 min at 4°C. The cell pellet was resuspended in a plating medium containing 10% fetal bovine serum and 0.1 mM bromodeoxyuridine and was pre-plated in tissue culture flasks two times for 20 min to reduce contaminating nonmyocytes, after which the cells were transferred into Primaria cell culture dishes (Becton Dickinson Labware, Mississauga, ON, Canada) and cultured for 48 h. The medium was replaced with a serum-free maintenance medium and incubated for another 24 h before being used for study. Approximately 95% of cells prepared by this method demonstrated sarcomeric myosin heavy chain staining, indicating relatively low nonmyocyte contamination (Rajapurohitam et al., 2003).
Drugs Used and Experimental Design. To first determine whether the activation of adenosine receptors inhibited PE (10 µM)-induced cardiomyocyte hypertrophy via KATP channels, we assessed the effect of KATP inhibition with the following pharmacological agents that were added 30 min before adenosine receptor activation: the mitoKATP blocker 5-HD (100 µM), the sarcKATP blocker HMR1098 (100 µM), or the nonspecific KATP blocker glibenclamide (50 µM). The following adenosine receptor agonists were then administered for a further 30 min, after which PE was administered for 24 h: the A1R agonist CPA (1 µM), the A2AR agonist CGS21680 (100 nM), or the A3R agonist IB-MECA (100 nM). For some experiments, the putative mitoKATP opener diazoxide (100 µM) was used as a positive control. All drugs were purchased from Sigma (Oakville, ON, Canada), with the exception of HMR1098, which was a kind gift from Sanofi-Aventis (Frankfurt, Germany).
Cell Surface Area Measurement. The cells were plated at a density of 1 x 106 cells/6-cm dish to obtain individually plated cells. At the end of the treatment period, the cells were washed twice with PBS, after which they were viewed using a Leica DMIL inverted microscope (Leica, Wetzlar, Germany) equipped with a Polaroid digital camera (Polaroid Corporation, Waltham, MA). For each sample (n = 1), eight random images were taken, and at least 40 individual cell surface area measurements were made using Mocha software.
Real-Time PCR. Myocytes were plated at a density of 6 x 106 cells/6-cm dish. After washing twice with PBS, RNA was isolated by adding 1 ml of TRIzol reagent (Invitrogen) to each dish. Five micrograms of total RNA was applied for reverse transcription by Superscript II reverse transcriptase (Invitrogen). One microliter from the 20-µl cDNA product was used for each PCR reaction. Real-time PCR was performed with a DNA Engine Opticon Real Time System (MJ Research, Waltham, MA) with the SYBR Green JumpStart Taq ReadyMix kit (Sigma) according to the manufacturer's instructions. Primer sequences for individual genes and PCR conditions are shown in Tables 1 and 2.
Fluorescence Measurement of Mitochondrial Membrane Potential and Mitochondrial Ca2+ Concentration. Cells were plated at 1 x 106 cells/well in 24-well dishes. After the cells were treated with PE for 30 min, the mitochondrial membrane potential (
m) was measured by loading cells with 10 µg/ml JC-1 (Molecular Probes, Eugene, OR) at 37°C for 15 min, and the mitochondrial Ca2+ concentration ([Ca2+]m) was monitored with the Ca2+ fluorophore Rhod-2 (Molecular Probes). The cardiomyocytes were loaded with 10 µg/ml Rhod-2 for 120 min at 4°C and then incubated for 30 min at 37°C in the culture media. This two-step cold loading/warm incubation protocol achieved exclusive loading of Rhod-2 into the mitochondria (Trollinger et al., 2000). Myocytes loaded with JC-1 or Rhod-2 were washed with PBS, and the fluorescence was measured by a Tecan multifunction microplate reader (Tecan, Durham, NC). JC-1 was excited at 488 nm, the red emission fluorescence was detected at 595 nm, and the green emission fluorescence was detected at 535 nm. The m is presented as a ratio of 595/535 nm compared with control cells. Rhod-2 was excited at 540 nm, with emission monitored at 605 nm.
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Fig. 1. Dose-dependent effect of A1R agonist CPA (A), the A2AR agonist CGS21680 (B), and the A3R agonist IB-MECA (C) on inhibition of 10 µM PE induced hypertrophy as assessed by cell surface area. Values indicate mean ± S.E.M. of n = 4. *, p < 0.05 versus control values obtained in the absence of any treatment.
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Confocal Fluorescence Imaging of m. Cells were plated at 3 x 106 cells/3.5-cm glass-bottomed dish precoated with type I collagen. After the cells were treated with PE for 30 min, the cells were washed with PBS, and JC-1 was added to each well to reach a final concentration as 10 µg/ml. The cells were incubated at 37°C for 30 min and then washed with PBS three times. Images were recorded on a Zeiss LSM 510 confocal microscope (Carl Zeiss GmbH, Jena, Germany). JC-1 was excited at 488 nm by an argon ion laser with red and green emissions detected at 568 and 510 nm, respectively.
Western Blotting for Phosphorylated and Total Akt. Cells were plated at a concentration of 6 x 106 cells/6-cm dish. After washing twice with PBS, the cells were scraped into 100 µl of lysis buffer (20 mM Tris, 150 mM NaCl, 1% Triton X-100, 10% glycerol, 2 mM EDTA, 2 mM EGTA, 50 mM NaF, 200 µM Na3VO4, 10 mM Na4P2O7, 40 mM 
-glycerophosphate, 10 µg/ml leupeptin, 1 µM pepstatin A, 1 mM PMSF, and 1 µM colyculin A). The lysate was transferred to 1.5-ml Eppendorf tubes, sonicated, and then centrifuged at 10,000g for 5 min at 4°C. The supernatant was transferred to a fresh tube, and the protein concentration was assayed by the Bradford protein assay kit (Bio-Rad, Mississauga, ON, Canada). Thirty micrograms of protein were loaded in 10% SDS-polyacrylamide gel electrophoresis and transferred to nylon membranes (Amersham, Little Chalfont, Buckinghamshire, UK). The membranes were blocked in 5% dry milk for 3 h with primary antibody for 2 h and secondary antibody for 1 h and then detected by enhanced chemiluminescence reagent (Amersham). After detection by phospho-Akt (Ser473) antibody, the same blot was stripped and reprobed by total Akt antibody to demonstrate equal sample loading. Antibodies against phospho-Akt (Ser473) and total Akt were purchased from Cell Signaling (Beverly, MA) and used at a 1:1000 dilution.
Statistical Analysis. All values in the figures and text are presented as mean ± S.E.M. Sample size per experiment is indicated under Results. Data were analyzed by one-way analysis of variance followed by a Tukey multiple comparison test using Prism (Graph-Pad Software Inc., San Diego, CA) with p < 0.05 considered to represent significant differences between groups.
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Results
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Influence of KATP Blockers on Antihypertrophic Effect of Adenosine Receptor Agonists. To identify the concentration of adenosine receptor agonists producing maximal inhibition of PE-induced hypertrophy, increasing concentrations of these agents were used to perform the cell surface area experiment. As shown in Fig. 1, 1 µM CPA, 100 nM CGS21680, and 100 nM IB-MECA produced maximal inhibition of PE-induced increase in cell surface area. Furthermore, our previous results have shown that the antihypertrophic effects of all three agonists can be completely reversed by their respective antagonists, thus suggesting a receptor-specific effect of these agonists (Gan et al., 2005). We next determined whether the inhibition of PE-induced cardiomyocyte hypertrophy by adenosine receptor agonists is mediated by KATP channels using primarily pharmacological approaches. As shown in Fig. 2, pharmacological blockers of KATP had diverse effects on the antihypertrophic effects of adenosine receptor agonists that, in general, reflected the nature of the adenosine agonist used. In the case of CPA, all three KATP blockers reversed the inhibitory effect of CPA against PE-induced hypertrophy, although the effect of 100 µM 5-HD was lower than that seen with either 100 µM HMR1098 or 50 µM glibenclamide. However, in the case of CGS21680 or IB-MECA, the antihypertrophic effect could be reversed by either 5-HD or glibenclamide, whereas the sarcKATP-specific blocker HMR1098 was without effect. Analysis of molecular markers of hypertrophy (ANP and c-fos mRNA expression) revealed generally identical responses to KATP inhibitors as shown in Figs. 3 and 4. Thus, inhibition of both ANP and c-fos up-regulation by CPA was abrogated by both HMR1098 and glibenclamide, whereas 5-HD was without effect. With respect to either CGS21680 or IB-MECA, both 5-HD and glibenclamide significantly reversed the antihypertrophic effects of the adenosine agonists vis-à-vis ANP and c-fos up-regulation (Figs. 3 and 4) akin to that seen with respect to cell surface area reported in Fig. 2. Overall, these data suggest that the antihypertrophic effect of the A1R agonist CPA is dependent on sarcKATP, whereas mitoKATP mediates the antihypertrophic influence of A2AR and A3R activation.
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Fig. 3. Effect of the KATP channel blockers 5-HD (100 µM), HMR1098 (100 µM), and glibenclamide (50 µM) on the antihypertrophic effects of the A1R agonist CPA (1 µM), the A2AR agonist CGS21680, and the A3R agonist IB-MECA (100 nM) as assessed by ANP expression. Hypertrophy was produced by 24-h exposure to 10 µM PE. Values indicate mean ± S.E.M. of n = 6.  , p < 0.05 versus control values obtained in the absence of any treatment; #, p < 0.05 versus PE; *, p < 0.05 versus PE + AR.
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Fig. 4. Effect of the KATP channel blockers 5-HD (100 µM), HMR1098 (100 µM), and glibenclamide (50 µM) on the antihypertrophic effects of the A1R agonist CPA (1 µM), the A2AR agonist CGS21680, and the A3R agonist IB-MECA (100 nM) as assessed by up-regulation of c-fos expression. Hypertrophy was produced by 24-h exposure to 10 µM PE. Values indicate mean ± S.E.M. of n = 6. , p < 0.05 versus control values obtained in the absence of any treatment; #, p < 0.05 versus PE; *, p < 0.05 versus PE + AR.
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The Effect of Adenosine Receptor Agonists on Mitochondrial Membrane Potential in Neonatal Cardiomyocytes. To obtain direct evidence that CGS21680 and IB-MECA inhibited PE-induced hypertrophy through opening mitoKATP channels, the m of these cells was measured. The cells were labeled with 10 µg/ml JC-1, and the fluorescence at 595 and 535 nm was measured by a fluorescence plate reader and imaged by confocal microscopy. Mitochondrial depolarization was indicated by a decrease of the ratio of F595/F535. Figure 5 shows the changes in m when cells were treated with adenosine receptor agonists and diazoxide with or without PE. Neither diazoxide nor adenosine receptor agonists alone exerted any effect on m compared with control cells (Fig. 5A, white bars). However, in the presence of PE (Fig. 5A, black bars), diazoxide significantly reduced m by 23% in cells exposed to PE. Likewise, both CGS21680 and IB-MECA significantly reduced m by 18 and 34%, respectively, in cells exposed to PE. In contrast, CPA was without effect on m.
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Fig. 5. The effect of diazoxide (100 µM) and adenosine receptor agonists on neonatal cardiomyocytes mitochondrial membrane potential in the presence or absence of 10 µM PE. A, quantitative JC-1 fluorescence values. The value of control was considered as 100%. Values indicate mean ± S.E.M. of n = 6. *, p < 0.05. B, representative confocal images of JC-1 staining. The decrease of red/green ratio indicates the decrease of mitochondrial membrane potential. Bar in the images, 50 µm.
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The Effect of Diazoxide and Adenosine Receptor Agonists on [Ca2+]m. We next examined treatments on [Ca2+]m in neonatal cardiomyocytes by Rhod-2 staining. As shown in Fig. 6A, PE significantly increased [Ca2+]m by 23%, whereas this was completely abrogated by diazoxide as well as CGS21680 and IB-MECA. In contrast, CPA was without effect. The data in Fig. 6B show that the mitochondrial Ca2+ uniport inhibitor ruthenium red inhibited the Ca2+ overload induced by 100 µM H2O2 in mitochondria, confirming that the Rhod-2 staining method is specific for mitochondrial Ca2+ measurement.
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Fig. 6. The effect of diazoxide (100 µM) and adenosine receptor agonists on neonatal cardiomyocytes mitochondrial Ca2+ uptake in the presence or absence of 10 µM PE measured by Rhod-2 staining (A). The value of the control was considered as 100%. Values indicate mean ± S.E.M. of n = 6. *, p < 0.05. The effect of mitochondrial Ca2+ uniport inhibitor ruthenium red on neonatal cardiomyocytes mitochondrial Ca2+ uptake with or without 100 µM H2O2 treatment measured by Rhod-2 staining (B). n = 4. *, p < 0.05; #, p < 0.01. Ruthenium red inhibited the Ca2+ overload induced by H2O2 in mitochondria indicated that this Rhod-2 staining method is specific for mitochondrial Ca2+ measurement.
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Effect of Activation of Adenosine Receptors on Akt Expression. To further address potential mechanisms underlying the antihypertrophic effects of adenosine receptor activation and the role of KATP, we determined the potential contribution of Akt activation that has been implicated as a major contributor to the cardiac hypertrophic program. We first determined whether PE or adenosine receptor agonists could activate Akt on their own. Interestingly, as shown in Fig. 7, a significant increase in Akt phosphorylation was observed 30 min after PE administration, whereas each of the adenosine receptors agonists increased Akt phosphorylation only 5 min after addition of the agent. As shown in Fig. 8, although adenosine receptor agonists had no effect on Akt phosphorylation at 30 min, all agents significantly prevented the effect of PE on Akt phosphorylation at this time point. However, this was unaffected by KATP blockers. In view of these findings, we next examined whether the antihypertrophic effects of mitoKATP channel openings can also be dissociated from Akt activation. As shown in Fig. 9, diazoxide had no effect on PE-induced Akt activation in neonatal cardiomyocytes.
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Fig. 7. Time-dependent effect of A1R agonist 1 µM CPA (square), the A2AR agonist 10 nM CGS21680 (triangle), the A3R agonist 10 nM IB-MECA (circle), and 10 nM phenylephrine (diamond) on Akt phosphorylation in neonatal cardiomyocytes. Representative western blots for phospho-Akt and Akt are shown above the chart. Values indicate mean ± S.E.M. of n = 3.
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Fig. 8. The effect of adenosine receptor activation on Akt phosphorylation induced by 30-min 10 µM PE treatment. Representative western blots for phospho-Akt and Akt are shown below the appropriate treatment groups. Values indicate mean ± S.E.M. of n = 3. *, p < 0.05 versus control values obtained in the absence of any treatment. #, p < 0.05 versus PE.
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Fig. 9. Lack of effect of 100 µM diazoxide (Dia) on PE-induced Akt phosphorylation. Values indicate mean ± S.E.M. of n = 3. *, p < 0.05 versus control.
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Discussion
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Our study demonstrates for the first time that the antihypertrophic effect of adenosine receptor activation in rat neonatal cardiomyocytes, at least with respect to PE-induced hypertrophy, is dependent on KATP activation and that distinct KATP channels mediate the actions of specific adenosine receptors. Based on our results, we propose that sarcKATP channels mediate the antihypertrophic effects of A1R activation, whereas mitoKATP channel activation mediates the antihypertrophic effects of both A2AR and A3R agonists. This reasoning is based on various lines of evidence. First, the overall role of KATP in mediating the antihypertrophic effect of adenosine receptor activation was strongly suggested by the ability of pharmacological inhibitors of the channels to abrogate the effect of A1R, A2AR, and A3R activation. Although the nonspecific KATP blocker glibenclamide reversed the effect of all three AR agonists, the sarcKATP-specific inhibitor HMR1098 was able to selectively prevent only the effects of the A1R agonist CPA. In contrast, the mitoKATP-specific blocker 5-HD had no effect on the antihypertrophic effect of CPA, whereas it blocked the effects of both the A2AR and A3R agonists. These differences were particularly evident in terms of molecular hypertrophic markers although surprisingly 5-HD partially reversed the antihypertrophic effect of CPA as determined by cell surface area although to a lesser degree than that seen with HMR1098. The basis for the diminished effect of 5-HD on cell surface area data compared with molecular indices of hypertrophy is uncertain, although it may reflect a nonspecific effect of 5-HD on cell size due to the drug's fatty acid moiety.
Mitochondrial KATP channel activation is associated with numerous effects, including membrane depolarization and changes in Ca2+ homeostasis (Holmuhamedov et al., 1998). Accordingly, to obtain further evidence that activation of A2AR and A3R opens mitoKATP channels, we assessed the changes in mitochondrial membrane potential and mitochondrial Ca2+ uptake in intact myocytes using the fluorescence dyes JC-1 and Rhod-2, respectively. Diazoxide was used as a positive control, although surprisingly, it was without effect on either mitochondrial membrane potential or mitochondrial Ca2+ content on its own. However, diazoxide was able to depolarize the membrane and decrease Ca2+ accumulation in the presence of PE, suggesting that the mitoKATP channel does not play an important role in mitochondrial homeostasis under normal condition, but its activation modulates the responses to cellular stimulation, such as that in response to PE. Our finding with diazoxide is in agreement with a study by Ishida et al. (2001), who showed that diazoxide was without direct effects on mitochondrial membrane potential or Ca2+ levels in ventricular myocytes but abrogated ouabain-induced mitochondrial Ca2+ levels that were associated with decreased JC-1 fluorescence. Interestingly, in our study, both the A2AR agonist CGS21680 and A3R agonist IB-MECA were also able to decrease the mitochondrial membrane potential as well as Ca2+ accumulation in PE-treated myocytes, similarly to that seen with diazoxide, although the A1R agonist CPA was without effect. It is important to note that mitochondrial Ca2+ uptake is driven by the mitochondrial membrane potential (Gunter and Pfeiffer, 1990). Therefore, it is likely that A2AR and A3R activation opens mitochondrial KATP and results in the decreases in the mitochondria membrane potential, thus reducing the driving force of Ca2+ influx and attenuating the mitochondria Ca2+ overload induced by PE. Thus, a reduction in "mitochondrial remodeling" may constitute an important contributor to the antihypertrophic effect of both A2AR and A3R activation. A brief depolarization of mitochondrial membrane may exert cardioprotection by preventing Ca2+ entry into the matrix. However, a prolonged reduction of mitochondrial membrane potential may indicate opening of the mitochondrial permeability transition pore. Indeed, we have demonstrated such a potential contribution of mitochondria to postinfarction responses in rats treated with a sodium-hydrogen exchange inhibitor (Javadov et al., 2005).
Although our data support the hypothesis that both A2AR and A3R activation inhibit PE-induced cardiac hypertrophy through the opening of mitoKATP channels in neonatal cardiomyocytes, the phenomenon is difficult to explain at present, particularly because the structure and pharmacological profile of these two receptors are quite different. Although it could be suggested that rat cardiomyocytes do not express the A3 receptor and that IB-MECA induces an unspecific effect through the A2A receptor rather than A3 receptor, we have found using Western blotting that the A1R, A2AR, and A3R are all expressed in neonatal cardiomyocytes (data not shown). Thus, it is unlikely that the effects of the receptor agonists were due to nonspecific effects of these agents, although this cannot be ruled out with certainty at this time.
A question that remains concerns the molecular and cellular bases for the antihypertrophic effect of adenosine receptor activation and contributing roles of KATP channels. Mitogen-activated protein kinase kinase/extracellular signal-regulated kinases 1 and 2 (ERK1/2) and phosphatidylinositol 3-kinase/Akt (also known as PKB) are important antiapoptotic signaling pathways that have been implicated recently in cardiac hypertrophy (Dorn and Force, 2005). However, we showed previously that ERK1/2 was not involved in the antihypertrophic effect of either diazoxide (Xia et al., 2004) or activation of adenosine receptors (Gan et al., 2005). In the current study, we focused on the potential contribution of Akt by determining the effects of treatments on Akt phosphorylation/activation. Indeed, PE induced a potent Akt up-regulation that reached maximum 30 min after PE addition, whereas each of the three adenosine receptor agonists induced a much earlier (5 min) and transient Akt up-regulation. This finding is generally similar to that reported by Germack et al. (2004), who demonstrated A1R and A3R, but contrary to our findings, not A2AR-dependent Akt activation in neonatal cardiomyocytes with peak up-regulation of Akt at 5 min with reversal to control by 30 min. Thus, adenosine receptor agonists seem to have complex effects on Akt activation, a direct early and transient direct activation, and an ability to prevent subsequent PE-induced Akt phosphorylation. The basis for these diverse effects of adenosine receptor agonists is uncertain at present, but the results suggest that prevention of Akt up-regulation is associated with the antihypertrophic effects of adenosine receptor agonists. However, KATP blockers that reversed the antihypertrophic effects of adenosine receptor agonists failed to reverse the inhibitory effects of adenosine agonists on Akt phosphorylation. Moreover, diazoxide, which prevents PE-induced hypertrophy (Xia et al., 2004), failed to inhibit PE-induced Akt up-regulation. These data suggest that KATP channel opening probably occurs downstream to Akt activation, which serves to explain why neither KATP blockers nor diazoxide exerted any effect on inhibition of Akt activation by adenosine receptor agonists. When taken together, our results suggest a potential role of Akt activation in mediating the hypertrophic effect of PE. The ability of adenosine receptor agonists to prevent Akt activation is encouraging in terms of implicating Akt in the antihypertrophic effect of adenosine receptor activation. However, our results clearly indicate that the degree of Akt phosphorylation can also be dissociated from the hypertrophic response in view of the inability of KATP blockers to reverse Akt inhibition by adenosine receptor agonists despite their ability to reverse the antihypertrophic effect of these agents.
In conclusion, our study shows an important role for KATP in mediating the antihypertrophic effects of multiple adenosine receptor subtype activation. Based on our results, we propose that the antihypertrophic effect of A1R activation is dependent on sarcKATP channel activity, whereas mitoKATP activity mediates the antihypertrophic effects of both A2A and A3 receptor activation. Although prevention of Akt phosphorylation was associated with the antihypertrophic effects of adenosine receptor activation, the precise role of this pathway needs to be studied further.
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Footnotes
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This work was supported by a grant from the Canadian Institutes of Health Research.
M.K. holds a Canada Research Chair in Experimental Cardiology.
Article, publication date, and citation information can be found at http://jpet.aspetjournals.org.
doi:10.1124/jpet.106.110494.
ABBREVIATIONS: sarcKATP, sarcolemmal ATP-sensitive potassium channel; mitoKATP, mitochondrial ATP-sensitive potassium channel; PE, phenylephrine; AR, adenonosine receptor; 5-HD, 5-hydroxydecanoic acid; HMR1098, 1-[[5-[2-(5-chloro-o-anisamido)ethyl]-2-methoxyphenyl]sulfonyl]-3-methylthiourea, sodium salt; CPA, N6-cyclopentyladenosine; CGS21680, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine; IB-MECA, N6-(3-iodobenzyl)adenosine-5'-methyluronamide; PBS, phosphate-buffered saline; PCR, polymerase chain reaction; JC-1, 5,5',6,6'-tetrachloro-1,1',3,3' tetraethylbenzimidazolylcarbocyanine iodide, chloride; Rhod-2, 1-[2-amino-5-(3-dimethylamino-6-dimethylammonio-9-xanthenyl)phenoxy]-2-(2-amino-5-methylphenoxy)ethane-N,N,N',N'-tetraacetic acid, chloride; Akt, serine/threonine kinase, protein kinase B; ANP, atrial natriuretic peptide.
Address correspondence to: Dr. Morris Karmazyn, Department of Physiology and Pharmacology, Schulich School of Medicine and Dentistry, Medical Sciences Building, University of Western Ontario, London, ON, Canada N6A 5C1. E-mail: Morris.Karmazyn{at}Schulich.uwo.ca
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