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Originally published In Press as doi:10.1074/jbc.M314238200 on July 15, 2004

J. Biol. Chem., Vol. 279, Issue 39, 40938-40945, September 24, 2004
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Direct Inhibition of Type 5 Adenylyl Cyclase Prevents Myocardial Apoptosis without Functional Deterioration*

Kousaku Iwatsubo{ddagger}, Susumu Minamisawa{ddagger}, Takashi Tsunematsu{ddagger}, Masamichi Nakagome{ddagger}, Yoshiyuki Toya{ddagger}, James E. Tomlinson§, Satoshi Umemura{ddagger}, Robert M. Scarborough§, Daniel E. Levy¶, and Yoshihiro Ishikawa{ddagger}||**

From the {ddagger}Departments of Physiology and Medicine, Yokohama City University School of Medicine, Yokohama 236-0004, Japan, §Portola Pharmaceuticals Inc., South San Francisco, California 94080, Scios, Inc., Fremont, California 94555, and the ||Departments of Cell Biology and Molecular Medicine, and Medicine (Cardiology), University of Medicine and Dentistry of New Jersey, Newark, New Jersey 07103

Received for publication, December 29, 2003 , and in revised form, July 12, 2004.


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Adenylyl cyclase, a major target enzyme of {beta}-adrenergic receptor signals, is potently and directly inhibited by P-site inhibitors, classic inhibitors of this enzyme, when the enzyme catalytic activity is high. Unlike {beta}-adrenergic receptor antagonists, this is a non- or uncompetitive inhibition with respect to ATP. We have examined whether we can utilize this enzymatic property to regulate the effects of {beta}-adrenergic receptor stimulation differentially. After screening multiple new and classic compounds, we found that some compounds, including 1R,4R-3-(6-aminopurin-9-yl)-cyclopentanecarboxylic acid hydroxyamide, potently inhibited type 5 adenylyl cyclase, the major cardiac isoform, but not other isoforms. In normal mouse cardiac myocytes, contraction induced by low {beta}-adrenergic receptor stimulation was poorly inhibited with this compound, but the induction of cardiac myocyte apoptosis by high {beta}-adrenergic receptor stimulation was effectively prevented by type 5 adenylyl cyclase inhibitors. In contrast, when cardiac myocytes from type 5 adenylyl cyclase knock-out mice were examined, {beta}-adrenergic stimulation poorly induced apoptosis. Our data suggest that the inhibition of {beta}-adrenergic signaling at the level of the type 5 adenylyl cyclase isoform by P-site inhibitors may serve as an effective method to prevent cardiac myocyte apoptosis induced by excessive {beta}-adrenergic stimulation without deleterious effect on cardiac myocyte contraction.


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
 REFERENCES
 
Adrenergic stimulation via the sympathetic nervous system is a major mechanism to regulate cardiac function (1). Norepinephrine released from the synaptic terminal binds to {beta}-adrenergic receptors ({beta}-AR),1 leading to the activation of adenylyl cyclase (AC) and thus the production of cAMP, which initiates a cascade of protein phosphorylation and regulation of many key signaling elements involved in muscle contraction and calcium handling. The immediate outcome of this stimulation is increased cardiac contractility and output. Pharmacological inhibition of this pathway, by the use of {beta}-AR antagonists, has been widely utilized to attenuate cardiac function in the clinical setting of treating high blood pressure for several decades. Recently, {beta}-AR antagonists have been used in the treatment of heart failure, i.e. in hearts with deteriorated cardiac function. This paradoxical usage of {beta}-AR antagonists in heart failure has been rationalized by the protection of the heart from excessive sympathetic stimulation. Under this pathological condition, it is well known that the sympathetic activity is increased markedly (2–4), leading to the remodeling of the heart (5) and, more importantly, the induction of cardiac myocyte apoptosis, as demonstrated by both in vivo and in vitro studies (6, 7). However, a major problem in introducing {beta}-AR antagonist therapy is the worsening of cardiac function (8).

It is believed that regulating {beta}-adrenergic signaling at the level of AC, instead of the receptor, may serve as an alternative to the common {beta}-AR regulating therapy, which is most exemplified by direct activation of AC in the treatment of acute heart failure (9–12). AC is made of multiple isoforms that differ in tissue distribution and biochemical properties (13), with each tissue containing a distinct set of AC isoforms. Although the importance of biochemical regulations, e.g. regulation by G{beta}{gamma} or kinases, has been investigated extensively, the importance of the difference in maximal catalytic activity among the AC isoforms has been less studied. Nevertheless the unique composition of AC isoforms likely confers distinct {beta}-AR signaling attributes to each tissue, with the dominant AC isoform playing the largest role. Type 5 AC is a dominant isoform in the heart which possesses the highest enzyme catalytic activity among the AC isoforms, and it is either poorly expressed or absent in the other peripheral tissues including lungs (14). These AC isoforms share multiple common properties, with a feature being the inhibition of catalytic activity by P-site inhibitors. This class of inhibitors comprises adenosine analogs (15) that inhibit AC activity by binding to the ATP substrate site (16). The mode of inhibition of AC by P-site inhibitors, unlike that of {beta}-AR by its antagonists, is either non- or uncompetitive with respect to the substrate ATP as shown by kinetic analysis (17). Importantly, P-site inhibition is potent when the catalytic activity of AC is high (e.g. during stimulation by the {alpha} subunit of stimulatory G protein (G{alpha}s)), but much less when AC is unstimulated or only mildly stimulated (10, 17).

In the current study, we have examined the AC isoform selectivity, in particular, type 5 AC selectivity, among P-site inhibitors (P-site inhibitors with metal chelating property and ribose-substituted P-site inhibitors), which have been described recently (10, 18) as well as classic inhibitors. We have also examined the effect of such inhibitors in regulating cardiac myocyte contractility and apoptosis induced by {beta}-AR stimulation. Finally, using myocytes from mice with targeted disruption of type 5 AC (AC5KO) (19, 20), we further validated the specificity of these inhibitors to type 5 AC and elucidated a principal role of this isoform, which has a very high catalytic activity and is dominantly expressed in the heart, in responding to {beta}-AR stimulation.


   EXPERIMENTAL PROCEDURES
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
RESULTS
 DISCUSSION
 REFERENCES
 
Reagents—All chemicals, including conventional P-site inhibitors, were purchased from Sigma unless otherwise specified. A series of P-site inhibitors with metal chelating property (PMC) were synthesized as described (18). PMC-1 is 5-(6-aminopurin-9-yl)-pentanoic acid hydroxyamide. PMC-2 is 4-(6-aminopurin-9-yl)-N-hydroxybutyramide. PMC-3 is 1S,4S-2-(4-(6-aminopurin-9-yl)-cyclopent-2-enyl)-N-hydroxyacetamide. PMC-4 is 1R,4S-(4-(6-aminopurin-9-yl)-cyclopent-2-enyl)acetic acid. PMC-5 is 1S,3S-4-(6-aminopurin-9-yl)-cyclopent-2-enecarboxylic acid hydroxyamide. PMC-6 is 1R,4R-3-(6-aminopurin-9-yl)-cyclopentane-carboxylic acid hydroxyamide. Ribose-substituted (RS) P-site ligands were purchased from Chem Ster Ltd. (Moscow, Russia). RS-1 is 2-amino-7,8-dihydro-7-(4-methoxyphenyl)-5(6H)-quinazolinone. RS-2 or NKY80 is 2-amino-7-(2-furanyl)-7,8-dihydro-5(6H)-quinazolinone. RS-3 is 2-amino-7,8-dihydro-7-phenyl-5(6H)-quinazolinone. RS-4 is 2-amino-7(4-chlorophenyl)-7,8-dihydro-5(6H)-quinazolinone.

Overexpression of the AC Isoforms and G{alpha}s in Insect Cells—Overexpression of each AC isoform or the G{alpha}s in insect cells was performed as described previously (12). High Five cells were washed twice with ice-cold phosphate-buffered saline (PBS) and homogenized in a buffer containing 50 mM Tris-HCl, pH 8.0, 1 mM EGTA, 1 mM EDTA, 1 mM dithiothreitol, 200 mM sucrose, and a protease inhibitor mixture containing 20 µg/ml 1-chloro-3-tosylamido-7-amino-L-2-heptanone, 10 µg/ml leupeptin, 0.1 mM phenylmethylsulfonyl fluoride, 50 units/ml egg white trypsin inhibitor, and 2 µg/ml aprotinin (buffer A). Cells were disrupted with a sonicator and centrifuged at 500 x g for 10 min at 4 °C. The supernatants were further centrifuged at 100,000 x g for 35 min at 4 °C. The resultant pellets were resuspended in the same buffer without EGTA (buffer B). For G{alpha}s preparation, we used the supernatant fraction after ultracentrifugation. The crude membranes and the G{alpha}s-rich supernatant were stored at -80 °C until use. Protein concentration was measured with the Bio-Rad protein assay system. We overexpressed the type 2, type 3, and type 5 AC isoforms because they represent each distinct subgroup within the AC family and can exhibit high catalytic activity when overexpressed in insect cells (12, 21, 22). The overexpression of recombinant AC isoform in insect cells increased the maximal catalytic activity by ~58-fold for type 2 AC, 46-fold for type 3 AC, and 330-fold for type 5 AC over that of control cell membranes as determined in the presence of 50 µM G{alpha}s·GTP{gamma}S·forskolin. Thus, under these conditions, each isoform was examined as the overwhelming AC activity in these cells.

Tissue Preparation—Development of AC5KO was described previously (20). AC5KO mice used in this study have been backcrossed to C57BL/6 for five generations. Mouse tissues were minced and homogenized with a Polytron for 3 x 10 s in buffer A followed by centrifugation at 500 x g for 10 min at 4 °C. The supernatants were centrifuged further at 100,000 x g for 35 min at 4 °C. The resultant pellets were resuspended in buffer B, and stored at -80 °C until use. Animals used in this study were maintained in accordance with the guidelines of the animal experiment committee of the Yokohama City University School of Medicine and New Jersey Medical School.

AC Assay—AC catalytic activity was measured as described previously (12). Briefly, the reaction mixtures contained 20 mM HEPES, pH 8.0, 0.5 mM EDTA, 0.1 mM ATP containing 1 x 106 cpm [{alpha}-32P]ATP, 0.1 mM cAMP, 1 mM creatine phosphate, 8 units/ml creatine phosphokinase, 5 mM MgCl2, and 4 µg (for insect cells) or 5–10 µg (for tissues) of membrane protein in a final volume of 100 µl. G{alpha}s-enriched supernatant obtained from insect cells overexpressing G{alpha}s was used in an amount that stimulated AC maximally in the presence of 1 µM GTP{gamma}S. Assays were performed at 30 °C for 15 min and terminated by the addition of 100 µl of 2% SDS. To monitor the recovery from the columns, 3H-labeled cAMP was used. cAMP was separated from ATP by passing through Dowex and alumina columns, and the radioactivity was measured by liquid scintillation counting.

cAMP-dependent Kinase Activity Assay—cAMP-dependent kinase activity was determined as described previously with some modifications (23). Briefly, H9C2 cells were maintained in Dulbecco's modified Eagle's medium with 10% fetal bovine serum and grown to 90% confluence in 10-mm dishes. Cells were washed with PBS twice and harvested gently. The cell suspensions were centrifuged at 500 x g for 3 min and resuspended in extraction buffer (25 mM Tris-HCl, pH 7.4, 0.5 mM EDTA, 0.5 mM EGTA, 10 mM {beta}-mercaptoethanol, 1 µg/µl leupeptin, 1 µg/µl aprotinin, 0.5 mM phenylmethylsulfonyl fluoride and 0.2 mM 3-isobutyl-1-methylxanthine (IBMX)). Cells were sonicated and centrifuged at 300 x g for 3 min. The supernatant was subjected to a cAMP-dependent kinase assay kit as recommended by the manufacturer (Promega, WI). cAMP-dependent kinase activity was determined by the addition of various cAMP concentrations in the presence or absence of 10 µM PMC-6.

Myocyte Preparation—Primary cultures of neonatal mouse cardiomyocytes were prepared from the heart of a 1-day-old mouse, as described previously with some modifications (24). Briefly, trypsinization and collagenization were performed, and cardiomyocytes were maintained at 37 °C in humidified air with 5% CO2. To reduce the number of contaminating non-muscle cells, dissociated cells were preplated on 100-mm culture dishes in minimum essential medium with 10% fetal bovine serum containing 1% penicillin-streptomycin for 3 h. The non-attached cardiomyocyte-rich fraction was plated on plastic dishes. The culture medium was changed 48 h after seeding to minimum essential medium containing 0.1% fetal bovine serum with 1% penicillin-streptomycin. At this point, >95% of the adherent cells displayed spontaneous contractile activity, indicative of a predominant population of cardiomyocytes. Neonatal Wister rat cardiomyocytes were prepared in a similar manner except for the use of Dulbecco's modified Eagle's medium.

Adult mouse ventricular myocytes were prepared as described previously with some modifications (25, 26). Briefly, the heart of a C57BL mouse at 10 weeks of age was excised, and the aorta was cannulated and mounted onto a Langendorff perfusion apparatus. The heart was perfused for 4 min in modified Joklik's minimal essential medium (Invitrogen) consisting of 113 mM NaCl, 4.7 mM KCl, 0.6 mM KH2PO4, 0.6 mM Na2HPO4, 1.2 mM MgSO4, 12 mM NaHCO3, 20 mM D-glucose, 10 mM HEPES, 30 mM taurine, 2 mM creatine, and 2 mM carnitine, pH 7.4. Perfusion was then switched to a modified Joklik's minimal essential medium plus 0.1% (w/v) collagenase type II (Worthington). After 10 min of enzymatic digestion, the atria and right ventricle were removed, the left ventricle was cut into several pieces, and the cells were dispersed by gentle agitation. Enzyme digestion was stopped by adding Joklik's minimal essential medium containing bovine calf serum, and 1.2 mM calcium was reintroduced gradually to the cells. Myocytes were centrifuged for 1 min at 180 x g and resuspended in minimum essential medium containing 5% bovine calf serum, 10 mM 2,3-butanedione monoxime, 2 mM L-glutamine, and 1% penicillin-streptomycin. Myocytes were counted, plated in a 10 µg/ml laminin-precoated glass cover, and incubated at 37 °C in humidified air with 5% CO2. After 1 h, medium was changed into minimum essential medium containing 0.1 mg bovine serum albumin, 10 mM 2,3-butanedione monoxime, 1 mg/liter insulin, 0.55 mg/liter transferrin, 0.67 µg/liter selenium, 2 mM L-glutamine and 1% penicillin-streptomycin. After 6 h, myocytes were exposed to isoproterenol (ISO) or ISO plus PMC-6 for 12 h and subjected to apoptosis assays.

[3H]Adenine Labeling and cAMP Accumulation Assay—cAMP accumulation assays in neonatal myocytes were performed with [3H]adenine as described previously with some modifications (12). Briefly, the cells were incubated with [3H]adenine (3 µCi/well) for 24 h. Cells were washed four times with 20 mM HEPES-balanced serum-free Dulbecco's modified Eagle's medium and pretreated with 20 mM HEPES-balanced serum-free Dulbecco's modified Eagle's medium containing 0.5 mM IBMX. Some experiments were performed without IBMX. Reactions were started by the addition of ISO and terminated by the addition of 12% (w/v) trichloroacetic acid, 0.25 mM ATP, and 0.25 mM cAMP. The [3H]ATP and [3H]cAMP were separated with single acidic alumina columns as described previously (10). The cAMP production was calculated as [3H]cAMP/([3H]cAMP + [3H]ATP) x 104.

Myocyte Shortening Measurement by Edge Detection—Myocyte shortening was examined as described previously (26). Adult mouse ventricular myocytes were prepared as described above. After digestion and agitation of ventricles, myocytes were filtered and washed, and 0.5 mM calcium was reintroduced. Isolated cardiomyocytes were transferred to a temperature-controlled perfusion chamber located on the stage of an inverted microscope (Nikon Diaphot-TMD, Tokyo, Japan). Cell shortening was measured using a video edge motion detector (IonOptix Inc., Boston) interfaced to a standard CCD camera (Myocam, IonOptix Inc., Boston), which was attached to the microscope. A 403 Nikon phase II objective was used. Cells were stimulated at 0.5 Hz with a 5-ms pulse duration by a electrostimulator (SEN-3301, Nihon Koden Inc., Tokyo, Japan). Edge detection measurements were performed on cardiomyocytes at 22 °C in continuously circulating Tyrode's solution (137 mM NaCl, 4.4 mM KCl, 0.5 mM MgCl2, 11.6 mM HEPES, 5 mM glucose) on cells. The concentration of calcium in Tyrode's solution was fixed at 2 mM.

Western Blot Analysis of Phospholamban—Western blot analysis of phospholamban was performed as described previously with some modifications (27). Briefly, neonatal mouse cardiomyocytes were incubated with an increasing concentration of ISO in the presence or absence of 10 µM PMC-6. Incubation was terminated by washing three times with ice-cold PBS containing 1 mM sodium pyrophosphate, 20 mM NaF, and 1 mM NaVO4. Cells were harvested and centrifuged at 1,000 x g for 3 min. The resultant pellet was resuspended in a homogenate buffer containing 50 mM Tris-HCl, pH 7.4, 500 mM sodium pyrophosphate, 5 mM NaF, 300 mM sucrose, 1 mM dithiothreitol, 1 mM EDTA, and protease inhibitors. Cellular suspension was sonicated and centrifuged at 100,000 x g for 60 min. The resultant pellet was resuspended in a homogenization buffer. After determination of protein concentration, SDS-PAGE and Western blotting were performed, followed by densitometric analysis. Anti-phosphorylated phospholamban (16-serine) and anti-phospholamban antibody were purchased from Badrilla (West Yorkville, UK).

Terminal Deoxynucleotidyl Transferase-mediated UTP Nick End Labeling (TUNEL) Assay—In situ labeling of fragmented DNA in cardiomyocytes was performed with the TACS 2-TdT Blue Apoptosis Detection Kit (Trevigen, Inc.) according to the manufacturer's instructions and as described previously (28). Briefly, cardiomyocytes on laminin-coated cover glass were fixed with 3.7% formaldehyde in PBS for 10 min and then incubated in 5 µg/ml proteinase K at room temperature for 20 min. The cells were incubated with 3% hydrogen peroxide for 10 min and washed with labeling buffer consisting of 50 mM Tris-HCl, pH 7.5, 5 mM CoCl2, 5 mM MnCl2, 60 µM 2-mercaptoethanesulfonic acid, and 0.05 mg/ml bovine serum albumin, followed by a 60-min incubation at 37 °C in labeling buffer containing 150 µM dATP, 150 µM dGTP, 150 µM dTTP, 5 µM biotinylated dCTP, and 40 units/ml terminal deoxynucleotidyltransferase enzyme. Nuclear blue staining was viewed under a light microscope. The percentage of TUNEL-positive myocytes (relative to total myocytes) was determined in a blinded manner by counting about 3,000–5,000 cells in randomly chosen fields/glass cover.

Analysis of DNA Fragmentation by ELISA—Histone-associated DNA fragments were quantified by the Cell Death Detection ELISA Kit (Roche Diagnostics) according to the manufacturer's instruction with minor modifications in lysis buffer (29). In brief, myocytes were collected by gentle scraping in warm PBS and pelleted by centrifugation (200 x g for 10 min). The supernatant, which may contain necrotic DNA that had leaked through the membrane, was discarded. The pelleted cells were dissociated and incubated with a cell lysis buffer containing 10 mM Tris-HCl, 1 mM EDTA, and 0.1% Triton X-100 at 4 °C for 30 min followed by centrifugation at 18,000 x g for 20 min. The resultant supernatant, which contained cytoplasmic histone-associated DNA fragments, was applied onto a streptavidin-coated microtiter plate. Subsequently, a mixture of biotin-labeled anti-histone antibody and peroxidase-conjugated anti-DNA antibody was added and incubated for 2 h. After removal of unbound antibodies by washing, the amount of nucleosomes was quantified by the peroxidase retained in the immune complex. The activity of peroxidase was determined photometrically with 2,2-azinodi-(3-ethylbenzthiazolinesulfonate) as substrate. The values from triplicate absorbance (at 405 nm) measurements were averaged and corrected by protein amount.

Caspase-3 Activity—Caspase-3 enzymatic activity was determined with the cysteine protease protein 32 assay kit (MBL, Nagoya, Japan), which detects the production of the chromophore p-nitroanilide after its cleavage from the peptide substrate DEVD-p-nitroanilide as described previously (30). In brief, myocytes were solubilized, and aliquots of lysates containing equal amounts of protein were reacted with 200 mM DEVD-p-nitroanilide at 37 °C for 2 h. The activity was measured in a microtiter plate reader at 405 nm.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
DISCUSSION
 REFERENCES
 
AC Isoform Selectivity among P-site Inhibitors—We examined the AC isoform selectivity among 10 new P-site inhibitors (6 PMC inhibitors and 4 RS inhibitors) (10, 18) as well as five classic inhibitors (3'-AMP, SQ22536, 2',5'-dd-Ado, Ara-Ade, and Ap(CH)2 pp). PMC inhibitors contain an adenine ring joined by a linker to a metal chelating hydroxamic acid (18), which forms a complex with magnesium bound in the active site of the enzyme. RS inhibitors possess a pharmacophore (=C2H-N1 = C6(NH2)-) in their structure, which is known to play an important role in inhibiting AC catalytic activity (10).

These inhibitors showed variable degrees of inhibition among type 2, type 3, and type 5 AC isoforms (Table I). In particular, among the new inhibitors, we found that PMC-6 very potently inhibited type 5 AC with a submicromolar IC50 value. The isoform selectivity of PMC-6 for type 5 AC appeared even greater than that of NKY80 (RS-2), a potent RS inhibitor (10). PMC-6 exhibited the lowest IC50 value (0.32 µM) for type 5 AC, which was lower by 50-fold than that of 14.8 µM NKY80 or 14.6 µM 3'-AMP, although the IC50 value was much higher (5.9 µM) when PMC-6 was examined with unstimulated type 5 AC activity (data not shown). The type 5 AC selectivity ratios of PMC-6 were 204 for type 2 AC and 34.7 for type 3 AC, which were higher than those of NKY80 (178 for type 2 AC and 15.3 for type 3 AC) and much higher than 3'-AMP (18 and 2.1 µM, respectively). It is interesting to note that the structural basis of PMC-6 was derived from 9-(cyclopentyl)adenine, a classic AC inhibitor. For comparison, 9-(cyclopentyl)adenine also inhibited type 5 AC in a similarly selective manner as PMC-6 (632 for type 2 AC and 79 for type 3 AC), as already reported by us (10) and others (31), although the IC50 value for type 5 AC (3.2 µM) was greater than that of PMC-6 (0.32 µM).


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  		TABLE I
Effect of various compounds on type 2, type 3, and type 5 AC Inhibitory effects of various compounds (various new and classic AC inhibitors) were evaluated using insect cell membranes overexpressing type 2, type 3, or type 5 AC. AC catalytic activity was measured in the presence of 5 mM MgCl2 with G{alpha}s·GTP{gamma}s·forskolin (50 µM). IC50 values (µM) for each AC isoform are shown (means ± S.E., n = 4). *, IC50 value was greater than 10 mM. The chemical structure of AC inhibitors are shown.

 
Some classic inhibitors showed even higher selectivity. 2',5'-dd-Ado (1.6 µM for type 5 AC, 253 µM for type 3 AC, and 2,382 µM for type 2 AC) and Ara-Ade (9.8 µM for type 5 AC, 375 µM for type 3 AC, and 7,202 µM for type 2 AC) inhibited type 5 AC much more selectively than 3'-AMP or Ap(CH)2 pp (17). SQ22536 showed a modest degree of selectivity to type 5 AC, which is in agreement with a previous report (10).

Kinetic Analysis—A Lineweaver-Burk plot analysis demonstrated that the mode of inhibition of PMC-6 was noncompetitive or mixed with respect to ATP (Fig. 1); this was unlike Ap(CH)2 pp, an adenine analog inhibitor of AC that strictly competes with the substrate ATP for the binding to the AC catalytic core site. We also examined whether PMC-6 interfered with the activity of cAMP-dependent kinase, another enzyme example that uses ATP as substrate. Activation of cAMP-dependent kinase by cAMP was unchanged in the presence or absence of PMC-6 (data not shown). These results suggest that PMC-6 is a new P-site inhibitor having demonstrated kinetic properties that define this class of inhibitors (32). Additionally, the inhibition of AC by this (PMC-6) and others (2',5'-dd-Ado and Ara-Ade) was highly selective for the type 5 AC isoform.



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  		FIG. 1.
Kinetic analysis of AC inhibitors. Double-reciprocal plots of the rate of type 5 AC catalytic activity against the substrate ATP are shown. Assays were performed in the presence of 100 nM or 1 µM PMC-6 and 100 µM Ap(CH)2 pp with 5 mM MgCl2, 50 µM forskolin, and 0.025–0.4 mM ATP. Data are the means ± S.E. of triplicate (PMC-6) or duplicate (Ap(CH)2 pp) determinations. All experiments were repeated three or more times with similar results.

 
We also examined the inhibitory effect of PMC-6 on tissue AC because tissue AC comprises multiple AC isoforms uniquely expressed in each organ. We thus examined the effect of PMC-6 on tissue AC catalytic activity using membrane preparations from mouse heart, lung, liver, and kidney (Table II). PMC-6 exhibited a greater degree of inhibition of AC activity in the heart than in the liver, kidney, or lung. At 1 µM, PMC-6 decreased AC activity by 55% from control in the heart but only by 28–38% in the other organs, defining a cardiac AC isoform composition, which was more sensitive to PMC-6 inhibition.


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  		TABLE II
Inhibitory effect of PMC-6 on AC activity among tissues Effects of PMC-6 were evaluated on membrane AC activity of liver, heart, kidney, and lung. Tissue AC catalytic activity was determined with 50 µM forskolin in the presence of PMC-6 at the indicated concentrations. The relative activities versus forskolin are shown. Data represent the means ± S.E. n = 4. *, p < 0.05, **, p < 0.01 versus control.

 
Effect on cAMP Accumulation in Intact Cells—We then examined the effect of the above inhibitors in intact cells because some P-site inhibitors may not penetrate cell membranes and thus cannot inhibit cAMP accumulation in intact cells. We compared the effect of the two most potent classic inhibitors (2',5'-dd-Ado and Ara-Ade), the two most potent new inhibitors (PMC-6 and NKY80), as well as SQ22536 and Ap(CH)2 pp, on cAMP accumulation in neonatal cardiomyocytes (Fig. 2). PMC-6 and 2',5'-dd-Ado potently and similarly decreased ISO-stimulated cAMP accumulation in a dose-dependent manner. In contrast, NKY80 and Ap(CH)2 pp did not inhibit cAMP accumulation even at 10 µM. NKY80, a potent type 5 AC inhibitor in membrane preparations (10), may not penetrate cellular membranes and was ineffective in intact cells.



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  		FIG. 2.
Inhibitory effect of various compounds on AC activity in intact cardiac myocytes. Effects of PMC-6 and other inhibitors were examined on cAMP accumulation in neonatal rat cardiac myocytes. Assays were performed with 0.5 mM IBMX in the presence or absence of inhibitors. Data are the percentage ± S.E. of 10 µM ISO, n = 4.

 
Effect of PMC-6 on Cardiac Myocyte Contraction—The above study demonstrated that PMC-6 selectively and potently inhibited type 5 AC catalytic activity as well as cAMP accumulation in intact cardiac myocytes. Accordingly, we examined the effect of PMC-6 on cardiac myocyte contraction. It should be noted that the stimulation of {beta}-AR with sympathomimetic amines, including ISO, enhances cardiac myocyte contractility through the production of cAMP and thus cAMP-dependent kinase-mediated phosphorylation of related proteins. However, the dose of ISO required for the maximal enhancement of cardiac contractility is much lower than that required for the maximal activation of cAMP production, suggesting the presence of a large cAMP reserve capacity by cardiac AC in regulating contractility (27, 33–37).

In accordance with these findings, we found that ISO at 10 nM only marginally (~10% of maximal) increased cAMP accumulation (Fig. 3), although this ISO concentration was enough to evoke both submaximal myocyte contraction (~80% of maximal contraction) (Fig. 4) and phosphorylation of phospholamban (Fig. 5a). PMC-6 did not inhibit cAMP accumulation induced by this concentration of ISO (10 nM) but did so at higher ISO concentrations (greater than 100 nM) (Fig. 3). In contrast, PMC-6, even at 10-5 M, did not inhibit ISO-induced enhancement of myocyte contractility (Fig. 4) or the phosphorylation of phospholamban (Fig. 5c). Taken together, PMC-6 did not inhibit the ISO effects at low ISO concentrations (cAMP accumulation, myocyte contraction, and phospholamban phosphorylation), suggesting that the inhibitory effect of PMC-6 was weak or that the inhibition was valid only in the presence of strong {beta}-AR stimulation and higher AC activity. This is in agreement with the mode of P-site inhibition.



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  		FIG. 3.
Dose-dependent effect of PMC-6 on cAMP accumulation in myocytes. cAMP accumulation in neonatal mouse cardiac myocytes was measured with an increasing ISO concentration in the presence or absence of PMC-6 for 10 min. Assays were performed without IBMX. Data are the means ± S.E. n = 4. *, p < 0.01 compared with control at each ISO concentration.

 



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  		FIG. 4.
Effect of PMC-6 on myocyte contractility. Adult mouse ventricular myocytes were isolated, and cell shortening was determined with an increasing ISO concentration in the presence or absence of PMC-6. Data are the means ± S.E. n = 25–29. *, p < 0.01 compared with basal at each ISO concentration. N.S., not significant.

 



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  		FIG. 5.
Effect of PMC-6 on phospholamban phosphorylation. Neonatal mouse cardiomyocytes were stimulated with an increasing ISO concentration (a and b) or 100 nM ISO in the presence or absence of 10 µM PMC-6 for 10 min (c and d) followed by immunoblotting for phospholamban (PLB) and phosphorylated phospholamban (p-PLB). Graphs show densitometric analysis of immunoblotting. Data are the percentage ± S.E. of 1 µM ISO (a and b) or 100 nM ISO (c and d).

 
Prevention of ISO-induced Cardiac Myocyte Apoptosis by P-site Inhibitors—An excessive degree of {beta}-AR stimulation may not occur in vivo under physiological conditions but may occur under pathological conditions with excessive sympathetic activity. Such strong stimulation is known to induce cardiac myocyte apoptosis, beyond regulating myocyte contractility, leading to myocardial myocyte loss and thus the deterioration of cardiac function. This phenomenon has been mimicked in various in vitro experiments using an excess amount of ISO stimulation (6, 38–40) and was examined similarly in this study. Neonatal cardiac myocytes treated with 100 µM ISO demonstrated a marked increase in the number of apoptotic myocytes (Fig. 6A). This increased apoptosis was successfully prevented by concurrent treatment with PMC-6 and 2',5'-dd-Ado, but not by Ap(CH)2 pp. PMC-6 and 2',5'-dd-Ado at 10-6 M effectively prevented myocyte apoptosis as measured by DNA fragmentation (Fig. 7) and caspase-3 activity (Fig. 8). When adult ventricular myocytes were used, ISO similarly increased the number of apoptotic myocytes as demonstrated by other investigators (39, 41, 42); PMC-6 successfully inhibited this apoptosis as well (data not shown).



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  		FIG. 6.
Prevention of ISO-induced cardiac myocyte apoptosis in TUNEL staining. A, neonatal rat cardiomyocytes were incubated with 100 µM ISO in the presence or absence of PMC-6, 2',5'-dd-Ado, or Ap(CH)2 pp at the indicated concentrations for 48 h. Data are expressed as percentage of the number of total nuclei. Results are the means ± S.E. from five to nine independent experiments. **, p < 0.01 compared with ISO. B, representative TUNEL staining photos of neonatal mouse cardiomyocytes of control myocytes (a), myocytes treated with 100 µM ISO (b), and myocytes treated with 100 µM ISO and 1 µM PMC-6 (c) are shown. The arrow indicates a representative blue-stained apoptotic nucleus.

 



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  		FIG. 7.
Prevention of ISO-induced cardiac myocyte apoptosis in ELISAs. Neonatal rat cardiomyocytes were incubated with 100 µM ISO in the presence or absence of PMC-6, 2',5'-dd-Ado, Ara-Ade, and Ap(CH)2 pp at the indicated concentrations for 48 h. DNA was extracted, and a DNA fragmentation ELISA was performed as described under "Experimental Procedures." Data are the means ± S.E. of eight independent experiments. **, p < 0.01 compared with ISO.

 



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  		FIG. 8.
Prevention of ISO-induced cardiac myocyte apoptosis in caspase-3 assays. Neonatal rat cardiomyocytes were incubated with 100 µM ISO in the presence or absence of PMC-6, 2',5'-dd-Ado, Ara-Ade, and Ap(CH)2pp at the indicated concentrations for 12 h. Cytosolic fraction was extracted, and caspase-3 activity was determined as described under "Experimental Procedures." Data are expressed as a percentage of control ± S.E. n = 6–10. *, p < 0.05, **, p < 0.01 compared with ISO.

 
ISO-induced Myocyte Apoptosis in AC5KO—The above findings indicated that the inhibition of cardiac AC activity by type 5-selective AC inhibitors prevented ISO-induced cardiac myocyte apoptosis, suggesting that this AC isoform, relative to the other isoforms, plays a greater role in inducing apoptosis in the heart.

We thus compared the effect of ISO stimulation on cardiac myocytes from mice with AC5KO versus wild type (WT) (Fig. 9). There was no difference in the cAMP accumulation at basal or at a low ISO concentration (100 nM) between AC5KO and WT. However, at higher ISO concentrations (>1 µM), cAMP accumulation was significantly lower in AC5KO than in WT. These findings are reminiscent of those in a previous experiment (Fig. 3) in which the inhibitory effect of PMC-6 did not become significant unless cells were stimulated by higher ISO concentrations. PMC-6 reduced cAMP accumulation in WT cardiomyocytes to a degree similar to that seen in AC5KO cardiomyocytes stimulated by a high ISO concentration. These results suggest the activation of type 5 AC by a high ISO concentration and its inhibition by PMC-6.



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  		FIG. 9.
ISO-induced cAMP accumulation in cardiac myocytes from AC5KO (KO). cAMP accumulation in mouse cardiac myocytes was determined with an increasing ISO concentration in the presence or absence of PMC-6 for 60 min. Our preliminary experiments demonstrated that ISO-stimulated cAMP accumulation reached the steady state at 60 min in the absence of IBMX (data not shown). Data are the means ± S.E., n = 4, *, p < 0.01 compared with WT.

 
The number of apoptotic cells increased dose-dependently with increasing ISO concentrations in WT, indicating that a much higher ISO concentration was required to induce maximal cardiac myocyte apoptosis than that for maximal myocyte contraction as shown in a previous experiment (Fig. 4). This increase in the number of apoptotic cells, however, was blunted in AC5KO (Fig. 10). Indeed, PMC-6 at 10-6 M effectively prevented myocyte apoptosis in WT to a degree similar to that observed in AC5KO in the absence of PMC-6. These results suggest that the genetic disruption and pharmacological inhibition of type 5 AC were similar in preventing myocardial apoptosis induced by {beta}-AR stimulation.



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  		FIG. 10.
ISO-induced apoptosis in cardiac myocytes from AC5KO (KO). Changes in apoptotic cells were determined by TUNEL staining. Neonatal mouse cardiomyocytes were incubated with ISO at the indicated concentrations in the presence or absence of PMC-6 for 24 h. Data are expressed as the percentage of total nuclei. Results are the means ± S.E. from four independent experiments. *, p < 0.01 compared with WT. **, p < 0.01 compared with 1 µM ISO in WT.

 

   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 EXPERIMENTAL PROCEDURES
 RESULTS
 DISCUSSION
REFERENCES
 
We have identified PMC-6 as a potent and selective inhibitor of type 5 AC with the lowest IC50 value among the inhibitors examined in this study. Some classic P-site inhibitors, such as 2',5'-dd-Ado, also inhibited type 5 AC with high selectivity. We have also demonstrated that PMC-6 did not inhibit cardiac myocyte contractility, but effectively prevented cardiac myocyte apoptosis. Indeed, type 5-selective P-site inhibitors that can inhibit cAMP accumulation in intact cells all prevented myocardial apoptosis effectively. Further, knocking out type 5 AC similarly attenuated the induction of apoptosis by ISO, mimicking the effect of PMC-6, but did not alter ISO-stimulated cAMP production. These findings strongly suggest that type 5 AC, a high catalytic activity isoform, plays a critical role in inducing cardiac myocyte apoptosis in response to excessive {beta}-AR stimulation, but only a minor role in cAMP production and myocyte contraction at rest, presumably because of the presence of multiple other AC isoforms in cardiac myocytes. These findings are reminiscent of our previous findings that the basal cardiac function of AC5KO mice was not decreased despite a decrease (30–40%) in the maximal cardiac membrane AC activity (20).

It is known that the inhibition of AC by P-site inhibitors is poor when AC is not fully stimulated, i.e. in the presence of weak {beta}-AR stimulation (10, 17). However, such weak {beta}-AR stimulation may be sufficient to evoke cardiac myocyte contraction fully (see Figs. 4 and 5). When an excessive degree of {beta}-AR stimulation is present, AC may be fully activated and thus becomes readily inhibitable by P-site inhibitors. Because type 5 AC is the dominant isoform in the heart and has very high catalytic activity (43), the inhibition of type 5 AC by PMC-6 may augment the impact of inhibiting AC signal in preventing apoptosis in cardiac myocytes. When only weak {beta}-AR stimulation is present, PMC-6 mediated inhibition also becomes weak (see under "Results"), and thus PMC-6 poorly inhibits myocyte contraction.

The magnitude of {beta}-AR stimulation required for maximal myocyte contraction is much lower than that required for maximal cAMP accumulation. This difference was originally demonstrated by Babich et al. (33) and has been confirmed by multiple investigators thereafter (27, 34–37) and in agreement with this study (Figs. 3 and 4). The original study used isolated rabbit hearts and found that the peak of cAMP elevation induced by ISO was much higher than the peak of ISO-induced contractive tension, indicating the presence of a large reserve capacity for cAMP accumulation. Perhaps, such reserve capacity may be required when the heart is failing, and thus an excessive degree of {beta}-AR stimulation is required to maintain cardiac contractility. The stimulation of {beta}-AR to such a high degree, however, may also induce cardiac myocyte apoptosis (6, 7). At this (or pathological) degree of {beta}-AR stimulation, a small increase in cAMP production appears sufficient to induce a marked increase in the degree of apoptosis (Fig. 10).

It is now known that the heart expresses multiple AC isoforms such as types 2, 3, 4, 6, 7, and 9 besides type 5 (20). These isoforms, although relatively low in maximal catalytic activity, may play an important role under physiological condition but little in pathological condition. In this regard, it was demonstrated recently that the transgenic overexpression of type 6 AC in the heart did not induce abnormal histological findings or deleterious changes (44), but that of type 5 AC did so (13). We do not know the exact molecular mechanisms underlying such differences. The affinity with G{alpha}s may differ among the AC isoforms and also change in the presence of additional activator(s) (12, 45). Subcellular localization of each AC isoform and its coupling to G proteins may play a role; it is now known that the molecules involved in {beta}-AR signaling are not distributed homogeneously within the plasma membrane, but distributed heterogeneously in membrane microdomains such as caveolae, and dynamically change their locations upon signal activation (46, 47). Such determination may have to await the development of antibody that can detect each AC isoform specifically in intact cells to identify its localization relative to other isoforms as well as G proteins and receptors.

We thereby propose that each AC isoform does not respond to {beta}-AR stimulation in an identical manner. Perhaps each isoform is activated to a variable degree depending on the intensity of {beta}-AR stimulation; this difference indeed may play a major role in the final output of {beta}-AR signaling. In this regard, our findings suggest that type 5 AC inhibitors may be useful to prevent apoptosis while preserving myocardial contractility. Such inhibitors may thus be used in the treatment of heart failure without a threat of worsening cardiac function, serving as an alternative in the future to the current {beta}-AR antagonist therapy.

It is only recently that investigators have realized that P-site inhibitors may be used to regulate AC in an isoform-specific manner (31). Because the regulation of the cAMP signal through enzyme subtypes, as exemplified by phosphodiesterase (48), has opened a new avenue for developing new pharmaco-therapy, our findings also suggest the feasibility of such efforts and the potential usage in therapy.


   FOOTNOTES
 
* The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact. Back

** To whom correspondence should be addressed: Cardiovascular Research Institute, Dept. of Cell Biology and Molecular Medicine, New Jersey Medical School, University of Medicine and Dentistry of New Jersey, 185 S. Orange Ave., Newark, NJ 07103. E-mail: ishikayo{at}umdnj.edu.

1 The abbreviations used are: {beta}-AR, {beta}-adrenergic receptor; AC, adenylyl cyclase; AC5KO, mice with targeted disruption of type 5 AC; Ap(CH)2 pp, {alpha},{beta}-methyleneadenosine 5'-triphosphate; Ara-Ade, 9-{beta}-D-arabinofuranosyl-adenine; 2',5'-dd-Ado; 2',5'-dideoxyadenosine; ELISA, enzyme-linked immunosorbent assay; GTP{gamma}S, guanosine 5'-O-(thiotriphosphate); IBMX, isobutylmethylxanthine; ISO, isoproterenol; PBS, phosphate-buffered saline; PMC, P-site inhibitor with metal chelating property; RS, ribose-substituted; SQ22536, 9-(tetrahydro-2-furanyl)-9H-purin-6-amine; TUNEL, terminal deoxynucleotidyl transferase-mediated UTP nick end labeling; WT, wild type. Back


   ACKNOWLEDGMENTS
 
We thank Drs. Hideaki Hori, Masahiko Hoshijima, and Toshitaka Yajima for editorial assistance and technical advice. We also thank Dr. Charles J. Homcy for encouragement and support throughout this project.



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 RESULTS
 DISCUSSION
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