Author + information
- Received September 27, 1999
- Revision received July 17, 2000
- Accepted September 11, 2000
- Published online January 1, 2001.
- Matthias Schäfer, PhD∗,
- Klaus Pönicke, PhD†,
- Ingrid Heinroth-Hoffmann, PhD†,
- Otto-Erich Brodde, PhD∗,
- Hans Michael Piper, MD, PhD∗ and
- Klaus-Dieter Schlüter, PhD∗,* ()
- ↵*Reprint requests and correspondence: Dr. Klaus-Dieter Schlüter, Physiologisches Institut, Aulweg 129, D-35392 Giessen, Germany
The study investigated whether β-adrenoceptor antagonists augment the hypertrophic response of cardiomyocytes evoked by norepinephrine.
In adult ventricular cardiomyocytes, stimulation of α- but not β-adrenoceptors induces myocardial hypertrophy. Natural catecholamines, like norepinephrine, stimulate simultaneously α- and β-adrenoceptors. We investigated whether β-adrenoceptor stimulation interferes with the hypertrophic response caused by α-adrenoceptor stimulation.
Adult ventricular cardiomyocytes isolated from rats were used as an experimental model. Hypertrophic parameters under investigation were stimulation of phenylalanine incorporation and protein mass, stimulation of 14C-uridine incorporation and RNA mass, and increases in cell shape.
Norepinephrine (0.01 to 10 μmol/liter) increased concentration-dependent phenylalanine incorporation; pEC50value was 5.9 ± 0.1 (n = 8). The α1-adrenoceptor antagonist prazosin (0.1 μmol/liter) suppressed norepinephrine-induced increase in rate of protein synthesis. Conversely, propranolol (1 μmol/liter) and the β1-adrenoceptor selective antagonists CPG 20712A (300 nmol/liter) or atenolol (1 μmol/liter) augmented increases in phenylalanine incorporation caused by norepinephrine. Addition of the β2-adrenoceptor antagonist ICI 118,551 (55 nmol/liter) did not influence the hypertrophic effect of norepinephrine. Atenolol augmented the norepinephrine-induced increases of all hypertrophic parameters investigated (i.e., protein mass, uridine incorporation, RNA mass, cell volume, and cross-sectional area). In the presence of norepinephrine, inhibition of β1-adrenoceptors increased the amount of protein kinase C-α and -δ isoforms translocated into the particulate fraction. The effect of pharmacological inhibition of β1-adrenoceptors could be mimicked by Rp-cAMPS (adenosine-3′, 5′-cyclic phosphorothiolate-Rp). The inhibitory effect of β1-adrenoceptor stimulation on the α-adrenoceptor-mediated effect persisted in cardiomyocytes isolated from hypertrophic hearts of rats submitted to aortic banding.
In isolated ventricular cardiomyocytes from rats, β1-adrenoceptor stimulation attenuates the hypertrophic response evoked by α1-adrenoceptor stimulation.
Increased plasma levels of catecholamines are commonly found under conditions leading to myocardial hypertrophy (1,2). Selective stimulation of α1-adrenoceptors, but not of β-adrenoceptors, increases cellular synthesis of protein and RNA in adult ventricular cardiomyocytes, which represent hallmarks of myocardial hypertrophy (3–5). Natural catecholamines stimulate both α1- and β1-adrenoceptors on ventricular cardiomyocytes. It is as yet unknown whether, in adult ventricular cardiomyocytes under simultaneous stimulation of α1- and β-adrenoceptors, the β-adrenoceptor stimulation can modulate the hypertrophic response to α1-adrenoceptor stimulation. This question was addressed in the study presented here.
A well-defined experimental model of cultured adult ventricular cardiomyocytes from rats was used (4). Ventricular cardiomyocytes from adult rats have previously been shown to possess functionally coupled β1-adrenoceptors (6); whether or not they also contain (few) β2-adrenoceptors is still a matter of debate (6,7). They also express functional α1-adrenoceptors but not α2-adrenoceptors (8).
Four series of experiments were performed. In the first series, the natural catecholamine norepinephrine, the main transmitter of the sympathetic nervous system, was used. Norepinephrine has a high affinity for α1- and β1- but not β2-adrenoceptors. It was tested if its effect on phenylalanine incorporation can be modified i) by presence of the β-adrenoceptor antagonist propranolol, ii) by the presence of the β2-adrenoceptor antagonists CPG 20712A or atenolol, or iii) by the presence of the β2-adrenoceptor antagonist ICI 118,551. In the second series of experiments, the effect of atenolol on the hypertrophic effect of norepinephrine was compared to the response of selective α1-adrenoceptor stimulation by phenylephrine. In addition to phenylalanine incorporation, effects on protein and RNA mass and cell volume were determined. In the third series of experiments, selective α1-adrenoceptor stimulation by phenylephrine was combined with stimulation of β-adrenoceptors or addition of dibutyryl-cyclo-AMP, to mimic β-adrenoceptor stimulation. In the fourth series of experiments, some of the experiments of the first series were repeated on cardiomyocytes isolated from hypertrophic myocardium of rats submitted to aortic banding.
Falcon tissue culture dishes were obtained from Becton-Dickinson (Heidelberg, Germany). Boehringer Mannheim (Mannheim, Germany) was the source for glutamine-free medium 199 and fetal calf serum (FCS). Cytsosine-β-d-arabinofuranoside, l-carnitine, creatine, taurine, l-phenylephrine hydrochloride, dl-isoproterenol hydrochloride, phorbol 12-myristate 13-acetate, procaterol, atenolol, and dibutyryl-cyclo-AMP were obtained from Sigma (Deisenhofen, Germany). l-Norepinephrine bitartrate was purchased from Serva (Heidelberg, Germany). The ICI 118,551 hydrochloride and CPG 20712A methasulfonate were obtained from RBI/Sigma (Deisenhofen, Germany). All other chemicals were of analytical grade.
Ventricular heart muscle cells were isolated from 200-g to 250-g male Wistar rats as previously described (9,10). Isolated cells were suspended in fetal calf serum-free culture medium and plated at a density of 1.4 × 105elongated cells/35-mm culture dish (Falcon type 3001; 14C-phenylalanine experiments) or 1.6 × 104elongated cells/22 mm culture dish (Falcon type 3815, 3H-phenylalanine experiments). The culture dishes had been preincubated overnight with 4% FCS in medium 199 to allow cell attachment (9). The basic culture medium consisted of medium 199 with Earle’s salts, 5 mmol/liter creatine, 2 mmol/liter l-carnitine, 5 mmol/liter taurine, 100 IU/ml penicillin, and 100 μg/ml streptomycin. To prevent growth of nonmyocytes, media were also supplemented with 10 μmol/liter cytosine-β-d-arabinofuranoside.
Four hours after plating, cultures were washed twice with culture medium to remove round and nonattached cells and supplied with FCS-free experimental media, in which cells were incubated for a 24-h period at 37°C. The experiments were carried out in basic culture medium (control), with additions of norepinephrine, phenylephrine, isoprenaline, atenolol, ICI 118,551, or dibutyrl-cyclo-AMP, at concentrations indicated. Ascorbic acid (100 μmol/liter) was added to all cultures as an antioxidant.
Incorporation of phenylalanine and uridine and changes in cellular protein and RNA mass
Incorporation of phenylalanine into cells was determined by exposing cultures to [3H]phenylalanine (0.5 μCi/ml) or l-[14C]phenylalanine (0.1 μCi/ml) for 20 to 24 h (11), and the incorporation of radioactivity into acid-insoluble cell mass was determined as described previously (4). We showed before that the incorporation of phenylalanine into cell protein is linear within the first 36 h (12). Nonradioactive phenylalanine (0.3 mmol/liter) was added to the medium to minimize variations in the specific activity of the precursor pool responsible for protein synthesis. Protein contents (13)and DNA contents (14)were determined according to previous reports. The RNA content was determined according to an earlier study (15).
Incorporation of uridine into cells was determined as described previously (12)by exposing cultures to l-[14C]uridine (0.1 μCi/ml) for 6 h and the incorporation of radioactivity into acid-insoluble cell mass was calculated. The radioactivity was determined in the aliquots used to quantify RNA mass as described above. We showed before that the incorporation of 14C-uridine into RNA is linear within the first 6 h (12).
Determination of cell volume and cross-sectional area
Myocyte growth was determined on phase-contrast micrographs recorded on tape using a CCD-video camera. Cell volumes were calculated by the following formula: Volume=(radius)2∗π∗length, assuming a cylindrical cell shape. Cross-sectional area was determined by the following formula: cross-sectional area=(radius)2∗π.
Aortic banding was performed in four week-old male Wistar-Kyoto rats exactly as recently described (16). Briefly, animals were anesthetized and the aorta was exposed through a midline abdominal incision distal to the coastal arch at the left side. The aorta was constricted with a cutton threat just proximal to the renal artery using a blunt wire (diameter 1 mm) to establish the diameter of the ligature. Subsequently, after infusing a solution of penicillin/streptomycin combination (Tardomyocel comp. III, Bayer AG, Leverkusen, Germany; 0.1 ml/kg) in the retroperitoneal cavity, the muscle layers were closed with absorbable suture and the skin with atraumatic silk suture. Controls were sham-operated, without placing the stenosis around the aorta. Both groups of animals were kept under the same condition and were used for experiments eight weeks after surgical intervention—that is, at 12 weeks of age. On the day of experiments, rats with an aortic banding and age-matched, sham-operated animals were anesthetized with pentobarbitone. The right carotid artery and the femoral artery were cannulated, and arterial pressure proximal and distal to the aortic constriction was measured with a pressure transducer (model W112, Hugo Sachs Electronic KG, Freiburg, Germany). The systolic blood pressure (carotid arterial pressure) in aortic banding rats (169 ± 13 mm Hg, n = 10) was significantly higher than in sham-operated animals (124 ± 9 mm Hg, n = 10). Differences between pre- and post-stenotic blood pressure measured in the carotid and the femoral artery were 52 ± 11 mm Hg (n = 10) in aortic banding rats and 1 ± 2 mm Hg (n = 10) in sham-operated animals.
Protein kinase C translocation
As a parameter of protein kinase C (PKC) activation its translocation into the particular fraction was investigated. Cardiomyocytes were treated for 5 min with norepinephrine with or without pretreatment with atenolol. Thereafter, the cultures were washed twice with ice-cold phosphate buffered solution (PBS), scraped off in PBS and centrifuged for 2 min at 12,000 g. The remaining pellet was redissolved in 100 μl of lysis buffer (composition in mmol/liter: Tris 20, EGTA (ethylene glycol-bis (β-amino ethyl ether) N,N,N′,N′-tetraacetic acid) 10, EDTA 2, sucrose 200, PMSF (phenylmethyl sulfononyl fluoride) 0.01, pH 7.4) and stored at −20°C. Thereafter, the solution was mixed vigorously and centrifuged again for 1 h at 38,000 g. The remaining pellet was used as the particular fraction, redissolved in 100 μl lysis buffer to which Triton X-100 was added (final concentration 0.1% [v/v]). The solution was incubated again for 2 h at 4°C. Thereafter, the solution was centrifuged again for 15 min with 12,000 gat 4°C. This supernatant was mixed with 20 μl Laemmli buffer. The samples were heated for 5 min at 95°C and used for gel electrophoresis. The samples were loaded on a 12.5% SDS-PAGE (sodium dodecyl sulfate-polyacrylamide gel electrophoresis) and after electrophoretic separation they were transferred on an immobilon-P membrane (Millipore, Bedford, Massachusetts). The bands were visualized and immunoblotted against antibodies directed against PKC-α and PKC-δ. The intensity of the final bands was analyzed densitometrically using Image-Quant software.
Data are given as means ± SEM from a different culture preparations. Statistical comparisons were performed by one-way analysis of variance (ANOVA) and use of the Student-Newman-Keuls test for post hoc analysis (17). Differences with p < 0.05 were regarded as statistically significant.
Influence of β-adrenoceptor antagonists on phenylalanine incorporation in the presence of norepinephrine
Norepinephrine (0.01 to 10 μmol/liter) caused concentration-dependent increases in phenylalanine incorporation (Fig. 1). The increase at 10 μmol/liter was about 45% above control, and the pEC50-value was 5.9 ± 0.1 (n = 8). The α1-adrenoceptor antagonist prazosin (0.1 μmol/liter) significantly suppressed norepinephrine-induced increase in protein synthesis (Fig. 1A). In contrast, the β-adrenoceptor antagonist propranolol (1 μmol/liter) significantly enhanced norepinephrine-induced increase in rate of protein synthesis (Fig. 1B). To study which β-adrenoceptor subtype might be involved in this suppression of norepinephrine-induced protein synthesis, experiments with the selective β1-adrenoceptor antagonist CPG 20712A (300 nmol/liter) and the selective β2-adrenoceptor antagonist ICI 118,551 (55 nmol/liter) were performed as shown in Figure 2. Both CPG 20712A and ICI 118,551 were used in concentrations at which they occupy less than 5% of β2-adrenoceptors or less than 10% of β1-adrenoceptors, respectively (18). In the presence of CPG 20712A, norepinephrine-induced phenylalanine incorporation was significantly enhanced, whereas ICI 118,551 did not affect it. Similar to CGP 20712A, the selective β1-adrenoceptor antagonist atenolol (1 μmol/liter) significantly augmented norepinephrine-induced increase in phenylalanine incorporation (Fig. 3).
Influence of atenolol on the hypertrophic responses caused by norepinephrine
The effects of phenylephrine (10 μmol/liter), norepinephrine (1 μmol/liter), or norepinephrine plus atenolol (1 μmol/liter) were compared in the next experimental series. Phenylephrine increased phenylalanine incorporation by 34%. Norepinephrine increased it by 22% in the absence and by 42% in the presence of atenolol (Fig. 4). The 24-h change in cellular protein mass, measured as protein/DNA ratio, was also determined (Fig. 4). Phenylephrine increased protein mass by 27%; norepinephrine alone had no significant effect. When norepinephrine was applied in the presence of atenolol, it caused an increase in protein mass (20%) comparable to that with phenylephrine.
The RNA synthesis in cardiomyocytes was estimated by determination of the 6-h incorporation of uridine into RNA (Fig. 5). In the presence of phenylephrine (10 μmol/liter), uridine incorporation was increased by 25%, and in the presence of norepinephrine (1 μmol/liter) by only 13%. When norepinephrine was administered in the presence of atenolol (1 μmol/liter), the increment in uridine incorporation became comparable to that seen in presence of phenylephrine. Similar changes were seen when the 24-h increase in total RNA mass, determined as RNA/DNA ratio, was measured (Fig. 5). Phenylephrine increased RNA mass by 13%, norepinephrine by only 7%, but norepinephrine combined with atenolol by 13%.
The influences on cell shapes of either norepinephrine or norepinephrine plus atenolol were also investigated. Compared to untreated control cells, norepinephrine (1 μmol/liter) increased cell volume by 6.4%; norepinephrine plus atenolol, however, increased it by 36.1% (Table 1). Cross-sectional area of the cells was increased by 16.0% compared to untreated control cells in the presence of norepinephrine, and by 46.0% in the presence of norepinephrine plus atenolol.
Intracellular signaling involved in the β1-/α-adrenoceptor cross-talk
The third series of experiments investigated whether β1-adrenoceptor stimulation influences the early events in α-adrenoceptor-mediated hypertrophy. As illustrated in Figure 6A, atenolol augmented the norepinephrine-induced translocation of PKC-α and -δ isoforms into the particulate fraction. The hypertrophic effects evoked by the selective α-adrenoceptor agonist phenylephrine (10 μmol/liter) were significantly reduced by β1-adrenoceptor stimulation (isoprenaline plus ICI 118,551) (Fig. 6B). In contrast, selective stimulation of β1-adrenoceptors did not antagonize the hypertrophic response evoked by direct stimulation of PKC—that is, by addition of phorbol myristate acetate (PMA, 100 nmol/liter) (Fig. 6B). Therefore, we conclude that stimulation of β1-adrenoceptors attenuates the activation of PKC caused by α-adrenoceptor stimulation.
The inhibitory effect of β-adrenoceptor stimulation on the initiation of protein synthesis was also studied in another set of experiments in which endothelin-1 (10 nmol/liter) was used to stimulate protein synthesis. These experiments were performed to investigate whether the inhibitory effect of β-adrenoceptor stimulation on protein synthesis can also be demonstrated for other agonists that stimulate protein synthesis via PKC activation. The hypertrophic effect evoked by endothelin (increase in phenylalanine incorporation to 119 ± 5%, p < 0.01 vs. control, n = 7) was blunted in co-presence of isoprenaline (93 ± 6%, p < 0.01 vs. endothelin alone, n = 7).
It was further investigated whether this inhibitory effect of β1-adrenoceptor stimulation is cAMP (cyclic adenosine monophosphate) dependent. Therefore, we performed experiments in which the β1-adrenoceptor-mediated inhibitory effect was antagonized on the postreceptor level by addition of Rp-cAMPS (adenosine-3′, 5′-cyclic phosphorothiolate-Rp), which augmented the hypertrophic response caused by norepinephrine. At 1 μmol/liter, norepinephrine did not affect protein synthesis. However, in co-presence of Rp-cAMPS phenylalanine incorporation was increased by 42% (Fig. 7). In addition, Rp-cAMPS also augmented the hypertrophic response evoked by phenylephrine in the presence of isoprenaline and ICI 118,551. In the presence of isoprenaline and ICI 118,551, phenylalanine increased protein synthesis by only 4%, but in co-presence of Rp-cAMPS this response was augmented to 38% (Fig. 7). Finally, the hypertrophic effect evoked by phenylephrine was markedly reduced by addition of dibutyrl-cyclic AMP (Fig. 7).
Influence of β-adrenoceptor antagonists on phenylalanine incorporation in the presence of norepinephrine in hypertrophied cardiomyocytes
In a last set of experiments, it was investigated whether the inhibitory effect of β1-adrenoceptor stimulation on the α-adrenoceptor-mediated growth effect is preserved in cardiomyocytes isolated from hearts with hypertrophy and β-adrenoceptor desensitization. For this purpose, cardiomyocytes from rats that had received aortic constriction for eight weeks were isolated, and growth response to norepinephrine was investigated in the absence and presence of propranolol. Hearts from rats that had undergone aortic banding showed increased left ventricular weight compared to sham-operated animals (Table 2A). In ventricular myocardium of these rats, activation of left ventricular adenylyl cyclase by isoprenaline was significantly reduced compared to sham-operated animals (Table 2A), indicating a desensitization of β-adrenoceptors. Similarly, the maximal increase in force of contraction of electrically driven left ventricular strips induced by isoprenaline was significantly lower than in strips of sham-operated animals (16). Cardiomyocytes isolated from animals with aortic banding showed a lower norepinephrine-induced increase in protein synthesis compared to those isolated from sham-operated animals. Nevertheless, presence of propranolol augmented the hypertrophic effect of norepinephrine in both animals with aortic banding and sham-operated animals (Table 2B).
Main findings of this study
The central question of this study was whether simultaneous stimulation of β-adrenoceptors can modulate the hypertrophic response to α1-adrenoceptor stimulation on adult ventricular cardiomyocytes. The results of the present study show that β1-adrenoceptor stimulation attenuates all investigated hypertrophy-related effects of α1-adrenoceptor stimulation.
Phenylalanine incorporation was determined for a one-day period. The changes described here in phenylalanine incorporation were paralleled by similar changes in cellular protein mass, indicating that they primarily represent changes in protein synthesis. Analogous considerations apply to changes in uridine incorporations that are accompanied by changes in RNA mass. In addition, cell volume and cross-sectional areas of the cells also increased, indicating hypertrophic growth of the cardiomyocytes.
Intracellular signals involved in the β-adrenoceptor-mediated inhibitory effect
In the present study, in adult rat ventricular cardiomyocytes, β1-adrenoceptor stimulation attenuated the hypertrophic response to α1-adrenoceptor stimulation. This conclusion is based on the following findings: a) The hypertrophic response to norepinephrine is enhanced by β1-adrenoceptor blockade by CPG 20712A or atenolol, but not affected by the highly selective β2-adrenoceptor antagonist ICI 118,551; b) the hypertrophic response to the α1-adrenoceptor agonist phenylephrine is significantly inhibited by isoprenaline in the presence of ICI 118,551 acting under these conditions solely at β1-adrenoceptors. This inhibitory effect of β1-adrenoceptor stimulation involves the cyclic-AMP-dependent protein kinase (PKA) system: thus, dibutyryl-cyclic-AMP can mimic the inhibitory effects of β1-adrenoceptor stimulation on phenylephrine-induced increase in rate of protein synthesis; moreover, the PKA-inhibitor Rp-cAMPS significantly enhanced norepinephrine-evoked increase in protein synthesis and abolished β1-adrenoceptor-mediated inhibition of phenylephrine-induced increases in rate of protein synthesis. Thus, taken together, in adult rat ventricular cardiomyocytes the hypertrophic response to norepinephrine is composed of two components: an α1-adrenoceptor-mediated stimulation and a β1-adrenoceptor-mediated inhibition. The β1-adrenoceptor-induced inhibition of protein synthesis is mediated by the cAMP/PKA activation pathway.
Additional experiments revealed that norepinephrine-evoked translocation of PKC (i.e., activation of PKC) was markedly enhanced by atenolol; thus, in rat cardiomyocytes β1-adrenoceptor stimulation attenuated activation of PKC, which is known to be an essential step in α1-adrenoceptor-mediated hypertrophic response (19). Conversely, when PKC was activated receptor-independently by the phorbolester PMA, β1-adrenoceptor stimulation failed to affect PKC translocation. This indicates that the interaction between α1- and β1-adrenoceptor signaling pathways occur at or above the level of PKC activation.
Comparison with other in vitro systems
Our results are in apparent contrast to studies on adult ventricular cardiomyocytes isolated from rabbits (5)or neonatal cardiomyocytes from rats (20)in which inhibition of β-adrenoceptors was found to reduce the increase in protein synthesis caused by norepinephrine. In those types of studies, however, cardiomyocytes were cultured under specific conditions in which the sole stimulation of β-adrenoceptors provokes a cellular hypertrophic effect of its own. Such a direct growth-promoting effect of β-adrenoceptor stimulation is not normally present in adult ventricular cardiomyocytes from rat that were used in the present study (4). It can appear when cells are cultured for prolonged time under specific culture conditions (12,21)—for example, in the presence of FCS. Our results demonstrate, however, that under basal conditions (e.g., without precultivation of cardiomyocytes with various agonists) the hypertrophic response caused by norepinephrine is reduced by simultaneous stimulation of β1-adrenoceptors.
In vivo relevance of the observed β-adrenoceptor-mediated effect
The pathophysiological relevance of this cross-talk between α1- and β1-adrenergic receptor stimulation in the hypertrophic response to norepinephrine in adult cardiomyocytes is at present only partly understood. There is no doubt that α1-adrenoceptor stimulation causes increases in the rate of protein synthesis. The β1-adrenoceptor stimulation can inhibit the hypertrophic response induced by norepinephrine (this study). Our experiments on rats with myocardial hypertrophy but not heart failure indicate that the β1-adrenergic attenuation of the α1-adrenergic growth effect is still active in compensated hypertrophy. In such a situation one should therefore expect an increase of hypertrophic growth upon use of β1-adrenoceptor blockers. It has indeed been reported that patients with moderate hypertension, but without cardiac failure, develop more cardiac hypertrophy when receiving atenolol, independent from its blood-pressure-lowering effect (22). The described mechanism may also explain why, in general, in experimental models associated with high blood pressure, atenolol was found less effective to initiate regression of hypertrophy as opposed to, e.g., angiotensin-converting enzyme (ACE) inhibitors (23).
Finally, β1-adrenoceptor stimulation can also induce cardiac myocyte apoptosis, and, by this, contribute to myocardial failure (24). In severe heart failure, β1-adrenoceptor-blocker might prevent cardiac myocyte apoptosis, and this might contribute to the beneficial effects in patients with chronic heart failure (reviewed in Bristow, 25). Moreover, under these conditions β-blockers might resensitize the desensitized β1-adrenoceptors in chronic heart failure (reviewed in Brodde et al., 26), thus improving the inhibitory effects of β1-adrenoceptor stimulation on the α1-adrenoceptor-mediated hypertrophic response. Hence, the influence of β1-adrenoceptor blockers on the extent of myocardial hypertrophy and heart failure might depend on the clinical situation.
☆ This study was supported by the Deutsche Forschungsgemeinschaft (DFG), grants SCHL 324/3-1 (to K.-D.S.), Pi 192/11-2 (to H.M.P.), and Br 526/6-1 (to O.-E.B.).
- angiotensin-converting enzyme
- ethylenediaminete acid
- ethylene glycol-bis(β-aminoethyl ether) N,N,N′,N′-tetraacetic acid
- fetal calf serum
- cyclic-AMP-dependent protein kinase
- protein kinase C
- phorbol myristate acetate
- phenylmethylsulfonyl fluoride
- adenosine-3′, 5′-cyclic phosphorothiolate-Rp
- sodium dodecyl sulfate–polyacrylamide gel electrophoresis
- Received September 27, 1999.
- Revision received July 17, 2000.
- Accepted September 11, 2000.
- American College of Cardiology
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