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Am J Physiol Heart Circ Physiol 280: H334-H343, 2001;
0363-6135/01 $5.00
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Vol. 280, Issue 1, H334-H343, January 2001

A partial agonist of the A1-adenosine receptor selectively slows AV conduction in guinea pig hearts

Lin Wu1, Luiz Belardinelli2, Jeffrey A. Zablocki2, Venkata Palle2, and John C. Shryock1

1 Department of Medicine, University of Florida, Gainesville, Florida 32610; and 2 CV Therapeutics, Palo Alto, California 94304


    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The use of full agonists of the A1-adenosine receptor (A1-ADOR) as antiarrhythmic agents is limited by their actions to cause high-grade atrioventricular (AV) block, profound bradycardia, atrial fibrillation, and vasodilation. It may be possible to avoid these undesired actions by use of partial agonists. We determined the effects of CVT-2759, a potential partial agonist of A1-ADORs, on guinea pig hearts. CVT-2759 (0.1-100 µM) increased the S-H interval of the isolated heart from 45 ± 1 to 60 ± 3 ms (P < 0.01) with a half-maximal effect at 3.1 µM. CVT-2759 did not cause second-degree AV block. CVT-2759 significantly attenuated the actions of the full agonists N6-cyclopentyladenosine and adenosine. CVT-2759 caused a moderate slowing of atrial rate by <= 13% and did not shorten the durations of either the atrial or the ventricular monophasic action potential. Coronary conductance was increased by CVT-2759 only at concentrations >10 µM. In contrast, CVT-2759 was a full agonist to decrease cAMP content of rat adipocytes and Fischer rat thyroid line 5 cells. Results of radioligand binding assays indicated that CVT-2759 stabilized a high-affinity, G protein-coupled state of the A1-ADOR in membranes prepared from rat adipocytes but not in membranes prepared from the guinea pig brain. The results suggest that a weak A1-ADOR agonist, such as CVT-2759, may be useful to slow AV nodal conduction and thereby ventricular rate without causing AV block, bradycardia, atrial arrhythmias, or vasodilation.

CVT-2759; FRTL-5; adipocyte; cyclopentyladenosine; iodotubercidin; atrioventricular


    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

A1-adenosine receptors (A1-ADOR) are G protein-coupled cell surface proteins, and their activation by adenosine leads to a number of important physiological responses (7, 11). In the heart, the negative chronotropic (slowing of heart rate) and dromotropic [slowing of atrioventricular (AV) nodal conduction] effects of adenosine are mediated by the A1-ADOR (3, 16). The intravenous administration of a bolus of adenosine causes a brief and reversible negative dromotropic response. Clinical applications of the negative dromotropic action of adenosine include the termination of supraventricular tachycardias and the differential diagnosis of tachyarrhythmias (2, 8-10, 29). Side effects of adenosine administration are common (29) and include severe bradycardia, high-grade AV block, atrial fibrillation (due to shortening of the atrial refractory period), cardiac arrest, and low blood pressure. These untoward effects are not a serious limitation to the clinical use of adenosine, which is administered primarily in a hospital setting and has only a transient action because of its short half-life in blood (18). However, undesired and life-threatening cardiovascular side effects are an impediment to the potential use of stable long-acting analogs of adenosine. With the use of a partial agonist of the A1-ADOR, it may be possible to moderately slow AV conduction time and control ventricular rate during episodes of atrial tachycardias without causing serious adverse cardiovascular effects. For this reason, we have investigated the actions of a novel putative partial agonist, CVT-2759, on guinea pig isolated hearts.

For any given receptor-mediated action of agonist on tissue, there is a maximum response that occurs when all receptors are occupied. The maximal effect, or efficacy, of an agonist depends on its effectiveness in binding to and activating a receptor, the efficiency of signaling from receptor to functional response, and the number of receptors. When all receptors are occupied, agonists causing a full effect are called full agonists, whereas those causing a partial effect are called partial agonists. A drug that is a partial agonist for one action may be a full agonist for another action in the same tissue or in a different tissue wherein the density of receptors and/or efficiency of signaling is greater. For example, we have previously observed (24) that the A1-ADOR partial agonist SHA-040 caused a 60% inhibition of isoproterenol-stimulated calcium current but only a 20% activation of potassium current in guinea pig atrial myocytes. These results were consistent with the finding (24) that the receptor reserve for the action of adenosine to inhibit isoproterenol-stimulated calcium current was greater than the receptor reserve for adenosine to increase potassium conductance in atrial myocytes. In addition, because the density of A1-ADORs in the heart is lower than that in some other tissues (e.g., the brain and adipose tissue), a partial agonist for an action on cardiac myocytes may be a full agonist of actions in these other tissues. Therefore, we have characterized the actions of the putative partial agonist CVT-2759 not only on cardiac tissues but also on three tissues or cells that are reported to have a relatively high density of A1-ADORs, namely the forebrain, adipocytes, and Fischer rat thyroid line 5 (FRTL-5) cells.


    METHODS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Chemicals

The A1-ADOR antagonists 8-cyclopentyl-1,3-dipropylxanthine (CPX) and 8-cyclopentyl-1,3-dimethylxanthine (CPT), the A1-ADOR agonists N6-cyclopentyladenosine (CPA), 2-chloro-N6-cyclopentyladenosine (CCPA), and N6-cyclohexyladenosine (CHA), the adenosine deaminase inhibitor erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), the adenosine kinase inhibitor iodotubercidin, and forskolin were purchased from Research Biochemicals (Natick, MA). CVT-2759, {[(5-{6-[(3R)oxolan-3-yl]amino}purin-9-yl)(3S,2R,4R)-3,4-di-hydroxyoxolan-2-yl]-methoxy}-N-methylcarboxamide, molecular weight 394.38, is a derivative of the selective A1-ADOR full agonist CVT-510 (see Ref. 23) and was synthesized at CV Therapeutics by coauthors J. A. Zablocki and V. Palle. Adenosine was purchased from Sigma Chemical (St. Louis, MO). The radioligand 8-cyclopentyl-1,3-dipropyl-[2,3-3H(N)]xanthine ([3H]CPX) was purchased from New England Nuclear (Boston, MA). Concentrated stock solutions (10-100 mM) of CVT-2759, CPX, CPT, CPA, CCPA, CHA, and forskolin were dissolved in dimethyl sulfoxide, stored as aliquots at -80°C, and diluted in physiological saline for use in experiments. The final content of dimethylsulfoxide in saline during experiments was not more than 0.1%. Adenosine and EHNA were dissolved in saline immediately before use.

Guinea Pig Isolated Perfused Hearts

Guinea pigs (Hartley) of either sex weighing 300-350 g were anesthetized with methoxyflurane and killed by decapitation. The chest was cut open, and the heart was quickly removed and rinsed in ice-cold modified Krebs-Henseleit (K-H) solution. The contents of the modified K-H solution were (in mM) 117.9 NaCl, 4.8 KCl, 2.5 CaCl2, 1.18 MgSO4, 1.2 KH2PO4, 0.5 Na2 EDTA, 0.14 ascorbic acid, 5.5 dextrose, 2.0 pyruvic acid (sodium salt), and 25 NaHCO3. The K-H solution was continuously gassed with 95% O2-5% CO2, and the pH was adjusted to a value of 7.4. To perfuse the heart by the Langendorff method, the transected aorta was slid onto a glass cannula and secured by a ligature. Retrograde perfusion of the aorta was initiated immediately at a constant flow of 10 ml/min with modified K-H solution warmed to 36.0 ± 0.5°C. A side port in the cannula was used to connect the perfusion line to a Gould pressure transducer for measurement of coronary perfusion pressure. Coronary perfusion pressure was continuously recorded on a strip chart (Gould RS3400, Cleveland, OH) throughout each experiment. Coronary conductance (in ml · min-1 · mmHg-1) was calculated as the ratio of coronary flow (10 ml/min) to perfusion pressure (in mmHg). To facilitate the exit of fluid from the left ventricle, the leaflets of the mitral valve were trimmed with fine spring-handled scissors. When appropriate, hearts were paced at a constant rate using external electrodes. After completion of dissection and instrumentation (see Measurement of Cardiac Electrical Activity) for measurement of either atrial rate, stimulus-to-His bundle (S-H) interval, monophasic action potentials, or coronary perfusion pressure, each heart was allowed to equilibrate for 20-40 min before the administration of drug. Experimental interventions were always preceded and followed by control measurements. Criteria for the exclusion of hearts from the study were 1) a coronary perfusion pressure of <50 mmHg, 2) absence of a stable coronary perfusion pressure during the equilibration period, and 3) inability to pace a heart at a constant rate throughout an experiment.

For electrical pacing of hearts, a bipolar Teflon-coated electrode was placed in the wall of the intra-atrial septum. Parts of the left and right atrial tissues, including the region of the sinoatrial node, were removed, both to decrease the spontaneous heart rate and to expose the atrial septum for electrode placement. Hearts were electrically paced at a fixed cycle length of 280-300 ms. Stimuli were provided by an interval generator (model 1830, WPI, Sarasota, FL) and delivered through a stimulus isolation unit (model 1880, WPI) as square wave pulses of 3 ms in duration and at least twice the threshold intensity.

Measurement of Cardiac Electrical Activity

Atrial rate. To measure spontaneous atrial rate in hearts that were not paced, a unipolar electrode was placed on the right atrium. A total of 10 atrial beats before, during, and after each intervention were recorded on chart paper moving at a rate of 50 mm/s, and the average atrial rate at each time was determined from these records.

S-H interval. Prolongation of the S-H interval was used as a measure of the negative dromotropic effect of A1-ADOR agonists on AV nodal conduction. Hearts were paced at a constant cycle length of 280-300 ms as described above. The His bundle electrogram was recorded from a unipolar electrode placed in the right side of the interatrial septum adjacent to the AV junction. The signal was displayed continuously in real time on an oscilloscope screen at a sweep rate of 10 ms/cm. The duration of time from the first pacing artifact to the maximum upward deflection of the His bundle signal was used as the S-H interval.

Monophasic action potentials. Monophasic action potential durations in atrial and ventricular regions of the heart were recorded using two pressure-contact Ag-AgCl electrodes (Langendorff Probe, EP Technologies, Sunnyvale, CA) placed on the surface of the left atrium and the inferior wall of the left ventricle, respectively. The signals from the His bundle, left atrium, and left ventricle were amplified and filtered by an isolated biological digital amplifier (ISODam, WPI) and displayed in real time on a computer screen. Signals were considered adequate if they were stable for at least 5-10 min and the amplitudes exceeded 10 mV. The data were digitized at 2 kHz by a DT-2801A digitizing board (Data Translation, Marlboro, MA) and were saved for analysis using the Snapshot data acquisition program (Snapshot Storage Scope, HEM Data, Southfield, MI). The duration of the atrial and ventricular monophasic action potentials were measured from onset of depolarization to either 50 or 90% of repolarization.

FRTL-5 Cells

FRTL-5 cells were obtained from the American Type Culture Collection (Rockville, MD) and cultured as described by Ambesi-Impiombato et al. (1) in Ham's F-12 medium (GIBCO-BRL, Grand Island, NY) with 14 mM sodium bicarbonate, 5% calf serum (GIBCO-BRL), and a six hormone mixture (1). The medium was changed twice weekly, and cells were subcultured once weekly. For experiments, cells were grown in 12-well culture plates until confluent. To initiate an experiment, the culture medium was removed, and warm (37°C) Hank's balanced saline solution was added and incubated with cells for 6 min. This solution was removed, and fresh solution containing 2 µM forskolin, 2 U/ml adenosine deaminase, and appropriate drugs (e.g., CVT-2759 and CPA) was added. Cells and medium were incubated at 37°C for an additional 6 min. The incubation medium was then removed, and 50 mM HCl was added to terminate the incubation and lyse the cells. cAMP content of the cell lysate was determined by radioimmunoassay as previously described (22).

Rat Epididymal Adipocytes

Adipocytes were isolated by collagenase digestion of epididymal fat pads removed from fed male Sprague-Dawley rats (350-400 g). Briefly, rats were anesthetized with methoxyflurane and decapitated. The epididymal fat pads were removed from the rat, and large blood vessels and adherent connective tissue were excised and discarded. The fat pads were placed in Krebs-Ringer-HEPES (KRH) buffer (pH 7.4) and cut with scissors into small pieces. The contents of KRH buffer were (in mM) 100 NaCl, 4.7 KCl, 1.19 MgSO4, 2.5 CaCl2, 1.18 KH2PO4, 5 dextrose, 5 sodium pyruvate, 3.6 NaHCO3, and 5 HEPES. The pieces of fat pad were rinsed once with fresh buffer and then placed into a 50-ml polypropylene tube with 25 ml of KRH buffer containing 1% (wt/vol) fatty acid-free BSA, 2 µM nicotinic acid (to inhibit lipolysis), and collagenase type 1 (1 mg/ml; Worthington Biochemical, Lakewood, NJ). Cells and buffer were incubated for 45-60 min at 37°C in a shaking water bath until the tissue was adequately digested into individual cells. The resultant suspension was poured through a nylon filter (pore size 210 µm) and then washed three times with fresh KRH buffer containing 1% BSA, allowing the cells to rise to the top of the suspension each time before removing the infranatent buffer (and contaminating cells). The final adipocyte suspension was diluted to achieve a cell density of ~20-30,000 cells/ml in KRH buffer containing 1% fatty acid-free albumin. The suspension was immediately pipetted in 100-µl aliquots to wells in a 24-well plastic culture plate placed in a shaker water bath maintained at 37°C. An additional 900 µl of KRH buffer containing 4% BSA, 30 nM isoproterenol, 1 mM ascorbic acid, 2 U/ml adenosine deaminase, and CVT-2759 (0.1 nM-10 µM) was added to each aliquot of cells. Cells and medium were incubated for 4 min at 37°C. Incubation was terminated by addition of 200 µl of 300 mM HCl to lyse the cells. cAMP content of cell lysates was determined as previously described (22).

Radioligand Binding

Membranes containing A1-ADORs for use in radioligand binding assays were prepared from guinea pig cerebral cortex or rat isolated epididymal adipocytes. Freshly isolated guinea pig brain cortical tissue was homogenized in ice-cold 50 mM Tris · HCl buffer (pH 7.4) using six up and down strokes of an ice-chilled Potter-Elvejhem tissue grinder and a motor-driven Teflon pestle. A crude membrane preparation was isolated by centrifugation of the homogenate at 15,000 g for 20 min at 4°C. The membrane pellet was resuspended in fresh Tris buffer and pelleted again by centrifugation. The final membrane pellet was suspended in Tris buffer to achieve a protein content of 1.1-1.4 mg/ml and divided into aliquots for storage at -80°C until needed for assays. Adipocytes were isolated from 10 epididymal fat pads and suspended in a homogenization buffer (pH 7.3) containing 250 mM sucrose, 20 mM HEPES, 1 mM EDTA, 10 µg/ml leupeptin, and 5 µg/ml pepstatin. Cells were homogenized using an ice-chilled Potter-Elvehjem tissue grinder and a motor-driven Teflon pestle. Cell membranes were separated from fat by centrifugation of the homogenate for 5 min at 1,000 g at 4°C. The infranate below the fat cake was removed and placed in a centrifuge tube. Membranes were collected by centrifugation of the infranate for 30 min at 20,000 g at 4°C. Membranes were resuspended in buffer (pH 7.1 at room temperature) containing 154 mM NaCl, 10 mM MgCl2, 50 mM HEPES, 1 mM EDTA, 5 µg/ml leupeptin, 5 µg/ml pepstatin, and 5 U/ml adenosine deaminase and stored as aliquots in liquid nitrogen until needed for assays.

To determine the affinity of CVT-2759 and CPA for A1-ADORs, membranes (10 µg protein), [3H]CPX (0.5-0.8 nM), adenosine deaminase (2 U/ml), and either CVT-2759 (10-11-10-4 M) or CPA (10-11-10-5 M) were incubated in a total volume of 300 µl of 50 mM Tris buffer (pH 7.4) for 3 h at room temperature. In some assays, 1 mM GTP and N-ethylmaleimide were added to the incubation medium to reduce high-affinity agonist binding to G protein-coupled receptors. Incubations were terminated by dilution of samples with ice-cold Tris buffer and immediate collection of membranes onto glass-fiber filters (Schleicher and Schuell, Keene, NH) by vacuum filtration using a cell harvester (Brandel, Gaithersburg, MD). Filters were washed quickly three times with ice-cold buffer to remove unbound radioligand. Filter disks containing trapped membranes and bound radioligand were placed in 4 ml of scintillation cocktail, and radioactivity of samples was quantified using a liquid scintillation counter. Assays to quantitate the affinities of CPA and CVT-2759 for A1-ADORs were conducted in parallel. Six and three determinations were done at each concentration of CVT-2759 and CPA, respectively. Specific binding of [3H]CPX was calculated as the difference between total radioligand bound and radioligand bound in the presence of the highest tested concentration of displacing ligand. Results of radioligand binding assays were analyzed using the Prism program from GraphPad (San Diego, CA). Values of the dissociation constant of a competitive inhibitor (Ki) for CVT-2759 and CPA were calculated from values of the IC50 for each ligand to displace the binding of [3H]CPX with the use of the Cheng-Prusoff equation. Values of the equilibrium dissociation constant for [3H]CPX binding to membranes prepared from the guinea pig brain and rat adipocytes were 1.4 and 0.4 nM, respectively, as determined by saturation binding assays.

HPLC of Adenosine

Samples (5 ml each) of the coronary effluent of isolated guinea pig hearts were collected during the last minute of a control or drug treatment period and stored at -80°C until analysis of adenosine content by HPLC. The HPLC system included a Spectra-Physics 8800 pump, 8490 ultraviolet detector, 4290 integrator, a Rainin injection valve, and a Luna 5-µm particle size C-18(2) column with a diameter of 4.6 mm and a length of 250 mm (Phenomenex, Torrance, CA). Samples and standards (10 pmol adenosine) of 100-µl volume were manually injected onto the column and eluted with a solution of 50 mM KH2PO4/K2HPO4 buffer, pH 6.5, with 15% methanol at a rate of 1.5 ml/min at room temperature. Adenosine content of samples was determined by quantification of peak height and comparison to the peak height recorded for a 10-pmol standard. A standard was assayed after analysis of every fifth sample. All samples were filtered (0.22 µm) before injection onto the HPLC column.

Data Analysis

All values are reported as means ± SE. Concentration-response and radioligand binding data were analyzed using GraphPad Prism version 2.01. When appropriate, the significance of differences among three or more individual mean values was determined by one-way ANOVA followed by Student-Newman-Keuls test. A P value <0.05 was considered to indicate a statistically significant difference.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

Actions of CVT-2759 and CPA on Guinea Pig Isolated Hearts

CVT-2759 slowed AV nodal conduction without causing second-degree AV block and caused minimal slowing of the atrial rate of the guinea pig isolated heart (Fig. 1). In contrast, the full agonist CPA caused markedly greater slowing of both AV nodal conduction and atrial rate than did CVT-2759 (Fig. 1). CVT-2759 increased the S-H interval of the electrically paced heart in a concentration-dependent manner by a maximum of 33%, from 45 ± 1 to 60 ± 3 ms (P < 0.01), with an EC50 value of 3.1 µM (95% confidence intervals, 2.1 and 4.7 µM) (Fig. 1A). CVT-2759 did not cause second-degree A-V block in any heart. The effect of CVT-2759 on the S-H interval appeared to reach a maximum at 30-100 µM. Low concentrations of CPA (<= 20 nM) caused stable, reversible increases of the S-H interval (Fig. 1A). Comparable submaximal responses (an increase of the S-H interval by 13 ms) occurred at CPA and CVT-2759 concentrations of 10 and 10,000 nM, respectively (Fig. 1A). Higher concentrations of CPA (>= 20 nM) caused the S-H interval to prolong rapidly, culminating in AV block. CPA (30 nM) increased the S-H interval from 44 ± 1 to 87 ± 2 ms (P < 0.01) and caused second-degree AV block in all hearts. CVT-2759 and CPA had markedly different effects on the atrial rate of isolated spontaneously beating hearts (Fig. 1B). CVT-2759 decreased the atrial rate by only 13%, from 195 ± 6 to 169 ± 7 beats/min (P < 0.05), whereas CPA decreased the atrial rate from 198 ± 15 to 1 ± 1 beats/min (Fig. 1B) with an EC50 value of 68 nM (95% confidence intervals, 57 and 80 nM).


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  		Fig. 1.   Concentration-response relationships for the negative dromotropic (increase of S-H interval; A) and negative chronotropic (decrease of atrial rate; B) actions of CVT-2759 and N6-cyclopentyladenosine (CPA). Experimental measurements of atrial rate and S-H interval were done on different hearts (see METHODS for protocols). A: each point represents the mean and SE of single determinations in each of 4-12 hearts for CVT-2759 and 4 hearts for CPA. B: each point represents the mean and SE of single determinations in each of 5 hearts for CVT-2759 and 4 hearts for CPA. *Second-degree atrioventricular (AV) block occurred in all hearts in the presence of 30 nM CPA

The A1-ADOR antagonist CPX (50 nM) significantly reversed the S-H interval prolongation induced by 10 µM CVT-2759 (data not shown). CVT-2759 (10 µM) caused an increase of 12 ms of the S-H interval from 46 ± 2 to 58 ± 2 ms (n = 7, P < 0.05). However, CVT-2759 (10 µM) only caused an increase of 4 ms of the S-H interval in the presence of 50 nM CPX [i.e., the S-H interval was reduced from 58 ± 2 to 50 ± 2 ms (n = 7, P < 0.05) by CPX in the continued presence of CVT-2759].

CVT-2759 also increased the coronary conductance of the guinea pig isolated heart. Analysis of concentration (9 concentrations from 0.01 to 100 µM)-response data from four experiments indicated that CVT-2759 at concentrations <= 10 µM did not significantly change the coronary conductance (data not shown). CVT-2759 increased coronary conductance in a concentration-dependent manner at concentrations between 10 and 100 µM. Values of coronary conductance increased from a control of 0.17 ± 0.01 to 0.185 ± 0.01 and 0.26 ± 0.02 ml · min-1 · mmHg-1 (n = 4, P < 0.05 vs. control) in the presence of 10 and 100 µM CVT-2759, respectively. From these data, a half-maximal response to CVT-2759 was calculated to occur at a concentration of 31 µM. However, because a maximal response to CVT-2759 may not have been achieved in these experiments due to limited solubility of CVT-2759, this estimate of the EC50 value for CVT-2759 to cause an increase of coronary conductance may be falsely low. Nonetheless, these data suggest that CVT-2759 is >= 10 times more potent as a negative dromotropic agent (i.e., to increase the S-H interval) than as a coronary vasodilator. The relatively selective A1-ADOR agonist CPA also increased coronary conductance of the guinea pig isolated heart. Both the potency and maximal effect of CPA were greater than those of CVT-2759. CPA increased coronary conductance in a concentration-dependent manner from 0.16 ± 0.01 to 0.33 ± 0.01 ml · min-1 · mmHg-1 (n = 4, P < 0.001). A half-maximal increase of coronary conductance occurred at 71 nM CPA (95% confidence limits, 59-86 nM). Thus CPA was at least 400 times more potent than CVT-2759 as a coronary vasodilator. The maximal increases of coronary conductance of 0.15 ± 0.02 and 0.17 ± 0.02 ml · min-1 · mmHg-1 caused by adenosine in a previous study (12) and CPA in this study were significantly greater than the 0.09 ± 0.01 ml · min-1 · mmHg-1 increase of coronary conductance caused by 100 µM CVT-2759 in the present study. Thus either CVT-2759 is not a full agonist to increase coronary conductance or a maximal response to CVT-2759 occurs at a higher concentration than was used here (100 µM).

CVT-2759 had no significant effect on the duration of either the ventricular or the atrial monophasic action potential in the guinea pig isolated heart. The durations of the ventricular monophasic action potential at 90% of repolarization in the absence and presence of 20 µM CVT-2759 were 158 ± 4 and 160 ± 7 ms, respectively (n = 4, P > 0.05). The durations of the atrial monophasic action potential at 50 and 90% of repolarization were 41 ± 3 and 66 ± 4 ms in the absence of CVT-2759 and 41 ± 3 and 74 ± 4 ms in the presence of 20 µM CVT-2759 (n = 4, P > 0.05). Because it is known that adenosine shortens the duration of the atrial action potential (3), a bolus (100 µl) of 1 mM adenosine was administered at the end of an experiment to confirm the responsiveness of each preparation. Adenosine shortened the duration of the atrial monophasic action potential at 90% of repolarization to 22 ± 5 ms (n = 4, P < 0.05 vs. control and CVT-2759).

Antagonistic Effect of CVT-2759 on Cardiac Responses Mediated by CPA and Adenosine

It is expected that a partial agonist will competitively antagonize the response to a full agonist (14). To confirm the partial agonist nature of CVT-2759, we measured responses of the guinea pig isolated heart to the full agonists CPA and adenosine in the absence and presence of 10 µM CVT-2759. As shown in Fig. 2, CPA significantly prolonged the S-H interval at concentrations of 10, 15, and 20 nM from a control value of 42 ± 1 ms to 59 ± 4, 81 ± 6, and 103 ± 8 ms, respectively. CVT-2759 (10 µM) partially reversed the S-H interval prolongation induced by CPA. The antagonism by 10 µM CVT-2759 of the S-H interval prolongation caused by CPA was greater in the presence of 20 nM CPA than in the presence of 15 or 10 nM CPA (Fig. 2).


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  		Fig. 2.   Attenuation by the partial agonist CVT-2759 (10 µM) of the increase of S-H interval caused by the full agonist CPA (10, 15, and 20 nM). In each experiment, a control S-H interval (0 nM CPA) and the response to a single concentration of CPA (in the absence and presence of 10 µM CVT-2759) were measured. Bars indicate the mean and SE of single determinations in each of 4 (presence of CPA) or 12 hearts (absence of CPA). *P < 0.05, **P < 0.01 compared with control by ANOVA and Dunnett's test. +P < 0.05, ++P < 0.01 compared with CPA alone by paired t-test.

Consistent with its antagonism of the effect of CPA, CVT-2759 shifted the concentration-response relationship for adenosine to prolong S-H interval significantly to the right (Fig. 3, n = 4, P < 0.01 by 2-way ANOVA). In the absence of CVT-2759, adenosine prolonged the S-H interval in concentration-dependent manner and caused second-degree AV block at a concentration of 6 µM. In the presence of 4 and 10 µM CVT-2759, adenosine caused second-degree AV block at concentrations of 8 and 20 µM (Fig. 3). Thus the value of pA2 for CVT-2759 (i.e., the concentration of CVT-2759 that would cause a twofold shift of the adenosine concentration-response relationship) appears to be >4 but <10 µM.


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  		Fig. 3.   Attenuation by CVT-2759 (4 and 10 µM) of the action of adenosine (ADO) to increase the S-H interval of the guinea pig isolated heart. In the presence of CVT-2759, the concentration-response relationship for adenosine to increase the S-H interval was shifted to the right. Points indicate the mean and SE of single determinations in each of 4-5 hearts. *AV block occurred at concentrations higher than those shown here.

If CVT-2759 antagonizes the actions of exogenous CPA and adenosine, then it should also antagonize the action of endogenous adenosine in the heart. Treatment of hearts with inhibitors of adenosine kinase and adenosine deaminase has been demonstrated to cause an increase of the interstitial adenosine concentration (25). Therefore, the adenosine kinase inhibitor iodotubercidin (1 µM) and the adenosine deaminase inhibitor EHNA (200 nM) were used in this study to cause an elevation of endogenous adenosine. Simultaneous administration of the two agents into the coronary perfusate of isolated hearts increased the adenosine concentration in the coronary effluent from 11 ± 2 to 65 ± 5 nM (n = 6, P < 0.01). Concomitantly, the S-H interval increased by 34 ± 6 ms from 46 ± 1 to 80 ± 6 ms (Fig. 4). In the presence of iodotubercidin and EHNA, 10 µM CVT-2759 decreased the S-H interval significantly by 16 ms from 80 ± 6 to 64 ± 2 ms (Fig. 4). The concentration of adenosine in the coronary effluent did not change during administration of 10 µM CVT-2759 and remained at 65 ± 9 nM (n = 6).


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  		Fig. 4.   Attenuation by CVT-2759 of the prolongation of S-H interval of the guinea pig isolated heart caused by the combination of iodotubercidin (ITU; 1 µM) and erythro-9-(2-hydroxy-3-nonyl)-adenine (EHNA; 200 nM). Simultaneous administration of ITU and EHNA significantly increased the concentration of adenosine in the coronary effluent (see text). Bars indicate values of the mean and SE of single determinations in each of 6 hearts. The S-H interval in the absence of drug (control) was 46 ± 1 ms. *P < 0.001 compared with control. **P < 0.01 compared with ITU + EHNA.

CVT-2759 is Full Agonist at Adenosine Receptors in FRTL-5 Cells and Rat Epididymal Adipocytes

A partial agonist of A1-ADORs in the heart may be a full agonist of A1-ADORs in tissues wherein either receptor density or receptor-to-response coupling efficiency is relatively high. Two such tissues are FRTL-5 cells (6) and rat epididymal adipocytes (21, 26). To determine whether CVT-2759 is a full agonist in FRTL-5 cells and in adipocytes, the effects of CVT-2759 on cAMP content of FRTL-5 cells and of adipocytes were compared with the effects of full agonists. CVT-2759 and CPA decreased the cAMP content of FRTL-5 cells in the presence of 2 µM forskolin and adenosine deaminase by up to 97 ± 1 and 98 ± 1%, respectively (Fig. 5A). Although the maximal effects of CVT-2759 and CPA were not significantly different, CVT-2759 was nearly 100-fold less potent than CPA (Fig. 5A). The values of EC50 for CVT-2759 and CPA to reduce the cAMP content of FRTL-5 cells were 35 and 0.4 nM, respectively. CVT-2759 and CPA also caused similar reductions of the cAMP content of rat isolated epididymal adipocytes in the presence of 30 nM isoproterenol and 2 U/ml adenosine deaminase (Fig. 5B). The values of EC50 for CVT-2759 and CPA in adipocyte experiments were 74 (95% confidence intervals, 44-127 nM) and 0.08 nM (95% confidence intervals, 0.073-0.086 nM), respectively (n = 5 experiments). The A1-ADOR agonists adenosine (3 µM), R-N6-(2-phenylisopropyl)-adenosine (0.1 µM), CCPA (0.1 µM), CHA (0.1 µM) and CVT-2759 (100 µM) caused similar maximal attenuation by 95, 98, 94, 93, and 95%, respectively, of the action of 30 nM isoproterenol on cAMP content of adipocytes (data not shown). These data indicate that CVT-2759 is a full agonist to reduce the cAMP content of both FRTL-5 cells and rat adipocytes.


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  		Fig. 5.   Actions of CVT-2759 and CPA to decrease the cAMP contents of Fischer rat thyroid line 5 (FRTL-5) cells (A) and rat epididymal adipocytes (B). FRTL-5 cells were incubated with 2 µM forskolin, 2 U/ml adenosine deaminase, and the indicated concentrations of either CVT-2759 or CPA. Adipocytes were incubated with 30 nM isoproterenol, 2 U/ml adenosine deaminase, 1 mM ascorbic acid, and either CVT-2759 or CPA. Symbols indicate the mean and SE of 6 determinations in each of 4 (A) or 5 (B) experiments.

Radioligand Binding to Determine Affinity of CVT-2759 for A1-ADOR

To determine whether CVT-2759 can distinguish high- and low-affinity states of the A1-ADOR (i.e., G protein-coupled and uncoupled receptors), the effect of 1 mM GTP on the reduction by CVT-2759 of [3H]CPX binding to both the guinea pig forebrain and rat adipocyte membranes was measured. For comparison, the reduction by the full agonist CPA of [3H]CPX binding was also measured in the absence and presence of 1 mM GTP. In principle, the uncoupling of a G protein-coupled receptor from G proteins is expected to reduce the affinity of a full agonist more than it reduces the affinity of a partial agonist (the affinity of an antagonist may be unchanged or increased when receptors are uncoupled). Assays of the reduction by CVT-2759 and CPA of [3H]CPX binding to guinea pig forebrain membranes were done in lieu of assays of binding to heart membranes, because binding of [3H]CPX to the latter preparation is too low to allow quantitation of the effect of GTP. CVT-2759 and CPA both reduced the binding of [3H]CPX to forebrain membranes (Fig. 6A). The affinity of CVT-2759 for A1-ADORs in guinea pig forebrain membranes was slightly but not significantly reduced by 1 mM GTP (Fig. 6A). The values of IC50 for CVT-2759 to reduce the binding of [3H]CPX in the absence and presence of GTP were 2.5 µM (95% confidence intervals, 1.9-3.3 µM) and 2.9 µM (95% confidence intervals, 1.9-4.5 µM), respectively [P = not significant (NS)]. In contrast, the values of IC50 for CPA to reduce the binding of [3H]CPX to forebrain membranes in the absence and presence of 1 mM GTP were 11-fold different: 2.5 nM (95% confidence intervals, 1.8-3.4 nM) and 28 nM (95% confidence intervals, 7.8-102 nM), respectively (P < 0.05). The monophasic reduction of the binding of [3H]CPX by CVT-2759 may be interpreted to indicate that CVT-2759 bound to G protein-coupled and -uncoupled A1-ADORs with a single low affinity. In contrast, CPA appeared to bind with a single high affinity to nearly all A1-ADORs in the absence of GTP, and its affinity was reduced when many receptors were uncoupled from G proteins in the presence of GTP. These results suggest that the G protein-bound state of the guinea pig brain A1-ADOR, which is presumed to have a high affinity for agonist, is better stabilized by CPA than by CVT-2759.


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  		Fig. 6.   A: reduction of the binding of 8-cyclopentyl-1,3-dipropyl-[2,3-3H(N)]xanthine ([3H]CPX) to guinea pig forebrain membranes caused by the A1-adenosine receptor agonists CVT-2759 and CPA in the absence and presence of 1 mM GTP. Values of the dissociation constant of a competitive inhibitor (Ki) and Hill slope for reduction by CVT-2759 of [3H]CPX binding in the absence and presence of GTP were 1.8 µM (95% confidence intervals, 1.4-2.3 µM) and 0.79 and 2.1 µM (95% confidence intervals, 1.4-3.2 µM) and 0.99, respectively. Values of Ki and Hill slope for reduction by CPA of [3H]CPX binding in the absence of GTP were 1.7 nM (95% confidence intervals, 1.3-2.3 nM) and 1.2. In the presence of GTP, the Hill slope was 0.53, and the value(s) of Ki for CPA were therefore calculated using a 2-site binding model with 73% of sites having a high affinity for CPA. Values of Ki for CPA for the high- and low-affinity sites in the presence of GTP were 10.4 and 882 nM, respectively. The mean values of concentrations of [3H]CPX in experiments with CVT-2759 and CPA were 0.58 and 0.66 nM, respectively. Symbols represent the mean and SE of 3-4 determinations in each of 5-6 experiments. B: reduction of the binding of 3[H]-CPX to rat adipocyte membranes caused by CVT-2759 and CPA in the absence and presence of 1 mM GTP. Values of Ki and Hill slope for reduction by CVT-2759 of [3H]CPX binding in the absence and presence of GTP were 62 nM (95% confidence intervals, 35-108 nM) and 0.87 and 3.3 µM (95% confidence intervals, 2.1-5.3 µM) and 1.0, respectively. Values of Ki and Hill slope for reduction by CPA of [3H]CPX binding in the absence and presence of GTP were 0.052 nM (95% confidence limits, 0.02-0.11 nM) and 0.88 and 0.52 nM (95% confidence limits, 0.40-0.66 nM) and 1.0, respectively. The mean values of concentrations of [3H]CPX in experiments with CVT-2759 and CPA were 0.75 and 0.80 nM, respectively. Assays were done as described in METHODS. Symbols represent the mean and SE of 3 (for CPA) or 6 (for CVT-2759) determinations in each of 4 (CPA) or 5 (CVT-2759) experiments.

Results of binding assays using rat adipocyte membranes were different from results of assays using guinea pig brain membranes (Fig. 6). Whereas the values of IC50 for CVT-2759 to reduce the binding of [3H]CPX to brain membranes in the absence and presence of GTP were not significantly different, the values of IC50 for CVT-2759 to reduce the binding of [3H]CPX to adipocyte membranes in the absence and presence of GTP were 53-fold different. The values of IC50 for CVT-2759 to reduce the binding of [3H]CPX in the absence and presence of 1 mM GTP were 0.18 µM (95% confidence intervals, 0.10-0.31 µM) and 9.5 µM (95% confidence intervals, 5.9-15.3 µM), respectively (Fig. 6B). These data indicate that CVT-2759 stabilized a G protein-coupled state of the rat adipocyte A1-ADOR. This finding is consistent with the observation that CVT-2759 is a full agonist for reduction of cAMP content of intact rat adipocytes. Reduction by CPA of binding of [3H]CPX to adipocyte membranes in the absence and presence of GTP was similar to reduction of binding of [3H]CPX to brain membranes. The values of IC50 for reduction by CPA of [3H]CPX binding to adipocyte membranes in the absence and presence of 1 mM GTP were 10-fold different: 0.15 and 1.5 nM, respectively, P < 0.05 (95% confidence limits, 0.07-0.32 and 1.2-2.0 nM, respectively). Thus GTP caused a greater reduction of the binding of CVT-2759 than that of CPA. A possible explanation for this unexpected finding is that the effect of GTP to uncouple rat adipocyte A1-ADOR from G proteins was incomplete and was antagonized more by the strong agonist CPA than by the weak agonist CVT-2759. Indeed, when adipocyte membranes were incubated with 1 mM GTP and 0.1 mM N-ethylmaleimide, a more complete uncoupling of A1ADOR from G proteins appeared to be achieved compared with that caused by GTP alone. The value of IC50 for reduction by CPA of [3H]CPX binding to adipocyte A1-ADOR was 22-fold higher in the presence of both GTP and N-ethylmaleimide than in the presence of GTP alone (33 vs. 1.5 nM, P < 0.05; data not shown). In contrast, the value of IC50 for reduction by CVT-2759 of [3H]CPX binding to adipocyte A1-ADOR was only 1.6-fold higher in the presence of GTP and N-ethylmaleimide than in the presence of GTP alone (15 vs. 9.5 µM, P = NS). Thus when rat adipocyte membranes are incubated with GTP, a stronger coupling of A1-ADOR to G proteins occurs in the presence of CPA than in the presence of CVT-2759.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES

The results of this study support the concept that a partial agonist of the A1-ADOR can be used for moderate and relatively selective slowing of AV nodal conduction. It was demonstrated that CVT-2759 slows AV nodal conduction but does not cause second-degree AV block when used at concentrations as high as 100 µM. The effect of CVT-2759 on the S-H interval appeared to reach a plateau at concentrations >10 µM. The implication of this finding is that CVT-2759 can be used to cause a moderate, predictable, and submaximal slowing of AV nodal conduction without the risk of AV block, even at high doses. The results also show that CVT-2759 attenuated the actions of the full agonists CPA and adenosine to cause AV block and conduction delay. This finding is consistent with the classification of CVT-2759 as a partial agonist for slowing of AV nodal conduction.

The effects of CVT-2759 on atrial rate and duration of atrial and ventricular myocardial action potentials in guinea pig isolated hearts were small or insignificant, respectively. CVT-2759 (100 µM) caused a 13% slowing of atrial rate, whereas the full agonist CPA reduced atrial rate to nearly zero. The absence of a significant effect of CVT-2759 on the duration of the atrial monophasic action potential contrasts with the reported effect of the full agonist CCPA (17). CCPA shortened the duration of the guinea pig atrial monophasic action potential by up to 64%; the EC50 for this action of CCPA was 44 nM (17). Because CVT-2759 did not shorten the duration of the atrial monophasic action potential, CVT-2759 may not contribute to the genesis of atrial fibrillation or flutter. Shortening of the atrial effective refractory period by adenosine facilitated the induction of atrial fibrillation and atrial flutter in dogs (13) and in humans (5, 20).

Several factors may contribute to the greater effect of CVT-2759 on AV nodal tissue than on atrial or ventricular tissues. It is possible that AV nodal tissue has a higher density of A1-ADORs than atrial tissue, and atrial tissue is reported to have a higher density of A1-ADORs than ventricular tissue (19). The density of inwardly rectifying K+ channels, which mediate much of the response to activation of A1-ADORs in the heart, is also greater in supraventricular than in ventricular myocardial cells (4). Activation of these channels by A1-ADOR agonists causes a greater reduction of action potential amplitude of AV nodal than of atrial cells (4). In addition, activation of A1-ADOR in AV nodal cells reduces the amplitudes of the "basal" L-type inward calcium current and the pacemaker current IF (4). Thus the effects of activating A1-ADOR in AV nodal cells may be greater and more numerous than the effects on other cardiac cells. Because CVT-2759 is a weak agonist, its effects on these other myocardial cells are likely to be less than those on AV nodal cells.

CVT-2759 (10-100 µM) increased coronary conductance of the guinea pig isolated heart, suggesting that at high concentrations CVT-2759 activated A2A-adenosine receptors. The potency of CVT-2759 to slow AV nodal conduction (EC50, 3.1 µM) was 10-fold greater than the potency of CVT-2759 to increase coronary conductance (EC50,>= 31 µM). CVT-2759 appears to be more selective than either adenosine or CPA for slowing of AV nodal conduction relative to increasing of coronary conductance. Equivalent increases of the S-H interval by 13 ms were caused by 10 nM CPA, 2,200 nM adenosine, and 10,000 nM CVT-2759. Equivalent increases of coronary conductance of 0.09 ml · min-1 · mmHg-1 were caused by 75 nM CPA, 90 nM adenosine (data from Ref. 12), and 100,000 nM CVT-2759. Thus the functional selectivities of CVT-2759, CPA, and adenosine for the A1-ADOR mediated action in the heart (i.e., the ratio of concentrations of agonist causing increase of coronary conductance and increase of S-H interval) are 10, 7.5, and 0.04. The selectivity of CVT-2759 for slowing of AV nodal conduction versus increasing of coronary conductance appears to be 400-fold greater than the selectivity of adenosine. As a consequence, it would be expected that CVT-2759 is much less likely than adenosine to cause a decrease of vascular resistance.

Several findings support the conclusion that CVT-2759 is a partial agonist of A1-ADORs in the AV node. In the presence of the full agonists CPA and adenosine, CVT-2759 acted as an antagonist. CVT-2759 significantly shortened the S-H interval prolongation and reversed the second-degree AV block induced by both CPA and exogenous adenosine and caused a rightward shift of the concentration-response curve for the action of adenosine on the S-H interval. Likewise, CVT-2759 attenuated the negative dromotropic action of endogenous adenosine (Fig. 4). This antagonistic effect of CVT-2759 to reduce responses to full agonists of the A1-ADOR indicates that the intrinsic efficacy of CVT-2759 to activate the A1-ADOR is less than the intrinsic efficacy of a full agonist. The similarity of values of EC50 for CVT-2759 to slow AV nodal conduction (3.1 µM; 95% confidence intervals, 2.1-4.7 µM) and of the concentration of CVT-2759 causing a twofold reduction of the negative dromotropic action of adenosine (between 4 and 10 µM, Fig. 3) suggests that the binding of CVT-2759 to A1-ADORs is responsible for both actions. In addition, the value of EC50 for CVT-2759 to slow AV nodal conduction (3.1 µM) and the value of Ki for CVT-2759 to displace binding of [3H]CPX to guinea pig A1-ADOR (1.8 µM) were not significantly different. Similar values of EC50 and Ki for CVT-2759 suggest that the relationship between occupancy of A1-ADOR by CVT-2759 and prolongation of AV nodal conduction time is linear. This result is expected for a partial agonist (i.e., there is no receptor reserve for a partial agonist).

The partial agonist CVT-2759 was also distinguished from the full agonist CPA in assays of competition with [3H]CPX for binding to A1-ADOR in guinea pig forebrain membranes. In the absence of GTP, distinct high- and low-affinity binding of CVT-2759 to the A1-ADOR was not observed, and the affinity of CVT-2759 for the A1-ADOR was not significantly reduced by 1 mM GTP (Fig. 6A). In contrast, the affinity of CPA for the guinea pig forebrain A1-ADOR was reduced significantly by 11 times when 1 mM GTP was present (Fig. 6). The results are consistent with the finding that full agonists detect more receptors in a high-affinity G protein-coupled state than do partial agonists (15).

Although CVT-2759 was a partial agonist of A1-ADORs in the guinea pig heart, it was a full agonist of these receptors in FRTL-5 cells and rat adipocytes (Fig. 5). Agonists of A1-ADORs are reported to decrease cAMP content of FRTL-5 cells and adipocytes with high potencies (6, 21). Thus it is not surprising that even a "weak" (low intrinsic efficacy) agonist such as CVT-2759 can cause sufficient stimulation of adenosine receptors to elicit full responses from adipocytes and FRTL-5 cells, wherein the density of adenosine receptors is known to be relatively high (26, 27). Therefore CVT-2759 would be expected to stimulate responses in thyroid and fat cells when administered to an intact animal, even at concentrations (e.g., 100 nM) that would cause minimal or no slowing of AV nodal conduction. This interpretation is consistent with the findings of van Schaick et al. (28), that partial agonists of the A1-ADOR were more potent in causing antilipolytic than cardiovascular effects. Another interpretation of our findings is that CVT-2759 is a partial agonist for activation of guinea pig A1ADORs but a full agonist for activation of rat A1-ADORs. This interpretation is consistent with the observation that the binding of CVT-2759 to rat adipocyte A1-ADORs was reduced by GTP, whereas the binding of CVT-2759 to guinea pig brain A1-ADORs was not (Fig. 6).

In conclusion, we have demonstrated that CVT-2759 is a partial agonist of the A1-ADOR in the guinea pig heart. CVT-2759 selectively prolonged cardiac AV nodal conduction time while causing minimal sinus bradycardia, shortening of the duration of the atrial monophasic action potential, or coronary vasodilation. CVT-2759 did not cause AV block even at high concentrations. CVT-2759 antagonized the negative dromotropic actions of the full agonists adenosine and CPA. CVT-2759 was found to be a full agonist at A1-ADORs in two rat tissues with a high density of receptors, FRTL-5 cells and adipocytes.


    ACKNOWLEDGEMENTS

We thank Becky Hamilton and Hui Xiu Liang for technical assistance with FRTL-5 cell and adipocyte experiments, and CV Therapeutics for the gift of CVT-2759.


    FOOTNOTES

This work was supported by National Heart, Lung, and Blood Institute Grant HL-56785.

Address for reprint requests and other correspondence: J. C. Shryock, Dept. of Medicine, Box 100277, Univ. of Florida, Gainesville, FL 32610 (E-mail: shryojc{at}medicine.ufl.edu).

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

Received 22 March 2000; accepted in final form 24 August 2000.


    REFERENCES
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Am J Physiol Heart Circ Physiol 280(1):H334-H343
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H. Fraser, Z. Gao, M. J. Ozeck, and L. Belardinelli
N-[3-(R)-Tetrahydrofuranyl]-6-aminopurine Riboside, an A1 Adenosine Receptor Agonist, Antagonizes Catecholamine-Induced Lipolysis without Cardiovascular Effects in Awake Rats
J. Pharmacol. Exp. Ther., April 1, 2003; 305(1): 225 - 231.
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 CirculationHome page
Y. Song, L. Wu, J. C. Shryock, and L. Belardinelli
Selective Attenuation of Isoproterenol-Stimulated Arrhythmic Activity by a Partial Agonist of Adenosine A1 Receptor
Circulation, January 1, 2002; 105(1): 118 - 123.
[Abstract] [Full Text] [PDF]

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