Experimental Procedures

Materials

Adenosine deaminase was purchased from Roche Molecular Biochemicals, Indianapolis, IN). [³H]ZM241385 was purchased from Tocris Cookson Ltd. (Langford, Bristol, UK). [³H]CPX was from PerkinElmer Life Science Products (Boston, MA). CGS21680, adenosine, NECA, R-PIA, phenylephrine, DMSO, rolipram, and HEK-hA_2AAdoR membranes were obtained from RBI/Sigma (Natick, MA). CVT-510, CVT-2995, CVT-3003, CVT-3006, CVT-3032, CVT-3033, CVT-3100, CVT-3101, CVT-3126, CVT-3127, CVT-3141, CVT-3144, CVT-3146, YT-146, and WRC0470 were synthesized by CV Therapeutics, Department of Medicinal and Bioorganic Chemistry, Palo Alto, CA (see for the chemical names of CVT compounds). HENECA (2-hexynyl-5′-N-ethylcarboxamidoadenosine) was a gift from Professor Gloria Cristalli of the University of Camerino, Camerino, Italy. Drug stock solutions (10 mM) were prepared in DMSO. Sprague-Dawley rats were purchased from Simonsen Laboratories (Gilroy, CA). Ketamine was purchased from Fort Dodge Animal Health (Fort Dodge, IA) and xylazine from Bayer (Shawnee Mission, KS). Succinyl cAMP-tyrosyl methyl ester was purchased from Sigma (St. Louis, MO) and iodinated in the presence of chloramine T.

Cell Culture and Membrane Preparation

Rat pheochromocytoma PC12 cells were obtained from the American Type Culture Collection (Rockville, MD) and grown in DMEM with 5% fetal bovine serum, 10% horse serum, 0.5 mM l-glutamine, 100 U/ml penicillin, 0.1 mg/ml streptomycin, and 2.5 μg/ml amphotericin. HEK-293 cells stably expressing recombinant human A_2BAdoRs (HEK-hA_2BAdoR) were grown in DMEM supplemented with 10% fetal bovine serum and 0.5 mg/ml G-418. CHO-K1 cells stably expressing the recombinant human A₁AdoR (CHO-hA₁AdoR) and A₃AdoR (CHO-hA₃AdoR) were grown as monolayers on 150-mm plastic culture dishes in Ham's F-12 media supplemented with 10% fetal bovine serum in the presence of 0.5 mg/ml G-418. Cells were cultured in an atmosphere of 5% CO₂, 95% air and maintained at 37°C.

Membranes from cultured cells and porcine striatum were prepared as described previously (Belardinelli et al., 1996). The protein concentration of membrane suspensions was determined by using the Bradford method (Bio-Rad, Richmond, CA) with bovine serum albumin as standard.

Competition Binding Assays

Competition assays were performed to determine the affinities (K_i) of CVT compounds for A₁ AdoRs ([³H]CPX binding sites on CHO-hA₁AdoR cell membranes), A_2AAdoRs([³H]ZM241385 binding sites on PC12 and HEK-hA_2AAdoR cell membranes), A_2BAdoR ([³H]CPX binding sites on HEK-hA_2BAdoR cell membranes), and A₃AdoR ([¹²⁵I]ABMECA binding sites on CHO-hA₃AdoR cell membrane). Membrane suspensions were incubated for 2 h at room temperature in 50 mM Tris-HCl buffer (pH 7.4) containing ADA (1 U/ml), Gpp(NH)p (100 μM), radioligand {either [³H]ZM241385 (∼1.5–5 nM), [³H]CPX (∼2.5–3.0 nM for A₁ and 30 nM for A_2B), or [¹²⁵I]ABMECA (1 nM)} and progressively higher concentrations of the competing agents. At the end of the incubation, free radioligand was separated from membrane-bound radioligand by filtration through Whatman GF/C glass fiber filters using a Brandel tissue harvester (Gaithersburg, MD). Triplicate determinations were performed for each concentration of unlabeled compound.

Determination of cAMP Accumulation

PC12 cells were rinsed three times with Hanks' balanced saline solution, detached using a cell lifter, and pelleted by centrifugation at 500g for 5 min. Aliquots of the cell suspension (0.1–0.2 mg of protein) were placed in microfuge tubes with 250 μl of Hanks' balanced saline solution containing rolipram (50 μmol/l) and warmed to 37°C. Appropriate drugs were added to the cell suspensions, and incubations were allowed to continue for 10 min. Tubes were placed in a boiling water bath for 5 min to terminate the incubation. The samples were then cooled to room temperature, diluted by the addition of 1 ml of 10 mM Tris-HCl buffer at pH 7.4, and then centrifuged for 2 min at 13,000g. The cAMP content of the supernatant was determined by a radioimmunoassay method as described previously (Belardinelli et al., 1996).

Isolated Perfused Heart Preparation

Rats of either sex weighing 230 to 260 g were used in this study. All animals received humane care according to the guidelines set forth in The Principles of Laboratory Animal Care formulated by the National Society for Medical research and the Guide for the Care and Use of Laboratory Animals prepared by the Institute of Laboratory Animal Resources and published by the National Institutes of Health (NIH Publication 86-23, revised 1996). Animals were anesthetized by intraperitoneal injection of a mixture of ketamine (100 mg/ml) and xylazine (20 mg/ml). The chest of each rat was opened and the heart quickly removed. The heart was briefly rinsed in ice-cold modified Krebs-Henseleit (K-H) solution containing 117.9 mmol/l NaCl, 4.8 mmol/l KCl, 2.5 mmol/l CaCl₂, 1.18 mmol/l MgSO₄, 1.18 mmol/l KH₂PO₄, 2.0 mmol/l pyruvate, 5.5 mmol/l glucose, 0.5 mmol/l Na₂EDTA, 1.4 mmol/l ascorbic acid, and 2.1 mmol/l NaHCO₃. The aorta was cannulated and the heart was perfused at a flow rate of 10 ml/min with modified K-H solution. The K-H solution (pH 7.4) was gassed continuously with 95% O₂ and 5% CO₂ and warmed to 35 ± 0.5°C. The heart was electrically paced at a fixed cycle length of 250 ms (240 beats/min) using a bipolar electrode placed in the left atrium. The electrical stimuli were generated by a Grass stimulator (model S48; W. Warwick, RI) and delivered through a stimuli isolation unit (model SIU5; Astro-Med, Inc., W. Warwick, RI) as square-wave pulses of 3 ms in duration and with an amplitude of at least twice the threshold intensity.

Coronary perfusion pressure (CPP) was measured using a pressure transducer that was connected to the aortic cannula via a T-connector positioned approximately 3 cm above the heart. Coronary perfusion pressure was monitored throughout an experiment and recorded either on a chart recorder (Gould recorder 2200S; Valley View, OH) or a computerized recording system (PowerLab/4S; ADInstruments Pty Ltd., Castle Hill, Australia). Only hearts with CPP ranging from 60 to 85 mm Hg (in the absence of drugs) were used in the study. Coronary conductance (in ml/min/mm Hg) was calculated as the ratio between coronary perfusion rate (10 ml/min) and coronary perfusion pressure.

A₁ adenosine receptor-mediated depression of A-V nodal conduction time by CVT-3033 and CVT-3146 (negative dromotropic effect) was measured. Atrial and ventricular surface electrograms were recorded from the isolated rat heart during constant atrial pacing. The effects of CVT-3146 and CVT-3033 on atrioventricular conduction time were determined as described previously by Jenkins and Belardinelli (1988).

Anesthetized Open-Chest Pig Preparation

Farm pigs weighing 22 to 27 kg were used in this study. Animals were anesthetized with ketamine (20 mg/kg i.m.) and sodium pentobarbital (15–18 mg/kg i.v.). Anesthesia was maintained with additional sodium pentobarbital (1.5–2 mg/kg i.v.) every 15 to 20 min. Animals were ventilated via a tracheotomy tube using a mixture of room air and 100% O₂. Tidal volume, respiratory rate and fraction of O₂ in inspired air were adjusted to maintain arterial blood gas and pH values. Core body temperature was monitored with an esophageal temperature probe and maintained at 37.0–37.5°C by use of a heating pad. Lactate Ringers solution was administered via an ear or femoral vein as an initial bolus of 300 to 400 ml followed by a continuous infusion at a rate of 5 to 7 ml/kg/h. A catheter was inserted into the femoral artery to monitor arterial blood pressure.

The heart was exposed through a median sternotomy and suspended in a pericardial cradle. Left ventricular pressure was measured with a 5F high-fidelity pressure sensitive tip transducer (Millar Instruments, Houston, TX) placed in the left ventricular cavity via an apical incision and secured with a purse string suture. A segment of the left anterior descending coronary artery, proximal to the origin of the first diagonal branch, was dissected free of surrounding tissue. A transit time perivascular flow probe (Transonic Systems Inc., Ithaca, NY) was placed around this segment to measure coronary blood flow (CBF). Proximal to the flow probe, a 24-gauge modified angiocatheter was inserted for intracoronary infusions. All hemodynamic data were continuously displayed on a computer monitor and fed through a 32-bit analog to digital converter into an online data acquisition computer with customized software (Augury, Coyote Bay Instruments, Manchester, NH). A_2A AdoR agonists were dissolved in DMSO to produce stock concentrations of 1–5 mM, which were diluted in 0.9% saline and infused at rates of 1–1.5 ml/min via the catheter. The A_2A AdoR agonists were administered intracoronary.

Experimental Protocols

Rat Isolated Perfused Hearts.

Effects of CVT-3146 and CVT-3033 on coronary conductance (A_2A effect) and atrioventricular (A-V) conduction time (A₁ effect).Hearts were instrumented for continuous recording of coronary perfusion pressure (A_2A response) and A-V conduction time (A₁ response). In each experiment, concentration-response relationships for CVT-3146 (n = 6 rats), and CVT-3033 (n = 5 rats) to increase coronary conductance and to prolong A-V conduction time were determined. After control measurements of CPP and A-V conduction time were made, progressively higher concentrations of either CVT-3146 (0.1–30 μM) or CVT-3033 (0.1–300 μM) were administered until maximal coronary vasodilation and A-V nodal conduction time were achieved. In separate rat hearts (n = 4), the effect of 100 to 400 nM CVT-510, an A₁ receptor agonist, on A-V nodal conduction time was determined for comparison with the effects of CVT-3146 and CVT-3033.

Coronary vasodilatory response to A_2A adenosine receptor agonists.

Concentration-response relationships for the effect of A_2A adenosine receptor agonists to increase coronary conductance were obtained. After control measurements of coronary perfusion pressure were recorded, progressive higher concentrations of an adenosine receptor agonist were administered until maximal coronary vasodilation was observed. The steady-state responses to each concentration of adenosine receptor agonist were recorded. Each heart was exposed to a single agonist.

Coronary vasodilatory effect of CVT-3146 in the absence and presence of adenosine receptor antagonists.

To determine which adenosine receptor subtype (A₁ or A_2A) mediated the coronary vasodilation caused by CVT-3146, the A₁-selective antagonist CPX and the A_2A-selective antagonist ZM241385 were used. Hearts (n = 6) were exposed to CVT-3146 (10 nM), and after the effect of this agonist reached steady state, CPX (final concentration, 60 nM) and then ZM241385 (60 nM) were added to the perfusate and the changes in CPP were recorded.

Relationship between affinity of various agonists for A_2A adenosine receptor and the reversal time of their effect to increase coronary conductance.

These experiments were performed to construct the relationship between the affinities of the various agonists for the A_2A adenosine receptor and the duration of their respective effects on coronary conductance. Boluses of various agonists were injected into the perfusion line of rat isolated perfused hearts (n = 4–6 for each agonist) and the time to 90% (t_0.9) reversal of the decrease in CPP was measured. The affinities of the various agonists for the A_2A adenosine receptor in pig striatum membranes were determined using radioligand binding assays, as described above. The values oft_0.9 for reversal of agonist-induced decreases in CPP were plotted against agonist affinities (pK_i) for the A_2A adenosine receptor.

Open-Chest Pig.

Relationship between affinity of various agonists for A_2A adenosine receptor and the reversal time of their effect to increase coronary conductance. Each experiment was preceded by a 30-min stabilization period following the completion of all instrumentation of the animal. Baseline hemodynamic data were then recorded and an intracoronary infusion of an A_2A AdoR agonist was initiated. Each infusion was maintained for 4 to 5 min to allow left anterior descending CBF to reach a steady state, after which the infusion was terminated. The times to recovery of CBF by 50% (t_0.5) and 90% (t_0.9) of the difference from peak effect to baseline CBF were recorded. Ten to 15 min after CBF returned to predrug values a second infusion with a different agonist was started. In preliminary studies it was found that the intracoronary infusion of adenosine receptor agonists produced varying degrees of systemic hypotension, and hence, in all subsequent experiments, phenylephrine was administered intravenously at dose of ∼1 μg/kg/min. Hemodynamic measurements were made prior to and following the initiation of the phenylephrine infusion. The phenylephrine infusion rate was adjusted during and following the infusions of the adenosine receptor agonists to maintain arterial blood pressure within 5 mm Hg of preinfusion values. The effect of a maximum of three different agonists was determined in each experiment.

Reversal Time of Adenosine Receptor Agonist-Mediated cAMP Accumulation in PC 12 Cells.

PC12 cells cultured in DMEM at 37°C were treated for 10 min with 1μM of each of the following adenosine receptor agonists in the presence of rolipram (50 μM): WRC0470, CGS21680, CVT-2995, CVT-3146, CVT-3033, and R-PIA. Each agonist caused a sustained and near maximal increase of cAMP content in PC12 cells. Subsequently, the A_2A antagonist SCH58261 (20 μM) was added and the time course of the decline of cAMP content was determined. The reversal time for cAMP content to decrease to half-maximal (t_0.5) was calculated and plotted against the affinity (pK_i) of each agonist for the A_2A adenosine receptor, as determined by competition radioligand binding assays.

Results

Radioligand Binding Studies.

The affinities of a series of CVT compounds for adenosine receptor subtypes (A₁, A_2A, A_2B, and A₃) were determined by use of radioligand binding assays. To facilitate comparison and avoid the complication of multiple affinity states caused by receptor coupling to G proteins, the competition binding studies for the A_2A and A₁ receptors were carried out in the presence of Gpp(NH)p (100 μM) to uncouple receptors from G proteins (Gao et al., 1999a). Hence, the K_i values reported herein represent the affinities of these compounds for receptors in their low-affinity states. The results of radioligand binding assays are presented in Fig. 1 and Table1. The affinities of 18 agonists for pig striatal A_2A receptors were determined initially (Table 1, column 1). Eight of these compounds (four novel compounds and four reference compounds) were studied further using additional binding assays (Fig. 1; Table 1). Two of these compounds (CVT-3146 and CVT-3033) were low-affinity A_2A agonists with moderate selectivity for the A_2A receptor. Furthermore, CVT-3146 and CVT-3033, at 10 μM, decreased by only ≤22% the specific binding of [³H]CPX or [¹²⁵I]ABMECA to membranes derived from HEK-293 or CHO-K1 cells expressing recombinant human A_2Band A₃ receptors, respectively (Fig.2).

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Figure 1

Competition of adenosine receptor agonists for binding sites labeled by [³H]ZM241385 or [³H]CPX in membranes prepared from HEK-hA_2AAdoR (A) and CHO-hA₁AdoR cells (B). [³H]ZM241385 (1.5–5 nM) or [³H]CPX (2.5–3.0 nM) and membrane suspensions were incubated with progressively increasing concentrations of unlabeled adenosine receptor agonists for 2 h at room temperature in 50 mM Tris-HCl buffer (pH 7.4) containing ADA (1 U/ml) and 100 μM Gpp(NH)p. Nonspecific binding of [³H]ZM241385 or [³H]CPX was determined in the presence of either 100 μM NECA or 1 μM CPX, respectively. Each point is the mean ± S.E.M. of data pooled from at least three experiments, each assayed in triplicate. Values ofK_i and pK_i are given in Table 1.

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Figure 2

Competition of CVT-3146 and CVT-3033 for binding sites labeled by [³H]CPX or [¹²⁵I]ABMECA in membranes prepared from HEK-hA_2BAdoR (A) and CHO-hA₃AdoR (B). [³H]CPX or [¹²⁵I]ABMECA and membrane suspensions were incubated with progressively increasing concentrations of CVT-3146 and CVT-3033 for 2 h at room temperature in 50 mM Tris-HCl buffer (pH 7.4) containing ADA (1 U/ml). Each point is the mean ± S.E.M. of data pooled from three experiments.

Functional Studies.

The potencies of CVT-2995, CVT-3032, CVT-3033, and CVT-3146 to increase the accumulation of cAMP in PC12 were determined and compared with that of the prototypical high-affinity A_2A agonist CGS21680. As illustrated in Fig. 3A, all compounds increased the cellular content of cAMP in a concentration-dependent manner. The rank order of potency of these AdoR agonists (Table2) to increase cAMP accumulation in PC12 cells is in good agreement with their rank order of affinity to bind to A_2A receptors (Table 1). Of note, the low-affinity A_2A agonists CVT-3032, CVT-3033, and CVT-3146 are not only less potent (10–15-fold) but also less efficacious in stimulating cAMP accumulation compared with CGS21680. The maximal responses induced by CVT-3146, CVT-3033, and CVT-3032 were 85, 63, and 65% of that induced by CGS21680, respectively. These data suggest that CVT-3146, CVT-3033, and CVT-3032 may be partial A_2A agonists in PC12 cells. To test this hypothesis, we determined whether CVT-3033 (1 μM) would antagonize the effect of the full agonist CGS21680 on cAMP accumulation in PC12 cells (Fig. 3B). CVT-3033 (1 μM) caused an approximate 5-fold shift to the right of the CGS21680 concentration-response curve. Accordingly, the potency of CGS21680 to increase cAMP content was decreased from 24 ± 4 to 106 ± 18 nM in the presence of CVT-3033. This finding further suggests that CVT-3033 is a partial agonist.

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Figure 3

Effects of adenosine receptor agonists on cAMP content in intact PC12 cells. A, PC12 cells were incubated for 10 min with various concentrations of adenosine receptor agonists in the presence of 50 μM rolipram. Cyclic AMP levels were determined as described under Experimental Procedures. Values are mean ± S.E.M. of results of three experiments performed in triplicate. B, displacement by the partial A_2A agonist CVT-3033 (1 μM) of the concentration-response relationship for the full agonist CGS21680 to increase cAMP accumulation in PC12 cells. PC12 cells were stimulated for 10 min with various concentrations of CGS21680 in the absence or presence of the partial agonist CVT-3033 (1 μM). Note that CVT-3033 (1 μM) caused an approximately 5-fold shift to the right of the CGS21680 concentration-response relationship. The basal cAMP content was 42.0 ± 4.7 pmol/min/mg. Values are mean ± S.E.M. of triplicate determinants from one representative experiment.

Next, we determined the effects of CVT-2995, CVT-3032, CVT-3033, and CVT-3146 on coronary conductance in rat isolated perfused hearts. A_2A adenosine receptors are known to mediate coronary vasodilation caused by adenosine and adenosine analogs (Belardinelli et al., 1998). As shown in Fig.4, adenosine, CGS21680, WRC0470, and the CVT compounds caused concentration-dependent increases in coronary conductance. The potencies (EC₅₀ values) of adenosine, CGS21680, WRC0470, and the CVT compounds to increase coronary conductance are summarized in Table 2. Some CVT compounds were more potent whereas others were less potent than adenosine to increase coronary artery conductance (Fig. 4A; Table 2). CGS21680, WRC0470, and CVT-2995 were the most potent agonists of this series (Table 2). The low-affinity agonist CVT-3146 was found to be approximately 10-fold more potent than adenosine but 10-fold less potent than the high-affinity agonists CGS21680 and WRC0470 to increase coronary conductance.

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Figure 4

Effects of adenosine receptor agonists on coronary conductance (A) and CCP (B and C) in rat isolated perfused heart. Concentration-response relationships for CGS21680, CVT-2995, WRC0470, CVT-3146, CVT-3033, CVT-3032, and CVT-3100 to increase coronary conductance of rat isolated perfused heart. Symbols and error bars are mean and S.E.M. of single determination from four to six hearts per agonist. Hearts were paced at a cycle length of 250 ms. Coronary conductance in the absence of drug was 0.17 ± 0.01 ml/min/mm Hg (mean ± S.E.M., n = 26). B, time course of decreases in CPP caused by infusion of CVT-3146 and CGS21680 alone (top) or combined (bottom). Top, analog record of the decreases in CPP caused by infusion of CVT-3146 (10 nM, 4 min) or CGS21680 (100 nM, 4 min) alone. In the same heart, both CVT-3146 and CGS21680 caused an equal maximal decrease in CPP. Bottom, analog record of the decrease in CPP caused by the infusion of CVT-3146 (10 nM) with or without additional infusion of CGS21680 (100 nM). The decrease in CPP caused by 10 nM CVT-3146 was maximal because an additional infusion of 100 nM CGS21680 did not cause a further decrease in CPP.

CVT3146 was found to be a full agonist as a coronary vasodilator. As shown in Fig. 4B (top), the maximal decrease in coronary perfusion pressure (used as an index of the coronary vasodilation) caused by CVT-3146 was identical to that caused by a supramaximal concentration of the full A_2A AdoR agonist CGS21680. Both 10 nM CVT-3146 and 100 nM CGS21680 decreased coronary perfusion pressure by 23 mm Hg. In addition, in the presence of 10 nM CVT-3146, CGS21680 (100 nM) did not cause a further decrease of the coronary perfusion pressure (Fig. 4B, bottom). These results strongly suggest that CVT-3146 is a full agonist to increase coronary artery conductance.

To demonstrate that coronary vasodilation observed in the presence of CVT-3146 is mediated by A_2A adenosine receptors, the effect of 10 nM CVT-3146 on coronary conductance was determined in the absence and presence of both CPX, a selective A₁ AdoR antagonist (Belardinelli et al., 1998) and ZM241385, a selective A_2A AdoR antagonist (Poucher et al., 1995), both at concentrations of 60 nM. As depicted in Fig. 5A, CVT-3146 significantly increased coronary conductance to 0.22 ± 0.01 ml mm Hg⁻¹ min⁻¹ from a baseline value of 0.16 ±0.02 ml mm Hg⁻¹min⁻¹. This increase in coronary conductance caused by CVT-3146 was not affected by 60 nM CPX but was completely reversed by 60 nM ZM241385. Furthermore, the inhibition by ZM241385 of an increase of coronary conductance caused by CVT-3146 was concentration-dependent (Fig. 5B).

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Figure 5

Increases of coronary conductance caused by CVT-3146 are mediated by A_2A adenosine receptors. A, increase in coronary conductance caused by CVT-3146 (10 nM) before and after infusions of the A₁ receptor antagonist CPX (60 nM) and the A_2A receptor antagonist ZM241385 (60 nM). Columns represent the mean ± S.E.M. of single measurements from six rat isolated perfused hearts. B, concentration-response relationship for inhibition by ZM241385 of the increase in coronary conductance caused by 10 nM CVT-3146 in rat isolated perfused heart. Symbols and error bars indicate the mean and S.E.M. of single measurements from four rat isolated perfused hearts.

Functional Selectivity of CVT-3146 and CVT-3033 for Adenosine Receptor Subtypes.

We selected the two low-affinity agonists CVT-3146 and CVT-3033 for further investigation of their functional selectivity for adenosine receptor subtypes. The potencies of CVT-3146 and CVT-3033 to cause coronary vasodilation (A_2AAdoR response) and prolongation of A-V nodal conduction time (A₁ AdoR response) were therefore determined in rat isolated perfused hearts. As shown in Fig.6A, CVT-3146 and CVT-3033 potently increased coronary conductance in a concentration-dependent manner, but did not prolong A-V nodal conduction time. In contrast, the A₁ AdoR full agonist CVT-510 (Snowdy et al., 1999) caused a significant prolongation of A-V nodal conduction time with an EC₅₀ value of 205 nM (pEC₅₀ = 6.69 ± 0.49, n = 4).

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Figure 6

Functional selectivity of CVT-3146 and CVT-3033 for adenosine receptor subtypes. A, concentration-response relationship for CVT-3146 and CVT-3033 to slow AV conduction time and to increase coronary conductance in rat isolated perfused hearts. Symbols and error bars indicate means ± S.E.M. of single determinations from each of three hearts. B, effect of CVT-3146 and CVT-3033 on cAMP content in intact HEK-293 cells. HEK-293 cells were incubated for 10 min with various concentrations of CVT-3146 or CVT-3033 in the presence of 50 μM rolipram. Cyclic AMP levels were determined as described underExperimental Procedures. Values are means ± S.E.M. of results of three experiments performed in triplicate.

Next, we determined the effects of CVT-3033 and CVT-3146 to increase cAMP accumulation in HEK-293 cells, a response that is mediated by endogenous A_2B adenosine receptors (Gao et al., 1999b; Cooper et al., 1997). NECA, a nonselective AdoR agonist, caused a concentration-dependent increase of cellular cAMP content. In contrast, neither CVT-3033 nor CVT-3146 had any detectable effects even at a high concentration of 100 μM (Fig. 6B). These results indicate that CVT-3033 and CVT-3146 have very weak, if any, interaction with A_2B receptors.

Time Course of Decrease of Functional Response to A_2AAgonists upon Termination of Drug Administration.

We set out to determine the relationship between the affinity of agonists for the A_2A receptor and the rate of return to baseline of agonist-mediated responses (coronary vasodilation in heart and cAMP accumulation in PC12 cells) upon termination of drug administration. All compounds were given as boluses into the perfusion line at their respective minimal concentrations that caused equally or near equally maximal increases in coronary conductance. Likewise, the onset and time to peak effect (i.e., maximal coronary vasodilation) were similar for all agonists. Although adenosine and the various agonists caused equal maximal increases in coronary conductance, the durations of their effects were markedly different. The duration of the effect of adenosine was the shortest followed by those of CVT-3033 and CVT-3146. The effects of CGS21680 and WRC0470 had the longest duration (Fig.7). The durations of the coronary vasodilations in rat isolated perfused heart caused by adenosine, the CVT compounds, and other agonists measured as the time to 50 and 90% (t_0.5 andt_0.9, respectively) reversal of the increases in coronary conductance after termination of drug administration are summarized in Table 3. The reversal time of coronary vasodilation correlated with the affinity of the adenosine derivatives for the A_2Areceptors. As shown in Fig. 7C, there was a significant (p < 0.05) inverse relationship (r = 0.87) between the affinity (pK_i) of the agonists for the A_2A AdoR (Table 1) and the reversal time (t_0.9) (Table 3) of the coronary vasodilation caused by the same agonists in rat isolated hearts.

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Figure 7

Relationship between affinity and reversal time of coronary vasodilation after termination of administration of A_2A agonists. A and B, time courses of the increases in coronary conductance in rat isolated perfused heart caused by CVT-3033 (A) and CVT-3146 (B), adenosine, and CGS21680 or WRC0470. The time courses and maximal increases in coronary conductance caused by CVT-3033, CVT-3146, and adenosine were similar. The durations of the effects caused by CGS21680 or WRC0470 were markedly longer than those caused by CVT-3033 or CVT-3146. C and D, linear regression analysis of the relationship between the pK_i and the reversal time (t_0.9) of coronary vasodilation for A_2A agonists. Binding affinities were determined by measurement of the displacement of specific binding of [³H]ZM241385 from membranes prepared from pig striatum (data from Table 1). The reversal times of the coronary vasodilations were determined in rat isolated perfused hearts (C) and open-chest anesthetized pigs (D). Each data point is the mean ± S.E.M. of pK_i and t_0.9values. r and n are the correlation coefficient and number of agonists, respectively.

As would be predicted from the results of experiments using rat isolated hearts, in experiments with in situ hearts of open-chest anesthetized pigs all CVT-compounds as well as CGS21680 and other A_2A AdoR agonists (i.e., WRC0470 and YT-146) caused significant increases in CBF. Selected doses of these compounds given as continuous (4–5 min) intracoronary infusions caused 3.1 to 3.8-fold increases in CBF (Table 4). Once it was established that all agonists caused comparable increases of CBF (i.e., “fold increase”) and caused little or no changes in heart rate and mean arterial blood pressure (data not shown), the reversal time of their coronary vasodilatory effects was determined. As summarized in Table 4 the t_0.5 andt_0.9 values for coronary vasodilation caused by the various A_2A AdoR agonists and CVT-compounds were different. The reversal times of the increase in CBF caused by the CVT-3146, CVT-3032, and CVT-3033 were shorter than those of CGS21680, WRC0470, or YT-146. More importantly, as depicted in Fig.7D, there was a significant (p < 0.05) inverse relationship (r = 0.93) between the affinity (pK_i) of the A_2AAdoR agonists for pig brain striatum A_2Areceptors and the reversal time (t_0.9) of coronary vasodilation in pig heart.

The reversal times of the cAMP responses to A_2Aagonists in PC12 cells were also determined. After an initial 10-min incubation with A_2A agonists alone, a supramaximal concentration of the A_2A antagonist SCH58261 was added and the rate of decline of cAMP was determined. Fig.8A shows the time course of the decline of agonist-stimulated cAMP accumulation following the addition of SCH58261. The apparent t_0.5 values of agonists were inversely related to their affinities for A_2A AdoRs, that is, the greater the agonist affinity, the slower the rate of decline of cAMP content upon application of the A_2A AdoR antagonist SCH58261 (Fig. 8A). As depicted in Fig. 8B, the relationship between the apparent t_0.5 and pK_i for the agonists was best fit by linear regression with a correlation coefficient (r value) of 0.84.

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Figure 8

Time course of the decline in cAMP accumulated during exposure to agonists following addition of the A_2AAdoR antagonist SCH58261. A, PC12 cells were stimulated with various adenosine receptor agonists in the presence of 50 μM rolipram at 37°C. After a 10-min incubation, SCH58261 (20 μM) was added and cAMP content was determined at the times indicated. ○, control; ■, CGS21680; ▵, CVT-2995; ▿, CVT-3033; ●, WRC0470; ▪, CVT-3146; ▴, R-PIA. B, linear regression analysis of the relationship between the t_1/2 (min) and pK_i for the agonists. Binding affinities were determined by measurement of the displacement of specific binding of [³H]ZM241385 from membranes prepared from PC12 cells. ■, CGS21680; ▵, CVT-2995; ▿; ●, WRC0470; ▪, CVT-3146; ▴, R-PIA. The data were taken from Table 1.

Discussion

We demonstrate that in the presence of a large receptor reserve, low-affinity A_2A agonists, such as CVT-3146 and CVT-3033, were capable of potently producing a maximal coronary vasodilation as were the high-affinity agonists CGS21680 and WRC0470. The coronary vasodilation induced by CVT-3033 and CVT-3146 was rapid in onset, but unlike the high-affinity agonists, the duration of their effect was remarkably shorter.

The two agonists CVT-3033 and CVT-3146 were selective for the A_2A versus the A₁, A_2B, and A₃ adenosine receptors in binding studies. The results of the functional studies were consistent with the greater affinity of these two agonists for A_2A receptor compared with the other AdoR subtypes: 1) CVT-3146 and CVT-3033 caused A_2A-mediated cAMP accumulation in PC12 cells but not in HEK-293 cells that express only endogenous A_2B AdoRs; and 2) both agonists caused coronary vasodilation but did not slow A-V nodal conduction (A₁ AdoR-mediated effect) in rat isolated perfused hearts. The lack of negative dromotropic effect of CVT-3033 and CVT-3146 is consistent with our observation that both agonists have greater affinity (i.e., >2- and >13-fold for CVT-3033 and CVT-3146, respectively) and efficacy for A_2A than A₁ AdoR, and the presence of a markedly greater receptor reserve for A_2A AdoR-mediated coronary vasodilation than for A₁ AdoR-mediated conduction slowing (Dennis et al., 1992; Shryock et al., 1998).

The coronary vasodilation caused by CVT-3146 was mediated by A_2A AdoRs. Evidence in support of this conclusion is based on the findings that coronary vasodilation caused by CVT-3146 was reversed by the selective A_2A AdoR antagonist ZM241385 but not by the A₁ AdoR antagonist CPX (Fig. 5), and the potency (K_B = 3.88 nM) of ZM241385 to antagonize the effect of CVT-3146 is similar to the potency of ZM241385 to antagonize the effect of the A_2A AdoR agonist CGS21680 (Poucher et al., 1995).

It is worth noting that the agonists tested were much more potent in causing an increase of coronary conductance in the heart than in stimulating cAMP accumulation in PC12 cells (Table 2). This is most likely due to a larger receptor reserve for A_2A-mediated coronary vasodilation in the heart. Consistent with this notion, we found that CVT-3146, a low-affinity A_2A agonist (K_i= 1269 nM), was a potent (EC₅₀ = 6.4 nM) and full agonist to produce coronary vasodilation, whereas in PC12 cells it appeared to be a weak (EC₅₀ = 291 nM) and partial agonist to cause cAMP accumulation. Nevertheless, the A_2A receptor reserve in cells (such as PC12 cells) or tissues other than coronary artery remain to be determined experimentally. Two lines of evidence provide strong support for the conclusion that CVT-3146 and CVT-3033 are full agonists as coronary vasodilators: 1) their maximal effects were identical to that of the prototypical full agonist CGS21680, and 2) the maximal effect of CVT-3146 was not increased by concomitant administration of a supramaximal concentration of CGS21680. On the other hand, in keeping with the notion that CVT-3146 and CVT-3033 are partial agonists in PC12 cells, we demonstrated that 1) both agonists caused submaximal cAMP accumulation compared with full agonist CGS21680; and 2) as would be expected from a partial agonist, CVT-3033, at a concentration of 1 μM, antagonized the effect of CGS21680.

The most important finding of this study is that the duration of A_2A AdoR agonist-mediated responses is inversely related to the agonist affinity for the receptor. This conclusion is based on independent but complementary findings. The affinities of agonists for A_2A receptors in membranes prepared from pig striatum and PC12 cells predicted rather well both the reversal time of the increases of coronary conductance in rat isolated perfused hearts, in open-chest pig preparations and the rate of decline of cAMP content of PC12 cells. The findings indicate the affinity of the agonist for the receptor can be a major determinant of the duration of the effect of an agonist. This interpretation is supported by the remarkable concordance between the reversal time of coronary vasodilation caused by the same agonists in rat isolated perfused hearts and in the anesthetized open-chest pig preparations. These are two markedly different preparations, that is, isolated hearts perfused with physiological saline solution, where metabolism of the agonists should be minimal, in contrast to whole animals where the metabolism and clearance of the agonists from the receptor are expected to affect the duration of the effect. This interpretation is further supported by the relationship between agonist affinity and reversal time of the cAMP response in PC12 cells in culture where the metabolism of the agonists is minimal, and hence, should not contribute at all to the rate of decline of agonist-induced cAMP accumulation.

Potential Implications.

The coronary vasodilator effect of adenosine is the basis for the use of this nucleoside in conjunction with radionuclide imaging of the heart to detect underperfused areas of myocardium (Verani, 1992, 1994). However, the pharmacological “stress” induced by adenosine or dipyridamole is associated with an overall high incidence of side effects that includes dyspnea, chest pain, and atrioventricular nodal block (Verani, 1992, 1994). Because a number of the side effects caused by adenosine appears to be mediated by receptor subtypes other than the A_2A, it is expected that selective agonists of the A_2Areceptor would cause less undesirable effects. Toward this goal, several potent, high-affinity and selective A_2Aadenosine receptor agonists have been synthesized since the discovery of the prototypical A_2A agonist CGS21680 (Hutchison et al., 1989). Some of them, including CGS21680, have been proposed to have advantages over adenosine and dipyridamole as coronary vasodilators during myocardial imaging using radionuclide agents because of their high selectivity for A_2Areceptors versus other adenosine receptor subtypes (He et al., 2000;Glover et al., 1996). However, these compounds may not be as ideal as originally thought for this application because of their long duration of action and activity as systemic vasodilators. Due to the presence of a large A_2A receptor capacity in the coronary artery, the high affinity and high efficacy of these agonists not only contributes to their high potency to cause coronary vasodilation (e.g., EC₅₀ ≤ 1 nM for CGS21680) but also results in a longer duration of action as demonstrated in this report. In addition, agonists with high affinity and high efficacy generally also have limited tissue/organ selectivity (Van Der Graaf et al., 1999). Untoward effects associated with activation of all available A_2A receptors in organs/tissues (e.g., peripheral vascular beds), including the intended target tissues, have been a major impediment for use of potent A_2A agonists as therapeutic and diagnostic agents. One way to overcome this problem relies on the premise that partial agonists with lower efficacy may display greater tissue/organ selectivity than full agonists (IJzerman et al., 1996; Kenakin, 1997). The potency of an agonist to induce a physiological response is determined not only by its affinity for the receptor but also by the receptor reserve for the particular response (Shryock et al., 1998). Hence, a low affinity and partial agonist for a response with low receptor reserve could function as a full and potent agonist in a system that has a high receptor reserve (e.g., A_2A receptors in coronary artery). Therefore, it is possible to achieve both tissue/organ selectivity and potency by use of a low-affinity, partial agonist in a target tissue such as the coronary artery where receptor reserve is high.

Taken together, it appears that a low-affinity partial A_2A agonist, such as CVT-3146 or CVT-3033, may be an ideal choice for use as a short-acting coronary vasodilator during pharmacological stress perfusion imaging. The low affinity of the A_2A agonist for the receptor, in conjunction with a large receptor reserve for coronary vasodilation, ensures that vasodilation is rapid both in onset and in termination. The low-affinity, low-efficacy agonists will cause minimal activation of A_2A receptors in cells with low expression levels of this receptor or low receptor coupling efficiency, and hence have functional selectivity for the coronary vasodilation. Consistent with this notion, we have observed that CVT-3146 (1 μg/kg), given as i.v. bolus injection to anesthetized closed-chest dogs, caused a 2.6-fold increase of coronary blood flow velocity, but only 1.1-fold increase of peripheral artery blood flow (Xu et al., 2000). Furthermore, as A_2A agonists are being developed for pharmacological vasodilation during myocardial radionuclide imaging for detection and prognosis of ischemic heart disease, the lack of an effect of either CVT-3146 or CVT-3033 to depress A-V nodal conduction, as demonstrated in Fig. 6, would make these compounds more suitable as pharmacological vasodilators than adenosine.

Conclusions

CVT-3033 and CVT-3146 are low-affinity A_2AAdoR agonists. Both are potent and full agonists to cause coronary vasodilation. The duration of their effect is severalfold shorter than that of the high-affinity agonists CGS21680 and WRC0470. Hence, these novel short-acting low affinity A_2A agonists may prove to be ideal pharmacological reagents for the detection of subcritical coronary artery stenoses in patients undergoing noninvasive myocardial perfusion imaging during pharmacological coronary vasodilation.