Ray A. Olsson

8-cyclopentyl-1,3-dipropylxanthine (DPCPX)-a selective high affinity antagonist radioligand for A1 adenosine receptors

SummaryThe properties of 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) as an antagonist ligand for A1 adenosine receptors were examined and compared with other radioligands for this receptor. DPCPX competitively antagonized both the inhibition of adenylate cyclase activity via A1 adenosine receptors and the stimulation via A2 adenosine receptors. The K1-values of this antagonism were 0.45 nM at the A1 receptor of rat fat cells, and 330 nM at the A2 receptor of human platelets, giving a more than 700-fold A1-selectivity. A similar Al-selectivity was determined in radioligand binding studies. Even at high concentrations, DPCPX did not significantly inhibit the soluble cAMP-phosphodiesterase activity of human platelets. [3H]DPCPX (105 Ci/mmol) bound in a saturable manner with high affinity to A1 receptors in membranes of bovine brain and heart, and rat brain and fat cells (KD-values 50–190 pM). Its nonspecific binding was about 1 % of total at KD, except in bovine myocardial membranes (about 10%). Binding studies with bovine myocardial membranes allowed the analysis of both the high and low agonist affinity states of this receptor in a tissue with low receptor density. The binding properties of [3H]DPCPX appear superior to those of other agonist and antagonist radioligands for the A1 receptor.

Intracoronary adenosine deaminase reduces canine myocardial reactive hyperemia.

We employed intracoronary infusions of calf intenstine adenosine deaminase (ADA) to test the hypothesis that adenosine regulates coronary blood flow during myocardial reactive hyperemia (RH). Infusions of 4.5 U ADA/min per kg body weight into the left circumflex coronary artery of 10 open-chest dogs reversibly reduced repayment of flow debt by 30–39% (P< 0.05) following 5-, 10-, 15-, 20-, and 30-second coronary occlusions, the percentage reduction being independent of occlusion length. ADA reduced peak RH flow rate (17%, P < 0.05) only after 5-second occlusions. Intracoronary infusions of [131]ADA in seven dogs produced interstitial ADA concentrations between 1.2 and 13.1 U/ ml in perfused myocardium and, In five of these dogs, 131I activity in the cardiac node was 1.8–35 times that of contiguous mediastinal tissue. Theophylline, a specific adenosine antagonist, reduced repayment of flow debt by 27–38% {P < 0.02) in eight dogs, an effect similar to that of ADA, In six other dogs, ADA plus theophylline did not reduce RH flow debt repayment below that produced by ADA alone. This experiment confirms the contribution of adenosine to myocardial RH but shows that this nucleoside accounts for but a third of volume flow. Other, as yet unidentified, factors are collectively more important.

2-aralkoxyadenosines: potent and selective agonists at the coronary artery A2 adenosine receptor.

A Langendorff guinea pig heart preparation served for the assay of agonist potency of a series of 26 2-aralkoxyadenosines at the A1 and A2 receptors of, respectively, the atrioventricular node (conduction block) and coronary arteries (vasodilation). All of the analogues are weak agonists at the A1 receptor, requiring concentrations greater than 9 microM to cause second degree heart block. At the A2 receptor 2-phenethoxyadenosine is the most potent of the 2-phenylalkyladenosines. The activity of ring-substituted (F, Cl, CH3, and OCH3) 2-phenethoxyadenosines increases ortho less than meta less than para. The EC50s of coronary vasoactivity of several para-substituted analogues are in the subnanomolar range. The most potent analogue, 2-[2-(4-methylphenyl)ethoxy]adenosine 19, has an EC50 for coronary vasodilation of 190 pM and an A1/A2 selectivity ratio of 44,000. Aryl groups such as thienyl, indoloyl, or naphthyl also support A2 agonist activity. Although 2-oxoadenosine is 3 times more vasoactive than 2-aminoadenosine, the activities of the phenyl derivatives are markedly different; 2-phenoxyadenosine is 23 times weaker than 2-(phenylamino)adenosine (CV-1808).

Structure-activity relationships for N6-substituted adenosines at a brain A1-adenosine receptor with a comparison to an A2-adenosine receptor regulating coronary blood flow

A series of 145 N6-substituted adenosines have been screened as inhibitors of the binding of [3H]cyclohexyladenosine to an A1-adenosine receptor in rat brain membranes and the results compared to the potencies of these analogs in increasing coronary blood flow via activation of an A2-adenosine receptor. The A1 receptor shows greater stereoselectivity in the N6 region of the receptor towards asymmetric aralkyl substituents, and shows greater bulk tolerance in the N6 region of the receptor such that it retains affinity for certain N6-tertiary alkyladenosines and N6-cycloalkyladenosines that are inactive at the coronary A2 receptor. At the A1 receptor, the most potent analogs have either aliphatic N6-substituents with four or more methylene residues or have an N6-halophenyl substituent. At the A2 receptor, the most potent analogs have an N6-phenethyl or similar heteroarylethyl substituent. Certain sets or series of analogs appear useful for identifying the subtypes of adenosine receptors involved in physiological functions.

Nitric oxide modulates coronary autoregulation in the guinea pig.

A guinea pig heart Langendorff preparation was used in the present study to test the hypothesis that the coronary endothelium modulates coronary autoregulation through the production of nitric oxide (NO). Pacing at 250 beats per minute and venting the left ventricle to ensure that the hearts did no external work were performed in an attempt to reduce the metabolic stimulus to coronary vasomotion and keep it constant. We measured the responses of coronary flow and oxygen metabolism to stepwise changes of the perfusion pressure over the range between 18 and 85 mm Hg. The hearts exhibited autoregulation between 25 and 55 mm Hg and active vasodilation at perfusion pressures above that range. Perfusion with 100 microM NG-nitro-L-arginine (NNLA), an inhibitor of NO synthase, decreased coronary flow over the entire range of perfusion pressures and abolished active vasodilation over 65 mm Hg, thus widening the autoregulatory range. The administration of 200 microM L-arginine, but not D-arginine, reversed the action of NNLA. Inhibition of the cyclooxygenase pathway by 10 microM indomethacin did not affect autoregulation. Perfusion with 1 nM arginine vasopressin, a direct smooth muscle constrictor, lowered coronary flow rate to the same extent as NNLA at 55 mm Hg but did not prevent the pressure-dependent increase in flow above that pressure. These observations suggest that 1) the coronary endothelium actively modulates coronary autoregulation through the production of NO but not prostanoids, 2) mechanical stress (shear stress and/or stretching secondary to vasodilation) may be the stimulus to NO production, especially above the autoregulatory range, and 3) autoregulatory tone is likely to be myogenic in origin rather than mediated by extrinsic vasoconstrictors.

In vivo imaging of adenosine A1 receptors in the human brain with [18F]CPFPX and positron emission tomography

The important roles played by the A(1) adenosine receptor (A(1)AR) in brain physiology and pathology make this receptor a target for in vivo imaging. Here we describe the distribution of A(1)ARs in the living human brain with PET, made possible for the first time by the highly potent and selective A(1)AR antagonist 8-cyclopentyl-3-(3-[(18)F]fluoropropyl)-1-propylxanthine ([(18)F]CPFPX). In vivo data demonstrate a rapid cerebral uptake, peaking at 2.9 +/- 0.6% injected dose/liter at 3.3 +/- 1.3 min, followed by a gradual washout. Consistent with the results of autoradiography, high receptor densities occurred in the putamen and the mediodorsal thalamus. Neocortical regions showed regional differences in [(18)F]CPFPX binding, with high accumulation in temporal > occipital > parietal > frontal lobes and a lower level of binding in the sensorimotor cortex. Ligand accumulation was low in cerebellum, midbrain, and brain stem. Metabolism of [(18)F]CPFPX is rapid outside the central nervous system, but the metabolites do not penetrate the blood-brain barrier. In conclusion, in vivo application of [(18)F]CPFPX, a highly potent and selective PET ligand, for the first time allows the imaging of A(1)ARs in the living human brain.

INHIBITION OF PLATELET AGGREGATION BY ADENOSINE RECEPTOR AGONISTS

Abstract2-(Ar)alkoxyadenosines, which are agonists selective for the A2AAR in PC 12 cell and rat striatum membranes, are also agonists at the A2AR coupled to adenylate cyclase (AC) that mediates the inhibition of platelet aggregation. A panel of twelve well-characterized adenosine analogues stimulated human platelet AC and inhibited ADP-induced platelet aggregation at sub- to low-micromolar concentrations with a potency ranking CGS 21680 < adenosine < R-PIA. There were significant correlations between the ECso of anti-aggregatory activity and either the ECso of stimulation of platelet and PC 12 cell AC (r2 = 0.66 and 0.67, respectively) or the K1 of inhibition of [3H]NECA binding to the rat striatum membranes (r2 = 0.75). Likewise, platelet AC stimulation correlated well with stimulation of PC 12 cell AC and with [3H]NECA binding (r2 = 0.94 and 0.91, respectively). Ten 2-(ar)alkoxyadenosines stimulated platelet AC at EC50s ranging between 0.16 and 2.3 μM and inhibited platelet aggregation at EC50s ranging between 2 and 30 μM. There were no correlations between the EC50s of anti-aggregatory activity and either the EC50s of the stimulation of platelet or PC 12 AC (r2 = 0.08 and 0.06, respectively) or with the K1 of the inhibition of [3H]NECA binding to the A2aAR in rat striatum (r2 = 0.02). The EC50s of the stimulation of platelet AC correlated with those of the stimulation of PC 12 AC (r2 = 0.48), and also with the K1 of [3H]NECA binding (r2 = 0.71). Each of the 23 adenosines completely inhibited platelet aggregation and thus, functionally, all behaved as full agonists. As stimulants of PC 12 cell AC, Group A and B analogues were equally efficacious. As stimulants of platelet AC, however, the efficacy relative to NECA ( = 1.0) of Group B analogues was significantly less than that of Group A analogues, 0.49 ± 0.2 vs. 0.72 ± 0.05, P±0.01. The partial agonist activity of Group B analogues at the platelet A2AR but full agonist activity at the PC 12 cell A2aAR, as well as the relatively low correlations between platelet AC stimulation and other indices of A2aAR agonist actlVlty, suggest the platelet receptor is not a typical A2aAR. Further, the lack of a correlation between the platelet anti-aggregatory and AC stimulatory activity suggests that (a) the 2-(ar)alkoxyadenosines might affect platelet aggregation by mechanisms other than AC stimulation or (b) that the stimulation of the platelet membrane AC by 2-(ar)alkoxy-adenosines does not correspond to the accumulation of cyclic AMP in intact platelets.

Compartmentalization of the adenosine pool of dog and rat hearts.

Studies in rat and dog hearts examined the hypothesis that the cardiac adenosine pool contains an intracellular compartment. Enzymatically dispersed rat cardiocytes contain 70 pmol adenosine/mg protein which is resistant to 10 U/ml adenosine deaminase (ADA). Incubating dog heart homogenates for 1 minute at 37°C with 10 U ADA/mL did not change adenosine levels perceptibly from the average control value of 1.28 nmol/g. Studies employing [3H]hypoxanthine arabinoside as an adenosine surrogate showed that this nucleoside penetrates into pericardia! superfusates, attaining concentrations equal to those in blood plasma by 30 minutes. Since blood, cardiac interstitium, and pericardial superfusate are three compartments in series, this validates the use of pericardial superfusates equilibrated for ⩾30 min as probes of cardiac interstitial composition. In eight dogs, pericardial superfusate adenosine concentration averaged 0.24 μM. Cardiac muscle adenosine content averaged 0.87 nmol/g, indicating that the interstitial compartment accounts for only 6% of the cardiac pool. Dog cardiac muscle contains a [3H]adenosine binding protein whose size, affinity for adenosine analogs, and ability to synthesize S-adenosylhomocysteine (AdoHcy) suggest it is S-adenosylhomocysteine hydrolase (SAH). Studies employing a dog erythrocyte model show that adenosine is bound to a protein in this cell; treatment with L-homocysteine greatly reduces the amount of adenosine recovered. The half-time for the dissociation of [3H]adenosine from SAH at 37°C is 2.5 hours, and in the presence of ADA is >6 hours. Thus, although adenosine bound to SAH can serve as a substrate for AdoHcy synthesis, this experiment does not support the idea that the dissociation of adenosine occurs to a physiologically significant extent. Thus, we are uncertain of the functional role of the intracellular adenosine compartment.

FUNCTIONAL CHARACTERIZATION OF ADENOSINE A2 RECEPTORS IN JURKAT CELLS AND PC12 CELLS USING ADENOSINE RECEPTOR AGONISTS

The effect of several adenosine analogues on cyclic AMP accumulation was examined in the rat phaeochromocytoma cell PC12 and in the human T-cell leukaemia cell Jurkat, selected as prototypes of cells predominantly expressing adenosine A2A or A2B receptors. Using the reverse transcription-polymerase chain reaction it was, however, demonstrated that the Jurkat cell and the PC12 cell express both A2A and A2B receptor mRNA, albeit in different relative proportions. In PC12 cells the concentration required for half-maximal response (EC50) for the full agonist 5′-N-ethyl-car-boxamidoadenosine (NECA) was 30 times lower than in Jurkat cells. There was no significant difference in the pA2 for the antagonist 5-amino-9-chloro-2-(2-furanyl)1,2,4-triazolo(1,5-C)quinazolinemonomethanesulphon-ate (CGS 15943) between the two cell types. In the presence of forskolin (1 μM in PC12 cells; 10 μM in Jurkat cells) the EC50 value for NECA was reduced two-to sixfold. Forskolin also increased the maximal cAMP accumulation twofold in PC12 cells and sevenfold in Jurkat cells. A series of 2-substituted adenosine analogues CV 1808 (2-phenylamino adenosine), CV 1674 [2-(4-methoxyphenyl)adenosine], CGS 21680 {2-[p-(2-carbonylethyl)phenylethylamino]-5′-N-ethyl-carboxamido adenosine}, and four 2-substituted isoguanosines, SHA 40 [2-(2-phenylethoxy)adenosine; PEA], SHA 91 [2-(2-cyclohexylethoxy)adenosine; CEA], SHA 118 {2-[2-(p-methylphenyl)ethoxy]adenosine; MPEA}, and SHA 125 (2-hexyloxyadenosine; HOA), all raised CAMP accumulation in PC12 cells, but had minimal or no effect in Jurkat cells. In the PC12 cells the addition of forskolin (1 μM) reduced the EC50 by a factor of 2 (CV 1808) to 12 (SHA 125). In Jurkat cells all the analogues gave a significant, but submaximal, cAMP response in the presence of forskolin (10 μM), but they were essentially inactive in its absence. The results show that a series of 2-substituted adenosine analogues can be used to discriminate between A2A and A2B receptors. The two receptor subtypes appear to coexist, even in clonal cells selected for typical pharmacology. A2 receptor pharmacology can therefore be complex.

Structure-activity relationships for 2-substituted adenosines at A1 and A2 adenosine receptors

A series of 55 2-alkyloxy-, 2-aryloxy- and 2-aralkyloxy-adenosines was screened as inhibitors of the binding of [3H]R-phenyl-isopropyladenosine to A1 adenosine receptors in rat cerebral cortical membranes, and of the binding of [3]N-ethylcarboxamidoadenosine to A2 adenosine receptors in rat striatal membranes and as agonists at A2 adenosine receptors coupled to adenylate cyclase in rat pheochromocytoma PC12 cell membranes. The activities are consonant with a hydrophobic binding site in the A2 receptors at a distance from the 2-position of the adenine ring corresponding to a spacer chain of -O-CH2-CH2-. These is little lateral steric tolerance in the region occupied by the spacer chain. Interaction with the hydrophobic binding site is greatest in the 2-alkyloxy series for 2-cyclohexylethoxy-, 2-cyclohexylpropoxy- and 2-cyclohexylbutoxyadenosines and in the 2-aralkoxy series for 2-phenylethoxy-, 2-(4-methylphenyl)ethoxy-, 2-(4-chlorophenyl)ethoxy-, and 2-naphthylethoxy-adenosine. The affinities of the 2-substituted adenosines for the rat cerebral cortical A1 receptors are not as markedly altered by structural changes and are in almost all cases two- to hundredfold less than the affinity of the 2-substituted adenosine for the rat striatal A2 receptor. There is excellent correspondence of the present data on rat A2 receptors with reported potencies of these 2-substituted adenosines as coronary vasodilators in guinea pig heart preparations.