Role of cyclic nucleotides in vasodilations of the rat thoracic aorta induced by adenosine analogues.

Although adenosine analogues such as 5'-N-ethylcarboxamidoadenosine (NECA) relax the rat thoracic aorta in a partially endothelium-dependent manner via adenosine A(2A) receptors, others such as N(6)-R-phenylisopropyladenosine (R-PIA) act via an endothelium-independent, antagonist-insensitive mechanism. The role of cyclic nucleotides in these relaxations was investigated in isolated aortic rings using inhibitors of adenylate and guanylate cyclases as well as subtype-selective phosphodiesterase inhibitors. The adenylate cyclase inhibitor 9-(tetrahydro-2-furanyl)-9H-purin-6-amine (SQ 22536; 100 microM) significantly inhibited responses to NECA, but not responses to R-PIA. The type IV (cyclic AMP-selective) phosphodiesterase inhibitor 4-[(3-butoxy-4-methoxyphenyl)methyl]-2-imidazolidinone (RO 20-1724; 30 microM) significantly enhanced responses to NECA and to a lesser extent those to R-PIA. The guanylate cyclase inhibitor 1H-[1,2,4]oxadiazolo[4,3a]quinoxalin-1-one (ODQ; 100 microM) significantly inhibited responses to NECA and acetylcholine but not responses to R-PIA. The selective phosphodiesterase V (cyclic GMP-selective) inhibitors, zaprinast (10 microM) and 4-[[3',4'-(methylenedioxy)benzyl]amino]-6-methoxyquinazoline (MMQ; 1 microM), had no significant effect on responses to either NECA or R-PIA, but enhanced responses to acetylcholine. These results are consistent with the effects of NECA being via activation of endothelial receptors to release NO which stimulates guanylate cyclase, as well as smooth muscle receptors coupled to stimulation of adenylate cyclase. The lack of effect of zaprinast and MMQ on responses to NECA are likely to be due to simultaneous activation of both adenylate and guanylate cyclases in the smooth muscle, as cyclic AMP reduces the sensitivity of phosphodiesterase V to inhibitors. These results also suggest that the effects of R-PIA are via neither of these mechanisms.     

7-Deaza-9-phenyladenines. A new class of adenosine receptor antagonists

A series of twelve 7-deaza-9-phenyladenines and of related 9-aralkyl-, 9-alkyl-, and 9-alkenyl-analogs and of 7-deaza-9-phenylhypoxanthines inhibited binding of [3H]phenylisopropyladenosine to rat brain A1-adenosine receptors and antagonized activation of adenylate cyclase elicited by interaction of N-ethylcarboxamidoadenosine with A2-adenosine receptors in rat pheochromocytoma PC12 cell membranes. A subset of seven compounds, encompassing the range of major structural variations, antagonized inhibition of adenylate cyclase elicited by interaction of R-phenylisopropyladenosine with A1-adenosine receptors in rat fat cell membranes. 7-Deaza-9-phenyladenine had a Ki value of 3 microM at the brain A1-receptor and a KB value of 17 microM at the PC12 A2-receptor and was thus about 5-fold more potent than theophylline at the former and nearly equipotent with theophylline at the latter. It had a KB value of 4.6 microM at the fat cell A1-receptor. The presence of methyl groups at the 7- and 8-positions reduced activity at all receptors several fold. Aryl substituents in a series of 7-deaza-7,8-dimethyl-9-phenyladenines did not have major effects on affinities for the brain A1- or the PC12 cell A2-adenosine receptors. The absence of the 9-phenyl substituent in the 7,8-dimethyl series reduced activity several fold, while replacement with arylalkyl (-CH2C6H4F), alkyl (-(CH2)5CH3) or alkenyl (-CH2CH = CH2) substituents had only modest effects on potency at the brain A1-receptor and the PC12 cell A2-receptor. 7-Deaza-7,8-dimethylhypoxanthine was nearly equipotent to the analogous 7-deazaadenine at the brain and fat cell A1-receptors, but was several fold more potent than the analogous 7-deazaadenine at the A2-receptor. 7-Deaza-7,8-dimethyl-9-(2,4-dibromophenyl)hypoxanthine was less potent than the analogous 7-deazaadenine at both the brain A1- and the PC12 cell A2-adenosine receptors. 7-Deaza-9-phenyl-7,8-benzohypoxanthine was the most potent of the present series of antagonists and was somewhat selective for the A2-adenosine receptor with a Ki of 0.9 microM at the brain A1-receptor, a KB of 1.4 microM at the fat cell A1-receptor, and a KB of 0.2 microM at the A2-receptor.     

Adenosine enhancement of adrenergic neuroeffector transmission in guinea-pig pulmonary artery.

1. Adenosine and its derivatives N6-[(R)-1-methyl-2-phenylethyl]adenosine (R-PIA) or 5'-N-ethyl-carboxamideadenosine (NECA) enhanced nerve-induced contractile responses and augmented the basal smooth muscle tone in transmurally stimulated isolated strips of the guinea-pig pulmonary artery. 2. Adenosine, R-PIA and NECA enhanced contractile responses induced by noradrenaline, whereas N6-[(S)-1-methyl-2-phenylethyl]-adenosine (S-PIA) was virtually inactive. 3. Adenosine, R-PIA and NECA inhibited the nerve stimulation evoked release of [3H]-noradrenaline. However, the total release of [3H]-noradrenaline during the periods of NECA application was increased. 4. The nucleoside effects were blocked by the adenosine receptor antagonist 8-p-sulphophenyltheophylline. 5. 8-p-Sulphophenyltheophylline inhibited nerve-induced contractions and lowered basal muscle tone in preparations not having received any exogenous purines. 6. It is suggested that the observed stimulatory effects on muscle tone and on contractile responses to transmural nerve stimulation are mainly due to action at postjunctional stimulatory A1 adenosine receptors. In addition, actions at prejunctional inhibitory A1 and stimulatory A2 adenosine receptors are evident in this preparation.     

9-Ethyladenine derivatives as adenosine receptor antagonists: 2- and 8-substitution results in distinct selectivities

9-Ethyladenine was used as the basis for a series of non-xanthine adenosine receptor antagonists at human adenosine receptors. The adenine-based compounds were substituted in 2- or 8-position with a variety of side chains including some aryl or arylalkynyl groups previously tested as 2-substituents in adenosine and 5'-N-ethylcarboxamidoadenosine (NECA) for their effect on agonist affinity. The affinity of the novel compounds was tested in radioligand binding assays (A1, A2A and A3) and inhibition of NECA-stimulated adenylyl cyclase activity (A2B) in membranes prepared from CHO cells stably transfected with the respective human receptor subtype. High affinity antagonists were identified for A1 (9-ethyl-8-phenyl-9H-adenine, compound 2; 6-(1-butylamino)-9-ethyl-8-phenyl-9H-purine, compound 3), A2A (8-ethoxy-9-ethyladenine; compound 8) and A3 (9-ethyl-8-phenylethynyl-9H-adenine, compound 5) with selectivities versus other receptor subtypes in the range of 10 to 600. These results demonstrate that adenine is a useful template for further development of high-affinity antagonists with distinct receptor selectivity profiles.     

Radioligand binding to adenosine receptors and adenosine uptake sites in different brain regions of normal and narcoleptic dogs

The present study compares the characteristics of radioligand binding to adenosine receptors and adenosine uptake sites in 100- and 50-day-old normal and narcoleptic dogs. Binding to A1 receptors was quantified using a selective A1 agonist ([3H]N6-[(R)-1-methyl-2-phenylethyl] adenosine, [3H]R-PIA) and an antagonist ([3H]dipropyl-8-cyclopentyl-xanthine, [3H]CPX). Differences in the binding of [3H]R-PIA and that of [3H]5'-ethylcarboxamide adenosine ([3H]NECA), which binds to both A1 and A2 receptors with similar affinities, were used to quantify A2 receptors. Nucleoside transport sites were labeled with [3H]nitrobenzylthioinosine ([3H]NBTI), a potent inhibitor of nucleoside transport systems. The present study offered no evidence that either adenosine A1 receptors and adenosine uptake sites in the frontal cortex or adenosine A2 receptors in the putamen were altered in narcoleptic dogs. However, we found that adenosine A1 receptors in the dog exist in different affinity states and that the affinity state in which the receptor is found depends on the brain region examined. A characterization of these low- and high-affinity sites was performed and results indicated that these sites cannot be explained by a single interaction of the A1 receptor with a single G-protein population.     

A new high affinity, iodinated adenosine receptor antagonist as a radioligand/photoaffinity crosslinking probe

A new high affinity antagonist photoaffinity crosslinking radioligand has been synthesized for use in studying adenosine receptors. This compound, PAPAXAC (8-[-4-[[[[[2-(4-aminophenylacetylamino)ethyl]amino]carbonyl]- methyl]oxy]phenyl]-1,3-dipropylxanthine), has been labeled with 125I by a chloramine T method. The radioligand [125I]PAPAXAC binds to A1 adenosine receptors from bovine cerebral cortex with high affinity (KD = 0.1 nM), appropriate stereoselectivity, and A1 adenosine receptor specificity. Binding is not perturbed by guanine nucleotides. Adenylate cyclase assays document that PAPAXAC is an antagonist capable of completely blocking the ability of N6-R-phenyl-2-propyladenosine (R-PIA) to inhibit adenylate cyclase activity via A1 adenosine receptors. [125I]PAPAXAC can be incorporated covalently into a peptide of Mr = 40,000 using the heterobifunctional crosslinking agent N-succinimidyl-6-(4'-azido-2'-nitrophenylamino)hexanoate. Covalent labeling can be inhibited with adenosine receptor ligands to demonstrate a potency series of R-PIA greater than S-PIA greater than NECA much greater than IBMX. Guanine nucleotides do not decrease covalent incorporation. These results suggest that antagonists such as [125I]PAPAXAC recognize the same A1 adenosine receptor-binding subunit as agonists, such as [125I]AZPNEA, which labels a similar Mr peptide with the same pharmacological potency series. This new antagonist photoaffinity crosslinking probe/radioligand should be of great utility in the molecular characterization of A1 adenosine receptors.     

Agonist activity of 2- and 5'-substituted adenosine analogs and their N6-cycloalkyl derivatives at A1- and A2-adenosine receptors coupled to adenylate cyclase.

The activity of N6-cycloalkyl derivatives of adenosine, 2-chloroadenosine, 5'-chloroadenosine and N-ethylcarboximidoadenosine (NECA) and of 2-fluoroadenosine and 5-methylthioadenosines were compared at the A1-adenosine receptor inhibitory to adenylate cyclase in rat fat cell membranes and at the A2A-adenosine receptors stimulatory to adenylate cyclase in rat PC12 cell membranes. The N6-cycloalkyl derivatives in all cases were more potent (4- to 23-fold) than the parent compound at the A1 receptor, and were less potent (1.6- to 11-fold) than the parent compound at the A2A receptor. N6-Cyclopentyl-5'-chloroadenosine was the most selective agonist (900-fold) for the A1 receptor, while 2-fluoroadenosine was the only agonist with some selectivity (4.8-fold) for the A2A receptor. 5'-Methylthioadenosine was a weak agonist at both adenosine receptors. A 2-fluoro derivative of 5'-methylthioadenosine was somewhat more potent. Affinities of these analogs for inhibition of binding of radioligands to rat brain A1 and A2A receptors are presented.     

Adenosine modulates the dopaminergic function in the nigro-striatal system by interacting with striatal dopamine dependent adenylate cyclase

Behavioral and pharmacological evidences suggest that dopaminergic mechanisms in striatum might be counteracted by adenosine or potentiated by its pharmacological antagonists methylxanthines. To test whether adenosine modulation of the dopaminergic function could be, at least in part, due to an interaction at the level of the adenylate cyclase complex, we studied the effects of the adenosine analog R-Phenyl-isopropil-adenosine (R-PIA) on basal and dopamine-sensitive adenylate cyclase in rat striatum. R-PIA, which interacts with both adenosine A1-inhibitory and A2-stimulatory receptors, dose-dependently inhibited the stimulation induced by dopamine, and seemed to utilize the same pool of enzyme linked to dopaminergic D1 receptors. Two experimental approaches leading to supersensitivity of striatal dopaminergic receptors, (i.e., 6-hydroxy-dopamine injection in substantia nigra and reserpine administration) also induced upregulation of adenosine-dependent adenylate cyclase in striatum, and altered R-PIA modulation of dopamine-sensitive adenylate cyclase. Conversely, after subchronic treatment with neuroleptics such as haloperidol or sulpiride, upregulation of 3H-Spiroperidol binding in striatum was not associated with changes of R-PIA dependent adenylate cyclase in this area. It is concluded that adenosine might modulate post-synaptic responses to dopamine via adenosine receptors which functionally interact with dopaminergic D1 receptors in striatum.     

Effect of adenosine receptor agonists and other compounds on cyclic AMP accumulation in forskolin-treated hippocampal slices

The effect of adenosine analogues and some putative neurotransmitters have been studied on cyclic AMP accumulation in rat hippocampal slices treated with the adenylate cyclase activator forskolin. The effects of PGE2 and histamine were potentiated by forskolin (0.1 microM). Isoprenaline and NECA had essentially additive effects with 0.1 microM forskolin and serotonin (above 10(-4) M) inhibited forskolin-stimulated cyclic AMP accumulation. The A1-adenosine receptor selective adenosine analogue R-PIA inhibited forskolin stimulated cyclic AMP accumulation in low doses and stimulated in high. NECA, adenosine and 2-chloroadenosine uniformly stimulated cyclic AMP accumulation. 2',5'-dideoxyadenosine inhibited, but only at high concentrations. Both the stimulatory and the inhibitory effects of R-PIA were antagonized by 8-phenyltheophylline (10 microM). Enprofylline (100 microM) selectively inhibited the stimulatory effect. In the presence of enprofylline both 2-chloroadenosine showed an inhibitory effect on cyclic AMP accumulation. It is concluded that the forskolin-treated rat hippocampal slice is a useful preparation to study both stimulatory and inhibitory effects of transmitters and modulators on adenylate cyclase. The results also show that the rat hippocampus has both A1-receptors that are linked to inhibition of cyclic AMP accumulation and A2-receptors that are linked to stimulation. Furthermore, enprofylline is shown to selectively antagonize the stimulatory response, revealing inhibitory effects of compounds such as 2-chloroadenosine and adenosine.     

A2A adenosine receptors from rat striatum and rat pheochromocytoma PC12 cells: characterization with radioligand binding and by activation of adenylate cyclase.

Binding assays and assays of activation of adenylate cyclase with the agonists 5'-N-ethylcarboxyamidoadenosine (NECA) and CGS21680 have been used to compare adenosine receptors in rat pheochromocytoma PC12 cells and in rat striatum. The [3H]NECA binding showed two components, whereas [3H]CGS21680 bound to one component in both tissues. The Kd value for the high affinity site labeled with [3H]NECA in PC12 cell membranes (2.3 nM) was lower than that in striatum (6.5 nM). The [3H]CGS21680 binding site showed a Kd value of 6.7 nM and 11.3 nM in PC12 cells and striatum, respectively. In the presence of GTP the KD values of [3H]NECA and [3H]CGS21680 for the high affinity site were increased severalfold, whereas the low affinity sites for [3H]NECA were no longer detected with filtration assays. A comparison of the ability of a series of agonists and antagonists to inhibit high affinity binding of [3H]NECA to A2 receptors in PC12 cell and striatal membranes indicated that agonists had higher affinities and antagonists had lower affinities in PC12 cells, compared with affinities in striatal membranes. Analysis of activation of adenylate cyclase in PC12 cell membranes suggested that the dose-dependent stimulation by NECA involved two components, whereas CGS21680 stimulated via one component. The maximal stimulation by NECA significantly exceeded that caused by CGS21680. In intact PC12 cells, NECA caused a greater accumulation of AMP than did CGS21680, as was the case in membranes. In striatal membranes, NECA and CGS21680 showed similar maximal stimulations of adenylate cyclase. Both NECA and CGS21680 were more potent in PC12 cell membranes than in striatal membranes, in agreement with binding data. However, in contrast to binding data, antagonists were not less potent versus stimulation of adenylate cyclase by NECA or CGS21680 in PC12 cell membranes, compared with striatal membranes. In toto, the results suggest that A2A receptors in striatum are virtually identical to the A2A receptors in PC12 cells. But, in addition to an A2A receptor, it appears that a lower affinity functional receptor, probably an A2B receptor, is present in PC12 cells and PC12 cell membranes, whereas such a functional low affinity receptor is not detectable in striatal membrane.     

Ribose-modified adenosine analogues as adenosine receptor agonists

Analogues of the potent adenosine receptor agonist (R)-N-(1-methyl-2-phenylethyl)adenosine (R-PIA), modified at N9, were prepared and evaluated for adenosine A1 and A2 receptor binding and in vivo central nervous system and cardiovascular effects. The modifications at N9 include deoxy sugars, 5'-substituted-5'-deoxyriboses, non-ribose sugars, sugar ring homologues, and acyclic sugar analogues. Most of the derivatives have poor affinity for adenosine receptors. Only minor modifications at C5' and C3' maintain potent binding. In general, those derivatives exhibiting in vivo behavioral or cardiovascular effects also have the highest affinity for adenosine receptors.     

2',3'-Dideoxy-N6-cyclohexyladenosine: an adenosine derivative with antagonist properties at adenosine receptors

The 2',3'-dideoxy analogue of the potent A1 receptor agonist, N6-cyclohexyladenosine (CHA), was synthesized as a potential antagonist for the A1 adenosine receptor. In studies on adenylate cyclase 2',3'-dideoxy-N6-cyclohexyladenosine (ddCHA) did not show agonist properties at A1 or at A2 receptors. However, it antagonized the inhibition by R-PIA of adenylate cyclase activity of fat cell membranes via A1 receptors with a Ki value of 13 microM. ddCHA competed for the binding of the selective A1 receptor antagonist, [3H]8-cyclopentyl-1,3-dipropylxanthine ([3H]DPCPX), to rat brain membranes with a Ki value of 4.8 microM; GTP did not affect the competition curve. In contrast to the marked stereoselectivity of the A1 receptor for the alpha- and the natural beta-anomer of adenosine, the alpha-anomer of ddCHA showed a comparable affinity for the A1 receptor (K1 value 13.9 microM). These data indicate that the 2'- and 3'-hydroxy groups of adenosine and its derivatives are required for agonist activity at and high affinity binding to A1 adenosine receptors and for the distinction between the alpha- and beta-forms.     

Activation of multiple sites by adenosine analogues in the rat isolated aorta.

1. The presence of A2 receptors mediating relaxation in the rat isolated aorta has been previously demonstrated. However, agonist dependency of the degree of rightward shift elicited by 8-sulphophenyltheophylline (8-SPT) led to the suggestion that the population of receptors in this tissue is not a homogeneous one. In this study we have re-examined the effects of 8-SPT in the absence and presence of the NO synthase inhibitor L-NAME (NG-nitro-L-arginine methyl ester) and investigated antagonism of responses by the potent A2a receptor ligands PD 115,199 (N-[2-dimethylamino)ethyl]-N-methyl-4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3 dipropyl-1H-purin-8-yl)) benzene sulphonamidexanthine), ZM 241385 (4-(2-[7-amino-2-(2-furyl) [1,2,4]-triazolo[2,3-a][1,3,5]triazin-5-yl amino]ethyl)phenol), and CGS 21680 (2-[p-(2-carboxyethyl)phenylamino]-5'-N-ethylcarboxamidoadenosine). We have also investigated the antagonist effects of BWA1433 (1,3-dipropyl-8-(4-acrylate)phenylxanthine) which has been shown to have affinity at rat A3 receptors. 2. Adenosine, R-PIA (N6-R-phenylisopropyl adenosine), CPA (N6-cyclopentyladenosine) and NECA (5'-N-ethylcarboxamidoadenosine) all elicited relaxant responses in the phenylephrine pre-contracted rat isolated aorta with the following potency order (p[A50] values in parentheses): NECA (7.07 +/- 0.11) > R-PIA (5.65 +/- 0.10) > CPA (5.05 +/- 0.12) > adenosine (4.44 +/- 0.12). 3. 8-SPT (10-100 microM) caused parallel rightward shifts of the E/[A] curves to NECA (pKB = 5.23 +/- 0.16). A smaller rightward shift of E/[A] curves to CPA was observed (pA2 = 4.85 +/- 0.17). However, no significant shifts of E/[A] curves to either adenosine or R-PIA were observed. 4. In the absence of endothelium E/[A] curves to NECA and CPA were right-shifted compared to controls. However, removal of the endothelium did not produce a substantial shift of adenosine E/[A] curves, and E/[A] curves to R-PIA were unaffected by removal of the endothelium. 5. In the presence of L-NAME (100 microM) E/[A] curves to NECA and CPA were right-shifted. However, no further shift of the CPA E/[A] curve was obtained when 8-SPT (50 microM) was administered concomitantly. The locations of curves to R-PIA and adenosine were unaffected by L-NAME (100 microM). 6. In the presence of PD 115,199 (0.1 microM) a parallel rightward shift of NECA E/[A] curves was observed (pA2 = 7.50 +/- 0.19). PD 115,199 (0.1 and 1 microM) gave smaller rightward shifts of E/[A] curves to R-PIA and CPA, but E/[A] curves to adenosine were not significantly shifted in the presence of PD 115,199 (0.1 or 1 microM). 7. The presence of ZM 241385 (3 nM-0.3 microM) caused parallel rightwad shifts of NECA E/[A] curves (pKB = 8.73 +/- 0.11). No significant shifts of E/[A] curves to adenosine, CPA or R-PIA were observed in the presence of 0.1 microM ZM 241385. 8. CGS 21680 (1 microM) elicited a relaxant response equivalent to approximately 40% of the NECA maximum response. In the presence of this concentration of CGS 21680, E/[A] curves to NECA were right-shifted in excess of 2-log units, whereas E/[A] curves to R-PIA were not significantly shifted. 9. BWA1433 (100 microM) caused a small but significant right-shift of the E/[A] curve to R-PIA yielding a pA2 estimate of 4.1 IB-MECA (N6-(3-iodo-benzyl)adenosine-5(1)-N-methyl uronamide) elicited relaxant responses which were resistant to blockade by 8-SPT (p[A]50 = 5.26 +/- 0.13). 10. The results suggest that whereas relaxations to NECA (10 nM-1 microM) are mediated via adenosine A2a receptors, which are located at least in part on the endothelium, R-PIA and CPA may activate A2b receptors on the endothelium and an additional, as yet undefined site, which is likely to be located on the smooth muscle and which is not susceptible to blockade by 8-SPT, PD 115,199 or ZM 241385. This site is unlikely to be an A3 receptor since the very small shift obtained in the presence of BWA1433 (100 microM), and the low potency of IB-MECA is not consistent with the affin     

Why are A(2B) receptors low-affinity adenosine receptors? Mutation of Asn273 to Tyr increases affinity of human A(2B) receptor for 2-(1-Hexynyl)adenosine

Adenosine A(2B) receptors are known as low-affinity receptors due to their modest-to-negligible affinity for adenosine and prototypic agonists. Despite numerous synthetic efforts, 5'-N-ethylcarboxamidoadenosine (NECA) still is the reference agonist, albeit nonselective for this receptor. In our search for higher affinity agonists, we developed decision schemes to select amino acids for mutation to the corresponding residues in the most homologous, higher affinity, human A(2A) receptor. One scheme exploited knowledge on sequence alignments and modeling data and yielded three residues, V11, L58, and F59, mutation of which did not affect agonist affinity. The second scheme combined knowledge on sequence alignments and mutation data and pointed to Ala12 and Asn273. Mutation of Ala12 to threonine did not affect the affinity for NECA, (R)-N(6)-(phenylisopropyl)adenosine (R-PIA), and 2Cl Ado. The affinity of the N273Y mutant for NECA and R-PIA and for the antagonists xanthine amine congener (XAC), ZM241385, and SCH58261 was also unaltered. However, this mutant had a slightly increased affinity for a 2-substituted adenosine derivative, CGS21680. This prompted us to investigate other 2-substituted adenosines, with selectivity and high affinity for A(2A) receptors. All four compounds tested had improved affinity for the N273Y receptor. Of these, 2-(1-hexynyl)adenosine had submicromolar affinity for the N273Y receptor, 0.18 +/- 0.10 microM, with a 61-fold affinity gain over the wt receptor. In addition, the non-NECA analog (S)-PHP adenosine had an affinity of 1.7 +/- 0.5 microM for the wt receptor. The high affinity of (S)-PHP adenosine for the wt receptor suggests that further modifications at the 2-position may yield agonists with even higher affinity for A(2B) receptors.     

Adenosine receptor-coupled adenylate cyclase in the caudate nucleus of the rat brain

5'-(N-Ethylcarboxamido)adenosine (NECA) and N6-[(R)-(phenylisopropyl)]adenosine (PIA) were incubated in an adenylate cyclase assay with a particulate fraction of caudate-putamen tissue of the rat, in order to examine the effect of kainic acid and a 6-hydroxydopamine-induced lesion on adenosine receptor coupled adenylate cyclase in vitro. There was an enhancement of formation of cyclic AMP induced by NECA, that was mediated by A2 adenosine receptors. Phenylisopropyl adenosine also stimulated adenylate cyclase in the striatum, with a maximum increase at 0.1 mM. At smaller concentrations, PIA inhibited the basal activity, which was previously described to be an effect mediated by A1 adenosine receptors. In caudate-putamen tissue from rats receiving a unilateral lesion, induced with kainic acid, basal and maximally NECA- and PIA-stimulated activity of adenylate cyclase was decreased. The maximum stimulatory effects of both substances were also significantly decreased, whereas no change of the inhibitory effect of PIA was observed. After unilateral lesion induced with 6-OHDA, basal and maximally NECA- and PIA-activated adenylate cyclase was increased; however, no inhibitory effect of PIA was seen. These results suggest that A2 adenosine receptor-coupled adenylate cyclase was located on neurones intrinsic to the neostriatum and probably postsynaptic to the dopaminergic input. The A1 adenosine receptors seem to be associated with the nigrostriatal pathway implying a presynaptic localization on dopaminergic afferents. In addition, since after both kainic acid- and 6-OHDA-induced lesions, respectively, in caudate-putamen tissue of the contralateral side, PIA no longer inhibited the activity of adenylate cyclase, contralateral structures also appeared to be involved in the regulation of A1 adenosine receptors.     

Demonstration of both A1 and A2 adenosine receptors in DDT1 MF-2 smooth muscle cells

Adenosine receptors of the A1 and A2 subtypes were characterized in membranes from DDT1 MF-2 smooth muscle cells. These cells possess a high density of A1 adenosine receptors (Bmax = 0.8-0.9 pmol/mg of protein), as measured by both agonist and antagonist radioligands. Agonists compete for [125I]N6-[2-(4-amino-3-iodophenyl)ethyl]-adenosine (A1 receptor-selective radioligand) binding with the following potency series: (R)-phenylisopropyladenosine [(R)-PIA] greater than 5'-N-ethylcarboxamide adenosine (NECA) greater than (S)-PIA, indicative of their interaction with A1 adenosine receptors. Agonist competition for [3H]8-(4-[[[(2-aminoethyl)amino]carbonyl)methyl)oxy]phenyl)-1, 3-dipropylxanthine [( 3H]XAC) (an antagonist radioligand for the A1 adenosine receptor) was described by a two-state model of 1.3 nM (high affinity state, KK) and 370 nM (low affinity state, KL), with 70% of the receptors in the high affinity state (RH). Addition of guanosine 5'-[beta, alpha-imido]triphosphate (100 microM) shifted the (R)-PIA competition curves to the right to lower affinities. Photoaffinity labeling with the agonist photoprobe [125I]N6-[2-(4-amino-3-iodophenyl) ethyl]adenosine indicates that the A1 adenosine receptor binding subunit is a Mr 38,000 protein. Adenosine receptor agonists [(R)-PIA, NECA, and (S)-PIA] inhibited isoproterenol-stimulated adenylate cyclase activity in DDT1 MF-2 cell membranes with IC50 values of 62, 538, and 750 nM, respectively. Inhibition of adenylate cyclase by (R)-PIA was attenuated by the A1 receptor antagonist XAC and following inactivation of Gi with pertussis toxin (100 ng/ml). Using a recently developed A2 adenosine receptor agonist radioligand 2-[4-(2-[( 4-aminophenyl]methylcarbonyl)ethyl) phenyl]ethylamino-5'-N-ethylcarboxamido adenosine (125I-PAPA-APEC), we have demonstrated the presence of A2 adenosine receptors in this cell line. Saturation curves with 125I-PAPA-APEC indicated the Bmax and Kd values to be 0.21 pmol/mg of protein and 4.0 nM, respectively. In competition experiments, NECA was more potent at inhibiting 125I-PAPA-APEC binding than (R)-PIA, with their respective IC50 values being 5.6 and 351 nM. The photolabeled A2 adenosine receptor migrated on sodium dodecyl sulfate-polyacrylamide gel electrophoresis with an Mr of 42,000. Finally, adenosine receptor agonists stimulated adenylate cyclase activity by approximately 2-3 fold with the following potency series: PAPA-APEC greater than or equal to NECA greater than (R)-PIA, indicative of their interaction at A2 receptors. These data represent the first demonstration of the presence of both A1 and A2 receptors in a single cell line, DDT1 MF-2 smooth muscle cells.     

Do adenosine A3 receptors exist?

1. It has been suggested that adenosine A1 receptors may be sub-divided into A1 and A3 types, based on the relative potencies of 5'-N-ethylcarboxamidoadenosine (NECA) and selected N6-substituted adenosine analogues. At A1 receptors (rat adipocytes) N6-phenylisopropyladenosine (PIA) was reported to be approx. 100-fold more potent than NECA, whereas the compounds were equipotent at A3 receptors (those in cardiac and neuronal preparations). 2. The study reported here has systematically evaluated this proposal, the rank orders of potency of NECA, R- and S-PIA, N6-cyclopentyladenosine (CPA) and N6-cyclohexyladenosine (CHA) being determined in rat adipocytes, guinea-pig ileum and rat and guinea-pig atria. 3. R-PIA, CHA and CPA were found to have consistent potencies relative to NECA across all 6 tissues, including rat adipocytes. The rank order was CPA greater than or equal to CHA, R-PIA greater than or equal to NECA greater than S-PIA. 4. We conclude that the relative potencies of these agonists do not support the concept that adenosine A1 receptors in rat adipocytes differ from those in neuronal and cardiac tissues.     

Effects of N-ethylmaleimide on adenosine receptors of rat fat cells and human platelets

N-Ethylmaleimide (NEM) differentially modified Ri adenosine receptors in rat fat cells and Ra adenosine receptors in human platelets. Pretreatment of rat fat cell membranes with NEM inhibited the binding of the agonist (-)N6-phenylisopropyl[3H]adenosine [( 3H]PIA), but did not affect the binding of the antagonist 1,3-diethyl-8-[3H]phenylxanthine [( 3H]DPX). The IC50-value for inhibition of [3H]PIA binding was 0.067 mM. Saturation of [3H]PIA binding revealed that NEM converts the high affinity form of the Ri receptor into a low affinity form. NEM also decreased the potency of agonists to displace [3H]DPX binding, as shown by a 74-fold shift of the Ki-value for (-)PIA, whereas antagonist-induced displacement remained unchanged. In addition, low concentrations of NEM (0.01-0.1 mM) attenuated the (-)PIA-induced inhibition of adenylate cyclase activity of rat fat cells. At higher concentrations (0.1-1 mM) NEM reduced basal and stimulated adenylate cyclase activities in rat fat cells and human platelets, presumably by inactivation of the catalytic unit. Radioligand binding of 5'-N-ethylcarboxamido[3H]-adenosine [( 3H]NECA) to Ra adenosine receptors of human platelet membranes was not changed by NEM at low radioligand concentrations. Saturation analysis of [3H]-NECA binding showed that NEM led to an apparent increase of agonist affinity with a concomitant decrease in total [3H]NECA binding sites. These results suggest that NEM reduces the affinity of Ri adenosine receptors, probably by affecting the inhibitory guanine nucleotide binding protein (Ni), whereas [3H]NECA binding sites are inversely affected.(ABSTRACT TRUNCATED AT 250 WORDS)     

Characterization of P1-purinoceptors on rat isolated duodenum longitudinal muscle and muscularis mucosae.

1. P1-purinoceptors mediating relaxation of the rat duodenum longitudinal muscle and contraction of the rat duodenum muscularis mucosae were characterized by the use of adenosine and its analogues, 5'-N-ethylcarboxamidoadenosine (NECA), N6-cyclopentyl-adenosine (CPA), N6-(phenylisopropyl)adenosine (R-PIA), 2-chloroadenosine (2-CADO) and 2-p-((carboxyethyl)phenethylamino)-5'-carboxamidoadenosine (CGS21680), as well as the P1-purinoceptor antagonist 8-phenyltheophylline (8-PT) and the A1-selective antagonist, 1,3-dipropyl-8-cyclopentylxanthine (DPCPX). 2. In the rat duodenum longitudinal muscle, the order of potency of the adenosine agonists was CPA > NECA > adenosine > CGS21680. DPCPX antagonized responses to CPA and NECA at a concentration of 1 nM suggesting that they are acting at A1 receptors. A Schild plot versus CPA gave a slope near to unity (slope = 0.955) and a pA2 of 9.8 confirming that CPA was acting via A1 receptors. Schild analysis for DPCPX versus NECA, however, gave a slope of 0.674 suggesting that NECA was acting on both A1 and A2 receptors. CGS21680, a selective A2a agonist, was much less potent than adenosine suggesting that the A2 receptors are of the A2b subtype. 3. In the rat duodenum muscularis mucosae, the order of potency of the adenosine agonists was NECA > or = R-PIA = CPA > 2-CADO > adenosine, and DPCPX antagonized responses to CPA and NECA at a concentration of 1 microM. CGS21680, at a concentration of 10 microM, had no effect on this tissue. This suggests the presence of A2 receptors in this tissue and that they are of the A2b subtype. 4. These results are in agreement with previous studies in the whole duodenum showing the presence of A1 and A2b receptors causing relaxation, and this shows that the longitudinal muscle dominates the response of the whole tissue. In addition, a contractile A2b receptor has been revealed on the muscularis mucosae, the first time this subtype has been reported to elicit an excitatory response in a smooth muscle preparation.     

Structure-activity relationships at human and rat A2B adenosine receptors of xanthine derivatives substituted at the 1-, 3-, 7-, and 8-positions

In the search for improved selective antagonist ligands of the A2B adenosine receptor, which have the potential as antiasthmatic or antidiabetic drugs, we have synthesized and screened a variety of alkylxanthine derivatives substituted at the 1-, 3-, 7-, and 8-positions. Competition for 125I-ABOPX (125I-3-(4-amino-3-iodobenzyl)-8-(phenyl-4-oxyacetate)-1-propylxanthine) binding in membranes of stably transfected HEK-293 cells revealed uniformly higher affinity (<10-fold) of these xanthines for human than for rat A2B adenosine receptors. Binding to rat brain membranes expressing A1 and A2A adenosine receptors revealed greater A2B selectivity over A2A than A1 receptors. Substitution at the 1-position with 2-phenylethyl (or alkyl/olefinic groups) and at N-3 with hydrogen or methyl favored A2B selectivity. Relative to enprofylline 2b, pentoxifylline 35 was equipotent and 1-propylxanthine 3 was >13-fold more potent at human A2B receptors. Most N-7 substituents did not enhance affinity over hydrogen, except for 7-(2-chloroethyl), which enhanced the affinity of theophylline by 6.5-fold to 800 nM. The A2B receptor affinity-enhancing effects of 7-(2-chloroethyl) vs 7-methyl were comparable to the known enhancement produced by an 8-aryl substitution. Among 8-phenyl analogues, a larger alkyl group at the 1-position than at the 3-position favored affinity at the human A2B receptor, as indicated by 1-allyl-3-methyl-8-phenylxanthine, with a K(i) value of 37 nM. Substitution on the 8-phenyl ring indicated that an electron-rich ring was preferred for A2B receptor binding. In conclusion, new leads for the design of xanthines substituted in the 1-, 3-, 7-, and 8-positions as A2B receptor-selective antagonists have been identified.