The role of receptor structure in determining adenosine receptor activity.

Adenosine produces a wide variety of physiological effects through the activation of cell surface adenosine receptors (ARs). ARs are members of the G-protein-coupled receptor family, and currently, four subtypes, the A1AR, A2AAR, A2BAR, and A3AR, are recognized. This review focuses on the role of receptor structure in governing various facets of AR activity. Ligand-binding properties of ARs are primarily dictated by amino acids in the transmembrane domains of the receptors, although a role for extracellular domains of certain ARs has been suggested. Studies have identified certain amino acids conserved amongst AR subtypes that are critical for ligand recognition, as well as additional residues that may differentiate between agonist and antagonist ligands. Receptor regions responsible for activation of Gs have been identified for the A2AAR. The location of these intracellular sites is consistent with findings described for other G-protein-coupled receptors. Site-directed mutagenesis has been employed to analyze the structural basis for the differences in the kinetics of the desensitization response displayed by various AR subtypes. For the A2AAR and A3AR, agonist-stimulated phosphorylation of the AR, presumably via a G-protein receptor kinase, has been shown to occur. For these AR subtypes, intracellular regions or individual amino acids that may be targets for this phosphorylation have been identified. Finally, the role of A1AR gene structure in regulating the expression of this AR subtype is reviewed.

Affinity chromatography of the bovine cerebral cortex A1 adenosine receptor.

An approximate 140-fold purification of the A1 adenosine receptor of bovine cerebral cortex has been obtained via affinity chromatography. The affinity column consists of Affi-Gel 10 coupled through an amide linkage to XAC, a high-affinity A1 adenosine receptor antagonist. As assessed by [3H]XAC binding, bovine brain membranes solubilized with the detergent CHAPS had a specific binding activity of 1.1 pmol/mg protein. Interaction of solubilized A1 adenosine receptors with the XAC-Affi-Gel was biospecific and 30% of the receptor activity was bound by the gel. Demonstration of [3H]XAC binding in the material eluted from the column with R-PIA required insertion of receptor into phospholipid vesicles. The specific activity of the affinity column purified receptor was 146 +/- 22 pmol/mg protein with typically 5-15% of the bound receptor recovered. The purified receptor displayed high-affinity antagonist binding and bound agonists with the potency order expected of the bovine brain A1 adenosine receptor: R-PIA greater than S-PIA greater than NECA. In purified preparations, the photoaffinity probe [125I]PAPAXAC-SANPAH specifically labelled a protein of molecular mass 38,000 which has previously been shown to be the A1 adenosine receptor binding subunit.

A role for central A3-adenosine receptors. Mediation of behavioral depressant effects.

The behavioral effects of a selective A3 adenosine receptor agonist 3-IB-MECA (N6-(3-iodobenzyl)-5'-N-methylcarboxamidoadenosine) in mice and the localization of radioligand binding sites in mouse brain were examined. Low levels of A3 adenosine receptors were detected in various regions of the mouse brain (hippocampus, cortex, cerebellum, striatum), using a radioiodinated, high-affinity A3-agonist radioligand [125I]AB-MECA (N6-(3-iodo-4-aminobenzyl)-5'-N-methylcarboxamidoadenosine). Scatchard analysis in the cerebellum showed that the Kd value for binding to A3 receptors was 1.39 +/- 0.04 nM with a Bmax of 14.8 +/- 2.1 fmol/mg protein. 3-IB-MECA at 0.1 mg/kg i.p. was a locomotor depressant with > 50% reduction in activity. Although selective A1 or A2a antagonists reversed locomotor depression elicited by selective A1 or A2a agonists, respectively, the behavioral depressant effects of 3-IB-MECA were unaffected. 3-IB-MECA also caused scratching in mice, which was prevented by coadministration of the histamine antagonist cyproheptadine. The demonstration of a marked behavioral effect of A3 receptor activation suggests that the A3 receptor represents a potential new therapeutic target.

Pharmacological characterization of novel A3 adenosine receptor-selective antagonists.

The effects of putative A3 adenosine receptor antagonists of three diverse chemical classes (the flavonoid MRS 1067, the 6-phenyl-1,4-dihydropyridines MRS 1097 and MRS 1191, and the triazoloquinazoline MRS 1220) were characterized in receptor binding and functional assays. MRS1067, MRS 1191 and MRS 1220 were found to be competitive in saturation binding studies using the agonist radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide) at cloned human brain A3 receptors expressed in HEK-293 cells. Antagonism was demonstrated in functional assays consisting of agonist-induced inhibition of adenylate cyclase and the stimulation of binding of [35S]guanosine 5'-O-(3-thiotriphosphate) ([35S]GTP-gamma-S) to the associated G-proteins. MRS 1220 and MRS 1191, with KB values of 1.7 and 92 nM, respectively, proved to be highly selective for human A3 receptor vs human A1 receptor-mediated effects on adenylate cyclase. In addition, MRS 1220 reversed the effect of A3 agonist-elicited inhibition of tumor necrosis factor-alpha formation in the human macrophage U-937 cell line, with an IC50 value of 0.3 microM.

Structure-activity relationships of 1,3-dialkylxanthine derivatives at rat A3 adenosine receptors.

1,3-Dialkylxanthine analogues containing carboxylic acid and other charged groups on 8-position substituents were synthesized. These derivatives were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells using the new radioligand [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-N-methyluronamide), and at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. A synthetic strategy for introducing multiple carboxylate groups at the 8-position using iminodiacetic acid derivatives was explored. The presence of a sulfonate, a carboxylate, or multiple carboxylate groups did not result in a significant enhancement of affinity at rat A3 receptors, although as previously observed an anionic group tended to diminish potency at A1 and A2a receptors. The rat A3 receptor affinity was not highly dependent on the distance of a carboxylate group from the xanthine pharmacophore. 2-Thio vs 2-oxo substitution favored A3 potency, and 8-alkyl vs 8-aryl substitution favored A3 selectivity, although few derivatives were truly selective for rat A3 receptors. 1,3-Dimethyl-8-(3-carboxypropyl)-2-thioxanthine was 7-fold selective for A3 vs A2a receptors. 1,3,7-Trimethyl-8-(trans-2-carboxyvinyl)xanthine was somewhat selective for A3 vs A1 receptors. For 8-arylxanthines affinity at A3 receptors was enhanced by 1,3-dialkyl substituents, in the order dibutyl > dipropyl > diallyl.

2-Substitution of N6-benzyladenosine-5'-uronamides enhances selectivity for A3 adenosine receptors.

Adenosine derivatives bearing an N6-(3-iodobenzyl) group, reported to enhance the affinity of adenosine-5'-uronamide analogues as agonists at A3 adenosine receptors (J. Med. Chem. 1994, 37, 636-646), were synthesized starting from methyl beta-D-ribofuranoside in 10 steps. Binding affinities at A1 and A2a receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells were compared. N6-(3-Iodobenzyl)adenosine was 2-fold selective for A3 vs A1 or A2a receptors; thus it is the first monosubstituted adenosine analogue having any A3 selectivity. The effects of 2-substitution in combination with modifications at the N6- and 5'-positions were explored. 2-Chloro-N6-(3-iodobenzyl)adenosine had a Ki value of 1.4 nM and moderate selectivity for A3 receptors. 2-Chloro-N6-(3-iodobenzyl)adenosine- 5'-N-methyluronamide, which displayed a Ki value of 0.33 nM, was selective for A3 vs A1 and A2a receptors by 2500- and 1400-fold, respectively. It was 46,000-fold selective for A3 receptors vs the Na(+)-independent adenosine transporter, as indicated in displacement of [3H]N6-(4- nitrobenzyl)-thioinosine binding in rat brain membranes. In a functional assay in CHO cells, it inhibited adenylate cyclase via rat A3 receptors with an IC50 of 67 nM. 2-(Methylthio)-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide and 2-(methylamino)-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide were less potent, but nearly as selective for A3 receptors. Thus, 2-substitution (both small and sterically bulky) is well-tolerated at A3 receptors, and its A3 affinity-enhancing effects are additive with effects of uronamides at the 5'-position and a 3-iodobenzyl group at the N6-position.

Selective ligands for rat A3 adenosine receptors: structure-activity relationships of 1,3-dialkylxanthine 7-riboside derivatives.

1,3-Dibutylxanthine 7-riboside has been found to be a partial agonist at A3 adenosine receptors (van Galen et al. Mol. Pharmacol. 1994, 45, 1101-1111). 1,3-Dialkylxanthine 7-riboside analogues modified at the 1-, 3-, and 8-purine positions and at the ribose 5'-position were synthesized. The nucleoside analogues were examined for affinity in radioligand binding assays at rat brain A3 adenosine receptors stably expressed in CHO cells, using the radioligand [[125I]-4-amino-3-iodobenzyl]adenosine-5'-N-methyluronamide (AB-MECA). Affinity was assayed at rat brain A1 and A2a receptors using [3H]PIA and [3H]CGS 21680, respectively. The affinity of xanthine 7-ribosides at A3 receptors depended on the 1,3-dialkyl substituents in the order: Pent > or = Bu >> Hx > Pr approximately Me. 1,3-Dipentylxanthine 7-riboside was slightly selective for A3 receptors (2-fold vs A1 and 10-fold vs A2a). 8-Methoxy substitution was tolerated at A3 receptors. 2-Thio vs 2-oxo substitution increased potency at all three subtypes and slightly increased A3 vs A1 selectivity. The 5'-uronamide modification, which was previously found to enhance A3 selectivity in N6-benzyladenosine derivatives, was also incorporated into the xanthine 7-ribosides, with similar results. The affinity of 1,3-dialkylxanthine 7-riboside 5'-uronamides at A3 receptors depended on the N-alkyluronamide substituent in the order: MeNH > EtNH >> NH2 >> Me2N. Affinity of the 5'-uronamides at A3 receptors was dependent on the 1,3-dialkyl substitution in the order: Bu > Pent > Hex. 1,3-Dibutylxanthine 7-riboside 5'-N-methylcarboxamide, with a Ki value of 229 nM at A3 receptors, was 160-fold selective for rat A3 vs A1 receptors and > 400-fold selective vs A2a receptors. This derivative acted as a full agonist in the A3 receptor-mediated inhibition of adenylate cyclase.

Interaction of 1,4-dihydropyridine and pyridine derivatives with adenosine receptors: selectivity for A3 receptors.

1,4-Dihydropyridine and pyridine derivatives bound to three subtypes of adenosine receptors in the micromolar range. Affinity was determined in radioligand binding assays at rat brain A1 and A2A receptors using [3H]-(R)-PIA [[3H]-(R)-N6-(phenylisopropyl)adenosine] and [3H]CGS 21680 [[3H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]-5'-(N-ethylcarbamoyl++ +) adenosine], respectively. Affinity was determined at cloned human and rat A3 receptors using [125I]AB-MECA [N6-(4-amino-3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine]. Structure-activity analysis at adenosine receptors indicated that sterically bulky groups at the 4-, 5-, and 6-positions are tolerated. (R,S)-Nicardipine, 12, displayed Ki values of 19.6 and 63.8 microM at rat A1 and A2A receptors, respectively, and 3.25 microM at human A3 receptors. Similarly, (R)-niguldipine, 14, displayed Ki values of 41.3 and 1.90 microM at A1 and A3 receptors, respectively, and was inactive at A2A receptors. A preference for the R- vs the S-enantiomer was observed for several dihydropyridines at adenosine receptors, in contrast with the selectivity at L-type Ca2+ channels. A 4-trans-beta-styryl derivative, 24, with a Ki value of 0.670 microM at A3 receptors, was 24-fold selective vs A1 receptors (Ki = 16.1 microM) and 74-fold vs A2A receptors (Ki = 49.3 microM). The affinity of 24 at L-type Ca2+ channels, measured in rat brain membranes using [3H]isradipine, indicated a Ki value of 0.694 microM, and the compound is thus nonselective between A3 receptors and L-type Ca2+ channels. Inclusion of a 6-phenyl group enhanced A3 receptor selectivity: Compound 28 (MRS1097; 3,5-diethyl 2-methyl-6-phenyl-4-(trans-2-phenylvinyl)-1,4(R,S)-dihydro-pyridin e-3, 5-dicarboxylate) was 55-fold selective vs A1 receptors, 44-fold selective vs A2A receptors, and over 1000-fold selective vs L-type Ca2+ channels. In addition, compound 28 attenuated the A3 agonist-elicited inhibitory effect on adenylyl cyclase. Furthermore, whereas nicardipine, 12, displaced radioligand from the Na(+)-independent adenosine transporter with an apparent affinity of 5.36 +/- 1.51 microM, compound 28 displaced less than 10% of total binding at a concentration of 100 microM. Pyridine derivatives, when bearing a 4-alkyl but not a 4-phenyl group, maintained affinity for adenosine receptors. These findings indicate that the dihydropyridines may provide leads for the development of novel, selective A3 adenosine antagonists.

Structure-activity relationships of 9-alkyladenine and ribose-modified adenosine derivatives at rat A3 adenosine receptors.

9-Alkyladenine derivatives and ribose-modified N6-benzyladenosine derivatives were synthesized in an effort to identify selective ligands for the rat A3 adenosine receptor and leads for the development of antagonists. The derivatives contained structural features previously determined to be important for A3 selectivity in adenosine derivatives, such as an N6-(3-iodobenzyl) moiety, and were further substituted at the 2-position with halo, amino, or thio groups. Affinity was determined in radioligand binding assays at rat brain A3 receptors stably expressed in Chinese hamster ovary (CHO) cells, using [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)adenosine-5'-(N-methyluronamide)), and at rat brain A1 and A2a receptors using [3H]-N6-PIA ((R)-N6-phenylisopropyladenosine) and [3H]CGS 21680 (2-[[[4-(2-carboxyethyl)-phenyl]ethyl]amino]-5'- (N-ethylcarbamoyl)adenosine), respectively. A series of N6-(3-iodobenzyl) 2-amino derivatives indicated that a small 2-alkylamino group, e.g., methylamino, was favored at A3 receptors. N6-(3-Iodobenzyl)-9-methyl-2-(methylthio)adenine was 61-fold more potent than the corresponding 2-methoxy ether at A3 receptors and of comparable affinity at A1 and A2a receptors, resulting in a 3-6-fold selectivity for A3 receptors. A pair of chiral N6-(3-iodobenzyl) 9-(2,3-dihydroxypropyl) derivatives showed stereoselectivity, with the R-enantiomer favored at A3 receptors by 5.7-fold. 2-Chloro-9-(beta-D-erythrofuranosyl)-N6-(3-iodobenzyl)adenine had a Ki value at A3 receptors of 0.28 microM. 2-Chloro-9-[2-amino-2,3-dideoxy-beta-D-5-(methylcarbamoyl)- arabinofuranosyl]-N6-(3-iodobenzyl)adenine was moderately selective for A1 and A3 vs A2a receptors. A 3'-deoxy analogue of a highly A3-selective adenosine derivative retained selectivity in binding and was a full agonist in the inhibition of adenylyl cyclase mediated via cloned rat A3 receptors expressed in CHO cells. The 3'-OH and 4'-CH2OH groups of adenosine are not required for activation at A3 receptors. A number of 2',3'-dideoxyadenosines and 9-acyclic-substituted adenines appear to inhibit adenylyl cyclase at the allosteric "P" site.

Novel N6-(substituted-phenylcarbamoyl)adenosine-5'-uronamides as potent agonists for A3 adenosine receptors.

A series of adenosine-5'-uronamide derivatives bearing N6-phenylurea groups have been synthesized and tested for their affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells. Some N6-arylcarbamoyl derivatives, N6-((2-chlorophenyl)carbamoyl)-, N6-((3-chlorophenyl)carbamoyl)-, and N6-((4-methoxyphenyl)carbamoyl)adenosine-5'-ethyluronamide (4l-n), were found to have affinity at A3 receptors in the low nanomolar range (Ki values < 10 nM). In CHO cells stably transfected with the rat A3 receptor, compound 4n was found to be a full agonist in inhibiting adenylate cyclase activity. The present study represents the first example of N6-acyl-substituted adenosine analogs having high affinity at adenosine receptors and, in particular, at the A3 receptor subtype.

Synthesis and biological activities of flavonoid derivatives as A3 adenosine receptor antagonists.

A broad screening of phytochemicals has demonstrated that certain flavone and flavonol derivatives have a relatively high affinity at A3 adenosine receptors, with Ki values of > or = 1 microM (Ji et al. J. Med. Chem. 1996, 39, 781-788). We have further modified the flavone structure to achieve a degree of selectivity for cloned human brain A3 receptors, determined in competitive binding assays versus [125I]AB-MECA[N6-(4-amino-3-iodobenzyl)adenosine-5'-(N-methylur onamide)]. Affinity was determined in radioligand binding assays at rat brain A1 and A2a receptors using [3H]-N6-PIA ([3H]-(R)-N6-phenylisopropyladenosine) and [3H]CGS21680 [[3H]-2-[[4-(2-carboxyethyl)phenyl]ethylamino]-5'-(N-ethylcarbamoyl++ +)adenosine], respectively. The triethyl and tripropyl ether derivatives of the flavonol galangin, 4, had Ki values of 0.3 - 0.4 microM at human A3 receptors. The presence of a 5-hydroxyl group increased selectivity of flavonols for human A3 receptors. The 2',3,4',7-tetraethyl ether derivative of the flavonol morin, 7, displayed a Ki value of 4.8 microM at human A3 receptors and was inactive at rat A1/A2a receptors. 3,6-Dichloro-2'-(isopropyloxy)-4'-methylflavone, 11e, was both potent and highly selective (approximately 200-fold) for human A3 receptors (Ki = 0.56 microM). Among dihydroflavonol analogues, the 2-styryl instead of the 2-aryl substituent, in 15, afforded selectivity for human A3 vs rat A1 or A2A receptors. The 2-styryl-6-propoxy derivative, 20, of the furanochromone visnagin was 30-fold selective for human A3 receptors vs either rat A1 or A2A receptors. Several of the more potent derivatives effectively antagonized the effects of an agonist in a functional A3 receptor assay, i.e. inhibition of adenylyl cyclase in CHO cells expressing cloned rat A3 receptors. In conclusion, these series of flavonoids provide leads for the development of novel potent and subtype selective A3 antagonists.

Selective A(3) adenosine receptor antagonists: water-soluble 3, 5-diacyl-1,2,4-trialkylpyridinium salts and their oxidative generation from dihydropyridine precursors.

A(3) adenosine receptor antagonists are sought for their potential antiinflammatory, antiasthmatic, and antiischemic properties. We have found that 3,5-diacyl-1,2,4-trialkyl-6-phenylpyridinium derivatives constitute a novel class of selective A(3) adenosine receptor antagonists. The structure-activity relationships of this class of antagonists, incorporating the 3-thioester, have been explored. The most potent analogue in this group was 2, 4-diethyl-1-methyl-3-(ethylsulfanylcarbonyl)-5-ethyloxycarbonyl -6-phe nylpyridinium iodide (11), which had an equilibrium inhibition constant (K(i)) value of 219 nM at human A(3) receptors (binding of [(125)I]AB-MECA (N(6)-(4-amino-3-iodobenzyl)-5'-N-methylcarbamoyladenosine)) expressed in Chinese hamster ovary (CHO) cells and >10 microM at rat brain A(1) and A(2A) receptors and at recombinant human A(2B) receptors. Compound 11 could be generated through oxidation of the corresponding 3,5-diacyl-1,2,4-trialkyl-6-phenyl-1,4-dihydropyridine, 24, with iodine or in the presence of rat brain homogenates. A 6-cyclopentyl analogue was shown to increase affinity at human A(3) receptors upon oxidation from the 1-methyl-1,4-dihydropyridine analogue, 25, to the corresponding pyridinium derivative, 23 (K(i) 695 nM), suggesting a prodrug scheme. Homologation of the N-methylpyridinium derivatives to N-ethyl and N-propyl at the 1-position caused a progressive reduction in the affinity at A(3) receptors. Modifications of the alkyl groups at the 2-, 3-, 4-, and 5-positions failed to improve potency in binding at A(3) receptors. The pyridinium antagonists are not as potent as other recently reported, selective A(3) receptor antagonists; however, they display uniquely high water solubility (43 mM for 11). Compound 11 antagonized the inhibition of adenylate cyclase elicited by IB-MECA in CHO cells expressing the human A(3) adenosine receptor, with a K(B) value of 399 nM, and did not act as an agonist, demonstrating that the pyridinium salts are pure antagonists.

Structure-activity relationships of N6-benzyladenosine-5'-uronamides as A3-selective adenosine agonists.

Adenosine analogues modified at the 5'-position as uronamides and/or as N6-benzyl derivatives were synthesized. These derivatives were examined for affinity in radioligand binding assays at the newly discovered rat brain A3 adenosine receptor and at rat brain A1 and A2a receptors. 5'-Uronamide substituents favored A3 selectivity in the order N-methyl > N-ethyl approximately unsubstituted carboxamide > N-cyclopropyl. 5'-(N-Methylcarboxamido)-N6-benzyladenosine was 37-56-fold more selective for A3 receptors. Potency at A3 receptors was enhanced upon substitution of the benzyl substituent with nitro and other groups. 5'-N-Methyluronamides and N6-(3-substituted-benzyl)adenosines are optimal for potency and selectivity at A3 receptors. A series of 3-(halobenzyl)-5'-N-ethyluronamide derivatives showed the order of potency at A1 and A2a receptors of I approximately Br > Cl > F. At A3 receptors the 3-F derivative was weaker than the other halo derivatives. 5'-N-Methyl-N6-(3-iodobenzyl)adenosine displayed a Ki value of 1.1 nM at A3 receptors and selectivity versus A1 and A2a receptors of 50-fold. A series of methoxybenzyl derivatives showed that a 4-methoxy group best favored A3 selectivity. A 4-sulfobenzyl derivative was a specific ligand at A3 receptors of moderate potency. An aryl amino derivative was prepared as a probe for radioiodination and receptor cross-linking.

Search for new purine- and ribose-modified adenosine analogues as selective agonists and antagonists at adenosine receptors.

The binding affinities at rat A1, A2a, and A3 adenosine receptors of a wide range of derivatives of adenosine have been determined. Sites of modification include the purine moiety (1-, 3-, and 7-deaza; halo, alkyne, and amino substitutions at the 2- and 8-positions; and N6-CH2-ring, -hydrazino, and -hydroxylamino) and the ribose moiety (2'-, 3'-, and 5'-deoxy; 2'- and 3'- O-methyl; 2'-deoxy 2'-fluoro; 6'-thio; 5'-uronamide; carbocyclic; 4'- or 3'-methyl; and inversion of configuration). (-)- and (+)-5'-Noraristeromycin were 48- and 21-fold selective, respectively, for A2a vs A1 receptors. 2-Chloro-6'-thioadenosine displayed a Ki value of 20 nM at A2a receptors (15-fold selective vs A1). 2-Chloroadenin-9-yl(beta-L-2'-deoxy-6'- thiolyxofuranoside) displayed a Ki value of 8 microM at A1 receptors and appeared to be an antagonist, on the basis of the absence of a GTP-induced shift in binding vs a radiolabeled antagonist (8-cyclopentyl-1,3-dipropyl-xanthine). 2-Chloro-2'-deoxyadenosine and 2-chloroadenin-9-yl(beta-D-6'-thioarabinoside) were putative partial agonists at A1 receptors, with Ki values of 7.4 and 5.4 microM, respectively. The A2a selective agonist 2-(1-hexynyl)-5'-(N-ethylcarbamoyl)adenosine displayed a Ki value of 26 nM at A3 receptors. The 4'-methyl substitution of adenosine was poorly tolerated, yet when combined with other favorable modifications, potency was restored. Thus, N6-benzyl-4'-methyladenosine-5'-(N-methyluronamide) displayed a Ki value of 604 nM at A3 receptors and was 103- and 88-fold selective vs A1 and A2a receptors, respectively. This compound was a full agonist in the A3-mediated inhibition of adenylate cyclase in transfected CHO cells. The carbocyclic analogue of N6-(3-iodobenzyl)adenosine-5'-(N-methyluronamide) was 2-fold selective for A3 vs A1 receptors and was nearly inactive at A2a receptors.

Identification of A2a adenosine receptor domains involved in selective coupling to Gs. Analysis of chimeric A1/A2a adenosine receptors.

Responses to adenosine are governed by selective activation of distinct G proteins by adenosine receptor (AR) subtypes. The A2aAR couples via Gs to adenylyl cyclase stimulation while the A1AR couples to Gi to inhibit adenylyl cyclase. To determine regions of the A2aAR that selectively couple to Gs, chimeric A1/A2aARs were expressed in Chinese hamster ovary cells and ligand binding and adenylyl cyclase activity analyzed. Replacement of the third intracellular loop of the A2aAR with that of the A1AR reduced maximal adenylyl cyclase stimulation and decreased agonist potency. Restricted chimeras indicated that the NH2-terminal portion of intracellular loop 3 was predominantly responsible for this impairment. Reciprocal chimeras composed primarily of A1AR sequence with limited A2aAR sequence substitution stimulated adenylyl cyclase and thus supported these findings. A lysine and glutamic acid residue were identified as necessary for efficient A2aAR-Gs coupling. Analysis of chimeric receptors in which sequence of intracellular loop 2 was substituted indicated that the nature of amino acids in this domain may indirectly modulate A2aAR-Gs coupling. Replacement of the cytoplasmic tail of the A2aAR with the A1AR tail did not affect adenylyl cyclase stimulation. Thus, selective activation of Gs is predominantly dictated by the NH2-terminal segment of the third intracellular loop of the A2aAR.

Age-related changes in responsiveness of the rat aorta to depolarizing and receptor-mediated contractile stimuli and to calcium antagonism.

This study was undertaken to determine the relative age-dependent responsiveness of the rat aorta to depolarizing (potassium) and receptor-activating (norepinephrine) contractile stimulants, and to the calcium antagonists propyl-methylenedioxyindene (pr-MDI) and nifedipine. Pr-MDI exhibits intracellular calcium antagonistic and calcium channel blocking properties in this tissue, while nifedipine acts principally as a calcium channel blocker. Thoracic strips from young (4-6 months old) and senescent (22-23 months old) Fischer F344 rats were contracted with KCl (10-60 mmol/l) or norepinephrine (10(-9)-5 X 10(-6) mol/l). Aortae from old rats were significantly more sensitive to norepinephrine than aortae from young rats, while the reverse was observed for KCl. Pr-MDI (10(-5)-10(-4) mol/l) significantly relaxed the aortic contractions induced by norepinephrine (10(-7) mol/l, a nondepolarizing concentration producing 88% of maximum response in young and old aortae) and by KCl (50 mmol/l, a depolarizing concentration producing 96% of maximum response in young and old aortae). However, there were no age-related differences in sensitivity to the relaxant effects of pr-MDI against either stimulant. Pr-MDI was more effective in relaxing KCl-induced contractions than those induced by norepinephrine. Similar results were obtained with nifedipine (10(-10)-10(-6) mol/l). These results indicate that senescence of the rat aorta is accompanied by an enhanced responsiveness of adrenergic alpha-receptor-mediated contraction, a reduced responsiveness to depolarizing stimuli, and no change in sensitivity to calcium antagonism.

Differential effects of calcium channel blockade and intracellular calcium antagonism on endothelium-dependent responses of the rat aorta to drugs.

The actions of many vasoactive drugs are mediated through, or modified by, the endothelium-derived relaxing (EDRF) and constricting (EDCF) factors. While EDRF appears to be nitric oxide, EDCF is a peptide or cyclooxygenase product. Using verapamil (a calcium channel blocker), propyl methylenedioxyindene (pr-MDI; an intracellular calcium antagonist), and sodium nitroprusside (which liberates nitric oxide from its molecular structure) as EDRF-independent pharmacological probes in rat aortic rings with and without endothelium, we attempted to provide additional insight into the role of extracellular [( Ca]o) and intracellular [( Ca]i) calcium in EDRF and EDCF release and action, and to explain some mechanisms underlying the modulatory effects of these endothelial factors on the actions of vasoactive drugs. The findings suggest that (1) the [Ca]o required for evoked EDRF release does not enter endothelial cells through verapamil-sensitive calcium channels; (2) mobilization of endoplasmic reticular [Ca]i by [Ca]o entering the endothelial cell may be the trigger for evoked EDRF release; (3) spontaneous release of EDRF appears to depend more on mobilization of [Ca]i than on influx of [Ca]o; (4) the action of EDRF on smooth muscle either does not require Ca or does not involve the mobilization of [Ca]i by [Ca]o; (5) Both EDRF and EDCF can modulate the actions of vasoactive drugs; (6) the EDCF of the rat aorta is not a cyclooxygenase product, and (7) the action of EDCF on vascular smooth muscle, and possibly its release from endothelial cells, are Ca-dependent.

Evaluation of the antiabortifacient and embryotoxic effects of methylenedioxyindene and methylenedioxyindan calcium antagonists.

Calcium channel blockers have been advocated as potential therapeutic agents in the management of premature labor. In the present study, the class of intracellular calcium antagonistic methylenedioxyindenes (MDIs) was investigated for potential antiabortifacient activity in mice. Pretreatment of pregnant mice from day 15 of gestation with the MDIs did not afford protection against the abortifacient effect of prostaglandin F2alpha administered from day 17 of gestation. The MDIs demonstrated embryotoxic and fetotoxic activity as shown by a significant increase in the incidence of resorptions and stillbirths. Similar embryotoxicity was previously reported for the calcium channel blockers. It appears doubtful that any of the calcium antagonists so far examined will be clinically useful in the management of premature labor.

PIP: 2 intracellular calcium antagonists with weak membrane calcium channel blocking activity were tested for antiabortifacient and embryotoxic effects in mice. The compounds were 2-n-propyl-3-dimethylamino-5,6-methylenedioxyindene (pr-MDI) and cis-2-n-butyl-3-dimethylamino-5,6-methylenedioxindan (cis-H-bu-MDI). Charles River CD-1 mice treated intramuscularly with saline (controls), or Pgf2alpha twice daily to induce premature abortion starting on day 17 of gestation, with 15 or 25 mg/kg cis-H-bu-MDI. or 70 mg/kg/pr-MDI. the LD50 for cis-H-bu-MDI was 75 mg/kg (single intramuscular dose). The compounds had no effect on premature delivery. They significantly decreased numbers of live-born pups, however, counted as the difference between implantation sites and recovered live or stillborn pups. There were no malformations, maternal toxicity or detrimental effects of survival or term-born progeny. These calcium antagonists appear to be of no value in the management of premature labor.

Adenosine receptors.

Our knowledge of A1 and A2ARs has grown dramatically since they were first defined by Burnstock. With the recent purification of the A1AR and the cDNA cloning of the A2AR, an even more rapid expansion of information regarding their structure, function, and regulation should now ensue. We may then be able to return to the intact cell and organ system and better understand the physiological role of these ubiquitous receptors.