Adenosine receptors and cancer.

The A(1), A(2A), A(2B) and A(3) G-protein-coupled cell surface adenosine receptors (ARs) are found to be upregulated in various tumor cells. Activation of the receptors by specific ligands, agonists or antagonists, modulates tumor growth via a range of signaling pathways. The A(1)AR was found to play a role in preventing the development of glioblastomas. This antitumor effect of the A(1)AR is mediated via tumor-associated microglial cells. Activation of the A(2A)AR results in inhibition of the immune response to tumors via suppression of T regulatory cell function and inhibition of natural killer cell cytotoxicity and tumor-specific CD4+/CD8+ activity. Therefore, it is suggested that pharmacological inhibition of A(2A)AR activation by specific antagonists may enhance immunotherapeutics in cancer therapy. Activation of the A(2B)AR plays a role in the development of tumors via upregulation of the expression levels of angiogenic factors in microvascular endothelial cells. In contrast, it was evident that activation of A(2B)AR results in inhibition of ERK1/2 phosphorylation and MAP kinase activity, which are involved in tumor cell growth signals. Finally, A(3)AR was found to be highly expressed in tumor cells and tissues while low expression levels were noted in normal cells or adjacent tissue. Receptor expression in the tumor tissues was directly correlated to disease severity. The high receptor expression in the tumors was attributed to overexpression of NF-kappaB, known to act as an A(3)AR transcription factor. Interestingly, high A(3)AR expression levels were found in peripheral blood mononuclear cells (PBMCs) derived from tumor-bearing animals and cancer patients, reflecting receptor status in the tumors. A(3)AR agonists were found to induce tumor growth inhibition, both in vitro and in vivo, via modulation of the Wnt and the NF-kappaB signaling pathways. Taken together, A(3)ARs that are abundantly expressed in tumor cells may be targeted by specific A(3)AR agonists, leading to tumor growth inhibition. The unique characteristics of these A(3)AR agonists make them attractive as drug candidates.

The interaction of N-formyl peptide chemoattractant receptors with the membrane skeleton is energy-dependent.

Desensitization of N-formyl peptide chemoattractant receptors (FPR) in human neutrophils is thought to be achieved by lateral segregation of receptors and G proteins within the plane of the plasma membrane resulting in an interruption of the signalling cascade. Direct coupling of FPR to membrane skeletal actin appears to be the basis of this process; however, the molecular mechanism is unknown. In this study we investigated the effect of energy depletion on formation of FPR-membrane skeleton complexes. In addition the effect of the protein kinase C inhibitor stauroporine and the phosphatase inhibitor okadaic acid on coupling of FPR to the membrane skeleton was studied. Human neutrophils were desensitized using the photoreactive agonist N-formyl-met-leu-phe-lys-N'-[125I]2(p-azidosalicylamido)ethyl-1,3' - dithiopropionate (fMLFK-[125I]ASD) after ATP depletion with NaF or after incubation with the respective inhibitors. The interaction of FPR with the membrane skeleton was studied by sedimentation of the membrane skeleton-associated receptors in sucrose density gradients. Energy depletion of the cells markedly inhibited the formation of FPR-membrane skeleton complexes. This does not appear to be related to inhibition of protein phosphorylation due to ATP depletion because inhibition of protein kinases and phosphatases had no significant effect on coupling of FPR to the membrane skeleton. We conclude, therefore, that coupling of FPR to the membrane skeleton is an energy-dependent process which does not appear to require modification of the receptor protein by phosphorylation.

G-protein-coupled A1 adenosine receptors in coated vesicles of mammalian brain: characterization by radioligand binding and photoaffinity labelling.

A1 adenosine receptors in coated vesicles have been characterized by radioligand binding and photoaffinity labelling. Saturation experiments with the antagonist 8-cyclopentyl-1,3-[3H]dipropyl-xanthine ([3H]DPCPX) gave a Kd value of 0.7 nM and a Bmax value of 82 +/- 13 fmol/mg protein. For the highly A1-selective agonist 2-chloro-N6-[3H]cyclopentyladenosine ([3H]CCPA) a Kd value of 1.7 nM and a Bmax value of 72 +/- 29 fmol/mg protein was estimated. Competition of agonists for [3H]DPCPX binding gave a pharmacological profile with R-N6-phenylisopropyladenosine (R-PIA) > CCPA > S-PIA > 5'-N-ethylcarboxamidoadenosine (NECA), which is identical to brain membranes. The competition curves were best fitted according to a two-site model, suggesting the existence of two affinity states. GTP shifted the competition curve for CCPA to the right and only one affinity state similar to the low affinity state in the absence of GTP was detected. The photoreactive agonist 2-azido-N6-125I-p-hydroxyphenylisopropyladenosine ([125I]AHPIA) specifically labelled a single protein with an apparent molecular weight of 35,000 in coated vesicles, which is identical to A1 receptors labelled in brain membranes. Therefore, coated vesicles contain A1 adenosine receptors with similar binding characteristics as membrane-bound receptors, including GTP-sensitive high-affinity agonist binding. Photoaffinity labelling data suggest that A1 receptors in these vesicles are not a processed receptor form. These results confirm that A1 receptors in coated vesicles are coupled to a G-protein, and it appears that the A1 receptor systems in coated vesicles and in plasma membranes are identical.

A carboxyl-terminal tail peptide of neutrophil chemotactic receptor disrupts its physical complex with G protein.

The binding of G protein to the N-formyl peptide receptor of human neutrophils was investigated with site-specific synthetic peptides. Peptide CT336(322) (322RALTEDSTQTSDTAT336) from the carboxyl-terminal tail region of the receptor competed with the receptor for binding to bovine Gi protein. The peptide competition was assayed by dissociation of a GTP-sensitive, rapidly sedimenting (7S) form of receptor-G protein complex as analyzed by velocity sedimentation on linear sucrose density gradients. An IC50 of 590 microM was determined for CT336(322) peptide. A control peptide, with the reverse sequence, rCT322(336) (336TATDSTQTSDETLAR322), did not perturb the sedimentation of the reconstituted receptor-G protein complex up to the highest tested concentration, 3 mM. Other peptides tested, corresponding to central portions of the predicted intracellular loop regions CII140(127) (127VLHPVWTQNHRTVS140) and CIII239(227) (227KIHKQGLIKSSRP239) of the receptor, failed to dissociate the reconstituted receptor-G protein complex. Control peptides from the extracellular region EII184(170) (170KTGTVACTFNFSPWT184) and an unrelated sequence matching a portion of neutrophil cytochrome b, CYT306(296) (296KVVITKVVTHPFKTIE306), were also ineffective. Our results suggest that the cytoplasmic tail of the formyl chemotactic peptide receptor is involved in its coupling to the signal-transducing G protein.

Synthesis, biological and modeling studies of 1,3-di-n-propyl-2,4-dioxo-6-methyl-8-(substituted) 1,2,3,4-tetrahydro [1,2,4]-triazolo [3,4-f]-purines as adenosine receptor antagonists.

A new series of potential adenosine receptor antagonists with a [1,2,4]-triazolo-[3,4-f]-purine structure bearing at the 1 and 3 position n-propyl groups have been synthesized, and their affinities at the four human adenosine receptor subtypes (A(1), A(2A), A(2B) and A(3)) have been evaluated. In this case the presence of n-propyl groups seems to induce potency at the A(2A) and A(3) adenosine receptor subtypes as opposed to our previously reported series bearing methyl substituents at the 1 and 3 positions. In particular the non-acylated derivative 17 showed affinity at these two receptor subtypes in the micromolar range. Indeed, preliminary molecular modeling investigations according to the experimental binding data indicate a modest steric and electrostatic antagonist-receptor complementarity.

A facile and novel synthesis of N(2)-, C(6)-substituted pyrazolo[3,4-d]pyrimidine-4 carboxylate derivatives as adenosine receptor antagonists.

An efficient synthetic procedure was adopted to synthesize a series of new molecules containing the pyrazolo[3,4-d]pyrimidine (PP) scaffold, which have been evaluated as promising human adenosine receptor (AR) antagonists. The effect of substitutions at the N(2), C(4) and C(6) positions of PPs on the affinity and selectivity towards the adenosine receptors were explored. Most of the pyrazolo[3,4-d]pyrimidine-4-carboxylates displayed from moderate to good affinity at the human A3AR (hA3AR), as indicated by the low micromolar range of Ki values (Ki hA3AR = 0.7-34 µM). In particular, compounds 60 and 62 displayed good affinity at the hA3AR (60, Ki hA3AR = 2.2 µM and 62, Ki hA3AR = 2.9 µM) and selectivity towards the other AR subtypes (60, >46-fold selective and 62, >34-fold selective, respectively). In view of these results, these novel PP analogues were docked both in the crystallographic structure of the hA2AAR and in a homology model of the hA3AR in order to support the structure activity relationship (SAR) analysis. These preliminary results demonstrated that pyrazolo[3,4-d]pyrimidine can be considered a promising scaffold to obtain new molecules with potent hA3AR antagonist activity.

High-affinity binding and lack of growth-promoting activity of 12(S)-hydroxyeicosatetraenoic acid (12(S)-HETE) in a human epidermal cell line.

The arachidonic acid metabolite 12-hydroxyeicosatetraenoic acid (12-HETE) is assumed to play an important role in skin physiology and pathophysiology. Specifically, it has recently been discussed as a growth promoting agent in keratinocytes. Our aim was to find out whether epidermal cells possess specific receptors for 12-HETE which would mediate the effects of this eicosanoid in skin, including the putative growth stimulating activity. We could identify specific binding sites for 12(S)-HETE on the human epidermal cell line SCL-II. The analysis of binding data revealed a single class of binding sites with a Kd of 2,6 nM and a Bmax of 216,000 sites per cell. The binding was saturable, readily reversible, and specific for 12(S)-HETE with lower affinities for other monoHETE. We failed to detect any significant proliferative activity of 12(S)-HETE in the same epidermal cell line, although we applied three independent methods for evaluation of cell growth and used a concentration of 12(S)-HETE which should enable an optimal receptor occupancy. Thus, epidermal cells possess high-affinity 12(S)-HETE binding sites which are likely to be involved in the effects of this eicosanoid in epidermis. However, biologic effects other than direct growth stimulation seem to be transduced by 12(S)-HETE receptors in epidermal cells which need further investigation.

Radiation inactivation analysis of the A1 adenosine receptor of rat brain. Decrease in radiation inactivation size in the presence of guanine nucleotide.

Radiation inactivation analysis of the binding of the A1 adenosine receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine to rat brain membranes yielded a radiation inactivation size of 58 kDa. In the presence of GTP gamma S this was reduced to 33 kDa, in good agreement with the size of the ligand-binding subunit detected after photoaffinity labelling. The data indicate that the structural association of A1 adenosine receptors with G-protein components is altered in situ in the presence of guanine nucleotides.

Water-soluble phosphate prodrugs of 1-propargyl-8-styrylxanthine derivatives, A(2A)-selective adenosine receptor antagonists.

Water-soluble prodrugs of potent, A(2A)-selective adenosine receptor (AR) antagonists were prepared. 8-(m-Bromostyryl)-3, 7-dimethyl-1-propargylxanthine (BS-DMPX, 11) and the analogous 8-(m-methoxystyryl)xanthine derivative (MS-DMPX, 5b) were used as starting points. It was found that polar functional groups suitable for the attachment of a prodrug moiety were tolerated on the styryl ring and even better on the 3-substituent. 8-(m-Hydroxystyryl)-DMPX (7) and 3-(3-hydroxypropyl)-8-(m-methoxystyryl)-1-propargylxanthine (5e, MSX-2) were the most potent and A(2A)-selective compounds and were selected for prodrug formation. For the preparation of 5e a new ring-closure method was applied. Treatment of 6-amino-1-(3-hydroxypropyl)-5-(m-methoxycinnamoylamino)-3-propa rgylur acil with hexamethyldisilazane at high temperature resulted in higher yields of the target xanthine than the standard ring-closure procedure using sodium hydroxide. Phosphate prodrugs were prepared by classical phosphorylation using phosphorus oxychloride and alternatively by using a phosphoramidite method. Phosphates of the aliphatic alcohol 5e could be obtained by both methods in similar yields. The phenolic compound 7, however, could be phosphorylated only by using the phosphoramidite method. The disodium salts of the phosphate prodrugs exhibited high water solubility (8-(m-methoxystyryl)-7-methyl-3-[3-O-phosphatylpropyl]-1- propargylxan thine disodium salt, 9b: 17 mM, 9 mg/mL). Prodrug 9b was found to be stable in aqueous solution (pH 7) but readily cleaved by phosphatases to liberate 5e (MSX-2). Compound 5e showed high affinity for rat A(2A) AR (K(i) = 8 nM), human recombinant A(2A) AR (K(i) = 5 nM), and human native A(2A) AR (K(i) = 15 nM) and was highly selective versus rat A(1) AR (110-fold), human recombinant A(2A) AR (500-fold), human A(2B) AR (>2000-fold), and human A(3) AR (>2000-fold).

Adenosine receptor agonists: synthesis and biological evaluation of 1-deaza analogues of adenosine derivatives.

In a search for more selective A1 adenosine receptor agonists, N6-[(R)-(-)-1-methyl-2-phenethyl]-1-deazaadenosine (1-deaza-R-PIA, 3a), N6-cyclopentyl-1-deazaadenosine (1-deazaCPA, 3b), N6-cyclohexyl-1-deazaadenosine (1-deazaCHA, 3c), and the corresponding 2-chloro derivatives 2a-c were synthesized from 5,7-dichloro-3-beta-D-ribofuranosyl-3H-imidazo[4,5-b]pyridine. On the other hand, N-ethyl-1'-deoxy-1'-(1-deaza-6-amino-9H-purin-9-yl)-beta-D-ribofuranu ronamide (1-deazaNECA, 10) was prepared from 7-nitro-3-beta-D-ribofuranosyl-3H-imidazo[4,5-b]pyridine, in an attempt to find a more selective A2 agonist. The activity of all deaza analogues at adenosine receptors has been determined in adenylate cyclase and in radioligand binding studies. 1-DeazaNECA proved to be a nonselective agonist at both subtypes of the adenosine receptor. It is about 10-fold less active than NECA but clearly more active than the parent compound 1-deazaadenosine as an inhibitor of platelet aggregation and as a stimulator of cyclic AMP accumulation. The N6-substituted 1-deazaadenosines largely retain the A1 agonist activity of their parent compounds, but lose some of their A2 agonist activity. This results in A1-selective compounds, of which N6-cyclopentyl-2-chloro-1-deazaadenosine (1-deaza-2-Cl-CPA, 2b) was identified as the most selective agonist at A1 adenosine receptors so far known. The activity of all 1-deaza analogues confirms that the presence of the nitrogen atom at position 1 of the purine ring is not critical for A1 receptor mediated adenosine actions.

2-Alkynyl derivatives of adenosine and adenosine-5'-N-ethyluronamide as selective agonists at A2 adenosine receptors.

In the search for more selective A2-receptor agonists and on the basis that appropriate substitution at C2 is known to impart selectivity for A2 receptors, 2-alkynyladenosines 2a-d were resynthesized and evaluated in radioligand binding, adenylate cyclase, and platelet aggregation studies. Binding of [3H]NECA to A2 receptors of rat striatal membranes was inhibited by compounds 2a-d with Ki values ranging from 2.8 to 16.4 nM. 2-Alkynyladenosines also exhibited high-affinity binding at solubilized A2 receptors from human platelet membranes. Competition of 2-alkynyladenosines 2a-d for the antagonist radioligand [3H]DPCPX and for the agonist [3H]CCPA gave Ki values in the nanomolar range, and the compounds showed moderate A2 selectivity. In order to improve this selectivity, the corresponding 2-alkynyl derivatives of adenosine-5'-N-ethyluronamide 8a-d were synthesized and tested. As expected, the 5'-N-ethyluronamide derivatives retained the A2 affinity whereas the A1 affinity was attenuated, resulting in an up to 10-fold increase in A2 selectivity. A similar pattern was observed in adenylate cyclase assays and in platelet aggregation studies. A 30- to 45-fold selectivity for platelet A2 receptors compared to A1 receptors was found for compounds 8a-c in adenylate cyclase studies.

7-Deazaadenines bearing polar substituents: structure-activity relationships of new A(1) and A(3) adenosine receptor antagonists.

A series of 28 new pyrrolo[2,3-d]pyrimidine-4-amines, pyrimido[4, 5-b]indole-4-amines, and tetrahydropyrimido[4,5-b]indole-4-amines was synthesized and their adenosine receptor affinity determined in radioligand binding assays at rat A(1) and A(2A) adenosine receptors (ARs). Selected compounds were additionally investigated in binding assays at recombinant A(3) ARs. The 2-phenyl residue in (R)-7-(1-methylbenzyl)-2-phenylpyrrolo[2,3-d]pyrimidine-4-amine (ADPEP, 1) and in the corresponding pyrimido[4,5-b]indole (APEPI, 3) could be bioisosterically replaced by heterocyclic rings, such as 2-thienyl and 4-pyridyl. The resulting compounds retained high affinity and selectivity for A(1) ARs. Judging from the investigation of selected compounds, it appears that they are also potent at human A(1) ARs and selective not only versus A(2A) ARs but also highly selective versus A(2B) and A(3) ARs. The p-pyridyl-substituted derivatives 11 and 27 (APPPI) may be interesting pharmacological tools due to their fluorescent properties. Pyrrolo[2,3-d]pyrimidine-4-amine derivatives which were simultaneously substituted at N7 and N(4), combining the substitution pattern of ADPEP (1) and DPEAP (2), showed very low affinity for A(1) ARs. This finding supports our previously published hypothesis of different binding modes for pyrrolopyrimidines, such as ADPEP (1) and DPEAP (2). DPEAP (2), a pyrrolo[2,3-d]pyrimidine-4-amine substituted at the amino group (N(4)), was found to exhibit high affinity for human A(3) ARs (K(i) = 28 nM), whereas N(4)-unsubstituted analogues were inactive. DPEAP (2) and related compounds provide new leads for the development of antagonists for the human A(3) AR.

Pyrazolo[4,3-e]1,2,4-triazolo[1,5-c]pyrimidine derivatives as highly potent and selective human A(3) adenosine receptor antagonists: influence of the chain at the N(8) pyrazole nitrogen.

An enlarged series of pyrazolotriazolopyrimidines previously reported, in preliminary form (Baraldi et al. J. Med. Chem. 1999, 42, 4473-4478), as highly potent and selective human A(3) adenosine receptor antagonists is described. The synthesized compounds showed A(3) adenosine receptor affinity in the sub-nanomolar range and high levels of selectivity evaluated in radioligand binding assays at human A(1), A(2A), A(2B), and A(3) adenosine receptors. In particular, the effect of the chain at the N(8) pyrazole nitrogen was analyzed. This study allowed us to identify the derivative with the methyl group at the N(8) pyrazole combined with the 4-methoxyphenylcarbamoyl moiety at the N(5) position as the compound with the best binding profile in terms of both affinity and selectivity (hA(3) = 0.2 nM, hA(1)/hA(3) = 5485, hA(2A)/hA(3) = 6950, hA(2B)/hA(3) = 1305). All the compounds proved to be full antagonists in a specific functional model where the inhibition of cAMP generation by IB-MECA was measured in membranes of CHO cells stably transfected with the human A(3) receptor. The new compounds are among the most potent and selective A(3) antagonists so far described. The derivatives with higher affinity at human A(3) adenosine receptors proved to be antagonists, in the cAMP assay, capable of inhibiting the effect of IB-MECA with IC(50) values in the nanomolar range, with a trend strictly similar to that observed in the binding assay. Also a molecular modeling study was carried out, with the aim to identify possible pharmacophore maps. In fact, a sterically controlled structure-activity relationship was found for the N(8) pyrazole substituted derivatives, showing a correlation between the calculated molecular volume of pyrazolo[4,3-e]1,2, 4-triazolo[1,5-c]pyrimidine derivatives and their experimental K(i) values.

Synergistic effects of calcium-mobilizing agents and adenosine on histamine release from rat peritoneal mast cells.

1. Adenosine and its metabolically stable analogue N-ethyl-carboxamidoadenosine (NECA) enhance histamine release from rat peritoneal mast cells when these are stimulated by calcium-mobilizing agents. NECA and adenosine shift the concentration-response curve of the calcium ionophore A23187 to lower concentrations. 2. The potencies of NECA or adenosine in enhancing A23187-induced histamine release are dependent on the level of stimulated release in the absence of adenosine analogues. At high levels of release their potencies are up to 20 times higher than at low levels. Consequently, averaged concentration-response curves of adenosine and NECA for enhancing histamine release are shallow. 3. The adenosine transport blocker S-(p-nitrobenzyl)-6-thioinosine (NBTI) has no effect by itself at low levels of stimulated histamine release, but abolishes the enhancing effect of adenosine. At high levels of release, however, NBTI alone enhances the release of histamine. 4. It is concluded that adenosine and calcium reciprocally enhance the sensitivity of the secretory processes to the effects of the other agent. The levels of intracellular adenosine obtained by trapping adenosine inside stimulated mast cells are sufficient to enhance histamine release substantially, suggesting that this effect may play a physiological and pathophysiological role.

Physical coupling of N-formyl peptide chemoattractant receptors to G protein is unaffected by desensitization.

Desensitization of N-formyl peptide chemoattractant receptors (FPR) in human neutrophils results in association of these receptors to the membrane skeleton. This is thought to be the critical event in the lateral segregation of receptors and guanyl nucleotide-binding proteins (G proteins) within the plane of the plasma membrane resulting in an interruption of the signaling cascade. In this study we probed the interaction of FPR with G protein in human neutrophils that were desensitized to various degrees. Human neutrophils were desensitized using the photoreactive agonist N-formyl-met-leu-phe-lys-N epsilon-[125I]2(p-azidosalicylamino)ethyl-1,3'- dithiopropionate (fMLFK-[125I]ASD). The interaction of FPR with protein was studied via a reconstitution assay and subsequent analysis of FPR-G protein complexes in sucrose density gradients. FPR-G protein complexes were reconstituted with solubilized FPR from partially and fully desensitized neutrophils with increasing concentrations of Gi purified from bovine brain. The respective EC50 values for reconstitution were similar to that determined for FPR from unstimulated neutrophils (Bommakanti RK et al., J Biol Chem 267: 7576-7581, 1992). We conclude, therefore, that the affinity of the interaction of FPR with G protein is not affected by desensitization, consistent with the model of lateral segregation of FPR and G protein as a mechanism of desensitization.

Carbamazepine distinguishes between adenosine receptors that mediate different second messenger responses.

The mechanism of the therapeutic and prophylactic effects of carbamazepine (CBZ) in affective psychoses is unknown but may in part be related to the potent competitive interaction of CBZ with adenosine-binding sites in the brain. The anticonvulsant and sedative properties of CBZ are reminiscent of the effects evoked by adenosine-agonists and contrast sharply with the opposite actions of adenosine-antagonists like caffeine. However, indirect evidence suggests an antagonist- rather than an agonist-like activity of CBZ at adenosine-receptors. We have used various model systems, in which adenosine receptor subtypes mediate different second messenger-responses, to investigate this-apparent paradox. CBZ was found to antagonize the A1-receptor-mediated inhibition of cyclic AMP accumulation in cultured astroblasts and in GH3-cells. Furthermore, CBZ also inhibits the adenosine-induced increase in the level of cyclic AMP in cultured astroblasts, which is mediated by low-affinity A2b-receptors. In contrast, CBZ does not block the inhibition elicited by adenosine-agonists of the agonist-induced increased formation of inositolphosphates in human neutrophils, which is mediated by high-affinity A2a-receptors. The specific antagonism by CBZ of A1- but not of high-affinity A2a-receptors was further supported by binding experiments using rat brain membranes. These results suggest that the paradox of CBZ's antagonistic effects at adenosine-receptors might be at least partially reconciled by a selective antagonistic action of CBZ at A1 receptors but not at high-affinity A2a-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.

Regulation of p42/p44 mitogen-activated protein kinase by the human adenosine A3 receptor in transfected CHO cells.

In this study we have investigated whether the human adenosine A3 receptor activates p42/p44 mitogen-activated protein kinase (MAPK) in transfected Chinese hamster ovary (CHO) cells (designated CHO-A3). The high affinity adenosine A3 receptor agonist IB-MECA (1-deoxy-1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-N-methyl-beta-D-ribofuranuronamide) stimulated time (peak activation occurring after 5 min) and concentration-dependent (pEC50=9.0+/-0.2) increases in p42/p44 MAPK in CHO-A3 cells. Adenosine A3 receptor-mediated increases in p42/p44 MAPK were sensitive to pertussis toxin and the MAPK kinase 1 inhibitor PD 98059 (2'-amino-3'-methoxyflavone). The broad range protein tyrosine kinase inhibitor genistein and the phosphatidylinositol 3-kinase inhibitors wortmannin and LY 294002 (2-(4-morpholinyl)-8-phenyl-4H-1-benzopyran-4-one) also blocked adenosine A3 receptor stimulation of p42/p44 MAPK. In contrast, inhibition of protein kinase C had no significant effect on adenosine A3 receptor-induced p42/p44 MAPK activation. IB-MECA (pEC50=10.1+/-0.2) also increased the expression of luciferase in CHO-A3 cells transiently transfected with a luciferase reporter gene containing the c-fos promoter. Furthermore, IB-MECA-induced increases in luciferase gene expression were sensitive to pertussis toxin, PD 98059, genistein, wortmannin and LY 294002. In conclusion, we have shown that the human adenosine A3 receptor stimulates p42/p44 MAPK and c-fos-mediated luciferase gene expression in transfected CHO cells.

Characterization of [3H]phenobarbital binding to rat brain membranes.

The binding of [3H]phenobarbital to rat brain membranes was studied in order to determine its characteristics and specificity. The binding reaction was rapid and occurred at sites of low affinity. (Kd = 700 microM) and very high density (Bmax = 2.7 nmol/mg protein). It was unaffected by temperature changes from 0 degrees C to 95 degrees C and was maximal at pH 5. Detergents in low concentrations markedly decreased the binding, apparently without solubilizing the binding sites. It is concluded that the binding of [3H]phenobarbital is a rather non-specific interaction with the plasma membrane.

Adenosine receptors and their ligands.

The regulatory actions of adenosine are mediated via four subtypes of G protein-coupled receptors distinguished as A1, A2A, A2B and A3 receptors. Their presence on basically every cell makes them an interesting target for the pharmacological intervention in many pathophysiological situations. A large number of ligands have been synthesized over the last two decades and provide agonists and antagonists that are more or less selective for the known receptor subtypes. In addition, many radioligands are available in tritiated or radioiodinated form. The comparative pharmacological characterization of all four human adenosine receptor subtypes revealed that some of the compounds thought to be selective from data in other species have unexpected potencies at human receptors. As a result, compounds that exhibit high affinity to only one subtype are an exception. Although the selection of ligands is immense, it is less than satisfying for most subtypes of adenosine receptors.