Microglia phenotype diversity.

Microglia, the tissue macrophages of the brain, have under healthy conditions a resting phenotype that is characterized by a ramified morphology. With their fine processes microglia are continuously scanning their environment. Upon any homeostatic disturbance microglia rapidly change their phenotype and contribute to processes including inflammation, tissue remodeling, and neurogenesis. In this review, we will address functional phenotypes of microglia in diverse brain regions and phenotypes associated with neuroinflammation, neurogenesis, brain tumor homeostasis, and aging.

Interaction between salsolinol (SAL) and thyrotropin-releasing hormone (TRH) or dopamine (DA) on the secretion of prolactin in ruminants.

We have recently demonstrated that salsolinol (SAL), a dopamine (DA)-derived compound, is present in the posterior pituitary gland and is able to stimulate the release of prolactin (PRL) in ruminants. The aim of the present study was to clarify the effect that the interaction of SAL with thyrotropin-releasing hormone (TRH) or DA has on the secretion of PRL in ruminants. A single intravenous (i.v.) injection of SAL (5mg/kg body weight (b.w.)), TRH (1microg/kg b.w.), and SAL plus TRH significantly stimulated the release of PRL in goats (P<0.05). The cumulative response curve (area under the curve: AUC) during 120min was 1.53 and 1.47 times greater after the injection of SAL plus TRH than either SAL or TRH alone, respectively (P<0.05). A single i.v. injection of sulpiride (a DA receptor antagonist, 0.1mg/kg b.w.), sulpiride plus SAL (5mg/kg b.w.), and sulpiride plus TRH (1microg/kg b.w.) significantly stimulated the release of PRL in goats (P<0.05). The AUC of PRL during 120min was 2.12 and 1.78 times greater after the injection of sulpiride plus TRH than either sulpiride alone or sulpiride plus SAL, respectively (P<0.05). In cultured bovine anterior pituitary (AP) cells, SAL (10(-6)M), TRH (10(-8)M), and SAL plus TRH significantly increased the release of PRL (P<0.05), but the additive effect of SAL and TRH detected in vivo was not observed in vitro. In contrast, DA (10(-6)M) inhibited the TRH-, as well as SAL-induced PRL release in vitro. All together, these results clearly show that SAL can stimulate the release of PRL in ruminants. Furthermore, they also demonstrate that the additive effect of SAL and TRH on the release of PRL detected in vivo may not be mediated at the level of the AP, but that DA can overcome their releasing activity both in vivo and in vitro, confirming the dominant role of DA in the inhibitory regulation of PRL secretion in ruminants.

Salsolinol is present in the bovine posterior pituitary gland and stimulates the release of prolactin both in vivo and in vitro in ruminants.

The aims of the present study were to determine whether salsolinol (SAL), a dopamine-related compound, is present in the bovine posterior pituitary (PP) gland, and to clarify the effect of SAL on the secretion of prolactin (PRL) in ruminants. SAL was detected in extract of bovine PP gland using high-pressure liquid chromatography with electrochemical detection (HPLC-EC). A single intravenous (i.v.) injection of SAL (5 and 10mg/kg body weight) significantly and dose-dependently stimulated the release of PRL in goats (P<0.05). Plasma PRL levels reached a peak 10min after the injection, then gradually returned to basal values in 60-80min. The PRL-releasing pattern was similar to that in response to sulpiride (a dopamine receptor antagonist). The intracerebroventricular (i.c.v.) injection of 1mg of SAL had no significant effect on the release of PRL in calves, however, 5mg significantly stimulated the release (P<0.05) with peak values reached 30-40min after the injection. Moreover, SAL significantly stimulated the release of PRL from cultured bovine anterior pituitary cells at doses of 10(-6) and 10(-5)M, compared to control cells (P<0.05). Taken together, our data clearly show that SAL is present in extract of the PP gland of ruminants, and has PRL-releasing activity both in vivo and in vitro. Therefore, this endogenous compound is a strong candidate for the factor having PRL-releasing activity that has been previously detected in extract of the bovine PP gland.

Caveolin-1 is transported to multi-vesicular bodies after albumin-induced endocytosis of caveolae in HepG2 cells.

Caveolae-mediated endocytosis is a highly regulated endocytic pathway that exists in parallel to other forms of clathrin-dependent and -independent endocytosis. Internalized caveolae accumulate in intermediate organelles called caveosomes. Here we addressed the further fate of internalized caveolae by inducing caveolae-mediated uptake of albumin by HepG2 cells. We followed the route of internalized caveolin-1 by immunogold labelling of ultrathin frozen sections and by Western blot analyses of purified membrane fractions. Long-term (1 and 3 hrs) albumin treatment resulted in the appearance of albumin-containing caveolae in special multi-caveolar complexes (consisting of multiple caveolae clustered together) connected to the plasma membrane and caveosome-like structures in the cytoplasm. In addition, numerous CD63 (LIMP-1) positive late endosomes/multi-vesicular bodies were found positive for caveolin-1, suggesting that upon albumin incubation, caveolin-1 is endocytosed and enters the degradative pathway. Surprisingly, the number of caveolae at the plasma membrane increased after addition of albumin. This increase was blocked by cycloheximide treatment, indicating that albumin internalization also stimulates de novo protein synthesis, which is necessary for new caveolae formation. Together, our results show that during long-term albumin uptake, caveolin-1 travels to late endosomes and is replaced by newly synthesized caveolin-1 at the plasma membrane.

MTD-PLS: a PLS-based variant of the MTD method. A 3D-QSAR analysis of receptor affinities for a series of halogenated dibenzoxin and biphenyl derivatives.

MTD-PLS, the Partial Least Squares (PLS) variant of the Minimum Topological Difference (MTD) method is described. In MTD-PLS, molecules are characterised not only by the occupancy or nonoccupancy of the hypermolecular vertices (as in classical MTD), but also by additional descriptors for each vertex: fragmental van der Waals volumes, fragmental hydrophobicities, partial atomic charges, etc. This method was applied to a series of 73 polyhalogenated derivatives of dibenzo-p-dioxine, dibenzofuran and biphenyl (induction of aryl hydrocarbon hydrolase and affinities to rat cytosolic receptor), previously studied by MTD. The separation of steric, hydrophobic, and electrostatic effects was achieved retranslating from the latent variable space into a linear combination of the initial structural variables. The MTD-PLS method yields more detailed results compared to classical MTD, indicating the importance of electrostatic effects at some substituent positions.

Regulation of G protein-coupled receptor-adenylyl cyclase responsiveness in human airway smooth muscle by exogenous and autocrine adenosine.

Adenosine is a mediator of bronchoconstriction in asthmatics and is believed to mediate its effects through adenosine receptor activation in inflammatory cells. In this study, we identify human airway smooth muscle (ASM) as a direct target of adenosine. Acute exposure of human ASM cultures to adenosine receptor (AR) agonists resulted in rapid accumulation of cyclic adenosine monophosphate (cAMP) with a pharmacologic profile consistent with A(2b)AR activation. Little or no evidence of A1AR or A3AR expression was suggested on acute addition of various AR ligands, although a low level of A1ARs was identified in radioligand binding studies. Treatment with adenosine deaminase suggested that human ASM cultures secrete adenosine that feeds back on A(2b)ARs and regulates basal cAMP levels as well as a small degree of A(2b)AR, beta(2)AR, and prostaglandin E(2) receptor desensitization. When subjected to chronic treatment with AR agonists or agents that enhance accumulation of endogenous, extracellular adenosine, a dual effect of A(2b)AR desensitization and adenylyl cyclase (AC) sensitization was observed. This AC sensitization was eliminated by pertussis toxin and partially reversed by the A1AR antagonist 8-cyclopentyl-1,3-dipropylxanthine, suggesting a contributory role for the A1AR. Overexpression of A1ARs and A(2b)ARs in human ASM cultures resulted in differential effects on basal, agonist-, and AC-mediated cAMP production. These data demonstrate that human ASM is a direct target of exogenous and autocrine adenosine, with effects determined by differential contributions of A(2b) and A1 adenosine receptors that are time-dependent. Accordingly, the relative distribution and activation of AR subtypes in ASM in vivo may influence airway function in diseases such as asthma and warrant consideration in therapeutic strategies that target ARs or alter nucleotide/ nucleoside levels in the airway.

Down-regulation of vascular endothelial growth factor expression after A(2A) adenosine receptor activation in PC12 pheochromocytoma cells.

Vascular endothelial growth factor (VEGF) is an endothelial cell mitogen that promotes angiogenesis during embryonic development and the progression of certain pathologies. This study examined the regulation of VEGF expression by adenosine receptor (AR) activation in PC12 rat pheochromocytoma cells. Treatment of cells with the AR agonist CGS21680 reduced the VEGF mRNA level to approximately 20% of that in control cells with an EC(50) value of 0.47 nM, indicative of mediation by the A(2A)AR. Down-regulation of VEGF mRNA by CGS21680 was abolished by pretreatment of cells with the AR antagonist ZM241385. Additionally, ZM241385 alone increased VEGF mRNA by 2.8-fold above basal. RNase protection assays indicated that CGS21680 down-regulated VEGF(121), VEGF(165), and VEGF(189) transcripts. VEGF protein secretion was similarly decreased by CGS21680. Under hypoxic conditions, VEGF mRNA expression was reduced by 85.7% after pretreatment with CGS21680. The down-regulation response appears to be mediated predominately by coupling of the A(2A)AR to G(s) because cholera toxin treatment also reduced VEGF expression. The decrease in VEGF mRNA steady-state levels after A(2A)AR activation is apparently due to a decrease in the VEGF gene transcription rate and not to a decrease in mRNA stability. Thus, depending on the cell type, adenosine may have an inhibitory effect on VEGF production, which may have implications in blood vessel development.

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.

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.

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.

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.

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.

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.

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.

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.

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.

Adenosine receptor subtypes: characterization and therapeutic regulation.

Adenosine receptors (ARs) are members of the G protein-coupled receptor family and mediate the multiple physiological effects of adenosine. Currently, four AR subtypes have been cloned: A1AR, A2aAR, A2bAR, and A3AR. All subtypes are distinctly distributed throughout the body and AR agonists and antagonists have potential therapeutic utility. Knowledge of AR amino acid structure has been utilized in mutagenesis studies to identify specific receptor regions that interact with distinct classes of ligands. Cloning of ARs has also permitted receptor regulatory processes such as desensitization to be studied in greater detail, in particular, the molecular mechanisms underlying this event. Cloning of the human A1AR has revealed that alternate splicing generates distinct receptor transcripts. The existence of a particular transcript in a tissue or cell apparently regulates the level of A1AR expression in the tissue. This review focuses on these aspects of AR structure and function and their therapeutic regulation.

Adenosine receptors: protein and gene structure.

Adenosine produces a wide variety of effects throughout the body via activation of cell surface adenosine receptors. Adenosine receptors belong to the family of seven transmembrane domain G protein-coupled receptors and four subtypes have been cloned from a variety of species: the A1AR, A2aAR, A2bAR and A3AR. With a knowledge of both the protein sequence of adenosine receptors and the structure of the A1AR gene, the function and regulation of these receptors can be further explored. Site-directed mutagenesis of the A1AR has resulted in the identification of amino acid residues in transmembrane domains 6 and 7 that are critical in both agonist and antagonist binding. The construction and analysis of A1/A3 chimeric receptors has also revealed regions of adenosine receptors important in ligand binding. These include the distal region of the second extracellular loop of adenosine receptors, which has a role in the binding of both agonist and antagonist ligands. A segment of the exofacial portion of the transmembrane domain 5 of adenosine receptors appears to be involved in the selective recognition of agonist ligands containing a substitution at the 5'-position of the ribose moiety. Isolation of the genomic sequence of the human A1AR, in combination with analysis of the transcript distribution in several tissues, indicates that alternative splicing of the human A1AR occurs in the 5'-untranslated region of the gene. Two distinct transcripts, containing either exons 3, 5 and 6 or exons 4, 5 and 6, exist with exons 3 and 4 apparently mutually exclusive. The exon 4, 5 and 6 transcript has been detected in all tissues that express the A1AR, while the exon 3, 5 and 6 mRNA is found in tissues that display a relatively high A1AR expression. Findings suggest that the presence of two ATG codons in exon 4, upstream of the translation start site, is involved in the repression of the A1AR expression in those tissues containing the exon 4, 5 and 6 transcript.

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.