Synthesis and structure-activity relationships of 3,7-dimethyl-1-propargylxanthine derivatives, A2A-selective adenosine receptor antagonists

A series of 8-substituted derivatives of 3,7-dimethyl-1-propargylxanthine (DMPX) was synthesized and investigated as A2A adenosine receptor antagonists. Different synthetic strategies for the preparation of DMPX derivatives and analogues were explored. A recently developed synthetic procedure starting from 3-propargyl-5,6-diaminouracil proved to be the method of choice for the preparation of this type of xanthine derivatives. The novel compounds were investigated in radioligand binding studies at the high-affinity adenosine receptor subtypes A1 and A2A and compared with standard A2A adenosine receptor antagonists. Structure-activity relationships were analyzed in detail. 8-Styryl-substituted DMPX derivatives were identified that exhibit high affinity and selectivity for A2A adenosine receptors, including 8-(m-chlorostyryl)-DMPX (CS-DMPX, Ki A2A = 13 nM, 100-fold selective), 8-(m-bromostyryl)-DMPX (BS-DMPX, Ki A2A = 8 nM, 146-fold selective), and 8-(3,4-dimethoxystyryl)-DMPX (Ki A2A = 15 nM, 167-fold selective). These and other novel compounds are superior to the standard A2A adenosine receptor antagonists KF17837 (4) and CSC (5) with respect to A2A affinity and/or selectivity.     

N6,C8-distributed adenosine derivatives as partial agonists for adenosine A1 receptors

The synthesis and biological evaluation of N6, C8-disubstituted derivatives of adenosine as potential partial agonists for adenosine receptors is described. Via three routes, two series of compounds were prepared, viz., N6-cyclopentyladenosine derivatives 3a-e and C8-(cyclopentylamino)adenosine analogs 3e and 9a-d, respectively. The X-ray structure determination of one of these compounds, N6-ethyl-8(cyclopentylamino)adenosine (9b), was carried out (orthorhombic, space group P2(1)2(1)2(1) (No. 19) with a = 11.039(3), b = 8.708(2), and c = 24.815(12) angstrom, Z=4,R1=0.0974,R2(W) = 0.2455). Due to intramolecular hydrogen bonding, the ribose moiety of this compound is in an anti conformation. The compounds were tested in vitro in radioligand binding studies, yielding their affinities for A1 and A2a adenosine receptors. All compounds appeared A1 selective, with affinities in the high nanomolar, low micromolar range. On A1 receptors the so-called GTP shift was also determined, i.e., the ratio between the affinities measured in the presence and absence of 1 mM GTP. All GTP shifts (values between 1.1 and 3.8) were lower than the GTP shift for CPA (6.0). This GTP shift appeared indicative for partial agonism in vivo, since the N6-cyclopentyladenosine derivatives showed lower intrinsic activities than the prototypic full agonist N6-cyclopentyladenosine on the decrease in heart rate in conscious, normotensive rats.     

Derivatives of the triazoloquinazoline adenosine antagonist (CGS 15943) having high potency at the human A2B and A3 receptor subtypes

The adenosine antagonist 9-chloro-2-(2-furanyl)[1,2,4]triazolo[1, 5-c]quinazolin-5-amine (CGS 15943) binds nonselectively to human A1, A2A, and A3 receptors with high affinity. Acylated derivatives and one alkyl derivative of the 5-amino group and other modifications were prepared in an effort to enhance A2B or A3 subtype potency. In general, distal modifications of the N5-substituent were highly modulatory to potency and selectivity at adenosine receptors, as determined in radioligand binding assays at rat brain A1 and A2A receptors and at recombinant human A3 receptors. In Chinese hamster ovary cells stably transfected with human A2B receptor cDNA, inhibition of agonist-induced cyclic AMP production was measured. An N5-(2-iodophenyl)acetyl derivative was highly selective for A2A receptors. An (R)-N5-alpha-methyl(phenylacetyl) derivative was the most potent derivative at A3 receptors, with a Ki value of 0.36 nM. A bulky N5-diphenylacetyl derivative, 13, displayed a Ki value of 0. 59 nM at human A3 receptors and was moderately selective for that subtype. Thus, a large, nondiscriminating hydrophobic region occurs in the A3 receptor in proximity to the N5-substituent. A series of straight-chain N5-aminoalkylacyl derivatives demonstrated that for A2B receptors the optimal chain length occurs with three methylene groups, i.e., the N5-gamma-aminobutyryl derivative 27 which had a pA2 value of 8.0 but was not selective for A2B receptors. At A1, A2A, and A3 receptors however the optimum occurs with four methylene groups. An N5-pivaloyl derivative, which was less potent than 27 at A1, A2A, and A3 receptors, retained moderate potency at A2B receptors. A molecular model of the 27-A2B receptor complex based on the structure of rhodopsin utilizing a "cross-docking" procedure was developed in order to visualize the environment of the ligand binding site.     

A novel class of adenosine A3 receptor ligands. 2. Structure affinity profile of a series of isoquinoline and quinazoline compounds

A series of 3-(2-pyridinyl)isoquinoline derivatives was synthesized as potential antagonists for the human adenosine A3 receptor by substitution of the 1-position. The compounds were obtained by various synthetic routes from 1-amino-3-(2-pyridinyl)isoquinoline. The affinity was determined in radioligand binding assays for rat brain A1 and A2A receptors and for the cloned human A3 receptor. A structure-activity relationship analysis indicated that a phenyl group when coupled by a spacer allowing conjugation on position 1 of the isoquinoline ring increased the adenosine A3 receptor affinity. In contrast, such a phenyl group directly bound to position 1 of the isoquinoline ring decreased affinity. Since the combination of a phenyl group together with a spacer raised adenosine A3 receptor affinity, various spacers were investigated. VUF8501 (N-[3-(2-pyridinyl)isoquinolin-1-yl]benzamidine (15) showed an affinity at the human adenosine A3 receptor of 740 nM. Substituent effects on the phenyl group were investigated by in vitro evaluation of a series of substituted benzamidines. Electron-donating groups at the para position of the benzamidine ring increased adenosine A3 receptor affinity. These investigations led to VUF8505 (4-methoxy-N-[3-(2-pyridinyl)isoquinolin-1-yl]benzamidine(22)), which is a moderately potent and selective ligand for the human adenosine A3 receptor with an affinity of 310 nM in our test system having negligible affinity for rat A1 and A2A receptors.     

Binding of the radioligand [3H]-SCH 58261, a new non-xanthine A2A adenosine receptor antagonist, to rat striatal membranes

1. The present study describes the binding to rat striatal A2A adenosine receptors of the new potent and selective antagonist radioligand, [3H]-5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazol o [1,5-c] pyrimidine, [3H]-SCH 58261. 2. [3H]-SCH 58261 specific binding to rat striatal membranes ( > 90%) was saturable, reversible and dependent upon protein concentration. Saturation experiments revealed that [3H]-SCH 58261 labelled a single class of recognition sites with high affinity (Kd = 0.70 nM) and limited capacity (apparent Bmax = 971 fmol mg-1 of protein). The presence of 100 microM GTP in the incubation mixture did not modify [3H]-SCH 58261 binding parameters. 3. Competition experiments showed that [3H]-SCH 58261 binding is consistent with the labelling of A2A striatal receptors. Adenosine receptor agonists competed with the binding of 0.2 nM [3H]-SCH 58261 with the following order of potency: 2-hexynyl-5'-N-ethyl carboxamidoadenosine (2HE-NECA) > 5'-N-ethylcarboxamidoadenosine (NECA) > 2-[4-(2-carboxyethyl)-phenethylamino]-5'-N-ethylcarboxamidoadenosi ne (CGS 21680) > 2-phenylaminoadenosine (CV 1808) > R-N6-phenylisopropyladenosine (R-PIA) > N6-cyclohexyladenosine (CHA) = 2-chloro-N6-cyclopentyladenosine (CCPA) > S-N6-phenylisopropyladenosine (S-PIA). 4. Adenosine antagonists inhibited [3H]-SCH 58261 binding with the following order: 5-amino-9-chloro-2-(2-furyl)-[1,2,4]-triazolo[1,5-c] quinazoline (CGS 15943) > 5-amino-8-(4-fluorobenzyl)-2-(2-furyl)-pyrazolo [4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine (8FB-PTP) = SCH 58261 > xanthine amine congener (XAC) = (E,18%-Z,82%)7-methyl-8-(3,4-dimethoxystyryl)-1,3-dipropylxanthine (KF 17837S) > 8-cyclopentyl-1,3-dipropylxanthine (DPCPX) > or = 8-phenyltheophylline (8-PT). 5. The Ki values for adenosine antagonists were similar to those labelled with the A2A agonist [3H]-CGS 21680. Affinities of agonists were generally lower. The A1-selective agonist, R-PIA, was found to be about 9 fold more potent than its stereoisomer, S-PIA, thus showing the stereoselectivity of [3H]-SCH 58261 binding. Except for 8-PT, the adenosine agonists and antagonists examined inhibited [3H]-SCH 58261 binding with Hill coefficients not significantly different from unity. 6. The present results indicate that [3H]-SCH 58261 is the first non-xanthine adenosine antagonist radioligand which directly labels A2A striatal receptors. High receptor affinity, good selectivity and very low non-specific binding make [3H]-SCH 58261 an excellent probe for studying the A2A adenosine receptor subtype in mammalian brain.     

Structure-activity relationships of 4-(phenylethynyl)-6-phenyl-1,4-dihydropyridines as highly selective A3 adenosine receptor antagonists

4-(Phenylethynyl)-6-phenyl-1,4-dihydropyridine derivatives are selective antagonists at human A3 adenosine receptors, with Ki values in a radioligand binding assay vs [125I]AB-MECA (N6-(4-amino-3-iodobenzyl)-5'-(N-methylcarbamoyl)adenosine) in the submicromolar range. In this study, structure-activity relationships at various positions of the dihydropyridine ring (the 3- and 5-acyl substituents, the 4-aryl substituent, and 1-methyl group) were probed synthetically. Using the combined protection of the 1-ethoxymethyl and the 5-[2-(trimethylsilyl)ethyl] ester groups, a free carboxylic acid was formed at the 5-position allowing various substitutions. Selectivity of the new analogues for cloned human A3 adenosine receptors was determined vs radioligand binding at rat brain A1 and A2A receptors. Structure-activity analysis at adenosine receptors indicated that pyridyl, furyl, benzofuryl, and thienyl groups at the 4-position resulted in, at most, only moderate selectivity for A3 adenosine receptors. Ring substitution (e.g., 4-nitro) of the 4-phenylethylnyl group did not provide enhanced selectivity, as it did for the 4-styryl-substituted dihydropyridines. At the 3-position of the dihydropyridine ring, esters were much more selective for A3 receptors than closely related thioester, amide, and ketone derivatives. A cyclic 3-keto derivative was 5-fold more potent at A3 receptors than a related open-ring analogue. At the 5-position, a homologous series of phenylalkyl esters and a series of substituted benzyl esters were prepared and tested. (Trifluoromethyl)-, nitro-, and other benzyl esters substituted with electron-withdrawing groups were specific for A3 receptors with nanomolar Ki values and selectivity as high as 37000-fold. A functionalized congener bearing an [(aminoethyl)amino]carbonyl group was also prepared as an intermediate in the synthesis of biologically active conjugates.     

Therapeutic window of serum haloperidol concentration in acute schizophrenia and schizoaffective disorder

Starting from 3-(3-chloro-1H-pyrazol-5-yl)-1H-quinoxalin-2-one (2) a series of substituted [1,2,4]triazolo[4,3-a]quinoxalines (3a-f) was prepared via a multistep reaction sequence. Affinities of the novel derivatives 3a-f for benzodiazepine as well as for adenosine A1- and A2A-receptors of rat brain were determined by radioligand binding assays. 1-Methyl-4-(3-chloro-1H-pyrazol-5-yl) derivative 3a exhibited submicromolar affinity for the benzodiazepine binding site of GABAA receptors (Ki = 340 nM) and was less potent at A1-(Ki = 7.85 microM) and A2A-(Ki = 1.43 microM) adenosine receptors (AR). Derivatives with larger substituents in the 1-position showed reduced binding to benzodiazepine and A2A-AR, but increased A1-AR affinity, the 2-thienylmethyl derivative 3f being the most potent and selective A1-AR ligand of the present series (Ki = 200 nM).     

Species differences in brain adenosine A1 receptor pharmacology revealed by use of xanthine and pyrazolopyridine based antagonists

1. The pharmacological profile of adenosine A1 receptors in human, guinea-pig, rat and mouse brain membranes was characterized in a radioligand binding assay by use of the receptor selective antagonist, [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). 2. The affinity of [3H]-DPCPX binding sites in rat cortical and hippocampal membranes was similar. Binding site affinity was higher in rat cortical membranes than in membranes prepared from guinea-pig cortex and hippocampus, mouse cortex and human cortex. pKD values (M) were 9.55, 9.44, 8.85, 8.94, 8.67, 9.39 and 8.67, respectively. The binding site density (Bmax) was lower in rat cortical membranes than in guinea-pig or human cortical membranes. 3. The rank order of potency of seven adenosine receptor agonists was identical in each species. With the exception of 5'-N-ethylcarboxamidoadenosine (NECA), agonist affinity was 3.5-26.2 fold higher in rat cortical membranes than in human and guinea-pig brain membranes; affinity in rat and mouse brain membranes was similar. While NECA exhibited 9.3 fold higher affinity in rat compared to human cortical membranes, affinity in other species was comparable. The stable GTP analogue, Gpp(NH)p (100 microM) reduced 2-chloro-N6-cyclopentyladenosine (CCPA) affinity 7-13.9 fold, whereas the affinity of DPCPX was unaffected. 4. The affinity of six xanthine-based adenosine receptor antagonists was 2.2-15.9 fold higher in rat cortical membranes compared with human or guinea-pig membranes. The rank order of potency was species-independent. In contrast, three pyrazolopyridine derivatives, (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352) and 6-oxo-3-(2-phenylpyrazolo[1,5-a]pyridin-3-yl)-1(6H)-pyridazinebutyric acid (FK838) exhibited similar affinity in human, guinea-pig, rat and mouse brain membranes. pKi values (M) for [3H]-DPCPX binding sites in human cortical membranes were 9.31, 7.52 and 7.92, respectively. 5. Drug affinity for adenosine A2A receptors was determined in a [3H]-2-[4-(2-carboxyethyl)phenethylamino]-5'-N-ethylcarboxamido ade nosine ([3H]-CGS 21680) binding assay in rat striatal membranes. The pyrazolopyridine derivatives, FK453, FK838 and FK352 exhibited pKi values (M) of 5.90, 5.92 and 4.31, respectively, compared with pKi values of 9.31, 8.18 and 7.57 determined in the [3H]-DPCPX binding assay in rat cortical membranes. These novel pyrazolopyridine derivatives therefore represent high affinity, adenosine A1 receptor selective drugs that, in contrast to xanthine based antagonists, exhibit similar affinity for [3H]-DPCPX binding sites in human, rat, mouse and guinea-pig brain membranes.     

Activation of hippocampal adenosine A3 receptors produces a desensitization of A1 receptor-mediated responses in rat hippocampus

The adenosine A3 receptor is expressed in brain, but the consequences of activation of this receptor on electrophysiological activity are unknown. We have characterized the actions of a selective adenosine A3 receptor agonist, 2-chloro-N6-(3-lodobenzyl)-adenosine-5'-N-methyluronamide (Cl-IB-MECA), and a selective A3 receptor antagonist, 3-ethyl-5-benzyl-2-methyl-4-phenylethynyl-6-phenyl-1, 4-(+/-)-dihydropyridine-3,5-dicarboxylate (MRS 1191), in brain slices from rat hippocampus. In the CA1 region, activation of A3 receptors had no direct effects on synaptically evoked excitatory responses, long-term potentiation, or synaptic facilitation. However, activation of A3 receptors with Cl-IB-MECA antagonized the adenosine A1 receptor-mediated inhibition of excitatory neurotransmission. The effects of Cl-IB-MECA were blocked by pretreatment with MRS 1191, which by itself had no effect on A1 receptor-mediated responses. The presynaptic inhibitory effects of baclofen and carbachol, mediated via GABA(B) and muscarinic receptors, respectively, were unaffected by Cl-IB-MECA. The maximal response to adenosine was unchanged, suggesting that the primary effect of Cl-IB-MECA was to reduce the affinity of adenosine for the receptor rather than to uncouple it. Similar effects could be demonstrated after brief superfusion with high concentrations of adenosine itself. Under normal conditions, endogenous adenosine in brain is unlikely to affect the sensitivity of A1 receptors via this mechanism. However, when brain concentrations of adenosine are elevated (e.g., during hypoxia, ischemia, or seizures), activation of A3 receptors and subsequent heterologous desensitization of A1 receptors could occur, which might limit the cerebroprotective effects of adenosine under these conditions.     

2-Alkynyl derivatives of adenosine-5'-N-ethyluronamide: selective A2 adenosine receptor agonists with potent inhibitory activity on platelet aggregation

A series of new 2-alkynyl and 2-cycloalkynyl derivatives of adenosine-5'-N-ethyluronamide (NECA) and of N-ethyl-1'-deoxy-1'-(6-amino-2-hexynyl-9H-purin-9-yl)-beta-D- ribofuranuronamide (1, HE-NECA), bearing hydroxy, amino, chloro, and cyano groups in the side chain, were synthesized. The compounds were studied in binding and functional assays to assess their potency for the A2 compared to A1 adenosine receptor. The presence of an alpha-hydroxyl group in the alkynyl chain of NECA derivatives accounts for the A2 agonist potency, leading to compounds endowed with sub-nanomolar affinity in binding studies. However, these analogues also possess good A1 receptor affinity resulting in low A2 selectivity. From functional experiments the 4-hydroxy-1-butynyl (6) and the 4-(2-tetrahydro-2H-pyranyloxy)-1-butynyl (16) derivatives appear to be very potent in inducing vasorelaxation without appreciable effect on heart rate. The new compounds were also tested as inhibitors of platelet aggregation induced by ADP. Introduction of an alpha-hydroxyl group in the alkynyl side chain caused a greater increase in antiaggregatory activity than either NECA or HE-NECA, resulting in the most potent inhibitors of platelet aggregation so far known in the nucleoside series. The presence of an alpha-quaternary carbon such as the 3-hydroxy-3,5-dimethyl-1-hexynyl (12) and the 3-hydroxy-3-phenyl-1-butynyl (15) derivatives markedly reduced the antiaggregatory potency without affecting the A2 affinity. The hydrophobicity index (k') of the new nucleosides barely correlated with the binding data, whereas high k' values were associated with increased A2 vs A1 selectivity but with reduced activity in all functional assays. Some of the compounds synthesized possess interesting pharmacological properties. Compounds having an appropriate balance between vasorelaxation and antiplatelet activity, if confirmed in vivo, deserve further development for the treatments of cardiovascular disorders.     

Selective labelling of bradykinin receptor subtypes in WI38 human lung fibroblasts

1. Binding of the B1 bradykinin receptor radioligand, [3H]-des-Arg10-kallidin (-KD) and the B2 receptor radioligand [3H]-bradykinin (-BK) was investigated in membranes prepared from WI38 human foetal lung fibroblasts. 2. One-site analysis of the saturation data for [3H]-des-Arg10-KD gave an equilibrium dissociation constant (KD) value of 0.51 +/- 0.12 nM and a maximum receptor density (Bmax) of 260 +/- 49 fmol mg-1 of protein. [3H]-des-Arg10-KD binding was displaced by ligands in the order: des-Arg10-KD > KD > > des-Arg9[Leu8]-BK > des-Arg9-BK > Hoe 140 > > BK, implying that it was binding selectively to B1 receptors. 3. One-site analysis of the binding of [3H]-BK to W138 membranes indicated that it had a KD value of 0.25 +/- 0.06 nM and a Bmax of 753 +/- 98 fmol mg-1 of protein. The potencies for displacement of [3H]-BK binding were: Hoe 140 > > BK = KD > > > des-Arg10-KD = des-Arg9[Leu8]-BK = des-Arg9-BK, which was consistent with binding to B2 receptors. 4. This is the first characterization of [3H]-des-Arg10-KD binding to include both kinetic and equilibrium data, and demonstrates that [3H]-des-Arg10-KD has a high affinity for human B1 bradykinin receptors and is sufficiently selective to be used as a radioligand for B1 receptors in human cells or tissues expressing an excess of B2 BK receptors.     

Binding affinity of adenosine receptor agonists and antagonists at human cloned A3 adenosine receptors

The A3 adenosine receptor is one of the four adenosine receptors which have thus far been identified. Cloning of the A3 receptor from animal species such as rat, sheep and human has shown that there are interspecies differences in its peripheral distribution, and binding affinity for various adenosine receptor ligands. The adenosine derivative, 4-aminobenzyl-5'-N-methylcarboxamidoadenosine (AB-MECA), is a potent A3 receptor agonist which is used as a reference drug. In this report we have characterized the binding of selected adenosine receptor agonists and antagonists to HEK 293 cells transfected with the human A3 adenosine receptor using [125I]AB-MECA as radioligand. HE-NECA and NECA were the most potent compounds showing Ki values in the low nanomolar range, while the recently discovered non-xanthine A2A receptor antagonists ZM 241385, SCH 58261 and SCH 63390 showed affinity values in the micromolar range. These data further indicate the need to examine the affinity of new adenosine receptor ligands directly in human A3 receptors.     

Defibrotide, a single-stranded polydeoxyribonucleotide acting as an adenosine receptor agonist

The binding of single-stranded polydeoxyribonucleotides to adenosine A1 and A2 receptors was investigated. Defibrotide, a natural substance with established anti-thrombotic and anti-ischaemic effects, displaced [3H]CHA (N6-cyclohexyl-adenosine) and [3H]NECA (5'-N-ethylcarboxamido-adenosine) concentration dependently, completely and competitively. Ki values of 371 +/- 68 and 688 +/- 115 micrograms/ml (mean +/- S.E.M. of 4-5 replications) were computed for adenosine A1 and A2 sites, respectively. Higher and lower molecular weight polydeoxyribonucleotides displayed comparable affinity, whereas a double-stranded polydeoxyribonucleotide and a polyanion with a negative charge comparable to that of defibrotide were inactive. Defibrotide did not affect the total number of binding sites in radioligand saturation experiments. Defibrotide relaxed the K(+)-contracted guinea-pig trachealis muscle (IC50 = 4001 micrograms/ml) about one-third as potently as the CHA-contracted preparation and as potently as the resting preparation. NECA, a mixed adenosine A1/A2 receptor agonist, behaved similarly. The effects were abolished by the adenosine A1/A2 receptor blocker 8-phenyltheophylline, but not by the selective A1 blocker, 1,3-dipropyl-8-(2-amino-4-chlorophenyl)-xanthine. These results demonstrate that defibrotide binds to adenosine receptors and triggers pharmacological responses comparable to those of a known agonist.     

Labeling of A2A adenosine receptors in human platelets by use of the new nonxanthine antagonist radioligand [3H]SCH 58261

The present study describes the binding to human platelet A2A adenosine receptors of the new potent and selective antagonist radioligand [3H]5-amino-7-(2-phenylethyl)-2-(2-furyl)-pyrazolo[4,3-e]-1,2,4-triazolo [1,5-c] pyrimidine ([3H]SCH 58261). Saturation experiments revealed that [3H]SCH 58261 labels a single class of recognition sites with high affinity (Kd = 0.85 nM), limited capacity (apparent Bmax = 85 fmol/mg of protein) and good specific binding (about 60%). [3H]SCH 58261 binding was not modulated by either the divalent cation Mg(+2) or guanine nucleotides. In competition experiments, a series of both adenosine agonists and antagonists inhibited [3H]SCH 58261 binding to A2A platelet receptors with rank order of potency and affinity similar to those observed in rat striatal membranes with the same radioligand. This confirms that the platelet A2A receptor is similar to that labeled in the brain striatum. Binding data were also found to be in good agreement with the results from functional studies such as A2A agonist-induced stimulation of adenylate cyclase or platelet aggregation inhibition. The present findings indicate that [3H]SCH 58261 is the first radioligand available for the characterization of the A2A receptor subtype in platelets.     

Pyrazolopyridine derivatives act as competitive antagonists of brain adenosine A1 receptors: [35S]GTPgammaS binding studies

The effects of adenosine receptor ligands and three novel pyrazolopyridine derivatives on guanosine-5'-O-(3-[35S]thio)triphosphate ([35S]GTPgammaS) binding to rat cerebral cortical membranes were examined. [35S]GTPgammaS binding was stimulated in a concentration dependent manner by several adenosine receptor agonists. The adenosine A2a receptor selective agonist, 2-p-(2-carboxyethyl)phenethylamino-5'-N-ethylcarboxamidoadenosine (CGS 21680), was ineffective confirming specificity for adenosine A1 receptor activation. 2-Chloro-N6-cyclopentyladenosine (CCPA; 10(-7) M)-stimulated [35S]GTPgammaS binding was inhibited by xanthine and pyrazolopyridine based adenosine receptor antagonists. The concentration-response curve for CCPA-stimulated [35S]GTPgammaS binding was shifted to the right with increasing concentrations of antagonist without significant changes in maximal response. Schild analyses determined pK(B) values of 8.97, 8.88, 8.21, 8.16, 7.79 and 7.65 for 8-cyclopentyl-1,3-dipropylxanthine (DPCPX), (R)-1-[(E)-3-(2-phenylpyrazolo[1,5a]pyridin-3-yl) acryloyl]-2-piperidine ethanol (FK453), 6-oxo-3-(2-phenylpyrazolo[1,5a]pyridin-3-yl)-1(6H)-pyridazinebutyric+ ++ acid (FK838), 9-chloro-2-(2-furyl)[1,2,4]triazolo-[1,5c]quinazolin-5-amine (CGS 15943), 8-cyclopentyl-1,3-methylxanthine (CPT) and (R)-1-[(E)-3-(2-phenylpyrazolo[1,5a]pyridin-3-yl) acryloyl]-piperidin-2-yl acetic acid (FK352), respectively. Schild slopes were close to unity, confirming that these novel pyrazolopyridine derivatives act as competitive antagonists at rat brain adenosine A1 receptors.     

Identification of a unique ligand which has high affinity for all four bombesin receptor subtypes

Four subtypes of bombesin receptors are identified (gastrin-releasing peptide receptor, neuromedin B receptor, the orphan receptor bombesin receptor subtype 3 (BB3 or BRS-3) and bombesin receptor subtype 4 (BB4)), however, only the pharmacology of the gastrin-releasing peptide receptor has been well studied. This lack of data is due in part to the absence of a general ligand. Recently we have discovered a ligand, 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) that binds to BRS-3 receptors. In this study we investigate its ability to interact with all four bombesin receptor subtypes. In rat pancreatic acini containing only gastrin-releasing peptide receptor and in BB4 transfected BALB cells, this ligand and 125I-[Tyr4]bombesin, the conventional gastrin-releasing peptide receptor ligand, gave similar results for receptor number, affinity for bombesin and affinity for the unlabeled ligand. In neuromedin B receptor transfected BALB cells, this ligand and 125I-[D-Tyr0]neuromedin B, the generally used neuromedin B receptor ligand, gave similar results for receptor number, neuromedin B affinity or the unlabeled ligand affinity. Lastly, in BRS-3 transfected BALB cells, only this ligand had high affinity. For all four bombesin receptors this ligand had an affinity of 1-8 nM and was equal or greater in affinity than any other specific ligands for any receptor. The unlabeled ligand is specific for gastrin-releasing peptide receptors on rat pancreatic acini and did not inhibit binding of 125I-cholecystokinin octapeptide (125I-CCK-8), 125I-vasoactive intestinal peptide (125I-VIP) or 125I-endothelin to their receptors. The unlabeled ligand was an agonist only at the gastrin-releasing peptide receptor in rat acini and did not interact with CCK(A) receptors or muscarinic M3 acetylcholine receptors to increase [3H]inositol phosphates. These results demonstrate 125I-[D-Tyr6,betaAla11,Phe13,Nle14]bombesin-(6-1 4) is a unique ligand with high affinity for all subtypes of bombesin receptors. Because of the specificity for bombesin receptors, this ligand will be a valuable addition for such pharmacological studies as screening for bombesin receptor agonists or antagonists and, in particular, for investigating BRS-3 cell biology, a receptor for which no ligand currently exists.     

Solubilized rabbit striatal A2a-adenosine receptors: stability and antagonist binding

The A2a-adenosine binding subunit from rabbit striatal membranes was solubilized using 1% 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate and was characterized using the antagonist radioligand [3H]8-[4-[[[[2-aminoethyl)amino]carbonyl]methyl]oxy] phenyl]-1,3-dipropylxanthine (XAC). The solubilized receptor was very stable, with 55% of the specific [3H]XAC binding remaining after storage for 15 days at 4 degrees C. The dissociation constant (Kd) for binding of [3H]XAC to solubilized A2 receptors was determined in saturation studies to be 4.0 nM, with a Bmax of 600 fmol/mg protein. Xanthine inhibitors displaced the specific binding of the adenosine antagonist [3H]XAC (in the presence of 50 nM 8-cyclopentyl-1,3-dipropylxanthine) at 25 degrees C, with Ki values consonant with the expected affinities at A2a receptors. Binding of [3H]XAC (1 nM) or the adenosine agonist [3H]2-(carboxyethylphenylethylamino)adenosine-5'-N-ethyl carboxamide (5 nM) to A2a receptors was diminished in the presence of 0.1 M Na+ in both membranes and solubilized preparations. Agonist binding was increased (by 280% for membranes and 180% for solubilized receptors), and antagonist binding was decreased in the presence of 10 mM Mg2+. Displacement of [3H]XAC by the agonist (R)-N6-phenylisopropyladenosine was biphasic, corresponding to high (IC50 = 188 nM, RH = 30%) and low (IC50 = 9730 nM, RL = 70%) affinity sites. Preincubation with 100 microM GTP (10 mM Mg2+) converted the high affinity binding to low affinity, suggesting that receptor and G-protein are dissociated by the guanine nucleotide. The solubilized receptor was more easily inactivated by exposure to the reducing agent dithiothreitol (IC50 = 3 mM) than in membranes (IC50 = 220 mM), suggesting increased accessibility of structurally essential disulfide bridges.     

des-AA-1,2,5[D-Trp8, IAmp9]somatostatin-14 allows the identification of native rat somatostatin sst1 receptor subtype

Somatostatin exerts multiple activities by interacting with at least five different receptor subtypes (sst[1-5]). The affinity of des-AA(1,2,5)-[D-Trp8, IAmp9]somatostatin-14 (CH-275) was studied by competition experiments using the non-selective radioligand [125I][Leu8, D-Trp22, Tyr25]somatostatin-28 in areas of the rat brain and pituitary known to express identified receptor subtypes. In the cerebellar nuclei and cerebral cortex, which possess the somatostatin sst1 receptor subtype, CH-275 exhibited a moderate affinity (IC50: 10-50 nM). Conversely, in the hippocampus, immature cerebellum and pituitary which contain different subsets of receptors mRNAs (sst[2-5]), the IC50 values were > 1 microM. These data indicate that CH-275 is an appropriate ligand for the identification of native rat somatostatin sst1 receptor subtype.     

Deficient muscle carnitine transport in primary carnitine deficiency

The effects of conditions that either increase or decrease heart rate on the pharmacological properties of adenosine receptors in cultured rat myocytes were examined. Levels of A1 adenosine receptors, following prolonged treatment with electrical stimulation (ES) or the antiarrhythmic drug amiodarone, were determined using radioligand binding with the specific A1 receptor antagonist [3H]1,3-dipropyl-8-cyclopentylxanthine (CPX). The effects of lowering temperature were also explored. Exposure to amiodarone for 4 days reduced the density of A1 receptors by 19% (from 24.7 +/- 0.4 to 20.09 +/- 0.3 fmol/dish) and inhibited the rate of contraction by 60% (from 188 +/- 16 to 76 +/- 30 beats/min), without changing the receptor affinity, protein content, creatine kinase (CK) activity or cell number. Electrical stimulation at 25 degrees C elevated the density of A1 adenosine receptors by 185% (from 4.1 +/- 0.4 to 11.69 +/- 2.1 fmol/dish). Four days of reduced temperature (from 37 degrees C to either 30 or 25 degrees C) lowered the density of A1 adenosine receptors by 69 or 86%, respectively (from 24.1 +/- 1.2 to 7.4 +/- 0.4 or 3.4 +/- 0.3 fmol/dish), with no significant change in the receptor affinity, activity of CK, or lactate dehydrogenase (LDH), protein content or cell number. The observed up- and down-regulation of A1 adenosine receptors in primary myocyte cultures in response to conditions that exogenously alter the rate of contraction, is indicative of the role of adenosine receptors in adaptation of heart cells to stress.     

Adenosine A1 receptors in cultured cerebellar granule cells: role of endogenous adenosine

Adenosine A1 receptors as well as other components of the adenylate cyclase system have been studied in cultured cerebellar granule cells. No significant changes in adenosine A1 receptor number, assayed by radioligand binding in intact cells, were detected from 2 days in vitro (DIV) until 7 DIV. Nevertheless, a decline in this parameter was detected at 9 DIV. The steady-state levels of alpha-Gg and alpha-Gi, detected by immunoblotting, showed similar profiles, increasing from 2 to 5 DIV and decreasing afterward. Forskolin-stimulated adenylate cyclase levels also showed an increase until 5 DIV, decreasing at 7 and 9 DIV. The adenosine A1 receptor analogue cyclopentyladenosine (CPA) was able to inhibit cyclic AMP accumulation at 2, 5, and 7 DIV but failed to do so at 9 DIV. This inhibition was prevented by the specific adenosine A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine. The presence of adenosine deaminase in the culture increased adenosine A1 receptor number during the period studied and induced recovery of the inhibitory effect of CPA, lost after 7 DIV. These data suggest that functional expression of adenosine A1 receptors and the other components of the adenylate cyclase system is subjected to regulation during the maturation of cultured cerebellar granule cells and demonstrates a key role for endogenous adenosine in the process.