A steric and electrostatic comparison of three models for the agonist/antagonist binding site on the adenosine A1 receptor.

Several models have been described in the literature to explain the similarity of interaction of adenosine receptor agonists and antagonists with the binding site of the receptor. Besides the superposition of the nitrogen atoms of adenosine and xanthine (the "standard" model), two other models have been described: one in which xanthine is rotated around its "horizontal" axis before superposition ("flipped") and one in which the adenosine N6-region and the xanthine C8-region are superimposed ("N6-C8"). In this study we compared the steric and electrostatic properties of these models. The flipped model tends to show higher percentages of overlap for the positive electrostatic potentials and the N6-C8 model yielded predominantly a slightly higher overlap for the negative electrostatic potentials, although these differences were rather small. Since the N6-region in adenosine and the C8-region in xanthine are coinciding in this model, the N6-C8 model yielded a much larger overlap in van der Waals volume than the other two models. The N6-C8 model seems therefore to be the more probable model, also because the interactive groups point in the same direction for both adenosine and xanthine analogues. We determined the geometries of both the adenosine N6-substituents and the xanthine 8-substituents in earlier studies. The N6-C8 model causes a coincidence of these separately determined conformations.     

Allosteric modulation of the rat adenosine A1 receptor: Differential effects on agonist and antagonist binding

The interaction of 2‐amino‐3‐benzoyl‐thiophene derivative, PD81,723, as well as other G protein‐coupled receptor (GPCR)‐modulating agents such as suramin (SUR), N‐ethylmaleimide (NEM), GTP, and NaCl with the rat adenosine A₁ receptor was investigated using kinetic, saturation, as well as displacement experiments of various agonists, partial agonists or antagonists. PD81,723 enhanced agonist (CPA, R‐PIA, NECA) binding ～2‐fold, while its effect on CPA binding was increased (4–11‐fold) with other modulators present. In contrast, binding of antagonists (DPCPX, CPT, N‐0840) was inhibited, while binding of partial agonists (8BCPA, MeSCPA) remained uninfluenced. The effect of PD81,723 is consistent with shifting A₁ receptors to an “active” (R*) state with high affinity for agonists and low for antagonists. Further, all described allosteric modulators influenced both agonist and antagonist binding. The IC₅₀ values observed for the agonist CPA, ranging from 4.7 nM in the presence of PD81,723 to a high value of 2.9 μM in the combined presence of NEM, NaCl, and GTP, represented a greater than 600‐fold affinity shift. We suggest that the latter micromolar IC₅₀ value may approximate CPA’s “true” affinity (K_A) for the rat adenosine A₁ receptor. Drug Dev. Res. 51:207–215, 2000. © 2001 Wiley‐Liss, Inc.     

NPC 205 is a potent and selective adenosine A1, receptor antagonist: Correlation among receptor binding,biochemical, and physiological assays

The chemical synthesis of 8-aryl-1,3-dialkyl derivatives of xanthine and the ability of these compounds to interact with adenosine A₁ and A₂ receptors in vitro (in receptor binding, biochemical, and physiological assays) are described. NPC 205 (1,3-dipropyl-8-[4-hydroxy- phenyl]xanthine) was the most potent and A₁-selective antagonist of the series. NPC 205 was twice as potent at PACPX (1,3-dipropyl-8-[2-amino-4-chlorophenyl]xanthine) and at least 20-fold more potent than 8-phenyltheophylline to inhibit specific binding of [³H]-cy-clohexyladenosine to guinea pig and rat brain membranes. Along with NPC 189 (1,3-dimethyl-8-[4-hydroxyphenyl]xanthine), NPC 200 (1,3-dipropyl-8-phenylxanthine), and PACPX, NPC 205 also was a more potent antagonist of A₁-receptor-mediated inhibition of adenylate cyclase (AC) in rat cortex than A₂ receptor-coupled stimulation of AC in PC-12 cell membranes. NPC 205 was, in fact, 30-fold more potent and fourfold more selective than PACPX for the AC-coupled A₁ receptor function and exhibited competitive interactions with A₁ receptors. In isolated guinea pig atria, NPC 205, NPC 189, NPC 200, and PACPX all reversed 5′N-ethyl-carboxamidoadenosine (NECA)-induced depression of tension and rate. In the Langendorff isolated perfused guinea pig heart preparation, NPC 205 was more potent than PACPX in reversing NECA-mediated depression in developed tension and rate (A₁-mediated) and, unlike PACPX, had no effect on coronary perfusion pressure (A₂-mediated). Neither NPC 205 or PACPX had significant competitive inhibitory effects on rat heart phosphodiesterase III activity in vitro. Together, the results suggest that NPC 205 is a highly potent, selective, and competitive antagonist of adenosine A₁ receptors and should be useful for examining the physiological significance of A₁ receptors in vitro and in vivo.     

Molecular modeling of adenosine receptors. I. The ligand binding site on the A1 receptor.

The amino acid sequence of the canine adenosine A1 receptor and the atomic coordinates of a structurally related protein, bacteriorhodopsin, were combined to generate a three-dimensional model for the adenosine A1 receptor. This model consists of seven amphipathic alpha-helices, forming a pore that has a rather distinct partition between hydrophobic and hydrophilic regions. Subsequently, a highly potent and selective ligand, N6-cyclopentyladenosine, was docked into this cavity. A binding site is proposed that takes into account the conformational characteristics of the ligand, obtained from indirect modeling studies by the 'active analog approach'. Moreover, it involves two histidine residues that were shown to be important for ligand coordination from chemical modification studies. Finally, the deduced binding site was used to model other potent ligands that could all be accommodated consistent with earlier biochemical and pharmacological findings.     

The effects of saponin on the binding and functional properties of the human adenosine A1 receptor.

1. Experiments with adenosine deaminase suggest that adenosine is present in membrane preparations from CHO cells bearing adenosine A1 receptors. 2. Pretreatment of the membranes (ca 0.6 mg protein ml-1) with the permeabilizing agent saponin (100 micrograms ml-1) or addition of saponin (10 micrograms ml-1) to the membranes (0.02-0.08 mg protein ml-1) in the assay, generates homogeneous low affinity agonist binding curves in the presence of GTP and an increased function, assessed by agonist stimulation of [35S]-GTP gamma S binding. The affinity constants for the binding of an agonist and an antagonist are not affected by this saponin treatment. Saponin facilitates the interaction of guanine nucleotides with receptor G-protein complexes, possibly by removing a permeability barrier to access of G-proteins by GTP. However, adenosine is still present in the binding assays after saponin treatment. 3. The agonist binding properties of the human A1 receptor have been characterized. In saponin pretreated membranes, 80-90% of the A1 receptors are capable of forming agonist-receptor-G protein complexes in the absence of GTP. These complexes have a 300-600 fold higher affinity than uncoupled receptors for N6-cyclohexyladenosine. 4. A very slow component is observed in the association and dissociation kinetics of the agonist [3H]-N6-cyclohexyladenosine ([3H]-CHA) and in the association but not dissociation kinetics of the antagonist [3H]-8-cyclopentyl-1,3-dipropylxanthine ([3H]-DPCPX). The slow association component of [3H]-DPCPX is essentially absent when incubations are carried out in the presence of GTP. The slow dissociation component of [3H]-CHA binding is rapidly disrupted by GTP. 5. It is hypothesized that long-lasting adenosine-receptor-G protein complexes are present in the CHO membrane preparations. The existence of these complexes, resistant to the action of adenosine deaminase but sensitive to GTP, may rationalize the observed kinetics and the increase in 3H-antagonist binding produced by GTP which has been observed in essentially all studies of A1 receptors and has been ascribed previously to precoupling of A1 receptors to G-proteins in the absence of agonists.     

A selective binding site for 3H-NECA that is not an adenosine A2 receptor

In homogenates of NG108-15 cells, adenosine analogues activate adenylate cyclase with the following order of potency: N-ethylcarboxamidoadenosine (NECA) greater than 2-chloroadenosine greater than N6-(L-phenylisopropyl)-adenosine (PIA) = cyclohexyladenosine = 2-phenylaminoadenosine. Adenosine receptor antagonists inhibit NECA-stimulated adenylate cyclase activity with the order of potency 3-isobutyl-1-methyl-xanthine (IBMX) greater than theophylline greater than caffeine. These data suggest that these ligands act at an adenosine A2 receptor. There is an apparently homogenous population of saturable 3H-NECA binding sites in homogenates of NG108-15 cells. These sites have an affinity for 3H-NECA of approximately 1 microM, and are present at a density of approximately 10 pmol/mg protein. Unlabelled NECA, 2-chloroadenosine, IBMX and theophylline displace 3H-NECA binding, with an order of potency that suggests that the 3H-NECA binding site may represent an adenosine A2 receptor. However, PIA, cyclohexyladenosine and 2-phenylaminoadenosine produce no detectable displacement of 3H-NECA binding at concentrations that produce a maximal stimulation of adenylate cyclase activity. Pretreatment of NG108-15 cells with either NECA or PIA produces a homologous desensitization of subsequent responses to all the adenosine analogues, with no effect on subsequent responses to a prostacyclin receptor agonist or NaF. This suggests that all the adenosine analogues examined activate an adenosine A2 receptor. Therefore, the 3H-NECA site at which PIA is inactive cannot represent this receptor.     

Pharmacological characterization and radioligand binding properties of a high-affinity, nonpeptide, bradykinin B1 receptor antagonist

Compound A (N-[2-[4-(4,5-dihydro-1H-imidazol-2-yl)phenyl]ethyl]-2-[(2R)-1-(2-napthylsulfonyl)-3-oxo-1,2,3,4-tetrahydroquinoxalin-2-yl]acetamide) is a member of a new class of aryl sulfonamide dihydroquinoxalinone bradykinin B1 receptor antagonists that should be useful pharmacological tools. Here we report on some of the pharmacological properties of compound A as well as the characterization of [35S]compound A as the first nonpeptide bradykinin B1 receptor radioligand. Compound A inhibited tritiated peptide ligand binding to the cloned human, rabbit, dog, and rat bradykinin B1 receptors expressed in CHO cells with Ki values of 0.016, 0.050, 0.56, and 29 nM, respectively. It was inactive at 10 microM in binding assays with the cloned human bradykinin B2 receptor. In functional antagonist assays with the cloned bradykinin B1 receptors, compound A inhibited agonist-induced signaling with activities consistent with the competition binding results, but had no antagonist activity at the bradykinin B2 receptor. Compound A was also found to be a potent antagonist in a rabbit aorta tissue bath preparation and to effectively block des-Arg9 bradykinin depressor responses in lipopolysaccharide-treated rabbit following intravenous administration. The binding of [35S]compound A was evaluated with the cloned bradykinin B1 receptors. In assays with human, rabbit, and dog receptors, [35S]compound A labeled a single site with Kd values of 0.012, 0.064, and 0.37 nM, respectively, and with binding site densities equivalent to those obtained using the conventional tritiated peptide ligands. Binding assays with the cloned rat bradykinin B1 receptor were not successful, presumably due to the low affinity of the ligand for this species receptor. There was no specific binding of the ligand detected in CHO cells expressing the human bradykinin B2 receptor. In assays with the cloned human bradykinin B1 receptor, the pharmacologies of the binding of [35S]compound A and [3H][Leu9]des-Arg10-kallidin were the same. The high signal-to-noise ratio obtained with [35S]compound A will allow this ligand to be a very useful tool for future investigations of the bradykinin B1 receptor.     

A model of the adrenergic beta-2 receptor and binding sites for agonist and antagonist.

From the known experimental data on adrenergic ligands and beta-2 receptor a model for the ligand binding sites was proposed and built. Agonist and antagonist have overlapping but different binding sites. The model correlates well with the structural information known from a series of adrenergic ligands.     

Rolofylline,an adenosine A1 receptor antagonist,inhibits osteoclast differentiation as an inverse agonist

BACKGROUND AND PURPOSE

Adenosine may be generated by hydrolysis of extracellular nucleotides by ectonucleotidases, including ectonucleoside triphosphate diphosphohydrolase 1 (CD39), ecto-5′-nucleotidase (CD73), nucleotide pyrophosphatase phosphodiesterase 1 (NPP-1) and tissue non-specific alkaline phosphatase (TNAP). Previous work from our laboratory has uncovered a critical role for adenosine A₁ receptors (A₁R) in osteoclastogenesis; blockade or deletion of these receptors diminishes osteoclast differentiation. Interestingly, selective A₁R agonists neither affect basal osteoclastogenesis nor do they reverse A₁R antagonist-mediated inhibition of osteoclastogenesis. In this study, we determined whether ectonucleotidase-mediated adenosine production was required for A₁R antagonist-mediated inhibition, and, when we saw no effect, determined whether A₁R was constitutively activated and the antagonist was acting as an inverse agonist to diminish osteoclast differentiation.

EXPERIMENTAL APPROACH

Osteoclast formation derived from wild-type, CD39 knockout (KO), CD73 KO, NPP-1 KO and TNAP KO mice was examined by tartrate-resistant acid phosphatase staining of receptor activator of NF-κB ligand–macrophage colony-stimulating factor-stimulated osteoclasts and osteoclast gene expression (Ctsk, Acp5, MMP-9 and NFATc1). Intracellular cAMP concentration was determined by elisa.

KEY RESULTS

Rolofylline inhibited osteoclast formation in a dose-dependent manner (IC₅₀ = 20–70 nM) in mice lacking all four of these phosphatases, although baseline osteoclast formation was significantly less in precursors from CD73 KO mice. Rolofylline (1 μM) stimulates cAMP production in bone marrow macrophages by 10.23 ± 0.89-fold.

CONCLUSIONS AND IMPLICATIONS

Based on these findings, we hypothesize that the A₁R is constitutively activated in osteoclast precursors, thereby diminishing basal AC activity, and that A₁R antagonists act as inverse agonists to release A₁R-mediated inhibition of basal AC activity and permit osteoclast differentiation. The constitutive activity of A₁R promotes osteoclast formation and down-regulation of this activity blocks osteoclast formation.     

Agomelatine and tianeptine antidepressant activity in mice behavioral despair tests is enhanced by DMPX,a selective adenosine A2A receptor antagonist,but not DPCPX,a selective adenosine A1 receptor antagonist

BackgroundAdenosine, an endogenous nucleoside, modulates the release of monoamines, e.g., noradrenaline, serotonin, and dopamine in the brain. Both nonselective and selective stimulation of adenosine receptors produce symptoms of depression in some animal models. Therefore, the main objective of our study was to assess the influence of a selective adenosine A₁ receptor antagonist (DPCPX) and a selective adenosine A_2A receptor antagonist (DMPX) on the activity of agomelatine and tianeptine.MethodsThe forced swim test (FST) and tail suspension test (TST) were performed to assess the effects of DPCPX and DMPX on the antidepressant-like activity of agomelatine and tianeptine. Drug serum and brain levels were analyzed using HPLC.ResultsCo-administration of agomelatine (20 mg/kg) or tianeptine (15 mg/kg) with DMPX (3 mg/kg), but not with DPCPX (1 mg/kg), significantly reduced the immobility time both in the FST and TST in mice. These effects were not associated with an enhancement in animals’ spontaneous locomotor activity. The observed changes in the mouse behavior after concomitant injection of DMPX and the tested antidepressant agents were associated with elevated brain concentration of agomelatine and tianeptine.ConclusionOur study shows a synergistic action of the selective A_2A receptor antagonist and the studied antidepressant drugs, and a lack of such interaction in the case of the selective A₁ receptor antagonist. The interaction between DMPX and agomelatine/tianeptine at least partly occurs in the pharmacokinetic phase. A combination of a selective A_2A receptor antagonist and an antidepressant may be a new strategy for treating depression.     

ZM 241385, an adenosine A(2A) receptor antagonist, inhibits hippocampal A(1) receptor responses

4-(2-[7-amino-2-(2-furyl?1,2,4?-triazolo?2,3a?-?1,3, 5?triazin-5-yl-amino]ethyl)phenol (ZM 241385) has been used as an antagonist of adenosine A(2A) receptors, exhibiting high selectivity over adenosine A(1) receptors. We now report that ZM 241385 (10-50 nM) attenuated the inhibitory action of N(6)-cyclopentyladenosine (10 nM) and R(-)-N(6)-phenylisopropyladenosine (R-PIA, 20 nM), two selective adenosine A(1) receptor agonists, on hippocampal population spike amplitude. This effect is unlikely to be a direct antagonism of adenosine A(1) receptor since the K(i) of ZM 241385 to displace [3H]PIA (2 nM) binding, from hippocampal membranes ranged from 0.8 to 1.9 microM. These results question the usefulness of ZM 241385 to define adenosine A(2A) receptors actions in functional studies.     

Effects of urea pretreatment on the binding properties of adenosine A1 receptors

The effect of denaturation and/or extraction of nonintegral membrane proteins by 7 M urea on the binding of the antagonist [3H]cyclopentyl-1,3-dipropylxanthine 8 dipropyl-2,3 ([3H]DPCPX), and the agonists adenosine, (-)-N6-(2-phenylisopropyl)-adenosine (R-PIA) and N6-cyclohexyladenosine (CHA), was investigated at human A1 adenosine receptors stably expressed in CHO cells. Pretreatment with urea caused a 56% reduction in membrane proteins. Compared to controls, the use of adenosine deaminase (ADA), 100 microM 5'-guanylylimidodiphosphate (Gpp(NH)p) or urea each caused equivalent increases in specific [3H]DPCPX binding. Neither the binding kinetics nor the affinity of [3H]DPCPX were significantly different in urea-pretreated compared to ADA-pretreated membranes. At 25 degrees C in ADA-pretreated membranes, the competition isotherms for R-PIA and CHA were characterized by two affinity states. Gpp(NH)p (100 microM) reduced, but did not abolish, the value of the high-affinity dissociation constant. Similar results were obtained after treatment with urea for R-PIA, whereas the high-affinity state for CHA was abolished. At 37 degrees C, urea pretreatment, but not 100 microM Gpp(NH)p, abolished high-affinity agonist competition binding. There was no significant effect of any of the treatments on the low-affinity agonist binding state. In urea-pretreated membranes, exogenously added adenosine competed according to a simple mass-action model with a pK(L) of 5.66+/-0.05 (n=3). Compared to the more common approaches of ADA treatment and/or use of guanine nucleotides, our findings suggest that urea pretreatment represents an inexpensive and useful approach for investigating the binding properties of adenosine A1 ligands (including adenosine) to the G protein-uncoupled form of the receptor.     

Istradefylline, a novel adenosine A2A receptor antagonist, for the treatment of Parkinson's disease

Dopamine replacement therapy effectively treats the early motor symptoms of Parkinson's disease (PD). However, its association with the development of motor complications limits its usefulness in late stages of the disease. Adenosine A(2A) receptors are localised to the indirect striatal output function and control motor behaviour. They are active in predictive experimental models of PD and appear to be promising as the first major non-dopaminergic therapy for PD. Istradefylline is a novel adenosine A(2A) receptor antagonist currently in Phase III clinical trials for efficacy in patients with PD; results from Phase II clinical trials demonstrated that it provides a clinically meaningful reduction in 'off' time and an increased 'on' time with non-troublesome dyskinesia in levodopa-treated patients with established motor complications, and is safe and well tolerated.     

The surmountable effect of FSCPX, an irreversible A1 adenosine receptor antagonist, on the negative inotropic action of A1 adenosine receptor full agonists in isolated guinea pig left atria

A₁ adenosine receptors (A₁ receptors) are widely expressed in mammalian tissues; therefore attaining proper tissue selectivity is a cornerstone of drug development. The fact that partial agonists chiefly act on tissues with great receptor reserve can be exploited to achieve an appropriate degree of tissue selectivity. To the best of our knowledge, the A₁ receptor reserve has not been yet quantified for the atrial contractility. A₁ receptor reserve was determined for the direct negative inotropic effect of three A₁ receptor full agonists (NECA, CPA and CHA) in isolated, paced guinea pig left atria, with the use of FSCPX, an irreversible A₁ receptor antagonist. FSCPX caused an apparently pure dextral displacement of the concentration–response curves of A₁ receptor agonists. Accordingly, the atrial A₁ receptor function converging to inotropy showed a considerably great, approximately 80–92 % of receptor reserve for a near maximal (about 91–96 %) effect, which is greater than historical atrial A₁ receptor reserve data for any effects other than inotropy. Consequently, the guinea pig atrial contractility is very sensitive to A₁ receptor stimulation. Thus, it is worthwhile considering that even partial A₁ receptor agonists, given in any indication, might decrease the atrial contractile force, as an undesirable side effect, in humans.     

腺苷A1受体阻断剂对学习记忆的影响及机制分析

目的探讨腺苷A1受体阻断剂对学习记忆的影响及其与胆碱能、氨基酸能神经的关系.方法采用避暗实验、分光光度法和HPLC法,观察腺苷A1受体特异性阻断剂8-环戊-1,3-二丙基黄嘌呤(DPCPX)对东莨菪碱(Scop)、2-氨基-5-磷戊酸(AP5)致小鼠记忆障碍及脑胆碱酯酶(AChE)活性、氨基酸水平的影响.结果 DPCPX可显著改善Scop致记忆障碍,但对AP5致记忆障碍无影响;在体内外高剂量DPCPX可显著抑制小鼠脑AChE活性;DPCPX icv可显著升高小鼠脑Glu和Asp含量,降低GABA含量,使脑内Glu/GABA比值显著升高.结论腺苷A1受体特异性阻断剂DPCPX可显著改善Scop而不能改善AP5致记忆障碍,在高剂量时可影响脑AChE活性和脑氨基酸水平、升高脑内Glu/GABA比值.     

Effects of benzodiazepine administration on A1 adenosine receptor binding in-vivo and ex-vivo.

The adenosine receptor has been implicated in the central mechanism of action of benzodiazepines. The specific binding of an A1-selective adenosine antagonist radioligand, [3H]8-cyclopentyl-1,3-dipropylxanthine, was measured in-vivo in mice treated with alprazolam (2 mg kg-1, i.p.), lorazepam (2 mg kg-1, i.p.) and vehicle. Binding studies were performed in-vivo and ex-vivo in mice receiving continuous infusion of alprazolam (2 mg kg-1 day-1), lorazepam (2 mg kg-1 day-1) and vehicle by mini-osmotic pumps for 6 days. Continuous infusion of alprazolam and lorazepam significantly decreased specific binding by 34 and 53%, respectively, compared with vehicle treatment (P less than 0.01). Single doses of alprazolam and lorazepam induced a similar trend in specific binding in-vivo (P = 0.07). There were no alterations in A1-receptor density (Bmax) or affinity (Kd) in cortex, hippocampus or brainstem in ex-vivo studies. Benzodiazepine treatment may diminish A1- receptor binding in-vivo by inhibiting adenosine uptake or by direct occupancy of the A1 adenosine receptor recognition site.     

Renal effects of FK453: A potent non-xanthine adenosine A1 receptor antagonist

The renal effects of FK453, a potent and selective non-xanthine adenosine A₁ receptor antagonist, were examined and compared with FR113452 (less active enantiomer of FK453), typical adenosine receptor antagonists, and diuretics. In rats FK453 possessed diuretic activity similar to 1, 3-dipropyl-8-cyclopentylxanthine (DPCPX, adenosine A₁ receptor antagonist), hydrochlorothiazide, and furosemide, but neither FR113452 nor CP66713 (an adenosine A₂ receptor antagonist) possessed diuretic activity. Urinary uric acid excretion in rats increased with FK453, but other drugs had no effect. These diuretic and uricosuric activities of FK453 were also observed in dogs. In anesthetized dogs, FK453 increased the renal blood flow (RBF), inulin clearance (Cin), and p-aminohippuric acid clearance (C_PAH). However, hydrochlorothiazide had no effect on RBF, Cin, and C_PAH. Furthermore, osmolar clearance experiments suggested that the renal site of action of FK453 was different from hydrochlorothiazide and furosemide. These results demonstrate that FK453 has diuretic activity and increases urinary uric acid excretion and suggest that the diuretic activity of FK453 is related to adenosine A₁ receptor antagonism; also, the diuretic mechanism of action and the renal site of action of FK453 are different from those of hydrochlorothiazide and furosemide. FK453 is a useful compound to clarify the physiological role of the adenosine A₁ receptor in the kidney. © 1995 Wiley-Liss, Inc.     

Allosteric modulators for the A1 adenosine receptor

Allosteric modulators of endogenous adenosine represent an alternative to direct acting adenosine agonists and nucleoside uptake blockers. These compounds can selectively enhance the response to adenosine in only those organs or localised areas of a given organ in which production of adenosine is increased. The present article is an overview of the recent patent literature related to allosteric modulators of adenosine function on the A₁ receptor. In particular, the compounds with improved potency as enhancers and reduced antagonist properties are mentioned. Among the reported compounds, two molecules appear to be of potential therapeutic utility. A synergistic combination of PD-81723 and cyclopentyladenosine, an allosteric enhancer and agonist, respectively, of the adenosine A₁ receptor, appeared to be effective to induce angiogenesis. Moreover, (2-amino-4,5,6,7-tetrahydro-benzo[b]thiophen-3-yl)-(4-chloro-phenyl)-methanone appears to be active for the treatment of neuropathic pain without co-administration of adenosine.     

Effects of a selective adenosine A1 receptor antagonist on the development of cyclosporin nephrotoxicity.

1. The clinical application of cyclosporin as an immunosuppressive agent is limited by its nephrotoxicity. 2. The effect of FK453, a selective A1-receptor antagonist, administered twice daily to rats at a dose of 100 mg kg-1 was assessed on the development of nephrotoxicity induced by cyclosporin (10 mg kg-1 i.p. daily) administered for 14 days. The effects of nifedipine administered twice daily (0.3 mg kg-1 s.c.) for 14 days, on cyclosporin nephrotoxicity were also studied. 3. Cyclosporin induced a 46.58% and 35.78% decline in glomerular filtration rate (GFR) and effective renal plasma flow (ERPF) respectively and a reduction of 16.69% in filtration fraction (FF). Co-administration of FK453 resulted in falls of 30.5%, 18.59% and 14.7% in GFR, ERPF and FF respectively, the former two significantly less than the falls seen with cyclosporin (CyA) alone (P < 0.05 vs CyA, ANOVA). 4. Nifedipine appeared to have a more pronounced protective effect resulting in a decline of only 20.91% in GFR, with no significant change in ERPF (increase of 0.93%) when co-administered with CyA. 5. These observations indicate adenosine plays a minor role in the pathophysiology of CyA nephrotoxicity.     

High affinity acylating antagonists for the A1 adenosine receptor: identification of binding subunit

Two isomeric isothiocyanate derivatives of the A1 adenosine receptor antagonist xanthine amine cogener (XAC) have been synthesized and found to be potent affinity labels (irreversibly bound ligands) for A1 adenosine receptors. The interaction of m- and p-isomers of 1,3-dipropyl-8-isothiocyanatophenyl(aminothiocarbonyl (2-aminoethylaminocarbonyl(4-methyloxy(phenyl]])-xanthine (DITC-XAC) with rat brain A1 receptors is of high affinity (EC50 = 27 and 52 nM, respectively) as determined by radioligand competition curves. These compounds reduced the number of A1 receptors (greater than 90% at 500 nM m-DITC-XAC) in brain membranes, without any change in the affinity of the remaining receptors for [125I]N6-2-(4-aminophenyl)ethyladenosine. Prior reaction of the isothiocyanate moiety with ethylenediamine did not alter the affinity of the XAC derivative for the A1 receptor but eliminated its ability to covalently incorporate into the receptor. Incubation of brain membranes with radiolabeled p- and m-DITC-XAC results in the specific labeling of a Mr 38,000 peptide. This labeling can be blocked with both an A1 adenosine receptor-specific agonist and an antagonist. This specific protein has the same molecular weight as the protein labeled with A1-selective photoaffinity probes. The much higher efficiency of incorporation of these affinity probes compared with photoaffinity probes should make them extremely useful for structural studies of A1 adenosine receptors.