Adenosine: a prototherapeutic concept in neurodegeneration.
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Adenosine↗
Biomedical subjects
K A Jacobson
Publications and source records attributed to K A Jacobson.
Chronic NMDA receptor stimulation: therapeutic implications of its effect on adenosine A1 receptors.
It is known that stimulation of adenosine A1 receptors has a modulatory effect on the excitability of postsynaptic NMDA receptors. Conversely, acute stimulation of NMDA receptors results in release of adenosine via calcium-independent mechanisms. These findings indicate a close functional relationship between these receptors. It is, therefore, possible that chronic, low level stimulation of the NMDA receptor may have a negative impact on these modulatory processes. To investigate this possibility, we have subjected C57BL mice either to an acute injection of a N6-cyclopentyladenosine (CPA, 0.01 mg/kg) or deoxycoformycin (1 mg/kg) followed by a convulsant dose of N-methyl-D-aspartate (NMDA) (60 mg/kg) or to chronic, low level (20 mg/kg i.p. daily) exposure to NMDA for 8 weeks. One day after the last injection of NMDA, animals were injected either with a convulsant dose of NMDA alone, or with either CPA at 0.001 or 0.01 mg/kg, or with 1 mg/kg deoxycoformycin followed 15 min later by 60 mg/kg NMDA. Neither CPA nor deoxycoformycin were protective when NMDA was given acutely at 60 mg/kg. Chronic treatment with NMDA alone or chronic administration of NMDA followed by 0.001 mg/kg CPA had no significant effect on mortality following a convulsant dose of NMDA. However, when the chronic regimen of NMDA was followed by either 0.01 mg/kg CPA or 1 mg/kg deoxycoformycin, mortality was reduced to 10% (CPA), or eliminated completely (deoxycoformycin). Moreover, combination of chronic NMDA treatment with either CPA (both doses) or deoxycoformycin produced a significant improvement in other measures, i.e., seizure onset, intensity of neurological impairment, and extension of time to death.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine↗
A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine selectively activates chloride efflux from human epithelial and mouse fibroblast cell lines expressing the cystic fibrosis transmembrane regulator delta F508 mutation.
Cystic fibrosis is an autosomal recessive disorder affecting chloride transport in pancreas, lung, and other tissues, which is caused by mutations in the cystic fibrosis transmembrane regulator (CFTR). The A1 receptor antagonist 8-cyclopentyl-1,3-dipropylxanthine (CPX) stimulates 36Cl- efflux from pancreatic CFPAC-1 cells which bear the delta F508 genotype common to most cases of cystic fibrosis [Eidelman et al. (1992) Proc. Natl. Acad. Sci. U.S.A. 89, 5562-5566]. By contrast, correction of the cystic fibrosis defect by retrovirus-mediated gene transfer renders the resulting CFPAC-PLJ-CFTR cells insensitive to CPX. We now report that CPX also activates chloride efflux from the CF tracheal epithelial cell line IB3-1 bearing a delta F508 allele, but not if the IB3-1 cells have been repaired by transfection of the wild-type CFTR gene. Similar results were obtained with recombinant NIH 3T3 cells, in which CPX activates 36Cl- efflux from cells expressing the CFTR (delta F508) gene product but not from 3T3 cells expressing the wild-type CFTR. In all three cell types expressing CFTR (delta F508), CPX was found to activate 36Cl- efflux in a dose-dependent manner over the concentration range of 1-30 nM and then gradually lose potency at higher CPX concentrations. Six CPX analogues, A1 receptor antagonists of affinity similar to that of CPX, were found to be much less effective than CPX at activating 36Cl- efflux from CFPAC-1 cells. These included 2-thio-CPX. CPT (8-cyclopentyl-1,3-dimethylxanthine),3,4-dehydro-CPX,3-F-CPX,3-1-CPX, and KW-3902 (8-noradamantyl-1,3-dipropylxanthine).(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Stimulation by alkylxanthines of chloride efflux in CFPAC-1 cells does not involve A1 adenosine receptors.
A series of 8-substituted derivatives of 1,3,7-alkylxanthines was synthesized as potential activators of chloride efflux from a human epithelial cell line (CFPAC) expressing the cystic fibrosis transmembrane regulator (CFTR) delta F508 mutation. Their interactions with rat brain A1 and A2a receptors were also studied in radioligand binding experiments. Substitution was varied at the xanthine 1-, 3-, 7- and 8-positions. 1,3-Dipropyl-8-cyclopentylxanthine (CPX) stimulated Cl- efflux in the 10(-8) M range, with a maximal effect reaching 200% of control and diminishing at higher concentrations. The potent adenosine antagonist 8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]oxy]phenyl]- 1,3-dipropylxanthine, nonselective at human A1 and A2a receptors, was inactive in Cl- efflux. 1,3-Diallyl-8-cyclohexylxanthine (DAX) was highly efficacious in stimulating chloride efflux with levels reaching > 300% of control, although micromolar concentrations were required. 1,3,7-Trimethyl-8-(3-chlorostyryl)xanthine, an A2a-selective adenosine antagonist, was only weakly active. Caffeine, which acts as an nonselective adenosine antagonist in the range of 10(-5) M, was active in Cl- efflux in the low nanomolar range but with low efficacy. Thus, among the xanthine derivatives of diverse structure, there was no correlation between potency in Cl- efflux and adenosine antagonism. Poly(A)+ RNA isolated from CFPAC-1 cells showed no hybridization to a human A1 receptor cDNA probe, excluding this receptor as a mediator of CPX-elicited Cl- efflux. Thus, this action of xanthines in stimulating Cl- efflux in CFPAC cells, which express a defective CFTR, represents a novel site of action apparently unrelated to adenosine receptors.
Animals↗
Site-directed mutagenesis identifies residues involved in ligand recognition in the human A2a adenosine receptor.
The A2a adenosine receptor is a member of the G-protein coupled receptor family, and its activation stimulates cyclic AMP production. To determine the residues which are involved in ligand binding, several residues in transmembrane domains 5-7 were individually replaced with alanine and other amino acids. The binding properties of the resultant mutant receptors were determined in transfected COS-7 cells. To study the expression levels in COS-7 cells, mutant receptors were tagged at their amino terminus with a hemagglutinin epitope, which allowed their immunological detection in the plasma membrane by the monoclonal antibody 12CA5. The functional properties of mutant receptors were determined by measuring stimulation of adenylate cyclase. Specific binding of [3H]CGS 21680 (15 nM) and [3H]XAC (4 nM), an A2a agonist and antagonist, respectively, was absent in the following Ala mutants: F182A, H250A, N253A, I274A, H278A, and S281A, although they were well expressed in the plasma membrane. The hydroxy group of Ser-277 is required for high affinity binding of agonists, but not antagonists. An N181S mutant lost affinity for adenosine agonists substituted at N6 or C-2, but not at C-5'. The mutant receptors I274A, S277A, and H278A showed full stimulation of adenylate cyclase at high concentrations of CGS 21680. The functional agonist potencies at mutant receptors that lacked radioligand binding were > 30-fold less than those at the wild type receptor. His-250 appears to be a required component of a hydrophobic pocket, and H-bonding to this residue is not essential. On the other hand, replacement of His-278 with other aromatic residues was not tolerated in ligand binding. Thus, some of the residues targeted in this study may be involved in the direct interaction with ligands in the human A2a adenosine receptor. A molecular model based on the structure of rhodopsin, in which the 5'-NH in NECA is hydrogen bonded to Ser-277 and His-278, was developed in order to visualize the environment of the ligand binding site.
Amino Acid Sequence↗
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.
Adenine↗
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↗
Presence of both A1 and A2a adenosine receptors in human cells and their interaction.
We have obtained pharmacological evidence for the expression of both A1- (inhibitory) and A2a- (stimulatory) adenosine receptors in cultured human foreskin fibroblasts and lung fibroblasts. The A1 receptors were sufficiently abundant in foreskin fibroblasts so that binding studies with a radioactively labeled specific ligand confirmed their existence. Both receptors were activated during the stimulation of cAMP accumulation and DNA synthesis by adenosine.
Adenosine↗
The effects of adenosine A3 receptor stimulation on seizures in mice.
We have previously shown that acute preischemic adenosine A3 receptor stimulation results in an increased postischemic damage, while chronic stimulation of this receptor diminishes it. Since several pathophysiological phenomena are common for both ischemia and seizures, we have explored the effect of acute and chronic administration of the adenosine A3 receptor selective agonist IB-MECA (N6-(3-iodobenzyl) adenosine-5'-N-methylcarboxamide) prior to seizures induced by N-methyl-D-aspartate (NMDA), pentamethylenetetrazole, or electric shock. At 100 micrograms/kg, acutely injected IB-MECA was protective in chemically but not electrically induced seizures. In chronic administration of IB-MECA, significant protection against chemically induced seizures was obtained in all studied measures, i.e., seizure latency, neurological impairment, and survival. Although threshold voltage was unchanged in electrically induced seizures, a chronic regimen of IB-MECA significantly reduced postepileptic mortality. Since the combination of an arteriole-constricting compound 48/80 and hypotension-inducing clonidine injected prior to NMDA results in a significant protection against seizures, and since acute stimulation of adenosine A3 receptor causes both arteriolar constriction and severe hypotension, there is a possibility that the protection obtained by the acutely administered drug may result from inadequate delivery of chemoconvulsants to the brain. It is, however, unknown whether the protective effect of chronically administered IB-MECA is related to the effect of the drug on blood flow, neuronal mechanisms, or both.
Adenosine↗
Regulation of adenosine receptors in cultured heart cells.
A1 adenosine receptors were studied on heart cells grown in cultures by the radioligand binding technique. Treatments with agents that accelerated heart rate for 3-4 days, caused an increase in the level of adenosine receptors. Treatments that attenuated heart rate, reduced the level of the receptors. Thus, the cardiac cells respond to environmental conditions affecting heart contraction so as to restore the basal rate of heart activity.
Animals↗
Molecular probes for muscarinic receptors: functionalized congeners of selective muscarinic antagonists.
The muscarinic agonist oxotremorine and the tricyclic muscarinic antagonists pirenzepine and telenzepine have been derivatized using a functionalized congener approach for the purpose of synthesizing high affinity ligand probes that are suitable for conjugation with prosthetic groups, for receptor cross-linking, fluorescent and radioactive detection, etc. A novel fluorescent conjugate of TAC (telenzepine amine congener), an n-decylamino derivative of the m1-selective antagonist, with the fluorescent trisulfonated pyrene dye Cascade Blue may be useful for assaying the receptor as an alternative to radiotracers. In a rat m3 receptor mutant containing a single amino acid substitution in the sixth transmembrane domain (Asn507 to Ala) the parent telenzepine lost 636-fold in affinity, while TAC lost only 27-fold. Thus, the decylamino group of TAC stabilizes the bound state and thus enhances potency by acting as a distal anchor in the receptor binding site. We have built a computer-assisted molecular model of the transmembrane regions of muscarinic receptors based on homology with the G-protein coupled receptor rhodopsin, for which a low resolution structure is known. We have coordinated the antagonist pharmacophore (tricyclic and piperazine moieties) with residues of the third and seventh helices of the rat m3 receptor. Although the decylamino chain of TAC is likely to be highly flexible and may adopt many conformations, we located one possible site for a salt bridge formation with the positively charged -NH3+ group, i.e. Asp113 in helix II.
Animals↗
Comparative molecular field analysis of selective A3 adenosine receptor agonists.
A series of 48 N6-benzyladenosine 5'-uronamide derivatives has been described recently as moderately selective A3 adenosine receptor agonists of nanomolar potency (Gallo-Rodriguez, C. et al. J. Med. Chem. 1994, 37, 636). Quantitative structure activity relationships in this series, including some novel derivatives, have been investigated using a Comparative Molecular Field Analysis (CoMFA), with emphasis on the N6-substituent. The resulting three dimensional pharmacophore model defines the steric and electronic factors which modulate in vitro affinities in binding to rat brain A3 adenosine receptors. The model indicates a positive correlation of affinity with the steric characteristics of the compounds (major factor), particularly toward the 3-position of the benzyl ring of N6-benzyl NECA, and a weak correlation with the electrostatic effects of the N6-substituent. A comparison of active and inactive compounds using volume maps showed that bulk at the 3-position of the benzyl ring of the molecule is conducive to high affinity at A3 receptors, while steric bulk at other positions of the benzyl ring leads to poor binding. t-Boc-amino acid conjugates of a 3-aminobenzyl derivative were synthesized to probe the steric and hydrophobic limitations at that position. We have discovered a subregion of the N6-benzyl binding pocket occupied by a 3-(L-prolylamino) group that is sterically disallowed at A3 receptors and allowed in A1 and A2a receptors. 6-N-Phenylhydrazino and 6-O-phenylhydroxylamino derivatives, incorporating major changes in electrostatic character of the ligand proximal to the purine, were predicted by the CoMFA model to have high A3 affinity. Such analogs were synthesized and found to be well tolerated at the A3 receptor binding site.
Adenosine↗
Potent agonist action of 2-thioether derivatives of adenine nucleotides at adenylyl cyclase-linked P2Y-purinoceptors.
1. Analogues of adenine nucleotides inhibited beta-adrenoceptor-stimulated cyclic AMP accumulation in C6 rat glioma cells with a pharmacological selectivity consistent with that for involvement of a P2Y-purinoceptor. 2. The inhibitory effect of adenine nucleotides was completely prevented by pretreatment of cells with pertussis toxin. 3. The capacity of a series of recently synthesized 2-thioether analogues of adenine nucleotides to inhibit cyclic AMP accumulation was examined. Several ATP analogues, e.g. 2-cyclohexylthio and 2-hexylthio ATP, inhibited cyclic AMP accumulation with EC50 values of approximately 30 pM. These values represent 100,000 fold increases in potency over ATP. 4. Analogues of ADP exhibited the same remarkable increase in potency relative to their natural congener and diphosphates were at least as potent as the corresponding triphosphates at the C6 cell P2Y-purinoceptor. 5. The relative potencies of a broad series of agonists at the C6 cell receptor did not correspond to the relative potencies of the same compounds for activation of P2Y-purinoceptors on turkey erythrocyte membranes. Some agonists, particularly 2-thioether derivatives were more potent for stimulation of the C6 cell receptor, whereas other agonists were more potent in the turkey erythrocyte system. 6. These results add further support to the view that the adenylyl cyclase-linked P2Y-purinoceptor of C6 rat glioma cells is a different subtype from the phospholipase C-linked P2Y-purinoceptor of turkey erythrocyte membranes and several mammalian tissues.
Adenine Nucleotides↗
CPX, a selective A1-adenosine-receptor antagonist, regulates intracellular pH in cystic fibrosis cells.
The selective A1-adenosine-receptor antagonist, 8-cyclopentyl-1,3-dipropylxanthine (CPX), has been reported to activate Cl- efflux from cystic fibrosis cells, such as pancreatic CFPAC-1 and lung IB3 cells bearing the cystic fibrosis transmembrane regulator(delta F508) mutation, but has little effect on the same process in cells repaired by transfection with wild-type cystic fibrosis transmembrane regulator (O. Eidelman, C. Guay-Broder, P. J. M. van Galen, K. A. Jacobson, C. Fox, R. J. Turner, Z. I. Cabantchik, and H. B. Pollard. Proc. Natl. Acad. Sci. USA 89: 5562-5566, 1992). We report here that CPX downregulates Na+/H+ exchange activity in CFPAC-1 cells but has a much smaller effect on cells repaired with the wild-type gene. CPX also mildly decreases resting intracellular pH. In CFPAC-1 cells, this downregulation is dependent on the presence of adenosine, since pretreatment of the cells with adenosine deaminase blocks the CPX effect. We also show that, by contrast, CPX action on these cells does not lead to alterations in intracellular free Ca2+ concentration. We conclude that CPX affects pH regulation in CFPAC-1 cells, probably by antagonizing the tonic action of endogenous adenosine.
Calcium↗
Quantification of the in vivo potency of the adenosine A2 receptor antagonist 8-(3-chlorostyryl)caffeine.
The purpose of the present study was to quantify the in vivo potency of the selective adenosine A2a antagonist CSC [8-(3-chlorostyryl)caffeine]. Four groups of conscious, normotensive rats received a continuous i.v. infusion of 0, 6, 12 and 24 micrograms/min/kg of CSC. During a steady-state infusion of CSC, the animals received 1000 micrograms/kg of the adenosine A2a receptor agonist CGS 21680C [the sodium salt of 2-p-(2-carboxyethyl) phenylethylamino-5'-N-ethylcarboxamidoadenosine] i.v. over 15 min. During the experiment, the mean arterial pressure and the heart rate were recorded continuously and arterial blood samples were taken for the analysis of drug concentrations. For each individual rat, the CGS 21680C-provoked reduction in blood pressure was related to the blood concentration of the agonist according to the sigmoidal Emax model. The presence of CSC produced a parallel shift of the concentration-hypotensive effect curve to the right, indicating competitive interaction of the compounds. Infusion of 0, 6, 12 and 24 micrograms/min/kg of CSC resulted in steady-state concentrations of 0, 85 +/- 7, 210 +/- 20 and 400 +/- 40 ng/ml, and apparent EC50 values of CGS 21680C based on free concentrations (EC50,u) of 4.8 +/- 1.1, 7.2 +/- 0.5, 32 +/- 6 and 57 +/- 10 ng/ml, respectively (mean +/- S.E., n = 6, 6, 5 and 6). The relationship between the CSC concentration and the apparent EC50 was quantified according to a competitive pharmacodynamic interaction model.(ABSTRACT TRUNCATED AT 250 WORDS)
Adenosine↗
G protein-dependent activation of phospholipase C by adenosine A3 receptors in rat brain.
The recently cloned G protein-coupled adenosine A3 receptor has been proposed to play a role in the pathophysiology of cerebral ischemia. Because phospholipase C activation occurs as a very early response to brain ischemia, we evaluated the ability of A3- selective and nonselective adenosine analogues to elicit phosphoinositide hydrolysis. In myo-[3H]inositol-labeled rat striatal and hippocampal slices, A3 agonists stimulated formation of [3H]inositol phosphates in a concentration-dependent manner. In striatum, the potency order was 2-chloro-N6-(3-iodobenzyl)- adenosine-5'-N-methyluronamide > or = N6-(3-iodobenzyl)- adenosine-5'-N-methyluronamide >> N-methyl-1,3-di-n-butylxanthine-7-beta-D-ribofuronamide > or = 5'-N-ethylcarboxamidoadenosine > or = N6-2-(4-aminophenyl)-ethyladenosine > N6-(p-sulfophenyl)-adenosine = 1,3-dibutylxanthine-7- riboside, which is identical to the potency order in binding studies at cloned rat A3 receptors. Stimulation of phospholipase C activity was abolished by guanosine-5'-O-(2-thiodiphosphate), confirming the involvement of a G protein-coupled receptor. Activation of phospholipase C was higher in the striatum than in the hippocampus, consistent with A3 receptor densities. Stimulation of phospholipase C activity by adenosine analogues was only modestly antagonized by xanthine derivatives and at much higher concentrations than needed for blocking adenosine A1, A2A, and A2b receptors. In the presence of an A1/A2 antagonist, a selective A3 in rat striation. Thus, stimulation of phospholipase C activity agonist only weakly inhibited forskolin-stimulated adenylyl cyclase activity represents a principal transduction mechanism for A3 receptors in mammalian brain, and perhaps A3 receptor-mediated increases of inositol phosphates in the ischemic brain contribute to neurodegeneration by raising intracellular calcium levels.
Adenosine↗
125I-4-(2-[7-amino-2-[2-furyl][1,2,4]triazolo[2,3-a][1,3,5] triazin-5-yl-amino]ethyl)phenol, a high affinity antagonist radioligand selective for the A2a adenosine receptor.
The A2a adenosine receptor (AR) mediates several important physiological effects of adenosine, including vasodilation and inhibition of platelet aggregation. Until recently, no antagonist radioligand of sufficient selectivity or affinity was available. We describe the synthesis and characterization by radioligand binding of 125I-4-(2-[7-amino-2-{2-furyl}-{1,2,4}triazolo{2,3-a}- {1,3,5}triazin-5-yl-amino]ethyl)phenol (125I-ZM241385) in membranes from two cell types that express A2a ARs. Membranes from Chinese hamster ovary (CHO) cells expressing a recombinant canine A2a AR bound 125I-ZM241385 with high affinity, and agonist competition experiments with 2-(p-carboxyethyl)-phenylamino-5'-N-carboxamidoadenosine, 5'-N-ethylcarboxamidoadenosine, and (-)-N6[(R)-phenylisopropyl]adenosine revealed a potency order characteristic of an A2a AR binding site. Membranes from bovine striatum, which contain a native A2a AR, also bound 125I-ZM214385 with similarly high affinity and also displayed a pharmacological profile for displacement of radioligand binding that was consistent with that of an A2a AR. Also, under conditions in which 125I-ZM241385 bound with high affinity to a recombinant rat A2a AR expressed in CHO cells, no specific binding was detectable in membranes from CHO cells expressing functional rat A1, A2b, or A3 ARs, indicating that over the range of concentrations used in radioligand binding assays, 125I-ZM214385 is a highly selective antagonist radioligand for study of A2a ARs within a given species.
Animals↗
Modelling the P2Y purinoceptor using rhodopsin as template.
The P2Y1 purinoceptor cloned from chick brain (Webb, T. et al (1993) FEBS Lett., 324, 219-225) is a 362 amino acid, 41 kDa protein. To locate residues tentatively involved in ligand recognition a molecular model of the P2Y purinoceptor has been constructed. The model was based on the primary sequence and structural homology with the G-protein coupled photoreceptor rhodopsin, in analogy to the method proposed by Ballesteros and Weinstein ((1995) Meth. Neurosci. 25, 366-428). Transmembrane helices were constructed from the amino acid sequence, minimized individually, and positioned in a helical bundle. The helical bundle was then minimized using the Amber forcefield in Discover (BIOSYM Technologies) to obtain the final model. Several residues that have been shown to be critical in ligand binding in other GPCRs are conserved in the P2Y1 purinoceptor. According to our model the side chains of these conserved residues are facing the internal cleft in which ligand binding likely occurs. The model also suggests four basic residues (H121 in TM3, H266 and K269 in TM6 and R299 in TM7) near the extracellular surface that might be involved in ligand binding. These basic residues might be essential in coordinating the triphosphate chain of the endogenous ligand adenosine 5'-triphosphate (ATP). Potential binding sites for agonists have been explored by docking several derivatives (including newly synthesized N6-derivatives) into the model. The N6-phenylethyl substituent is tolerated pharmacologically, and in our model this substituent occupies a region predominantly defined by aromatic residues such as F51 (TM1), Y100 (TM2) and F120 (TM3). The dimethylated analogue of ATP, N6,N6-dimethyl-adenosine 5'-triphosphate, is less well tolerated pharmacologically, and our model predicts that the attenuated activity is due to interference with hydrogen bonding capacity to Q296 (TM7).
Amino Acid Sequence↗