Receptors In Rat Brain Membranes Research Articles

Inverse agonist properties of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide HCl (SR141716A) and 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1 H-pyrazole-3-carboxylic acid phenylamide (CP-272871) for the CB 1 cannabinoid receptor

Two subtypes of cannabinoid receptors are currently recognized, CB 1, found in brain and neuronal cells, and CB 2, found in spleen and immune cells. We have characterized 1-(2-chlorophenyl)-4-cyano-5-(4-methoxyphenyl)-1 H-pyrazole-3-carboxylic acid phenylamide (CP-272871) as a novel aryl pyrazole antagonist for the CB 1 receptor. CP-272871 competed for binding of the cannabinoid agonist 3H-labeled (−)-3-[2-hydroxy-4-(1,1-dimethylheptyl)-phenyl]-4-[3-hydroxypropyl]cyclohexan-1-ol ([ 3H]CP-55940) at the CB 1 receptor in rat brain membranes with a K d value 20-fold greater than that of N-(piperidin-1-yl)-5-(4-chlorophenyl)-1-(2,4-dichlorophenyl)-4-methyl-1 H-pyrazole-3-carboxamide HCl (SR141716A). CP-272871 also competed for binding with the aminoalkylindole agonist 3H-labeled ( R)-(+)-[2,3-dihydro-5-methyl-3-[(4-morpholinyl)methyl]pyrrolo[1,2,3- de]1,4-benzoxazin-6-yl](1-naphthyl)methanone ([ 3H]WIN-55212–2), as well as the aryl pyrazole antagonist [ 3H]SR141716A. Inverse agonist as well as antagonist properties were observed for both SR141716A and CP-272871 in signal transduction assays in biological preparations in which the CB 1 receptor is endogenously expressed. SR141716A augmented secretin-stimulated cyclic AMP (cAMP) accumulation in intact N18TG2 neuroblastoma cells, and this response was reversed by the agonist desacetyllevonantradol. CP-272871 antagonized desacetyllevonantradol-mediated inhibition of adenylyl cyclase in N18TG2 membranes, and increased adenylyl cyclase activity in the absence of agonist. SR141716A and CP-272871 antagonized desacetyllevonantradol-stimulated 35S-labeled guanosine-5′- O-(γ-thio)-triphosphate ([ 35S]GTPγS) binding to brain membrane G-proteins, and decreased basal [ 35S]GTPγS binding to G-proteins. K + enhanced CP-272871 and SR141716A inverse agonist activity compared with Na + or NMDG + in the assay. These results demonstrated that the aryl pyrazoles SR141716A and CP-272871 behave as antagonists and as inverse agonists in G-protein-mediated signal transduction in preparations of endogenously expressed CB 1 receptors.

6beta-Acyloxy(nor)tropanes: affinities for antagonist/agonist binding sites on transfected and native muscarinic receptors.

A series of esters of 6beta-hydroxynortropane and the N-methyl analogue 6beta-tropanol were synthesized and screened versus binding of an antagonist (quinuclidinyl benzilate) and an agonist (oxotremorine-M) at sites on human m(1)-, m(2)-, m(3)-, and m(4)-muscarinic receptors in transfected cell membranes and on native M(1)-muscarinic receptors in rat brain membranes and native M(2)-muscarinic receptors in rat heart membranes. Most 6beta-acyloxy(nor)tropanes had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at transfected m(1)- and native M(1)-receptors, indicative of agonist activity. 6beta-Acetoxynortropane had K(i) values versus oxotremorine-M binding at m(1)-, m(2)-, and m(4)-receptors ranging from 4 to 7 nM. N-Methylation reduced affinity greatly as did increasing the size of the acyl moiety. The affinity of 6beta-benzoyloxynortropane and other analogues with larger acyl moieties was little affected by N-methylation or in some cases was increased. 6beta-Acyloxy(nor)tropanes and classical muscarinic agonists, such as muscarine and oxotremorine, had higher affinity versus oxotremorine-M binding compared to quinuclidinyl benzilate binding at native M(2)-muscarinic receptors of heart, but not at transfected m(2)-muscarinic receptors. Antagonist/agonist binding ratios were not obtained for transfected m(3)-receptors, since significant oxotremorine-M binding could not be detected. 6beta-Acyloxy(nor)tropane, two other (nor)tropanes, and the classical muscarinic agonists had higher affinity versus agonist binding compared to antagonist binding for transfected m(4)-receptors. The antagonist/agonist binding ratio method is clearly not always reliable for predicting agonist activity at muscarinic receptors.

Azido- and isothiocyanato-substituted aryl pyrazoles bind covalently to the CB1 cannabinoid receptor and impair signal transduction.

3-Azidophenyl- and 3-isothiocyanatophenyl-and 2-(5'-azidopentyl)- and 2-(5'-isothiocyanatopentyl)pyrazoles were synthesized to determine whether these compounds could behave as covalently binding ligands for the CB1 cannabinoid receptor in rat brain membranes. Heterologous displacement of [3H]CP55940 indicated that the apparent affinity of these compounds for the CB1 receptor was similar to that of the parent compound, SR141716A, with the exception of the 3-isothiocyanato derivatives, which showed a 10-fold loss of affinity. The 3-azidophenyl and 3-isothiocyanatophenyl compounds behaved as antagonists against the cannabinoid agonist desacetyllevonantradol in activation of G proteins [guanosine 5'-O-(y-[35S]thio)triphosphate ([35S]GTPgammaS) binding] and regulation of adenylyl cyclase. The 2-(5'-azidopentyl)- and 2-(5'-isothiocyanatopentyl)pyrazoles were poor antagonists for [35S]GTPgammaS binding, and both compounds failed to antagonize the cannabinoid regulation of adenylyl cyclase. After incubation with the isothiocyanato analogues or UV irradiation of the azido analogues, the 3-substituted aryl pyrazoles formed covalent bonds with the CB1 receptor as evidenced by the loss of specific binding of [3H]CP55940. In the case of the isothiocyanato analogues, the log concentration-response curve for cannabinoid-stimulated [35S]GTPgammaS binding was shifted to the right, indicating that loss of receptors compromised signal transduction capability. These irreversibly binding antagonists might be useful tools for the investigation of tolerance and receptor down-regulation in both in vitro and in vivo studies.

Characterization of the Picrotoxin Site of GABAA Receptors

AbstractThis protocol describes an in vitro assay for characterization of the picrotoxin site of GABAA receptors in rat brain membranes using various radioligands. Methods and representative data for Scatchard analysis (Kd, Bmax determination), association kinetics, dissociation kinetics and competition assays (IC50, Ki determination) are included in the protocol.

Effects of antisense oligonucleotides on brain delta-opioid receptor density and on SNC80-induced locomotor stimulation and colonic transit inhibition in rats.

1. To reduce the density of delta-opioid receptor protein, five antisense phosphorothioate oligodeoxynucleotides (aODN), targeting the three exons of rat delta-opioid receptor mRNA (DOR), were injected twice daily for 4 days or continuously infused for 7 days into brain lateral ventricles (i.c.v.) of Sprague-Dawley rats. Rats acting as controls were infused or injected with a mismatch sequence (mODN) of each aODN. The density of opioid receptors in rat brain membranes was measured by saturation binding experiments using selective ligands for delta, mu and kappa opioid receptors. 2. aODNs injected twice a day for 4 days left rat brain delta-opioid receptor density unchanged. The ODN targeting the DOR nucleotide sequence 280 - 299 (aODN280 - 299, exon 2), decreased brain delta-opioid receptor density significantly more than aODNs targeting exon 1 (aODN239 - 258), exon 2 (aODN361 - 380), or exon 3 (aODN741 - 760) (to 52% vs 79, 72, and 68%). None of the aODNs to the DOR changed the brain density of mu- or k-opioid receptors. 3. When in a novel environment (but not when kept in their home cages), the locomotor activity of aODN280 - 299 treated rats was significantly lower than that of saline or mODN treated rats. The delta-opioid agonist SNC80 (5 mg kg-1, s.c.) significantly and potently stimulated locomotion and delayed colonic propulsion in saline- and mODN-infused rats, but left motor behaviour and colonic transit of delta-knockdown rats unchanged. 4. The baseline nociceptive threshold and the antinociceptive response to morphine were unchanged in delta-knockdown rats.

Characterization of [ 125I]AR-M100613, a high-affinity radioligand for δ opioid receptors

AR-M100613 ([I]-Dmt-c[- d-Orn-2-Nal- d-Pro- d-Ala-]) is the iodinated analog of a cyclic casomorphin previously shown to be a potent antagonist at the δ opioid receptor. Specific [ 125I]AR-M100613 binding to rat whole brain membranes was saturable, reversible, and best fit to a one-site model (K d = 0.080 ± 0.008 nM, B max = 45.2 ± 4.4 fmol/mg protein). [ 125I]AR-M100613 binding was displaced with high affinity by the δ opioid receptor ligands SNC-80, Deltorphin II and DPDPE but not the μ or κ-selective receptor ligands DAMGO and U69593. Residual non-selective binding of [ 125I]AR-M100613 to μ opioid receptors is blocked by the addition of CTOP to the assay buffer. [ 35S]GTPγS binding assays indicate that AR-M100613 is a potent, selective, and reversible antagonist for δ opioid receptors in rat brain membranes. The high-affinity, high specific activity, low nonspecific binding and antagonist profile of [ 125I]AR-M100613 favor its use as a radiochemical probe for δ opioid receptors.

Synthesis and biological activity of a new series of N6-arylcarbamoyl, 2-(Ar)alkynyl-N6-arylcarbamoyl, and N6-carboxamido derivatives of adenosine-5'-N-ethyluronamide as A1 and A3 adenosine receptor agonists.

A new series of 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ +-b earing N-arylureas or N-arylcarboxamido groups at the purine 6 position and N-arylureas combined with halogens or alkynyl chains at the 2 position have been synthesized and tested for affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors expressed in CHO cells. The derivatives contained the 5' substituent found in the potent, nonselective agonist 1-(6-amino-9H-purin-9-yl)-1-deoxy-N-ethyl-beta-D-ribofuranuronamide++ + (NECA). While the carboxamido derivatives (9-13) showed affinity for A1 receptors, the urea derivatives (30-45) showed different degrees of affinity and selectivity for the A3 adenosine receptor subtype. In particular the derivative bearing a p-sulfonamidophenyl-urea at the 6 position, 31 showed a high affinity (Ki = 9 nM) and selectivity for the A3 receptors compared to that of the reference compound 1-[6-[[(3-iodophenyl)methyl]amino]-9H-purin-9-yl]-1-deoxy-N-methyl-be ta-D-ribofuranuronamide (IB-MECA). Furthermore, the importance of the stereochemistry in the interaction of these ligands at the rat A3 adenosine receptors has been evaluated by introducing a chiral chain at the 6 position. The introduction of halogens or alkynyl chains at the purine 2 position of selected ureas did not give the expected enhancement of potency at A2A and/or A3 receptors but rather showed a dramatic reduction of A2A affinity, resulting in compounds with good A2A/A3 selectivity. For example, the 2-(3-hydroxy-3-phenyl-1-propyn-1-yl)-6-(4-methoxyphenylurea) derivative 61 showed the capability to bind simultaneously to A1 and A3 receptor subtypes, excluding the A2A receptor. Compound 31 was shown to be an agonist, 9-fold more potent than NECA, at A3 receptors in rat RBL-2H3 mast cell membranes through stimulation of binding of [35S]GTP-gamma-S.

125I-Tyr 1]biphalin binding to opioid receptors of rat brain and NG108-15 cell membranes

Mono iodinated analogues of biphalin [(Tyr-D-Ala-Gly-Phe-NH-) 2], both nonradioactive [I-Tyr 1]biphalin and radioactive [ 125I-Tyr 1]biphalin have been synthesized. The radioligand binding profiles of these compounds for two types of tissues, rat brain membranes, and NG108-15 cell membranes were identical to the parent biphalin. This is additional evidence for the hypothesis that biphalin behaves like a monomeric ligand and that only one intact tyrosine is necessary for high biological activity. The second tyrosine could be used for successful radioiodination which may greatly simplify biochemical and pharmacological studies of biphalin. The results of receptor binding studies show that the binding of both biphalin and [I-Tyr 1]biphalin to the δ and μ opioid receptors are not independent. [ 125I-Tyr 1]Biphalin binds to δ receptors as shown in NG108-15 cell membranes. Nevertheless, [ 125I]biphalin binding to δ receptors in rat brain membranes was hardly evident and μ receptor binding predominated or at least was much more readily detectable in this preparation.

5-(N-oxyaza)-7-substituted-1,4-dihydroquinoxaline-2,3-diones: novel, systemically active and broad spectrum antagonists for NMDA/glycine, AMPA, and kainate receptors.

A group of 5-aza-7-substituted-1,4-dihydroquinoxaline-2,3-diones (QXs) and the corresponding 5-(N-oxyaza)-7-substituted QXs were prepared and evaluated as antagonists of ionotropic glutamate receptors. The in vitro potency of these QXs was determined by inhibition of [3H]-5,7-dichlorokynurenic acid ([3H]DCKA) binding to N-methyl-D-aspartate (NMDA)/glycine receptors, [3H]-(S)-alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding to AMPA receptors, and [3H]kainate ([3H]KA) binding to KA receptors in rat brain membranes. 5-(N-Oxyaza)-QXs 12a-e all have low micromolar or submicromolar potency for NMDA/glycine receptors and low micromolar potencies for AMPA and KA receptors. QXs 12a-e display 2-12-fold selectivity for NMDA/glycine receptors compared to AMPA receptors, and approximately 2-fold difference between AMPA and KA potency. In contrast to other QXs that either show high selectivity for NMDA (such as ACEA 1021) or AMPA (such as NBQX) receptors, these molecules are broad spectrum antagonists of ionotropic glutamate receptors. 7-Nitro-5-(N-oxyaza)-QX (12e) is the most potent inhibitor among 12a-e, having IC50 values of 0.69, 1.3, and 2.4 microM at NMDA, AMPA, and KA receptors, respectively. In functional assays on glutamate receptors expressed in oocytes by rat cerebral cortex poly(A+) RNA, 7-chloro-5-(N-oxyaza)-QX (12a) and 7-nitro-5-(N-oxyaza)-QX (12e) have Kb values of 0.63 and 0.31 microM for NMDA/glycine receptors, and are 6- and 4-fold selective for NMDA over AMPA receptors, respectively. 5-(N-Oxyaza)-7-substituted-QXs 12a-e all have surprisingly high in vivo potency as anticonvulsants in a mouse maximal electroshock-induced seizure (MES) model. 7-Chloro-5-(N-oxyaza)-QX (12a), 7-bromo-5-(N-oxyaza)-QX (12b), and 7-methyl-5-(N-oxyaza)-QX (12c) have ED50 values of 0.82, 0.87, and 0.97 mg/kg i.v., respectively. The high in vivo potency of QXs 12a-e is particularly surprising given their low log P values (approximately -2.7). Separate studies indicate that QXs 12a and 12e are also active in vivo as neuroprotectants and also have antinociceptive activity in animal pain models. In terms of in vivo activity, these 5-(N-oxyaza)-7-substituted-QXs are among the most potent broad spectrum ionotropic glutamate antagonists reported.

Helix-inducing alpha-aminoisobutyric acid in opioid mimetic deltorphin C analogues.

The achiral symmetric alpha-aminoisobutyric acid (Aib) replaced the critical N-terminal residues of the amphibian skin opioid deltorphin C (H-Tyr-D-Ala-Phe-Asp-Val-Val-Gly-NH2) without detriment to the physicochemical requirements for delta opioid receptor recognition. Substitutions by the alpha, alpha-dialkyl amino acid in place of D-Ala2 or Phe3, or both, exhibited high delta receptor affinity (Ki delta = 0.12-3.6 nM) and 5-9-fold greater selectivity (Ki mu/Ki delta = 5000-8500) than the parent compound. This is the first definitive demonstration that the D-chirality of alanine and the aromaticity of phenylalanine are replaceable by an achiral alpha, alpha-dialkylated residue without detrimental effects on ligand binding. Incorporation of the mono-alpha-alkyl amino acid L- or D-Ala at the third position also produced highly selective delta ligands (Ki mu/Ki delta = 2000-3500), albeit with reduced delta affinities (Ki delta = 6-15 nM). Replacement of the anionic residue Asp4 by Aib yielded an opioid peptide that fit two-site binding models for the delta receptor (eta = 0.763; P < 0.0001) and displayed dual high affinity for both delta and mu receptors, emphasizing the repulsive effect by a negative charge at mu receptor sites and the insignificance of Asp for delta affinity. Molecular dynamics conformation analyses suggested that Aib residues caused distinct changes in deltorphin C secondary structure when substituted for D-Ala2, Asp4, and simultaneously D-Ala2 and Phe3 but not when substituted for Phe3. These conformational changes might be critical factors for the proper orientation of reactive constituents of residues in the N-terminal region of deltorphin C. Disparities between binding data and functional bioassays of [Aib3] indicated that Phe3 was required for bioactivity in mouse vas deferens but not for interaction with delta opioid receptors in rat brain membranes.

Thermodynamic parameters of opioid binding in the presence and absence of G-protein coupling.

We have investigated the thermodynamic parameters of various opioid ligands interacting with their receptors in rat brain membranes. Affinity constants (Ka), enthalpy and entropy values were obtained from homologous displacement experiments performed at 0, 24 and 33 degrees C. It was found that all the opioid agonists tested ([3H]dihydromorphine (DHM) mu alkaloid; [3H]DAMGO mu peptide; [3H]deltorphin-B delta peptide) display endothermic binding accompanied with a large entropy increase, regardless of their chemical structure (alkaloid or peptide), or of their mu or delta receptor selectivity. In contrast, binding of the antagonist naloxone is exothermic, mainly enthalpy driven. Na+ or Mg2+ results only in quantitative changes of the thermodynamic parameters. In the presence of the GTP-analog Gpp(NH)p; or Gpp(NH)p + Na+; or Gpp(NH)p + Na- + Mg2+ the affinity of DHM binding dramatically decreases which might reflect functional uncoupling of the receptor-ligand complex and G-proteins. This altered molecular interactions are also indicated by curvilinear van't Hoff plot and entropy increase. It is concluded that the thermodynamic analysis provides means of determining the underlying driving forces of ligand binding and helps to delineate its mechanism.

Novel N6-(substituted-phenylcarbamoyl)adenosine-5'-uronamides as potent agonists for A3 adenosine receptors.

A series of adenosine-5'-uronamide derivatives bearing N6-phenylurea groups have been synthesized and tested for their affinity at A1 and A2A adenosine receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells. Some N6-arylcarbamoyl derivatives, N6-((2-chlorophenyl)carbamoyl)-, N6-((3-chlorophenyl)carbamoyl)-, and N6-((4-methoxyphenyl)carbamoyl)adenosine-5'-ethyluronamide (4l-n), were found to have affinity at A3 receptors in the low nanomolar range (Ki values < 10 nM). In CHO cells stably transfected with the rat A3 receptor, compound 4n was found to be a full agonist in inhibiting adenylate cyclase activity. The present study represents the first example of N6-acyl-substituted adenosine analogs having high affinity at adenosine receptors and, in particular, at the A3 receptor subtype.

The amphiphilic peptide adenoregulin enhances agonist binding to A1-adenosine receptors and [35S]GTPγS to brain membranes

1. Adenoregulin is an amphilic peptide isolated from skin mucus of the tree frog, Phyllomedusa bicolor. Synthetic adenoregulin enhanced the binding of agonists to several G-protein-coupled receptors in rat brain membranes. 2. The maximal enhancement of agonist binding, and in parentheses, the concentration of adenoregulin affording maximal enhancement were as follows: 60% (20 microM) for A1-adenosine receptors, 30% (100 microM) for A2a-adenosine receptors, 20% (2 microM) for alpha 2-adrenergic receptors, and 30% (10 microM) for 5HT1A receptors. High affinity agonist binding for A1-, alpha 2-, and 5HT1A-receptors was virtually abolished by GTP gamma S in the presence of adenoregulin, but was only partially abolished in its absence. Magnesium ions increased the binding of agonists to receptors and reduced the enhancement elicited by adenoregulin. 3. The effect of adenoregulin on binding of N6-cyclohexyladenosine ([3H]CHA) to A1-receptors was relatively slow and was irreversible. Adenoregulin increased the Bmax value for [3H]CHA binding sites, and the proportion of high affinity states, and slowed the rate of [3H]CHA dissociation. Binding of the A1-selective antagonist, [3H]DPCPX, was maximally enhanced by only 13% at 2 microM adenoregulin. Basal and A1-adenosine receptor-stimulated binding of [35S]GTP gamma S were maximally enhanced 45% and 23%, respectively, by 50 microM adenoregulin. In CHAPS-solubilized membranes from rat cortex, the binding of both [3H]CHA and [3H]DPCPX were enhanced by adenoregulin. Binding of [3H]CHA to membranes from DDT1 MF-2 cells was maximally enhanced 17% at 20 microM adenoregulin. In intact DDT1 MF-2 cells, 20 microM adenoregulin did not potentiate the inhibition of cyclic AMP accumulation mediated via the adenosine A1 receptor. 4. It is proposed that adenoregulin enhances agonist binding through a mechanism involving enhancement of guanyl nucleotide exchange at G-proteins, resulting in a conversion of receptors into a high affinity state complexed with guanyl nucleotide-free G-protein.

Binding thermodynamics of serotonin to rat-brain 5-HT 1a, 5HT 2a and 5-HT 3 receptors

The thermodynamic parameters ΔG° , ΔH° and Δs° of the binding equilibrium of serotonin to 5-HT 1A, 5-HT 2A and 5-HT 3 rat-brain membrane receptors have been determined by means of affinity constant measurements at six temperatures in the range 0 –35 ° C and van't Hoff plots. At variance with 5-HT 1A and 5-HT 3, the binding at the 5-HT 2A receptors is strongly endothermic and entropy-driven. Comparison with the results obtained by other authors on 5-HT 2A receptors in rats and humans suggests that the observed differences can be explained by a single amino acid difference in the receptor sequence between these two species.

The selective 5-HT 1A antagonist radioligand [ 3H]WAY 100635 labels both G-protein-coupled and free 5-HT 1A receptors in rat brain membranes

The tritiated derivative of the novel silent 5-HT 1A receptor antagonist WAY 100635 [ N-(2-(4-(2-methoxyphenyl)-1-pineperazinyl)ethyl)- N-(2-pridinyl) cyclohexane carboxamide] was tested as a potential radioligand of 5-HT 1A receptors in the rat br brain. Binding assays with membranes from various brain regions showed that [ 3H]WAY 100635 specifically bound to a homogenous population of sites, with a K d of 0.10nM. The regional distribution of [ 3H]WAY 100635 specific binding sites, as assessed in membrane binding assays and by autoradiography of labelled brain sections, superimposed exactly over that of 5-HT 1A receptors specifically labelled by [ 3 H]8- hydroxy-2-( di-n- propylamino) tetralin ([ 3H]-8-OH-DPAT). Futhermore, the positive correlation ( r = 0.96 A) between the respective p K l values of a large series of ligands as inhibitors of the specific binding of [ 3H]WAY 100635 and [ 3H]8-OH-DPAT in hippocampal membranes indicated that their pharmacological properties were similar. Nevertheless, marked differences also existed between [ 3H]8-OH-DPAT and [ 3H]WAY 100635 specific binding, as the former was inhibited by 1–100 μM GTP and GppNHp, whereas the latter was enhanced by these guanic nucleotides. In contrast, Mn 2+ (1–10 mM) increased the specific binding of [ 3H]8-OH-DPAT, but inhibited that of [ 3H]WAY 100635. Treatment of membranes with N-ethylmalcimide (1–5 mM) markedly reduced their capacity to specifically bind [ 3H]8-OH-DPAT, but slightly increased (at 1 mM) or did not affect (at 5 mM) their [ 3H]WAY 100635 specific binding capacity. Finally, the B max of [ 3H]WAY 100635 specific binding sites was regularly 50–60% higher than that of [ 3H]8-OH-DPAT in the same membrane preparations from various brain regions (hippocampus, septum, cerebral cortex). These data are compatible with the idea that whereas [ 3H]8-OH-DPAT only binds to G-protein-coupled 5-HT 1A receptors, [ 3H]WAY 100635 is a high affinity ligand of both G-protein-coupled and free 5-HT 1A receptor binding subunits in brain membranes.

Kinetic analysis of ligand-receptor association data that display an overshoot phenomenon.

A binding overshoot was frequently observed in the time course of association of diazepam with rat brain membrane receptors shortly after the start of the interaction. Such time profiles most likely reflect the "receptor switch" mechanism, assuming an equilibrium between two forms of a receptor (R and R*) that possess different affinities to the ligand (L) in question. Similar effects could be caused by the presence of a slowly dissociating competitor. The kinetics of these mechanisms were verified by simulation of theoretical time courses. A computer program for simulation of the time course, and estimation of rate constants of the individual reaction steps, was developed and is described in this communication. It employs the Euler-Cauchy integration for simulation of theoretical time courses. Optimised estimates of the rate constants were computed by simultaneous random variation of parameters within a pre-set interval. Stable solutions can be obtained for this system, thus enabling evaluation of equilibrium constants defined by the model. The source code is available in Turbo-Pascal. It can be used, after re-writing the rate equations, for fitting of similar kinetic models to suitable experimental data.

2-Substitution of N6-benzyladenosine-5'-uronamides enhances selectivity for A3 adenosine receptors.

Adenosine derivatives bearing an N6-(3-iodobenzyl) group, reported to enhance the affinity of adenosine-5'-uronamide analogues as agonists at A3 adenosine receptors (J. Med. Chem. 1994, 37, 636-646), were synthesized starting from methyl beta-D-ribofuranoside in 10 steps. Binding affinities at A1 and A2a receptors in rat brain membranes and at cloned rat A3 receptors from stably transfected CHO cells were compared. N6-(3-Iodobenzyl)adenosine was 2-fold selective for A3 vs A1 or A2a receptors; thus it is the first monosubstituted adenosine analogue having any A3 selectivity. The effects of 2-substitution in combination with modifications at the N6- and 5'-positions were explored. 2-Chloro-N6-(3-iodobenzyl)adenosine had a Ki value of 1.4 nM and moderate selectivity for A3 receptors. 2-Chloro-N6-(3-iodobenzyl)adenosine- 5'-N-methyluronamide, which displayed a Ki value of 0.33 nM, was selective for A3 vs A1 and A2a receptors by 2500- and 1400-fold, respectively. It was 46,000-fold selective for A3 receptors vs the Na(+)-independent adenosine transporter, as indicated in displacement of [3H]N6-(4- nitrobenzyl)-thioinosine binding in rat brain membranes. In a functional assay in CHO cells, it inhibited adenylate cyclase via rat A3 receptors with an IC50 of 67 nM. 2-(Methylthio)-N6-(3-iodobenzyl)-adenosine-5'-N-methyluronamide and 2-(methylamino)-N6-(3-iodobenzyl)adenosine-5'-N-methyluronamide were less potent, but nearly as selective for A3 receptors. Thus, 2-substitution (both small and sterically bulky) is well-tolerated at A3 receptors, and its A3 affinity-enhancing effects are additive with effects of uronamides at the 5'-position and a 3-iodobenzyl group at the N6-position.

Alkyl‐modified side chain variants of anatoxin‐a: A series of potent nicotinic agonists

AbstractThe potent nicotinic agonist anatoxin‐a has a semi‐rigid structure amenable to chemical synthesis and modification, making it an attractive candidate for exploring the structure‐activity relationships of ligands at nicotinic acetylcholine receptors. Racemic anatoxin‐a and a series of three analogues with one or more methine or methylene units added to the acetyl sidechain were synthesised and designated homoanatoxin, propylanatoxin, and isopropylanatoxin. In competition binding assays on two neuronal nicotinic receptors in rat brain membranes, labelled with [3H]nicotine and [125I]αbungarotoxin, the analogues retained or exceeded the potency of the parent structure. Ki values for anatoxin‐a, homoanatoxin, propylanatoxin, and isopropylanatoxin were 19, 5.5, 24 and 7.5 nM, respectively, at the [3H]nicotine site and 900, 340, 40, and 120 nM, respectively, at the [125I]αbungarotoxin site. Thus propylanatoxin appears to show a preference for the latter receptor site. Functional potencies of homoanatoxin and isopropylanatoxin were determined at neuronal α7 nicotinic receptors reconstituted in Xenopus oocytes, by two electrode voltage clamp recording. EC50 values of 0.72 μM and 0.66 μM were determined, similar to that previously published for (+)anatoxin‐a. Molecular modelling of anatoxin‐a and its alkyl‐modified analogues shows them to have very similar energy profiles, consistent with their similar potencies. In the preferred (low energy) conformation, the enone moiety adopts an s‐trans arrangement, although this configuration does not fit the classical pharmacophore model for nicotinic ligands. This series of alkyl‐modified analogues based on anatoxin‐a provides some novel potent nicotinic agonists and with propylanatoxin, receptor subtype selectivity may be emerging. This rational approach to ligand design also enables us to explore further the requirements for ligand recognition through the application of computational chemistry. © 1994 Wiley‐Liss, Inc.

Effects of anandamide on cannabinoid receptors in rat brain membranes

Anandamide (arachidonylethanolamide) is a compound recently isolated from porcine brain as a putative endogenous ligand at cannabinoid receptors. The present studies examined the effects of anandamide on cannabinoid receptor binding sites and adenylyl cyclase in rat brain membranes. Receptor binding experiments, conducted at 25° for 90 min, apparently resulted in significant degradation of anandamide, since anandamide (10 μM) had little effect on [ 3H]WIN 55212-2 binding in cerebellar membranes. Addition of the general serine protease inhibitor phenylmethylsulfonyl fluoride (PMSF) protected against this degradation, resulting in an ic 50 value of 90 nM for anandamide versus [ 4H]WIN 55212-2 binding. Anandamide inhibited adenylyl cyclase in cerebellar membranes in a GTP-dependent manner, exhibiting a maximal inhibition level slightly less than that of WIN 55212-2 and CP-55,940, with an ic 50 value of 1.9 μM. The effect of anandamide on adenylyl cyclase was region-specific, with maximal inhibition occurring in cerebellum and striatum. These results suggest that anandamide acts at G-protein-coupled cannabinoid receptors in brain with properties similar to those of exogenous cannabinoids.

Unsaturated fatty acids induce inhibition of the A1-adenosine receptor in rat brain membranes.

We examined the effect of fatty acids (FA) and some other bioactive lipids on A1-adenosine receptor (A1-AR) binding in rat brain membranes using the selective agonist (3H)-N6-cyclohexyladenosine [(3H)CHA]. A significant reduction in ligand binding was observed at micromolar concentrations of unsaturated fatty acid with the following potency: oleic < arachidonic < decosaenoic < linoleic < linolenic acid. The other tested compounds: lysophospholipids, platelet-activating factor (PAF) and GM1-ganglioside were without effect on A1-AR expression. The inhibition with unsaturated FA was non-competitive and partially reversed by albumin. The A1-AR agonist binding inhibition evoked by unsaturated FA in many respects is similar to that observed previously following ischaemia and may be contributory to the increased excitability of post-ischaemic brain.