3H]-MRE 2029-F20, a selective antagonist radioligand for the human A2B adenosine receptors

MRE 2029-F20 [N-benzo[1,3]dioxol-5-yl-2-[5-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-1-methyl-1H-pyrazol-3-yloxy]-acetamide] is a selective antagonist ligand of A2B adenosine receptors. For use as a radioligand, 1,3-diallyl-xanthine, the precursor of [3H]-MRE 2029-F20, was synthesized, and tritiated on the allyl groups. [3H]-MRE 2029-F20 bound to human A2B receptors expressed in CHO cells showed a KD value of 1.65+/-0.10 nM and Bmax value of 36+/-4 fmol/mg protein. [3H]-MRE2029-F20 represents a useful tool for the pharmacological characterization of human A2B adenosine receptor subtype.     

Subchronic Treatment with Methamphetamine and Phencyclidine Differentially Alters the Adenosine A1 and A2A Receptors in the Prefrontal Cortex,Hippocampus, and Striatum of the Rat

Subchronic treatment with MAP (4.6 mg/kg, i.p., once daily for 11 days) significantly decreased the K_d, but not B_max, values of [³H]1,3-dipropyl-8-cyclopentylxanthine ([³H]DPCPX) binding to adenosine A₁ receptors in the prefrontal cortex and hippocampus, but not striatum, of rat brain. However, subchronic treatment with PCP (10 mg/kg, i.p., once daily for 11 days) did not alter the K_d and B_max values of [³H]DPCPX binding to adenosine A₁ receptors in these three regions. Subchronic treatment with MAP or PCP did not alter the B_max and K_d values of [³H]2-p-(2-carboxyehyl)phenethylamino-5-N-ethylcarboxyamidoadenosine ([³H]CGS21680) binding to adenosine A_2A receptors in the striatum. Furthermore, subchronic treatment with MAP or PCP significantly decreased the specific binding of [³H]CGS21680 to adenosine A_2A receptors in the hippocampus, but not in the prefrontal cortex. Thus, these results suggest that MAP and PCP may produce differential effects on the adenosine A_2A receptors, but not adenosine A₁ receptors in rat brain.     

1,3-Dipropyl-8-(1-phenylacetamide-1H-pyrazol-3-yl)-xanthine derivatives as highly potent and selective human A(2B) adenosine receptor antagonists

A new series of 1,3-dipropyl-8-(1-phenylacetamide-1H-pyrazol-3-yl)-xanthine derivatives has been identified as potent A(2B) adenosine receptor antagonists. The products have been evaluated for their binding affinities for the human A(2B), A(1), A(2A), and A(3) adenosine receptors. N-(4-chloro-phenyl)-2-[3-(2,6-dioxo-1,3-dipropyl-2,3,6,7-tetrahydro-1H-purin-8-yl)-5-methyl-pyrazol-1-yl] (11c) showed a high affinity for the human A(2B) adenosine receptor K(i)=7nM and good selectivity (A(1), A(2A), A(3)/A(2B)>140). Synthesis and SAR of this novel class of compounds is presented herein.     

Solubilized Rat Brain Adenosine Receptors Have Two Binding Sites For 1,3-Dipropyl-8-Cyclopentylxanthine

Abstract

The use of [³H]-L-phenylisopropyladenosine ([³H]-L-PIA) and 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) in binding assays to solubilized preparations from rat brain membranes suggested the presence of an A₁, and a non-A₁ non-A₂ adenosine receptor may be equivalent to the A₃ subtype.     

Characterization of a low affinity binding site for N6-substituted adenosine derivatives in rat testis membranes

Abstract

The binding characteristics of radiolabeled N⁶-(cyclohexyl)adenosine ([³H]CHA), N⁶-(R-phenylisopropyl)adenosine ([³H]R-PIA), 5′-N-ethylcarboxamidoadenosine ([³H]NECA), and 2-[4-(2-carboxyethyl)phenyl]ethyl-amino-5′-N-ethylcarboxamidoadenosine ([³H]CGS 21680), to rat testis membranes were investigated. Specific binding of [³H]CGS 21680, a selective agonist for the A_2a adenosine receptor, was very modest whilst the nonselective agonist [³H]NECA bound to rat testis membranes showing high binding capacity. At least two types of binding sites for [³H]NECA could be identified in rat testis membranes: high affinity sites and high capacity sites. Selective agonists for the At adenosine receptor, [³H]CHA and [³H]R-PIA bound with high affinity to a single class of binding sites. This high affinity binding site showed the typical pharmacological specificity of the A₁ adenosine receptor with a potency order for agonists of CHA R-PIA > NECA > N⁶-(S-phenylisopropyl)adenosine (S-PIA). In order to detect the presence of the A₃ adenosine receptor in these membranes we selectively blocked the A₁ receptor with a large molar excess of a xanthine antagonist, either 1,3-dipropyl-8-cyclopentylxanthine (DPCPX) or xanthine amine congener (XAC). In the presence of an antagonist a low affinity binding site for [³H]CHA and [³H]R-PIA was detected. This low affinity binding site showed a different pharmacological specificity than the high affinity binding site. In fact the potency order for agonists was CHA NECA = R-PIA > S-PIA. This finding suggests that the low affinity binding site represents the A₃ adenosine receptor.     

Characterization of human and rodent native and recombinant adenosine A2B receptors by radioligand binding studies

Adenosine A_2B receptors of native human and rodent cell lines were investigated using [³H]PSB-298 [(8-{4-[2-(2-hydroxyethylamino)-2-oxoethoxy]phenyl}-1-propylxanthine] in radioligand binding studies. [³H]PSB-298 showed saturable and reversible binding. It exhibited a K_D value of 60 ± 1 nM and limited capacity (B_max = 3.511 fmol per milligram protein) at recombinant human adenosine A_2B receptors expressed in human embryonic kidney cells (HEK-293). The addition of sodium chloride (100 mM) led to a threefold increase in the number of binding sites recognized by the radioligand. The curve of the agonist 5′-N-ethylcarboxamidoadenosine (NECA) was shifted to the right in the presence of NaCl, while the curve of the antagonist PSB-298 was shifted to the left, indicating that PSB-298 may be an inverse agonist at A_2B receptors. Adenosine A_2B receptors were shown to be the major adenosine A₂ receptor subtype on the mouse neuroblastoma x rat glioma hybrid cell line NG108-15 cells. Binding studies at rat INS-1 cells (insulin secreting cell line) demonstrated that [³H]PSB-298 is a selective radioligand for adenosine A_2B binding sites in this cell line.     

Enhancement of [m-methoxy3H]MDL100907 binding to 5HT2A receptors in cerebral cortex and brain stem of streptozotocin induced diabetic rats

5-Hydroxytryptamine_2A (5-HT_2A) receptor kinetics was studied in cerebral cortex and brain stem of streptozotocin (STZ) induced diabetic rats. Scatchard analysis with [³H] (±) 2,3dimethoxyphenyl-1-[2-(4-piperidine)-methanol] ([³H]MDL100907) in cerebral cortex showed no significant change in maximal binding (B_max) in diabetic rats compared to controls. Dissociation constant (K_d) of diabetic rats showed a significant decrease (p < 0.05) in cerebral cortex, which was reversed to normal by insulin treatment. Competition studies of [³H]MDL100907 binding in cerebral cortex with ketanserin showed the appearance of an additional low affinity site for 5-HT_2A receptors in diabetic state, which was reversed to control pattern by insulin treatment. In brain stem, scatchard analysis showed a significant increase (p < 0.05) in B_max accompanied by a significant increase (p < 0.05) in K_d. Competition analysis in brain stem also showed a shift in affinity towards a low affinity State for 5-HT_2A receptors. All these parameters were reversed to control level by insulin treatment. These results show that in cerebral cortex there is an increase in affinity of 5-HT_2A receptors without any change in its number and in the case of brain stem there is an increase in number of 5HT_2A receptors accompanied by a decrease in its affinity during diabetes. Thus, from the results we suggest that the increase in affinity of 5-HT_2A receptors in cerebral cortex and upregulation of 5-HT_2A receptors in brain stem may lead to altered neuronal function in diabetes.     

Tritium-labeled agonists as tools for studying adenosine A<Subscript>2B</Subscript> receptors

A selective agonist radioligand for A_2B adenosine receptors (A_2BARs) is currently not available. Such a tool would be useful for labeling the active conformation of the receptors. Therefore, we prepared BAY 60-6583, a potent and functionally selective A_2BAR (partial) agonist, in a tritium-labeled form. Despite extensive efforts, however, we have not been able to establish a radioligand binding assay using [³H]BAY 60-6583. This is probably due to its high non-specific binding and its moderate affinity, which had previously been overestimated based on functional data. As an alternative, we evaluated the non-selective A_2BAR agonist [³H]NECA for its potential to label A_2BARs. [³H]NECA showed specific, saturable, and reversible binding to membrane preparations of Chinese hamster ovary (CHO) or human embryonic kidney (HEK) cells stably expressing human, rat, or mouse A_2BARs. In competition binding experiments, the AR agonists 2-chloroadenosine (CADO) and NECA displayed significantly higher affinity when tested versus [³H]NECA than versus the A_2B-antagonist radioligand [³H]PSB-603 while structurally diverse AR antagonists showed the opposite effects. Although BAY 60-6583 is an A_2BAR agonist, it displayed higher affinity versus [³H]PSB-603 than versus [³H]NECA. These results indicate that nucleoside and non-nucleoside agonists are binding to very different conformations of the A_2BAR. In conclusion, [³H]NECA is currently the only useful radioligand for determining the affinity of ligands for an active A_2BAR conformation.     

Characterization of the Binding of a Novel Non-Xanthine Adenosine Antagonist Radioligand, [3H]CGS 15943, to Multiple Affinity States of the Adenosine A1 Receptor in the Rat Cortex

Abstract

The use of xanthine adenosine receptor antagonists such as 1,3-dipropyl-8-phenylxanthine (DPX) as radioligands for the characterization of adenosine receptor Pharmacology have been limited by their high lipophilicity, low specific activity, and their general lack of selectivity and affinity for adenosine receptors. Recent attempts to address the technical problems associated with this class of compounds has resulted in the development of several xanthine derivatives (e.g. the functionalized xanthine congeners [³H]XCC and [³H]XAC², and [³H]CPX³) which bind with high and selective affinity to the adenosine A₁ receptor subtype. Based on efforts to optimize non-xanthine adenosine receptor antagonists, CGS 15943, a derivative of the pyrazoloquinazoline benzodiazepine receptor inverse agonist CGS 8216⁵, represents the first reported non-xanthine structure that potently blocks adenosine receptors⁶. CGS 15943 has nanomolar affinity for both A₁ and A₂ receptor subtypes⁶. However, in contrast to many of the xanthine adenosine receptor antagonists, CGS 15943 is not a phosphodiesterase inhibitor and does not interact with adenosine transporter sites⁶. This compound is a potent and selective adenosine receptor antagonist in vivo ⁷ with a solubility/affinity ratio of greater than 1000⁷. In the present studies, the binding of [³H]CGS 15943 to the adenosine A₁ receptor was characterized.     

Excitatory and Inhibitory Effects of A1 and A2A Adenosine Receptor Activation on the Electrically Evoked [3H]Acetylcholine Release from Different Areas of the Rat Hippocampus

Abstract: The modulation by adenosine analogues and endogenous adenosine of the electrically evoked release of [³H]acetylcholine ([³H]ACh) was compared in subslices of the three areas of the rat hippocampus (CA1, CA3, and dentate gyrus). The mixed A₁/A₂ agonist 2-chloroadenosine (CADO; 2–10 µM) inhibited, in a concentration-dependent manner, the release of [³H]ACh from the three hippocampal areas, being more potent in the CA1 and CA3 areas than in the dentate gyrus. The inhibitory effect of CADO (5 µM) on [³H]ACh release was prevented by the A₁ antagonist 1,3-dipropyl-8-cyclopentylxanthine (DPCPX; 50 nM) in the three hippocampal areas and was converted in an excitatory effect in the CA3 and dentate gyrus areas. The A_2A agonist CGS-21680 (30 nM) produced a greater increase of the evoked release of [³H]ACh in the CA3 than in the dentate gyrus areas, whereas no consistent effect was found in the CA1 area or in the whole hippocampal slice. The excitatory effect of CGS-21680 (30 nM) in the CA3 area was prevented by the adenosine receptor antagonist 3,7-dimethyl-1-propargylxanthine (10 µM). Both adenosine deaminase (2 U/ml) and DPCPX (250 nM) increased the evoked release of [³H]ACh in the CA1 and CA3 areas but not in the dentate gyrus. The amplitude of the effect of DPCPX and adenosine deaminase was similar in the CA1 area, but in the CA3 area DPCPX produced a greater effect than adenosine deaminase. It is concluded that the electrically evoked release of [³H]ACh in the three areas of the rat hippocampus can be differentially modulated by adenosine. In the CA1 area, only A₁ inhibitory receptors modulate ACh release, whereas in the CA3 area, both A_2A excitatory and A₁ inhibitory adenosine receptors modulate ACh release. In the dentate gyrus, both A₁ inhibitory and A_2A excitatory adenosine receptors are present, but endogenous adenosine does not activate them.     

Characteristics of Binding of [3H]WAY100635 to Rat Hippocampal Membranes

Kinetic analysis of binding of [³H][N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide ([³H]WAY100635) to 5-HT_1A receptors in rat hippocampal membranes has revealed complex regulation mechanism for this radioligand. Saturation binding experiments revealed that [³H]WAY100635 binds to a single class of receptors with very high apparent affinity (K _D = 87 ± 4 pM, B _max = 15.1 ± 0.2 fmol/mg protein). The binding was almost irreversible, as the dissociation rate constant obtained k _off = (7.8 ± 1.1) × 10⁻³ min⁻¹, means that equilibrium with this radioligand cannot be achieved before 7.5 h incubation at 25°C. Systematic association kinetic studies of [³H]WAY100635 binding revealed sharp reaction acceleration at higher radioligand concentration, proposing mechanism of positive cooperativity. The affinities of antagonists determined from competition with [³H]WAY100635 did not coincide with their abilities to inhibit 5-HT-dependent activation of [³⁵S]GTPγS binding probably due to the ligand’s kinetic peculiarities. Thus, [³H]WAY100635 appears to be an excellent tool for determining receptor binding sites, but its applicability in equilibrium studies is strongly limited.     

Species Comparison of Adenosine Receptor Subtypes in Brain and Testis

We aimed at comparing the binding characteristics of adenosine A₁ and A_2A receptors (A₁Rs and A_2ARs) in high-expressing cerebral areas, the cortex and striatum respectively, of human, bovine and rat brain. Adenosine A₃ receptor (A₃R) binding was studied in rat and bovine testis. Results confirmed species differences in AR saturation-displacement binding parameters. To investigate A₃Rs in CNS, we carried out immunoblot in human brain, resolving two signals, a 52 KDa band with the highest density in hippocampus and a 48 KDa one, slightly more expressed in cortex. Subsequently, A₃R binding was performed by [¹²⁵I]-4-aminobenzyl-5′-N-methylcarboxamidoadenosine ([¹²⁵I]-AB-MECA) in human hippocampus, revealing an high affinity population of sites and another non saturable component. [¹²⁵I]-AB-MECA first site displacement by N⁶ (3-iodobenzyl)adenosine-5′-N-methyluronamide (IB-MECA) and 1,3-dipropyl-8-cyclopenthyl-xanthine (DPCPX) distinguished two affinity sites, being only in part identified as A₃Rs. Therefore, A₃Rs result clearly expressed by Western blot in human brain, but their full CNS characterization needs further investigation.     

GTP and IAP Shifts Adenosine A1-Agonist Binding in Smooth Muscle Membranes to the Low Affinity State,Similar to the one Found in Intact Cells

Abstract

Adenosine A₁ receptors in the smooth muscle cell line DDT₁ MF-2 were characterized by radioligand binding using the antagonist [³H]-8-cyclopentyl-1,3-dipropylxanthine ([³H]DPCPX) as the ligand. Binding properties of adenosine agonists and antagonists to both intact cells and membranes were investigated.     

Characterisation of the Binding of [3H]WAY-100635, a Novel 5-Hydroxytryptamine1A Receptor Antagonist, to Rat Brain

Abstract: The specific binding of [³H]WAY-100635 {N-[2-[4-(2-[O-methyl-³H]methoxyphenyl)-1-piperazinyl]ethyl]-N-(2-pyridinyl)cyclohexane carboxamide trihydrochloride} to rat hippocampal membrane preparations was time, temperature, and tissue concentration dependent. The rates of [³H]WAY-100635 association (k₊₁ = 0.069 ± 0.015 nM^?1 min^?1) and dissociation (k_?1 = 0.023 ± 0.001 min^?1) followed monoexponential kinetics. Saturation binding isotherms of [³H]WAY-100635 exhibited a single class of recognition site with an affinity of 0.37 ± 0.051 nM and a maximal binding capacity (B_max) of 312 ± 12 fmol/mg of protein. The maximal number of binding sites labelled by [³H]WAY-100635 was ～36% higher compared with that of 8-hydroxy-2-(di-n-[³H]-propylamino)tetralin ([³H]8-OH-DPAT). The binding affinity of [³H]WAY-100635 was significantly lowered by the divalent cations CaCl₂ (2.5-fold; p < 0.02) and MnCl₂ (3.6-fold; p < 0.05), with no effect on B_max. Guanyl nucleotides failed to influence the K_D and B_max parameters of [³H]WAY-100635 binding to 5-HT_1A receptors. The pharmacological binding profile of [³H]WAY-100635 was closely correlated with that of [³H]8-OH-DPAT, which is consistent with the labelling of 5-hydroxytryptamine_1A (5-HT_1A) sites in rat hippocampus. [³H]WAY-100635 competition curves with 5-HT_1A agonists and partial agonists were best resolved into high- and low-affinity binding components, whereas antagonists were best described by a one-site binding model. In the presence of 50 µM guanosine 5′-O-(3-thiotriphosphate) (GTPγS), competition curves for the antagonists remained unaltered, whereas the agonist and partial agonist curves were shifted to the right, reflecting an influence of G protein coupling on agonist versus antagonist binding to the 5-HT_1A receptor. However, a residual (16 ± 2%) high-affinity agonist binding component was still apparent in the presence of GTPγS, indicating the existence of GTP-insensitive sites.     

Affinities of muscarinic drugs for [3H]N-methylscopolamine (NMS) and [3H]oxotremorine (OXO) binding to a mixture of M1−M4 muscarinic receptors: Use of NMS/OXO-M ratios to group compounds into potential agonist,partial agonist,and antagonist classes

The relative affinities of various muscarinic drugs in the antagonist ([³H]N-methyl scopolamine ([³H]NMS)) and agonist ([³H]Oxotremorine-m ([³H]OXO-M)) binding assays using a mixture of tissues containing M₁–M₄ receptor subtypes have been determined. [³H]NMS bound with high affinity (K_d=25±5.9 pM; n=3) and to a high density (B_max=11.8±0.025 nmol/g wet weight) of muscarinic receptors. [³H]OXO-M appeared to bind to two binding sites with differing affinities (K_d1=2.5±0.1 nM; K_d2=9.0±4.9 M; n=4) and to a different population of binding sites (B_max1=5.0±0.26 nmol/g wet weight; B_max2=130±60 nmol/g wet weight). Well known antagonists exhibited high affinity for [³H]NMS binding but a lower affinity for [³H]OXO-M binding. The opposite was true for acetylcholine and other known agonists. However, pilocarpine and McN-A-343 had similar affinities for sites labeled by both radioligands. Using the ratios of antagonist-to-agonist binding affinities, it was possible to group compounds into apparently distinct full agonist (ratios of 180–665; e.g. carbachol, muscarine, OXO-M, OXO-S and arecoline), partial agonist (ratios of 14–132; e.g. McN-A-343, pilocarpine, aceclidine, bethanechol, OXA-22 and acetylcholine) and antagonist (ratios of 0.22–1.9; e.g. atropine, NMS, pirenzepine, methoctramine, 4-DAMP and p-fluorohexahydrosialo-difenidol) classes. These data suggest that the NMS/OXO-M affinity ratios using a mixture of M₁–M₄ muscarinic receptors may be a useful way to screen and group a large number of compounds into apparent agonist, partial agonist, and antagonist classes of cholinergic agents.     

Pharmacological and biochemical characteristics of partially purified GABAB receptor

Pharmacological and biochemical characteristics of the partially purified -aminobutyric acid (GABA)_B receptor using baclofen affinity column chromatography have been examined. The Scatchard analysis of [³H]GABA binding to the purified GABA_B receptor showed a linear relationship and the K_D and B_max values were 60 nM and 118 pmol/mg of protein, respectively. Although GTP and Mg²⁺ did not affect on the GABA_B receptor binding, Ca²⁺ significantly increased [³H]GABA binding to the purified GABA_B receptor in a dose-dependent manner and showed its maximum effect at 2 mM. The enhancement of the binding by Ca²⁺ was found to be due to the increase of Bmax by the Scatchard analysis. The treatments with pronase and trypsin significantly decreased the binding of [³H]GABA, but phospholipase A₂ had no significant effect on the binding. In addition, treatment with glycosidases such as glycopeptidase A and -galactosidase significantly decreased the binding of [³H]GABA to the purified GABA_B receptor. These results suggest that purification of the solubilized GABA_B receptor by the affinity column chromatography may result in the functional uncoupling of GABA_B receptor with GTP-binding protein. Furthermore, the present results suggest that cerebral GABA_B receptor may be a glycoprotein and membrane phospholipids susceptible to phospholipase A₂ treatment may not be involved in the exhibition of the binding activity.Special issue dedicated to Dr. Eugene Roberts.     

Recombinant Human NMDA Homomeric NR1 Receptors Expressed in Mammalian Cells Form a High-Affinity Glycine Antagonist Binding Site

Abstract: The cDNA NMDAR1 (NR1) encodes a single polypeptide that forms a receptor-channel complex with electrophysiological and pharmacological properties characteristic of the N-methyl-d -aspartate receptor. Homomeric NR1 recombinant receptors expressed in Xenopus oocytes show functional responses with low levels of conductance. In this study we have characterized, by radioligand binding techniques, the pharmacological properties of homomeric receptors of two human NR1 isoforms (NR1a and NR1e, which differ in their C-terminal region), transiently expressed in human embryonic kidney 293 cells. The glycine site antagonist (±)-4-(trans)-2-carboxy-5,7-dichloro-4-[³H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([³H]L-689,560) bound to NR1a- and NR1e-transfected cells with high affinity (K_D = 3.29 and 1.61 nM, respectively). B_max values for NR1a- and NR1e-transfected cells were 3.82 and 1.69 pmol/mg of protein, respectively, and Hill coefficients were close to unity. K_i values for glycine site antagonists inhibiting [³H]L-689,560 binding to NR1e-transfected cells were similar to those observed with rat brain membranes. Affinity values for agonists and partial agonists were four- to 16-fold weaker, indicating that the glycine site of homomeric NR1 receptors is in an antagonist-preferring state. K_i values obtained with NR1a-transfected cells were approximately twofold lower than those obtained with NR1e-transfected cells. High-affinity binding to NR1-transfected cells was not observed with the transmitter recognition site radioligands l -[³H]glutamate and d,l -(ε)-2-[³H]amino-4-propyl-5-phosphono-3-pentanoic acid ([³H]CGP-39653) or the ion-channel radioligand [³H]dizocilpine ([³H]MK-801). These results indicate that although transfection of mammalian cells with homomeric NR1 recombinant receptors does not appear to result in functional receptors, a glycine binding site is formed that may have a physiological role if present in vivo.     

NAD-299 ANTAGONISES 5-HT-STIMULATED AND SPIPERONE-INHIBITED [35S]GTPγS BINDING IN CLONED 5-HT1A RECEPTORS

ABSTRACT

In Chinese Hamster Ovary (CHO) cells expressing cloned human 5-hydroxy-tryptamine_1A (5-HT_1A) receptors, (R)-3-N,N-dicyclobutylamino-8-fluoro-[6-³H]-3,4-dihydro-2H-1-benzopyran-5-carboxamide ([³H]NAD-299) exhibited high affinity (K_d = 0.16?nM) and labeled 34% more receptors than 8-hydroxy-2-([2,3-³H]di-n-propylamino)tetralin ([³H]8-OH-DPAT). NAD-299 behaved as a silent antagonist in [³⁵S]GTPγS binding similar to N-tert-butyl-3-(4-(2-methoxyphenyl)-piperazin-1-yl)-2-phenylpropanamide (WAY-100635) and (S)-5-fluoro-8-hydroxy-2-(di-n-propylamino)tetralin ((S)UH-301). 5-HT and 5-carboxamidotryptamine (5-CT) stimulated [³⁵S]GTPγS binding 2.5-fold while spiperone and methiothepin inhibited [³⁵S]GTPγS binding 1.4-fold. Furthermore, NAD-299 antagonised both the 5-HT stimulated and the spiperone inhibited [³⁵S]GTPγS binding to basal levels. The K_iL/K_iH ratios for spiperone (0.66), methiothepin (0.39), WAY-100635 (0.32), (S)UH-301 (0.94), NAD-299 (1.29), NAN-190 (1.23), (S)pindolol (5.85), ipsapirone (13.1), buspirone (24.6), (±)8-OH-DPAT (47.3), flesinoxan (55.8), 5-HT (200) and 5-CT (389) correlated highly significantly with the intrinsic activity obtained with [³⁵S] GTPγS (r = 0.97).     

Interactions Between the Glutamate and Glycine Recognition Sites of the N-Methyl-d-Aspartate Receptor from Rat Brain, as Revealed from Radioligand Binding Studies

Abstract: The N-methyl-d -aspartate (NMDA) receptor possesses two distinct amino acid recognition sites, one for glutamate and one for glycine, which appear to be allosterically linked. Using rat cortex/hippocampus P₂ membranes we have investigated the effect of glutamate recognition site ligands on [³H]glycine (agonist) and (±)4-trans-2-car-boxy-5,7-dichloro-4-[³H]phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline ([³H]l -689,560; antagonist) binding to the glycine site and the effect of glycine recognition site ligands on l -[³H]glutamate (agonist), dl -3-(2-carboxypiperazin-4-yl)-[³H]propyl-1 -phosphonate ([³H]-CPP; “C-7” antagonist), and cis-4-phosphonomethyl-2-[³H]piperidine carboxylate ([³H]CGS-19755; “C-5” antagonist) binding to the glutamate site. “C-7” glutamate site antagonists partially inhibited [³H]l -689,560 binding but had no effect on [³H]glycine binding, whereas “C-5” antagonists partially inhibited the binding of both radioligands. Glycine, d -serine, and d -cycloserine partially inhibited [³H]CGS-19755 binding but had little effect on l -[³H]-glutamate or [³H]CPP binding, whereas the partial agonists (+)-3-amino-1-hydroxypyrrolid-2-one [(+)-HA-966], 3R-(+)cis-4-methyl-HA-966 (l -687,414), and 1-amino-1-carboxycyclobutane all enhanced [³H]CPP binding but had no effect on [³H]CGS-19755 binding, and (+)-HA-966 and l -687,414 inhibited l -[³H]glutamate binding. The association and dissociation rates of [³H]l -689,560 binding were decreased by CPP and d -2-amino-5-phosphonopentanoic acid (“C-5”). Saturation analysis of [³H]l -689,560 binding carried out at equilibrium showed that CPP had little effect on the affinity or number of [³H]l -689,560 binding sites. These results indicate that complex interactions occur between the glutamate and glycine recognition sites on the NMDA receptor. In addition, mechanisms other than allosterism may underlie some effects, and the possibility of a steric interaction between CPP and [³H]l -689,560 is discussed.     

Pertussis toxin-sensitive G proteins mediate the inhibition of basal phosphoinositide metabolism caused by adenosine A1 receptors in rat hippocampal slices

The adenosine A₁ receptor selective agonist, N ⁶-cyclopentyladenosine (CPA, 300 nM) inhibited basal accumulation of [³H]inositol phosphates ([³H]InsPs), but not the total levels of membrane [³H]-phosphoinositides, in rat hippocampal slices. This action of CPA was not significantly modified when synaptic transmission was blocked with tetrodotoxin (TTX, 200 nM) but was prevented in slices pre-incubated with pertussis toxin (PTX, 5 g/mL) for 12-16 hr. Neither PTX nor TTX, when applied in the absence of CPA, influenced basal [³H]InsPs accumulation. It is concluded that the inhibition of the basal phosphatidylinositol metabolism by adenosine A₁ receptor activation is independent of neurotransmission and involves a PTX-sensitive G protein, probably of the G_i/G_o family.