32P]2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate ([32P]MRS2500), a novel radioligand for quantification of native P2Y1 receptors

Analysis of the P2Y family of nucleotide-activated G-protein-coupled receptors has been compromised by the lack of selective high-affinity, high-specific-radioactivity radioligands. We have pursued quantification of the P2Y1 receptor through the development of a series of selective P2Y1 receptor antagonists. Recently, we synthesized 2-iodo-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate (MRS2500), a selective, competitive antagonist that exhibits a Ki of 0.8 nM in competition-binding assays with [3H]MRS2279. A 3'-monophosphate precursor molecule, MRS2608, was radiolabeled at the 5' position with 32P using polynucleotide kinase and [gamma32P]ATP to yield [32P]MRS2500. [32P]MRS2500 bound selectively to Sf9 insect cell membranes expressing the human P2Y1 receptor (Sf9-P2Y1), but did not detectably bind membranes expressing other P2Y receptors. P2Y1 receptor binding to [32P]MRS2500 was saturable with a KD of 1.2 nM. Agonists and antagonists of the P2Y1 receptor inhibited [32P]MRS2500 binding in Sf9-P2Y1 membranes with values in agreement with those observed in functional assays of the P2Y1 receptor. A high-affinity binding site for [32P]MRS2500 (KD=0.33 nM) was identified in rat brain, which exhibited the pharmacological selectivity of the P2Y1 receptor. Distribution of this binding site varied among rat tissues, with the highest amount of binding appearing in lung, liver, and brain. Among brain regions, distribution of the [32P]MRS2500 binding site varied by six-fold, with the highest and lowest amounts of sites detected in cerebellum and cortex, respectively.Taken together, these data illustrate the synthesis and characterization of a novel P2Y1 receptor radioligand and its utility for examining P2Y1 receptor expression in native mammalian tissues.     

2-Substitution of adenine nucleotide analogues containing a bicyclo[3.1.0]hexane ring system locked in a northern conformation: enhanced potency as P2Y1 receptor antagonists

Preference for the northern (N) ring conformation of the ribose moiety of adenine nucleotide 3',5'-bisphosphate antagonists of P2Y(1) receptors was established by using a ring-constrained methanocarba (a bicyclo[3.1.0]hexane) ring as a ribose substitute (Nandanan et al. J. Med. Chem. 2000, 43, 829-842). We have now combined the ring-constrained (N)-methanocarba modification with other functionalities at the 2-position of the adenine moiety. A new synthetic route to this series of bisphosphate derivatives was introduced, consisting of phosphorylation of the pseudoribose moiety prior to coupling with the adenine base. The activity of the newly synthesized analogues was determined by measuring antagonism of 2-methylthio-ADP-stimulated phospholipase C (PLC) activity in 1321N1 human astrocytoma cells expressing the recombinant human P2Y(1) receptor and by using the radiolabeled antagonist [(3)H]2-chloro-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bisphosphate 5 in a newly developed binding assay in Sf9 cell membranes. Within the series of 2-halo analogues, the most potent molecule at the hP2Y(1) receptor was an (N)-methanocarba N(6)-methyl-2-iodo analogue 12, which displayed a K(i) value in competition for binding of [(3)H]5 of 0.79 nM and a K(B) value of 1.74 nM for inhibition of PLC. Thus, 12 is the most potent antagonist selective for the P2Y(1) receptor yet reported. The 2-iodo group was substituted with trimethyltin, thus providing a parallel synthetic route for the introduction of an iodo group in this high-affinity antagonist. The (N)-methanocarba-2-methylthio, 2-methylseleno, 2-hexyl, 2-(1-hexenyl), and 2-(1-hexynyl) analogues bound less well, exhibiting micromolar affinity at P2Y(1) receptors. An enzymatic method of synthesis of the 3',5'-bisphosphate from the corresponding 3'-monophosphate, suitable for the preparation of a radiophosphorylated analogue, was explored.     

2-Chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate is a selective high affinity P2Y(1) receptor antagonist

1. We reported previously that bisphosphate derivatives of adenosine are antagonists of the P2Y(1) receptor and that modification of the ribose in these analogues is tolerated in the P2Y(1) receptor binding pharmacophore. 2. Here we delineate the pharmacological activity of one such non-nucleotide molecule, 2-chloro N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate (MRS2279), in which the ribose is replaced by a cyclopentane ring constrained in the (N)-conformation by a cyclopropane moiety. 3. MRS2279 antagonized 2MeSADP-stimulated inositol phosphate formation in turkey erythrocyte membranes with competitive kinetics (pK(B)=7.75). High affinity competitive antagonism by MRS2279 was also observed at the human P2Y(1) receptor (pK(B)=8.10) stably expressed in 1321N1 human astrocytoma cells. Antagonism was specific for the P2Y(1) receptor since MRS2279 had no effect on activation of the human P2Y(2), P2Y(4), P2Y(6), or P2Y(11) receptors by their cognate agonists. 4. MRS2279 also did not block the capacity of ADP to act through the Gi/adenylyl cyclase linked P2Y receptor of platelets to inhibit cyclic AMP accumulation. 5. In contrast, the P2Y(1) receptor is known to be obligatory in the process of ADP-induced platelet aggregation, and MRS2279 competitively inhibited ADP-promoted platelet aggregation with an apparent affinity (pK(B)=8.05) similar to that observed at the human P2Y(1) receptor heterologously expressed in 1321N1 cells. 6. Taken together these results illustrate selective high affinity antagonism of the P2Y(1) receptor by a non-nucleotide molecule that should prove useful for pharmacological delineation of this receptor in various tissues.     

Inhibition of platelet function by administration of MRS2179, a P2Y1 receptor antagonist

The effects of a potent P2Y1 receptor antagonist, N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179) on adenosine-5'-diphosphate (ADP)-induced platelet aggregation in vitro, ex vivo and on the bleeding time in vivo were determined. In suspensions of washed platelets, MRS2179 inhibited ADP-induced platelet shape change, aggregation and Ca2+ rise but had no effect on ADP-induced inhibition of adenylyl cyclase. Binding studies using the new radioligand [33P]MRS2179 showed that washed human platelets displayed 134+/-8 binding sites per platelet with an affinity (Kd) of 109+/-18 nM. Finally, intravenous injection of MRS2179 resulted in inhibition of rat platelet aggregation in response to ADP and prolonged the bleeding time, in rats or mice, as compared to controls. These results suggest this potent P2Y1 receptor antagonist to be a promising tool to evaluate the in vivo effects of pharmacologically targeting the P2Y1 receptor with a view to antithrombotic therapy.     

Agonist binding and Gq-stimulating activities of the purified human P2Y1 receptor

The human P2Y1 receptor (P2Y1-R) was purified after high-level expression from a recombinant baculovirus in Sf9 insect cells. Quantification by protein staining and with a radioligand binding assay using the high-affinity P2Y1-R antagonist [3H]MRS2279 ([3H]2-chloro-N6-methyl-(N)-methanocarba-2'-deoxyadenosine 3',5'-bis-phosphate) indicated a nearly homogenous preparation of receptor protein. Ki values determined in [3H]MRS2279 binding assays for antagonists with the purified P2Y1-R were in good agreement with the Ki and KB values determined for these molecules in membrane binding and activity assays, respectively. Availability of P2Y1-R in purified form allowed direct determination of nucleotide agonist affinities under conditions not compromised by nucleotide metabolism/interconversion, and an order of affinities of 2-methylthio-ADP (2MeSADP) > ADP = 2-methylthioATP = adenosine-5'-O-(3-thio)triphosphate = adenosine-5'-O(2-thiodiphosphate) > ATP was obtained. The signaling activity of the purified P2Y1-R was quantified after reconstitution in proteoliposomes with heterotrimeric G proteins. Steady-state GTP hydrolysis in vesicles reconstituted with P2Y1-R and Galpha(q)beta(1)gamma(2) was stimulated by the addition of either 2MeADP or RGS4 alone and was increased by up to 50-fold in their combined presence. EC50 values of agonists for activation of the purified P2Y1-R were similar to their respective Ki values determined in radioligand binding experiments with the purified receptor. Moreover, ATP exhibited 20-fold higher EC50 and Ki values than did ADP and was a partial agonist relative to ADP and 2MeSADP under conditions in which no metabolism of the nucleotide occurred. Both RGS4 and PLC-beta1 were potent and efficacious GTPase-activating proteins for Galphaq and Galpha11 in P2Y1-R-containing vesicles. These results illustrate that the binding and signaling properties of the human P2Y1-R can be studied with purified proteins under conditions that circumvent the complications that occur in vivo.     

3H]MRS 1754, a selective antagonist radioligand for A(2B) adenosine receptors

MRS 1754 [N-(4-cyanophenyl)-2-[4-(2,3,6,7-tetrahydro-2,6-dioxo-1,3-dipropyl-1H-purin-8-yl)-phenoxy]acetamide] is a selective antagonist ligand of A(2B) adenosine receptors. This is the least well-defined adenosine receptor subtype, and A(2B) antagonists have potential as antiasthmatic drugs. For use as a radioligand, MRS 1754, a p-cyanoanilide xanthine derivative, was tritiated on the propyl groups in a two-step reaction using a p-carboxamido precursor, which was dehydrated to the cyano species using trifluoroacetic anhydride. [3H]MRS 1754 (150 Ci/mmol) bound to recombinant human A(2B) adenosine receptors in membranes of stably transfected HEK-293 cells. Specific binding was saturable, competitive, and followed a one-site model, with a K(D) value of 1.13 +/- 0.12 nM and a B(max) value of 10.9 +/- 0.6 pmol/mg protein. Specific binding utilizing 0.7 nM [3H]MRS 1754 was > 70% of total binding. The affinity calculated from association and dissociation binding constants was 1.22 nM (N = 4). Binding to membranes expressing rat and human A(1) and A(3) adenosine receptors was not significant, and binding in membranes of HEK-293 cells expressing human A(2A) receptors was of low affinity (K(D) > 50 nM). The effects of cations and chelators were explored. Specific binding was constant over a pH range of 4.5 to 6.5, with reduced binding at higher pH. The pharmacological profile in competition experiments with [3H]MRS 1754 was consistent with the structure-activity relationship for agonists and antagonists at A(2B) receptors. The K(i) values of XAC (xanthine amine congener) and CPX (8-cyclopentyl-1,3-dipropylxanthine) were 16 and 55 nM, respectively. NECA (5'-N-ethylcarboxamidoadenosine) competed for [3H]MRS 1754 binding with a K(i) of 570 nM, similar to its potency in functional assays. Thus, [3H]MRS 1754 is suitable as a selective, high-affinity radioligand for A(2B) receptors.     

2,2'-Pyridylisatogen tosylate antagonizes P2Y1 receptor signaling without affecting nucleotide binding

The effect of 2,2'-pyridylisatogen tosylate (PIT) on the human P2Y(1) receptor and on other recombinant P2Y receptors has been studied. We first examined the modulation by PIT of the agonist-induced accumulation of inositol phosphates. PIT blocked 2-methylthio-ADP (2-MeSADP)-induced accumulation of inositol phosphates in 1321N1 astrocytoma cells stably expressing human P2Y(1) receptors in a non-competitive and concentration-dependent manner. The IC(50) for reduction of the maximal agonist effect was 0.14microM. In contrast, MRS2179, a competitive P2Y(1) receptor antagonist, parallel-shifted the agonist concentration-response curve to the right. PIT also concentration-dependently blocked the P2Y(1) receptor signaling induced by the endogenous agonists, ADP and ATP. A simple structural analogue of PIT was synthesized and found to be inactive as a P2Y(1) receptor antagonist, suggesting that the nitroxyl group of PIT is a necessary structural component for P2Y(1) receptor antagonism. We next examined the possible modulation of the binding of the newly available antagonist radioligand for the P2Y(1) receptor, [3H] MRS2279. It was found that PIT (0.01-10microM) did not inhibit [3H] MRS2279 binding to the human P2Y(1) receptor. PIT (10microM) had no effect on the competition for [3H] MRS2279 binding by agonists, ADP and ATP, suggesting that its antagonism of the P2Y(1) receptor may be allosteric. PIT had no significant effect on agonist activation of other P2Y receptors, including P2Y(2), P2Y(4), P2Y(6), P2Y(11) and P2Y(12) receptors. Thus, PIT selectively and non-competitively blocked P2Y(1) receptor signaling without affecting nucleotide binding.     

Antiaggregatory activity in human platelets of potent antagonists of the P2Y 1 receptor

Activation of the P2Y(1) nucleotide receptor in platelets by ADP causes changes in shape and aggregation, mediated by activation of phospholipase C (PLC). Recently, MRS2500(2-iodo-N(6)-methyl-(N)-methanocarba-2'-deoxyadenosine-3',5'-bisphosphate) was introduced as a highly potent and selective antagonist for this receptor. We have studied the actions of MRS2500 in human platelets and compared these effects with the effects of two acyclic nucleotide analogues, a bisphosphate MRS2298 and a bisphosphonate derivative MRS2496, which act as P2Y(1) receptor antagonists, although less potently than MRS2500. Improved synthetic methods for MRS2500 and MRS2496 were devised. The bisphosphonate is predicted to be more stable in general in biological systems than phosphate antagonists due to the non-hydrolyzable CP bond. MRS2500 inhibited the ADP-induced aggregation of human platelets with an IC(50) value of 0.95 nM. MRS2298 and MRS2496 also both inhibited the ADP-induced aggregation of human platelets with IC(50) values of 62.8 nM and 1.5 microM, respectively. A similar order of potency was observed for the three antagonists in binding to the recombinant human P2Y(1) receptor and in inhibition of ADP-induced shape change and ADP-induced rise in intracellular Ca(2+). No substantial antagonism of the pathway linked to the inhibition of cyclic AMP was observed for the nucleotide derivatives, indicating no interaction of these three P2Y(1) receptor antagonists with the proaggregatory P2Y(12) receptor, which is also activated by ADP. Thus, all three of the bisphosphate derivatives are highly selective antagonists of the platelet P2Y(1) receptor, and MRS2500 is the most potent such antagonist yet reported.     

Characterization of the cAMP binding site of purified S-adenosyl-homocysteine hydrolase from bovine kidney

The enzyme S-adenosyl-homocysteine hydrolase (AdoHcyase) which catalyzes the reversible hydrolysis of AdoHcy to adenosine and homocysteine is an adenosine binding protein. In the present study we examined the characteristics of [(3)H]cAMP binding to purified AdoHcyase from bovine kidney in comparison with the high affinity adenosine binding site of AdoHcyase. AdoHcyase exhibits one [(3)H]cAMP binding site with an affinity of K(d)=23.1+/-1.1nM and a B(max) of 116.6+/-3.8pmol/mg protein. Binding of [(3)H]cAMP obeyed a monophasic reaction with a k(+1) value of 0.035min/M. The dissociation of AdoHcyase-[(3)H]cAMP complex exhibited a time- and temperature-dependent character. After a 240min incubation at 0 degrees only 5-10%, however, at 20 degrees 90% were displaceable. Adenosine and cAMP displace each other with similar affinities of EC(50) 57nM vs. EC(50) 65nM. 2'-Deoxyadenosine, N(6)-methyladenosine, and NECA displace 25nM [(3)H]cAMP and 10nM [(3)H]adenosine with EC(50) values of 94, 90 and 80nM, respectively. All other nucleosides studied, adenine, inosine, adenosine-2',3'-dialdehyde, 2-chloroadenosine, aristeromycin, and adenine nucleotides were only week competitors for [(3)H]cAMP and [(3)H]adenosine. These compounds displace [(3)H]cAMP and [(3)H]adenosine with equal potencies. Our data indicate that the binding site for nanomolar concentrations of cAMP and adenosine at the AdoHcyase appears to be identical. The physiological implications of a cAMP binding site at the AdoHcyase remain to be established.     

Pharmacological profiles of the metabotropic glutamate receptor ligands

Metabotropic glutamate receptors (mGluRs) are a family of G-protein coupled receptors that are expressed in the central and peripheral nervous systems. The purpose of this study was to compare the ligand binding selectivity profiles of the mGluR agonist [(3)H]L-AP4 and the novel radiolabeled phenylglycine antagonist [(3)H]CPPG at all eight rat mGluR subtypes expressed in transfected human embryonic kidney cells. At a concentration of 30 nM [(3)H]L-AP4, no specific binding was detected in membranes expressing the group I receptors mGluR1a or mGluR5a, or in membranes expressing the group II mGluRs, mGluR2 and mGluR3. Among the group III mGluRs, specific [(3)H]L-AP4 binding was detected in cells expressing mGluR4a and mGluR8a but not in cells expressing mGluR6 or mGluR7a. The binding of [(3)H]CPPG showed an exceptional pattern of selectivity amongst the mGluR subtypes; at a concentration of 20 nM [(3)H]CPPG, a high level of specific binding was seen in membranes containing mGluR8a but not in any of the other mGluR subtypes. The affinity constant (K(D)) calculated for [(3)H]CPPG binding to mGluR8a was 183 nM. In competition experiments, the phosphono-substituted phenylglycine congeners including MPPG, (RS)-PPG, and unlabeled CPPG were the most potent inhibitors of [(3)H]CPPG binding while non-phosphonated compounds such as L-glutamate and MCPG were substantially less potent. These results demonstrate that [(3)H]L-AP4 and [(3)H]CPPG can be used as probes to selectively label group III mGluRs and that CPPG and related phenylglycine derivatives are useful for studying differences in the ligand recognition sites of highly homologous mGluRs.     

(3)H]ZM241385--an antagonist radioligand for adenosine A(2A) receptors in rat brain

Binding of the novel adenosine A(2A) receptor-selective antagonist radioligand [2-(3)H]-4-(2-[7-amino-2-(2-furyl)(1,2,4)triazolo(2,3-a)(1,3,5,)triazin-5-yl amino]ethyl)phenol ([(3)H]ZM241385) was examined using particulate preparations and frozen sections of rat brain. In membranes from the rat striatum, binding was saturable, reversible and temperature-dependent. Analysis of saturation isotherms indicated that [(3)H]ZM241385 bound with high affinity (K(d) of 0.84 nM), high density (1680 fmol mg protein(-1)) and with a high proportion of specific binding (93% at 1 nM radioligand). Examination of competition profiles indicated that [(3)H]ZM241385 bound to sites with an A(2A) adenosine receptor-like rank order. The presence of guanosine 5'-(3-thio)-triphosphate failed to alter either [(3)H]ZM241385 binding or agonist competition for [(3)H]ZM241385 binding. Autoradiographic analysis of [(3)H]ZM241385 binding to frozen sections of rat brain indicated specific binding to the rat striatum of similar affinity (K(d) of 0.43 nM) and susceptibility to adenosine receptor ligands. At 2 nM [(3)H]ZM241385, specific binding comprised 95+/-1% total binding. In the hippocampus and frontal cortex, binding of [(3)H]ZM241385 failed to saturate and was of lower density. Taken together, these results indicate that [(3)H]ZM241385 should prove to be a useful radioligand in the characterisation of adenosine A(2A) receptors.     

Differential [(3)H]idazoxan and [(3)H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) binding to imidazoline I(2) receptors in human postmortem frontal cortex.

[(3)H]2-(2-benzofuranyl)-2-imidazoline (2-BFI) and [(3)H]idazoxan are the most used tools to characterise imidazoline I(2) receptors. We evaluated the binding of both radioligands to human postmortem frontal cortex membranes. Saturation binding analyses revealed that [(3)H]idazoxan (in the presence of 2 microM efaroxan to avoid radioligand binding to alpha(2)-adrenoceptors and imidazoline I(1) receptors) and [(3)H]2-BFI bound with high affinity to an apparent single population of sites. However, in competition studies whereas [(3)H]idazoxan (10 nM) binding was displaced monophasically by idazoxan and 2-BFI, both drugs displayed biphasic curves for [(3)H]2-BFI (1 nM). The proportion of the low-affinity binding site increased from 17% to 25% when 10 nM [(3)H]2-BFI was displaced by idazoxan. Amiloride inhibited [(3)H]2-BFI (10 nM) binding with low affinity and in a monophasic way. These data indicate that [(3)H]2-BFI recognises in human postmortem brain membranes a second binding site different from the imidazoline I(2) receptors labelled by [(3)H]idazoxan.     

Involvement of imidazoline receptors in the centrally acting muscle-relaxant effects of tizanidine

BIBN4096BS [[R-(R,(R*,S*)]-N-[2-[[5-amino-1-[[4-(4-pyridinyl)-1-piperazinyl]carbonyl] pentyl]amino]-1-[(3,5-dibromo-4-hydroxyphenyl)methyl]-2-oxoethyl]-4-(1,4-dihydro-2-oxo-3(2H)-quinazolinyl)-,1-Piperidinecarboxamide] is a selective calcitonin gene-related peptide (CGRP) receptor antagonist with a picomolar affinity to the CGRP receptor in human neuroblastoma SK-N-MC cells. Here, we describe the characterisation of the binding properties of the tritiated radioanalogue of BIBN4096BS in SK-N-MC cells as well as in marmoset tissue. [(3)H]BIBN4096BS showed reversible and saturable binding to SK-N-MC cells with a K(D) of 0.045 nM. In competition experiments, [3(H)]BIBN4096BS is concentration-dependently displaced from SK-N-MC cell membranes by BIBN4096BS as well as by the endogenous ligand CGRP and its analogues with the rank order of affinity BIBN4096BS>human alpha-CGRP=human beta-CGRP>[Cys(Et)(2,7)]human alpha-CGRP>adrenomedullin (high affinity site)=human alpha-CGRP-(8-37)=human beta-CGRP-(8-37)>calcitonin=amylin. In the marmoset cortex, saturable [(3)H]BIBN4096BS binding was observed with a K(D) of 0.077 nM. CGRP showed biphasic competition of [(3)H]BIBN4096BS binding, whilst BIBN4096BS monophasically displaced its radioanalogue with a K(i) of 0.099 nM. These data, using [(3)H]BIBN4096BS, confirm the high affinity of this novel antagonist for the primate CGRP receptor and demonstrate furthermore that this radioligand is a useful tool to study CGRP receptor pharmacology.     

Inhibition of ATP-induced glutamate release by MRS2179 in cultured dorsal spinal cord astrocytes

It was reported that ATP, an excitatory chemical mediator, exerts its effects by activation of the P2X (ligand-gated cationic channels) and P2Y (G protein-coupled receptors) purinoceptors in the nervous system. In the present work, we used confocal laser scanning microscopy and high-performance liquid chromatography to assess the role of the P2Y1 receptor in ATP-evoked Ca2+ mobilization and glutamate release from cultured dorsal spinal cord astrocytes. ATP (0.01-100 micromol/l) produces a dose-dependent rise in the Ca2+ relative fluorescence intensity in cultured astrocytes. N6-methyl-2'-deoxyadenosine-3',5'-bisphosphate (MRS2179, 0.01-100 micromol/l), a P2Y1-specific antagonist, could dose-dependently inhibit ATP-evoked Ca2+ mobilization. In addition, 100 micromol/l ATP caused glutamate efflux from cultured dorsal spinal cord astrocytes in a time-dependent manner. 100 micromol/l MRS2179 significantly inhibited the glutamate efflux induced by ATP, which suggests that P2Y1 receptor activation is responsible for the ATP-induced glutamate efflux from astrocytes. Taken together, our results demonstrate that P2Y1 receptor plays an important role in modulating the function of astrocytes, which raises the possibility that MRS2179, a potent P2Y1-specific antagonist, may become a potential drug in treating many chronic neurological diseases characterized by astrocytic activation in the nervous system.     

Discrimination in 5-HT3 receptor binding in murine brain and cultured cell preparations

One-hundred ninety-one ligands were screened at 5-HT(3) receptors in membranes from rat brain and NCB20 cells for their ability to displace the selective, high-affinity 5-HT(3) receptor antagonist, [(125)I]DAIZAC ([(125)I]( S)-5-chloro-3-iodo-2-methoxy- N-(1-azabicyclo[2.2.2]oct-3-yl)benzamide). Thirty-seven compounds having structures related to benzamide, dibenzepine, serotonin, phenylbiguanide, or arylpiperzine were selected for more extensive displacement studies in membranes from rat and mouse brains, from two cultured cell preparations expressing heteromeric mouse-derived 5-HT(3) receptor proteins (NCB20 and NG108-15 cell lines), and from recombinant Sf9 cells expressing homomeric 5-HT(3A) receptors. [(125)I]DAIZAC bound specifically to a single site in each of the five tissue preparations with high affinity ( K(D) 0.12-0.19 nM). The densities of [(125)I]DAIZAC-labeled 5-HT(3) receptors were 7.4-7.5 fmol/mg protein in membranes from murine brain, and 38, 99, and 1588 fmol/mg protein in membranes from cultured NCB20, NG108-15, and recombinant Sf9 cells, respectively. The affinity of substituted benzamides ( n=10) was similar in all five tissue preparations. The affinity of dibenzepines ( n=17) was significantly higher in membranes from cultured cells as compared to membranes from rat and mouse brain, but similar in the two brain membrane preparations, and in each of the cultured cell membrane preparations. Serotonin-, phenylbiguanide-, and quipazine-analogs ( n=10), which typically function as 5-HT (5-hydroxytryptamine) agonists, exhibited significantly higher apparent p K(i) values in membranes from rat brain and Sf9 recombinant cells than in membranes from the three preparations expressing heteromeric mouse-derived 5-HT(3) receptor proteins ( F=7.52, P<0.001). These findings confirm that there are both species and cell-type dependent differences in binding to 5-HT(3) receptors, and that care must be taken when comparing results between experimental paradigms that utilize different sources of 5-HT(3) receptors.     

Distribution and function of beta-adrenoceptors in different chambers of the canine heart

1 [(3)H]-clonidine binds reversibly to membranes prepared from regions of guinea-pig kidney.2 Higher levels of binding were obtained in the membranes prepared from renal cortex (2.15 +/- 0.27 pmol/g wet wt.) than renal medulla (0.53 +/- 0.07 pmol/g wet wt.) or papilla (0.14 +/- 0.06 pmol/g wet wt.; n = 4).3 Scatchard analysis performed by addition of unlabelled clonidine (1 to 30 pmol) gave figures for the dissociation constant (K(d)) for the binding of [(3)H]-clonidine to renal cortical membranes of 9.0 +/- 0.8 nM and B(max) of 21.6 +/- 1.7 pmol/g wet wt. (n = 4). Hill plots of these data gave gradients close to unity, indicating a lack of co-operative site interactions.4 The monovalent cations, sodium and potassium, and the divalent cation, calcium, produced concentration-dependent decreases in [(3)H]-clonidine binding to membranes prepared from renal cortex, the EC(50)s being respectively 25 mM, 37 mM and 23 mM.5 At low concentrations the divalent cations, magnesium (1 mM) and manganese (0.1 mM), produced enhancement of [(3)H]-clonidine binding. At higher concentrations (>10 mM) both divalent cations inhibited binding.6 Scatchard analysis of [(3)H]-clonidine binding performed in the presence of sodium (100 mM), magnesium (1 mM) or manganese (0.1 mM) revealed that the alterations in binding are primarily due to changes in apparent affinity rather than a change in the number of binding sites. Sodium (100 mM) produced a change in the K(d) from 7.0 +/- 0.4 nM (n = 8) to 42.3 +/- 27.5 nM (n = 3), whereas magnesium (1 mM) decreased the K(d) to 6.0 +/- 0.9 nM and manganese (0.1 mM) to 4.0 +/- 1.0 nM (n = 3).7 The results indicate that [(3)H]-clonidine labels a binding site that has properties resembling an alpha(2)-adrenoceptor, located in the renal cortex. The changes produced by the addition of monovalent and divalent cations are entirely due to changes in the apparent affinity of [(3)H]-clonidine binding.     

Novel antagonists acting at the P2Y(1) purinergic receptor: synthesis and conformational analysis using potentiometric and nuclear magnetic resonance titration techniques

The human P2Y(1) receptor is widely distributed in many tissues and has a classical structure of a G protein-coupled receptor. Activated by adenosine-5'-diphosphate (ADP), this receptor is essential for platelet aggregation. In the present paper, we describe the synthesis of novel P2Y(1) antagonists that could be of interest at least as tools to define the physiological roles of the P2Y(1) receptor, at best as new antithrombotic agents. Thus, we prepared the 2,N(6)-dimethyl-2'-deoxyadenosine-3',5'-bisphosphate derivative, 1e. The biological activity was demonstrated by the ability of compound 1e to inhibit ADP-induced platelet aggregation, shape change, and intracellular calcium rise. This compound was a full antagonist at the P2Y(1) receptor with a pA(2) value of 7.11 +/- 0.11 and was found to be 4-fold more potent than the reference N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate (1a, pA(2) = 6.55 +/- 0.05), revealing the potency-enhancing effects of the 2-methyl group. The better activity of 1e as compared to 1a was analyzed using both potentiometric and nuclear magnetic resonance titration techniques, which highlighted specific conformational features of this compound. These results clearly indicate the preference for both compounds for an anti conformation at the N-glycosyl linkage. Furthermore, the percentage of S conformer of 1e is close to that of 1a, which is nearly 70% at pH = 2.8 and increases dramatically when pH increases. From the macroprotonation constants, it can be noted that compound 1e is significantly more basic than 1a. This is indeed expected for the N1 adenine nitrogen due to the electron-donating character of the methyl moiety. By considering the microconstants of the phosphate groups, the higher basicity of P3 and P5 for 1e may be due to the decrease in the local dielectric constant induced by the substitution of the hydrogen atom by a more lipophilic methyl group. Thus, it may be suggested that the gain in activity of 1e when compared to the reference compound 1a would result from its gain in basicity rather than steric and conformational modifications. The synthesis of the first selective radioligand acting at the P2Y(1) receptor ([(33)P]-N(6)-methyl-2'-deoxyadenosine-3',5'-bisphosphate, 17) is also reported and will be used in the future for efficient screening needed for drug optimization.     

Characterization of a mutant sulfonylurea receptor SUR2B with high affinity for sulfonylureas and openers: differences in the coupling to Kir6.x subtypes.

ATP-dependent K(+) channels are composed of pore-forming subunits of the Kir6.x family and of sulfonylurea receptors (SURs). SUR1, expressed in pancreatic beta-cells, has a higher affinity for sulfonylureas, such as glibenclamide, than SUR2B, expressed in smooth muscle. This difference is mainly caused by serine 1237 in SUR1 corresponding to tyrosine 1206 in SUR2B. To increase the affinity of SUR2B for glibenclamide, the mutant SUR2B(Y1206S) was constructed. In whole-cell patch-clamp experiments, glibenclamide inhibited the channel formed by coexpression of mutant SUR2B with Kir6.1 or 6.2 in human embryonic kidney cells with IC(50) values of 2.7 and 13 nM, respectively (wild-type, 43 and 167 nM). In intact cells, [(3)H]glibenclamide bound to mutant SUR2B with a K(D) value of 4.7 nM (wild-type, 32 nM); coexpression with Kir6.1 or 6.2 increased affinity by 4- and 8-fold, respectively. Binding of the opener [(3)H]P1075 to SUR2B(Y1206S) was the same as to wild-type and was unaffected by coexpression. In cells, the ratio of glibenclamide:P1075 sites was approximately 1:1; in membranes, it varied with the MgATP concentration. Heterologous competition curves were generally biphasic; the shape of the curve depended on the Kir-subtype. The effects of coexpression were weakened or abolished when binding assays were conducted in membranes. It is concluded that the mutation Y1206S increases the affinity of SUR2B for and the channel sensitivity toward glibenclamide by 7- to 15-fold. The interaction of glibenclamide (but not opener) with mutant SUR2B is modified by coexpression with Kir6.x in a manner depending on the Kir subtype and on the integrity of the cell.     

Inhibition of acetylcholine muscarinic M(1) receptor function by the M(1)-selective ligand muscarinic toxin 7 (MT-7)

MT-7 (1 - 30 nM), a peptide toxin isolated from the venom of the green mamba Dendroaspis angusticeps and previously found to bind selectively to the muscarinic M(1) receptor, inhibited the acetylcholine (ACh)-stimulated [(35)S]-guanosine-5'-O-(3-thio)triphosphate ([(35)S]-GTPgammaS) binding to membranes of Chinese hamster ovary (CHO) cells stably expressing the cloned human muscarinic M(1) receptor subtype. MT-7 failed to affect the ACh-stimulated [(35)S]-GTPgammaS binding in membranes of CHO cells expressing either the M(2), M(3) or M(4) receptor subtype. In N1E-115 neuroblastoma cells endogenously expressing the M(1) and M(4) receptor subtypes, MT-7 (0.3 - 3.0 nM) inhibited the carbachol (CCh)-stimulated inositol phosphates accumulation, but failed to affect the CCh-induced inhibition of pituitary adenylate cyclase activating polypeptide (PACAP) 38-stimulated cyclic AMP accumulation. In both CHO/M(1) and N1E-115 cells the MT-7 inhibition consisted in a decrease of the maximal agonist effect with minimal changes in the agonist EC(50) value. In CHO/M(1) cell membranes, MT-7 (0.05 - 25 nM) reduced the specific binding of 0.05, 1.0 and 15 nM [(3)H]-N-methylscopolamine ([(3)H]-NMS) in a concentration-dependent manner, but failed to cause a complete displacement of the radioligand. Moreover, MT-7 (3 nM) decreased the dissociation rate of [(3)H]-NMS by about 5 fold. CHO/M(1) cell membranes preincubated with MT-7 (10 nM) and washed by centrifugation and resuspension did not recover control [(3)H]-NMS binding for at least 8 h at 30 degrees C. It is concluded that MT-7 acts as a selective noncompetitive antagonist of the muscarinic M(1) receptors by binding stably to an allosteric site.     

Pharmacology and autoradiography of human DP prostanoid receptors using [(3)H]-BWA868C, a DP receptor-selective antagonist radioligand

1. A potent and highly selective DP prostanoid receptor antagonist radioligand, [(3)H]-cyclohexyl-N-BWA868C (3-benzyl-5-(6-carboxyhexyl)-1-(2-cyclohexyl-2-hydroxyethyl-amino) hydantoin, ([(3)H]-BWA868C)), has been generated for receptor binding and autoradiographic studies. 2. Specific [(3)H]-BWA868C binding to human platelet membranes achieved equilibrium within 60 min at 23 degrees C and constituted up to 95% of the total binding. The association (K(+1)) and dissociation (K(-1)) rate constants of binding were 0.758+/-0.064 min(-1), mmol and 0.0042+/-0.0002 min(-1), respectively, yielding dissociation constants (K(D)s) of 5.66+/-0. 44 nM (n=4). 3. Specific [(3)H]-BWA868C bound to DP receptors with a high affinity (K(D)=1.45+/-0.01 nM, n=3) and to a finite, saturable number of binding sites (B(max)=21.1+/-0.6 nmol g(-1) wet weight). 4. DP receptor class prostanoids (e.g. ZK118182, BW245C, BWA868C, PGD(2)) exhibited high (nanomolar) affinities for [(3)H]-BWA868C binding, while prostanoids selective for EP, FP, IP and TP receptors showed a low (micromolar) affinity. 5. Specific DP receptor binding sites were autoradiographically localized on the ciliary epithelium/process, longitudinal and circular ciliary muscles, retinal choroid and iris in human eye sections using [(3)H]-BWA868C. While [(3)H]-PGD(2) yielded similar quantitative distribution of DP receptors as [(3)H]-BWA868C, the level of non-specific binding observed with [(3)H]-PGD(2) was significantly greater than that observed with [(3)H]-BWA868C. 6. It is concluded that [(3)H]-BWA868C is a high-affinity and very specific DP receptor radioligand capable of selectively labelling the DP receptor. [(3)H]-BWA868C may prove useful for future homogenate-based and autoradiographic studies on the DP receptor.