Competition Curves Research Articles

Biodistribution, blood half-life, and receptor binding of a somatostatin-dextran conjugate.

Derivatives of somatostatin (sms) are attracting increasing interest as part of the treatment of several cancer diseases expressing sms receptors (srs). Radiolabeled sms analogs can additionally be used for systemic radiotherapy and for diagnostic investigations. Glycosylated sms-14 (sms-dextran70) was characterized regarding in vitro srs binding, biodistribution, and blood half-life in mice. Rat brain cortex membranes (expressing srs 2) were used for the srs binding study. Tyr3-Octreotide was used as positive control. The binding data were analyzed by competition curve analysis. In the biodistribution study, the Bolton-Hunter reagent was used for the radioiodination of sms-dextran70. Organs and blood were collected at different time-points and the percentage of the injected dose per gram of tissue (%ID/g) was calculated. The conjugate was administered subcutaneously (sc). The sms-dextran70 showed high srs binding affinity (i.e., in the same nanomolar range as the reference ligand Tyr3-octreotide (IC50 approximately 2.5 nM). The blood half-life was approx 27 h after reaching maximum blood concentration 24 h postinjection. Because of the molecular weight of the conjugate (i.e., approx 75,000) being above the kidney threshold for dextran (i.e., 50,000), the digestion and excretion is assumed to be mainly through the hepatobiliary system. Increased uptake was seen in the adrenals, which are receptor-positive organs. Some accumulation was seen in the stomach, indicating certain deiodination of the conjugate label. The sms-dextran70 showed promising properties and its clinical relevance is currently being evaluated in clinical phase I-II studies.

Modulation of Blood Glucose by Melatonin: A Direct Action on Melatonin Receptors in Mouse Hepatocytes

Melatonin receptors were studied in isolated mouse hepatocytes using the 2[¹²⁵I]iodomelatonin binding assay. The binding of 2[¹²⁵I]iodomelatonin to hepatocytes isolated from the mouse using collagenase was stable, saturable, reversible and of high affinity. The equilibrium dissociation constant (K_d) obtained from saturation studies was 10.0 ± 0.4 pmol/l (n = 16), which was comparable to the K_d obtained from kinetics studies (6.9 ± 1.2 pmol/l, n = 3), and the maximum number of binding sites (B_max) was 2.9 ± 0.4 fmol/mg protein (n = 16). The relative order of potency of indoles in competing for 2[¹²⁵I]iodomelatonin binding was 2-iodomelatonin > 2-phenylmelatonin > 6-chloromelatonin > melatonin > 6-hydroxymelatonin > N-acetylserotonin, indicating that the binding was mediated by the ML₁ receptor subtype. The linear Rosenthal plots, the close proximity of the Hill coefficient to unity and the monophasic competition curves suggest that a single class of 2[¹²⁵I]iodomelatonin binding sites is present in the mouse hepatocytes. Guanosine 5′-O-(3-thiotriphosphate) dose-dependently inhibited 2[¹²⁵I]iodomelatonin by lowering the affinity of binding, while no inhibitory effects of adenosine nucleotides were observed, suggesting that the binding sites are G-protein linked. Western immunoblotting was used to identify the melatonin receptor subtype in mouse hepatocytes using anti-Mel_1a and anti-Mel_1b. Hepatocyte membrane extract reacted with anti-Mel_1b but not anti-Mel_1a giving a peptide-blockable band of 36 kD, supporting the hypothesis that the melatonin receptors in mouse hepatocytes are of the Mel_1b subtype. Melatonin injection and a high plasma glucose level affected 2[¹²⁵I]iodomelatonin binding in the whole mouse liver homogenates. Plasma glucose was elevated by mid-light intraperitoneal injection of melatonin (4 and 40 mg/kg body weight) in a dose-dependent manner with maximum elevation achieved 1 h after injection. 2[¹²⁵I]Iodomelatonin binding at this time showed increased K_d with no changes in B_max. When the plasma glucose returned to normal within 2 h, the binding remained lowered with increased K_d but no changes in B_max. Elevation of plasma glucose by 2-deoxyglucose injection (500 mg/kg), on the other hand, decreased the binding by decreasing the B_max without affecting the K_d. Suppression of plasma glucose by insulin injection (3 IU/kg) did not change the binding. Thus, melatonin may act directly on the liver to elevate the plasma glucose level, and changes in plasma glucose level itself may in turn affect hepatic melatonin binding.

Autoradiographic Comparison of [3H]-Clonidine Binding to Non-Adrenergic Sites and α2-Adrenergic Receptors in Human Brain

Clonidine is a partial agonist at brain α2-adrenoceptors (α2AR), but also has high affinity (KD = 51 nM) in homogenate binding assays for non-adrenergic imidazoline-binding sites (I-sites; imidazoline receptors). Herein, an autoradiographic comparison of [3H]-clonidine binding to I-sites and α2AR in sections of human brain is reported. For I-sites, the adrenergic component of 50 nM [3H]-clonidine binding was masked with either 60 μM norepinephrine (NE; α2AR agonist) or 12.5 μM methoxy-idazoxan (MIDX; selective α2AR antagonist), whereas the remaining non-adrenergic sites were studied by displacement with 20 μM cirazoline. Levels of [3H]-clonidine binding to α2AR and I-sites, determined in adjacent tissue sections, were positively correlated across 27 brain regions (p = 0.0003; r2 = 0.385). The principal olivary nucleus and the rostral portion of the ventrolateral medulla had highest ratios of I-sites: α2AR (>4:1). Quantitative transepts drawn across hippocampal images revealed α2AR enrichments in the CA-1 and inner molecular layers of the dentate gyrus—areas not enriched in I-sites. Competition curves were generated for I-sites in caudate sections using 10 ligands known to distinguish between I1 and I2 subtypes. The rank-order of affinities were cirazoline > harmane > BDF6143 > idazoxan = tizanidine (affinities of agmatine, efaroxan, moxonidine, NE, and oxymetazoline were too low to be reliable). Only the endogenous I-site ligand, harmane, had a monophasic displacement curve at the non-adrenergic sites (Ki = 521 ± 12 nM). In conclusion: 1) the distribution of non-adrenergic [3H]-clonidine binding sites in human brain sections was correlated with, but distinct from α2AR; and 2) the affinities of these sites was distinct from α1AR, α2AR, I1 or I2 sites as previously defined in membrane binding assays. The properties of this non-adrenergic [3H]-clonidine binding site are consistent with I-sites previously labeled by [3H]-cirazoline in rat brain.

Evidence that the σ 1 receptor is not directly coupled to G proteins

Sigma (σ) receptors have been implicated in psychosis, cognition, neuroprotection, and locomotion in the central nervous system. The signal transduction mechanisms for σ receptors have not been fully elucidated. In this study, we examined the possible coupling between σ 1 receptors and heterotrimeric guanine nucleotide-binding proteins (G proteins) in rodent brain. In σ 1 receptor-rich cerebellar membrane preparations, the competitive binding curves of two σ 1 agonists, (+)pentazocine and 1 S,2 R-(−)- cis- N-[2-(3,4-dichlorophenyl)ethyl]- N-methyl-2-(1-pyrrolidinyl)cyclohexylamine (BD737), were unaffected by the addition of 10 μM guanosine-5′- O-(γ-thio)-triphosphate (GTPγS). Neither (+)pentazocine (1–100 μM) nor BD737 (0.01–10 μM) stimulated GTPase activities significantly above basal levels in agonist-stimulated GTPase activity assays in cerebellar membranes. Furthermore, when using the method of agonist-stimulated [ 35S]GTPγS binding as assessed by autoradiography, we did not observe significant stimulation of [ 35S]GTPγS binding in rat brain sections by either (+)pentazocine or BD737. The above results demonstrate that the σ 1 receptor is not likely be directly coupled to G proteins.

Separation of p-xylene from C 8 aromatics on binder-free hydrophobic adsorbent of MFI zeolite. I. Studies on static equilibrium

The static equilibrium of C 8 aromatics in liquid phase was studied on a binder-free MFI zeolite adsorbent, which possesses perfect framework and high hydrophobicity. The selectivity factor for para-xylene/ethylbenzene and para-xylene/ ortho-xylene is determined as 7.95 and 104, respectively. A series of desorbents matched with adsorbents such as benzene, toluene, butylbenzene, monochlorobenzene, para-diethylbenzene and para-dichlorobenzene were investigated as well. The adsorbent can be regenerated and used without controlling the content of water. The isotherms for each isomer of C 8 aromatics and the desorbents possess the adsorption characteristics of Langmuir type. The saturation adsorption capacities of the adsorbates were determined. Unusually, no adsorption of meta-xylene on the adsorbent was found, which is much different from those observed on the zeolite of ZSM-5. The competitive adsorption curves of the multi-component feed were also investigated. The high shape selectivity of separation is related to the perfect structure of the adsorbent.

Expression of nitric oxide synthases in subcutaneous adipose tissue of nonobese and obese humans

Studies have shown evidence of production of nitric oxide (NO) in adipose tissue, as well as inhibition of lipolysis by NO. We have analyzed nitric oxide synthase (NOS) expression in subcutaneous adipose tissue from 13 nonobese and 18 obese male subjects. Using a competitive reverse transcription polymerase chain reaction method, endothelial (eNOS) and inducible (iNOS), but not neuronal (nNOS), nitric oxide synthase mRNA expression was detected in isolated fat cells and pieces of adipose tissue. Tissue mRNA levels for eNOS were 3,814 +/- 825 and 5,956 +/- 476 amol/mg RNA (P = 0.043), and for iNOS 306 +/- 38 and 332 +/- 48 amol/mg RNA, for nonobese and obese individuals, respectively. Western blotting revealed similar eNOS protein levels in isolated fat cells and adipose tissue pieces. Protein levels for eNOS in nonobese and obese individuals, respectively, were (in optical density [OD] units per mm(2) per 100 microgram of total protein) 0.11 +/- 0.08 and 2.80 +/- 1.30 (P = 0.043). iNOS protein was detectable, but not measurable, at low levels in a subset of obese patients (3 of 10). iNOS protein levels could not be detected in nonobese individuals. Hormone-sensitive lipase (HSL), the key regulating enzyme in lipolysis, is reduced in obesity. The expression of HSL protein in subcutaneous adipose tissue was studied in the same subset of patients; in agreement with previous results, HSL levels were reduced in obese subjects: 4.64 +/- 1.10 and 1.27 +/- 0.35 (P = 0.012) in nonobese and obese subjects, respectively. In conclusion, this study shows that eNOS and iNOS, but not nNOS, are present in human subcutaneous adipose tissue. Gene expression and protein levels of eNOS are increased, whereas HSL protein levels are decreased in obesity. It is speculated that increased NO production, preferably by eNOS, and decreased HSL levels may cause decreased subcutaneous adipose tissue lipolysis in obesity. synthases in subcutaneous adipose tissue of nonobese and obese humans.

Site-directed mutagenesis studies of human A 2A adenosine receptors : Involvement of glu 13 and his 278 in ligand binding and sodium modulation

To provide insights into interactions between ligands and A 2A adenosine receptors, site-directed mutagenesis was used to test the roles of a glutamic acid residue in the first transmembrane domain (Glu13) and a histidine residue in the seventh transmembrane domain (His278). The two residues, which have been suggested to be closely linked in molecular modeling studies, were mutated to glutamine (E13Q) and tyrosine (H278Y), respectively. Saturation experiments revealed that [ 3H]ZM241385 (4-{2-[7-amino-2-(2-furyl)-1,2,4-triazolo[1,5- a][1,3,5]triazin-5-yl-amino]ethyl}phenol) bound wild-type and mutant receptors in membranes from COS-7 cells expressing human A 2A adenosine receptors with high affinity and low non-specific binding. It was found from the competition experiments that the affinity of the A 2A adenosine receptor agonists for the mutant receptors was 3- to 200-fold lower than for the wild-type receptor. Among antagonist competitors of binding at E13Q and H278Y mutant receptors, there was variation in the affinity depending on their different structures, although changes were relatively minor (<3-fold) except in the case of theophylline, whose affinity was decreased approximately 20 times on the H278Y mutant. The possible involvement of the two residues in sodium ion regulation was also tested. The agonist competition curves for [ 3H]ZM241385 were shifted to the right in both wild-type and mutant receptors in the presence of 1 M sodium ions, but the extent of shift (2- to 27-fold) in wild-type receptor was generally larger than for the mutant receptors. Sodium ions also decreased [ 3H]ZM241385 dissociation from both wild-type and mutant receptors, being more influential on the former than the latter. The results suggest that the two closely linked residues Glu13 and His278 in A 2A adenosine receptor are most important for agonist recognition and are partly responsible for the allosteric regulation by sodium ions.

Pharmacology of [ [formula omitted]] R(+)-7-OH-DPAT binding in the rat caudate-putamen

Dopamine D 3 receptors may be involved in drug addiction and in disorders such as schizophrenia and Parkinson’s disease. To determine the pharmacological properties of dopamine D 3 receptors in the rat caudate-putamen, we have investigated R(+)-[ 3 H ]7-hydroxy- N, N-di- n-propyl-2-aminotetralin ([ 3 H ] R(+)-7-OH-DPAT) binding to membrane preparations from the rat caudate-putamen. Kinetic analyses showed that [ 3 H ] R(+)-7-OH-DPAT binding reached equilibrium in approximately 1 h and that both association and dissociation curves were composed of at least two components. Likewise, saturation curves showed at least two binding components with a combined B max value of about 600 fmol/mg protein, which is three times higher than what is present in the subcortical limbic area. Competition curves were performed with agonists such as R(−)-propylnorapomorphine, dopamine, PD 128907, quinpirole, and bromocriptine, and antagonists such as haloperidol, raclopride, clozapine, GR 218231x, remoxipride, and U99194A. These experiments revealed that [ 3 H ] R(+)-7-OH-DPAT binding could be resolved into three specific binding sites ( R 1– R 3) and one nonspecific binding site, with R 1– R 2 probably representing D 3 receptor binding and the minor R 3 representing D 2 receptor binding. The low affinities of (±)-8-OH-DPAT and 1,3-di(2-tolyl)guanidine to inhibit [ 3 H ] R(+)-7-OH-DPAT binding indicate negligible involvement of 5-HT 1A or sigma binding sites, respectively. The pharmacological profile of [ 3 H ] R(+)-7-OH-DPAT (2 nM) binding in the caudate-putamen was similar to that of dopamine on [ 125 I ]iodosulpride binding in the cerebellar lobule X, which contain D 3 but not D 2 receptors. Mg 2+ increased and GTP and Na + decreased the binding of [ 3 H ] R(+)-7-OH-DPAT, suggesting a coupling of endogenous D 3 receptors to G proteins. Taken together, these results suggest that dopamine D 3 receptors display multiple agonist binding states, and that D 3 receptors are present in high concentrations in the rat caudate-putamen. These results may have implications for the physiological and pathological roles of dopamine D 3 receptors in the brain.

A galpha(s) carboxyl-terminal peptide prevents G(s) activation by the A(2A) adenosine receptor.

The molecular mechanisms of interaction between G(s) and the A(2A) adenosine receptor were investigated using synthetic peptides corresponding to various segments of the Galpha(s) carboxyl terminus. Synthetic peptides were tested for their ability to modulate binding of a selective radiolabeled agonist, [(3)H]2-[4-(2-carboxyethyl)phenylethylamino]-5'-N-ethylcarboxam idoade nosine ([(3)H]CGS21680), to A(2A) adenosine receptors in rat striatal membranes. The Galpha(s) peptides stimulated specific binding both in the presence and absence of 100 microM guanosine-5'-O-(3-thiotriphosphate) (GTPgammaS). Three peptides, Galpha(s)(378-394)C(379)A, Galpha(s)(376-394)C(379)A, and Galpha(s)(374-394)C(379)A, were the most effective. In the presence of GTPgammaS, peptide Galpha(s)(374-394)C(379)A increased specific binding in a dose-dependent fashion. However, the peptide did not stabilize the high-affinity state of the A(2A) adenosine receptor for [(3)H]CGS21680. Binding assays with a radiolabeled selective antagonist, [(3)H]5-amino-7-(2-phenylethyl)-2-(2-furyl)pyrazolo[4, 3-e]-1,2,4-triazolo[1,5-c]pyrimidine ([(3)H]SCH58261), showed that the addition of the Galpha(s) peptide modified the slope of the 5'-N-ethylcarboxamidoadenosine (NECA) competition curve, suggesting modulation of receptor affinity states. In the presence of GTPgammaS, the displacement curve was right-shifted, whereas the addition of Galpha(s)(374-394)C(379)A caused a partial left-shift. Both curves were fitted by one-site models. This same Galpha(s) peptide was also able to disrupt G(s)-coupled signal transduction as indicated by inhibition of the A(2A) receptor-stimulated adenylyl cyclase activity without affecting either basal or forskolin-stimulated enzymatic activity in the same membrane preparations. Shorter peptides from Galpha(s) and Galpha(i1/2) carboxyl termini were not effective. NMR spectroscopy showed the strong propensity of peptide Galpha(s)(374-394)C(379)A to assume a compact carboxyl-terminal alpha-helical conformation in solution. Overall, our results point out the conformation requirement of Galpha(s) carboxyl-terminal peptides to modulate agonist binding to rat A(2A) adenosine receptors and disrupt signal transduction.

Speed-up in fractal image coding: comparison of methods

Fractal image compression has received much attention from the research community because of some desirable properties like resolution independence, fast decoding, and very competitive rate-distortion curves. Despite the advances made, the long computing times in the encoding phase still remain the main drawback of this technique. So far, several methods have been proposed in order to speed-up fractal image coding. We address the problem of choosing the best speed-up techniques for fractal image coding, comparing some of the most effective classification and feature vector methods--namely Fisher, Hurtgen, and Saupe--and a new feature vector coding scheme based on the block's mass center. Furthermore, we introduce two new coding schemes combining Saupe with Fisher, and Saupe with mass center coding scheme. Experimental results demonstrate both the superiority of feature vector techniques on classification and the effectiveness of combining Saupe and the mass center coding scheme, an approach that exhibits the best time-distortion curves.

Characterization of Proexosite I on Prothrombin

Activation of prothrombin by factor Xa is accompanied by expression of regulatory exosites I and II on the blood coagulation proteinase, thrombin. Quantitative affinity chromatography and equilibrium binding studies with a fluorescein-labeled derivative of the exosite I-specific peptide ligand, hirudin(54-65) ([5F]Hir(54-65) (SO(3)(-)), were employed to identify and characterize this site on human and bovine prothrombin and its expression on thrombin. [5F]Hir(54-65)(SO(3)(-)) showed distinctive fluorescence excitation spectral differences in complexes with prothrombin and thrombin and bound to human prothrombin and thrombin with dissociation constants of 3.2 +/- 0.3 micrometer and 25 +/- 2 nm, respectively, demonstrating a 130-fold increase in affinity for the active proteinase. The bovine proteins similarly showed a 150-fold higher affinity of [5F]Hir(54-65)(SO(3)(-)) for thrombin compared with prothrombin, despite a 2-5-fold lower affinity of the peptides for the bovine proteins. Unlabeled, Tyr(63)-sulfated and nonsulfated hirudin peptides bound competitively with [5F]Hir(54-65)(SO(3)(-)) to human and bovine prothrombin and thrombin, exhibiting similar, 40-70-fold higher affinities for the proteinases, although nonsulfated Hir(54-65) bound with 7-17-fold lower affinity than the sulfated analog. These studies characterize proexosite I for the first time as a specific binding site for hirudin peptides on both human and bovine prothrombin that is present in a conformationally distinct, low affinity state and is activated with a approximately 100-fold increase in affinity when thrombin is formed.

A chicken leptin-specific radioimmunoassay

Recombinant chicken leptin was used to produce an antiserum in order to develop a specific and sensitive radioimmunoassay (RIA) for chicken leptin in plasma and serum. We have used either murine or chicken leptin as tracer and competition curves were performed using recombinant chicken leptin. Variations in leptin plasma levels in different chicken strains and various nutritional states were correlated with the physiological status. Leptin plasma concentrations were regulated by the nutritional state with higher levels in the fed state as compared to the fasted state (3.36 ± 0.13 versus 2.78 ± 0.11 ng/ml) and being dependent upon the age. Higher leptin levels were found in 22 week-old as compared to 15 week-old layer chickens (2.709 ± 0.172 versus 1.478 ± 0.102 ng/ml). We have also shown that the multispecies leptin RIA kit (LINCO Inc.) underestimated leptinemia compared to the chicken leptin- specific RIA reported here. In conclusion the RIA developed in the present study is specific to the chicken and thus may be considered as powerful tool for investigating the physiological significance of leptin in chickens.

The size and/or configuration of the cycloalkane D′ ring in pentacyclic progesterone derivatives are crucial for their high-affinity binding to a protein in addition to progesterone receptor in rat uterine cytosol☆

[ 3H]labeled progesterone and a number of its 16α,17α-cycloalkano derivatives with an additional three to six-membered D′ ring were investigated for mutual competition and equilibrium binding to proteins from rat uterine cytosol. The interaction of all studied [ 3H]ligands with proteins was characterized by comparable affinity ( K d in nM region) and apparent homogeneity in terms of affinity. At the same time, the concentrations of binding sites for ligands bearing 16α,17α cyclopentano, cyclohexano, or cyclohexeno substituents were several-fold higher than those for progesterone or 16α,17α-cyclopropanoprogesterone. In mutual competition experiments, when [ 3H]progesterone or [ 3H]16α,17α-cyclopropanoprogesterone were used, the curves of ‘bound radioactivity—log of competitor concentration’ for all compounds studied were parallel and corresponded to a model of ‘one protein—two ligands.’ However, when [ 3H]ligands with bulky 16α,17α-substituents (with the possible exception of cyclohexene derivative) were used, competitive curves for various ligands had different appearances and fell into two groups. Parallel curves for derivatives with 5 or 6 carbons in D′ ring described by a model of ‘one protein—two ligands’ formed the 1st group. The 2nd group comprised curves for progesterone or 16α,17α-cyclopropanoprogesterone that had lower slopes and could be described by a model of ‘two proteins—two ligands.’ Taken together, the results suggest the presence in rat uterine cytosol, of a protein in addition to progesterone receptor capable of discriminating between ligands with no or small 16α,17α-cycloalkano substituents and ligands with more bulky substituents.

Transducer abstraction: A novel approach to the detection of partial agonist efficacy in radioligand binding studies

The properties of the ternary complex model (TCM) of drug action at G protein-coupled receptors (GPCRs) were examined, using theoretical computer simulations, with regard to the predicted effects of the presence of a fixed concentration of one agonist on the competition binding profile of another. Subsequently, the binding properties of the full muscarinic acetylcholine receptor (mAChR) agonists acetylcholine (ACh) and carbachol (CCh), and the partial agonists pilocarpine and McN-A-343, were investigated in competition experiments against [ 3H] N-methylscopolamine using homogenate preparations from Chinese hamster ovary cells, stably expressing the human M 1 or M 2 mAChR. At the M 2 mAChR, all agonists displayed biphasic binding curves and were readily modulated by the non-hydrolyzable GTP analogue, Gpp(NH)p, in accordance with previously established experimental observations. In contrast, agonist binding at the M 1 mAChR showed no significant change in the presence of Gpp(NH)p, even in the case of a full agonist. This phenomenon precludes using the “GTP-shift” to assess agonist efficacy at the M 1 mAChR. When the ACh competition curves were reconstructed in the presence of graded concentrations of either a full or a partial agonist, a significant redistribution of the fraction of the high-affinity state recognized by ACh was observed. However, when the procedure was repeated using the antagonist, atropine, no significant effect on the fraction of either the high or low affinity ACh binding components at the mAChR was observed. Taken together, these results indicate that changes in the profile of full agonist binding isotherms, when constructed in the presence of a partial agonist, may be more sensitive indicators of partial agonist efficacy than regular assays that directly measure partial agonist binding.

Identification of a Binding Site on the Type II Activin Receptor for Activin and Inhibin

Type II activin receptors (ActRII and ActRIIB) are single-transmembrane domain serine/threonine kinase receptors that bind activin to initiate the signaling and cellular responses triggered by this hormone. Inhibin also binds type II activin receptors and antagonizes many activin effects. Here we describe alanine scanning mutagenesis of the ActRII extracellular domain. We identify a cluster of three hydrophobic residues (Phe(42), Trp(60), and Phe(83)) that, when individually mutated to alanine in the context of the full-length receptor, cause the disruption of activin and inhibin binding to ActRII. Each of the alanine-substituted ActRII mutants retaining activin binding maintains the ability to form cross-linked complexes with activin and supports activin cross-linking to the type I activin receptor ALK4. Unlike wild-type ActRII, the three mutants unable to bind activin do not cause an increase in activin signaling when transiently expressed in a corticotroph cell line. Together, our results implicate these residues in forming a critical binding surface on ActRII required for functional interactions with both activin and inhibin. This first identification of a transforming growth factor-beta family member binding site may provide a general basis for characterizing binding sites for other members of the superfamily.

Evaluation of [11C]GB67, a novel radioligand for imaging myocardial alpha 1-adrenoceptors with positron emission tomography.

Dysfunction of the sympathetic nervous system underlies a number of myocardial disorders. Positron emission tomography (PET) offers a way of assessing receptor function non-invasively in humans, but there are no PET radioligands for assessing myocardial alpha-adrenoceptors. GB67, a structural and pharmacological analogue of the alpha 1-adrenoceptor antagonist prazosin, was labelled with positron-emitting carbon-11 (t1/2 = 20.4 min) by 11C-methylation of N-desmethylamido-GB67 (GB99). [11C]GB67 was injected intravenously into conscious rats. Serial arterial blood samples were taken. Rats were killed and tissues removed to determine radioactivity. The percentages of unchanged [11C]GB67 and its radioactive metabolites in plasma and tissues were assessed by HPLC. Plasma clearance of radioactivity was rapid. Myocardial uptake was maximal at 1-2 min and decreased slowly during 60 min. Predosing with adrenoceptor antagonists demonstrated selectivity for myocardial alpha 1-adrenoceptors. GB67 and prazosin blocked uptake of radioactivity; the non-selective antagonist, phentolamine, partially blocked uptake; the alpha 2-adrenoceptor antagonist, RX 821002, only blocked uptake at high dose and the beta-adrenoceptor antagonist, CGP 12177, had no effect. Additionally, injection of prazosin at 20 min after radioligand displaced radioactivity. In vivo competition curves obtained by injecting [11C]GB67 with varying amounts of either unlabelled GB67 or its precursor GB99 were fitted to a competitive binding model to provide estimates of the maximum number of binding sites (Bmax) and half saturation doses (K) for myocardium. Assuming a tissue protein content of 10%, the values of Bmax [approximately 13 pmol.(g tissue)-1[ were similar to those ]50-170 fmol.(mg protein)-1] reported for myocardial alpha 1-adrenoceptors assessed in vitro. Both GB67 and its precursor GB99 had high affinity for alpha 1-adrenoceptors [KGB67 = 1.5 nmol.(kg body weight)-1, KGB99 = 4.8 nmol.(kg body weight)-1]. HPLC demonstrated four radioactive metabolites in plasma. [11C]GB67 was 80% of the radioactivity at 5 min and 50% at 45 min. No radioactive metabolites were detected in myocardium up to 60 min after injection. [11C]GB67 was assessed in two male human volunteers. PET demonstrated high myocardial uptake. The profile of radioactive metabolites in plasma was comparable to that in the rat, although metabolism was slower in humans. Thus, [11C]GB67 is a promising radioligand for assessing alpha 1-adrenoceptors in human myocardium with PET.

Stereoselective Synthesis and Receptor Binding of Conformationally Restricted and Flexible 2,4-Disubstituted 1,3-Dioxanes Derived from Benzomorphans

The key steps in the stereoselective synthesis of the tricyclic aminomethyl derivatives 19 and 20 and the aminoethyl substituted 1,3-dioxanes 24 and 25 are nucleophilic addition of aryllithium intermediates to the nitroalkene 13, intramolecular transacetalization of the addition products 15 and 16 (only for the tricyclic derivatives 19 and 20) and subsequent reduction of the nitro group. The affinities of the secondary and tertiary amines 19c,d, 20c,d, 24c,d, and 25c,d for the ion channel binding site of the NMDA receptor, for μ-, κ-, and σ-receptors have been investigated. In the group of tricyclic compounds only 19d shows remarkable σ-receptor affinity (Ki = 21.6/1.10 μM). In the 1,3-dioxane series the moderate μ- (Ki = 27.8 μM) and κ-receptor affinity (Ki = 36 μM) as well as the high σ-receptor affinity (Ki = 3.3 μM) of the (S,S,S)-configurated methylamine 24c should be emphasized. The pentan-1-ol 26, the side product isolated during the synthesis of 24c, is of particular interest because of its considerable affinity to μ- (Ki = 16.0 μM), κ- (Ki = 2.8 μM), and σ-receptors (Ki = 14.5/1.26 μM). The biphasic competition curves obtained during σ-receptor binding studies of 19d and 26 (two Ki values) may be explained by different interaction with σ-receptor subtypes.

Molecular and pharmacological characterization of the canine brainstem alpha-2A adrenergic receptor.

This study characterizes the alpha2-adrenergic receptors present in canine brainstem. Radioligand binding and reverse transcriptase-polymerase chain reaction (RT-PCR) experiments were performed in canine brainstem to identify the receptors present and determine the pharmacological properties of these receptors. The pKi values derived from radioligand competition curves for a number of adrenergic receptor agents at the four alpha2-adrenergic receptor subtypes were compared to the canine brainstem. The pKi values at the canine brainstem alpha2-adrenergic receptor were consistent with the presence of the alpha2A-adrenergic receptor. To determine whether the canine brainstem expressed the message for the alpha2A-adrenergic receptor, RT-PCR was performed with specific primers for the four subtypes of alpha2-adrenergic receptors. In the canine brainstem, only the primers corresponding to a region in the human alpha2A-adrenergic receptor produced a PCR product. No bands were detected in the canine brainstem lanes with the alpha2B-, alpha2C-, or alpha2D-receptor primers. These data suggest that the canine brainstem contains the alpha2A-adrenergic receptor.

Stereoselective synthesis and receptor binding of conformationally restricted and flexible 2,4-disubstituted 1,3-dioxanes derived from benzomorphans.

The key steps in the stereoselective synthesis of the tricyclic aminomethyl derivatives 19 and 20 and the aminoethyl substituted 1,3-dioxanes 24 and 25 are nucleophilic addition of aryllithium intermediates to the nitroalkene 13, intramolecular transacetalization of the addition products 15 and 16 (only for the tricyclic derivatives 19 and 20) and subsequent reduction of the nitro group. The affinities of the secondary and tertiary amines 19c,d, 20c,d, 24c,d, and 25c,d for the ion channel binding site of the NMDA receptor, for mu-, kappa-, and sigma-receptors have been investigated. In the group of tricyclic compounds only 19d shows remarkable sigma-receptor affinity (Ki = 21.6/1.10 microM). In the 1,3-dioxane series the moderate mu- (Ki = 27.8 microM) and kappa-receptor affinity (Ki = 36 microM) as well as the high sigma-receptor affinity (Ki = 3.3 microM) of the (S,S,S)-configurated methylamine 24c should be emphasized. The pentan-1-ol 26, the side product isolated during the synthesis of 24c, is of particular interest because of its considerable affinity to mu- (Ki = 16.0 microM), kappa- (Ki = 2.8 microM), and sigma-receptors (Ki = 14.5/1.26 microM). The biphasic competition curves obtained during sigma-receptor binding studies of 19d and 26 (two Ki values) may be explained by different interaction with sigma-receptor subtypes.

Using a radioalloster to test predictions of the cooperativity model for gallamine binding to the allosteric site of muscarinic acetylcholine M(2) receptors.

The muscarinic M(2) receptor contains an orthosteric and an allosteric site. Binding of an allosteric agent may induce a shift alpha of the equilibrium dissociation constant K(D) of a radioligand for the orthosteric site. According to the cooperativity model, the K(A) of alloster binding is expected to be shifted to an identical extent depending on whether the orthosteric site is occupied by the orthoster or not. Here, the novel radioalloster [(3)H]dimethyl-W84 (N,N'-bis[3-(1,3-dihydro-1, 3-dioxo-4-methyl-2H-isoindol-2-yl)propyl]-N,N,N',N'-tetramethyl-1, 6-hexanediaminium diiodide) was applied to directly measure the K(A) shift induced for the prototype allosteric modulator gallamine by binding of N-methylscopolamine (NMS) to the orthosteric site of porcine heart M(2) receptors (4 mM Na(2)HPO(4), 1 mM KH(2)PO(4), pH 7.4; 23 degrees C; data are means +/- S.E.). First, in the common way, the concentration-dependent inhibition by gallamine of [(3)H]NMS equilibrium binding was measured and analyzed using the cooperativity model, which yielded for the affinity of gallamine binding at free receptors a pK(A)= 8.35 +/- 0.09 and a cooperativity factor alpha = 46 (n = 5). The dissociation constant for gallamine binding at NMS-occupied receptors was predicted as p(alpha. K(A)) = 6.69. Labeling of the allosteric site by [(3)H]dimethyl-W84 allowed the measure of competitive displacement curves for gallamine. The K(i) for gallamine at free receptors amounted to pK(i,-NMS) = 8.27 +/- 0.39 (n = 5), which is in line with the prediction of the cooperativtiy model. In the presence of 1 microM NMS, to occupy the orthosteric site, gallamine displaced [(3)H]dimethyl-W84 with pK(i, +NMS) = 6.60 +/- 0.19 (n = 3). Thus, the NMS-induced pK(i) shift amounted to 47, which matches the predicted value of alpha = 46. These results validate the cooperativity model.