Stereospecific Binding Research Articles

Stereospecific recognition sites for [ 3H]inositol(1,4,5)-trisphosphate in particulate preparations of rat cerebellum

A very high density of stereospecific binding sites for inositol(1,4,5)P 3 have been identified in rat cerebellar membranes using [ 3H]inositol(1,4,5)P 3 and a rapid centrifugation step to separate free and bound ligand. Binding was shown to be rapid and reversible and of relatively high affinity (K D 23 nM). Incubations were carried out at 4° and under these conditions HPLC analysis demonstrated that there was no significant metabolism of [ 3H]-(1,4,5)P 3 in the presence or absence of ATP over 15 min. The specificity of the site has been carefully evaluated using both natural and novel synthetic inositol phosphates. The stereospecificity is very marked with the D-, DL- and L-isomers of Ins(1,4,5)P 3 showing a 1:4:2000 ratio of affinity for the binding site. D-Ins(2,4,5)P 3 was the only other phosphate to show relatively high affinity (K D 1500 nM). HPLC-pure Ins(1,3,4)P 3 and Ins(1,3,4,5)P 4 were substantially weaker and Ins(1,4)P 2, Ins-2-P 1, Ins-1-P 1, Ins(1,2)-cyclic P 1 and inositol were totally inactive at concentrations <50 μM. These data are discussed in relation to a putative receptor on the endoplasmic reticulum by which Ins(1,4,5)P 3 can initiate the release of bound Ca 2+.

Demonstration of specific E-type prostaglandin receptors using enriched preparations of canine parietal cells and [ 3H] misoprostol free acid

High-affinity, E-type prostaglandin binding sites in enriched canine parietal cell preparations were identified with [ 3H] misoprostol free acid, a prostaglandin E 1 analogue. Saturable, reversible, and highly stereospecific binding was identified, with approximately 8,000 binding sites per cell. Prostaglandin I and F bound weakly, and cimetidine and histamine did not bind. The results indicate that [ 3H] misoprostol free acid binds to E-type prostaglandin receptors, which suggests that the ulcer-healing inhibition of gastric acid secretion by misoprostol results from its interaction with a specific E-type prostaglandin receptor.

The gaba/benzodiazepine receptor — chloride ionophore complex: Nature and modulation

Bruun-Meyer, Sturla E.: The GABA/Benzodiazepine Receptor-Chloride Ionophore Complex: Nature and Modulation. Prog. Neuro-Psychopharmacol. & Biol. Psychiat. 1987, 11 : 365–387 1. 1. A high affinity, saturable, stereospecific binding site for Benzodiazepines has been found to be functionally and possibly structurally related to a GABA receptor-chloride ionophore complex. 2. 2. There are both central(CNS) as well as “peripheral” binding sites, involving multiple organs. 3. 3. Evidence strongly suggests that mutually exclusive Benezodiazepine agonists and antagonists bind to the same receptor, possibly in an agonist-antagonist-inverse agonist continuum. 4. 4. The search for an endogenous ligand has been inconclusive and the question of such a substance remains open. 5. 5. Although the relationship between this receptor and the Limbic System remains unclear, it seems certain that the Benzodiazepine receptor plays an important role in the modulation of Limbic System excitability.

High affinity stereospecific binding of [3H] cocaine in striatum and its relationship to the dopamine transporter.

A high affinity (KD 35 nM) binding site for [3H]cocaine is detected in rat brain striatum present at 2-3 pmol/mg protein of synaptic membranes. This binding is displaced by cocaine analogues with the same rank order as their inhibition of [3H]dopamine ([3H]DA) uptake into striatal synaptosomes (r = 0.99), paralleling the order of their central stimulant activity. The potent DA uptake inhibitors nomifensine, mazindol, and benztropine are more potent inhibitors of this high affinity [3H]cocaine binding than desipramine and imipramine. Cathinone and amphetamine, which are more potent central stimulants than cocaine, displace the high affinity [3H]cocaine binding stereospecifically, but with lower potency (IC50 approximately equal to 1 microM) than does cocaine. It is suggested that the DA transporter in striatum is the putative "cocaine receptor." Binding of [3H]cocaine, measured in 10 mM Na2HPO4-0.32 M sucrose, pH 7.4 buffer, is inhibited by physiologic concentrations of Na+ and K+ and by biogenic amines. DA and Na+ reduce the affinity of the putative "cocaine receptor" for [3H]cocaine without changing the Bmax, suggesting that inhibition may be competitive. However, TRIS reduces [3H]cocaine binding noncompetitively while Na+ potentiates it in TRIS buffer. Binding of [3H]mazindol is inhibited competitively by cocaine. In phosphate-sucrose buffer, cocaine and mazindol are equally potent in inhibiting [3H]mazindol binding, but in TRIS-NaCl buffer cocaine has 10 times lower potency. It is suggested that the cocaine receptor in the striatum may be an allosteric protein with mazindol and cocaine binding to overlapping sites, while Na+ and DA are allosteric modulators, which stabilize a lower affinity state for cocaine.

Cyclophilin binds to the region of cyclosporine involved in its immunosuppressive activity

Although several cytosolic proteins including calmodulin and cyclophilin have been shown to bind cyclosporine, the direct involvement of these proteins in the immunosuppressive activity of cyclosporine remains to be established. In the present study, a quantitative immunoassay for cyclophilin was developed which made it possible to compare its relative affinity for cyclosporine and any of its analogues. The binding of cyclophilin to cyclosporine coated on a solid phase was revealed by anti-cyclophilin rabbit antiserum followed by antiglobulin-enzyme conjugate. This reaction could be inhibited by addition of free cyclosporine or certain cyclosporine analogues. By studying the binding of cyclophilin to more than fifty cyclosporine derivatives modified singly on each of the eleven amino acid residues, it could be shown that cyclophilin binds to the residues of cyclosporine known to be critical for its immunosuppressive activity. These data identify cyclophilin as a highly discriminating stereospecific binding protein for cyclosporine.

Binding sites for [ 3H]SCH 23390 in retina: properties and possible relationship to dopamine D 1-receptors mediating stimulation of adenylate cyclase

Bovine, rat and chick embryo retinal membranes contain high affinity, saturable and stereospecific binding sites for the selective dopamine D 1-receptor antagonist, [ 3H]SCH 23390 ( R-(+)-8-chloro-2,3,4,5-tetrahydro-3-methyl-5-phenyl-1H-3-benzazepin-7-ol). Saturation studies and Scatchard analyses showed a single class of [ 3H]SCH 23390 binding sites with K d (apparent dissociation constant) values of 0.5–1.4 nM for the different species studied. A high ratio of specific to non-specific binding was found over a wide range of radioligand concentrations. The B max (binding site number) for [ 3H]SCH 23390 in calf retina was 307 ± 38 fmol/mg protein, significantly greater than B max values previously obtained for binding of [ 3H]spiroperidol and [ 3H]ADTN (2-amino-6,7-dihydroxy-1,2,3,4-tetrahydronaphthalene) to dopamine D 2-receptors in calf retina. Relative affinities ( K i values) of dopamine antagonists for calf retinal [ 3H]SCH 23390 binding sites were similar to those reported for [ 3H]SCH 23390 binding in rat striatum and also were in general agreement with potencies for antagonism of retinal dopamine-stimulated adenylate cyclase. However, they differed markedly from the relative affinities for retinal D 2-receptor sites. Additional data indicated that SCH 23390 did not bind significantly to retinal D 2-or serotonergic receptors and had 30- to 80-fold less affinity for α 2-noradrenergic than for the [ 3H]SCH 23390 sites. Competition studies indicated a high degree of selectivity for dopamine agonists over other agonists for [ 3H]SCH 23390 binding sites with K i values in the range expected for interaction with dopamine receptors mediating stimulation of adenylate cyclase. Affinity for dopamine was decreased in the presence of the GTP analogue, Gpp(NH)p. In the presence of sodium ions the affinities of dopamine agonists for [ 3H]SCH 23390 binding sites were markedly and selectively decreased; the sensitivity to dopamine for stimulation of adenylate cyclase activity was also decreased in the presence of sodium ions. Modulation by sodium ions was previously observed for D 2- but not for a D 1-receptor interaction. It is proposed that [ 3H]SCH 23390 binds to a unique class of receptors, most likely D 1-receptors coupled to adenylate cyclase in retina. [ 3H]SCH 23390 provides a potent new tool for study of these receptors. In retina D 1-receptors positively coupled to cyclase as well as D 2- and other receptors that may be negatively coupled to cyclase, appear to be regulated by sodium ions as well as by guanine nucleotides.

Stereospecific binding of the antidepressant rolipram to brain protein structures.

The characteristics for the binding of the selective cAMP phosphodiesterase inhibitor and antidepressant agent rolipram to brain and peripheral organs were investigated. (+/-)-[3H]Rolipram equilibrium binding and Scatchard analysis revealed saturable, reversible, stereospecific, Mg2+-dependent and heat-sensitive binding with an apparent Hill number of 1. Binding was detected both to membrane-bound and soluble sites, with dissociation constants Kd of 1.2 and 2.4 nM, respectively, and binding site concentrations (Bmax) of 19.3 and 23.6 pmol/g rat forebrain. The (-)-enantiomer of rolipram was ca. 20 times more effective than the (+)-enantiomer in displacing (+/-)-[3H]rolipram from membranes. Rolipram bound to brain tissue of all mammalian species tested including man, while tissue from bird and fish showed less binding. Organs other than brain exhibited only negligible binding. Only specific cAMP phosphodiesterase inhibitors (ICI 63.197, Ro 20-1724) were potent competitors, while rolipram itself was inactive in a variety of receptor binding assays of neuroactive ligands. The kinetics of (-)-[3H]rolipram binding to the particulate fraction revealed a complex association and dissociation behaviour. The nature of the rolipram binding protein(s) is not clear, but the low affinity binding site evident from binding kinetics may represent a rolipram-sensitive phosphodiesterase isoenzyme also common to some peripheral organs, while the high affinity binding site(s) may be related to PDE isoenzymes more confined to the central nervous system.

Receptor-independent sequestration of beta-adrenergic ligands by alveolar type II cells.

Numerous studies indicate that synthesis and secretion of surfactant by type II pneumocytes are modulated by the interaction of beta-adrenergic agonists with specific cell surface receptors. Two 125I-labeled beta-adrenergic ligands, l-iodopindolol (IPIN) and l-iodocyanopindolol (ICYP), were thus employed to investigate the properties of type II cell beta-receptors. Saturable, high-affinity, stereospecific binding to crude membrane fractions from whole rat lungs was exhibited by both ligands (IPIN, KD 283 pM, Bmax 508 fmol/mg protein; ICYP, KD 18 pM, Bmax 404). Type II cell membranes obtained by N2 cavitation also revealed stereospecific, saturable, high-affinity binding. In intact cells (37 degrees C) however, rapid, highly concentrative (cell/media greater than 1000), nonspecific ligand uptake compromised estimates of specific binding (specific/total binding less than 0.1). Total ligand uptake was inhibited at 4 degrees C, by decreasing pH within the physiological range (7-8) and by the lysosomotropic compound chloroquine (50-200 microM), without a detectable change in specific binding. Other basic drugs were also inhibitory at similar concentrations; acidic drugs had no effect. Even at 4 degrees C, specific binding remained low, as IPIN and ICYP were displaced less than 30% by l-alprenolol (1 microM). Physicochemical properties of IPIN and ICYP considered with the above studies suggest that passive ligand entry into intact pneumocytes and subsequent trapping of the protonated species in a cellular compartment of low pH may account for high nonspecific ligand uptake.

Glucostatic regulation of hypothalamic and brainstem [3H](+)-amphetamine binding during food deprivation and refeeding.

Saturable and stereospecific binding sites for [3H](+)-amphetamine have been identified in rat hypothalamus and structure-activity studies suggest that these sites may mediate the anorectic actions of amphetamine and related phenylethylamines. In order to determine whether these binding sites may be involved in the physiological control of appetite, [3H](+)-amphetamine binding was measured in crude synaptosomal membranes prepared from various brain regions following food deprivation and refeeding. Rats deprived of food for 24 to 72 h exhibited time-dependent reductions in body weight, blood glucose concentration, and specific [3H](+)-amphetamine binding in hypothalamus and brainstem. No change in specific [3H](+)-amphetamine binding was found in the frontal cortex, striatum, cerebellum, or liver prepared from these same animals. The decrease in specific [3H](+)-amphetamine binding in both the hypothalamus and brain stem following food deprivation was rapidly and completely reversed by allowing animals brief access to food or a 10% glucose solution. The changes in [3H](+)-amphetamine binding in hypothalami and brainstem of food-deprived and refed rats were also highly correlated with blood glucose concentration. Further, parenteral administration of D-glucose, but not L-glucose, to either fed or food-deprived rats significantly increased the number of [3H](+)-amphetamine binding sites in hypothalamus. These data suggest that the [3H](+)-amphetamine binding site in the hypothalamus and (or) brainstem may function in the 'glucostatic' regulation of appetite.

Cardiac sarcoplasmic reticulum contains a low-affinity site for phenylalkylamines.

The distribution of the bovine cardiac binding sites for the organic calcium-channel blockers was studied. Crude microsomal membranes were separated into three fractions, which contained mainly membranes derived from sarcolemma, 'junctional' sarcoplasmic reticulum containing transversal tubuli, and free sarcoplasmic reticulum. The high-affinity binding site for the dihydropyridines, determined in the presence of nitrobenzylthioinosine, was enriched 12-fold and 17-fold in sarcolemma and junctional sarcoplasmic reticulum. The binding sites for the phenylalkylamines, determined with [3H]verapamil or [3H](-)desmethoxyverapamil, were enriched 1.5-3.4-fold in sarcolemma and junctional sarcoplasmic reticulum but 6-10-fold in free sarcoplasmic reticulum. The phenylalkylamine-binding site, present in free sarcoplasmic reticulum, was partially destroyed by chymotrypsin or phospholipase A2 and C treatment. Specific binding was proportional to the concentration of the added membrane protein. The binding of (-)desmethoxyverapamil was half-maximally inhibited by 6.5 mM calcium chloride and was optimal in the presence of 5 mM EGTA. In three out of five preparations (-)desmethoxyverapamil bound to a single site with an apparent Kd value of 191 +/- 42.8 nM and a density of 34.5 +/- 7.7 pmol/mg protein. In two out of five preparations an additional high-affinity site (Kd approximately 0.67 nM) was detected. The low-affinity site bound other phenylalkylamines, but stereospecific binding of phenylalkylamines was not observed. Binding of phenylalkylamines to the low-affinity site was inhibited by some but not all calmodulin 'antagonists'. Furthermore dihydropyridines did not affect the binding of (--)desmethoxyverapamil suggesting that the low-affinity site differs considerably from the high-affinity sarcolemmal site. These results suggest that free sarcoplasmic reticulum contains a binding site for phenylalkylamines at a relative high density, which is not related to the high-affinity site present in the voltage-dependent calcium channel.

(-)[125I]iodopindolol binding to beta-adrenergic receptors in tissues of the rat with particular reference to the characterization in liver.

The high affinity beta-adrenergic antagonist (-)[125I]iodopindolol was used to characterize and quantitate beta-adrenergic binding in liver and other tissues of the rat. Saturable, stereospecific binding with typical characteristics of beta-adrenergic receptors was demonstrated in all tissues examined (liver, lung, heart, kidney, fat and brain). Unlike the case with other radioactive ligands that have been used to measure beta-adrenergic receptors, assays were possible in crude homogenates as well as in purified membranes. Specific binding was defined as [125I]iodopindolol displaced by 0.1 mM isoproterenol. The percentage of [125I]iodopindolol specifically bound was greater than with other labelled antagonist ligands and it ranged from 95-60% of total binding over [125I]iodopindolol concentrations of 15-1000 pM. beta-Adrenergic binding could be quantitated in liver of animals of all ages whereas previously quantitation in liver was possible only using rats less than 2 months of age. Binding capacities ranged from a low of approximately 6 fmol/mg in liver particles precipitated at 4500 g to approximately 550 fmol/mg in 4500 g lung particles. The Kd of binding obtained by kinetic analysis was similar in all tissues. [125I]Iodopindolol is a nearly ideal ligand for the quantitation of beta-adrenergic receptors in various tissues of the rat. Because of its high affinity, stability and availability at high specific activity, this ligand should be especially useful in physiological studies requiring the accurate quantitation of receptor capacities.

PAF-acether specific binding sites: 1. quantitative SAR study of PAF-acether isosteres

PAF-acether is a phospholipid which mediates a range of biological activities including anaphylaxis and shock. Stereospecific binding sites exist in platelets and lung. In a series of two articles, P. Braquet and J.J. Godfroid describe these sites, their biological control and the medicinal chemistry which has led to the design of specific antagonists. As discussed in Part 1 below, QSAR studies of PAF-acether isosteres have yielded much information about the nature of the binding site itself, and a putative conformation is put forward.

Interaction of ethanol with opiate receptors

Addition of ethanol to rat brain homogenate containing opiate receptors inhibits at a concentration of 50 mM the stereospecific binding of 3H-naloxone at 37 degrees C but not at 0 degree C, with the ID50 being 462 mM under these conditions. The temperature-dependent inhibition of the ligand binding suggests that ethanol does not compete with naloxone for specific binding sites of opiate receptors and changes the structure of lipids in biological membranes. Scatchard's analysis has demonstrated that apart from a decrease in the number of highly affinity binding sites of 3H-naloxone, the total amount of the binding sites remains unchanged both in the presence and absence of ethanol and constitutes 453 and 549 fmol/mg protein. It is assumed that ethanol might interconvert highly and low-affinity binding sites. Analysis of the effect of ethanol on 3H-naloxone binding with opiate receptors contained by synaptic membranes obtained from animals with varying predisposition to voluntary alcoholization has shown that ethanol inhibits to a greater degree ligand binding with membranes obtained from rats predisposed to alcoholization. The possibility of the involvement of receptors in the biochemical mechanisms by which the initial alcoholic motivation is effected is under discussion.

Effects of heavy metal cations, sulfhydryl reagents and other chemical agents on striatal D 2 dopamine receptors

To investigate aspects of the biochemical nature of the membrane-bound D 2 dopamine receptor, rat striatal homogenates were pretreated with heavy metal cations and a variety of other chemical agents, and their effects on D 2 receptor density were subsequently determined using a standard [ 3H]spiperone binding assay. Preincubation of striatal membranes in the presence of 3 mM Mn 2+, Fe 2+, Co 2+, EDTA, or ascorbate enhanced subsequently measured stereospecific binding of [ 3H]spiperone compared to control (e.g. control: B max = 140 mfoles/mg protein, K D = 0.21 nM; Mn 2+-treated: B max = 253 fmoles/mg protein, K D = 0.20 NM). Another group of metal cations, is that Zn 2+, Cd 2+, Cu 2+, Hg 2+ and CH 3Hg +, all of which have significant −SH reactivity, as well as the −SH alkylating agent N-ethylmaleimide (NEM), caused a decrease in the specific binding sites. Pretreatment with 3mM Cd 2+ or Cu 2+ resulted in a 40–60% reduction in the subesequently measured stereospecific binding of [ 3H]-spiperone, whereas 1 mM Hg 2+ or 3 mM NEM completely abolished specific [ 3Hspiperone binding. The effect of Hg 2+ could not be reversed by washing the membranes, nor by further incubation of the membranes in the presence of excess EDTA or 2,3-dimercapto-1-propanesulfonic acid (DMPS). Further incubation in the presence of 3 mM dithioerythritol (DTE) resulted in the regeneration of about 40% of lost sites. Agents which enhanced receptor density, such as Mn 2+ or EDTA, could not antagonize the effect of Hg 2+, nor could the mercury-chelating agent DMPS, when added to crude homogenates prior to Hg 2+. Ascorbate protected 25–35% of specific binding sites by virtue of its ability to reduce Hg 2+ to insoluble Hg +. Only 3 mM DTE afforded afforded complete protection against 1 mM Hg 2+. Prior formation of the spiperone/receptor complex also demonstrated considerable ability to protect receptors from destruction by Hg 2+. Preincubation of striatal membranes in the presence of 0.5 mM spiperone protected about 80% of sites from the subsequent addition of 1 mM Hg 2+. A major conclusion of these studies is that one or more free -SH groups on or adjacent to the active site may be a requirement for specific antagonist binding to the membrane-bound D 2 receptor. Occlusion of these -SH groups by sulfhydryl reagents results in partial to complete abolition of subsequently measured specific 3H-antagonist binding. Only agents which can regenerate free -SH groups, such as DTE, are able to induce any recovery in specific binding sites. Other substances, such as Mn 2+, EDTA and ascorbate which, on their own, cause an enhanced density of specific antagonist binding sites are generally ineffective in preventing the action of sulfhydryl binding agents. Their enhancing effect is probably due to the protection of receptors from normal degradation during incubation and to an improved recovery of receptor sites after centrifugation procedures.

Stoichiometry and kinetics of the interaction of prostaglandin H synthase with anti-inflammatory agents.

We have examined the kinetics, stoichiometry, and chemical nature of the interaction of three anti-inflammatory agents (indomethacin, flurbiprofen, and meclofenamic acid) with pure ovine prostaglandin H synthase. The kinetics of the interaction with the synthase for each of the three agents, monitored by the decrease in cyclooxygenase activity, was consistent with the model proposed by Rome and Lands (Rome, L.H., and Lands, W.E.M. (1975) Proc. Natl. Acad. Sci. U.S.A. 72, 4863-4865): a rapid and reversible initial binding, followed by a first-order decay of the synthase-inhibitor complex. A relatively stable form of the cyclooxygenase, which had 4-10% of the initial activity, was the eventual product of this decay process. The dissociation constants evaluated for the initial binding were 1.7 +/- 1.5 microM for indomethacin, 0.2 +/- 0.1 microM for flurbiprofen, and 0.08 +/- 0.06 microM for meclofenamic acid. The values of the first order rate constants for the subsequent decay process were 14.9 +/- 11.3 min-1 for indomethacin, 3.4 +/- 0.7 min-1 for meclofenamic acid, and 16.6 +/- 6.2 min-1 for flurbiprofen. In repeated titrations of the cyclooxygenase with the three agents, 1.3 +/- 0.3 mol of indomethacin, 1.2 +/- 0.1 mol of meclofenamic acid, and 1.2 +/- 0.1 mol of S-(+)-flurbiprofen/mol of synthase dimer were found to result in maximal inhibition of the enzyme. Racemic flurbiprofen required 2.4 +/- 0.3 mol/mol synthase dimer for full effect, and the R-(-)-isomer was not inhibitory. Inhibition of the cyclooxygenase activity by these agents thus appears to result from a stereospecific binding to only one of the subunits of the synthase. Intact indomethacin could be recovered quantitatively after prolonged incubation (in stoichiometric quantities) with the synthase had resulted in maximal inhibition of the cyclooxygenase activity. The time-dependent effect of indomethacin on the cyclooxygenase is thus likely to involve a conformational change in the synthase rather than a covalent interaction.

High-affinity stereospecific binding of cyclodiene insecticides and γ-hexachlorocyclohexane to γ-aminobutyric acid receptors of rat brain

The γ-aminobutyric acid (GABA)/benzodiazepine/picrotoxin receptor complex of rat brain was identified by the specific binding of three radiolabeled ligands in the same membrane preparation: [ 3H]flunitrazepam ([ 3H]Flu) for the benzodiazepine receptor site, [ 3H]muscimol for the GABA receptor site, and t-[ 35S]butylbicyclophosphorothionate ([ 35S]TBPS) for the picrotoxinin site. Seven cyclodiene insecticides, γ-hexachlorocyclohexane (γ-BHC), β-BHC, and hexachlorobenzene had no significant effect on the specific binding of [ 3H]Flu or [ 3H]muscimol. But the cyclodienes and γ-BHC were very potent in displacing [ 35S]TBPS binding with IC 50 values ranging from 30 n M for endrin and endosulfan I to 500 n M for aldrin. Inhibition of [ 35S]TBPS binding by endrin was competitive. Binding was stereospecific since the more toxic stereoisomer of three pairs of compounds was more potent with the following IC 50's (in n M): endrin, 30; dieldrin, 100; endosulfan I, 30; endosulfan II, 60; γ-BHC, 150; β-BHC, > 10,000. Also the more toxic dieldrin epoxide and heptachlor epoxide had IC 50's of 100 and 70n M, respectively, compared to 500 and 400 n M for the less toxic aldrin and heptachlor, respectively. The results suggest that the ionic channel of the GABA receptor in mammalian brain is a primary target for the toxic action of γ-BHC and cyclodienes.

ChemInform Abstract: 3-(1-INDOLINYL)BENZYLAMINES: A NEW CLASS OF ANALGESIC AGENTS

An extensive series of 3-(1-indolinyl)benzylamines and related compounds was synthesized and tested for analgesic activity. After a detailed study of structure-activity relationships, 3-(1-indolinyl)benzylamine (2b) was selected for further investigation as the most interesting member of this novel class of compounds. It was active in both the phenylquinone writhing and tail-flick assays for analgesic activity. No motor deficits were observed in the rotorod test, and 2b was found to be free of any other effects on the central nervous system. The compound did not bind to opiate receptors, since it was inactive in inhibiting the stereospecific binding of [3H]naloxone in rat brain homogenates. Thus, 3-(1-indolinyl)benzylamine represents a novel analgesic with an unusual chemical structure and biological profile.

Muscarinic cholinergic receptors in pancreatic acinar carcinoma of rat.

The active enantiomer of tritiated quinuclidinyl benzilate (3H(-)QNB) was used as a ligand to evaluate the muscarinic receptors. The 3H(-)QNB binding characteristics of muscarinic cholinergic receptors obtained from normal and neoplastic tissues were studied to determine changes in receptor properties during neoplastic transformation. Saturable and stereospecific binding sites for 3H(-)QNB are present in homogenates of rat pancreatic adenocarcinoma. The proportions of high- and low-affinity agonist binding sites are similar for neoplastic and normal tissues. The density of muscarinic receptors is higher in neoplastic (200 femtomoles/mg protein) than in normal pancreatic homogenates (80 femtomoles/mg protein). The muscarinic binding sites of the neoplastic and fetal pancreas show similar KD values which are higher than those observed for normal pancreas.

Formula omitted]: The radioligand of choice for pett studies of the dopamine receptor in human brain

N-methylspiroperidol, the amide N-methyl analogue of the neuroleptic spiroperidol, was radiolabeled with fluorine-18, and its distribution in the baboon brain was studied using positron emission transaxial tomography. Stereospecific binding was demonstrated in the striatum (but not in the cerebellum) by pretreatment with (-)- or (+)-butaclamol. The kinetic distribution was similar to that of [ 18F]spiroperidol, but the absolute striatal uptake (in percent of administered dose) was at least two-fold higher. Analysis of baboon blood at 10 min after injection indicated that less than half of the radioactivity in the plasma was due to unchanged radioligand. Analysis of the metabolic stability of [ 18 F]-N- methylspiroperidol in rat brain for 4 hr indicated that, like [ 18F]spiroperidol, it is very stable to metabolic transformation in the rat central nervous system. Striatal uptake and retention in the rat was five-fold higher for [ 18 F]-N- methylspiroperidol than for [ 18F]spiroperidol. These results suggest that [ 18 F]-N- methylspiroperidol is an ideal choice for studies of the dopamine receptor in humans.

Altered binding properties of β-adrenergic receptors and lack of coupling to adenylate cyclase in P815 mastocytoma cells

P185, a murine mastocytoma cell line, possesses β-adrenergic binding sites as assessed by using [ 3H]dihydroalprenolol (antagonist) and [ 3H]hydroxybenzylisoproterenol (agonist). The number of binding sites per cell was 29 000 for the agonist and 75 000 for the antagonist, as determined by direct binding assays and inhibition experiments on intact cells. On membrane preparations from the same cells, binding of alprenolol was only displaceable by antagonists, while stereospecific binding of hydroxybenzylisoproterenol was only displaceable by agonists. The P815 membranes also possessed an adenylate cyclase stimulated by Gpp(NH)p and NaF but not by l-isoproterenol. The intracellular cAMP level of intact cells was not modulated by l-isoproterenol or by l-epinephrine, but was increased by forskolin. These results suggest that the β-adrenergic receptor of P815 mastocytoma cells is non-functional. This may explain the failure of agonists to stimulate adenylate cyclase activity in these cells.