The Biological Consequences of Replacing d‐Ala in Biphalin with Amphiphilic α‐Alkylserines

Biphalin, a synthetic opioid peptide with a broad affinity for all opioid receptors (δ, μ, and κ) and high antinociceptive activity, has been under extensive study as a potential analgesic drug. This study presents the synthesis and biological properties of four new analogues of biphalin containing amphiphilic α‐alkylserines in position 2 and 2′. The incorporation of bulky α,α‐disubstituted amino acids in the peptide chain using standard peptide chemistry is often unsuccessful. We synthesized depsipeptides, and then, the desired peptides were obtained by internal O,N‐migration of the acyl residue from the hydroxyl to the amino group under mild basic conditions. The potency and selectivity of the new analogues were evaluated by a competitive receptor‐binding assay in the rat brain using [³H]DAMGO (a μ ligand) and [³H]DELT (a δ ligand). Their binding affinity is strongly dependent on the chirality of α‐alkylserine, as analogues containing (R)‐α‐alkylserines displayed higher μ receptor affinity and selectivity than those incorporating the (S)‐isomers.     

Dehydro-enkephalins. III. Synthesis and biological activity of [ΔAla2, Leu5]-enkephalin

[ΔAla², Leu⁵]-enkephalin has been prepared and shown to be more active than the parent saturated enkephalin in a binding assay using rat brain membranes and [³H] dihydromorphine as a tracer. In a comparison of potencies against [³H] dihydromorphine and [³H]-[d -Ala², d -Leu⁵]-enkephalin as tracers, [ΔAla², Leu⁵]-enkephalin showed preference for μ opiate receptors, possibly due to the hydrophobicity of the ΔAla² residue. A synthetic tetrapeptide enkephalin [ΔAla²]-desLeu⁵-enkephalin had weak activity and high selectivity for the μ receptors. O-Acylation of a serine residue in the peptide was achieved by coupling between the peptide and a carboxylic acid using DCC and a catalytic amount of 4-dimethylaminopyridine.     

Further biochemical characterization of [3H]dihydroazapetine binding to α-adrenoceptor — related proteins from the rat vas deferens*

The binding of [³H]dihydroazapetine, a reversible blocker of the α-adrenoceptor, has been characterized in a membrane preparation from the rat vas deferens. Binding is rapid, reversible and shows evidence of saturation. Postganglionic sympathetic denervation of the tissue does not decrease binding to the subcellular fraction. Drugs known to act on the α-adrenoceptor are specific inhibitors of [³H]dihydroazapetine binding while those acting on other receptors are without effect. Scatchard plot analysis suggests that two specific binding components are present, one of which has a similar dissociation constant for [³H]dihydro-azapetine to that determined pharmacologically. [³H]Dihydroazapetine binding is lowest in the physiological pH range and tends to decrease with increasing temperature in the range of 25 to 60°. Calcium, magnesium, sodium and potassium inhibit binding of [³H]dihydroazapetine with the divalent ions being approximately 100 times more potent. Studies with group selective reagents indicate that a disulphide bridge, and sulphydryl and carbonyl groups, are at or near the binding site and are necessary for binding. The site is protein possibly with involvement of phospholipids. Specific binding of [³H]dihydroazapetine is inhibited by low concentrations of local anaesthetics suggesting that the binding site of the antagonist may be related to the ‘calcium mobilization’ site of the α-adrenoceptor.     

Synthesis and binding properties of specific photoaffinity ligands for μ and δ opioid receptor subtypes

We report the synthesis and binding properties of specific photoaffinity ligands for μ and δ opioid receptor subtypes. These ligands are derived from DAGO: Tyr-D-Ala-Gly-NMePhe-Gly-ol, a μ selective probe and DTLET: Tyr-D-Thr-Gly-Phe-Leu-Thr, a δ selective probe by modifying the Phe 4 residue. These modifications are: i) a nitro group on the para position of Phe ring as Phe(4 NO₂) or Nip, ii) an azido group as Phe(4 N₃) or AZ. Pharmacological responses on mouse vas deferens (δ sites) and guinea pig ileum (μ sites), as well as competition experiments with [³H] DAGO and [³H] DTLET on crude rat brain membranes have been performed. The nitro group on the phenyl ring of the Phe residue preserves the affinity and selectivity of each probe: NipDAGO for the μ sites, NipDTLET for the δ ones. However the nitro probes do not appear to be photo-activable by u.v. irradiation. Likewise, azidation of the phenyl ring of the Phe residue does not change the receptor selectivity of each probe, but AZDAGO has less affinity than its parent molecule DAGO, while AZDTLET has more affinity than DTLET. These compounds are photoactivable and provide an efficient tool to characterize and isolate the different receptor subtypes, especially the δ site.     

Opioid receptor selectivity of peptide models of β-endorphin*

Two peptides, designed to contain structural models of the proposed hydrophilic linker domain (residues 6-12) and amphiphilic α-helical domain (residues 13-29) in β-endorphin, have been tested for their abilities to mimic the opioid receptor selectivity profile of the natural hormone. In competitive binding assays employing guinea-pig brain membranes, both peptides displayed a much higher affinity for μ- and δ-opioid receptors than for κ opioid receptors. Relative to β-endorphin, the peptide models were 2-3 times more potent in the μ and κ receptor binding assays, and about equipotent in the δ receptor binding assay. In guinea-pig ileum assays, one peptide was equipotent to β-endorphin and the other was twice as potent. Like β-endorphin, their actions on this tissue were highly sensitive to naloxone antagonism, indicating that they were mediated by μ receptors and not κ receptors. In view of the design of the two peptide models, and their minimal homology to the natural hormone, these results provide additional evidence in support to our proposal for the functional conformation of β-endorphin.     

Potential Irreversible Ligands for Opioid Receptors. Cinnamoyl Derivatives of β-Naltrexamine

Cinnamoyl derivatives of β-naltrexamine (β-NTA) have been prepared and evaluated as potential irreversible opioid antagonists. In receptor binding assays, isolated tissue preparations and mouse antinociception assays the p-methylcinnamoyl derivative BU42 was similar to the standard opioid ligand β-funaltrexamine (β-FNA). The main features were reversible κ agonism and irreversible μ antagonism. Surprisingly the p-chlorocinnamoyl derivative BU59 showed only modest competitive antagonist activity in-vivo despite appearing to bind irreversibly to μ receptors in the guinea-pig ileum (GPI) preparation. BU60, the dihydrocinnamoyl analogue of BU59, like BU59 displayed reversible κ agonism in GPI but in mouse antinociception assays its agonism was mediated by κ and β receptors rather than κ. The surprising changes of profile attributable to substitution in the aromatic ring of the cinnamoylamido group in this small series suggests that a larger range of substituted cinnamoylamido derivatives should be studied to further elucidate the effects of Michael acceptor activity and other factors.     

α-Flupenthixyl chloride: Binding profile to dopaminergic,serotonergic, adrenergic,and cholinergic neuro-receptors

The binding of the putative affinity ligand α-flupenthixyl chloride (FPT-Cl), a derivative of the neuroleptic α-flupenthixol (FPT), to rat striatal dopamine, α₁-adrenergic, and muscarinic cholinergic recognition sites, as well as to serotonin (5-HT) receptors and to the presynaptic 5-HT transport complex, was examined in vitro. FPT and FPT-Cl were relatively potent at inhibiting the binding of [³H]-flupenthixol and [³H]-spiroperidol in striatal tissue preparations as well as [³H]/spiroperidol and [³H]-WB-4101 in cortex with K_i values in the 10–20 nM range. With the exception of the muscarinic cholinergic sites (³H-quinuclidinyl-benzilate [³H-QNB]), where FPT-Cl was more potent than FPT, the K_i values for FPT-Cl were, in general, slightly greater than those for FPT for other neuro-receptors tested. FPT-Cl was found to be essentially inactive at 5-HT binding sites labeled with [³H]-LSD and at the 5-HT transport complex labeled with [³H]-imipramine, with lC₅₀ values for both exceeding 5μM. It is concluded that the transformation of FPT to FPT-Cl is not severely detrimental to its dopamine receptor binding characteristics, suggesting that in the presence of appropriate antagonists for masking other receptors, FPT-Cl may represent a useful affinity ligand to label dopamine receptors.     

Two distinct α1-adrenergic receptor sites in rat liver: Differential binding of (−)-[3H]norepinephrine, [3H]prazosin and [3H]dihydroergocryptine: Effects of guanine nucleotides and proteolysis; implications for a two-site model of α-receptor regulation

(?)-[³H]Norepinephrine, [³H]prazosin and [³H]dihydroergocryptine bind to rat liver plasma membranes in a manner indicating a selective interaction with α₁-adrenergic receptors. All three ligands display monophasic saturation with a single component on Scatchard analysis. The binding capacities of (?)-[³H]norepinephrine, [³H]prazosin and [³H]dihydroergocryptine are 340 ± 70 fmol/mg of protein 760 ± 40 fmol/mg of protein and 1200 ± 300 fmol/mg of protein, respectively. Differential drug potencies in competing for (?)-[³H]norepinephrine and [³H]prazosin binding sites suggest that these two ligands label two distinct binding sites at the α₁-adrenergic receptor, while [³H]dihydroergocryptine labels both sites. Guanine nucleotides lower the apparent affinity of (?)-[³H]norepinephrine for its binding site, without affecting the number of sites or the binding of [³H]prazosin and [³H]dihydroergocryptine. Incubation of membranes with α-chymotrypsin slightly reduces the binding of [³H]prazosin and [³H]dihydroergocryptine, but causes a 2-fold increase in (?)-[³H]norepinephrine binding. Both the number of (?)-[³H]norepinephrine binding sites and the affinity are increased. Following proteolysis, (?)-[³H]norepinephrine binding still occurs to a single class of sites, but is no longer affected by guanine nucleotides. The effect of α-chymotrypsin is abolished by pretreatment of membranes with the irreversible α-adrenergic antagonist phenoxybenzamine. We propose that in rat liver, (?)-[³H]norepinephrine labels the physiologically active form of the α-adrenergic receptor while [³H]prazosin binds to a precursor form of the active receptor, and/or to an α₁-adrenergic binding site not coupled to an effector system.     

EFFECT OF THYROID STATUS ON β-ADRENORECEPTORS AND MUSCARINIC RECEPTORS IN THE RAT LUNG

• 1 The effects of thyroid status on the specific binding of the muscarinic ligand (–)-[³H] quinuclidinyl benzilate (QNB) and of the β-adrenoreceptor ligand (–)-[³H] dihydroalprenolol (DHA) in the adult rat lung were investigated.
• 2 The specific binding of (–)-[³H] quinuclidinyl benzilate (QNB) to lung membranes was saturable and the equilibrium dissociation constant (K_D) determined from Scatchard analysis was 54 pM. Kinetic analysis of the binding of [³H] QNB yielded a K_D of 42 pM. [³H] QNB binding was inhibited by muscarinic agonists and antagonists, the order of their potency was l-hyoscyamine>atropine>scopolamine>oxotremorine>carbachol. These data were consistent with [³H] QNB binding to the muscarinic receptor.
• 3 Adult male rats treated for 2 weeks with the antithyroid agent 3-amino-1,2,4-triazole (ATZ) showed a 52% and 80% reduction in the serum concentration of triiodothyronine (T₃) and thyroxine (T₄) respectively. These hypothyroid rats also had a 39% decrease in the concentration of lung β-adrenoreceptors and a 37% decrease in the concentration of lung muscarinic receptors as compared to euthyroid controls. Concurrent treatment of rats with ATZ and T₄ for 2 weeks resulted in a reduction of 15% and 20% in the concentration of lung β-adrenoreceptors and muscarinic receptors respectively. The K_D values for [³H] DHA and [³H] QNB binding did not change with the ATZ or ATZ + T₄ treated groups.
• 4 Administration of T₄ (500 μg/kg/day) to male rats for 12 days did not result in any significant change in the concentration of either β-adrenoreceptors or muscarinic receptors compared to euthyroid controls. No change in the K_K values for [³H] DHA or [³H] QNB binding were detected.
• 5 The results show that hypothyroid rats have a reduced lung concentration of both β-adrenoreceptors and muscarinic receptors whereas in hyperthyroid rats these receptors do not significantly change from euthyroid controls.

     

Methadone is a Non‐Competitive Antagonist at the α4β2 and α3* Nicotinic Acetylcholine Receptors and an Agonist at the α7 Nicotinic Acetylcholine Receptor

Nicotine–methadone interactions have been studied in human beings and in various experimental settings regarding addiction, reward and pain. Most methadone maintenance treatment patients are smokers, and methadone administration has been shown to increase cigarette smoking. Previous in vitro studies have shown that methadone is a non‐competitive antagonist at rat α3β4 nicotinic acetylcholine receptors (nAChR) and an agonist at human α7 nAChRs. In this study, we used cell lines expressing human α4β2, α7 and α3* nAChRs to compare the interactions of methadone at the various human nAChRs under the same experimental conditions. A [³H]epibatidine displacement assay was used to determine whether methadone binds to the nicotinic receptors, and ⁸⁶Rb⁺ efflux and changes in intracellular calcium [Ca²⁺]_i were used to assess changes in the functional activity of the receptors. Methadone displaced [³H]epibatidine from nicotinic agonist‐binding sites in SH‐EP1‐hα7 and SH‐SY5Y cells, but not in SH‐EP1‐hα4β2 cells. The K_i values for methadone were 6.3 μM in SH‐EP1‐hα7 cells and 19.4 μM and 1008 μM in SH‐SY5Y cells. Methadone increased [Ca²⁺]_i in all cell lines in a concentration‐dependent manner, and in SH‐EP1‐hα7 cells, the effect was more pronounced than the effect of nicotine treatment. In SH‐EP1‐hα4β2 cells, the effect of methadone was negligible compared to that of nicotine. Methadone pre‐treatment abolished the nicotine‐induced response in [Ca²⁺]_i in all cell lines expressing nAChRs. In SH‐EP1‐hα4β2 and SH‐SY5Y cells, methadone had no effect on the ⁸⁶Rb⁺ efflux, but it antagonized the nicotine‐induced ⁸⁶Rb⁺ ion efflux in a non‐competitive manner. These results suggest that methadone is an agonist at human α7 nAChRs and a non‐competitive antagonist at human α4β2 and α3* nAChRs. This study adds further support to the previous findings that opioids interact with nAChRs, which may underlie their frequent co‐administration in human beings and might be of interest to the field of drug discovery.     

3H]Dihydroergocryptine binding to alpha-adrenergic receptors of human platelets. A reassessment using the selective radioligands [3H]prazosin, [3H]yohimbine, and [3H]rauwolscine

Which subtype(s) of the alpha-adrenergic receptor occurs on human platelets? Studies of platelet responsiveness to adrenergic compounds and indirect radioligand binding studies addressing this question have yielded contradictory conclusions. These binding studies employed the ligand [³H]dihydroergocryptine ([³H]DHE), an alpha-adrenergic antagonist that does not select between alpha₁- and alpha₂-adrenergic receptors and that also binds to other receptor types in some tissues. To determine the subtype of the platelet alpha-adrenergic receptor, we have examined the binding to intact human platelets of [³H]prazosin (alpha₁-selective), [³H]yohimbine (alpha₂-selective), and [³H]rauwolscine (alpha₂-selective), and we have compared the binding of these selective radioligands with that of [³H]DHE. [³H]Yohimbine and [³H]rauwolscine both bound with high affinity (K_D = 2.7 and 4.6 nM, respectively) to an equal number and a single class (Hill coefficient ～1.0) of sites (～300 per platelet), but [³H]yohimbine yielded lower nonspecific binding than did [³H]rauwolscine. In paired experiments, [³H]DHE bound to 1.5 times as many (phentolamine-displaceable) sites as did [³H]yohimibine or [³H]rauwolscine. Unlabeled vohimbine and epinephrine competed for fewer [³H]DHE binding sites than did phentolamine. Thus, in addition to binding to the alpha₂-adrenergic receptors identified by [³H]yohimbine and [³H]rauwolscine, [³H]DHE seems to bind to other sites on human platelets. The nature of these sites is not clear. We found that [³H]prazosin did not identify alpha₁-adrenergic receptors on platelets, and that phenoxybenzamine only inhibited [³H]yohimbine and [³H]DHE binding at higher concentrations than usually observed for alpha₁-adrenergic receptors. We conclude that (1) all alpha-adrenergic sites on human platelets are of the alpha₂ subtype, (2) [³H]DHE may bind to additional, as yet ill-defined, sites in addition to those sites identified by [³H]yohimbine and [³H]rauwolscine, and (3) [³H]yohimbine is the preferred antagonist radioligand for studying the alpha₂-adrenergic receptors on human platelets.     

Proton NMR Conformational Analysis of Cyclic β-Casomorphin Analogues of the Type Tyr-cyclo[-Nω-D-Orn-Xaa-Yaa-Gly-]

A conformational study of the cyclic β-casomorphin-5 analogues H-Tyr-cyclo[-D-Orn-2-Nal-Pro-Gly-] ( 1 ) (μ-selective agonist; 2-Nal = 2-naphthylalanine), H-Tyr-cyclo[-D-Orn-2-Nal-D-Pro-Gly-] ( 2 ) (mixed μ agonist/δ antagonist) and H-Tyr-cyclo[-D-Orn-Phe-D-Pro-Gly-] ( 3 ) (highly potent μ and δ agonist) has been carried out using ¹H NMR spectroscopy. A complete assignment of the proton resonances of the three pentapeptides has been achieved. Compound 1 was shown to exist in two conformations, a major one (90%) characterized by a cis amide bond between 2-Nal³ and Pro⁴, and a minor one (10%) showing cis amide bonds both between D-Orn² and 2-Nal³ and between 2-Nal³ and Pro⁴. Peptides 2 and 3 each showed only one conformer with all-trans peptide bonds in both cases. Temperature dependence studies of the amide proton chemical shifts indicated the existence of several intramolecular hydrogen bonds in the case of compounds 2 and 3 but not in the case of peptide 1. The backbone conformations of 2 and 3 were found to be similar, both being characterized by two consecutive γ turns around the D-Pro⁴ and D-Orn² residues, respectively, and by a D-Orn²-CO←HN^δ-D-Orn² hydrogen bond. Altogether, the overall backbone conformation and the preferred side chain conformation were found to be roughly similar for the three title peptides. For all three compounds a close proximity between the aromatic moiety of the 3-position residue (2-Nal or Phe) and the D(or L)-Pro⁴ residue was established on the basis of ROESY experiments. The examination of low energy conformations obtained in molecular modelling studies by taking into account the various experimentally found NMR parameters (NOEs, vicinal H,H coupling constants, torsion angles, H-bonds) led to proposals of the solution conformation for each peptide. These conformations are in close agreement with a pharmacophore model for μ opioid receptor binding compounds.     

Effects of venlafaxine given repeatedly on α1‐adrenergic,dopaminergic and serotonergic receptors in rat brain

Venlafaxine (VEN), a representative of a new class of antidepressants (serotonin and noradrenaline reuptake inhibitors, SNRI), administered repeatedly affects—as was demonstrated by us previously—the behavioural responsiveness of α₁‐adrenergic, dopaminergic (D₂ and D₃) and serotonergic systems to their agonists. In the present study we aimed to find out whether parallel changes in the binding to the respective receptors also occurred. The experiment was carried out on male Wistar rats. VEN was administered in a dose of 10 mg/kg once or repeatedly (14 days, twice daily). The obtained results showed that VEN did not change the binding (B_max and K_D) of α₁‐adrenergic receptors to [³H]‐prazosin in the cerebral cortex, having increased only its displacement by phenylephrine. The binding (B_max and K_D) to D₁ and D₂ receptors in the limbic forebrain and the striatum was not affected by repeated venlafaxine when [³H];‐SCH 23390 and [³H]‐spiperone, respectively, were used as ligands. When [³H]‐quinpirole was used as a ligand, the binding was enhanced in the striatum, the nucleus accumbens (shell and core) and islands of Calleja. VEN also increased the binding of [³H]‐7‐OH‐DPAT to D₃ receptors in islands of Calleja and the nucleus accumbens (shell). In the serotonergic system, a decrease in the density of 5‐HT_1A receptors was observed in the hippocampus, whereas no changes occurred in the binding of 5‐HT₂ receptors in the cortex. Thus VEN given repeatedly enhanced the binding (of the ligands that are agonists) to dopamine D₂ and D₃ receptors. Weaker effects were observed in the α₁‐adrenergic and the serotonergic systems. Copyright © 1999 John Wiley & Sons, Ltd.     

Syntheses,opioid binding affinities,and potencies of dynorphin A analogues substituted in positions 1, 6, 7, 8 and 10

Structural, stereochemical, stereoelectronic and conformational requirements for biological activity of dynorphin A_1–11-NH₂ analogues at opioid receptors were explored by substitution of Tyr¹, Arg⁶, Arg⁷, Ile⁸ and Pro¹⁰ with other amino acid residues. Interestingly, substitution of Tyr¹ with N^α-Ac-Tyr^l, D-Tyr¹, Phe¹ or p-BrPhe¹ led to analogues that were quite potent at κ opioid receptors, and additional substitution of Ile⁸with D-Ala⁸ and/or Pro¹⁰ with D-Pro¹⁰ retained high potency in brain binding assay: [N^α-Ac-Tyr¹]- (1), [D-Tyr¹]- (2) [Phe¹]- (3), [Phe¹. D-Ala⁸]- (5), [p-BrPhe¹, D-Ala^s]- (6), [Phe¹, D-Pro¹⁰]- (7) and [Phe¹, D-Ala⁸, D-Pro¹⁰]-Dyn A_1–11-NH₂ (8) had IC₅₀(nM) binding affinities of 13.2, 18.6, 1.64, 1.26, 1.84, 2.44 and 1.62 nM, respectively. The D-Phe¹ analogue 4, however, was only weakly active (610 nM). All of the analogues except 4 were modestly selective for κ vs. μ guinea pig brain opioid receptor (11- to 88–fold) and quite selective for κ vs. δ receptors (65–576). However, all of the analogues appeared to have very low or essentially no activity in the guinea pig ileum and mouse vas deference functional bioassays, and one analogue, 5, appeared to have weak antagonist activities. On the other hand, if constrained amino acids such as β-methylphenylalanine or 1,2,3,4-tetrahydroisoquinoline carboxylic acid, and hydroxyproline were placed in the 1 position, inactive analogues or analogues with greatly reduced potency and biological activity were obtained (compounds 12–14). It had previously been suggested that the Arg⁶ and Arg⁷ residues were critical for biological activity. However, when we replace either one of these residues, [Nle⁶]Dyn A_1–11 (9) and [Nle⁷]Dyn A_1–11-NH₂ (10) were both highly potent binders in κ receptor binding studies (IC₅₀= 0.95 and 0.43 nM, respectively), and interestingly also were potent in μ and δ binding studies. Furthermore, both of the analogues were modestly potent in the GPI and MVD assays (94, 65 nM; 31, 81 nM, respectively). These results demonstrate that basic residues at positions 6 and 7 in dynorphin are not very important for binding to κ opioid receptors. Finally, many of the compounds reported here showed high selectivity for central vs. peripheral κ opioid receptors, with compound 4 being the most selective (63 000-fold).     

[3H]5′-N-Ethylcarboxamidoadenosine selectively labels the low affinity adenosine binding protein,adenotin, on intact chinese hamster ovary cells

The ability of [³H]5′-N-ethylcarboxamidoadenosine (NECA) to specifically bind recognition sites on intact Chinese hamster ovary (CHO) cells was examined in the present study. Saturation experiments indicated that [³H]NECA bound with moderate affinity (Kd = 400 nM) and large capacity (apparent Bmax = 3.2 pmol/10⁵ cells) to intact CHO cells. No specific binding to these cells was observed with the A₁-selective agonist 20 nM [³H]cyclohexyladenosine or with the A₂-selective agonist 20 nM [³H]CGS 21680. Competition studies revealed that close structural analogs of NECA and the xanthine phosphodiesterase inhibitor 3-isobutyl-1-methylxanthine (IBMX) inhibited 20 nM [³H]NECA binding with moderate affinity (IC₅₀s 0.5–15 μM). Adenosine also showed weak activity (IC₅₀ = 100 μM) for inhibiting [³H]NECA binding. However, a wide variety of prototypic adenosine receptor agonists and antagonists did not significantly interact with these [³H]NECA recognition sites on CHO cells. [³H]NECA binding to CHO cell membranes was not sensitive to guanine nucleotides and NECA did not stimulate cAMP formation. These results are consistent with the previously demonstrated ability of [³H]NECA to bind low affinity adenosine binding proteins (adenotin proteins), as well as, adenosine receptors in a variety of mammalian tissues. The present results further indicate that [³H]NECA selectively labels in adenotin-like recognition site on intact CHO cells in the absence of detectable binding to high affinity adenosine receptors. © 1993 Wiley-Liss, Inc.     

Further characterization of [3H]ifenprodil binding in rat brain

The present study was undertaken to characterize [³H]ifenprodil binding in rat brain. [³H]ifenprodil showed saturable, high-affinity binding at 4°C. Specific binding, defined with 10 μM ifenprodil as a competitor, was inhibited biphasically by the s receptor ligands, GBR 12909, 1,3-di-o-tolylguanidine (DTG), and (+)-3-(3-hydroxyphenyl)-N-propylpiperidine ((+)-3-PPP). At 4°C, 3 μM GBR 12909, which inhibited about 50% of specific binding of [³H]ifenprodil, was used to mask σ receptors. Under these conditions, specific binding of [³H]ifenprodil was inhibited potently by ifenprodil, SL 82.0715, poly(l-arginine), poly(l-lysine), neomycin, ruthenium red, spermine, arcaine and spermidine. In the presence of 3 μM GBR 12909, Zn²⁺ and Mg²⁺ partially inhibited specific binding of [³H]ifenprodil at 4°C. In contrast, in the absence of GBR 12909, at 37°C specific binding of [³H]ifenprodil was partially inhibited by Zn²⁺, but not by Mg²⁺. The anatomical distribution of [³H]ifenprodil binding at 4°C (GBR 12909 included) in rat brain closely paralleled that of [³H]MK-801 (dizocilpine) binding (r = 0.971, P < 0.005). Without GBR 12909, specific [³H]ifenprodil binding at 37°C was inhibited potently by σ ligands. In the presence of 3 μM GBR 12909, [³H]ifenprodil binding at 4°C was highest in synaptosomal and myelin fractions; however, without GBR 12909, [³H]ifenprodil binding at 37°C was highest in microsomal and myelin fractions, consistent with the subcellular distribution of σ receptors. The results suggest that, in the presence of 3 μM GBR 12909, at 4°C, [³H]ifenprodil binds to sites that are sensitive to polyamines and related compounds; and that without GBR 12909, at 37°C, [³H]ifenprodil interacts with σ receptors in rat brain.     

Melatonin MT1 and MT2 receptors display different molecular pharmacologies only in the G-protein coupled state

BACKGROUND AND PURPOSE

Melatonin receptors have been extensively characterized regarding their affinity and pharmacology, mostly using 2-[¹²⁵I]-melatonin as a radioligand. Although [³H]-melatonin has the advantage of corresponding to the endogenous ligand of the receptor, its binding has not been well described.

EXPERIMENTAL APPROACH

We characterized [³H]-melatonin binding to the hMT₁ and hMT₂ receptors expressed in a range of cell lines and obtained new insights into the molecular pharmacology of melatonin receptors.

KEY RESULTS

The binding of [³H]-melatonin to the hMT₁ and hMT₂ receptors displayed two sites on the saturation curves. These two binding sites were observed on cell membranes expressing recombinant receptors from various species as well as on whole cells. Furthermore, our GTPγS/NaCl results suggest that these sites on the saturation curves correspond to the G-protein coupled and uncoupled states of the receptors, whose pharmacology was extensively characterized.

CONCLUSIONS AND IMPLICATIONS

hMT₁ and hMT₂ receptors spontaneously exist in two states when expressed in cell lines; these states can be probed by [³H]-melatonin binding. Overall, our results suggest that physiological regulation of the melatonin receptors may result from complex and subtle mechanisms, a small difference in affinity between the active and inactive states of the receptor, and spontaneous coupling to G-proteins.     

In-vivo Binding of (+)-[3H]PN 200–110 to Peripheral Tissues and Brain of Spontaneously Hypertensive Rats: Effect of Lacidipine

Abstract— The time-course of dihydropyridine receptor occupancy by lacidipine and its relationship with pharmacological activity has been studied in spontaneously hypertensive rat (SHR), as measured by the inhibition of specific (+)-[³H]PN 200–110 binding in-vivo. After oral administration of doses active in reducing blood pressure, lacidipine did not show tissue target differences in respect to binding sites labelled by (+)-[³H]PN 200–110 in cerebral cortex, heart, ileum, bladder and thoracic aorta. The relative occupancy of receptors in heart 60 min after oral administration of 1 mg kg^?1 lacidipine was 75%. After 12 h, when lacidipine was still effective in reducing blood pressure in SHR, a low (15%) but detectable proportion of receptors was still occupied by the drug. The percentage decrease of blood pressure was linear with the percentage of receptor occupancy obtained by different doses of lacidipine; that is, there was a close correspondence between ED25 for decrease in blood pressure (0·33 mg kg^?1) and ED25 for inhibition of (+)-[³H]PN 200–110 specific binding in the heart (0·36 mg kg^?1). The long-lasting effect of lacidipine on blood pressure might be explained by its selective interaction with dihydropyridine binding sites labelled in-vivo by (+)-[³H]PN 200–110.     

Labeling in vivo of sigma receptors in mouse brain with [3H]-(+)-SKF 10,047: Effects of phencyclidine, (+)- and (−)-N-allylnormetazocine,and other drugs

The sigma receptor, so named because of the distinct pharmacolgical profile produced by its prototypic agonist SKF 10,047 (N-allynormetazocine), is believed to mediate mania and psychotomimetic effects in man. While this sigma receptor has received extensive biochemical and pharmacological characterization in vitro, little information is available on the nature of the sigma site in vivo. In the present study, we examined the binding of [³H]-(+)-SKF 10,047 to sigma receptors in mouse brain in vivo and to sigma receptors in mouse and guinea pig brain in vitro and determined the relative potencies of various drugs in displacing this ligand. Mice were injected with 5 μCi of [³H]-(+)-SKF 10,047 into the tail vein. After various time intervals, the mice were decapitated; their brains were rapidly removed, weighed, and homogenized in 50 mM Tris-HCI buffer, pH 7.7; and total and particulate bound radioactivity were determined. Specifically bound [³H]-(+)-SKF 10,047 in the particulate fraction was defined as the difference in total radioactivity in the particulate fraction obtained from vehicle-injected mice minus the radioactivity in the particulate fraction from haloperidol (2 mg/kg i.p.)-injected mice. Specifically bound [³H]-(+)-SKF 10,047 in the particulate fraction reached peak levels of 30 min after i.v. injection and constituted 90–95% of the total particulate radioactivity. Labeling of the sigma sites could be blocked in vivo by injecting mice i.p. with the drug 30 min before the i.v. injection of the ³H-ligand. Under these conditions, (+)-SKF 10,047, (+)-3-PPP, cyclazocine, pentazocine, and haloperidol were found to be the most potent compounds in reducing specific [³H]-(+)-SKF 10,047 binding. Neuroleptics such as thioridazine and chlorpromazine had good potency, while clozapine, spiperone, and sulpiride were very weak inhibitors in vivo. Specific [³H]-(+)-SKF 10,047 binding was also reduced in vivo by imipramine, dl-propranolol, bupropion, rimcazole, and phenoxybenzamine, but was not reduced by apomorphine and naloxone at doses of 50 mg/kg i.p. Phencyclidine, m-NH₂-PCP and (?)-3-PPP were only weak inhibitors of (+)-SKF 10,047 binding in vivo. The relative potencies of these agents obtained in vivo correspond well with their relative affinities obtained in vitro in mouse and guinea pig brain for displacing [³H]-(+)-SKF 10,047 but not with their relative affinities for displacing TCP in guinea pig brain. Comparison of the dose-response curves for the drug revealed the presence of perhaps two sites labeled by [³H]-(+)-SKF 10,047 in vivo.     

Long-Term Alterations in Benzodiazepine,Muscarinic and α-Amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) Receptor Density Following Continuous Cocaine Administration

Abstract: Animal models of clinical phenomena, such as stimulant-induced psychosis have focused primarily on persisting alterations that develop in brain after chronic stimulant administration. The present study utilized autoradiographic measures to examine changes in the density of benzodiazepine ([³H] flunitrazepam), muscarinic ([³H] quinuclidinyl benzilate), and non-NMDA glutamatergic (³H α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid: AMPA) receptor binding in rats 21 days following two exposures to cocaine administered continuously for 5 days via subcutaneous pellets. A marked, selective increase in [³H] flunitrazepam binding in both the lateral and medial habenula nucleus was observed. Reduced [³H] quinuclidinyl benzilate binding was observed in various brain areas, including large decreases in the anterior cingulate cortex and ventral thalamus. A reduction in [³H]AMPA binding was observed in the ventral striatum and was suggested in the nucleus accumbens. [³H] Flunitrazepam binding was also examined 12 hr following a single 5 day cocaine exposure to determine if the long-term habenular changes were evident at acute withdrawal. No alterations in [³H] flunitrazepam binding were observed in the habenula or any other structure analyzed at this time point. The relation of these results to persisting alterations in mesocorticolimbic pathways and previous findings of cocaine-induced degeneration in lateral habenula circuitry is discussed.