Rat Brain Membrane Homogenates Research Articles

Investigation of receptor binding and functional characteristics of hemopressin(1–7)

The orally active, α-hemoglobin derived hemopressin (PVNFKFLSH, Hp(1–9)) and its truncated (PVNFKFL, Hp(1–7) and PVNFKF, Hp(1–6)) and extended ((R)VDPVNFKFLSH, VD-Hp(1–9) and RVD-Hp(1–9)) derivatives have been postulated to be the endogenous peptide ligands of the cannabinoid receptor type 1 (CB1). In an attempt to create a versatile peptidic research tool for the direct study of the CB1 receptor–peptide ligand interactions, Hp(1–7) was radiolabeled and in vitro characterized in rat and CB1 knockout mouse brain membrane homogenates. In saturation and competition radioligand binding studies, [3H]Hp(1–7) labeled membrane receptors with high densities and displayed specific binding to a receptor protein, but seemingly not to the cannabinoid type 1, in comparison the results with the prototypic JWH-018, AM251, rimonabant, Hp(1–9) and RVD-Hp(1–9) (pepcan 12) ligands in both rat brain and CB1 knockout mouse brain homogenates. Furthermore, functional [35S]GTP γS binding studies revealed that Hp(1–7) and Hp(1–9) only weakly activated G-proteins in both brain membrane homogenates. Based on our findings and the latest literature data, we assume that the Hp(1–7) peptide fragment may be an allosteric ligand or indirect regulator of the endocannabinoid system rather than an endogenous ligand of the CB1 receptor.

Further Characterization of Hemopressin Peptide Fragments in the Opioid and Cannabinoid Systems.

Hemopressin, so-called because of its hypotensive effect, belongs to the derivatives of the hemoglobin α-chain. It was isolated from rat brain membrane homogenate by the use of catalytically inactive forms of endopeptidase 24.15 and neurolysin. Hemopressin has antihyperalgesic features that cannot be prevented by the opioid receptor antagonist, naloxone. In the present study, we investigated whether hemopressin (PVNFKFLSH) and its C-terminally truncated fragment hemopressin 1-7 (PVNFKFL) have any influence on opioid-dependent signaling. Peptides have been analyzed using G-protein-stimulating functional and receptor bindings in this experimental setup. These 2 compounds efficiently activated the G-proteins, and naloxone slightly blocked this stimulation. At the same time, they were able to displace radiolabeled [3H]DAMGO, a selective ligand for μ-opioid system, at micromolar concentrations. Displacement caused by the heptapeptide was more modest compared with hemopressin. Experiments performed on cell lines overexpressing μ-opioid receptors verified the opioid activity of both hemopressins. Moreover, the CB1 cannabinoid receptor antagonist, AM251, significantly decreased their G-protein stimulatory effect. Here, we further confirm that hemopressins can modulate CB1 receptors and can have a slight modulatory effect on the opioid system.

Synthesis and biological evaluations of novel endomorphin analogues containing α-hydroxy-β-phenylalanine (AHPBA) displaying mixed μ/δ opioid receptor agonist and δ opioid receptor antagonist activities.

A novel series of endomorphin-1 (EM-1) and endomorphin-2 (EM-2) analogues was synthesized, incorporating chiral α-hydroxy-β-phenylalanine (AHPBA), and/or Dmt(1)-Tic(2) at different positions. Pharmacological activity and metabolic stability of the series was assessed. Consistent with earlier studies of β-amino acid substitution into endomorphins, multiple analogues incorporation AHPBA displayed high affinity for μ and δ opioid receptors (MOR and DOR, respectively) in radioligand competition binding assays, and an increased stability in rat brain membrane homogenates, notably Dmt-Tic-(2R,3S)AHPBA-Phe-NH2 (compound 26). Intracerebroventricular (i.c.v.) administration of 26 produced antinociception (ED50 value (and 95% confidence interval)=1.98 (0.79-4.15) nmol, i.c.v.) in the mouse 55°C warm-water tail-withdrawal assay, equivalent to morphine (2.35 (1.13-5.03) nmol, i.c.v.), but demonstrated DOR-selective antagonism in addition to non-selective opioid agonism. The antinociception of 26 was without locomotor activity or acute antinociceptive tolerance. This novel class of peptides adds to the potentially therapeutically relevant collection of previously reported EM analogues.

Synthesis and pharmacological evaluation of novel N-alkyl/aryl substituted thiazolidinone arecoline analogues as muscarinic receptor 1 agonist in Alzheimer's dementia models

Earlier we have reported the effect of arecoline thiazolidinone and morpholino arecoline analogues as muscarinic receptor 1 agonist in Alzheimer's dementia models. To elucidate further our SAR study on the chemistry and muscarinic receptor binding efficacy, a series of novel N-alkyl/aryl substituted thiazolidinone arecoline analogues 6( a– m) were designed and synthesized from 3-pyridine carboxaldehyde by reacting with different amines in the presence of γ-ferrite as catalyst and subjected to in vitro muscarinic receptor binding studies using male Wistar rat brain membrane homogenate and extended to in vivo pharmacological evaluation of memory and learning in male Wistar rats. Derivative 6j having diphenylamine moiety attached to nitrogen of thiazolidinone showed significant affinity for the M1 receptor binding.

Tritium labelling and degradation studies of Dmt1‐endomorphin 2

AbstractTwo tritiated derivatives of Dmt1‐endomorphin 2 (Dmt1‐EM2) were prepared by the catalytic tritiodehalogenation of 3′,5′‐diiodo‐Dmt1‐EM2 and the saturation of 3,4ΔPro2‐Dmt1‐EM2, resulting in [3′,5′‐3H2]Dmt1‐EM2 and [3H2]Pro2‐Dmt1‐EM2 with specific activities of 2.88 TBq/mmol (77.8 Ci/mmol) and 1.95 TBq/mmol (52.8 Ci/mmol), respectively. 3′,5′‐Diiodo‐Dmt1‐EM2 was synthesized by the chloramine T method from Dmt1‐EM2. 3,4ΔPro2‐Dmt1‐EM2 was synthesized by using the Merrifield solid‐phase method. The distributions of the tritium in the labelled peptides were investigated by reversed‐phase high‐performance liquid chromatography after acidic hydrolysis. The stability of Dmt1‐EM2 in a rat brain membrane homogenate was also determined. Copyright © 2007 John Wiley & Sons, Ltd.

Low concentrations of ethanol do not affect radioligand binding to the δ-subunit-containing GABA A receptors in the rat brain

In the present study, we investigated the co-localization pattern of the δ subunit with other subunits of GABA A receptors in the rat brain using immunoprecipitation and Western blotting techniques. Furthermore, we investigated whether low concentrations of ethanol affect the δ-subunit-containing GABA A receptor assemblies in the rat brain using radioligand binding to the rat brain membrane homogenates as well as to the immunoprecipitated receptor assemblies. Our results revealed that δ subunit is not co-localized with γ 2 subunit but it is associated with the α 1, α 4 or α 6, β 2 and/or β 3 subunit(s) of GABA A receptors in the rat brain. Ethanol (1–50 mM) neither affected [ 3H]muscimol (3 nM) binding nor diazepam-insensitive [ 3H]Ro 15-4513 (2 nM) binding in the rat cerebellum and cerebral cortex membranes. However, a higher concentration of ethanol (500 mM) inhibited the binding of these radioligands to the GABA A receptors partially in the rat cerebellum and cerebral cortex. Similarly, ethanol (up to 50 mM) did not affect [ 3H]muscimol (15 nM) binding to the immunoprecipitated δ-subunit-containing GABA A receptor assemblies in the rat cerebellum and hippocampus but it inhibited the binding partially at a higher concentration (500 mM). These results suggest that the native δ-subunit-containing GABA A receptors do not play a major role in the pharmacology of clinically relevant low concentrations of ethanol.

Effect of novel arecoline thiazolidinones as muscarinic receptor 1 agonist in Alzheimer's dementia models

The discovery of cholinergic deficit in Alzheimer's disease (AD) patient's brain has triggered research efforts, using cholinomimetic approaches for their efficacy in AD therapy. Various therapies may be of potential clinical use in AD. Among these are cholinergic agents, which include muscarinic agonists, acetylcholinesterase inhibitors, and acetylcholine releasing agents. One of the muscarinic agonists tested in AD is arecoline and its bioisosters, which are widely explored as muscarinic receptor 1 agonist (M1 receptor agonist) in AD research. In this regard, five-membered heterocyclic ring system attached arecoline basic nucleus ( N-methyl tetrahydropyridines) at third position has been extensively researched on. The present research involved synthesis of arecoline thiazolidinones 5( a– j) by using dipolar addition of 3-aminopyridine and alkyl/aryl carboxaldehydes in presence of gamma ferrite as catalyst. The resulting products were methylated and reduced to get desired products. Subsequently the synthesized arecoline thiazolidinones were subjected to in vitro muscarinic receptor binding studies using male Wistar rat brain (cerebral cortex) membrane homogenate and extended this in vitro study to in vivo pharmacological evaluation of memory and learning in male Wistar rats. Four derivatives ( 5a– 5c and 5e) showed considerable M1 receptor binding affinity ( in vitro) and elicited beneficial effects in vivo memory and learning models (Rodent memory evaluation, plus and Y maze studies).

Synthesis of enzymatically resistant nociceptin‐related peptides containing a carbamic acid residue

Nociceptin, a 17-amino acid peptide (FGGFTGARKSARKLANQ, N/OFQ), is the endogenous ligand of the nociceptin/orphanin FQ (NOP) receptor. This receptor-ligand system is involved in various physiological as well as pathophysiological mechanisms, but owing to the peptidic structure, it is rapidly degraded by enzymes. The enzymatic digestion of nociceptin involves mainly aminopeptidases and yields Noc(2-17)-OH and other smaller fragments. We aimed at increasing the enzymatic stability against aminopeptidases in the case of peptide Noc(1-13)-NH(2), which possesses the minimum sequence capable of interacting with the NOP receptor. Therefore we developed a new procedure for the synthesis of peptides with the carbamic acid residue [...-NH-CH(R)-CO-NH-CO-NH-CH(Q)-CO-.]. A set of four carbamic acid-nociceptin derivatives were produced. The carbamic acid residue was incorporated into the inner part of the peptides, building on solid phase, by using a suitable dipeptide fragment with carbamic acid residue produced by a simple and efficient three-step solution phase procedure. Enzymatic stability of carbamic acid peptides was studied in the presence of aminopeptidase M (AP-M) and in rat brain membrane homogenate. The receptor-binding properties were also studied by radioligand binding assay on crude rat brain membranes and the activity of the ligands were analyzed on isolated mouse vas deferens (MVD) tissues. We found that incorporation of the carbamic acid residue into the N-terminal part of nociceptin significantly increases the resistance against AP-M. We observed the decrease of binding affinities to the NOP receptor in case of the peptides modified in the N-terminal portion. Consequently, the incorporation of the carbamic acid residue into peptides can be proposed as a promising and reasonable tool for increasing enzymatic stability, where the native molecule is less sensitive for carbamic acid residue-related structural change.

Characterization of a new neuropeptide Y Y5 agonist radioligand: [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP.

In order to optimally characterize a class of neuropeptide Y (NPY) receptors expressed in a tissue enriched with multiple subtypes (Y1, Y2, Y4 and Y5) and to establish its detailed distribution, it is critical to use highly selective and specific probes that possess very low non-specific binding. In that context, we recently reported on the development of [125I][hPP(1-17), Ala31, Aib32]NPY as Y5 receptor radioligand. However, the non-specific binding obtained with this radioligand was too high to allow for detailed receptor autoradiography studies [Br. J. Pharmacol. 139 (2003) 1360]. Iodinated [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP may represent a better Y5 radioligand in that regard. Accordingly, [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding was investigated in rat brain membrane homogenates and its specificity and selectivity established in rat Y1, Y2, Y4 and Y5 transfected HEK293 cells. No specific binding was detected in HEK293 cells transfected with the rat Y1, Y2 or Y4 receptors, while saturable binding was observed in cells transfected with the rat Y5 receptor cDNA and in rat brain membrane homogenates (KD of 0.5-0.7 nM). Competition binding experiments performed in rat brain membrane homogenates demonstrated that specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding was competed with nanomolar affinities by Y5 agonists and antagonists such as [Leu31,Pro34]PYY, PYY(3-36), [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP, [Ala31, Aib32]NPY, [hPP(1-17), Ala31, Aib32]NPY, CGP71683A and JCF109, but not by Y1 (BIBP3226 and BIBO3304), Y2 (BIIE0246) and Y4 (GR231118) ligands. Non-specific binding was also lower than that reported for [125I][hPP(1-17), Ala31, Aib32]NPY. Interestingly, detailed analysis of competition binding curves obtained with [Leu31, Pro34]PYY, hPP, PYY(3-36) and [cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP against specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP sites were best fitted to a two-site model. Additionally, receptor autoradiography studies revealed the presence of specific [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP binding sites in the lateral septum and area postrema while other brain regions contained much lower levels of specific binding. Taken together, these data suggest that [125I][cPP(1-7), NPY(19-23), Ala31, Aib32, Gln34]hPP represents a useful tool to study the unique feature of the Y5 receptor subtype.

Development and characterization of a highly selective neuropeptide Y Y5 receptor agonist radioligand: [125I][hPP1-17, Ala31, Aib32]NPY.

(1) The existence of multiple classes of neuropeptide Y (NPY) receptors (Y(1), Y(2), Y(4), Y(5) and y(6)) is now well established. However, one of the major difficulties in the study of these various receptor subtypes is the current lack of highly selective probes to investigate a single receptor class. Up to most recently, this was particularly true for the Y(4) and Y(5) subtypes. (2) [hPP(1-17), Ala(31), Aib(32)]NPY, the first highly selective Y(5) agonist, was iodinated using the chloramine T method and purified by high-pressure liquid chromatography. (3) Binding performed in rat brain homogenates revealed that equilibrium was reached after 120 min (t(1/2)=21 min) and 60 min (t(1/2)=12 min) at 25 and 100 pM [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY, respectively. (4) Isotherm saturation binding experiments demonstrated that [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY binds to an apparent single population with high-affinity (K(D) of 1.2 and 1.7 nM) and low-capacity (B(max) of 14+/-3 fmol/100,000 cells and 20+/-5 fmol/mg protein) sites in Y(5) receptor HEK293-transfected cells and rat brain membrane homogenates, respectively. No specific [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY binding sites could be detected in Y(1), Y(2) or Y(4) receptors transfected HEK293 cells, demonstrating the high selectivity of this ligand for the Y(5) subtype. (5) Competition binding experiments performed in rat brain membrane homogenates and Y(5)-receptor transfected HEK293 cells demonstrated that specific [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY binding was competed with high affinity by Y(5) agonists and antagonists such as [Ala(31), Aib(32)]NPY, [hPP(1-17), Ala(31), Aib(32)]NPY, hPP, CGP71683A and JCF109, but not by Y(1) (BIBP3226), Y(2) (BIIE0246) and Y(1)/Y(4) (GR231118) preferential ligands. (6) Taken together, these data demonstrate that [(125)I][hPP(1-17), Ala(31), Aib(32)]NPY is the first highly selective Y(5) radioligand to be developed. This new probe should prove most useful for further detailed studies of the molecular and pharmacological properties of this receptor subtype in brain and peripheral tissues.

Appetite-boosting property of pro-melanin-concentrating hormone(131-165) (neuropeptide-glutamic acid-isoleucine) is associated with proteolytic resistance.

Melanin-concentrating hormone (MCH) is a cyclic neuropeptide, with a major role in stimulation of feeding behavior in mammals. MCH signals in the brain occur via two seven-transmembrane G protein-coupled receptors, namely MCH1 (SLC-1, MCH(1), MCH-R1, or MCH-1R) and MCH2 (SLT, MCH(2), MCH-R2, or MCH-2R). In this study, we demonstrate that the pro-MCH(131-165) peptide neuropeptide-glutamic acid-isoleucine (NEI)-MCH is more potent than MCH in stimulating feeding in the rat. Using rat MCH1-expressed human embryonic kidney 293 cells, we show that NEI-MCH exhibits 5-fold less affinity in a binding assay and 2-fold less potency in a cAMP assay than MCH. A similar 7- to 8-fold shift in potency was observed in a Ca(2+)(i) assay using rat MCH1 or human MCH2-transfected Chinese hamster ovary cell models. This demonstrates that NEI-MCH is not a better agonist than MCH at either of the MCH receptors. Then, we compared the proteolysis resistance of MCH and NEI-MCH to rat brain membrane homogenates and purified proteases. Kinetics of peptide degradation using brain extracts indicated a t(1/2) of 34.8 min for MCH and 78.5 min for NEI-MCH with a specific pattern of cleavage of MCH but not NEI-MCH by exo- and endo-proteases. Furthermore, MCH was found highly susceptible to degradation by aminopeptidase M and endopeptidase 24.11, whereas NEI-MCH was fully resistant to proteolysis by these enzymes. Therefore, our results strongly suggest that reduced susceptibility to proteases of NEI-MCH compared with MCH account for its enhanced activity in feeding behavior. NEI-MCH represents therefore the first MCH natural functional "superagonist" so far described.

Total synthesis of cyclic ADP-carbocyclic-ribose, a stable mimic of Ca2+-mobilizing second messenger cyclic ADP-ribose.

The synthesis of cyclic ADP-carbocyclic-ribose (cADPcR, 4) designed as a stable mimic of cyclic ADP-ribose (cADPR, 1), a Ca2+-mobilizing second messenger, was achieved using as the key step a condensation reaction with the phenylthiophosphate-type substrate 14 to form an intramolecular pyrophosphate linkage. The N-1-carbocyclic-ribosyladenosine derivative 16 was prepared via the condensation between the imidazole nucleoside derivative 17, prepared from AICA-riboside (19), and the readily available optically active carbocyclic amine 18. Compound 16 was then converted to the corresponding 5' '-phosphoryl-5'-phenylthiophosphate derivatives 14. Treatment of 14 with AgNO3 in the presence of molecular sieves (3 A) in pyridine at room temperature gave the desired cyclization product 32 in 93% yield, and subsequent acidic treatment provided the target cADPcR (4). This represents a general method for synthesizing biologically important cyclic nucleotides of this type. 1H NMR analysis of cADPcR suggested that its conformation in aqueous medium is similar to that of cADPR. cADPcR, unlike cADPR, was stable under neutral and acidic conditions, where under basic conditions, it formed the Dimroth-rearranged N6-cyclized product 34. cADPcR was also stable in rat brain membrane homogenate which has cADPR degradation activity. Furthermore, cADPcR was resistant to the hydrolysis by CD38 cADPR hydrolase, while cADPR was rapidly hydrolyzed under the same conditions. When cADPcR was injected into sea urchin eggs, it caused a significant release of Ca2+ in the cells, an effect considerably stronger than that of cADPR. Thus, cADPcR was identified as a stable mimic of cADPR.

(3)H](2S,4R)-4-Methylglutamate: a novel ligand for the characterization of glutamate transporters.

[(3)H](2S,4R)-4-Methylglutamate ([(3)H]4MG), used previously as a ligand for low-affinity kainate receptors, was employed to establish a binding assay for glutamate transporters (GluTs), as 4MG has also been shown to have affinity for the glial GluTs, GLT1 and GLAST. In rat brain membrane homogenates in the presence of Na(+) ions at 4 degrees C, specific binding of [(3)H]4MG was rapid and saturable (t(1/2) approximately 15 min), representing > 90% of total binding. Dissociation of [(3)H]4MG occurred in a biphasic manner, however, saturation studies and Scatchard analysis indicated a single site of binding (n(H) = 0.85) and a K(d) of 6.2 +/- 0.8 microM with a B(max) of 111.8 +/- 23.8 pmol/mg protein. Specific binding of [(3)H]4MG was Na(+)-dependent and inhibited by K(+) and HCO(3-). Pharmacological inhibition with compounds acting at GluTs revealed that Glu, D- and L-aspartate, L-serine-O-sulfate and Ltrans-pyrrolidine-2,4-dicarboxylate fully displaced specific binding. Drugs having preferential affinity for GLT1, kainate, dihydrokainate and Lthreo-3-methylglutamate, all inhibited approximately 40% of specific binding. The inhibition pattern of L-serine-O-sulfate in the presence of a saturating concentration of dihydrokainate was suggestive of [(3)H]4MG also labelling GLAST. 6-Cyano-7-nitroquinoxaline, a kainate receptor antagonist, and a range of Glu receptor agonists and antagonists failed to significantly inhibit [(3)H]4MG binding. The pharmacological profile of binding of [(3)H]4MG resembled that found for [(3)H]D-aspartate, a ligand specific for GluTs, reinforcing the hypothesis that [(3)H]4MG was labelling GluTs in this assay. Together, these data illustrate the development of an efficient, economic binding assay that is suitable for the characterization of different subtypes of GLuTs.

Evidence for the Differential Distribution of at Least Two Adrenomedullin Receptor Subtypes in the Rat Brain

We recently reported that specific [I]human Adrenomedullin13-52 (hADM13-52) binding sites are very discretely distributed in the rat brain[1]. Furthermore, in rat brain membrane homogenates, we suggested the existence of at least two populations of [I]hADM13-52 receptor binding sites[2]. In the present study, we investigated the differential distribution of specific [I]hADM1352 sites that are sensitive and insensitive to hADM22-52 in the rat brain. Adjacent coronal rat brain sections were incubated with 35 pM [I]hADM13-52 in the presence of increasing concentrations of hADM22-52. As previously reported, specific [I]hADM13-52 sites were observed in the choroid and linings of ventricles, basal amygdaloid nucleus, neuronal lobe of the pituitary gland, trigeminal nucleus, and cerebellum. Specific [I]hADM13-52 binding is fully inhibited by 1 μM hADM22-52 in all these brain regions except in the cerebellum. In fact, hADM22-52 was able to inhibit only 70% of specific [I]hADM13-52 binding sites in the rat cerebellum. These data suggest for the first time that the rat cerebellum contains at least two populations of ADM receptors, one that is sensitive to hADM22-52 while the other is resistant. This characterization of cerebellar [I]hADM13-52 / hADM22-52 -insensitive sites is currently under investigation. Supported by grant from the CIHR to RQ.

BIIE0246, a potent and highly selective non-peptide neuropeptide Y Y(2) receptor antagonist.

1. BIIE0246, a newly synthesized non-peptide neuropeptide Y (NPY) Y(2) receptor antagonist, was able to compete with high affinity (8 to 15 nM) for specific [(125)I]PYY(3 - 36) binding sites in HEK293 cells transfected with the rat Y(2) receptor cDNA, and in rat brain and human frontal cortex membrane homogenates. 2. Interestingly, in rat brain homogenates while NPY, C2-NPY and PYY(3 - 36) inhibited all specific [(125)I]PYY(3 - 36) labelling, BIIE0246 failed to compete for all specific binding suggesting that [(125)I]PYY(3 - 36) recognized, in addition to the Y(2) subtype, another population of specific NPY binding sites, most likely the Y(5) receptor. 3. Quantitative receptor autoradiographic data confirmed the presence of [(125)I]PYY(3 - 36)/BIIE0246-sensitive (Y(2)) and-insensitive (Y(5)) binding sites in the rat brain as well as in the marmoset monkey and human hippocampal formation. 4. In the rat vas deferens and dog saphenous vein (two prototypical Y(2) bioassays), BIIE0246 induced parallel shifts to the right of NPY concentration-response curves with pA(2) values of 8.1 and 8.6, respectively. In the rat colon (a Y(2)/Y(4) bioassay), BIIE0246 (1 microM) completely blocked the contraction induced by PYY(3 - 36), but not that of [Leu(31), Pro(34)]NPY (a Y(1), Y(4) and Y(5) agonist) and hPP (a Y(4) and Y(5) agonist). Additionally, BIIE0246 failed to alter the contractile effects of NPY in prototypical Y(1) in vitro bioassays. 5. Taken together, these results demonstrate that BIIE0246 is a highly potent, high affinity antagonist selective for the Y(2) receptor subtype. It should prove most useful to establish further the functional role of the Y(2) receptor in the organism.

(125)I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y(1) and Y(4) receptors.

GR231118 (also known as 1229U91 and GW1229), a purported Y(1) antagonist and Y(4) agonist was radiolabelled using the chloramine T method. [(125)I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [(125)I]-GR231118 binds with very high affinity (K(d) of 0.09 - 0.24 nM) in transfected HEK293 cells with the rat Y(1) and Y(4) receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y(2) or Y(5) receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [(125)I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y(1), but not rat Y(4), receptor cDNA. Autoradiographic studies demonstrated that [(125)I]-GR231118 binding sites were fully inhibited by the Y(1) antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [(125)I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [(125)I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y(4) receptor subtype. In summary, [(125)I]-GR231118 is a new radiolabelled probe to investigate the Y(1) and Y(4) receptors; its major advantage being its high affinity. Using highly selective Y(1) antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [(125)I]-GR231118 to the Y(1) receptor allowing for the characterization and visualization of the purported Y(4) subtype. British Journal of Pharmacology (2000) 129, 37 - 46

Evidence that the inhibition of luteinizing hormone secretion exerted by central administration of neuropeptide Y (NPY) in the rat is predominantly mediated by the NPY-Y5 receptor subtype.

A number of studies have indicated that neuropeptide Y (NPY) is a central regulator of the gonadotropic axis, and the Y1 receptor was initially suggested to be implicated. As at least five different NPY receptor subtypes have now been characterized, the aim of the present study was to reinvestigate the pharmacological profile of the receptor(s) mediating the inhibitory action of NPY on LH secretion by using a panel of NPY analogs with different selectivity toward the five NPY receptor subtypes. When given intracerebroventricularly (icv) to castrated rats, a bolus injection of native NPY (0.7-2.3 nmol) dose-dependently decreased plasma LH. Peptide YY (PYY; 2.3 nmol) was as potent as NPY, suggesting that the Y3 receptor is not implicated. Confirming previous data, the mixed Y1, Y4, and Y5 agonist [Leu31,Pro34]NPY (0.7-2.3 nmol) inhibited LH release with potency and efficacy equal to those of NPY. Neither the selective Y2 agonist C2-NPY (2.3 nmol) nor the selective Y4 agonist rat pancreatic polypeptide affected plasma LH, excluding Y2 and Y4 subtypes for the action of NPY on LH secretion. The mixed Y4-Y5 agonist human pancreatic polypeptide (0.7-7 nmol) as well as the mixed Y2-Y5 agonist PYY3-36 (0.7-7 nmol) that displayed very low affinity for the Y1 receptor, thus practically representing selective Y5 agonists in this system, decreased plasma LH with potency and efficacy similar to those of NPY, indicating that the Y5 receptor is mainly involved in this inhibitory action of NPY on LH secretion. [D-Trp32]NPY, a selective, but weak, Y5 agonist, also inhibited plasma LH at a dose of 7 nmol. Furthermore, the inhibitory action of NPY (0.7 nmol) on LH secretion could be fully prevented, in a dose-dependent manner (6-100 microg, icv), by a nonpeptidic Y5 receptor antagonist. This antagonist (60 microg, icv) also inhibited the stimulatory action of NPY (0.7 nmol) on food intake. The selectivity of PYY3-36, human PP, [D-Trp32]NPY, and the Y5 antagonist for the Y5 receptor subtype was further confirmed by their ability to inhibit the specific [125I][Leu31,Pro34]PYY binding to rat brain membrane homogenates in the presence of the Y1 receptor antagonist BIBP3226, a binding assay system that was described as being highly specific for Y5-like receptors. With the exception of [D-Trp32]NPY, all analogs able to inhibit LH secretion were also able to stimulate food intake. Taken together, these results indicate that the Y5 receptor is involved in the negative control by NPY of the gonadotropic axis.

Biphasic modulation of GABA(A) receptor binding by steroids suggests functional correlates.

Neuroactive steroids and other positive modulators of GABA(A) receptors showed regional variation in both the efficacy and potency for modulation of [35S]TBPS binding to rat brain membrane homogenates, with biphasic concentration-dependence. GABA present in the binding assays prevented the enhancement phase of the steroid concentration-dependence plot while the antagonists bicuculline and RU5135 prevented the inhibition phase. Using recombinant GABA(A) receptors, expressed in insect cell line Sf9 using baculovirus, enhancement by steroids of [35S]TBPS binding was sensitive to the presence of the gamma2 subunit and the nature of the alpha subunit (alpha1 beta2 gamma2S > alpha1 beta2, alpha6 beta2, alpha6 beta2 gamma2S, and alpha6 beta2 delta). As in cerebellum, addition of RU5135 reduced the inhibitory phase and revealed a small enhancement of TBPS binding by neuroactive steroids. The subunit-dependent interactions of steroid and GABA site ligands are consistent with a three-state model in which the receptor mono-liganded by GABA or steroid has a different affinity for TBPS than the resting state, and the receptor biliganded by GABA, steroid, or both has little affinity for TBPS.

Radioiodinated analogs of xylamine: N‐(2‐chloroethyl)‐N‐ethyl‐2‐[125I]iodobenzylamine and N,N‐diethyl‐2‐[125I]iodobenzylamine as potential tools for monoamine uptake exploration by spect

AbstractIn order to improve the scintigraphy and radiotherapy of neuroendocrine tumors we synthesized two radioiodinated benzylamines [N‐(2‐chloroethyl)‐N‐ethyl‐2‐[125I]iodobenzylamine and N,N‐diethyl‐2‐[125I]iodobenzylamine], analogs of xylamine [N‐(2‐chloroethyl)‐N‐ethyl‐2‐methylbenzylamine]. Xylamine is an irreversible inhibitor of uptake and accumulation of noradrenaline. The two unlabelled iodinated derivatives [N‐(2‐chloroethyl)‐N‐ethyl‐2‐iodobenzylamine and N,N‐diethyl‐2‐iodobenzylamine] were synthesized, purified and checked by HPLC, NMR and mass spectrography. Their affinity for the noradrenaline transporter was determined in vitro on rat brain membrane homogenates with [3H]nisoxetine. Radioiodination was performed by iodide for bromide nucleophilic exchange from brominated precursors. The N,N‐diethyl‐2‐[125I]iodobenzylamine was obtained directly from N,N‐diethyl‐2‐bromobenzylamine. Radiosynthesis of N‐(2‐chloroethyl)‐N‐ethyl‐2‐[125I]iodobenzylamine required three steps. A new brominated precursor [N‐ethyl‐N‐(2‐bromobenzyl)glycine ethyl ester] which was stable for radiolabelling and suitable for reduction to N‐(2‐hydroxyethyl)‐N‐cthyl‐2‐[125I]iodobenzylamine was synthesized. N‐(2‐Hydroxyethyl)‐N‐ethyl‐2‐[125I]iodobenzylamine was converted to N‐(2‐chloroethyl)‐N‐ethyl‐2‐[125I]iodobenzylamine in the presence of an excess of thionyl chloride. Radioiodinated derivatives were purified and checked by HPLC.

Nonpeptide peptidomimetic antagonists of the neuropeptide Y receptor: benextramine analogs with selectivity for the peripheral Y2 receptor.

We synthesized a new series of benextramine analogs as neuropeptide Y (NPY) functional group mimetics and tested them for N-[propionyl-3H]NPY ([3]NPY) displacement activity in rat brain membrane homogenates and for NPY receptor antagonist activity in the rat femoral artery. The tetraamine, carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-1,6-hexanediamine (15) was equipotent with benextramine (based on comparison of the relevant IC50's) in a rat brain [3H]NPY displacement assay, suggesting that the disulfide is not a necessary feature of benextramine's [3H]NPY displacement activity, although this analog maintained selectivity for the benextramine-sensitive binding site population. The bis(N,N-dialkylguanyl) disulfide and carbon analogs 14a-c were 3-4 times more potent than their respective controls in displacing [3H]NPY from rat brain membrane homogenates with IC50's ranging from 15 to 18 microM and maintained selectivity for the benextramine-sensitive, Y1 binding site population. However, the activity of the carbon analog N,N'-bis[6-[N-(2-naphthylmethyl)amino]hexyl]-N,N'-(1,6- hexanediyl)diguanidine tetrahydrochloride (14b) showed a different profile in a femoral artery vasoconstriction assay; at 1.0 nM, this analog shifted the concentration-effect curve of the Y2-selective agonist NPY13-36 to the right (pA2 = 9.2; Kd = 0.63 nM) without a significant change in the maximum effect, while even at 1.0 mM it had no effect on the vasoconstrictive activity of the Y1-selective agonist [Leu31,Pro34]NPY. Thus, the bis(N,N-dialkylguanidine) analogs of benextramine are selective, competitive antagonists of the postsynaptic NPY receptor in the femoral artery.