Rat Cortical Membranes Research Articles

Comparison [3H]-flumazenil binding parameters in rat cortical membrane using different separation methods, filtration and centrifugation

Radioligand receptor binding assays are a common method to evaluate the affinity of newly synthesized benzodiazepine ligands for the receptor. [(3)H]-flumazenil is an antagonist of benzodiazepine receptors and is generally used as a radioligand. In this study, the binding parameters of [(3)H]-flumazenil to rat cortical membranes were evaluated using two separation methods: filtration with GF/C filters and centrifugation. Additionally, the effects of vacuum pressure, exposure time to the cocktail, and geometry on the filtration method were studied. The binding parameters of [(3)H]-flumazenil (Kd and Bmax) were determined through saturation studies using two methods. The results from this study showed that the filtration method is time consuming and requires more steps to be completed. Because filtration causes partial elution of bound [(3)H]-flumazenil into the liquid scintillation cocktail, the results are not reproducible, which result in inaccurate estimation of the binding parameters. The centrifugation method in contrast to filtration is straightforward and produces reproducible as well as reliable results, all of the steps are performed in a single polypropylene tube, which eliminates the loss of tissue and avoids other systematic errors associated with transfer and handling.

Anti-Alcohol and Anxiolytic Properties of a New Chemical Entity, GET73.

N-[(4-trifluoromethyl)benzyl]4-methoxybutyramide (GET73) is a newly synthesized compound structurally related to the clinically used, alcohol-substituting agent, gamma-hydroxybutyric acid (GHB). The present study was designed to assess whether GET73 may share with GHB the capacity to reduce alcohol intake in rats. Additionally, the effect of treatment with GET73 on anxiety-related behaviors and cognitive tasks in rats was investigated. A series of in vitro binding assays investigated the capacity of GET73 to bind to the GHB binding site and multiple other receptors. GET73 (10−9–10−3 M) failed to inhibit [3H]GHB binding at both high- and low-affinity GHB recognition sites in rat cortical membranes. GET73 displayed minimal, if any, binding at dopamine, serotonin, GABA, and glutamate receptors in membranes from different rat brain areas. Acute treatment with low-to-moderate, non-sedative doses of GET73 (5–50 mg/kg, i.g. or i.p.) (a) reduced alcohol intake and suppressed “alcohol deprivation effect” (a model of alcohol relapse) in selectively bred, Sardinian alcohol-preferring (sP) rats, (b) exerted anxiolytic effects in Sprague-Dawley (SD) and sP rats exposed to the Elevated Plus Maze test, and (c) tended to induce promnestic effects in SD rats exposed to a modified water version of the Hebb–Williams maze test. Although the mechanism of GET73 action is currently unknown, the results of the present study suggest that GET73 has a multifaceted pharmacological profile, including the capacity to reduce alcohol drinking and anxiety-related behaviors in rats.

Molecular and neurochemical evaluation of the effects of etizolam on GABAA receptors under normal and stress conditions.

The thienobenzodiazepine derivative etizolam (CAS 40054-69-1, 6-(o-chlorophenyl)-8-ethyl-1-methyl-4H-s-triazolo-(3,4-c)thienol(1 ,4) diazepine) is a potent anxiolytic with a pharmacological profile similar to that of classical benzodiazepines. In order to rationalize the therapeutic use of etizolam, its pharmacodynamics properties on GABAA receptors were investigated by a comparative study with other ligands on human recombinant GABAA as well as rat brain native receptors. Etizolam inhibited in a concentration-dependent manner [3H]flunitrazepam (CAS 1622-62-4) binding to rat cortical membranes, with an affinity of 4.5 nmol/l greater than that of alprazolam (CAS 28981-97-7) (7.9 nmol/l). Ethizolam enhanced GABA-induced Cl- currents in oocytes expressing human cloned GABAA receptors. With alpha 1 beta 2 gamma 2S subunit combination, etizolam produced a 73% increase in GABA-induced currents with an EC50 of 92 nmol/l. At the same receptor type, alprazolam showed a higher degree of potentiation and potency (98%, EC50 56 nmol/l). At alpha 2 beta 2 gamma 2S or alpha 3 beta 2 gamma 2S subunit constructs, the effects of etizolam were similar to those of alprazolam. Flumazenil (CAS 78755-81-4) completely blocked both etizolam and alprazolam effects on GABA-induced currents. Etizolam, administered i.p., was uneffective in changing ex vivo t-[35S]butylbicyclophosphorothionate ([35S]-TBPS) binding to rat cerebral cortex, whereas alprazolam and abecarnil (CAS 111841-85-1) significantly reduced this parameter. However, etizolam similarly to abecarnil and alprazolam, antagonized isoniazid-induced increase (61%) in [35S]-TBPS binding to rat cortical membranes. Further, etizolam inhibited in a dose-dependent manner basal acetylcholine release from both hippocampus and prefrontal cortex, and reversed foot-shock-induced increase of basal acetylcholine release to a control level. Altogether, these results suggest that etizolam may have a reduced intrinsic activity, at least at specific subpopulations of GABAA receptors. This property, together with the pharmacokinetic indication of a short-acting drug, may characterize etizolam as a ligand endowed with less side-effects typical of full agonits such as diazepam (CAS 439-14-5) and alprazolam. Finally, given its marked efficacy under conditions of GABAergic deficit, etizolam may represent a possible drug of choice with reduced liability to produce tolerance and dependence after long-term treatment of anxiety and stress syndromes.

Characterization of COR627 and COR628, Two Novel Positive Allosteric Modulators of the GABAB Receptor

The potential efficacy of GABA(B) receptor agonists in the treatment of pain, drug addiction, epilepsy, cognitive dysfunctions, and anxiety disorders is supported by extensive preclinical and clinical evidence. However, the numerous side effects produced by the GABA(B) receptor agonist baclofen considerably limit the therapeutic use of this compound. The identification of positive allosteric modulators (PAMs) of the GABA(B) receptor may constitute a novel approach in the pharmacological manipulation of the GABA(B) receptor, leading to fewer side effects. The present study reports the identification of two novel compounds, methyl 2-(1-adamantanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR627) and methyl 2-(cyclohexanecarboxamido)-4-ethyl-5-methylthiophene-3-carboxylate (COR628), which act as GABA(B) PAMs in 1) rat cortical membranes and 2) in vivo assay. Both compounds potentiated GABA- and baclofen-stimulated guanosine 5'-O-(3-[(35)S]thio)-triphosphate binding to native GABA(B) receptors, while producing no effect when given alone. GABA concentration-response curves in the presence of fixed concentrations of COR627 and COR628 revealed an increase of potency of GABA rather than its maximal efficacy. In radioligand binding experiments [displacement of the GABA(B) receptor antagonist, 3-N-[1-((S)-3,4dichlorophenyl)-ethylaminol]-2-(S)hydroxypropyl cyclo-hexylmethyl phosphinic acid ([(3)H]CGP54626)], both COR627 and COR628 increased the affinity of high- and low-affinity binding sites for GABA, producing no effect when administered alone up to a concentration of 1 mM. In vivo experiments indicated that pretreatment with per se ineffective doses of COR627 and COR628 potentiated the sedative/hypnotic effect of baclofen. In conclusion, COR627 and COR628 may represent two additional tools for use in investigating the roles and functions of positive allosteric modulatory binding sites of the GABA(B) receptor.

Synthesis and characterization of [125I]2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide as novel imaging probe for glycine transporter 1

In this study, 2-iodo substituted 1-methylpiperidin-2-yl benzamide derivatives were synthesized and evaluated as candidate SPECT imaging agents for glycine transporter 1 (GlyT1). In JAR cells, which predominantly express GlyT1, 2-iodo N-[(S)-{(S)-1-methylpiperidin-2-yl}(phenyl)methyl]3-trifluoromethyl-benzamide (5) showed excellent inhibitory activity of [3H]glycine uptake (IC50=2.4nM). Saturation assay in rat cortical membranes revealed that [125I]5 had a single high affinity binding site with a Kd of 1.54nM and a Bmax of 3.40pmol/mg protein. In vitro autoradiography demonstrated that [125I]5 showed consistent accumulation with GlyT1 expression. The in vitro binding was greatly inhibited by GlyT1 inhibitors but not by other site ligands, which suggested the high specific binding of [125I]5 with GlyT1. In the biodistribution and ex vivo autoradiography studies using mice, [125I]5 showed high blood–brain barrier permeability (1.68–2.17% dose/g at 15–60min) and similar regional brain distribution pattern with in vitro results. In addition, pre-treatment of GlyT1 ligands resulted in significant decrease of [125I]5 binding in the GlyT1-rich regions. This preliminary study demonstrated that radio-iodinated 5 is a promising SPECT imaging probe for GlyT1.

Effect of some cationic amphiphilic drugs on phospholipid methylation in the central nervous system of rats.

Imipramine, desipramine, citalopram and chlorpromazine in concentrations which corresponded with their concentration in the central nervous system of rats after pharmacological doses, potentiated phospholipid methylation in the synaptic cortical membranes of naive rats in-vitro. Chronic administration of imipramine, desipramine or citalopram induced changes in the activity of phospholipid methyltransferases since none of these drugs stimulated phospholipid methylation in the synaptic cortical membranes of rats treated with these antidepressants for two weeks. In contrast, chronic treatment with chlorpromazine did not change the sensitivity of phospholipid methyltransferases to the stimulating effect of chlorpromazine, whereas addition of haloperidol to the synaptic cortical membranes of rats treated chronically with haloperidol led to a decrease of phospholipid methylation.

Effect of Imipramine on the Membrane Anisotropy and on the Phospholipid Methylation in the Central Nervous System of the Rat

An ex-vivo and in-vitro study of the effects of imipramine on the membrane anisotropy and phospholipid methylation in the rat cortical membranes was carried out. A comparative study of the membrane fluidity in various brain regions indicated different basal anisotropy of the areas and different reaction of these membranes to imipramine. It was found that imipramine when given to rats chronically (14 x 10 mg kg-1, i.p.) or added externally to the cortical membranes of naive rats or rats treated with a single dose of imipramine (10 mg kg-1, i.p.) decreased the anisotropy of cortical membranes. Chronic imipramine produced some changes of the membrane architecture in the cortex, whereas imipramine in different concentrations did not fluidize these membranes in-vitro. Imipramine in concentrations corresponding to its mean concentration in the rat brain after administration at a dose of 10 mg kg-1 i.p., potentiated phospholipid methylation in the cortical membranes of naive rats and rats receiving imipramine in a single dose of 10 mg kg-1 i.p. in an in-vitro study, whereas the prolonged administration of imipramine decreased the sensitivity of phospholipid methyltransferases to the stimulating effect of the drug in-vitro.

Effects of N-aralkyl substitution of beta-agonists on alpha- and beta-adrenoceptor subtypes: pharmacological studies and binding assays.

The pharmacological and binding properties of four beta-adrenomimetic drugs with N-alkyl substitutions (isoprenaline, terbutaline, salbutamol and soterenol) were compared with those of four corresponding drugs with N-aralkyl substitutions (protokylol, ME 506, salmefamol and zinterol). BD-40 A, a very powerful beta 2-agonist with a related chemical structure, was also included in this study. The beta 1- and beta 2-activities of these drugs were determined on guinea-pig atria and trachea, their alpha-adrenolytic activity was measured on rat aorta and their affinities (Ki) for alpha 1- and alpha 2-adrenoceptors on rat cortical membranes were assessed using [3H]prazosin and [3H]yohimbine. In this group of beta-agonists, substitution of the N-alkyl by an N-aralkyl group had a variable effect on the beta 2-selectivity whereas alpha-adrenolytic properties were always enhanced. An increase of the affinities (Ki) for both alpha 1- and alpha 2-adrenoceptors was found but the effect was much more pronounced for alpha 1-adrenoceptors. These results indicated that the alpha-adrenolytic activity observed with the N-aralkyl beta-agonists was selective for alpha 1-adrenoceptors.

A rapid filtration assay for the glycine binding site on the NMDA receptor in rat cortical membranes using [3H]dichlorokynurenic acid.

A filtration binding assay using [3H]dichlorokynurenic acid to label the glycine binding site on the N-methyl-D-aspartic acid receptor has been evaluated on rat cortical membranes. This ligand binds to a single population of binding sites following mass action kinetics with a KD of 29 nM and a capacity of 5.73 pmol (mg protein)-1. The pharmacological specificity of the binding site is identical to that previously reported for this binding site using [3H]glycine as a radioligand. Agonists showed lower affinity and antagonists higher affinity when [3H]dichlorokynurenic acid was used compared with [3H]glycine. The higher affinity of [3H]dichlorokynurenic acid compared with [3H]glycine make it the more suitable compound with which to label the glycine site.

Phenobarbitone enhances [35S]TBPS binding to extensively washed rat cortical membranes.

Pentobarbitone and phenobarbitone enhance [35S]TBPS binding to extensively washed rat cortical membranes with ED50 values of 15 and 120 microM, respectively, indicative of an interaction with the chloride ionophore part of the GABA-receptor different from the TBPS binding site, but allosterically coupled to this site.

Selective Interactions of Valeriana officinalis Extracts and Valerenic Acid with [3H]Glutamate Binding to Rat Synaptic Membranes

Although GABA neurotransmission has been suggested as a mechanism for Valeriana officinalis effects, CNS depression can also be evoked by inhibition of ionotropic (iGluR) and metabotropic glutamate receptors (mGluR). In this study, we examined if aqueous valerian extract interacted with glutamatergic receptors. Freshly prepared aqueous valerian extract was incubated with rat cortical synaptic membranes in presence of 20 nM [3H]Glutamate. Aqueous valerian extract increased [3H]Glutamate binding from 1 × 10−7 to 1 × 10−3 mg/mL. In the presence of (2S,1′S,2′S)-2-(Carboxycyclopropyl)glycine (LCCG-I) and (2S,2′R,3′R)-2-(2′,3′-Dicarboxycyclopropyl)glycine (DCG-IV), Group II mGluR agents, valerian extract markedly decreased [3H]Glutamate binding, while (2S)-2-amino-3-(3,5-dioxo-1,2,4-oxadiazolidin-2-yl) propanoic acid) (quisqualic acid, QA), Group I mGluR agonist, increased [3H]Glutamate binding. At 0.05 mg/mL aqueous valerian extract specifically interacted with kainic acid NMDA and AMPA receptors. Valerenic acid, a marker compound for Valeriana officinalis, increased the [3H]Glutamate binding after 1.6 × 10−2 mg/mL, and at 0.008 mg/mL it interacted only with QA (Group I mGluR). The selective interactions of valerian extract and valerenic acid with Group I and Group II mGluR may represent an alternative explanation for the anxiolytic properties of this plant.

Millimolar Mn2+ influences agonist binding to 5-HT1A receptors by inhibiting guanosine nucleotide binding to receptor-coupled G-proteins

Manganese is an essential trace element but its overexposure causes poisoning (called manganism) that shares several symptoms with Parkinson's disease, but with a mechanism that is still not well understood: in addition to involvement of the dopaminergic system, both serotonergic and peptiergic systems have been implicated. In the present report we have studied the influence of Mn2+ on 5-HT1A receptor signaling complexes in rat brain and found that Mn2+ in millimolar concentration caused an increase of high-affinity agonist binding to rat hippocampal membranes in comparison with experiments in the presence of Mg2+, but not in rat cortical membranes and in Sf9 cell membranes expressing 5-HT1A receptors and Gi1 heterotrimers. Activation of G proteins with 30μM GTPγS turned all 5-HT1A receptors in these preparations into a low-affinity state for agonist binding in the presence of 1mM Mg2+, but not in the presence of 1mM Mn2+ in rat hippocampal membranes. However, if 1μM GTPγS was used for G protein activation, a substantial amount of high affinity agonist binding was detected in the presence of Mn2+ also in cortical membranes and Sf9 cells, but not with Mg2+ or EDTA. Comparison of the abilities of GDP and GTPγS to modulate high affinity agonist binding to 5-HT1A receptors indicated that both nucleotides were almost 10-fold less potent in the presence of MnCl2 compared to MgCl2. This means that by inhibiting guanosine nucleotide binding to G proteins in complex with 5-HT1A receptors, Mn2+ acts as an enhancer for agonist binding and signal transduction. As the influence of Mn2+ resembles the hypersensitivity of dopaminergic system in Parkinsonial models, it can be proposed that at least some symptoms of manganism are connected with a change of signal transduction complex caused by manganese-nucleotide complexes.

Levels of Gsα (short and long), Gα olf and Gβ (common) subunits, and calcium-sensitive adenylyl cyclase isoforms (1, 5/6, 8) in post-mortem human brain caudate and cortical membranes: Comparison with rat brain membranes and potential stoichiometric relationships

The levels of expression of Gsα (short and long), Gα olf and Gβ (common) subunits, and calcium-sensitive adenylyl cyclases isoforms (AC1, 5/6, and 8) in human brain cortical and caudate membranes were quantified by western blot analysis in order to establish their contribution to the patterns of AC functioning. Both areas expressed Gsα long (52 kDa) with values ranging from about 1400 ng/mg of membrane protein in cerebral cortex to close to 600 ng/mg of membrane protein in caudate nucleus. In contrast, Gsα short and Gsα olf were expressed separately, Gsα short in cortical membranes with values around 500 ng/mg of membrane protein and Gα olf in caudate membranes with values around 1300 ng/mg of membrane protein. Quantitative measurements of Gβ, revealed a similar expression level in cortical and caudate membranes (5444 ± 732 versus 5511 ± 394 ng/mg protein; p = 0.966). The B max values of GTPγS-dependent [ 3H]-forskolin binding show the following descending order: rat striatal membranes > rat cortical membranes = human caudate membranes > human cortical membranes. Therefore, as measured immunochemically and by [ 3H]-forskolin binding, there seems to be a vast excess of Gsα subunits over catalytic units of AC. The highest levels of AC5/6 expression were detected in caudate membranes. AC8 was little expressed, and there were no significant differences in the relative values between both human brain regions. Finally, the levels of the AC1 isoform were significantly lower in caudate than in cortical membranes. It is concluded that these stoichiometric data contribute nonetheless to explain the significant differences observed in signalling capacities through the AC system in both human brain regions.

Δ9‐Tetrahydrocannabivarin suppresses in vitro epileptiform and in vivo seizure activity in adult rats

We assessed the anticonvulsant potential of the phytocannabinoid Δ⁹-tetrahydrocannabivarin (Δ⁹-THCV) by investigating its effects in an in vitro piriform cortex (PC) brain slice model of epileptiform activity, on cannabinoid CB1 receptor radioligand-binding assays and in a generalized seizure model in rats. Δ⁹-THCV was applied before (10 μm Δ⁹-THCV) or during (10-50 μm Δ⁹-THCV) epileptiform activity induced by Mg²(+) -free extracellular media in adult rat PC slices and measured using multielectrode array (MEA) extracellular electrophysiologic techniques. The actions of Δ⁹-THCV on CB1 receptors were examined using [³H]SR141716A competition binding and [³⁵S]GTPγS assays in rat cortical membranes. Effects of Δ⁹-HCV (0.025-2.5 mg/kg) on pentylenetetrazole (PTZ)-induced seizures in adult rats were also assessed. After induction of stable spontaneous epileptiform activity, acute Δ⁹ -THCV application (≥ 20 μm) significantly reduced burst complex incidence and the amplitude and frequency of paroxysmal depolarizing shifts (PDSs). Furthermore, slices pretreated with 10 μm Δ⁹-THCV prior to induction of epileptiform activity exhibited significantly reduced burst complex incidence and PDS peak amplitude. In radioligand-binding experiments, Δ⁹-THCV acted as a CB1 receptor ligand, displacing 0.5 nm [³H]SR141716A with a Ki∼290 nm, but exerted no agonist stimulation of [³⁵S]GTPγS binding. In PTZ-induced seizures in vivo, 0.25 mg/kg Δ⁹-THCV significantly reduced seizure incidence. These data demonstrate that Δ⁹-THCV exerts antiepileptiform and anticonvulsant properties, actions that are consistent with a CB1 receptor-mediated mechanism and suggest possible therapeutic application in the treatment of pathophysiologic hyperexcitability states.

Enhancement of agonist binding to 5-HT 1A receptors in rat brain membranes by millimolar Mn 2+

Manganese in millimolar concentration caused increase in specific binding of [ 3H]8-OH-DPAT to rat hippocampal membranes up to 44% in comparison with experiments in the presence of Mg 2+, while no significant differences were found in rat cortical membranes. Similar increase in high-affinity agonist binding sites by Mn 2+ was found in displacement curves of 8-OH-DPAT, where antagonist [ 3H]WAY100635 was used as reporter ligand. The removal of bivalent ions with EDTA caused full loss of high-affinity binding of agonists, but not for antagonists. Therefore it was hypothesized, that the effect of Mn 2+- and Mg 2+-ions was modulated through their action on different G-proteins. Results showed that efficient coupling of G-protein and 5-HT 1A receptors is crucial to modify Mg 2+ and Mn 2+ effects, whereas Mn 2+ is more potent stabilizer of agonist high-affinity binding, especially when GTPγS is present. Using Sf9 cells as model system, we have shown that G i1 proteins are required to modulate Mn 2+-dependent high-affinity agonist binding to 5-HT 1A receptors, but further studies are necessary to find the cofacors of Mn 2+ modulation to signal transduction.

Antidepressants Targeting the Serotonin Reuptake Transporter Act via a Competitive Mechanism

Although several antidepressants (including fluoxetine, imipramine, citalopram, venlafaxine, and duloxetine) are known to inhibit the serotonin transporter (SERT), whether or not these molecules compete with 5-hydroxytryptamine (serotonin) (5-HT) for binding to SERT has remained controversial. We have performed radioligand competition binding experiments and found that all data can be fitted via a simple competitive interaction model, using Cheng-Prusoff analysis (Biochem Pharmacol 22:3099-3108, 1973). Two different SERT-selective radioligands, [(3)H]N,N-dimethyl-2-(2-amino-4-cyanophenyl thio)-benzylamine (DASB) and [(3)H]S-citalopram, were used to probe competitive binding to recombinantly expressed human SERT or native SERT in rat cortical membranes. All the SERT inhibitors that we tested were able to inhibit [(3)H]DASB and [(3)H]S-citalopram binding in a concentration-dependent manner, with unity Hill coefficient. In accordance with the Cheng-Prusoff relationship for a competitive interaction, we observed that test compound concentrations associated with 50% maximal inhibition of radiotracer binding (IC(50)) increased linearly with increasing radioligand concentration for all ligands: 5-HT, S-citalopram, R-citalopram, paroxetine, clomipramine, fluvoxamine, imipramine venlafaxine, duloxetine, indatraline, cocaine, and 2-beta-carboxy-3-beta-(4-iodophenyl)tropane. The equilibrium dissociation constant of 5-HT and SERT inhibitors were also derived using Scatchard analysis of the data set, and they were found to be comparable with the data obtained using the Cheng-Prusoff relationship. Our studies establish a reference framework that will contribute to ongoing efforts to understand ligand binding modes at SERT by demonstrating that 5-HT and the SERT inhibitors tested bind to the serotonin transporter in a competitive manner.

Pharmacological characterization of 3H‐HYDIA a new tritiated ligand selective for mGlu2/3 receptors

Metabotropic glutamate receptor 2 and 3 have been involved in dementia, depression and schizophrenia and selective ligands are under development. This study contains the pharmacological characterization of a new radioligand 3H‐HYDIA: the selective and competitive mGlu2/3 antagonist (1S,2R,3R,5R,6S)‐2‐amino‐3‐hydroxy‐3‐tritio‐bicyclo[3.1.0]hexane‐2,6‐dicarboxylic acid RO0653479‐002‐002. This radioligand was generated after the observation that the introduction of the R‐configurated 3‐hydroxy‐group was sufficient to turn the potent mGlu2/3 agonist LY354740 into a selective mGlu2/3 antagonist (see Woltering T. J. et al. ChemMedChem 2007). The in vitro binding of 3H‐HYDIA was characterized in recombinant rat and human mGlu2 and mGlu3, in rat cortical and hippocampal membranes as well as in rat brain sections. Ionotropic glutamate receptor ligands, group I and III selective mGlu receptor ligands and negative allosteric modulators for group II mGlus did not inhibit 3H‐HYDIA binding. Several known mGluII ligands e.g. LY341495, LY354740, MGS0039, (2S,2′R,3′R)‐2‐(2′,3′‐dicarboxycyclopropyl)glycine, glutamate, completely inhibited the specific binding. This radioligand, a unique molecule interacting at the ortosteric binding site of the receptors, may serve as useful tools for the study of the molecular determinants of activity in the glutamate binding pocket of mGlu2/3.

Dantrolene antagonizes the glycineB site of the NMDA receptor

In this study we tested the hypothesis that dantrolene, an established inhibitor of the skeletal muscle isoform of the ryanodine receptor, may interfere with activity of NMDA receptors in neurons. We assessed the effects of dantrolene on [(3)H]MK-801 and [(3)H]glycine binding to isolated rat cortical membranes. Dantrolene inhibited [(3)H]MK-801 binding in the presence of 100 microM NMDA with an IC(50) of 58.4 microM. The IC(50) value increased to 99.6, 343.0 and 364.6 microM in the presence of 10, 30 and 50 microM glycine, respectively, suggesting that dantrolene competes with glycine for binding site at the NMDA receptor complex. A binding assay using [(3)H]glycine confirmed this supposition: dantrolene inhibited strychnine-insensitive glycine binding in a dose-dependent way. Thus, our results show that dantrolene at concentrations of 50-100 microM and higher blocks the glycine binding site of the NMDA receptor complex and in this way inhibits activation of the NMDA ion channel. These data reveal a new mechanism of dantrolene action in neuronal tissue. Our results also suggest that the neuroprotective effect of dantrolene may be at least partly explained by its activity as a non-competitive antagonist of NMDA receptors.

P.2.d.024 Differential sensitivity to SSRIs in the tail suspension test in NMRI and C57 mice: Relation to brain 5-HT levels

6-Nitroquipazine has been known as one of the most potent and selective inhibitors of serotonin transporter in vitro and in vivo. Nine derivatives of 6-nitroquipazine were synthesized and tested for their potential abilities to displace [3H]citalopram binding to the rat cortical membranes.

Structural Requirements of Maxadilan, a Non-Mammalian Potent Vasodilatory Peptide, for Interaction with the PAC-1 Receptor from Rat Brain Using a Synthetic Mini-Library

Although there is no amino acid sequence similarity between maxadilan (Maxa) and pituitary adenylate cyclase activating polypeptide (PACAP), our synthetic Maxa was found to bind PACAP specific receptors (PAC-1 receptors) with a high affinity, but low potency for the accumulation of cAMP in PC12 cells. Competitive binding studies of 125I-PACAP-27 to rat cortical membranes allowed exploration of the structural requirements for this interaction using mini-libraries constructed by solid-phase peptided synthesis, that include disulfide isomers, N-, C- and middle segment deleted peptides and analogs. Maxa as well as PACAP38 inhibited the specific binding of 125I-PACAP-27 with IC50 values of 3.89 and 4.90 nM, respectively. The most potent derivative of our synthetic Maxa-analogs with an IC50 value of 1.99 nM was Maxa[1–23 + 43–61, S–S14–51 Ala1,5] which consists of N- (position 1–23) and C- (position 43-61) terminal linear fragments cross-linked by a disulfide bridge between positions 14 and 51. This peptide did not increase intracellular cAMP, at a concentration of 100 nM, but inhibited cAMP accumulation induced by 1 nM PACAP-27 in PC12 cells, whereas wild Maxa increased intracellular cAMP although it was weaker than PACAP-27. Our data suggest deletion of the middle segment between residues 24–42 affords some derivatives that behave as low affinity antagonists.