Rat Cerebral Cortex Membranes Research Articles

Changes in [3H]-ouabain and [3H]-neurotensin binding to rat cerebral cortex membranes after administration of antipsychotic drugs haloperidol and clozapine

Evidences indicate the relationship between neurotensinergic and dopaminergic systems. Neurotensin inhibits synaptosomal membrane Na+, K+-ATPase activity, an effect blocked by SR 48692, antagonist for high affinity neurotensin receptor (NTS1) type. Assays of high affinity [3H]-ouabain binding (to analyze K+ site of Na+, K+-ATPase) show that in vitro addition of neurotensin decreases binding. Herein potential interaction between NTS1 receptor, dopaminergic D2 receptor and Na+, K+-ATPase was studied. To test the involvement of dopaminergic D2 receptors in [3H]-ouabain binding inhibition by neurotensin, Wistar rats were administered i.p.with antipsychotic drugs haloperidol (2mg/kg) and clozapine (3, 10 and 30mg/kg). Animals were sacrificed 18h later, cerebral cortices harvested, membrane fractions prepared and high affinity [3H]-ouabain binding assayed in the absence or presence of neurotensin at a 10 micromolar concentration. No differences versus controls for basal binding or for binding inhibition by neurotensin were recorded, except after 10mg/kg clozapine. Rats were administered with neurotensin (3, 10y 30μg, i.c.v.) and 60min later, animals were sacrificed, cerebral cortices harvested and processed to obtain membrane fractions for high affinity [3H]-ouabain binding assays. Results showed a slight but statistically significant decrease in binding with the 30μg neurotensin dose. To analyze the interaction between dopaminergic D2 and NTS1 receptors, [3H]-neurotensin binding to cortical membranes from rats injected with haloperidol (2mg/kg, i.p.) or clozapine (10mg/kg) was assayed. Saturation curves and Scatchard transformation showed that the only statistically significant change occurred in Bmax after haloperidol administration. Hill number was close to the unit in all cases. Results indicated that typical and atypical antipsychotic drugs differentially modulate the interaction between neurotensin and Na+, K+-ATPase. At the same time, support the notion of an interaction among dopaminergic and neurotensinergic systems and Na+, K+-ATPase at central synapses.

Allosteric effect of serotonin and mianserin on the kinetics of specific [3H]-ligand binding to adrenergic and muscarinic receptors in the rat cerebral cortex membranes

The effects of serotonin receptor activation (by serotonin) and inhibition (by mianserin) on the properties of the α1-, α2-adrenoreceptors, and muscarinic cholinergic receptors in subcellular membrane fractions from the rat cerebral cortex were studied. Experimental data on the kinetics of specific antagonists binding to adrenergic and muscarinic receptors were analyzed by graphical and mathematical methods. The results suggest the presence of allosteric (cross-talk) interaction. In the control, α1- and α2-adrenoreceptors were represented by a single pool, and muscarinic receptors, by two pools. Two pools of adrenoreceptors with different affinity were detected against the background of serotonin. It was found that mianserin induces the formation of two pools of only (α2-receptors and muscarinic receptors are represented by two pools differing in the main parameters, such as dissociation constants and adrenoreceptor concentrations, in the control and experimental groups. It was shown that the allosteric effect of serotonin and mianserin is manifested in the inhibition of muscarinic receptors. It was assumed that the adrenergic and cholinergic receptors exist as dimers. The interaction between the adrenergic, cholinergic, and serotonergic systems is likely to be implemented at the cell membrane level.

Serotoninergic agonist and antagonist are modulators of the α2-adrenoceptor activity in rat brain cortex membranes

The influence of activation and inhibition of serotonin receptors by serotonin (5HT) and miancerin on binding of specific nonselective α2-antagonist [3H]RX821002 in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction for α2-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of the [3H]RX821002 binding to α2-adrenoceptors were as follows: Kd = 1.57 ± 0.276 nM, Bmax = 7.24 ± 1.63 fmol/mg protein, n = 2. In the case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters Kd1 = 0.82 ± 0.06; Kd2 = 2.65 ± 0.22 nM; Bm1 = 1.65 ± 0.23; Bm2 = 4.20 ± 0.11 fmol/mg protein; n = 2. The affinity of high-affinity receptors increased twofold and the affininty of low-affinity receptors decreased by 69% as compared to control values. The concentration of high-affinity receptors decreased 4.4-fold, and of low-affinity, 1.7-fold. The value of maximal reaction (Bmax) decreased by 20%. In the case of miancerin-induced inhibition of 5HT-receptors the character of ligand binding also changed; two pools of receptors were detected with the following parameters: Kd1 = 0.48 ± 0.09; Kd2 = 3.79 ± 0.71 nM; B1 = 0.63 ± 0.17; B2 = 4.75 ± 0.21 fmol/mg protein; n = 2. The affinity of high-affinity receptors pool increased by 70% and that of low-affinity receptors decreased by 76% as compared to control values. The concentration of active high-affinity and low-affinity α2-adrenoceptors decreased by 70% and 141%, respectively. The total amount of the receptors (Bmax) decreased by 26%. The data suggest that α2-adrenoceptors in rat cerebral cortex exist as dimers. Modulatory effects of serotonin and miancerin on specific antagonist binding to α2-adrenoceptors may be accomplished by altering the character and binding parameters of the nonselective α2-antagonist [3H]RX821002.

Serotoninergic agonists and antagonists are the modulators of α1-Adrenoceptor activity in rat brain cortical membranes

The effects of activation and inhibition of serotonin receptors by serotonin (5-HT) and mianserin on the specific nonselective α1-antagonist [3H]prazosine binding in rat cerebral cortex membranes was studied. It was shown that the ligand-receptor interaction of α1-adrenoceptors corresponded to the model suggesting the presence of one pool of receptors and the binding of two ligand molecules to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were as follows: Kd =1.85 ± 0.16 nM, Bmax = 31.1 ± 0.3 fmol/mg protein, n = 2. In case of activation of 5HT-receptors by serotonin, the character of ligand binding was different: two pools of receptors were detected with the parameters Kd1 = 0.61 ± 0.04, Kd2 = 3.82 ± 0.15 nM, Bm1 = 6.6 ± 0.7, Bm2 = 25.6 ± 0.4 fmol/mg protein, n = 2. The sensitivity of the high-affinity pool increased threefold and the sensitivity of the low-affinity pool decreased twofold as compared to the control. The value of maximal reaction (Bmax) did not change. In the case of inhibition of 5HT-receptors by mianserin, radioactive ligand is bound to α1-adrenoceptors according to the same model as in the control conditions. The affinity of α1-adrenoceptors to [3H]prazosine decreases twofold and the concentration increases (Kd = 3.97 ± 0.12 nM, Bmax = 40.0 ± 0.5 fmol/mg protein). The data suggest that α1-adrenoceptors in rat cerebral cortex exist as a dimer. The modulatory effects of serotonin and mianserin on the specific binding of [3H]prazosine to α1-adrenoceptors was detected, manifesting itself as changes in the binding parameters and in the general character of ligand-receptor interactions.

The histamine autoreceptor is a short isoform of the H3 receptor

BACKGROUND AND PURPOSE The histamine H(3) receptor was identified as the autoreceptor of brain histaminergic neurons. After its cloning, functional H(3) receptor isoforms generated by a deletion in the third intracellular loop were found in the brain. Here, we determined if this autoreceptor was the long or the short isoform. EXPERIMENTAL APPROACH We hypothesized that the deletion would affect H(3) receptor stereoselectivity. The effects of the enantiomers of two chiral ligands, N(α)-methyl-α-chloromethylhistamine (N(α) Me-αClMeHA) and sopromidine, were investigated on cAMP formation at the H(3(445)) and H(3(413)) receptor isoforms, common to all species. They were further compared with their effects at autoreceptors. They were also compared on [(35)S]GTPγ[S] binding to membranes of rat cerebral cortex, striatum and hypothalamus, the richest area in autoreceptors. KEY RESULTS The stereoselectivity of N(α) Me-αClMeHA enantiomers as agonists was similar at the H(3(413)) receptor isoform and autoreceptors, but lower at the long isoform. While (S) sopromidine did not discriminate between the isoforms, (R) sopromidine was an antagonist at the H(3(413)) receptor isoform and autoreceptors, but a full agonist at the long isoform. In rat brain, stereoselectivity of N(α) Me-αClMeHA was higher in the hypothalamus than in cerebral cortex or striatum, whereas the opposite pattern was found for sopromidine. CONCLUSIONS AND IMPLICATIONS The pharmacological profiles of H(3) receptor isoforms differed markedly, showing that the function of autoreceptors was fulfilled by a short isoform, such as the H(3(413)) receptor. Development of drugs selectively targeting autoreceptors might enhance their therapeutic efficacy and/or decrease incidence of side effects.

Hypnotic effects and GABAergic mechanism of licorice (Glycyrrhiza glabra) ethanol extract and its major flavonoid constituent glabrol

Licorice (Glycyrrhiza glabra, GG) is one of the most frequently used herbal medicines worldwide, and its various biological activities have been widely studied. GG is reported to have neurological properties such as antidepressant, anxiolytic, and anticonvulsant effects. However, its hypnotic effects and the mechanism of GG and its active compounds have not yet been demonstrated. In this study, GG ethanol extract (GGE) dose-dependently potentiated pentobarbital-induced sleep and increased the amount of non-rapid eye movement sleep in mice without decreasing delta activity. The hypnotic effect of GGE was completely inhibited by flumazenil, which is a well-known γ-aminobutyric acid type A-benzodiazepine (GABAA-BZD) receptor antagonist, similar to other GABAA-BZD receptor agonists (e.g., diazepam and zolpidem). The major flavonoid glabrol was isolated from the flavonoid-rich fraction of GGE; it inhibited [3H] flumazenil binding to the GABAA-BZD receptors in rat cerebral cortex membrane with a binding affinity (Ki) of 1.63μM. The molecular structure and pharmacophore model of glabrol and liquiritigenin indicate that the isoprenyl groups of glabrol may play a key role in binding to GABAA-BZD receptors. Glabrol increased sleep duration and decreased sleep latency in a dose-dependent manner (5, 10, 25, and 50mg/kg); its hypnotic effect was also blocked by flumazenil. The results imply that GGE and its flavonoid glabrol induce sleep via a positive allosteric modulation of GABAA-BZD receptors.

Calcium antagonism and structure-affinity relationships of terfenadine, a histamine H1 antagonist, and some related compounds.

Calcium channel affinity of terfenadine and its optical isomers was determined by the displacement of [3H]nitrendipine on rat cerebral cortex membranes. Terfenadine showed a pKd of 6.36 +/- 0.03 whereas its R(+)-isomer (VUF4567) had a pKd value of 6.39 +/- 0.03 and the S(-)-isomer (VUF4568) had a pKd of 6.40 +/- 0.04. The same affinity between the enantiomers suggests that the binding domain on the membrane is not sterically restricted towards the part of the molecule in which the chiral centre is present. The characteristics of terfenadine in regulating [3H]nitrendipine binding were similar to those of some other diphenyl-alkylamine type calcium antagonists. It allosterically altered the binding affinity for nitrendipine and acted at the same site linked to the calcium channel as gallopamil. A structure-affinity relationship among a group of terfenadine analogues is discussed.

Alpha 1-adrenoceptors: the ability of various agonists and antagonists to discriminate between two distinct [3H]prazosin binding sites.

Recently, it has been demonstrated that two distinct alpha 1-adrenoceptor binding sites showing high and low affinity for WB-4101 (2-(2,6-dimethoxyphenoxy)ethyl-aminomethyl-1,4-benzodioxane) and 5-methyl-urapidil can be distinguished. In the present study we examined the ability of several agonists and antagonists to discriminate between these alpha 1-adrenoceptor binding sites. [3H]Prazosin binding to membranes of rat liver, heart, cerebral cortex and hippocampus was inhibited monophasically by butanserine, I-BE 2254 (2-(3-(4-hydroxy-3-iodophenyl)ethylaminomethyl)tetralone-hydrochloride), prazosin, rauwolscine and verapamil. In contrast, competition curves of adrenaline, oxymetazoline, amidephrine and YM-12617 (5-[2-[[2-(o-ethoxy-phenoxy)ethyl]-amino]propyl]-2- methoxybenzenesulfonamide HCl) were best described by a model of two binding sites. Chloroethylclonidine (CEC), a compound shown to irreversibly eliminate binding sites with low affinity for WB-4101, increased the proportion of high affinity binding sites for oxymetazoline and amidephrine, whereas the binding data for prazosin and adrenaline remained unchanged. These results indicate that amidephrine, oxymetazoline and YM-12617, but not the other drugs tested discriminate between different alpha 1-adrenoceptor recognition sites labelled by [3H]prazosin.

Pharmacological characterisation of the GlyT-1 glycine transporter using two novel radioligands

Inhibitors of the glycine transporter GlyT-1 are being developed as potential treatments for schizophrenia. Here we report on the use of two novel radioligands, [ 3H]-SB-733993 and [ 3H]-GSK931145, for the characterisation of GlyT-1 in both cells and native tissue. Binding was evaluated in membranes either from HEK293 cells expressing recombinant human GlyT-1 (hGlyT-1) or from rat cerebral cortex. Specific binding of both [ 3H]-SB-733993 and [ 3H]-GSK931145 to hGlyT-1 HEK293 cell membranes and rat cerebral cortex membranes was saturable and comprised >90% of total binding. K d and B max values for the two radioligands were fairly similar, with K d values of 1–2 nM and B max values of around 7000 fmol/mg protein in hGlyT-1 membranes and 3000 fmol/mg protein in rat cortex membranes. Association of [ 3H]-SB-733993 was faster, with binding reaching equilibrium within 30 min compared with 90 min for [ 3H]-GSK931145. Dissociation was also much slower for [ 3H]-GSK931145 than for [ 3H]-SB-733993, with 50% of specific binding being dissociated by approximately 40 min and 5 min, respectively. Autoradiography studies with [ 3H]-GSK931145 showed widespread distribution of binding in rat brain, with generally higher binding in caudal compared with rostral areas. Initial studies in human frontal cortex membranes showed clear specific binding of [ 3H]-GSK931145, though with much lower density ( B max 570 fmol/mg protein) and slightly lower affinity (K d 4.5 nM) compared with rat cortex. A human brain autoradiography study showed higher specific binding in cerebellum compared with frontal cortex. All GlyT-1 inhibitors tested, as well as glycine itself, competed fully for the binding of both [ 3H]-SB-733993 and [ 3H]-GSK931145 in both hGlyT-1 and rat cortex membranes. Studies on the effect of varying NaCl concentration showed that [ 3H]-SB-733993 binding was reduced by >90% in the absence of added Na + ions, whilst [ 3H]-GSK931145 binding was unaffected. Glycine produced concentration-dependent decreases in binding affinity of both radioligands without major changes in B max values, suggesting that both [ 3H]-SB-733993 and [ 3H]-GSK931145 bind to sites on GlyT-1 that are orthosteric to the site at which glycine itself binds. Overall, these results show that both [ 3H]-SB-733993 and [ 3H]-GSK931145 are useful radioligands for studies on GlyT-1 in both cell lines and native tissues, with [ 3H]-GSK931145 being the radioligand of choice for further studies on GlyT-1 expression and pharmacology.

Muscarinic cholinoceptor agonists and antagonists are modulators of the activity of α1-adrenoceptors in the membranes of rat cerebral cortex

The effects of activation and inhibition of muscarinic cholinoceptors by carbachol and atropine on the binding of specific nonselective α1-antagonist [3H]prazosine in synaptosomal membranes of rat cerebral cortex have been studied. It has been shown that the ligand-receptor interaction of α1-adrenoceptors corresponds to the model suggesting the presence of a single receptor pool and the binding of two ligand molecules to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were as follows: Kd = 1.56 ± 0.17 nM, Bmax = 30.25 ± 1.78 fmol/mg protein, n = 2. Upon inhibition of muscarinic cholinoceptors by atropine or their activation by carbachol, the radiolabelled ligand is bound to α1-adrenoceptors according to the same model but at n = 1. In the presence of atropine, the sensitivity of α1-adrenoceptors to [3H]prazosine decreases more than twofold (Kd = 3.52 ± 0.36 nM) and the concentration of the active receptors is 36% lower (Bmax = 19.45 ± 1.46 fmol/mg protein). Carbachol does not reduce the affinity of adrenoceptors to the ligand, while the concentration of active receptors decreases like in the case of atropine. It is supposed that α1-adrenoceptors in the membranes of rat cerebral cortex exist as dimers. The modulating effects of atropine and carbachol on the binding of specific antagonist by α1-adrenoceptors are exhibited as changes in the general character of binding (monomerization of α1-adrenoceptors) and as inhibitory effect on the [3H]prazosine binding parameters.

Synthesis and biological activity of the calcium modulator ( R) and ( S)-3-methyl 5-pentyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate

An efficient total synthesis of ( R) and ( S)-3-methyl 5-pentyl 2,6-dimethyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3,5-dicarboxylate in high optical purities is reported. The useful step is the resolution of racemic 2, 6-dimethyl-5-methoxycarbonyl-4-(3-nitrophenyl)-1,4-dihydropyridine-3-carboxylic acid by using commercially available Cinchona alkaloids cinchonidine and quinidine as the resolving agents. Under the optimum conditions, the optical purities for R- and S-enantiomers are extremely high (ee >99.5%). The further dihydropyridine receptor binding activity assay shows that the S-enantiomer is more potent than R-enantiomer both in rat cardiac (approximately 19 times) and cerebral cortex membrane (12 times).

Allosteric modulation of the [3H]quinuclidinyl benzylate binding to M-cholinoreceptors in rat cerebral cortex by adrenotropic agents

The allosteric effects of adrenotropic drugs and the membranotropic agent cocaine on the kinetics of [3H]quinuclidinyl benzylate ([3H]QNB) binding to muscarine cholinoceptors of synaptosomal membranes of rat cerebral cortex were studied. In control, the best results were obtained for the model that assumes the existence of two receptor pools (with high and low affinity) with calculated parameters of the activity (K d), amount (B max), and mono- to dimer receptors ratio (n). For the high-affinity receptors these parameters were K d1 = 0.18 ± 0.08 nM, B m1 = 221.2 ± 56.7 fmol/mg protein, n 1 = 2, and for low-affinity receptors, K d2 = 1.33 ± 0.11 nM, B m2 = 748.7 ± 53.3 fmol/mg protein, n 2 = 2. Allosteric modulation of the activity of specific neurotransmitter receptors can be accomplished by changing the receptor affinity and amount as well as the proportion of mono- and dimer receptors. Under control conditions, muscarine receptors exist as dimers. In the presence of α-adrenoreceptor agonist noradrenaline and β-adrenoreceptor antagonist propranolol, only one pool of the dimer muscarine receptors remains. The number of binding sites for noradrenaline and propranolol decreases approximately by 40% and 20%, respectively. The agonist of α2-adrenoreceptors clonidine, the antagonist of α2-adrenoreceptors yohimbine, and a membranotropic agent cocaine change the ligand binding so that only one receptor pool remains but some of the dimer receptors become monomeric (1 < n < 2). The amount of binding sites reduces by 20%, 25%, and 45% for clonidine, yohimbine, and cocaine, respectively.

Comparative characteristics of binding kinetics of specific antagonists by α1- and α2-adrenoceptors in rat cerebral cortex membranes

The binding of specific nonselective α1- and α2-adrenoceptor antagonists [3H]prazosine and [3H]RX821002 has been studied on rat cerebral cortex synaptosomal membranes. It is shown that for α1-adrenoceptors the ligand-receptor interaction corresponds to the model assuming the presence of one pool of receptors and binding of two ligand molecules to the receptor. The parameters of [3H]prazosine binding to α1-adrenoceptors were: Kd= 1.56 ± 0.17 nM, Bmax = 30.25 ± 1.78 fmol/mg protein, n = 2. The parameters of [3H]RX821002 binding to α2-adrenoceptors were: Kd = 1.94 ± 0.08 nM, Bmax = 12.77 ± 3.17 fmol/mg protein, n = 2. For α2 -adrenoceptors the ligand-receptor interaction corresponded to the same model. For α1 - and α2-adrenoceptor antagonists the dissociation constants (Kd) are approximately equal (1.56 ± 0.17 and 1.94 ± 0.08 nM, respectively), but the concentration of α2-adrenoceptors is two times lower than that of α1-adrenoceptors ( 12.77 ± 3.17 and 30.25 ± 1.78 fmol/mg protein, respectively). The efficiency (E = Bmax/2Kd) of the ligand binding to α1-adrenoceptors is 2.3 times higher than that to α2-adrenoceptors (7.46 ± 1.32 and 3.29 ± 0.68 fmol/mg protein/nM, respectively. The data suggest that α1- and α2 -adrenoceptors in rat cerebral cortex exist as dimers.

Comparison of the binding affinity of CGP-12177A at recombinant rat α 1D-adrenoceptors expressed in BHK-21 cell membranes and α 1-adrenoceptors present in rat cerebral cortex membranes

Recent in vitro studies, performed in rat aorta, mesenteric and intrapulmonary arteries, and human pulmonary artery, demonstrated that the β-adrenoceptor ligand CGP-12177A (4-[3-[(1,1-dimethylethyl)amino]-2-hydroxypropoxy]-1,3-dihydro-2H-benzimidazol-2-one) is also provided with antagonist or partial agonist properties at α 1-adrenoceptors. These observations were supported by estimates of CGP-12177A binding affinity at α 1-adrenoceptors, which have been always performed in rat cerebral cortex membranes, as a surrogate of vascular tissue. Since α 1D-adrenoceptors are predominant in both rat aorta and mesenteric artery, in the present study, we measured, for the first time, the binding affinity of CGP-12177A at recombinant rat α 1D-adrenoceptors expressed in BHK-21 cell membranes. CGP-12177A binding affinity was also determined in rat cerebral cortex membranes, where various α 1-adrenoceptor subtypes are present. By means of [ 3H]prazosin binding competition experiments, we found that CGP-12177A bound to α 1D-adrenoceptor-expressing BHK-21 cell membranes, with a binding affinity (p K i = 5.39 ± 0.27) almost identical to that measured in cerebral membranes (p K i = 5.44 ± 0.07), indicating that it is a non-subtype selective α 1-adrenoceptor ligand. Moreover, CGP-12177A binding affinity was very close to its functional affinity evaluated in rat aorta in terms of antagonist potency against phenylephrine-induced contraction (p K B = 5.65 ± 0.07). In conclusion, our results demonstrate that, in order to evaluate CGP-12177A binding affinity at aorta and mesenteric artery α 1-adrenoceptors, estimates in rat cerebral membranes are as reliable as those in recombinant rat α 1D-adrenoceptors, since both values are very close to CGP-12177A functional affinities in isolated vessels.

Kinetics of [3H]prazosin and [3H]dihydroalprenolol binding to α1- and β-adrenoreceptors in rat brain cortex membranes

The binding of nonselective α1- and β-adrenoreceptor antagonists [3H]prazosin and [3H]dihydroalprenolol ([3H]DHA) to rat cerebral cortex synaptosomal membranes has been studied. It is found that ligand-receptor interactions of α1-adrenoreceptors fit into a single receptor pool model, which assumes the binding of two ligand molecules to one receptor molecule. The parameters of [3H]prazosin binding to α1-adrenoreceptors are as follows: K d = 2.58 ± 0.20 nM; B m = 2.95 ± 1.12 fmol/mg protein; Hill coefficient, n = 2. For β-adrenoreceptors, ligand-receptor interactions fit into a model assuming the presence of two receptor pools in the same effector system and binding of two ligand molecules to one receptor molecule. The corresponding parameters of the [3H]DHA binding to β-adrenoreceptors are as follows: K d1 = 0.74 ± 0.09 nM; K d2 = 7.63 ± 0.70 nM; B m1 = 25 ± 2 fmol/mg, B m2 = 48 ± 2 fmol/mg, n 1 = 2; n 2 = 2. We suggest that in rat cerebral cortex membranes α-and β-adrenoreceptors exist as dimers.

The Expression of NMDA Receptor Subunits in Cerebral Cortex and Hippocampus is Differentially Increased by Administration of Endobain E, a Na+, K+-ATPase Inhibitor

Previous studies showed that endobain E, an endogenous Na+, K+-ATPase inhibitor, decreases dizocilpine binding to NMDA receptor in isolated membranes. The effect of endobain E on expression of NMDA receptor subunits in membranes of rat cerebral cortex and hippocampus was analyzed by Western blot. Two days after administration of 10 mul endobain E (1 microl = 29 mg fresh tissue) NR1 subunit expression enhanced 5-fold and 2.5-fold in cerebral cortex and hippocampus, respectively. NR2A subunit expression increased 2-fold in cerebral cortex and 1.5-fold in hippocampus. The level of NR2B subunit raised 3-fold in cerebral cortex but remained unaltered in hippocampus. NR2C subunit expression was unaffected in either area. NR2D subunit enhanced 1.6 and 2.1-fold for cerebral cortex and hippocampus, respectively. Results indicate that endogenous Na+, K+-ATPase inhibitor endobain E differentially modifies the expression of NMDA receptor subunits.

Thermodynamic analysis of ligands at cholecystokinin CCK2 receptors in rat cerebral cortex.

Several studies using radioligand binding assays, have shown that measurement of thermodynamic parameters can allow discrimination of agonists and antagonists (Weiland et al., 1979; Borea et al., 1996a). Here we investigate whether agonists and antagonists can be thermodynamically discriminated at CCK(2) receptors in rat cerebral cortex. The pK(L) of [(3)H]-JB93182 in rat cerebral cortex membranes was determined at 4, 12, 21 and 37 degrees C in 50 mM Tris-HCl buffer (buffer B pH 6.96; containing 0.089 mM bacitracin). pK(I) values of ligands of diverse chemical structure and with differing intrinsic activity (alpha), as defined by the lumen-perfused rat and mouse stomach bioassays, were determined in buffer B at 4, 12, 21 and 37 degrees C. [(3)H]-JB93182 labelled a homogeneous population of receptors in rat cerebral cortex at 4, 12, 21 and 37 degrees C and the pK(L) and B(max) were not altered by incubation temperature. [(3)H]-JB93182 binding reached equilibrium after 10, 50, 90 and 220 min at 37, 21, 12 and 4 degrees C, respectively. pK(I) values for R-L-365,260, R-L-740,093, YM220, PD134,308 and JB95008 were higher at 4 degrees C than at 37 degrees C. There was no effect of temperature on pK(I) values for pentagastrin, CCK-8S, S-L-365,260, YM022, PD140,376 and JB93242. CCK(2) receptor agonists and antagonists at rat CCK(2) receptors cannot be discriminated by thermodynamic analysis using [(3)H]-JB93182 as the radioligand.

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.

In vitro and antinociceptive profile of HON0001, an orally active NMDA receptor NR2B subunit antagonist

The analgesic activity and side effect liabilities of a novel NR2B antagonist, 7-hydroxy-6-methoxy-2-methyl-1-(2-(4-(trifluoromethyl)phenyl)ethyl)-1,2,3,4-tetrahydroisoquinoline hydrochloride (HON0001) were investigated. HON0001 inhibited [ 3H]MK-801 binding to rat brain membranes in a biphasic manner, with IC 50 values of 54.68 ± 4.96 nM and 46.48 ± 5.85 μM for high- and low-affinity sites, respectively. HON0001 inhibited [ 3H]ifenprodil binding to membranes of rat cerebral cortex with an IC 50 value of 57.01 ± 3.4 nM, consistent with the results obtained for high-affinity sites of [ 3H]MK-801 binding. HON0001 exhibited no or negligible affinity for other receptors, transporters and ion channels, while HON0001 had a moderate agonistic activity at μ-opioid receptors and affinity for dopamine D 1 receptors. HON0001 exhibited an analgesic effect in carrageenan-induced mechanical hyperalgesia and in the Seltzer model of partial sciatic nerve ligation following oral administration. In contrast, unlike MK-801, HON0001 did not affect spontaneous locomotor activity, rotarod performance and step-through latency in a passive avoidance task even at doses much higher than antinociceptive doses. HON0001 exhibited excellent brain penetration with a brain-to-plasma ratio of 34.5. These findings show that HON0001 is an orally active NR2B antagonist and that it may be useful for treating patients with neuropathic and other conditions without causing the side effects often observed with currently available non-subtype selective NMDA receptor antagonists.

Cannabidiol is an allosteric modulator at mu- and delta-opioid receptors

The mechanism of action of cannabidiol, one of the major constituents of cannabis, is not well understood but a noncompetitive interaction with mu opioid receptors has been suggested on the basis of saturation binding experiments. The aim of the present study was to examine whether cannabidiol is an allosteric modulator at this receptor, using kinetic binding studies, which are particularly sensitive for the measurement of allosteric interactions at G protein-coupled receptors. In addition, we studied whether such a mechanism also extends to the delta opioid receptor. For comparison, (-)-Delta9-tetrahydrocannabinol (THC; another major constituent of cannabis) and rimonabant (a cannabinoid CB1 receptor antagonist) were studied. In mu opioid receptor binding studies on rat cerebral cortex membrane homogenates, the agonist 3H-DAMGO bound to a homogeneous class of binding sites with a KD of 0.68+/-0.02 nM and a Bmax of 203+/-7 fmol/mg protein. The dissociation of 3H-DAMGO induced by naloxone 10 microM (half life time of 7+/-1 min) was accelerated by cannabidiol and THC (at 100 microM, each) by a factor of 12 and 2, respectively. The respective pEC50 values for a half-maximum elevation of the dissociation rate constant k(off) were 4.38 and 4.67; 3H-DAMGO dissociation was not affected by rimonabant 10 microM. In delta opioid receptor binding studies on rat cerebral cortex membrane homogenates, the antagonist 3H-naltrindole bound to a homogeneous class of binding sites with a KD of 0.24+/-0.02 nM and a Bmax of 352+/-22 fmol/mg protein. The dissociation of 3H-naltrindole induced by naltrindole 10 microM (half life time of 119+/-3 min) was accelerated by cannabidiol and THC (at 100 microM, each) by a factor of 2, each. The respective pEC50 values were 4.10 and 5.00; 3H-naltrindole dissociation was not affected by rimonabant 10 microM. The present study shows that cannabidiol is an allosteric modulator at mu and delta opioid receptors. This property is shared by THC but not by rimonabant.