Rat Cerebellum Membranes Research Articles

Coronaridine congeners potentiate GABAA receptors and induce sedative activity in mice in a benzodiazepine-insensitive manner

To determine whether (+)-catharanthine induces sedative- or anxiolytic/anxiogenic-like activity in male mice, proper animal paradigms were used. The results showed that (+)-catharanthine induces sedative-like activity in the 63–72 mg/Kg dose range in a flumazenil-insensitive manner, but neither this effect nor anxiolytic/anxiogenic-like activity was observed at lower doses. To determine the underlying molecular mechanism of the sedative-like activity, electrophysiological and radioligand binding experiments were performed with (+)-catharanthine and (±)-18-methoxycoronaridine [(±)-18-MC] on GABAA (GABAARs) and glycine receptors (GlyRs). Coronaridine congeners both activated and potentiated a variety of human (h) GABAARs, except hρ1. (+)-Catharanthine-induced potentiation followed this receptor selectivity (EC50's in μM): hα1β2 (4.6 ± 0.8) > hα2β2γ2 (12.6 ± 3.8) ~ hα1β2γ2 (14.4 ± 4.6) indicating that both α1 and α2 are equally important, whereas γ2 is not necessary. (+)-Catharanthine was >2-fold more potent and efficient than (±)-18-MC at hα1β2γ2. (+)-Catharanthine also potentiated, whereas (±)-18-MC inhibited, hα1 GlyRs with very low potency. Additional [3H]-flunitrazepam competition binding experiments using rat cerebellum membranes clearly demonstrated that these ligands do not bind to the benzodiazepine site. This is supported by the observed activity at hα1β2 (lacking the BDZ site) and similar effects between α1- and α2-containing GABAARs. Our study shows, for the first time, that (+)-catharanthine induced sedative-like effects in mice, and coronaridine congeners potentiated human α1β2γ2, α1β2, and hα2β2γ2, but not ρ1, GABAARs, both in a benzodiazepine-insensitive fashion, whereas only (+)-catharanthine slightly potentiated GlyRs.

Arachidonoyl amino acids and arachidonoyl peptides: Synthesis and properties

N-Arachidonoyl (AA) derivatives of amino acids (glycine, phenylalanine, proline, valine, gamma-amino butyric acid (GABA), dihydroxyphenylalanine, tyrosine, tryptophan, and alanine) and peptides (Semax, MEHFPGP, and PGP) were synthesized in order to study the biological properties of acylamino acids. The mass spectra of all the compounds at atmospheric pressure electrospray ionization display the most intense peaks of protonated molecular ions; the detection limits for these compounds are 10 fmol per sample. AA-Gly showed the highest inhibitory activity toward fatty acid amide hydrolase from rat brain (IC50 6.5 microM) among all the acylamino acids studied. AA-Phe, AA-Tyr, and AA-GABA exhibited a weak but detectable inhibitory effect (IC50 55, 60, and 50 microM, respectively). The acylated amino acids themselves, except for AA-Gly, were stable to the hydrolysis by this enzyme. All the arachidonoylamino acids inhibited cabbage phospholipase D to various degrees; AA-GABA and AA-Phe proved to be the most active (IC50 20 and 27 microM, respectively). Attempts to detect the biosynthesis of AA-Tyr in homogenates of rat liver and nerve tissue showed no formation in vitro of either this acylamino acid or AA-dopamine and AA-Phe, the products of its metabolism. The highest contents of these metabolites were detected in liver homogenate and in the brain homogenate, respectively. Acylamino acids exert no cytotoxic effect toward the glioma C6 cells. It was shown that N-acylation of Semax with arachidonic acid results in enhancement of its hydrolytic stability and increases its affinity for the sites of specific binding in rat cerebellum membranes. The English version of the paper: Russian Journal of Bioorganic Chemistry, 2006, vol. 32, no. 3; see also http://www.maik.ru.

Novel 17β-Substituted Conformationally Constrained Neurosteroids that Modulate GABAA Receptors

The goal of this study was to develop a series of allopregnanolone analogues substituted by conformationally constrained 17beta side chains to obtain additional information about the structure-activity relationship of 5alpha-reduced steroids to modulate GABA(A) receptors. Specifically, we introduced alkynyl-substituted 17beta side chains in which the triple bond is either directly attached to the 17beta-position or to the 21-position of the steroid skeleton. Furthermore, we investigated the effects of C22 and C20 modification. The in vitro binding affinity for the GABA(A) receptor of the new analogues was measured by allosteric displacement of the specific binding of [(3)H]4'-ethynyl-4-n-propyl-bicycloorthobenzoate (EBOB) to GABA(A) receptors on synaptosomal membranes of rat cerebellum. An allosteric binding model that has been successfully applied to ionotropic glycine receptors was employed. The most active derivative is (20R)-17beta-(1-hydroxy-2,3-butadienyl)-5alpha-androstane-3-ol (20), which possesses low nanomolar potency to modulate cerebellar GABA(A) receptors and is 71 times more active than the control compound allopregnanolone. Theoretical conformational analysis was employed in an attempt to correlate the in vitro results with the active conformations of the most potent of the new analogues.

Synthesis and evaluation of [ 123I]labelled analogues of the partial inverse agonist Ro 15-4513 for the study of diazepam-insensitive benzodiazepine receptors

The imidazobenzodiazepines ethyl 8-iodo-5,6 dihydro-5-methyl-6-oxo-4H-imidazo[1,5a][1,4] benzodiazepine-3-carboxylate 1 and tert-butyl 8-iodo-5,6 dihydro-5-methyl-6-oxo-4H-imidazo [1,5a][1,4] benzodiazepine-3-carboxylate 2 were prepared to study the diazepam-insensitive (DI) benzodiazepine receptor (BZR) subtype. The [ 123I] analogues were prepared via iododestannylation reactions in radiochemical yields of 70–80% and a specific activity >2,500 Ci/mmol. The tert-butyl analogue [ 123I]-2 exhibited nanomolar affinity for BZRs in homogenate membranes of rat cerebellum with K d values for the diazepam-sensitive (DS) and DI receptors of 3.18 ± 0.58 and 13.55 ± 2.72 nM, respectively. The B max for cerebellar DS and DI receptors were 1,276 ± 195 and 518 ± 26 fmol/mg protein, respectively.

Localization and pharmacological characterization of somatostatin sst2 sites in the rat cerebellum.

Radioligand binding studies were performed in membranes of rat cerebellum using [125I]-[Tyr3]octreotide ([125I]204-090) to characterize the nature of cerebellar somatostatin receptors. Saturation experiments suggest the presence of a single class of binding sites with high affinity, pKd = 9.53 +/- 0.11, but low receptor density, Bmax = 12.7 +/- 1.0 fmol/mg protein. The pharmacological profile of [125I]204-090 sites in cerebellar membranes was established using a range of ligands known to interact with SSTR-2 (now called sst2) and other somatostatin (SRIF) receptors. SRIF analogues such as octreotide (SMS 201-995), seglitide (MK 678) and somatuline (BIM 23014) displayed very high affinity for cerebellar [125I]204-090 binding sites. The data were compared to results obtained using the same ligand in rat cerebral cortex membranes known to represent sst2 binding. The pharmacological characteristics of the cerebellar sites were in close correlation with those of the cerebral cortex (r = 0.976, n = 19, p < 0.001) and CHO-cells expressing human recombinant sst2 receptor (r = 0.977, n = 19, p < 0.001). By contrast, there was very little correlation between cerebellar binding and published affinities for rat sst5 receptors (r = 0.465), for which octreotide has also high affinity. In vitro autoradiographic studies performed in cerebellar slices using [125I]204-090 demonstrated the presence of binding sites in the molecular layer of the rat cerebellum. In situ hybridization studies using sst2 receptor mRNA selective oligoprobes confirmed the presence of sst2 receptor mRNA in the rat cerebellum. Together, the present data demonstrate the presence of a low density of SRIF receptors in the molecular layer of the adult rat cerebellum which are best characterized as sst2. This is the first pharmacological characterization and localization of sst2 receptors in the adult rat cerebellum.

Characterisation of stereospecific binding sites for inositol 1,4,5‐trisphosphate in airway smooth muscle

1. A 'P2' membrane fraction of bovine tracheal smooth muscle displays high affinity (KD 3.8 +/- 0.2 nM), saturable (Bmax 1003 +/- 170 fmol mg-1 protein) and reversible binding of D-myo[3H]-inositol 1,4,5-trisphosphate ([3H]-Ins(1,4,5)P3). 2. This binding site shows strict stereo- and positional specificity for the D-Ins(1,4,5)P3 isomer with L-Ins(1,4,5)P3, DL-Ins(1,3,4,5)P4 and D-Ins(1,3,4)P3 displacing [3H]-Ins(1,4,5)P3 with Ki values of 20 microM, 0.35 microM and 2.4 microM, respectively. 3. Specific binding of [3H]-Ins(1,4,5)P3 is enhanced at alkaline pH values (maximal at pH 7.75) and, in distinct contrast to [3H]-Ins(1,4,5)P3 binding in rat cerebellum membranes, is not inhibited by Ca2+ (5-500 microM). 4. Heparin displaces [3H]-Ins(1,4,5)P3 specific binding with an IC50 of 7.6 +/- 1.0 micrograms ml-1. 5. Comparative studies demonstrated specific [3H]-Ins(1,4,5)P3 binding in bovine cardiac atrial preparations (Bmax 75 +/- 5 fmol mg-1 protein) and very low specific [3H]-Ins(1,4,5)P3 binding in bovine cardiac ventricle and skeletal muscle membranes (less than or equal to 25 fmol mg-1 protein).

The effect of external calcium and pH on inositol trisphosphate-mediated calcium release from cerebellum microsomal fractions.

Binding of D-myo-inositol 1,4,5-trisphosphate (InsP3) to rat cerebellum membranes has previously been shown to be stimulated by alkaline pH and inhibited by low concentrations of Ca2+ [Worley, Baraban, Suppatopone, Wilson & Snyder (1987) J. Biol. Chem. 262, 12132-12136]. In the present study, Scatchard analysis of InsP3 binding to cerebellum microsomes indicates that the effects of Ca2+ and pH are exerted through changes in the apparent affinity of the receptor without effects on maximal binding. The influence of extravesicular Ca2+ and pH on InsP3-mediated 45Ca2+ release was investigated. Extravesicular Ca2+ inhibited InsP3-mediated Ca2+ release. The inhibitory effect of Ca2+ was most marked when a sub-optimal concentration of InsP3 was used. An increase in extravesicular pH produced a decrease in the concentration of InsP3 that yielded half-maximal Ca2+ release. Regulation of the affinity of the InsP3 receptor by Ca2+ and pH can qualitatively account for the observed effects of these factors on InsP3-mediated Ca2+ release. Feedback inhibition of InsP3 binding by Ca2+ could provide a mechanism to generate Ca2+ oscillations, particularly under hormonal conditions that produce sub-optimal elevations of InsP3 concentration.

Specific binding of 36Cl- ions to rat cerebellar membranes: effect of GABA and S-100 protein.

36Cl- ions display specific binding to rat cerebellum membranes. Although this binding at a 136Cl-1 of 2 X 10(-7) M is only 18% of total binding, it shows several interesting characteristics. It is higher in a GABA receptor rich region such as the cerebellum than in the cerebral cortex. It is higher in synaptic than in total membranes of the cerebral cortex. 36Cl- binding to cerebellar membranes in inhibited by 10(-4) M picrotoxin and by 10(-4) M GABA, the GABA effect being antagonized by bicuculline. All these characteristics appear to point out that 36Cl- specifically binds to GABAA receptor associated Cl- channels in their closed state. The brain specific antigen S-100 also is able to inhibit 36Cl- binding to rat cerebellar membranes.

Interactions of putatively irreversible antagonists with β1 − and β2 − adrenergic receptors

Three compounds that have been suggested to irreversibly inactivate β-adrenergic receptors were studied: NHNPNBE [ N-(2-hydroxy-3-[1-napthoxy]-propyl)- N-bromoacetylethylenediamine], BAAM (bromoacetylalprenololmenthane), and Ro 3-7894 [1-(5-chloracetylaminobenzfuran-2-yl)-2-isopropylaminoethanol]. Membranes of rat cerebral cortex were used as a source of predominantly β 1 − adrenergic receptors and membranes of rat cerebellum were used as a source of predominantly β 2 − adrenergic receptors. β− Adrenergic receptor binding sites were studied by Scatchard analysis of saturation isotherms of specific [ 125I]-pindolol ([ 125I]PIN) binding. NHNPNBE added to the incubation medium competitively inhibited specific [125 I]PIN binding in both cerebellum and cerebral cortex with K l values of 1–2 μM. in each tissue. After washout of membranes pretreated with NHNPNBE for 30 min at 37°, no loss of specific [ 125I]PIN binding sites was observed in either cerebellum or cortex except at very high concentrations (30–100 μM). Ro 3-7894 caused a simple competitive inhibition of specific [ 125I]PIN binding in rat cerebellar membranes with a K I , of approximately 14 μM, an effect which was reversed completely by washing. In cerebral cortex, Ro 3-7894 added to the incubation medium apparently decreased the density of [ 125I]PIN binding sites with an IC 50 around 1 μM. This effect was reversed after washing the membranes twice. However, in the presence of Ro 3-7894 some Scatchard plots showed a slight curvature. Further saturation of the [ 125I]PIN binding sites in cerebral cortex showed that the inhibition by Ro 3-7894 was competitive but with a high- and low-affinity component, consistent with Ro 3-7894 being a β 1-selective competitive antagonist. Ro 3-7894 was also β 1ft-selective in other tissues. BAAM added to the incubation medium competitively inhibited specific [ 125I]PIN binding in both cerebellum and cortex with K l values of 0.006 to 0.03 μM, but was about 5-fold more potent in cerebellum. After treatment of membranes with higher concentrations of BAAM for 30 min at 37° and washing twice, there was a dose-dependent decrease in the density of specific [ 125I]PIN binding sites with ic 50 values of approximately 0.3 μM in both tissues. Similar effects were observed in rat heart. These data suggest that NHNPNBE is a simple competitive antagonist at both β 1 − and β 2-adrenergic receptors except at very high concentrations. Ro 3-7894 is a β 1-selective competitive antagonist with no apparent irreversible effects. BAAM is a slightly β 2-selective competitive antagonist which can also irreversibly decrease the density of both β 1- and β 2-adrenergic receptor binding sites at relatively low concentrations.

Irreversible blockade of beta-adrenergic receptors with a bromoacetyl derivative of pindolol.

A potent irreversible beta-adrenergic derivative of pindolol possessing a chemically reactive group (Br-AAM-pindolol) was synthesized. This compound devoid of agonist properties, competed for all (3H)-dihydroalprenolol (3H-DHA) binding sites in C6 glioma cell and rat cerebellum membranes. Pretreatment of C6 glioma cell membranes with Br-AAM-pindolol and subsequent washing resulted in a time- and dose-dependent blockade of beta-adrenergic receptors. A 50% blockade was achieved in the presence of 1.6 nM Br-AAM-pindolol. This blockade occurs specifically at the beta-adrenergic receptor level, as: 1) it induced a decrease of maximal isoproterenol stimulated adenylate cyclase activity with no modification of basal and sodium fluoride stimulated activity and 2) decreases of (3H)-DHA binding and stimulation of adenylate cyclase activity by the agonist were suppressed in the presence of isoproterenol, a beta-adrenergic agonist. Furthermore, Br-AAM-pindolol treatment did not affect (3H)-diazepam binding in C6 glioma cell membranes. Pretreatment of C6 glioma cells with Br-AAM-pindolol also reduced the response of adenylate cyclase to isoproterenol and the number of beta-adrenergic receptors. The blockade of beta-adrenergic receptors of C6 glioma cells by Br-AAM-pindolol was non-competitive, whereas the blockade obtained with AM-pindolol, a derivative of pindolol devoid of alkylating properties, was competitive. The irreversible blockade of beta-adrenergic receptors by Br-AAM-pindolol in rat erythrocyte membranes was substantiated by the demonstration that no recovery of beta-adrenergic receptors occurred during long term incubation of the membranes (48 h) following Br-AAM-pindolol treatment and subsequent washing.(ABSTRACT TRUNCATED AT 250 WORDS)

Distinction of benzodiazepine agonists from antagonists by photoaffinity labelling of benzodiazepine receptors in vitro

When membranes of rat cerebellum are exposed to UV light in the presence of flunitrazepam this ligand can be incorporated into one of the assumed 4 benzodiazepine binding sites of the GABA-benzodiazepine receptor complex. This irreversible incorporation of flunitrazepam, in contrast to reversible binding of this substance, leads to conformational changes of the remaining 3 benzodiazepine binding sites which result in a decreased affinity of benzodiazepine agonists, but not of benzodiazepine antagonists. The investigation of the affinity of drugs for [ 3H]benzodiazepine antagonist binding before and after photoaffinity labelling of benzodiazepine receptors with flunitrazepam can therefore be used as a sensitive and simple test to distinguish between agonists and antagonists in vitro.

Effect of picrotoxin on benzodiazepine receptors and GABA receptors with reference to the effect of Cl − ion

Picrotoxin does not by itself affect [ 3H] diazepam binding to synaptosomal membranes of rat cerebellum; however, picrotoxin stimulated the binding in the presence of Cl − ion or Cl − ion plus low concentrations of GABA. On the other hand, in the presence of GABA at concentrations higher than 1 × 10 −6 M, picrotoxin inhibited [ 3H]diazepam binding. This inhibition seems to be the result of reduced GABA binding, which occurred in the presence of picrotoxin and Cl − ion. These results may indicate that benzodiazepine receptors, GABA receptors, and the Cl − ionophore are closely associated with each other.

Lectin cytochemistry of carbohydrates on cell membranes of rat cerebellum.

Wheat germ agglutinin (WGA), Ricinis communis agglutinin (RCA) and Lens culinaris (LC) lectins have been used to characterize carbohydrates of neuronal membrane systems in rat and chick cerebellum. WGA and RCA both label Golgi membrane cisternae on the side of the membrane facing the cisternal space but they do not label other elements of the granular and agranular endoplasmic reticulum (ER). WGA labels the plasma membrane generally and WGA binding sites are concentrated within the synaptic cleft and at specialized sites along the axolemma at the node of Ranvier. RCA labelling of plasma membranes is sparse. Neuraminidase treatment of tissue slices prior to lectin cytochemistry does not alter the Golgi membrane distribution of WGA and RCA binding sites and other ER membranes remain unlabelled. The plasma membrane staining with WGA is decreased and that with RCA is increased after neuraminidase pretreatment. The pattern of lectin labelling with LC resembles that seen with concanavalin A (con A) in that all elements of the ER within cell bodies label on the side of the membrane facing the cisternal space. A major difference when compared to con A labelling is that the hypolemmal cisternae lying adjacent to the plasma membrane of Purkinje cell axons and dendrites do not label with LC. Collectively, these results suggest that elements of the ER of Purkinje cells are heterogeneous with respect to their lectin binding properties in a manner which is consistent with the formation of more complex oligosaccharides on membranes of the cell periphery.