Brain Membrane Preparations Research Articles

Increased methylation of chloroform extractable products in brain membrane preparations after propranolol injection to the rat

1. 1. One and four hours after acute or chronic (8 days) intraperitoneal injection of propranolol to rat at doses up to 25 mg/kg, a β-adrenergic receptor blockage occurred in brain homogenates, as monitored by [ 3H]dihydroalprenolol binding. 2. 2. A dose-dependent increased methylation of chloroform extractable products was observed (acute treatment: 18% increase, chronic treatment: 136% increase for the dose of 25 mg/kg). 3. 3. The implication of β-adrenergic receptors in this effect was studied and this revealed that: 3.1. (a) acute intraperitoneal injection of the less active propranolol stereoisomer (+) did not produce any effect; 3.2. (b) the chronic intraperitoneal injection of salbutamol, a β-adrenergic receptor agonist, induced a decreased methylation of chloroform extractable products (62% decrease for the dose of 2.5 mg/kg). 4. 4. It is concluded that injection of β-adrenergic antagonist to the rat increases basal methyltransferase activity in the brain. This induces a decreased sensitivity of the enzyme activity to noradrenaline.

Interaction of formamidine pesticides with insect neural octopamine receptors: Effects on radioligand binding and cyclic AMP production

AbstractThe formamidines chlordimeform, demethylchlordimeform (DCDM), amitraz and BTS‐2727l were tested for their respective abilities to inhibit [3H]mianserin binding in membrane preparations of cockroach brain and, in the same tissue, to stimulate octopamine‐sensitive receptors coupled to adenylate cyclase. Analysis of [3H]mianserin binding indicated negative cooperativity between two binding sites with respective Kd and Bmax values of 3.82 mM and 0.886 pmol (mg protein)−1 for a high‐affinity site and 218 nM and 13.56pmol (mg protein)−1 for a low‐affinity site. DCDM, BTS‐27271 and amitraz inhibit [3H]mianserin binding with IC50 values similar to those obtained with octopamine and the μ‐adrenergic antagonist, phentolamine, whereas chlordimeform is a poor competitor for the binding sites. Similarly DCDM, BTS‐27271 and amitraz elevate cyclic AMP production in brain membrane preparations in a dose‐dependent manner with Ka values of 0.32 uM, 1.5 uM and 3.4 uM respectively, whereas chlordimeform was again without effect. The formamidine‐mediated responses were fully additive with the evaluation of cyclic AMP obtained using the maximal concentration of dopamine but not with octopamine‐mediated increases; thus the formamidine effects appear to be expressed through partial agonism of octopamine receptors that are coupled to adenylate cyclase.

5-Hydroxytryptamine 2 (5-HT 2) structure-function relationships of the nitro and amino phenylpiperazines on intact human platelets

Nitro and amino phenylpiperazines were synthesized to study the agonist and antagonist activities of the phenylpiperazines at the human platelet 5-hydroxytryptamine 2 (5-HT 2) receptor. Amplification of ADP-induced aggregation and binding competition experiments with [ 3H]ketanserin were used to evaluate receptor interactions in this system. All the monosubstituted phenylpiperazines were antagonists despite the wide variation in electronic and hydrophobic properties. The parent compound, unsubstituted phenylpiperazine (PP), had the lowest affinity for the [ 3H]ketanserin binding site. The intensely electron-withdrawing NO 2 substituent increased binding affinity at all ring positions and this activity correlated with antagonist potency in platelet aggregation studies (rank order: 4-NO 2PP >3-NO 2-PP > 2-NO 2-PP). NH 2 substitution decreased binding affinity at the 4- and 2-positions compared with the analogous NO 2 substituted compounds; however, evaluation of NH 2-PP antagonist potency in aggregation studies was complicated due to slow association with the receptor. To compare the activities of the phenylpiperazines at other 5-HT sites, binding competition experiments were performed using [ 3H]5-HT in bovine brain membranes. The rank order of the affinities for the NO 2 substituted compounds was distinctly different from that determined with platelets, reflecting the heterogeneous composition of 5-HT receptor subtypes in the brain membrane preparations. The platelet aggregation experiments demonstrated that marked alteration of the electronic and hydrophobic properties of phenylpiperazine by ring substitution did not impart 5-HT 2 agonist activity. By contrast, 5-HT 2 antagonist activity appeared to be enhanced markedly by electron-withdrawing resonance effects which decreased the electron density at the 1'-piperazine nitrogen. This enhancement appeared to be specific for the 5-HT 2 receptor subtype.

Increased Methylation of Chloroform Extractable Products and CTP: Cholinephosphate Cytidylyltransferase in Brain Membrane Preparations from Triethyltin‐Intoxicated Rats

Rats were chronically intoxicated with triethyltin in the drinking water (0.002%) for a period of 15 days. Starting with day 5 of the intoxication period a decrease in the body weight was observed and, in parallel, the development of a cerebral oedema could be followed by measuring white matter density. At the same time, an increase of phosphatidylethanolamine-N-methyltransferase and cholinephosphate cytidylyltransferase activities was noted. This increase might be a compensatory mechanism for counteracting the membrane damages induced by triethyltin.

Chronic imipramine reduces [ 3H]SCH 23390 binding and DA-sensitive adenylate cyclase in the limbic system

[ 3H]SCH 23390 binding and dopamine (DA)-stimulated adenylate cyclase activity were measured in brain membrane preparations from rats chronically treated with imipramine (10 mg/kg twice daily for 14 days). [ 3H]SCH 23390 binding sites were decreased by 27% in the limbic system but by only 14% in the striatum. The responsiveness of adenylate cyclase to DA was reduced by 38% in the limbic system but was not modified in the striatum. Concomitant treatment with α-methyltyrosine (α-MPT) (50 mg/kg daily for 14 days) prevented the imipramine-induced reduction in both [ 3H]SCH 23390 binding sites and the responsiveness of adenylate cyclase to DA.

Diversity and novel pharmacological properties of Ca2+ channels in Drosophila brain membranes.

Binding studies as well as affinity labelling and immunoblot techniques were used to identify and characterize the receptors for Ca2+ channel blockers in Drosophila brain membranes. Despite structural analogies with mammalian receptors, Drosophila binding sites for phenylalkylamines and 1,4-dihydropyridines, unlike those described in skeletal and cardiac muscle, were found to be located on separate Ca2+ channels. Single-channel bilayer recordings from reconstituted membranes revealed the presence of eight distinct cobalt-sensitive Ba2+-conducting channels in Drosophila brain membrane preparations. In good agreement with binding studies, the most frequently observed Ca2+ channel type (Ba2+ conductance of 13 pS) was extremely sensitive to phenylalkylamines but not affected by micromolar concentrations of 1,4-dihydropyridines. Distinct 1,4-dihydropyridine-sensitive and phenylalkylamine-insensitive channels were also identified. They had unitary Ba2+ conductances of 21 and 31 pS. A detailed analysis of drug action showed that both 1,4-dihydropyridines and phenylalkylamines first increased channel open state probability before fully blocking channel activity. Other types of channels have been identified with unitary Ba2+ conductances of 9, 41, 53, 64 and 81 pS. They were insensitive to the previously described organic Ca2+ channel blockers. The Drosophila system seems to be a unique model to analyse the properties of several different types of Ca2+ channels and particularly those of channel types that are uniquely blocked by phenylalkylamines or uniquely blocked by 1,4-dihydropyridines.

Synthesis and evaluation of optically pure [ 3H]-(+)-pentazocine, a highly potent and selective radioligand for σ receptors

Tritium-labeled (+)-pentazocine ([ 3H]- 1b) of specific activity 26.6 Ci/mmol was synthesized in 3 steps starting with (+)-normetazocine ( 2) of defined optical purity. [ 3H]- 1b has been characterized as a highly selective ligand for labeling of σ receptors. Competition data revealed that [ 3H]- 1b could be displaced from guinea pig brain membrane preparations with a number of commonly used σ receptor ligands. [ 3H]- 1b exhibited saturable, enantioselective binding with a K d of 5.13±0.97 nM and a B max of 1146±122 fmol/mg protein. Phencyclidine (PCP) displaced [ 3H]- 1b with low affinity while MK-801 was inactive, thus indicating insignificant activity at the PCP-binding site; apomorphine failed to displace [ 3H]- 1b indicating lack of dopamine receptor cross-reactivity. Since the affinity of [ 3H]- 1b is about 6 times that of the two commonly employed σ ligands ((+)-3-[ 3H]PPP and [ 3H]DTG) and since it is more selective for σ receptors than the benzomorphan [ 3H]SKF-10,047, it represents the first example of a highly selective benzomorphan based σ receptor ligand. [ 3H]- 1b should prove useful for further study of the structure and function of σ receptors.

Synthesis and characterization of a series of diarylguanidines that are noncompetitive N-methyl-D-aspartate receptor antagonists with neuroprotective properties.

Four diarylguanidine derivatives were synthesized. These compounds were found to displace, at submicromolar concentrations, 3H-labeled 1-[1-(2-thienyl)cyclohexyl]piperidine and (+)-[3H]MK-801 from phencyclidine receptors in brain membrane preparations. In electrophysiological experiments the diarylguanidines blocked N-methyl-D-aspartate (NMDA)-activated ion channels. These diarylguanidines also protected rat hippocampal neurons in vitro from glutamate-induced cell death. Our results show that some diarylguanidines are noncompetitive antagonists of NMDA receptor-mediated responses and have the neuroprotective property that is commonly associated with blockers of the NMDA receptor-gated cation channel. Diarylguanidines are structurally unrelated to known blockers of NMDA channels and, therefore, represent a new compound series for the development of neuroprotective agents with therapeutic value in patients suffering from stroke, from brain or spinal cord trauma, from hypoglycemia, and possibly from brain ischemia due to heart attack.

Evidence of mitochondrial involvement in scrapie infection

Two cDNA libraries were constructed from brain membrane and cytoskeletal preparations purified from scrapie-infected hamster brains. Four recombinants strongly preferential to the scrapie cytoskeletal preparation were identified by the differential hybridization of 7,000 recombinants. These clones were not, however, preferential to total nucleic acids extracted from scrapie-infected hamster brains. DNA sequence analysis revealed all four clones to have significant sequence similarities to the mouse mitochondrial genome. This correlation led us to consider a mitochondrial association with scrapie infectivity. Brain mitochondria were purified by sucrose gradient density centrifugation and found to contain high infectivity. Removal of mitochondrial outer membranes by osmotic shock or digitonin treatment resulted in no detectable loss of titer.

Distribution and Pharmacological Properties of the GABAA/ Benzodiazepine/Chloride Ionophore Receptor Complex in the Brain of the Fish Anguilla anguilla

In the present study, we characterized the distribution and the pharmacological properties of the different components of the GABAA receptor complex in the brain of the eel (Anguilla anguilla). Benzodiazepine recognition sites labeled "in vitro" with [3H]flunitrazepam ([3H]FNT) were present in highest concentration in the optic lobe and in lowest concentration in the medulla oblongata and spinal cord. A similar distribution was observed in the density of gamma-[3H]aminobutyric acid ([3H]GABA) binding sites. GABA increased the binding of [3H]FNT in a concentration-dependent manner, with a maximal enhancement of 45% above the control value, and, vice versa, diazepam stimulated the binding of [3H]GABA to eel brain membrane preparations. The density of benzodiazepine and GABA recognition sites and their reciprocal regulation were similar to those observed in the rat brain. In contrast, the binding of the specific ligand for the Cl- ionophore, t-[35S]butylbicyclophosphorothionate ([35S]TBPS), to eel brain membranes was lower than that found in the rat brain. In addition, [35S]TBPS binding in eel brain was less sensitive to the inhibitory effects of GABA and muscimol and much more sensitive to the stimulatory effect of bicuculline, when compared with [35S]TBPS binding in the rat brain. Moreover, the uptake of 36Cl- into eel brain membrane vesicles was only marginally stimulated by concentrations of GABA or muscimol that significantly enhanced the 36Cl- uptake into rat brain membrane vesicles. Finally, intravenous administration of the beta-carboline inverse agonist 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylic acid methyl ester (20 mg/kg) and of the chloride channel blocker pentylenetetrazole (80 mg/kg) produced convulsions in eels that were antagonized by diazepam at doses five to 20 times higher than those required to produce similar effects in rats. The results may indicate a different functional activity of the GABA-coupled chloride ionophore in the fish brain as compared with the mammalian brain.

Developmental expression of reactivity to monoclonal antibodies generated against olfactory epithelia

The developmental expression of immunocytochemical reactivity to 3 monoclonal antibodies (Mabs Neu 4, Neu 5, and Neu 9) that were generated against adult rat olfactory epithelium was examined in olfactory tissues of embryonic rats. Tissues examined included the nasal olfactory epithelium, nerve, and olfactory bulb, as well as vomeronasal epithelium and nerve. Reactivity patterns of these Mabs in adult rats have been described previously (Hempstead and Morgan, 1985a). All 3 Mabs show reactivity on the cell surfaces of neurons, axons, and dendrites of the olfactory epithelium proper. Neu 5 alone shows reactivity on the dendritic knobs, site of transduction of the olfactory stimuli. These reactivities appear early, suggesting developmentally significant roles for the antigens to these Mabs. For Neu 5 and Neu 9 initial reactivity occurs on outgrowing olfactory axons at E13. Dendritic and perikaryal reactivities begin appearing at E14. For Neu 4 initial reactivity occurs simultaneously on olfactory neuronal perikarya, axons, and dendrites at E14. Reactivity also occurs on cells that migrate from the olfactory epithelium and are associated with the olfactory nerves. Within the developing olfactory bulb, Neu 5 behaves as a general cell-surface marker. Neu 4 and Neu 9, however, show enhanced reactivity in the glomerular layer after the onset of synaptogenesis. Reactivity is also seen in the nasal respiratory epithelium and in the vomeronasal epithelia and nerve. Neu 5 and several antibodies to rat neural cell adhesion molecules (N-CAMs) show similar, although not identical, immunohistochemical staining patterns. They also react with the same bands in Western blots of brain membrane preparations. Western blots of Neu 5-reactive material also show developmental and spatial correlations of apparent molecular-weight distributions expected of N-CAM-like components as well.

Effect of ethanol on hippocampal GABA receptors in the rat brain

The effect of ethanol on GABA receptors was studied in hippocampal slices and membrane preparations of the rat brain. In slice preparation, ethanol enhanced the GABA inhibition of the population spikes evoked in CA 1 pyramidal cells by Schaffer collateral stimulation. The effect of ethanol was dose-dependent being observed with minimal concentration of 70 mM. In 3H-GABA binding experiments, ethanol enhanced the binding to fresh membranes with no change in the affinity. Maximal stimulation was observed at ethanol concentration of 70 mM. In hippocampal slices or membrane preparations obtained from ethanol- or barbital-administered rats chronically, ethanol failed to enhance GABA function. Thus, the present study demonstrated the involvement of GABA in central effect of ethanol. Also suggested in GABA was the involvement in the development of cross-tolerance between ethanol and barbiturates.

Characterization of the Neuronal Receptor for Basic Fibroblast Growth Factor and Comparison to Receptors on Mesenchymal Cells

The receptor for basic fibroblast growth factor (bFGF) was characterized in highly enriched cultures of fetal hippocampal neurons. Two major components of binding could be distinguished. One component comprising about 70% of total binding was removed by 2 M NaCl, and by analogy to other cells could be presumptively attributed to glycosaminoglycans. The remaining 30% of binding which was resistant to 2 M NaCl reflects a high affinity receptor. Scatchard analysis indicated that the two components have Kd values of 500 and 120 pM, and densities of 165,000 and 35,000-60,000 sites/neuron, respectively. Cross-linking 125I-bFGF to neuronal cultures with disuccinimidyl suberate labeled a major membrane protein of 135 kDa and a minor protein of 85 kDa. Examination of neuronal cultures derived from multiple brain regions and membrane preparations from fetal brain suggested that the larger protein was widely distributed. The neuronal bFGF receptor was not blocked by heparin at concentrations up to 100 micrograms/ml. Twelve synthetic peptide fragments of bFGF were examined to determine the domain of bFGF interacting with the neuronal receptor. Inhibition was observed chiefly with a peptide including the sequence 103-146. The properties of the neuronal bFGF receptor were compared directly to those of the receptors characterized on BHK cells and other mesenchymal cells. Specific differences observed between neuronal and mesenchymal bFGF receptors are discussed.

Effect of sodium on [3H]ethylketocyclazocine binding to opioid receptors in frog brain membranes

The specific binding of [3H]ethylketocyclazocine to frog brain membrane preparation was enhanced in the presence of sodium ions administered as NaCl, both at 0 degree C and at room temperature. The optimal NaCl concentration was 25 mM at 0 degree C and 50 mM at 24 degrees C. MgCl2 inhibited the [3H]ethylketocyclazocine binding. Two binding sites (high and low affinity) were established with [3H]ethylketocyclazocine as ligand by equilibrium binding studies. Addition of NaCl increased the Bmax of the low-affinity site more than that of the high-affinity site at both temperatures. Affinities were higher at 0 degree C than at 24 degrees C. The KD values were not significantly influenced by sodium ions. The dissimilarities between the rat and frog brain opioid receptors in [3H]ethylketocyclazocine binding are attributed to the different lipid composition of the two membranes.

Distribution of basic fibroblast growth factor binding sites in various tissue membrane preparations from adult guinea pig

In order to localize a rich source of basic FGF receptor, we examined the distribution of basic FGF binding sites in brain, stomach, lung, spleen, kidney, liver and intestine membrane preparations from adult guinea pig. Comparative binding studies using iodinated basic FGF showed that a specific binding was detected in all the membrane preparations tested. Scatchard plots from iodinated basic FGF competition experiment with native basic FGF in various membrane preparations, suggested the presence of one class of binding sites in some tissues such as liver, kidney, spleen, lung, stomach, and intestine with an apparent dissociation constant (appKD) value ranging from 4 to 7.5 nM and the existence of a second class of higher affinity sites in brain membranes with appKD value of 15 pM. Characterization of these basic FGF high affinity interaction sites was performed using a cross-linking reagent. These results show for the first time that specific interaction sites for basic FGF are widely distributed, suggesting that this growth factor might play a role in the physiological functions of a number of adult organs.

Monoclonal antibodies against the S2-serotonin receptor from rat brain that cross-react with dopamine and opiate receptors

Balb/c mice were immunized with rat striatal integral membrane proteins. After hybridization of splenocytes with myeloma cells, hybridoma lines secreting antibodies against serotonin, dopamine and opiate receptors were detected by inhibition of ligand binding to brain membrane preparations. Antibodies from two positive lines, Mab/a9 and Mab/a18, were able to inhibit ligand binding to the S2-serotonin (Kd range: 10–100 nM), the μ-opiate (Kd range: 0.4–3 μM) and the δ-opiate receptors (Kd range: 0.7–1.1 μM), while Mab/a9 was also found to inhibit ligand binding to the D2/D4-dopamine receptor (Kd ∼ 50 nM). An apparent molecular mass of 60 kDa could be ascribed to the δ-opiate receptor and apparent molecular masses of 29 and 36 kDa to the μ-opiate receptor by ligand elution from immuno-precipitates.

Reconstitution of opioid receptor and GTP-binding protein.

The signal transduction mechanism of opioid receptors was studied in reconstitution experiments with GTP-binding protein. μ-Opioid receptor exclusively purified from rat brains was reconstituted with purified Gi or Go in phosphatidylcholine vesicles. The μ-agonist-displacement activity of antagonist binding was markedly increased by reconstitution with Gi or Go. The μ-agonist stimulated G-protein activities in both reconstituted vesicles. From the experiment that the recovery of agonist-stimulation of G-protein activity was additive when Gi and Go were simultaneouly reconstituted in brain membrane preparations, it is suggested that μ-receptors exist in μi and μo, separately coupled to Gi and Go, respectively.

Central mu, delta, and kappa opioid binding sites, and brain and pituitary beta-endorphin and met-enkephalin in genetically obese (ob/ob) and lean mice

The equilibrium dissociation constants and maximal binding capacities of 3H-dihydromorphine (DHM), 3H-D-Ala 2-D-leu 3 -enkephalin (DADL), and 3H-dynorphin A(1–8) for their respective mu, delta, and kappa opiate binding sites were studied in brain membrane preparations from lean and genetically obese-hyperglycaemic (Aston ob/ob) mice. The concentration of kappa binding sites was 2.7 fold higher in obese compared with lean mouse brain ( 231 ±44.6 versus 83.3±10.3 fmoles 3 H-dynorphin/mg protein respectively. mean ± SEM ). The concentration of delta binsing sites in obese was 1.6 fold that in lean mouse brain ( 94.5±8.6 vesus 59.5±6.5 fmoles 3 H-DADL/mg protein ). In contrast, the concentration of brain mu receptors was 40% lower in obese compared with lean mice ( 20.8±2.19 and 34.8±3.1 fmoles 3 H-DHM/mg protein respectively ). Binding affinities of delta and kappa sites for their respective ligands were not significantly different inlean V. obese mice. However, for mu sites, lean mouse binding data showed two affinities, one was not significantly different from obese (0.35nM) the second was lower (1.18nM) for DHM. Increases of approximately 5 fold and 3 fold in the brain content of beta-endorphin and met-enkephalin respectively, and no differences in brain dynorphin levels, were demonstrated in obese mice compared with lean controls. In obese mice, pituitary beta-endorphin content was 9 fold higher, met-enkephalin 4 fold higher and dynorphin 12 fold higher than in lean mice. The striking differences in opioid binding-site characteristics and in endogenous opioid peptide levels in obese compared with lean mice may contribute to the hyperphagia and, directly or indirectly, to the development of hyperglycaemia and hyperinsulinaemia in obese mice.

Characterization and quantitative autoradiographic distribution of [ 3H]acetylcholine muscarinic receptors in mammalian brain. Apparent labelling of an M 2-like receptor sub-type

[ 3H]Acetylcholine receptor binding characteristics (under muscarinic conditions) have been investigated using membrane binding assays and in vitro receptor autoradiography. In rat, guinea-pig and monkey brain membrane preparations, [ 3H]acetylcholine binds with high affinity (25–50 nM) to an apparently single class of sites which is differentially distributed across brain regions. The ligand selectivity pattern reveals that the potency of (−)quinuclidinyl benzylate is greater than (>) atropine > scopolamine > oxotremorine > carbamylcholine > pirenzepine > methylcarbamyl-choline = nicotine in competing for [ 3H]acetylcholine binding sites, indicating that [ 3H]acetylcholine selectively binds to muscarinic sites under these incubation conditions. Moreover, the low potency of pirenzepine suggests that [ 3H]acetylcholine does not label a significant proportion of the M 1 receptor sub-type but most likely binds to putative M 2-like receptor sites. This hypothesis is also supported by the autoradiographic distribution of [ 3H]acetylcholine binding sites in all species studied here. High densities of [ 3H]acetylcholine binding sites are seen in various nuclei of the medulla and pons, certain thalamic nuclei, medial septum, laminae III, V and VI of the cortex and just above the pyramidal cell layer of the hippocampus. Such localization is much different from that seen with the non-selective antagonist [ 3H]quinuclidinyl benzylate and the selective M 1 receptor ligand [ 3H]pirenzepine, although it resembles that of the selective M 2 receptor antagonist [ 3H]AF-DX 116. Thus, [ 3H]acetylcholine apparently mostly binds with high affinity mainly to non-M 1 muscarinic receptor types in mammalian brain tissues. Moreover, the ligand selectivity pattern and in vitro receptor autoradiographic data suggest that at low concentrations (10–20 nM) most of [ 3H]acetylcholine labelled sites are of the M 2-like receptor class.

Ro 15-4513, a partial inverse agonist for benzodiazepine recognition sites, has proconflict and proconvulsant effects in the rat

The present report describes the effects of Ro 15-4513 (ethyl-8-azido-5,6-dihydro-5-methyl-6-oxo-4H-imidazo-(1,5-a)(1,4)-benzodiazepine-3- carboxylate) in the conflict test, on convulsions induced by isoniazid and DMCM (methyl-6,7-dimethoxy-4-ethyl-β-carboline-3-carboxylate) and on the binding of [ 3H]γ-aminobutyric acid ([ 3H]GABA) to rat brain membrane preparations. Ro 15-4513 produced a dose-dependent proconflict effect that was prevented by the administration of the benzodiazepine antagonist, Ro 15-1788. In addition, Ro 15-4513 was not convulsant per se but enhanced the convulsions produced by isoniazid and completely blocked the convulsions induced by full inverse agonist, DMCM. In vitro, Ro 15-4513, like ethyl-β-carboline-3-carboxylate (βCCE), antagonized the increase in [ 3H]GABA binding induced by diazepam. The results indicate that Ro 15-4513 is anxiogenic and interacts with benzodiazepine recognition sites as a partial inverse agonist.