Crude Synaptic Membranes Research Articles

GABAergic synapses supramolecular organization and biochemical regulation

Extraneurally released gamma-aminobutyric acid (GABA) interacts with specific recognition sites associated with proteins located in postsynaptic neuronal membranes that function as chloride (Cl −)ionophores. As a result of the interaction between GABA and the recognition sites, C1 − ionophores are opened causing an influx or an efflux of Cl −, depencling on the values of the Cl − equilibrium potential and of the membrane potential. Hyperpolarization or depolari/ation will result from inward or outward Cl − fluxes, respectively. Independently of the change in conductivity elicited by GABA, this amino acid transmitter will reduce the effectiveness of the sodium ion (Na +) excitatory potential. In attempts to elucidate the molecular mechanism, whereby benzodiazepines facilitate the action of GABA on membrane conductance without changing the activity of Cl − or other ionophore, a basic protein (GABA-modulin, GM) has been isolated from rat brain which is similar in structure to the small molecular weight myelin basic protein, found in rodent brain. While GABA-modulin is located in synaptosomes, the small molecular weight myelin basic protein is located in the myelin fraction: more important, GABA-modulin inhibited the high affinity bincling of GABA to crude synaptic membranes while the basic myelin protein did not. Also, amino acid composition and molecular weight differentiate the two proteins. The GABA-modulin can be phosphorylated with different stoichiometry by cyclic AMP-dependent protein kinase (4 mol PO 4 −3) or Ca 2+-dependent protein kinase (1 mol PO 4 −3). Only cyclic AMP-dependent phosphorylation inhibited the action of GABA-modulin on GABA bincling.

Influences of isoproterenol pretreatment on cerebral cortical bindings of [3H]clonidine and [3H]dihydroalprenolol in infant and adult rats.

Specific bindings of [3H]clonidine and [3H]dihydroalprenolol [( 3H]DHA) to crude synaptic membranes were measured after cerebral cortical slices or synaptic membranes were pre-incubated with isoproterenol, 200 microM at 37 degrees C for 40 min, in 7- and 70-day-old rats. Isoproterenol caused a significant increase of the Bmax value of [3H]clonidine binding sites at both days 7 and 70, without changing the Kd. Scatchard analysis of [3H]clonidine binding to adult synaptic membranes which was examined by using a wide range of [3H]clonidine concentration (0.05-15 nM), showed that the Bmax in only high-affinity binding sites was 4-fold increased by pretreatment of synaptic membranes with isoproterenol 200 microM. In contrast, the Bmax of [3H]DHA binding sites was significantly reduced by isoproterenol at 7 and 70 days. These results suggest that the possible interrelationship between beta- and alpha 2-adrenoceptors which exists in synaptic membranes, matures by day 7 in the cerebral cortex of rats.

Modulation of neuronal serotonin uptake by a putative endogenous ligand of imipramine recognition sites.

Imipramine inhibits the serotonin uptake by binding with high affinity to regulatory sites of this uptake located on axons that release serotonin. The number of imipramine recognition sites located on crude synaptic membrane preparations is reduced by two daily injections of imipramine or desmethylimipramine for 3 weeks. When the binding sites for [3H]imipramine are down-regulated the Vmax of the neuronal uptake of serotonin is increased. Moreover, in minces prepared from the brain hippocampus of rats receiving imipramine in a dose regimen that reduces the number of [3H]imipramine recognition sites, the efficiency of imipramine as a blocker of the serotonin uptake is diminished. Hence the high-affinity binding sites for [3H]imipramine may have a physiological role in modulation of serotonin reuptake. Probably this is mediated by an endogenous effector of these regulatory sites. A nonpeptidic constituent of rat brain capable of displacing [3H]imipramine from its high-affinity binding site and of inhibiting the serotonin uptake in a dose-related manner has been extracted and its partial purification is described.

Selective decrease in the affinity of D2 dopamine receptor for agonist induced by islet-activating protein, pertussis toxin, associated with ADP-ribosylation of the specific membrane protein of bovine striatum

When the crude synaptic membrane preparations from bovine striatum were treated with islet-activating protein (IAP), one of the pertussis toxins, a protein with a molecular weight of about 40,000 was ADP-ribosylated. In parallel with this ADP-ribosylation, there was a decrease in D2 dopamine receptor affinity for agonist, while the affinity for antagonist remained unaltered. Addition of GTP to nontreated membranes also resulted in a decrease in the affinity of D2 receptor for agonist, and there was no further reduction of affinity for agonist with addition of GTP to the IAP-treated membranes. As IAP specifically acts on the guanine nucleotide regulatory protein which mediates the inhibition of adenylate cyclase activity (Ni), our findings indicate a possible molecular interaction between the brain D2 dopamine receptor and Ni.

Regulation by calcium ions of glutamate receptor binding in hippocampal slices

Hippocampal slices were incubated in a Krebs-bicarbonate buffer with various concentrations of calcium and [3H]glutamate receptor binding was measured in crude synaptic membranes derived from these slices. Increasing the calcium concentration from 0 to 2.5 mM resulted in a 2.2-fold increase in the maximal number of the Na-independent [3H]glutamate binding sites without changes in their affinity for [3H]glutamate. This effect was totally blocked by the addition of the protease inhibitor leupeptin (50 μM) to the slice incubation medium. No effect was observed on the Na-dependent [3H]glutamate binding nor on the Na-independent [3H]glutamate binding measured in the presence of a concentration of calcium of 250 μM. Increasing the calcium concentration also resulted in an increased proteolytic activity which was inhibited by about 70% by the addition of leupeptin. Finally, increasing the calcium concentration induced the degradation of high-molecular weight proteins, the microtubule-associated proteins (MAPs) and the 220 000 dalton doublet protein corresponding to fodrin. Both effects were partially prevented by the addition of leupeptin in the slice incubation medium. These results indicate that the same calcium-dependent processes which were previously shown to regulate [3H]glutamate receptor binding to hippocampal membranes occur in the hippocampal slice preparation, and they suggest a mechanism by which fluctuations in calcium levels can activate a calcium-dependent proteinase, the degradation of cytoskeletal-associated proteins and the unmasking of additional glutamate receptors. The participation of such processes in various forms of plasticity is discussed.

Evidence of enrichment in glycine receptors of crude synaptic membranes from rat spinal cord following Triton X-100 treatment

Treatment of synaptic membranes from rat brainstem and spinal cord with the nonionic detergent Triton X-100 at 1–10 μl/mg protein caused a marked increase in glycine receptor ( 3H)strychnine binding expressed per mg of residual membrane protein. The effect was maximal (220±6% of control) at 5 μl Triton/mg protein, while higher concentrations caused progressive loss of strychnine binding ability of membranes (27±6% at 25 μl Triton/mg protein). The increase in strychnine binding caused by low Triton X-100 reflected an increase in membrane B max, the k D being unaffected by the treatment. The affinity of glycine analogues for receptor sites was not appreciably affected by the detergent either. The findings suggest an enrichment of the synaptic membrane preparation in glycine receptors, caused by the solubilization by Triton of membrane constituents not related to the receptor sites.

4-Aminobutyric acid methyl ester hydrochloride, a precursor of 4-aminobutyric acid

4-Aminobutyric methyl ester hydrochloride (GME) is able to cross the blood-brain barrier after intracardiac administration to the rat. GME has an LD 50 of 1300 mg/kg in mice and 950 mg/kg in rats, exhibits an antiaggressive effect and is able to decrease isoniazid-induced convulsions in the rat. GME is hydrolyzed to 4-aminobutyric acid (GABA) by brain homogenates, acts as an inhibitor of GABA binding to crude synaptic plasma membranes, activates the release and inhibits the uptake of GABA by rat synaptosomes and acts as a competitive inhibitor of the so-called GABAse system in vitro.

Phosphorylation induces a decrease in the biological activity of the protein inhibitor (GABA-modulin) of gamma-aminobutyric acid binding sites.

gamma-Aminobutyric acid (GABA)-modulin is a brain protein of Mr 16,500 that down-regulates the high-affinity binding site for GABA which is located in crude synaptic membranes. This protein can be phosphorylated in vitro by the catalytic subunit of cAMP-dependent protein kinase and by a partially purified preparation of calmodulin-sensitive Ca2+-dependent protein kinase. The GABA-modulin sites that are phosphorylated by the two enzymes are different, as revealed by HPLC analysis of tryptic digests. The capacity of GABA-modulin to decrease the number of sites that bind [3H]muscimol was completely abolished by phosphorylation of this protein with the cAMP-dependent protein kinase but not with the Ca2+-dependent enzyme. GABA-modulin present in crude synaptic membranes prepared from rat cortex also was shown to be phosphorylated by endogenous protein kinases activated by cAMP, Ca2+ and calmodulin, and Ca2+ and phosphatidylserine. These results suggest a potentially important role for protein kinase and GABA-modulin in the regulation of the number of GABA recognition sites.

Phospholipid methylation increases [3H]diazepam and [3H]GABA binding in membrane preparations of rat cerebellum.

The effect of phospholipid methylation on both [3H]diazepam and [3H]GABA ( [3H]gamma-aminobutyric acid) binding to crude synaptic plasma membrane from rat cerebellum has been studied. S-Adenosylmethionine (SAM) stimulates [3H]methyl group incorporation into membrane phospholipids and enhances [3H]diazepam binding by increasing the apparent Bmax. Conversely, inhibition of [3H]methyl group transfer from [3H]SAM to phospholipids by preincubation with SAM at 0 degrees C or with SAH abolishes the increase of binding. After preincubation with SAM, analysis of the GABA binding reveals the presence of binding sites with high affinity, a property absent in control membranes preincubated without SAM. Among the neurotransmitter bindings tested, only those of GABA and benzodiazepine in the cerebellum and beta-adrenergic ligands in the cerebral cortex are enhanced upon stimulation of phospholipid methyltransferase activity. [3H]Dihydromorphine, [3H]dihydro-alpha-ergokryptine and [3H]spiroperidol bindings are not affected by SAM. The present data suggest an involvement of phospholipid methylation in regulation of both [3H]GABA and [3H]-diazepam binding.

Differences in the regulatory adaptation of the 5HT 2 recognition sites labelled by 3H-mianserin or 3H-ketanserin

In crude synaptic membranes prepared from rat brain the sites occupied by 3H-spiroperidol that are displaced by μM concentrations of serotonin (5HT) have been termed 5HT 2 receptors ( Peroutka and Snyder, 1980). Since the 3H-spiroperidol displaced by 5HT is also displaced very effectively (IC 50 in the nM range) by ketanserin and mianserin it was suggested that spiroperidol, mianserin and ketanserin are labelling 5HT 2 receptors. Data are presented showing that the 3H-ketanserin and 3H-mianserin bound to crude synaptic membrane in the presence of a H 1 receptor blocker are not labelling the same recognition site. Hence from this standpoint the recognition site marked by 3H-mianserin and 3H-ketanserin is not identical. The possibility that allosteric effects are operative in some of these ligand displacements should be entertained.

Brain membrane disordering by administration of a single ethanol dose

There is a bulk of evidence that ethanol exerts an important direct effect on biological membranes, especially in the central nervous system, during chronic administration. Whether membranes are affected after an acute and subacute ethanol administration remains to be demonstrated. Crude synaptic membrane fluidity (checked by fluorescence polarization) together with ( Na + + K +)ATPase activity were therefore examined 18 hours after a single oral ethanol administration (5 g/kg bwt.) to naive rats or to rats previously intubated with ethanol repeatedly during 4 days (increasing the daily dose from 7 to 10 g/kg). The sensitivity of both parameters to different concentrations of ethanol added in vitro (0.175 M–1.400 M) was also determined. Although no changes in the basal intrinsic fluidity were found, (Na + + K +)ATPase activity increased slightly after administration of ethanol to naive as well as to short-term ethanol intoxicated rats. The fluidizing as well as the ATPase inhibiting effects following the addition of ethanol in vitro were markedly increased 18 hours after ethanol administration to naive rats. Such an hypersensitization seems not to be related to an unspecific stress or to changes in body temperature and was no longer apparent in short-term ethanol intoxicated rats. Disappearance of the acute ethanol induced hypersensitization with further ethanol administration may represent the first stage of tolerance acquisition.

Supersensitivity of the GABA receptor in the frog spinal cord, as induced by kainic acid

1. In the isolated frog spinal cord perfused with kainic acid (KA, 5 × 10 −4M) containing Ringer's solution, within 2 hr there were increases in the amplitude of the dorsal root depolarization, as induced by the GABA-agonists. 2. KA perfusion produced increases in the specific binding of [ 3H]muscimol to crude synaptic membranes and incubation with KA for 3 hr did not increase [ 3H]muscimol binding. 3. [ 3H]GABA was released from KA-treated spinal cord slices in the presence of high K +. 4. KA-induced supersensitivity of the dorsal root to GABA may relate to direct actions on primary afferent terminals and not to denervation of GABAergic neurons.

Characteristics of GABAB receptor binding sites on rat whole brain synaptic membranes.

1 Saturable binding of (+/-)-[3H]-baclofen and [3H]-gamma- aminobutyric acid ([3H]-GABA) to rat brain crude synaptic membranes has been examined by means of a centrifugation assay. 2 The binding of [3H]-baclofen could be detected in fresh or previously frozen tissue and was dependent on the presence of physiological concentrations of Ca2+ or Mg2+ although a lower affinity Na+ -dependent component could also be observed. Both components probably reflect binding to receptor recognition sites. 3 The saturable portion of bound [3H]-baclofen formed 20.3 +/- 6.9% of total bound ligand. This could be displaced by GABA (IC50 = 0.04 microM), (-)-baclofen (0.04 microM) and to a much lesser extent by (+)-baclofen (33 microM). Isoguvacine, piperidine-4-sulphonic acid and bicuculline methobromide were inactive (up to 100 microM) and muscimol was only weakly active (IC50 = 12.3 microM). 4 Saturable binding of [3H]-GABA increased on adding CaCl2 or MgSO4 (up to 2.5 mM and 5.0 mM respectively) to the Tris-HCl incubation solution. This binding (GABAB site binding) was additional to the bicuculline-sensitive binding of GABA (GABAA site binding) and could be completely displaced by (-)-baclofen (IC50 = 0.13 microM). 5 Increasing the Ca2+ concentration (0 to 2.5 mM) increased the binding capacity of the membranes without changing their affinity for the ligand. 6 The binding of [3H]-GABA to GABAB sites could be demonstrated in fresh as well as previously frozen membranes with a doubling of the affinity being produced by freezing. Further incubation with the non-ionic detergent Triton-X-100 (0.05% v/v) reduced the binding capacity by 50%. 7 The pharmacological profile of displacers of [3H]-GABA from GABAB sites correlated well with that for [3H]-baclofen displacement. A correlation with data previously obtained in isolated preparations of rat atria and mouse vas deferens was also apparent. 8 It is concluded that [3H]-baclofen or [3H]-GABA are both ligands for the same bicuculline-insensitive, divalent cation-dependent binding sites in the rat brain.

Modulation of brain adenylate cyclase activity by polysaturated fatty acids

Effects of polyunsaturated fatty acids, mainly linoleic acid (LA), on the adenylate cyclase (AC) system in the brain were i-ir.ed by using slices and crude synaptic membranes of ebral cortex as preparations.

The regulatory mechanism in histamine H2 receptor binding of the rat brain : Effects of Cu2+, dithiothreitol and ascor-bate

To identify the regulatory mechanism of histamine H2 receptors in the CNS, influences of Cu2+, dithiothreitol (DTT) and ascor-bate on [3H]Cimetidine binding to crude synaptic membranes were investigated in the male Wistar rat brain. Fifty μM CuCl2 increased 3-fold specific [3H]Cimetidine binding compared to the control. In the presence of 50 μM CuCl2, Scatchard plots showed an upward curve, which was divided into two types of binding sites : high affinity (KD=1.97 nM) and low affinity (KD=21.6 nM). The inhibitory potency (IC50) of metiamide on the binding was not affected by 50 μM CuCl2 (control, 0.16 μM ; CuCl2, 0.14 μM), although the inhibitory effect of mepyramine up to 0.1 mM was not observed in the presence of CuCl2 compared to that (IC50=0.1 mM) in the control. In the presence of 50 μM CuCl2, 0.1 - 30 μM DTT and 10 μM - 10 mM ascorbate increased the binding in a concentration-dependent manner, but both drugs showed no effect in the absence of CuCl2. These results suggest that [3H]Cimetidine binding sites indicate histamine H2 receptors that are specifically increased by Cu2+ and that dithiothreitol and ascorbate enhance the increasing effect of Cu2+. In contrast, influence of Cu2+ on GTP regulatory protein is also suggested from a finding that 50 μM CuCl2 reduced the affinity of histamine to [ 3H]Cimetidine binding sites.

High specific binding of [3H]GABA and [3H]muscimol to membranes from dendrodendritic synaptosomes of the rat olfactory bulb.

Olfactory bulbs contain dendrodendritic synapses, which occur between granule cells and mitral cells, and gamma-aminobutyric acid (GABA) is thought to act as an inhibitory neurotransmitter at these synapses. Synaptosomes derived from the dendrodendritic synapses of the olfactory bulb were shown previously to contain considerable L-glutamate decarboxylase activity. The subcellular distribution and binding parameters of [3H]GABA and [3H]muscimol binding sites have now been determined in the rat olfactory bulb. Of all fractions examined, crude synaptic membranes (CSM) prepared from the dendrodendritic synaptosomes were shown to have the highest specific binding activity and accounted for nearly all of the total binding activity for both ligands. The specific binding activities for [3H]GABA and for [3H]muscimol were greatly increased after treating the CSM with 0.05% Triton X-100. Binding was shown to be Na+-independent, reversible, pharmacologically specific, and saturable. High- and low-affinity sites were detected for both ligands, and both classes of sites had appreciably lower KD values for muscimol (KD1 = 3.1 nM, KD2 = 25.1 nM) than for GABA (KD1 = 8.6 nM; KD2 = 63.7 nM). The amounts of the high-affinity binding sites for muscimol and GABA were similar (Bmax = 1.7 and 1.5 pmol/mg protein, respectively). The results of the present experiments indicate that the GABA and muscimol binding sites represent the GABA postsynaptic receptor, presumably on mitral cell dendrites, and provide further support for the hypothesis that GABA functions as a neurotransmitter at the dendrodendritic synapses in the olfactory bulb.

Down-regulation of β-adrenergic receptors following repeated injections of desmethylimipramine: Permissive role of serotonergic axons

The injection of desmethylimipramine (DMI) twice daily for 3 weeks reduced the density of β-adrenergic receptor recognition sites located in crude synaptic membranes prepared from the cortex and hippocampus and attenuated the stimulation of the membrane-bound adenylate cyclase by isoproterenol. Both actions were abolished if prior to treatment with desmethylimipramine the serotonergic axons were destroyed by an intraventricular injection of 5,7-dihydroxytryptamine. These results show that the down-regulation of β-adrenergic receptors elicited by repeated injections of desmethylimipramine occurs only if the serotonergic axons are intact.

Isolation, characterization, and purification to homogeneity of a rat brain protein (GABA-modulin).

gamma-Aminobutyric acid (GABA)-modulin is a brain neuropeptide that appears to modulate specific high-affinity (20 nM) GABA recognition sites in brain. When added to crude synaptic membranes this peptide inhibits binding of [3H]GABA to the high-affinity site and prevents facilitation of [3H]diazepam binding elicited by GABA. GABA-modulin has been purified to homogeneity by ammonium sulfate precipitation, gel chromatography, and reverse-phase HPLC. Homogeneity was confirmed by a variety of means, including chromatography under four different HPLC conditions, two different polyacrylamide gel electrophoreses, and end group analysis. Purified GABA-modulin contains approximately 126 amino acids and has a molecular weight of 16,500. The GABA-modulin molecule contains an abundance of hydrophilic basic residues, and neither cysteine nor GABA is present. End group analyses of GABA-modulin showed that histidine is the free COOH terminus and the NH2 terminus is blocked. GABA-modulin specifically blocked both [3H]GABA binding to synaptic membranes (IC50, 0.5 microM) and GABA-stimulated [3H]diazepam binding; the binding of [3H]GABA to low-affinity sites was not affected.

Brain membrane disordering related to acute ethanol administration in naive and short-term ethanol-intoxicated rats.

Crude synaptic membrane fluidity (checked by fluorescence polarization) together with (Na+ + K+) ATPase activity were examined 18 hours after a single oral ethanol administration (5 g/kg bwt.) to naive rats and to rats previously intubated with ethanol repeatedly during 4 days. The sensibility of both parameters to different concentrations of ethanol added in vitro (0.175 M-1.400 M) was also determined. Although no changes in the basal intrinsic fluidity were found, (Na+ + K+)ATPase activity increased slightly in both conditions. The fluidizing as well as the ATPase inhibiting effects following the addition of ethanol in vitro were markedly increased 18 hours after ethanol administration to naive rats. This hypersensitization was no longer apparent in rats pretreated with ethanol during 4 days. The acute ethanol-induced hypersensitization found in naive rats appears not to be related to an unspecific stress or to changes in body temperature. The disappearance of this hypersensitization in short-term alcohol-intoxicated animals may represent the first stage of tolerance acquisition.

Neuronal location of [formula omitted] binding sites in rat and guinea-pig brain

N 6- cyclohexyl[ 3H]adenosine , ([ 3H]CHA), was used to label adenosine a 1 receptors in crude synaptic membranes prepared from rat and guinea pig brain. The density of [ 3H]CHA binding sites was highest in the rat hippocampus and cerebellum and in the guinea-pig hippocampus. A microdissection of coronal sections of guinea-pig hippocampus revealed that the specific binding capacity for [ 3H]CHA in area CA-1 was 20–30% higher than in area CA-3, dentate gyrus and subiculum. Selective neuronal lesions of serotonergic, noradrenergic and cholinergic afferents to the hippocampus failed to alter [ 3H]CHA binding to hippocampal membranes. These results suggest that [ 3H]CHA binding sites are not associated with axons or terminals of these neurones in the hippocampus. Intrahippocampal injection of kainic acid reduced the number of [ 3H]CHA recognition sites by 30% with no alteration in the affinity of [ 3H]CHA for these receptors. Thus, a significant portion of A 1 receptors may be associated with intrinsic neurones of the hippocampus which do not appear to be innervated by noradrenergic, cholinergic or serotonergic axons.