Crude Synaptic Membranes Research Articles

Vinconate, a cognitive enhancer, and PI turnover-phospholipase C systems in the brain

The molecular mechanisms for the stimulation of inositol 1-phosphate (IPI) formation by vinconate were investigated using preparations of rat brain. Vinconate (10−8–10−3 M) dose-dependently inhibited the binding of [3H]quinuclidinyl benzilate ([3H]QNB) to muscarinic acetylcholine receptors and its IC50 value for [3H]QNB binding was 1.7 × 10−5 M. The rightward shift of carbachol displacement curve of [3H]QNB binding by GTP (10−4 M) was completely abolished by Vinconate (10−5 M). Carbachol (10−8–10−2 M) increased [3H]IP1 formation in a dose-dependent manner and the carbachol-induced [3H]IP1 formation was significantly accentuated by Vinconate (10−5 M). The enhancement of [3H]IP1 accumulation by Vinconate was inhibited by approximately 50% in the presence of atropine (10−5 M), although phentolamine and ketanserin had no effects on the stimulatory effect of Vinconate on [3H]IP1 formation. Vinconate showed no alteration in the binding of [3H]guanosine 5′-(β,γ-imino)triphosphate ([3H]Gpp(NH)p) to the crude synaptic membranes. The enhancement of phosphatidylinositol 4,5-biphosphate (PIP2)-specific phospholipase C (PLC) activity by GTP was unaffected in the presence of 10-3 M Vinconate, whereas Vinconate alone dose-dependently enhanced the activities of both PIP2-specific and cytosolic PLC. These results suggest that Vinconate may induce the facilitation of phosphatidylinositide (PI) turnover via the stimulation of muscarinic acetylcholine receptors, the enhancement of coupling between muscarinic acetylcholine receptors and GTP-binding protein, and the direct activations of PIP2-specific and cytosolic PLC.

Characterization of α, β-Methylene ATP Binding Sites in Mouse Crude Synaptic Membranes

Since ATP has been reported to be a potent excitatory transmitter in the mammalian central nervous system (CNS), we studied the neurochemical characters of the binding sites of α, β-methylene ATP, an agonist of P 2X receptors, in mouse crude synaptic membranes. ATP and its related compounds inhibited [ 3H] α, β-methylene ATP binding in a concentration-dependent manner. The potency order in the inhibition of the binding was as follows; α, β-methylene ATP = ADP βS > ATP γS > ATP ≥ ADP > β, γ-methylene ATP ⪢ UTP > 2-methylthio ATP. And adenosine did not affect the binding. The order was different from those reported in peripheral tissues. And Sr 2+, Ca 2+, Mg 2+, and Cd 2+ enhanced the binding. These results suggest that α, β-methylene ATP binding sites in CNS have different characters from those in peripheral tissues.

Role of protein kinase C in desensitization of spinal delta-opioid-mediated antinociception in the mouse.

1. Receptor phosphorylation and down-regulation by protein kinases may be a key event initiating desensitization. The present studies were designed to investigate the effect of a potent protein kinase C (PKC) activator, phorbol 12,13-dibutyrate (PDBu), on antinociception induced by intrathecal (i.t.) administration of a selective delta-opioid receptor agonist [D-Ala2] deltorphin II in the male ICR mouse and on the specific binding of [3H]-[D-Ser2, Leu5]enkephalin-Thr6 (DSLET), a delta-opioid receptor ligand, in the crude synaptic membrane of the spinal cord. 2. Intrathecal (i.t.) pretreatment with PDBu at low doses, which injected alone did not affect the basal tail-flick latency, dose-dependently attenuated the antinociception induced by i.t. administration of [D-Ala2]deltorphin II. The attenuation of i.t.-administered [D-Ala2] deltorphin II-induced antinociception by PDBu was reversed in a dose-dependent manner by i.t. concomitant pretreatment with a specific PKC inhibitor, calphostin C. 3. In the binding experiment, incubation of the crude synaptic membrane of the spinal cord for 2 h at 25 degrees C with PDBu (0.03 to 10 microM) caused a dose-dependent inhibition of the [3H]-DSLET binding. Scatchard analysis of [3H]-DSLET binding revealed that PDBu at 10 microM displayed a 30.7% reduction in the number of [3H]-DSLET binding sites with no significant change in affinity, compared with the non-treatment control, indicating that the activation of membrane-bound PKC by PDBu causes a decrease in the number of specific delta-opioid agonist binding sites. 4. An i.t. injection of [D-Ala2]deltorphin II produced an acute antinociceptive tolerance to the antinociceptive effect of a subsequent i.t. challenge of [D-Ala2]deltorphin II. Concomitant pretreatment with calphostin C markedly prevented the development of acute tolerance to the i.t.-administered [D-Ala2]deltorphin II-induced antinociception. On the other hand, a highly selective protein kinase A (PKA) inhibitor, KT5720, did not have any effect on the development of acute tolerance to [D-Ala2]deltorphin II antinociception. 5. These findings suggest that a loss of specific delta-agonist binding by the activation of PKC by PDBu is involved in the PDBu-induced antinociceptive unresponsiveness to delta-opioid receptor agonist in the mouse spinal cord. Based on the acute tolerance studies, we propose that PKC, but not PKA, plays an important role in the process of homologous desensitization of the spinal delta-opioid receptor-mediated antinociception.

Effect of hypothyroidism on the subcellular distribution of Ca2+/calmodulin-stimulated protein kinase II in chicken brain during posthatch development.

In developing chicken brain Ca2+/calmodulin-stimulated protein kinase II (CaMPK-II) changes from being primarily cytosolic to being primarily particulate during the protracted maturation period. To investigate whether thyroid hormone levels may be involved in regulating this subcellular redistribution, we raised chickens from 1 day posthatching on food soaked in 0.15% (wt/vol) propylthiouracil (PTU) plus 0.05% (wt/vol) methimazole (MMI). This produced a mild hypothyroidism specifically during the maturation period and resulted in a 67% reduction in the levels of free triiodothyronine (T3) at 42 days. The concentrations of alpha- and beta-CaMPK-II in cytosol (S3) and crude synaptic membrane (P2M) fractions from forebrain were measured by three methods: Ca2+/calmodulin- or ZN2+-stimulated autophosphorylation or binding of biotinylated calmodulin. By all three methods hypothyroid animals showed a marked retardation of the redistribution of both subunits of CaMPK-II: an increase in the concentration of the enzyme in S3 and a corresponding decrease in P2M with no overall change in the total amount of enzyme and little apparent change in the concentration of other proteins. In both fractions, there was a parallel change in the Ca2+/calmodulin-stimulated phosphorylation of endogenous protein substrates but no change in the basal or cyclic AMP-stimulated protein phosphorylation. Supplementing the PTU/MMI-treated diet with thyroxine (0.5 ppm) prevented all of the observed changes.

Characterization and subcellular localization of L-[3H] carnitine binding sites in rat brain.

In the present study, we investigated the existence of a binding site for L-carnitine in the rat brain. In crude synaptic membranes, L-[3H]carnitine bound with relatively high affinity (KD = 281 nM) and in a saturable manner to a finite number (apparent Bmax value = 7.3 pmol/mg of protein) of binding sites. Binding was reversible and dependent on protein concentration, pH, ionic strength, and temperature. Kinetic studies revealed a Koff of 0.018 min-1 and a Kon of 0.187 x 10(-3) min-1 nM-1. Binding was highest in spinal cord, followed by medulla oblongata-pons > or = corpus striatum > or = cerebellum = cerebral cortex = hippocampus = hypothalamus = olfactory bulb. L-[3H]Carnitine binding was stereoselective for the L-isomers of carnitine, propionylcarnitine, and acetylcarnitine. The most potent inhibitor of L-[3H]carnitine binding was L-carnitine followed by propionyl-L-carnitine. Acetyl-L-carnitine and isobutyryl-L-carnitine showed an affinity approximately 500-fold lower than that obtained for L-carnitine. The precursor gamma-butyrobetaine had negligible activity at 0.1 mM. L-Carnitine binding to rat crude synaptic membrane preparation was not inhibited by neurotransmitters (GABA, glycine, glutamate, aspartate, acetycholine, dopamine, norepinephrine, epinephrine, 5-hydroxytryptamine, histamine) at a final concentration of 0.1 mM. In addition, the binding of these neuroactive compounds to their receptors was not influenced by the presence of 0.1 mM L-carnitine.(ABSTRACT TRUNCATED AT 250 WORDS)

Calcium and Membrane Binding Properties of Bovine Neurocalcin δ Expressed in Escherichia coli

Calcium and Membrane Binding Properties of Bovine Neurocalcin δ Expressed in Escherichia coli

Inhibitory effects of pentamidine on N-methyl-D-aspartate (NMDA) receptor/channels in the rat brain.

Effects of pentamidine, a therapeutic drug for Pneumocystis carinii pneumonia (PCP) in acquired immunodeficiency syndrome (AIDS), on specific bindings of [3H](+)-5-methyl-10,11-dihydro-5H- dibenzo[a,d]cyclohepten-5,11-imine maleate (MK-801) and [3H]nitrendipine were investigated in crude synaptic membranes (CSM) of rat brain. Pentamidine inhibited [3H]MK-801 binding but did not change [3H]nitrendipine binding, although neither binding was inhibited by 3'-azido-2',3'-dideoxythymidine or 2',3'-dideoxycytidine (inhibitors for reverse transcriptase of HIV-1), or FK-506 or cyclosporin A (immunosuppressants). In Triton X-100-treated CSM (post-synaptic density-rich fractions), the inhibitory effect of pentamidine on [3H]MK-801 binding was partially prevented by addition of spermine and NMDA plus glycine (Gly). Electrophysiological experiments showed that pentamidine also inhibited Ca(2+)-current evoked by NMDA plus Gly in Xenopus oocytes injected with rat brain mRNA. These results suggest that pentamidine is a potent inhibitor for NMDA receptor/channels.

Effects of thyroxine and its related compounds on cerebral gaba receptors: Inhibitory action of benzodiazepine recognition site in GABA A receptor complex

The effects of thyroxine and its related derivatives on γ-aminobutyric acid (GABA) receptors in the rat brain were examined. d-Thyroxine strongly inhibited [ 3H]flunitrazepam binding to benzodiazepine receptor in crude synaptic membrane from the rat brain. The Scatchard analysis of the [ 3H]flunitrazepam binding in the presence of d-thyroxine indicated the decreases in the affinity and maximum number of binding site. Furthermore, d-thyroxine inhibited the enhancing effect of flunitrazepam on GABA-stimulated 36Cl − influx into membrane vesicles, although GABA-stimulated 36Cl − influx alone was not affected by d-thyroxine. On the other hand, the effects of thyroxine and its related derivatives on cerebral GABA B receptor binding were not noted. These results suggest that d-thyroxine may be a drug which is able to modulate the function of GABA A receptor complex via the inhibitory action on benzodiazepine recognition site.

Purification of the synaptic vesicle-binding protein physophilin. Identification as 39-kDa subunit of the vacuolar H(+)-ATPase.

Physophilin is a 36-kDa polypeptide originally identified in synaptic plasma membrane fractions, which binds to synaptic vesicles and has been implicated in vesicle docking and/or exocytosis during neurotransmitter release. Here we report on the purification, amino acid sequence analysis, and subcellular localization of physophilin. Physophilin was enriched from detergent extracts of crude synaptic plasma membranes by a combination of cation exchange and lentil-lectin chromatography. Sequence analysis of peptides generated after sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed that physophilin is identical to the 39-kDa subunit (Ac39) of the vacuolar H(+)-ATPase. This was confirmed further by Western blot analysis with an Ac39-specific antiserum and by vesicle binding assays with recombinant Ac39 protein. Subcellular fractionation showed that Ac39 is enriched in synaptic vesicles, with lesser amounts being present in synaptic plasma membrane fractions. These results argue against a docking role of physophilin/Ac39 in synaptic vesicle exocytosis.

GABA A receptors in mouse cortical homogenates are phosphorylated by endogenous protein kinase A

Biochemical, molecular, and electrophysiological studies suggest that phosphorylation of β subunits of the GABA A receptor (GaR) by exogenous protein kinase A inactivates the receptor channels. We have developed a method which for the first time allows the study of GaR phosphorylation in brain tissues by endogenous PKA. Desalted homogenates or crude synaptic membranes from mouse cerebral cortex were incubated with [γ- 32P] ATP and 8-Br-cAMP or chlorophenylthio-cAMP. Extracts from these incubations were immunoprecipitated by polyclonal antibodies against native GaR and analyzed by SDS-gel electrophoresis and autoradiography. In both homogenates and membranes, cAMP-dependent incorporation of 32P was observed for a 57-kDa peptide, and to a lesser extent 51- to 53-kDa peptides. Phosphorylation of affinity-purified GaR by the catalytic subunit of PKA also produced a major 57-kDa phosphopeptide and a minor 51-kDa phosphopeptide. Limited digestion by S. aureus V-8 protease of the 57-kDa phosphopeptide from the desalted homogenates or from purified receptors produced a major 32P-labeled fragment of 11 kDa, suggesting that the phosphorylation site is similar to that shown previously to reduce GaR function. The phosphorylation of GaRs in homogenates was time dependent and blocked by H-89 or protein kinase inhibitor 5–24, specific inhibitors of protein kinase A. Prolonged incubations resulted in dephosphorylation of the 57-kDa phosphoprotein by a microcystin-LR sensitive phosphatase. In cortical homogenates the level of cAMP-dependent phosphorylation of the 57-kDa GaR peptide was more than 5 times that obtained with washed synaptic membranes. However, assays of PKA using the heptamer kemptide as substrate showed that the specific activity in the particulate fraction was 57% that of the homogenate. This suggests that GaRs on synaptic membranes are preferentially phosphorylated by a cytoplasmic form of protein kinase A. By comparing the [ 3H] flunitrazepam-photolabeled 53-kDa GaR subunit with the 51–57 kDa [ 32P] peptides from cortical homogenates, the molar ratio of [ 32P]/[ 3H] was estimated at 0.43, suggesting that a substantial fraction of the GaR pool is phosphorylated under these conditions.

Specific [ 3H] l-glutamate binding and [ 3H] d-aspartate release in the hippocampus of rat after pentylenetetrazol kindling and long-term potentiation

In the present study, the time-course of changes in glutamate binding and aspartate release in the rat hippocampus following tetanization of perforant pathway and of pentylenetetrazol-induced kindling was investigated. The K + (48 mM)-stimulated [ 3H] d-aspartate release from hippocampal slices and the specific [ 3H] l-glutamate binding to crude synaptic membranes of the hippocampus were measured 1, 4 and 24 h after the last tetanization train or one, five and nine weeks after the last pentylenetetrazol injections, respectively. The tetanization of the fascia dentata was followed by a significant increase in K +-stimulated [ 3H] d-aspartate release from hippocampal slices only 1 h after the last tetanization train, but the specific [ 3 H] l-glutamate binding was enhanced 4 h afterwards. No further deviations from controls have been seen 24 h after tetanization. After pentylenetetrazol kindling, the K +-stimulated amino acid release from hippocampal slices was not changed. However, at one week as well as at five and nine weeks after the pentylenetetrazol kindling the specific [ 3H] l-glutamate binding in the hippocampus was significantly increased by about 50% compared to controls. From these findings it can be assumed that after long-term potentiation induction presynaptic transmitter release and post- (and/or pre-)synaptic glutamate binding are elevated with a different time course only in the early phase of potentiation. In contrast, kindling results in a long lasting increase in activity state of glutamatergic transmission. Therefore, it can be concluded that differences of the synaptic excitability following long-term potentiation and kindling are reflected by a different activation of glutamatergic system in the central nervous system.

Association and agonistic action of DN-2327, a novel isoindoline derivative, on GABA B receptor in brain

The association and action of DN-2327, 2-(7-chloro-1,8-naphthyridin-2-yl)-3-[(1,4-dioxa-8-azaspiro[4,5]dec-8-yl)carbonylmethyl]isoindolin-1-one, on the γ-aminobutyric acid (GABA) B receptor in rat brain have been examined. DN-2327 inhibited the binding of [ 3H]GABA to GABA B receptor in crude synaptic membrane obtained from rat brain. The Scatchard analysis of [ 3H]GABA binding to GABA B receptor indicated that DN-2327 induced the decrease in affinity of both high and low affinity binding sites without changing the B max values. The forskolin-stimulated adenylate cyclase activity in slices from rat cerebral cortex was significantly suppressed by the addition of DN-2327. Furthermore, this inhibition by DN-2327 was eliminated by the simulataneous additions of 2-hydroxy saclofen or CGP 55845A, GABA B receptor antagonists. These results suggest that DN-2327 may have not only a high association with GABA B receptor but also an agonistic action on the receptor.

3H](+)-Pentazocine binding to rat brain σ1 receptors

[ 3H](+)-Pentazocine binding has been characterized in the rat brain. It binds to a single population of binding sites with affinity of about 7 nM and density of 280 fmol/mg protein. [ 3H](+)-Pentazocine binding is not enriched in the crude synaptic membrane, being about 1 6 of what we found in the crude membrane preparation. The binding, like that for other σ ligands, was enriched in the microsomal and nuclear fractions. The inhibition by haloperidol, proadifen and d-fenfluramine was the same in the crude synaptic membrane, nuclear and microsomal fractions, suggesting that [ 3H](+)-pentazocine binds to a homogeneous protein in the different subcellular fractions. Our pharmacological characterization using 45 different drugs suggests that the [ 3H](+)-pentazocine binding site in rat brain differs from other σ ligands, like N-propyl-3-(3-hydroxyphenyl)piperidine ([ 3H](+)-3PPP), N, N′-di( o-tolyl)guanidine ([ 3H]DTG) and (+)- N-allylnormetazocine ([ 3H](+)-SKF10,047). [ 3H](+)-Pentazocine binding in rat brain is inhibited by σ compounds and some cytochrome P450 ligands, like proadifen and 1-[2-[bis(4-fluorophenyl) methoxy]ethyl]-4-[3-phenylpropyl] piperazine (GBR 12909), although with considerably lower potency than reported for other σ ligands. Other inhibitors are some serotonin uptake blockers or their metabolites and phenylalkylamines.

Characterization of the calcium and chloride [ 3H]glutamate binding site in crude synaptic membranes from human brain tissue

1. The specific binding properties of [ 3H]glutamate to crude synaptic membranes (CSM) from postmortem human brain were studied. 2. Equilibrium binding analysis of [ 3H]glutamate binding to CSM from human brain cortex revealed a K D = 110 ± 12 nM and a B max = 27 ± 4 pmol/mg protein). 3. Calcium increased the number of binding sites, B max = 44 ± 6 pmol/mg protein, without a significant change in the affinity constant, K D = 95 ± 10 nM. 4. The dissociation constant of the [ 3H]glutamate bound to human CSM was 4.0 ± 0.4 min −1 ( n = 3). 5. The relative potencies of glutamate analogs and 2-amino-4-phosphonobutyric acid (APB) to compete for the glutamate binding sites, in human CSM, were glutamate > quisqualate = ibotenic acid > APB ⪢ α-amino-3-hydroxy-5-methyl-4-isoxozolepropionate acid. 6. The glutamate specific binding in CSM from postmortem human brain was particularly rich in the gyrus hippocampus, nucleus accumbens, thalamus and frontal cortex. 7. This glutamate binding protein is related, probably, to a presynaptic neurosecretory pathway.

Neurotoxicity of Nonionic Low-Osmolar Contrast Media

Mennini T, Bernasconi P, Fiori MG. Neurotoxicity of nonionic low-osmolar contrast media: a receptor binding study. Invest Radiol 1993;28:821-827. rationale and objectives. The use of the newest nonionic, water-soluble, low-osmolar radiographic contrast media (CM) is still associated with occasional adverse reactions affecting the neural tissues. Because these CM display lipophilic potential in their interactions with biological membranes when diffusing within the brain parenchyma, they could affect neurotransmitter binding to the receptors. Two representative nonionic CM, iopamidol and iohexol, were studied to assess whether CM-related neurotoxicity derived from their interactions with specific receptors on neural membranes. methods. Binding assays were carried out in vitro on crude total membrane or crude synaptic membrane preparations from selected brain areas (cortex, striatum, hippocampus, cerebellum). The concentrations of CM and reference drugs that reduce specific binding of each ligand by 50% of its maximum value (IC50) were determined using radioligands to the receptors of the most common neurotransmitters in the central nervous system, including excitatory amino acids. results. Neither iopamidol nor iohexol inhibited the (3H) ligand binding to any kind of receptor up to very high concentrations (100 μM). conclusions. The nonionic, low-osmolar CM did not influence the normal functions of neural membranes in our model. This suggests that occasional neurotoxic effects do not occur as a consequence of specific action on brain receptors. These CM may have an indirect, postmembrane site of action.

Neurotoxicity of Nonionic Low-Osmolar Contrast Media

The use of the newest nonionic, water-soluble, low-osmolar radiographic contrast media (CM) is still associated with occasional adverse reactions affecting the neural tissues. Because these CM display lipophilic potential in their interactions with biological membranes when diffusing within the brain parenchyma, they could affect neurotransmitter binding to the receptors. Two representative nonionic CM, iopamidol and iohexol, were studied to assess whether CM-related neurotoxicity derived from their interactions with specific receptors on neural membranes. Binding assays were carried out in vitro on crude total membrane or crude synaptic membrane preparations from selected brain areas (cortex, striatum, hippocampus, cerebellum). The concentrations of CM and reference drugs that reduce specific binding of each ligand by 50% of its maximum value (IC50) were determined using radioligands to the receptors of the most common neurotransmitters in the central nervous system, including excitatory amino acids. Neither iopamidol nor iohexol inhibited the (3H) ligand binding to any kind of receptor up to very high concentrations (100 microM). The nonionic, low-osmolar CM did not influence the normal functions of neural membranes in our model. This suggests that occasional neurotoxic effects do not occur as a consequence of specific action on brain receptors. These CM may have an indirect, postmembrane site of action.

GABA A receptor impairment in the genetic absence epilepsy rats from Strasbourg (GAERS): an immunocytochemical and receptor binding autoradiographic study

Some aspects of the GABA and cholinergic systems have been investigated in the cortex and thalamus of GAERS Wistar rats, a model of petit-mal epilepsy, and in a non-epileptic control strain. GABA and its synthetic enzyme, glutamic acid decarboxylase (GAD), were located by immunocytochemistry; the GABA A receptors were evaluated by autoradiography of GABA-enhanced 3H-flunitrazepam binding and by immunocytochemistry using specific antibodies against the β 2-β 3 subunits of GABAP A receptor protein. GABA and GAD immunocytochemistry did not show up any difference in density or distribution of immunoreactive elements (fibers, terminals and neurons) between epileptic and control animals, but autoradiographic and immunocytochemical studies showed a decreased enhancement of 3H-flunitrazepam binding and of β 2-β 3 subunits of GABA A receptor in the sensorimotor cortex and anterior thalamic areas of the epileptic strain. No differences were found in benzodiazepine receptors in the two strains. GABA B receptors were measured as 3H-baclofen binding in a crude synaptic membrane preparation and there was no difference between epileptic and control animals. Choline acetyltransferase, the synthetic enzyme for acetylcholine, and muscarinic receptor subtypes (M 1 and M 2), visualized respectively by an immunocytochemical procedure and binding autoradiography, did not differ in epileptic and normal rats. The data suggest an impairment of the ‘GABA A system’ in restricted brain regions of epileptic rats, due to a reduction of receptor β 2-β 3 subunits and coupling to benzodiazepine receptors despite the normal synthesis and location of the neurotransmitter.

Identification of GABAA/benzodiazepine receptors on clathrin-coated vesicles from rat brain.

To investigate the subcellular compartments that are involved in the endocytosis and intracellular trafficking of GABAA/benzodiazepine receptors, we have studied the distribution and properties of clonazepam-displaceable binding of [3H]flunitrazepam to membrane fractions from rat brain. The microsomal fraction was subjected to density centrifugation and gel filtration to isolate clathrin-coated vesicles. Homogeneity of the coated-vesicle fraction was demonstrated by using electron microscopy and by analysis of clathrin subunits and clathrin light-chain kinase. Vesicles exhibiting specific binding of [3H]flunitrazepam eluted from the sieving gel as a separate peak, which was coincident with that for coated vesicles. Scatchard analysis of equilibrium binding of [3H]flunitrazepam to coated vesicles yielded a KD value of 21 +/- 4.7 nM and a Bmax value of 184 +/- 28 fmol/mg. The KD value for coated vesicles was 12-19-fold that found with microsomal or crude synaptic membranes. This low-affinity benzodiazepine receptor was not identified on any other subcellular fraction and thus appears to be a novel characteristic of coated vesicles. The Bmax value for coated vesicles, expressed per milligram of protein, corresponded to 16 and 115% of that found for crude synaptic and microsomal membrane fractions, respectively. Because the trafficking of neurotransmitter receptors via clathrin-coated vesicles is most likely to occur through endocytosis, the data suggest that an endocytotic pathway may be involved in the removal of GABAA/benzodiazepine receptors from the neuronal surfaces of the rat brain. This mechanism could play a role in receptor sequestration and down-regulation that is produced by exposure to GABA and benzodiazepine agonists.

ES-242-2, -3, -4, -5, -6, -7, and -8, novel bioxanthracenes produced by Verticillium sp., which act on the N-methyl-D-aspartate receptor.

Verticillium sp. SPC-15898 was found to produce novel metabolites, designated ES-242-2-(-)8, which were structurally related to ES-242-1. These compounds were isolated from the culture broth and the physico-chemical and biochemical properties were examined. ES-242-2-(-)8 inhibited [3H]thienyl cyclohexypiperidine ([3H]TCP) binding to rat crude synaptic membranes (CSM) with IC50 values of 0.116, 2.9, ca. 2.9, 25.3, 1.0, 59, 24, and 13 microM, respectively. None of these compounds showed inhibitory effects against the binding of [3H]kainate to its receptor, which is another subtype of the excitatory amino acid receptor.

Monoclonal antibody to GABA binding protein, a possible GABA b receptor

A monoclonal antibody has been raised against a partially purified preparation for the GABA b receptor. The antibody recognized a protein of about 80 kDa in bovine brain synaptic membrane. Immunoabsorbent agarose beads conjugated with the antibody were able to remove, without visible changes in electrophoresed profiles of total proteins, over 90% of the baclofen suppressive GABA binding activity (designated herein, GABA b receptor binding activity) in the solubilized synaptic membrane fraction. Moreover, the addition of GB-1 antibody directly inhibited the GABA binding activity in the crude synaptic membrane fraction. These results indicate that the monoclonal antibody obtained here recognizes the GABA binding protein, or more specifically a GABA b receptor.