Main Immunogenic Region of Torpedo Electroplax and Human Muscle Acetylcholine Receptor: Localization and Microheterogeneity Revealed by the Use of Synthetic Peptides

Most anti-nicotinic acetylcholine receptor (AChR) antibodies in myasthenia gravis are directed against an immunodominant epitope or epitopes [main immunogenic region (MIR)] on the AChR alpha-subunit. Thirty-two synthetic peptides, corresponding to the complete Torpedo alpha-subunit sequence and to a segment of human muscle alpha-subunit, were used to map the epitopes for 11 monoclonal antibodies (mAbs) directed against the Torpedo and/or the human MIR and for a panel of anti-AChR mAbs directed against epitopes on the alpha-subunit other than the MIR. A main constituent loop of the MIR was localized within residues alpha 67-76. Residues 70 and 75, which are different in the Torpedo and human alpha-subunits, seem to be crucial in determining the binding profile for several mAbs whose binding to the peptides correlated very well with their binding pattern to native Torpedo and human AChRs. This strongly supports the identification of the peptide loop alpha 67-76 as the actual location of the MIR on the intact AChR molecule. Residues 75 and 76 were necessary for binding of some mAbs and irrelevant for others, in agreement with earlier suggestions that the MIR comprises overlapping epitopes. Structural predictions for the sequence segment alpha 67-76 indicate that this segment has a relatively high segmental mobility and a very strong turning potential centered around residues 68-71. The most stable structure predicted for this segment, in both the Torpedo and human alpha-subunits, is a hairpin loop, whose apex is a type I beta-turn and whose arms are beta-strands. This loop is highly hydrophilic, and its apex is negatively charged. All these structural properties have been proposed as characteristic of antibody binding sites. We also localized the epitopes for mAbs against non-MIR regions. Among these, the epitope for a monoclonal antibody (mAb 13) that noncompetitively inhibits channel function was localized within residues alpha 331-351.     

Decrease in the Function of the γ-Aminobutyric Acid-Coupled Chloride Channel Produced by the Repeated Administration of Pentylenetetrazol to Rats

The acute administration of pentylenetetrazol (PTZ; 25-75 mg/kg i.p.) failed to modify the specific binding of t-[35S]butylbicyclophosphorothionate ([35S]TBPS) to membrane preparations from the cerebral cortex of the rat. In contrast, the repeated administration of PTZ (30 mg/kg i.p., three times a week for 12 weeks) reduced by 26% the density of [35S]TBPS binding sites without modifying the dissociation constant. This effect was observed 3 days after the last PTZ administration. A parallel reduction of gamma-aminobutyric acid (GABA)-stimulated 36Cl- uptake was measured in the cerebral cortex of PTZ-treated rats 3 days after the last injection. The repeated administration of PTZ produced sensitization to the drug, or chemical kindling. In fact, no convulsions were observed in the first week of treatment, but all the animals became sensitized to PTZ by the 12th week. The results are consistent with the hypothesis that chronic treatment with PTZ at a subconvulsant dose causes a decrease in GABA-coupled chloride channel activity that may be related to the chemical kindling produced by this compound.     

α1-Adrenergic Receptor Mediates Arachidonic Acid Release in Spinal Cord Neurons Independent of Inositol Phospholipid Turnover

The alpha 1-adrenergic receptor has been shown to mediate the release of arachidonic acid in FRTL5 thyroid cells and MDCK kidney cells. In primary cultures of spinal cord cells, norepinephrine stimulated release of arachidonic acid (from neurons only) and turnover of inositol phospholipids (from neurons and glia) via alpha 1-adrenergic receptors. These two responses were dissociated by treatment with phorbol ester and pertussis toxin, which inhibited production of inositol phosphates with no appreciable effect on release of arachidonic acid. Extracellular calcium was required for release of arachidonic acid, but not for production of inositol phosphates. The calcium channel blockers nifedipine and verapamil inhibited release of arachidonic acid only. However, 8-(N,N-diethylamino)octyl-3,4,5-trimethoxybenzoate (TMB-8), a compound that blocks intracellular calcium release, diminished production of inositol phosphates, but had little effect on release of arachidonic acid. These results suggest that alpha 1-adrenergic receptors couple to release of arachidonic acid in primary cultures of spinal cord cells by a mechanism independent of activation of phospholipase C, possibly via the activation of phospholipase A2.     

M1 Muscarinic Acetylcholine Receptor in Cultured Rat Neostriatum Regulates Phosphoinositide Hydrolysis

Abstract: : Muscarinic acetylcholine receptor expression and function in cultured rat neostriatal neurons were examined. All experiments were performed on intact neurons grown in vitro for 12-14 days. The muscarinic antagonist N-[³H]methylscopolamine ([³H]NMS) binds to a single site in cultures with a K_D of 89 pM and a B_max of 187 fmol/mg of protein, or 32,000 sites/neuron. Competition studies using [³H]NMS were performed to determine what receptor sur > types were present. Nonlinear analysis of competition curves was best described with a single binding site for atropine, pirenzepine, and AF-DX 116 {11-[[2-[(diethylamino)-methyl]-1-piperidinyl]acetyl]-5,11-dihydro-6H-pyrido[2,3-b][1,4]benzodiazepine-6-one}, with K_i values of 0.6, 62, and 758 nM, respectively. These results indicate that the muscarinic receptors present in neostriatal cultures are of the M₁subtype, having high affinity for pirenzepine and low affinity for AF-DX 116. In contrast with antagonists, carbachol displaced [³H]NMS from two sites with K_i values of 6.5 and 147 μM, with the higher-affinity form predominant (83% of sites). The M₁ receptor subtype was linked to phosphoinositide turnover. Carbachol stimulated the formation of phosphoinositides with an EC₅₀ of 37 μM and was antagonized by atropine. At equimolar doses, pirenzepine was more potent than AF-DX 116 at antagonizing the response.     

Discrete Distributions of Adenosine Receptors in Mammalian Retina

Binding sites for both the adenosine A1 receptor agonists [3H]phenylisopropyladenosine and [3H]cyclohexyladenosine and the mixed A1-A2 agonist N-[3H]ethylcarboxamidoadenosine [( 3H]NECA) were localized in rabbit and mouse retinas using autoradiographic techniques. These two classes of agonists bound to very different regions of mammalian retinas. A1 agonist binding was localized to the inner retina, particularly over the inner plexiform layer. The binding of [3H]NECA was observed primarily over the retinal pigmented epithelium and the outer and inner segments of photoreceptors. [3H]NECA labeling was not affected either by including a low concentration of unlabeled A1 agonist or by pretreating tissue with N-ethylmaleimide to inhibit ligand binding at A1 sites. While virtually all of the [3H]NECA binding was displaced by an excess of unlabeled NECA, displacement with antagonist or a large excess of cyclohexyladenosine revealed that approximately 30% of the [3H]NECA binding was at non-A1,A2 sites. The majority of the binding in the outer retina thus labeled A2 receptor sites. The unique localizations of the two classes of adenosine receptors suggest different functions in visual processing.     

Regional Dissociation of Cyclic AMP and Inositol Phosphate Formation in Response to Thyrotropin-Releasing Hormone in the Rat Brain

The present study was undertaken to define effects of thyrotropin-releasing hormone (TRH) on formation of cyclic AMP (cAMP) and inositol phosphates (IPs) in rat brain regions. The brain of male Wistar rats was dissected into seven discrete regions, and each region was sliced. The slices were incubated in Krebs-Henseleit glucose buffer containing varying doses of TRH. TRH caused a significant and consistent increase in cAMP level, but not in formation of IPs, in the hypothalamus, striatum, and midbrain. TRH stimulated formation of IPs in the cerebellum, where the tripeptide did not change the cAMP level. In contrast, formation of neither cAMP nor IPs was affected by TRH in the cortex, hippocampus, or pons-medulla. These data suggest that TRH possesses two distinct types of brain intracellular signaling systems, which vary with brain regions.     

Differential Binding Properties of the Peripheral-Type Benzodiazepine Ligands [3H]PK 11195 and [3H]Ro 5-4864 in Trout and Mouse Brain Membranes

High-affinity binding sites for [3H]PK 11195 have been detected in brain membranes of rainbow trout (Salmo gairdneri) and mouse forebrain, where the densities of receptors were 1,030 and 445 fmol/mg of protein, respectively. Ro 5-4864 (4'-chlorodiazepam) was 2,200-fold less potent as a competitor of [3H]PK 11195 binding in the piscine than the murine membranes. Investigation of the regional distribution of these sites in trout yielded a rank order of density of spinal cord greater than olfactory bulb = optic tectum = rhombencephalon greater than cerebellum greater than telencephalon. This site in trout shared some of the characteristics of the peripheral-type benzodiazepine receptor (PTBR) (also known as the mitochondrial benzodiazepine receptor) in rodents, i.e., high affinity for PK 11195 and the endogenous ligand protoporphyrin IX, but was unique in the low affinity of Ro 5-4864 (41 microM) and diazepam and the relatively high affinity of the calcium channel ligand diltiazem and two central benzodiazepine ligands, CGS 8216 and CGS 9896. The differential affinity for the two prototypic PTBR ligands in trout is similar to that previously observed in calf and human brain membranes. Structural differences for the trout sites are indicated by the relative inability of diethyl pyrocarbonate to modify histidine residues of the binding site in trout as compared with mouse membranes. Heterogeneity of binding of the two prototypic PTBR ligands in mouse brain membranes was indicated by additivity studies, equilibrium competition experiments, and saturation isotherms, which together support the hypothesis that Ro 5-4864 discriminates between two [3H]PK 11195 binding sites having high (nanomolar) and low (micromolar) affinity, respectively.     

Direct Evidence That Excitotoxicity in Cultured Neurons Is Mediated via N-Methyl-D-Aspartate (NMDA) as well as Non-NMDA Receptors

Cultured GABAergic cerebral cortex neurons were exposed to the excitatory amino acid (EAA) L-glutamate, kainate (KA), N-methyl-D-aspartate (NMDA), or RS-alpha-amino-3-hydroxy-5-methyl-4-isoxazolopropionate (AMPA). To ensure a constant glutamate concentration in the culture media during the exposure periods, the glutamate uptake inhibitor L-aspartic acid beta-hydroxamate was added at 500 microM to the cultures that were exposed to glutamate. Each of these EAAs was able to induce neurotoxicity. It was not possible to reduce or prevent glutamate-induced cytotoxicity by blocking only one of the glutamate receptor subtypes with either the NMDA receptor antagonist D-(-)-2-amino-5-phosphonopentanoate (APV) or with one of the specific non-NMDA antagonists 6-cyano-7-nitroquinoxaline-2,3-dione (CNQX) and 6,7-dinitroquinoxaline-2,3-dione (DNQX). However, if the cultures were exposed simultaneously to glutamate and the antagonists in combination, i.e., APV plus CNQX or APV plus DNQX, the toxicity was completely prevented. Furthermore, CNQX and DNQX were shown to be selective blockers of cytotoxic phenomena induced by non-NMDA glutamate agonists with no effect on NMDA-induced cell death. Likewise, APV prevented NMDA-induced cell death without affecting the KA- or AMPA-induced neurotoxicity. It is concluded that EAA-dependent neurotoxicity is induced by NMDA as well as non-NMDA receptors.     

Reduced Glycine Stimulation of [3H]MK-801 Binding in Alzheimer''s Disease

The novel N-methyl-D-aspartate receptor channel ligand (+)-[3H]5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5, 10-imine maleate ([3H]MK-801) has been utilized to label this receptor in human brain tissue. Characteristics of [3H]MK-801 binding to well-washed membranes from 17 control subjects and 16 patients with Alzheimer's disease were determined in frontal, parietal, and temporal cerebral cortex and cerebellar cortex. In control tissue the pharmacological specificity of the binding of this substance is entirely consistent with the profile previously reported for rat brain. Binding could be stimulated by the addition of glutamic acid to the incubation medium; addition of glycine produced further enhancement which was not prevented by strychnine. The specificity of the effects of these and other amino acids on the binding was the same as in the rat. In Alzheimer's disease significantly less binding was observed in the frontal cortex under glutamate- and glycine-stimulated conditions. This appears to be associated with a reduced affinity of the site whereas the pharmacological specificity of the site remained unchanged. The effect did not appear to be due to differences in mode of death between Alzheimer's disease and control subjects and is unlikely to be related to factors for which the groups were matched. In contrast, binding was not altered in the absence of added amino acids and presence of glutamate alone. These results imply that in the cerebral cortex the agonist site and a site in the cation channel of the receptor are not selectively altered, but that their coupling to a strychnine-insensitive glycine recognition site is impaired.     

A Glycine Site Associated with N-Methyl-d-Aspartic Acid Receptors: Characterization and Identification of a New Class of Antagonists

Membranes from rat telencephalon contain a single class of strychnine-insensitive glycine sites. That these sites are associated with N-methyl-D-aspartic acid (NMDA) receptors is indicated by the observations that [3H]glycine binding is selectively modulated by NMDA receptor ligands and, conversely, that several amino acids interacting with the glycine sites increase [3H]N-[1-(2-thienyl)cyclohexyl]piperidine ([3H]TCP) binding to the phencyclidine site of the NMDA receptor. The endogenous compound kynurenate and several related quinoline and quinoxaline derivatives inhibit glycine binding with affinities that are much higher than their affinities for glutamate binding sites. In contrast to glycine, kynurenate-type compounds inhibit [3H]TCP binding and thus are suggested to form a novel class of antagonists of the NMDA receptor acting through the glycine site. These results suggest the existence of a dual and opposite modulation of NMDA receptors by endogenous ligands.