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.     

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.     

Comparison of [125I]Iodolysergic Acid Diethylamide Binding in Human Frontal Cortex and Platelet Tissue

The human platelet contains a functional 5-hydroxytryptamine (5-HT) receptor that appears to resemble the 5-HT2 subtype. In this study, we have used the iodinated derivative [125I]iodolysergic acid diethylamide ([125I]iodoLSD) in an attempt to label 5-HT receptors in human platelet and frontal cortex membranes under identical assay conditions to compare the sites labelled in these two tissues. In human frontal cortex, [125I]iodoLSD labelled a single high-affinity site (KD = 0.35 +/- 0.02 nM). Displacement of specific [125I]iodoLSD binding indicated a typical 5-HT2 receptor inhibition profile, which demonstrated a significant linear correlation (r = 0.97, p less than 0.001, n = 17) with that observed using [3H]ketanserin. However, [125I]iodoLSD (Bmax = 136 +/- 7 fmol/mg of protein) labelled significantly fewer sites than [3H]ketanserin (Bmax = 258 +/- 19 fmol/mg of protein) (p less than 0.001, n = 6). In human platelet membranes, [125I]iodoLSD labelled a single site with affinity (KD = 0.37 +/- 0.03 nM) similar to that in frontal cortex. The inhibition profile in the platelet showed significant correlation with that in frontal cortex (r = 0.96, p less than 0.001, n = 16). We conclude that the site labelled by [125I]iodoLSD in human platelet membranes is biochemically similar to that in frontal cortex and most closely resembles the 5-HT2 receptor subtype, although the discrepancy in binding capacities of [125I]iodoLSD and [3H]ketanserin raises a question about the absolute nature of this receptor.     

Alteration of opioid receptors in seizure-susceptible El mouse brain

The distribution density of opioid receptors in the brain of El mice (seizure-susceptible strain) was examined to determine the relation between seizures and the opioid system. Saturation curves and Scatchard plots of [³H]2-d-alamine-5-d-leucine enkephalin binding revealed that the opioid delta receptor density in adult El mice during interictal periods was significantly increased in the cerebral cortex, hippocampus, and septal area. It was further shown that the concentration of such receptors in 25-day-old El mice that had no seizures was also significantly increased in the hippocampus and septal area, with no changes in apparent affinities, as compared with in the corresponding regions in ddY mice (seizure-nonsusceptible strain; the mother strain of El). Such up-regulation of opioid receptors in the El mouse brain could result from deficits in endogenous opioid peptides, which could be associated with the pathogenesis of seizure diathesis in the El mouse.