Interference of S-Nitrosoglutathione with the Binding of Ligands to Ionotropic Glutamate Receptors in Pig Cerebral Cortical Synaptic Membranes

The interactions of S-nitrosoglutathione (GSNO) with the ionotropic glutamate receptors were studied on synaptic membranes isolated from the pig cerebral cortex. GSNO displaced the binding of [³H]glutamate, 3-[(R)-2-carboxypiperazin-4-yl][³H]propyl-1-phosphonate ([³H]CPP), a competitive N-methyl-D-aspartate (NMDA) antagonist, and [³H]kainate, with IC₅₀ values in the low micromolar range. It failed to displace (S)-5-fluoro-[³H]willardiine, a selective agonist of 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) receptors. Reduced and oxidized glutathione were almost as effective as GSNO in glutamate and CPP binding. Of the three, GSNO was the most potent in kainate binding. They all stimulated [³H]dizocilpine binding in a concentration-dependent manner. This effect was additive to that of glycine and not mimicked by NO donors such as S-nitroso-N-acetylpenicillamine, 5-amino-3-morpholinyl-1,2,3-oxadiazolium chloride (SIN-1) and nitroglycerin. We assume that GSNO may act as an endogenous ligand at the NMDA and non-NMDA classes of glutamate receptors. In this manner it may facilitate NO transfer and target its delivery to specific sites in these receptors.     

Involvement of a Disulfide Bond in the Binding of Flunitrazepam to the Benzodiazepine Receptor from Bovine Cerebral Cortex

Abstract: The effects of chemical modification of a disulfide bond(s) (-SS-) or sulfhydryl group(s) (-SH) on the [³H]-flunitrazepam ([³H]FNZ) binding to membrane-bound or immunoprecipitated benzodiazepine (BZD) receptors (BZD-R) from bovine cerebral cortex were examined. Reduction of -SS- with dithiothreitol (DTT) brought about a reversible, time- and dose-dependent inhibition of [³H]FNZ binding to the membrane-bound BZD-R. Alkylation of the membranes with the -SH-modifying reagent iodoacetamide (IAA) or 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB) produced a slight inhibition of [³H]FNZ binding in a dose-dependent manner. Scatchard analysis of saturation curves of [³H]FNZ binding in the presence and absence of 5 m M DTT revealed changes in affinity without modification in the maximal binding capacity, thus indicating a competitive mode of interaction. DTT pretreatment of both the membrane-bound and the immunoprecipitated BZD-R led to [³H]FNZ binding inhibition. Consistent with the modification of a binding site is the observation that reduction of -SS- does not bear on the binding affinity, but rather reduces the number of sites. Complete protection from DTT inhibition of [³H]FNZ binding by FNZ (an agonist) or by Ro 15–1788 (an antagonist) suggests the presence of -SS- at, or very close to, the BZD recognition binding site. No protection against IAA or DTNB inhibition was provided by FNZ. Photoaffinity labeling experiments with [³H]FNZ revealed a clear-cut band of 50 kDa in native and alkylated membranes but an extremely weak label in 5 m M DTT/IAA-treated membranes. The present results provide evidence for the participation of a disulfide bond in the recognition binding site of the bovine cerebral cortex BZD-R.     

Membrane Potentials in Cell-free Preparations from Guinea Pig Cerebral Cortex: Effect of Depolarizing Agents and Cyclic Nucleotides

The distribution of [3H]triphenylmethylphosphonium ion between the medium and vesicular entities was examined in a cell-free, particulate preparation from guinea pig cerebral cortex. This distribution followed the Nernst relationship with regard to the external potassium ion concentration and, in physiological media, indicated the maintenance of a mean trans-membrane potential ranging from -58 to -78 mV. The neurotoxins batrachotoxin, veratridine, and grayanotoxin I, partially depolarized the preparation. Tetrodotoxin blocked the depolarization by batrachotoxin, veratridine, and gray-anotoxin I. The depolarization by these neurotoxins was potentiated by the presence of anemone toxin II and presumably reflected the response of vesicular components of neuronal origin. An additional potassium-sensitive depolarization probably represented the response of vesicular components of glial origin with an apparent transmembrane potential of -8 to -35 mV. No correlation could be demonstrated between changes in transmembrane potential and stimulation of cyclic AMP generation by a variety of agents in this preparation.     

Decrease of Clonidine Binding Affinity to α2-Adrenoceptor by ADP-Ribosylation of 41,000-Dalton Proteins in Rat Cerebral Cortical Membranes by Islet-Activating Protein

The IC50 value for inhibition of specific [3H]yohimbine binding to rat cerebral cortical membranes by clonidine was increased, and the Hill coefficient (nH) approached unity in the presence of 150 microM GTP. Pretreatment of membranes with islet-activating protein (IAP) in the presence of NAD caused an increase in IC50 and nH values for clonidine compared with control membranes in the absence of GTP, the addition of which was without effect. Scatchard analysis showed that the Bmax value of the high-affinity component in [3H]clonidine binding was decreased by pretreatment with IAP/NAD. GTP in a concentration range of 0.1 microM-1 mM caused a significant elevation of [3H]yohimbine binding. In IAP/NAD-pretreated membranes, however, [3H]yohimbine binding was no longer affected by GTP, although IAP/NAD significantly (p less than 0.01) increased [3H]yohimbine binding compared to control. IAP ADP-ribosylated 41,000 dalton proteins of cerebral cortical membranes. From these results, it can be suggested that inhibitory guanine nucleotide regulatory protein with Mr 41,000 couples to alpha 2-adrenoceptors to regulate binding affinity of agonists and antagonists in membranes of the rat cerebral cortex.     

Characterization of Na+-Independent GABA and Flunitrazepam Binding Sites in Preparations of Synaptic Membranes and Postsynaptic Densities Isolated from Canine Cerebral Cortex and Cerebellum

The binding of [3H]GABA and [3H]flunitrazepam was performed with synaptic membranes and post-synaptic densities (PSDs) isolated from canine cerebral cortex and cerebellum. Two GABA binding sites were found with cerebral cortex membranes but only one with cerebellar membranes. PSDs isolated from these showed only single binding sites, with cerebellar PSDs exhibiting lower KD values and a larger concentration of sites than did cerebral cortex PSDs. In the case of flunitrazepam, only one binding site was found for all four preparations, with cerebellar PSDs having twice the concentration of sites of cerebral PSDs. Photoaffinity labeling of the flunitrazepam receptor in PSDs resulted in the binding to a 51,000 Mr protein in both cases, with cerebellar PSDs again showing an increased concentration over that found in cerebral cortex PSDs. Based on this work, and on earlier work of ourselves and of others, we conclude that both populations of isolated PSDs contain inhibitory sites, but that the intact PSDs in both preparations are derived from Gray type I, probably excitatory, synapses, and that the inhibitory sites are found in the broken-up material in the PSD fractions which are derived from Gray type II, probably inhibitory, synapses.     

Age-Related Loss of t-[35S]Butylbicyclophosphorothionate Binding to the γ-Aminobutyric AcidA Receptor-Coupled Chloride Ionophore in Rat Cerebral Cortex

Muscimol and t-butylbicyclophosphorothionate (TBPS) are known to label two distinct sites within the gamma-aminobutyric acidA (GABAA) receptor complex, i.e., the GABA recognition site and the chloride ionophore, respectively. Age-dependent changes in the specific binding of [3H]muscimol and [35S]TBPS were compared in membranes prepared from the cerebral cortex of rats, 2-800 days old. Perinatal (day 2) binding of muscimol and TBPS represented 8 and 20% of the respective values for adults (day 180). After the first week, muscimol binding increased more rapidly than TBPS binding. Levels near those of adults were reached at day 20 and remained practically unchanged in adulthood (day 180). In aged (780-day-old) rats, the binding of TBPS was significantly reduced, whereas muscimol binding did not change compared with adult values. This decrease of TBPS binding derived from a reduced density of binding sites, rather than from affinity changes. The allosteric responsiveness of TBPS binding to exogenous GABA was also reduced in aged animals. These findings indicate an age-related change in the molecular (structural) organization of the GABAA receptor-chloride ionophore complex in rat cerebral cortex.     

β-Amyloid (25-35) or Substance P Stimulates [3H]MK-801. Binding to Rat Cortical Membranes in the Presence of Glutamate and Glycine

Abstract: Micromolar concentrations of β-amyloid (25–35) or substance P stimulated [³H] MK-801 binding in the presence of low concentrations of glutamate (1 γM) and glycine (0.02 γM). Unlike polyamines spermine and spermidine, neither β-amyloid (25–35) nor substance P increased [³H] MK-801 binding in the presence of maximally stimulating concentrations of glutamate and glycine. 5,7-Dichloro-kynurenic acid, CGS-19755, and arcaine completely inhibited the stimulated [³H] MK-801 binding. There was an apparent decreased potency of the [³H] MK-801 binding inhibition curve for 5,7-dichlorokynurenic acid, but not CGS-19755 or arcaine, in the presence of either β-amyloid (25–35) or substance P. The compounds do not appear to act through the strychnine-insensitive glycine binding site because neither β-amyloid (25–35) nor substance P displaced [³H] glycine binding. Full-length β-amyloid (1-40), up to 10 γM, did not stimulate [³H] MK-801 binding. Concentrations >10 γM could not be tested because they formed large aggregate precipitates in the assay. The data indicate that β-amyloid (25–35) or substance P does not stimulate [³H] MK-801 binding at either the N-methyl-D-aspartate, glycine, or polyamine binding sites. Furthermore, the nonpeptide substance P receptor (NK,) antagonist, CP-96,345, did not block β-amyloid (25–35)- or substance P-stimulated [³H] MK-801 binding. Therefore, the effect is not due to an interaction between the substance P receptors and the N-methyl-D-aspartate receptor-operated ionophore. Finally, if these observations can be verified using single-channel recording techniques, they may have implications in the pattern of selective neuronal loss observed in patients with neurodegenerative processes such as Alzheimer's, Parkinson's, and Huntington's diseases.