No abstract

The in vitro binding of a new subtype‐selective NMDA receptor antagonist, [3H]Ro 25‐6981, to rat brain membranes and sections was characterized. The compound bound to a single site on the membranes with a KD of 3 nM and a Bmax of 1.6 pmol/mg of protein. Specific binding, defined with a new NR2B‐specific antagonist, Ro 04‐5595 [1‐(4‐chlorophenyl)‐2‐methyl‐6‐methoxy‐7‐hydroxy‐1,2,3,4‐tetrahydroisoquinoline], at 10 µM, was fully inhibited by several compounds with the following rank order of affinities—Ro 25‐6981 > CP‐101,606 > Ro 04‐5595 = ifenprodil ≫ eliprodil > haloperidol > spermine > spermidine > MgCl2 > CaCl2—and partially inhibited by competitive glutamate recognition site antagonists. A high density of binding sites was detected, radioautographically, in several layers of the cerebral cortex, in the hippocampus, dentate gyrus, tuberculum olfactorium, caudate putamen, medium densities in the globus pallidus, thalamus, spinal cord dorsal horn, and motoneurons, whereas the cerebellum, pons, and medulla were, with a few exceptions, e.g., locus coeruleus, poorly labeled. Overall, the distribution of [3H]Ro 25‐6981 binding sites correlated well with that of NR2B (but not NR2A) transcripts, revealed by in situ hybridization histochemistry. The high affinity of [3H]Ro 25‐6981 for NR2B‐containing receptors renders this compound the ligand of choice to study the regulation of NR2B‐containing receptor expression.

No abstract

Abstract:We have investigated the binding properties of [ 3 H]quisqualate to rat metabotropic glutamate (mGlu) 1a and 5a receptors and to rat and human brain sections. Saturation isotherms gave K D values of 27 Ϯ 4 and 81 Ϯ 22 nM for mGlu1a and mGlu5a receptors, respectively. Several compounds inhibited the binding to mGlu1a and mGlu5a receptors concentration-dependently. (S)-4-Carboxyphenylglycine, (S)-4-carboxy-3-hydroxyphenylglycine, and (R,S)-1-aminoindan-1,5-dicarboxylic acid, which completely inhibited [ 3 H]quisqualate binding to the mGlu5a receptor, were inactive in a functional assay using this receptor. The distribution and abundance of binding sites in rat and human brain sections were studied by quantitative receptor radioautography and image analysis. Using 10 nM [ 3 H]quisqualate, a high density of binding was detected in various brain regions with the following rank order of increasing levels: medulla, thalamus, olfactory bulb, cerebral cortex, spinal cord dorsal horn, olfactory tubercle, dentate gyrus molecular layer, CA1-3 oriens layer of hippocampus, striatum, and cerebellar molecular layer. The ionotropic component of this binding could be inhibited by 30 M kainate, revealing the distribution of mGlu1ϩ5 receptors. The latter were almost completely inhibited by the group I agonist (S)-3,5-dihydroxyphenylglycine. The binding profile correlated well with the cellular sites of synthesis and regional expression of the respective group I receptor proteins revealed by in situ hybridization histochemistry and immunohistochemistry, respectively. Key Words: [ 3 H]Quisqualate binding-Metabotropic glutamate 1a and 5a receptors-Radioautography. J. Neurochem. 75, 2590 -2601 (2000).Eight G protein-coupled metabotropic glutamate (mGlu) receptors have been cloned to date (for review, see Pin et al., 1999). On the basis of their sequence similarities, signal transduction, and agonist rank order of potency, these receptors have been subdivided into three groups. Group I mGlu receptors, for which several splice variants have been identified , are activated by the weak group I-selective agonists (S)-3,5-dihydroxyphenylglycine (DHPG) and (S)-3-hydroxyphenylglycine (3HPG) and the potent nonselective agonist quisqualate (Aramori and Nakanishi, 1992). This group comprises mGlu1, which is selectively blocked noncompetitively by 7-(hydroxyimino)cyclopropa [b]chromen1a-carboxylate ethyl ester (CPCCOEt) (Litschig et al., 1999), and mGlu5, which is selectively blocked, noncompetitively, with high potency by methylphenylethynylpyridine . Although precise physiological roles cannot yet be assigned to mGlu1 and mGlu5 receptors (potent, selective agonists/antagonists with good brain penetration are not yet widely available), group I receptors have, nevertheless, been implied in psychiatric and neurological diseases (see Bordi and Ugolini, 1999).

The distribution and abundance of specific binding sites (=95% of total) in sections of rat CNS, revealed by quantitative receptor radioautography and image analysis, indicated a very discrete localization. Highest binding values were observed in cortical layers (binding in layers 1 and 2> binding in layers 3-6), hippocampal formation, striatum, dorsal septum, reticular thalamic nucleus, cerebellar molecular layer, and spinal cord dorsal horn. At 1 nM, the values for specific binding were highest in the cortical layers 1 and 2 and lowest in the brainstem (=2.6 and 0.4 pmol/mg of protein, respectively). Ro 48-8587 is a potent and selective AMPA receptor antagonist with improved binding characteristics (higher affinity, selectivity, and specific binding) compared with those previously reported. Key Words: a-Amino-3-hydroxy-5-methylisoxazole-4-propionate-9-lmidazol-1 -yl-8-nitro-2,3, 5,6-tetrahydro- High-affinity a-aniino-3-hydroxy-5-methylisoxazole-4-propionate (AMPA) receptors are ligand-gated cation channels for L-glutalnate, the major excitatory transmitter in the mammalian CNS. Their activation mediates fast transmission at glutamatergic synapses. The receptors, heterooligomeric proteins composed of the subunits G1uR1 -4 with flip and flop forms (Hollmann and Heinemann, 1994;Bettler and Mulle, 1995;Barnard, 1997), play key roles in CNS physiology and pathology. AMPA receptor subunits G1uR1 -4 are differentially regulated during ontogeny, and in adult rat brain they have only partially overlapping distributions (Keinanen et al