dizocilpine-maleate and 4-trans-2-carboxy-5-7-dichloro-4-phenylaminocarbonylamino-1-2-3-4-tetrahydroquinoline

The purpose of this study was to clarify the in vitro pharmacological profile and the in vivo activity of (3S)-7-chloro-3-[2-((1R)-1-carboxyethoxy)-4-aminomethylphenyl]aminocarbonylmethyl-1,3,4,5-tetrahydrobenz[c,d]indole-2-carboxylic acid hydrochloride (SM-31900). SM-31900 inhibited the binding of [3H]glycine and [3H]5,7-dichlorokynurenic acid, radioligands for the N-methyl-D-aspartate (NMDA) receptor glycine-binding site, to rat brain membranes in a competitive manner, with K(i) values of 11+/-2 and 1.0+/-0.1 nM, respectively, and completely prevented the binding of [3H]dizocilpine (MK-801), a radioligand for the NMDA receptor channel site. In cultures of rat cortical neurons, SM-31900 markedly prevented the neuronal cell death induced by transient exposure to glutamate, in a concentration-dependent manner. Its neuroprotective potency was much stronger than those of other glycine-binding site antagonists (4-trans-2-carboxy-5,7-dichloro-4-phenylaminocarbonylamino-1,2,3,4-tetrahydroquinoline (L-689,560), 5,7-dichlorokynurenic acid, and 7-chlorokynurenic acid). Furthermore, SM-31900 showed anticonvulsant activity when administered systemically, unlike other antagonists. These data indicate that SM-31900 is a systemically active antagonist with high affinity for the NMDA receptor glycine-binding site.

Topics: Aminoquinolines; Animals; Binding, Competitive; Brain; Cells, Cultured; Dizocilpine Maleate; Dose-Response Relationship, Drug; Excitatory Amino Acid Antagonists; Fetus; Glutamic Acid; Indoles; Kynurenic Acid; Male; Neurons; Neuroprotective Agents; Neurotoxins; Radioligand Assay; Rats; Rats, Sprague-Dawley; Rats, Wistar; Receptors, Glycine; Receptors, N-Methyl-D-Aspartate; Seizures

Repeated injections of a subconvulsant dose of pentylenetetrazole (PTZ, 30 mg/kg IP three times weekly for 13 injections) in Wistar and hooded Lister rats resulted in kindled seizures, the extent of which varied between strains. Wistar rats achieved stage 4 of clonic-tonic seizures, whereas hooded Lister rats only reached stage 2 of convulsive waves axially through the body. Rats were killed 10 days after their final injection, and radioligand binding was used to measure the expression of NMDA receptors in cortex and hippocampus using [3H]MK-801 and [3H]L-689,560, the latter binding specifically to the NR1 subunit. [3H]Ro 15-1788 measured expression of GABA(A)-benzodiazepine binding sites containing alpha1, alpha2, alpha3, or alpha5 subunits. Specific analysis of GABA(A) receptors containing the alpha5 subunit, which are preferentially localized in the hippocampus, was assessed with [3H]L-655,708. In the cortex, there was no effect of strain or treatment on the K(D) or B(max) of any of the ligands. Similarly, there was no effect of strain or treatment on hippocampal [3H]L-689,560 or [3H]Ro 15-1788 binding. However, in the hippocampus there was a significant, albeit modest, effect of treatment on the B(max) of [3H]MK-801 binding and the B(max) and K(D) of [3H]L-655,708 binding, i.e., PTZ-treated rats had fewer [3H]MK-801 and [3H]L-655,708 binding sites (NMDA and alpha5-containing GABA(A) receptors, respectively), but, these reductions were significant only in the relatively seizure-insensitive hooded Lister strain. This suggests that the increased susceptibility to kindling in Wistar rats is not directly related to alterations in the expression of NMDA or GABA(A) receptors.

Topics: Aminoquinolines; Animals; Cerebral Cortex; Convulsants; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Flumazenil; Hippocampus; Imidazoles; Kindling, Neurologic; Male; Pentylenetetrazole; Radioligand Assay; Rats; Rats, Wistar; Receptors, GABA-A; Receptors, N-Methyl-D-Aspartate; Species Specificity; Time Factors

Several studies have suggested that polyamines modulate the interaction of glycine with the NMDA receptor. We have further investigated the effects of polyamines using the NMDA receptor glycine site antagonist [(E)-3-(2-phenyl-2-carboxyethenyl)-4,6-dichloro-1H-indole-2-carbox ylic acid] ([3H]MDL 105,519). [3H]MDL 105,519 binding assays were performed using well washed membranes prepared from frozen rat brains. The polyamines spermine and spermidine increased the fraction of non-specific binding (determined by the addition of 1 mM glycine) in the [3H]MDL 105,519 binding assay from 40-60% when spermine or spermidine concentration was increased from 1 to 100 microM. Polyamine agonists spermine, spermidine and 1,5-(diethylamino)piperidine (30 microM) did not have a significant effect on displacement of [3H]MDL 105,519 binding by glycine or glycine site antagonists. Similarly, the polyamine antagonist arcaine did not have a significant effect on displacement of [3H]MDL 105,519 binding by glycine or glycine site antagonists. However, spermidine significantly depressed the potency of MDL 105,519 in displacing [3H]dizocilpine binding. These data suggest that [3H]MDL 105,519 may preferentially bind to a polyamine insensitive form of the NMDA receptor.

Topics: Aminoquinolines; Animals; Binding Sites; Binding, Competitive; Brain; Cell Membrane; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Glycine; Indoles; Kynurenic Acid; Polyamines; Rats; Receptors, N-Methyl-D-Aspartate; Spermidine; Spermine; Tritium

Transfection of mouse L(tk-) cells with human N-methyl-D-aspartate (NMDA) receptor subunit cDNAs under the control of a dexamethasone-inducible promoter has been used to generate two stable cell lines expressing NR1a/NR2A receptors and a stable cell line expressing NR1a/NR2B receptors. The cell lines have been characterised by northern and western blot analyses, and the pharmacology of the recombinant receptors determined by radioligand binding techniques. Pharmacological differences were identified between the two NMDA receptor subtypes. The glutamate site antagonist D, L-(epsilon)-2-[3H]amino-4-propyl-5-phosphono-3-pentanoic acid ([3H]CGP 39653) had high affinity for NR1a/NR2A receptors (KD = 3.93 nM) but did not bind to NR1a/NR2B receptors. Glycine site agonists showed a 2.6-5.4-fold higher affinity for NR1a/NR2B receptors. Data from radioligand binding studies indicated that one of the cell lines, NR1a/NR2A-I, expressed a stoichiometric excess of the NR1a subunit, which may exist as homomeric assemblies. This observation has implications when interpreting data from pharmacological analysis of recombinant receptors, as well as understanding the assembly and control of expression of native NMDA receptors.

Topics: 2-Amino-5-phosphonovalerate; Aminoquinolines; Animals; Binding Sites; Blotting, Northern; Cell Line; Dizocilpine Maleate; Excitatory Amino Acid Antagonists; Gene Expression; Glutamine; Glycine; Humans; Mice; Radioligand Assay; Rats; Receptors, N-Methyl-D-Aspartate; Recombinant Proteins; Tritium

KCl (20-100 mM) and N-methyl-D-aspartate (NMDA, 100-1,000 microM) produce concomitant concentration-dependent increases in the release of previously captured [14C]acetylcholine and [3H]spermidine from rat striatal slices in vitro. The effects of NMDA (300 microM) on striatal [14C]acetylcholine and [3H]spermidine release were blocked with equal potencies by the competitive NMDA antagonist CGP 37849, the glycine site antagonist L-689,560, and the NMDA channel blocker dizocilpine. In contrast, although NMDA-evoked [14C]acetylcholine release was antagonized by ifenprodil (IC50 = 5.3 microM) and MgCl2 (IC50 = 200 microM), neither compound antagonized the NMDA-evoked release of [3H]spermidine at concentrations up to 100 microM (ifenprodil) or 1 mM (MgCl2). Distinct NMDA receptor subtypes with different sensitivities to magnesium and ifenprodil therefore exist in the rat striatum.

Topics: 2-Amino-5-phosphonovalerate; Acetylcholine; Aminoquinolines; Animals; Carbon Radioisotopes; Corpus Striatum; Dizocilpine Maleate; Dose-Response Relationship, Drug; In Vitro Techniques; Kinetics; Magnesium; Magnesium Chloride; N-Methylaspartate; Piperidines; Rats; Receptors, N-Methyl-D-Aspartate; Spermidine; Tritium