Sensitization and desensitization of the NMDA receptor complex. Implications for therapy.
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Animals↗
Biomedical subjects
P Skolnick
Publications and source records attributed to P Skolnick.
Synthesis, resolution, and biological evaluation of the four stereoisomers of 4-methylglutamic acid: selective probes of kainate receptors.
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Animals↗
Synthesis of novel imidazobenzodiazepines as probes of the pharmacophore for "diazepam-insensitive" GABAA receptors.
The syntheses of a series of novel imidazobenzodiazepines and their affinities for diazepam sensitive (DS) and diazepam insensitive (DI) GABAA receptors are described. Imidazobenzodiazepines belong to one of the very few chemical families which exhibit high to moderate potency for DI GABAA receptors. Although imidazobenzodiazepines such as Ro 15-4513, 20, are the most potent DI GABAA receptor ligands described to date, their selectivity for DI versus DS GABAA receptors is only marginal. Previous structure-activity relationship (SAR) studies of imidazobenzodiazepines have indicated that the 3- and 8-positions are critical for high-affinity binding to DI GABAA receptors (J. Med. Chem. 1993, 36, 479-490. J. Med. Chem. 1993, 36, 1001-1006. J. Med. Chem. 1993, 36, 1820-1830). In order to determine why the ester function is critical to high affinity at the DI site, we have synthesized several derivatives which have substituents other than an ester at the C(3) position including 3-alkyl-, 3-alkylketo-, 3-alkyl ether, and 3-dialkylamino-substituted imidazobenzodiazepines. The SAR analysis of these compounds when combined with that of several pyrazoloquinolinones indicates that interactions at H1 and L1 as well as interactions at H2 anti to the imidazole N(2) and at a lipophilic pocket (labeled LDi) about the 3-position are required in order for imidazobenzodiazepines to exhibit selectivity and high affinity for DI GABAA receptors. Furthermore, the imidazobenzodiazepines substituted with an electron-donating group (alkoxy function) at position 8 revealed that the change of the substituent at C(8) from an electron-withdrawing to a donating function did not substantially alter either ligand affinity or selectivity for DI GABAA receptors. Thus, a pharmacophore is proposed for DI GABAA receptor ligands, which is characterized by the requirement of a lipophilic pocket LDi about the C(3) position of imidazobenzodiazepines. Using this model, two pyrazoloquinolinone derivatives were designed and synthesized. Their affinities and selectivities for DI GABAA receptors are consistent with those predicted by the DI GABAA receptor pharmacophore. In addition, examination of the in vitro binding data of 3-alkyl ether analogs confirms that the anti conformation of the ester group at the C(3) position of imidazobenzodiazepines (Ro15-4513, 20 series) is preferred at both DI and DS GABAA receptors. This constitutes the first evidence (other than molecular modeling) to support the auxillary involvement of H2 at the DI site and is important with regard to the synthesis of other DI GABAA receptor selective ligands in the future.(ABSTRACT TRUNCATED AT 400 WORDS)
Affinity Labels↗
Computer-aided molecular modeling, synthesis, and biological evaluation of 8-(benzyloxy)-2-phenylpyrazolo[4,3-c]quinoline as a novel benzodiazepine receptor agonist ligand.
Using computer-aided conformational analysis, based on molecular dynamics simulation, cluster analysis, and Monte Carlo techniques, we have designed and synthesized compounds in which a benzyloxy substituent has been incorporated into a series of pyrazoloquinoline benzodiazepine receptor (BZR) ligands. Earlier studies had shown that the benzyloxy group could act as part of the agonist pharmacophoric determinant in the beta-carboline ring system. Furthermore, the agonist beta-carboline had been correlated with a binding site orientation and volume fit for an agonist 6-phenylimidazobenzodiazepine carboxylate. The present study was undertaken to determine whether the benzyloxy substituent could be used as an agonist pharmacophoric descriptor for the phenylpyrazolo[4,3-c]quinolin-3-one BZR ligands. The results of a determination of GABA shift ratios for the synthetic ligands indicate that 8-(benzyloxy)-2-phenylpyrazolo[4,3-c]quinolin-3-one can be predicted to be an agonist at the BZR.
Animals↗
Distinct mechanisms for Ca2+ entry induced by OKT3 and Ca2+ depletion in Jurkat T cells.
Ca2+ influx triggered by antigen binding to T cell receptors (TCR) is an early event in T cell activation. An additional Ca2+ influx induced by depletion of intracellular Ca2+ (CDCI) has been characterized in human Jurkat T cells that is both temporally and mechanistically distinct from TCR-mediated Ca2+ influx (TCRCI). Both TCRCI and CDCI were insensitive to voltage-gated Ca2+ channel antagonists (e.g., nifedipine, verapamil, and omega-conotoxin G) and pertussis toxin, yet were voltage-sensitive and inhibited by SKF 96365 (a receptor-gated Ca2+ channel blocker) and cholera toxin. However, TCRCI but not CDCI was associated with a significant increase in inositol phosphate (IP chi) levels and inhibited by phorbol ester, while CDCI but not TCRCI was inhibited by Sr2+, forskolin (FSK), and 1,9-dideoxy FSK in a cAMP-independent fashion. Moreover, TCR stimulation did not deplete thapsigargin-sensitive Ca2+ stores, suggesting that TCRCI is not merely a consequence of Ca2+ depletion. These results indicate that Ca2+ entry following the depletion of intracellular Ca2+ stores or TCR stimulation occur through distinct cellular mechanisms coexisting in Jurkat T cells.
8-Bromo Cyclic Adenosine Monophosphate↗
Contribution of "diazepam-insensitive" GABAA receptors to the alcohol antagonist properties of Ro 15-4513 and related imidazobenzodiazepines.
Both in vivo and in vitro studies have shown that Ro 15-4513 can antagonize many of the pharmacologic actions of ethanol. In contrast to many benzodiazepine receptor (BzR) ligands, Ro 15-4513 binds with high affinity to a novel GABAA receptor subtype, referred to as "diazepam-insensitive" (DI). This study examined the contribution of DI GABAA receptors to the modulation of ethanol-induced sleep time by Ro 15-4513 and related imidazobenzodiazepines [e.g., Ro 19-4603, Ro 16-6028, and ZG-63 (t-butyl-8-chloro-5,6-dihydro-5-methyl-6-oxo-imidazo[1,5,a] [1,4]benzodiazepine-3-carboxylate)] that possess high affinities for this GABAA receptor subtype. Ro 15-4513 (0.6-5 mg/kg) significantly reduced ethanol (3.5 g/kg, i.p.) sleep time in mice (p < 0.001, analysis of variance). This effect was not blocked by BzR antagonists ZK 93426 (5 mg/kg) and Ro 14-7437 (5 mg/kg), which possess low affinities for DI but bind with high affinities to other "diazepam-sensitive" (DS) GABAA receptor isoforms. Although Ro 19-4603 (2.5 mg/kg) also reduced ethanol sleep time (p < 0.01), this effect was attenuated by coadministration of ZK 93426 (2.5 mg/kg). Ro 16-6028 (2.5 mg/kg) prolonged (p < 0.01) ethanol sleep time. However, in the presence of either Ro 19-7437 (5 mg/kg) or ZK 93426 (2.5 mg/kg) ethanol-induced sleep time was reduced to values approaching those obtained with ethanol in the presence of Ro 15-4513. A low dose (2.5 mg/kg) of ZG-63 did not significantly affect alcohol sleep time. However, in the presence of ZK 93426, ZG-63 increased sleep time (p < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)
Alcohol Deterrents↗
Antidepressant-like actions of the polyamine site NMDA antagonist, eliprodil (SL-82.0715).
Functional N-methyl-D-aspartate (NMDA) antagonists including competitive antagonists, glycine partial agonists, and use-dependent channel blockers exhibit antidepressant-like actions in preclinical models. The present study examined the effects of eliprodil (SL-82.0715), an NMDA antagonist acting at polyamine sites, in behavioral and neurochemical tests predictive of antidepressant activity. In mice, eliprodil produced a dose-dependent reduction in immobility in the forced swim test, but was inactive in the tail suspension test. Chronic treatment with eliprodil produced both a significant downregulation of beta-adrenoceptors and a reduction in the potency of glycine to inhibit [3H]5,7-dichlorokynurenic acid binding to strychnine-insensitive glycine receptors in neocortical membranes. In toto, these data indicate that like other NMDA antagonists, eliprodil possesses antidepressant-like actions in preclinical tests predictive of clinical efficacy.
Adrenergic Uptake Inhibitors↗
Morphine inhibits the development of allogeneic immune responses in mouse lymph node.
Morphine and related opiates are often administered to relieve post-operative and chronic pain following transplantation surgery. Opiates have been shown to suppress a variety of immune parameters in both animal models and man. In the present study, we investigated whether morphine affects allogeneic immune responses by injecting C57BL/6 mice in the footpad with allogeneic spleen cells and examining changes in the draining popliteal lymph node (PLN). Morphine (administered as subcutaneous implants) had profound inhibitory effects on the development of alloreactivity manifested as a suppression of: (1) lymph node hyperplasia, (2) mixed lymphocyte reactivity (MLR) in PLN cells and (3) the number of CD4+ and Thy 1.2 lymphoid subsets. These inhibitory effects of morphine were abolished or dramatically reduced by co-administration of the opiate antagonist, naltrexone, indicating that suppression of allo-sensitization was opiate receptor mediated. In toto, these findings demonstrate that morphine administration interferes with the development of allogeneic immune response in mouse lymph node through an opiate receptor mediated mechanism.
Animals↗
Neuroprotective actions of 1-aminocyclopropanecarboxylic acid (ACPC): a partial agonist at strychnine-insensitive glycine sites.
1-Aminocyclopropanecarboxylic acid is a high affinity ligand with partial agonist properties at strychnine-insensitive glycine sites associated with the N-methyl-D-aspartate subtype of glutamate receptors. Since occupation of these sites appears required for operation of N-methyl-D-aspartate, receptor coupled cation channels, it was hypothesized that a glycine partial agonist could function as an N-methyl-D-aspartate antagonist. This hypothesis was examined by evaluating the in vivo and in vitro neuroprotective actions of 1-aminocyclopropanecarboxylic acid. 1-Aminocyclopropanecarboxlic acid (150-600 mg kg-1) administered to gerbils five minutes following twenty minutes of forebrain ischemia significantly improved seven day survival; the optimal dose (300 mg kg-1) increased 7 days survival > 4-fold, from 20% to 92%. Survival of hippocampal CA1 neurons (quantitated 7 days post-ischemia) was significantly (approximately 3-fold) increased by the 600 mg kg-1 dose. Seven day survival was not significantly increased when the interval between reperfusion and drug administration (300 mg kg-1) was increased from 5 to 30 min. In cerebellar granule cell cultures, NMDA combined with a saturating concentration of glycine (10 microM) resulted in a 500% increase in cGMP levels. cGMP levels were increased by 100% over basal when NMDA was combined with a saturating (10 microM) concentration of ACPC, indicating that in this measure, the efficacy of ACPC relative to glycine was approximately 0.2. Consistent with previous findings, 1-aminocyclopropanecarboxylic acid significantly reduced glutamate-induced neurotoxicity in cerebellar granule cell cultures. ACPC was most effective in blocking neurotoxicity at glutamate concentrations producing low to moderate levels of cell death.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
NMDA antagonist properties of the putative antiaddictive drug, ibogaine.
Both anecdotal reports in humans and preclinical studies indicate that ibogaine interrupts addiction to a variety of abused substances including alcohol, opiates, nicotine and stimulants. Based on the similarity of these therapeutic claims to recent preclinical studies demonstrating that N-methyl-D-aspartate (NMDA) antagonists attenuate addiction-related phenomena, we examined the NMDA antagonist properties of ibogaine. Pharmacologically relevant concentrations of ibogaine produce a voltage-dependent block of NMDA receptors in hippocampal cultures (Ki, 2.3 microM at -60 mV). Consistent with this observation, ibogaine competitively inhibits [3H]1-[1-(2-thienyl)-cyclohexyl]piperidine binding to rat forebrain homogenates (Ki, 1.5 microM) and blocks glutamate-induced cell death in neuronal cultures (IC50, 4.5 microM). Moreover, at doses previously reported to interfere with drug-seeking behaviors, ibogaine substitutes as a discriminative stimulus (ED50, 64.9 mg/kg) in mice trained to discriminate the prototypic voltage-dependent NMDA antagonist, dizocilpine (0.17 mg/kg), from saline. Consistent with previous reports, ibogaine reduced naloxone-precipitated jumping in morphine-dependent mice (ED50, 72 mg/kg). Although pretreatment with glycine did not affect naloxone-precipitated jumping in morphine-dependent mice, it abolished the ability of ibogaine to block naloxone-precipitated jumping. Taken together, these findings link the NMDA antagonist actions of ibogaine to a putative "antiaddictive" property of this alkaloid, its ability to reduce the expression of morphine dependence.
Animals↗
100 years of ibogaine: neurochemical and pharmacological actions of a putative anti-addictive drug.
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Animals↗
Different subunit requirements for volatile and nonvolatile anesthetics at gamma-aminobutyric acid type A receptors.
The ability of volatile (halothane and isoflurane) and nonvolatile (alphaxalone and pentobarbital) general anesthetics to modulate radioligand binding to gamma-aminobutyric acid (GABA)A receptors was examined in an immortalized cell line (WSS-1) expressing rat alpha 1 and gamma 2 subunits. Volatile anesthetics enhance [3H]flunitrazepam binding to WSS-1 cells in a concentration-dependent manner, with potencies and efficacies comparable to those found with native GABAA receptors. Transfection of these cells with cDNAs encoding rat beta 2 or beta 3 subunits had a significant influence on anesthetic efficacy but not potency in this assay. Thus, transfection with the beta 2 subunit reduced the efficacy of both isoflurane and halothane, whereas transfection with the beta 3 subunit increased the efficacy of isoflurane but not halothane, compared with values obtained in WSS-1 cells. In contrast, alpha-xalone (an anesthetic steroid) had no effect, whereas at high concentrations pentobarbital (an anesthetic barbiturate) produced a modest inhibition of [3H]flunitrazepam binding to GABAA receptors in WSS-1 cells. Transfection of WSS-1 cells with cDNAs encoding either beta 2 or beta 3 subunits resulted in a concentration-dependent enhancement of [3H]flunitrazepam binding by these nonvolatile anesthetics. Moreover, pentobarbital was significantly more potent in enhancing [3H]flunitrazepam binding to WSS-1 cells transfected with the beta 2 subunit, compared with the beta 3 subunit. The difference in subunit requirements between volatile and nonvolatile anesthetics for enhancement of [3H]flunitrazepam binding indicates that these classes of agents affect GABAA receptor function at distinct loci. These studies also provide evidence that the beta subunit is required for these nonvolatile anesthetics to positively modulate GABAA receptors.
Anesthetics↗
Sustained exposure to 1-aminocyclopropanecarboxylic acid, a glycine partial agonist, alters N-methyl-D-aspartate receptor function and subunit composition.
Partial agonists at the strychnine-insensitive glycine sites coupled to N-methyl-D-aspartate (NMDA) receptors reduce both glutamate-induced neurotoxicity in vitro and ischemia-induced neurodegeneration in vivo. Paradoxically, sustained exposure of cultured cerebellar granule cell neurons to glycinergic ligands, including glycine and the glycine partial agonists (+/-)-3-amino-1-hydroxy-2-pyrrolidone, 1-aminocyclopropanecarboxylic acid (ACPC), and D-cycloserine, attenuates the neuroprotective effects of (+/-)-3-amino-1-hydroxy-2-pyrrolidone and ACPC. In the present study, we investigated the mechanisms responsible for this attenuated neuroprotection. Three NMDA receptor-mediated responses were examined after sustained exposure to ACPC: glutamate-induced neurotoxicity, NMDA-stimulated increases in cGMP levels, and NMDA-stimulated increases in [Ca+2]i. Consistent with previous findings, coincubation with ACPC blocked glutamate-induced neurotoxicity, whereas sustained (24 hr) exposure to ACPC attenuated its protective effects. Moreover, sustained exposure to ACPC caused an apparent approximately 2-fold increase in the potency of both glutamate to act as neurotoxin and NMDA to stimulate cGMP formation. Sustained exposure to ACPC also increased NMDA-stimulated [Ca+2]i approximately 3-fold compared with control granule cell cultures but did not affect basal [Ca+2]i. This apparent increase in glutamate sensitivity may be attributable to a change in NMDA receptor subunit composition as sustained ACPC exposure resulted in a approximately 2.5-fold increase in NMDA receptor 2C RNA levels, without concomitant changes in the amounts of RNA encoding the NMDA receptor 2A, 2B, or 1 subunit. This is the first demonstration that sustained exposure to a glycinergic ligand can alter the expression of RNAs encoding NMDA receptor subunits. Because glycinergic ligands are potential clinical candidates, these results may have important implications for the treatment of neurodegenerative disorders.
Amino Acids↗
1-Aminocyclopropanecarboxylic acid protects against dynorphin A-induced spinal injury.
Lumbar subarachnoid injection of dynorphin A causes an ischemia-induced neuronal degeneration and persistent hindlimb paralysis. The protective effects of a variety of competitive and non-competitive N-methyl-D-aspartate (NMDA) receptor antagonists indicate that activation of the NMDA receptor complex is essential for dynorphin A-induced spinal cord injury. 1-Aminocyclopropanecarboxylic acid (ACPC) is a high affinity, partial agonist at strychnine-insensitive glycine receptors associated with the NMDA receptor complex. Pretreatment of rats with ACPC (100 and 200 mg/kg, i.p., 30 min prior to dynorphin A) significantly eliminated the persistent hindlimb motor deficits and neuropathological changes produced by 20 nmol of this peptide. The neuroprotective effects of ACPC (100 mg/kg, i.p.) were abolished by parenteral administration of glycine (800 mg/kg, 30 min prior to ACPC), consistent with other in vivo and in vitro studies indicating that the pharmacological actions of ACPC are effected through strychnine-insensitive glycine receptors. When given instead as six daily injections (200 mg/kg, i.p.) followed by an injection-free day, ACPC also significantly improved neurological recovery following dynorphin-A injection. These results support earlier indications that: (1) activation of the NMDA receptor complex plays a critical role in mediating dynorphin A-induced rat spinal cord injury; (2) ACPC provides an effective means of antagonizing excitotoxic phenomena; and (3) chronic administration of ACPC can elicit a persistent change in the NMDA receptor complex.
Amino Acids↗
Volatile anesthetics bidirectionally and stereospecifically modulate ligand binding to GABA receptors.
Pharmacologically relevant concentrations of volatile anesthetics can bidirectionally modulate radioligand binding to GABAA receptors. In mouse cerebral cortex, halothane (a prototypic volatile anesthetic) increased [3H]muscimol (a GABA receptor agonist) binding while inhibiting the binding of a GABA receptor antagonist ([3H]SR 95531). These bidirectional effects of inhalational anesthetics on ligand binding to GABA receptors are effected through changes in the Bmax with no significant alterations in the KD of these radioligands. Moreover, the concentration dependent, bidirectional modulation of radioligand binding to GABA receptors by volatile anesthetics exhibited stereoselectivity. Thus, (+)-isoflurane was about twice as potent as the (-)-enantiomer in enhancing [3H]muscimol binding and approximately 50% more potent as an inhibitor of [3H]SR 95531 binding, respectively. The demonstration of a bidirectional, stereospecific modulation of radioligand binding to GABA receptors by inhalational agents is consistent with the presence of specific recognition sites for inhalational anesthetics on the GABAA receptor complex.
Animals↗
Internucleosomal DNA fragmentation in gerbil hippocampus following forebrain ischemia.
Internucleosomal DNA fragmentation, the characteristics feature of programmed cell death, was demonstrated in gerbil hippocampus following 10 min of forebrain ischemia. Quantitative analysis revealed the presence of DNA fragments as early as 12 h after ischemia, reaching a maximum at 48 h. Measurable DNA fragmentation was still present in 3/3 subjects 96 h after the ischemic insult. In situ staining of hippocampus demonstrated pronounced DNA fragmentation that was localized in the CA1 region. The localization of fragmented DNA to the CA1 is consistent with the vulnerability of this layer to ischemic insult, and indicates that DNA fragmentation may be associated with the delayed loss of CA1 neurons in this model of forebrain ischemia.
Animals↗
Synthesis of benzo-fused benzodiazepines employed as probes of the agonist pharmacophore of benzodiazepine receptors.
The synthesis and in vitro evaluation of benzo-fused benzodiazepines 1-6 are described. These "molecular yardsticks" were employed to probe the spatial dimensions of the lipophilic pocket L2 in the benzodiazepine receptor (BzR) cleft and to determine the effect of occupation of L2 with respect to agonist activity. Of the new analogs synthesized, the 7,8-benzo-fused benzodiazepine 6 displayed moderately high affinity for the BzR (IC50 = 55 nM) and exhibited both anticonvulsant (ED50 approximately 15 mg/kg) and muscle relaxant (ED50 approximately 15 mg/kg) activity. As expected, 2 and 4 interacted with the repulsive regions of interaction, S1 and S2, and exhibited low affinities for BzR. The rigid nature of these molecular yardsticks (especially 6, Figure 7) has been employed to probe the depth of L2. Moreover, in the case of 6 full occupation of L2 has resulted in an increase in the muscle relaxant effect at the expense of the anticonvulsant/anxiolytic effect.
Animals↗
Drugs acting at the strychnine-insensitive glycine receptor do not induce HSP-70 protein in the cingulate cortex.
The potential for compounds acting at the strychnine-insensitive glycine receptor to injure neurons was examined using induction of a 70 kDa heat shock protein (HSP-70) as a marker. HSP-70 was consistently detected in retrosplenial and cingulate cortices after MK-801 but not glycine drug treatment. Elsewhere in the cortex, mild diffuse HSP-70 immunoreactivity was detected following 7-chlorokynurenic acid. Following HA-966, intense hippocampal HSP-70 immunoreactivity was observed. These findings indicate that even after very high doses, drugs acting at the strychnine-insensitive glycine receptor are less likely to injure cingulate cortical neurons than other classes of NMDA antagonists.
Amino Acids↗