Diffuse mesenteric amyloidosis.
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Abdominal Pain↗
Biomedical subjects
J A Kemp
Publications and source records attributed to J A Kemp.
Ro 25-6981, a highly potent and selective blocker of N-methyl-D-aspartate receptors containing the NR2B subunit. Characterization in vitro.
The interaction of Ro 25-6981 with N-methyl-D-aspartate (NMDA) receptors was characterized by a variety of different tests in vitro. Ro 25-6981 inhibited 3H-MK-801 binding to rat forebrain membranes in a biphasic manner with IC50 values of 0.003 microM and 149 microM for high- (about 60%) and low-affinity sites, respectively. NMDA receptor subtypes expressed in Xenopus oocytes were blocked with IC50 values of 0.009 microM and 52 microM for the subunit combinations NR1C & NR2B and NR1C & NR2A, respectively, which indicated a >5000-fold selectivity. Like ifenprodil, Ro 25-6981 blocked NMDA receptor subtypes in an activity-dependent manner. Ro 25-6981 protected cultured cortical neurons against glutamate toxicity (16 h exposure to 300 microM glutamate) and combined oxygen and glucose deprivation (60 min followed by 20 h recovery) with IC50 values of 0.4 microM and 0.04 microM, respectively. Ro 25-6981 was more potent than ifenprodil in all of these tests. It showed no protection against kainate toxicity (exposure to 500 microM for 20 h) and only weak activity in blocking Na+ and Ca++ channels, activated by exposure of cortical neurons to veratridine (10 microM) and potassium (50 mM), respectively. These findings demonstrate that Ro 25-6981 is a highly selective, activity-dependent blocker of NMDA receptors that contain the NR2B subunit.
Animals↗
A novel mechanism of activity-dependent NMDA receptor antagonism describes the effect of ifenprodil in rat cultured cortical neurones.
1. Ifenprodil is a selective, atypical non-competitive antagonist of NMDA receptors that contain the NR2B subunit with an undefined mechanism of action. Ifenprodil is neuroprotective in in vivo models of cerebral ischaemia but lacks many of the undesirable side-effects associated with NMDA antagonist. 2. Using whole-cell voltage-clamp recordings, we have studied the mechanism of inhibition of NMDA-evoked currents by ifenprodil in rat cultured cortical neurones in the presence of saturating concentrations of glycine. 3. Ifenprodil antagonized NMDA receptors in an activity-dependent manner, whilst also increasing the receptor affinity for glutamate recognition-site agonists. Ifenprodil inhibition curves against 10 and 100 microM NMDA-evoked currents yielded IC50 values of 0.88 and 0.17 microM, respectively. Thus, the apparent affinity of ifenprodil for the NMDA receptor is increased in an NMDA concentration-dependent manner. 4. Currents evoked by 0.3 and 1 microM NMDA were potentiated to approximately 200% of control levels in the presence of 3 microM ifenprodil. Thus, with increasing concentration of NMDA the effect of ifenprodil on NMDA-evoked currents changed from one of potentiation to one of increasing inhibition. 5. These results are predicted by a reaction scheme in which ifenprodil exhibits a 39- and 50-fold higher affinity for the agonist-bound activated and desensitized states of the NMDA receptor, respectively, relative to the resting, agonist-unbound state. Furthermore, ifenprodil binding to the NMDA receptor results in a 6-fold higher affinity for glutamate site agonists. 6. This represents a novel mechanism of NMDA receptor antagonism that, together with the subunit selectivity, probably contributes to the attractive neuropharmacological profile of this and related compounds.
Animals↗
Modulation of voltage-gated calcium channels by orphanin FQ in freshly dissociated hippocampal neurons.
Orphanin FQ (OFQ) has recently been reported to be an endogenous ligand for the opioid-like LC132 receptor. The effect of OFQ on high voltage-gated calcium channels (VGCCs) was examined in freshly dissociated rat pyramidal neurons using the whole-cell configuration of the patch-clamp technique. High-threshold Ba2+ currents were reversibly inhibited by OFQ. The depression of the currents was associated with a slowed rate of activation and a change in the activation I-V relationship at step potentials higher than +30 mV. In concentration-response experiments, a mean (+/-SEM) pEC50 value of 7.0 +/- 0.07 and a Hill coefficient of 1.5 +/- 0.08 (n = 5) were obtained. The near-maximum inhibition of the Ba2+ currents by OFQ (1 microM) amounted to 31 +/- 2.2% of control (n = 15). Opioid receptors could not account for the effects of OFQ on VGCCs, because naloxone, a broad spectrum mu-, delta-, and kappa-receptor antagonist, did not reduce the effectiveness of OFQ. When GTP-gamma-S was included in the pipette, the depression of the currents by OFQ was irreversible, whereas currents from neurons preincubated with pertussis toxin were not inhibited by OFQ, consistent with the involvement of a PTX-sensitive G-protein. When selective blockers of VGCCs were used, it was demonstrated that all subtypes of VGCCs were affected by OFQ. In conclusion, the effect of OFQ on VGCCs expressed in hippocampal CA3 and CA1 neurons may play an important role in the regulation of hippocampal cell excitability and neurotransmitter release.
Animals↗
Electrophysiological characterisation of the antagonist properties of two novel NMDA receptor glycine site antagonists, L-695,902 and L-701,324.
The pharmacological effects of two novel N-methyl-D-aspartate (NMDA) receptor glycine site antagonists, L-701,324 and L-695,902 were examined on whole-cell voltage-clamped cells and compared to a prototypic antagonist, 7-chlorokynurenic acid. Both L-701,324 and L-695,902 non-competitively antagonised NMDA responses elicited in rat cultured cortical neurones, this was shown to be due to a competitive interaction at the glycine co-agonist site on the receptor complex (Kb values: 19 nM and 2.6 microM, respectively). Inhibition curves for the antagonism of responses to combined applications of NMDA and glycine showed that both antagonists were devoid of any intrinsic activity i.e. "full" antagonists and were, therefore, capable of completely abolishing inward currents. Despite this fact, both of these novel antagonists apparently modulated glutamate affinity for its recognition site-a property hitherto associated only with glycine site partial agonists. Human recombinant NMDA receptors comprising NR1a/NR2A and NR1a/NR2B subunits expressed in mouse fibroblast cells were also used to determine whether L-701,324 and L-695,902 were capable of discriminating between subtypes of NMDA receptor. Inhibition curves to each antagonist showed no difference in affinity for either of these subunit assemblies (mK1 values L-701,324 = 0.005 microM on both assemblies; L-695,902 = 4.37 and 3.7 microM on NR1a/NR2A and NR1a/NR2B, respectively). Kinetic analysis of the off-rates of antagonism with L-701,324 revealed that the high affinity of this compound compared to 7-chlorokynurenic acid were attributable to an exceptionally slow dissociation of the antagonist from the receptor.
Animals↗
Beta-amyloid-induced cell toxicity: enhancement of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide-dependent cell death.
In an attempt to understand the cause of neurodegeneration in Alzheimer's disease, the toxic effects of beta-amyloid (Abeta) peptides have been widely studied. At high micromolar concentrations Abeta peptides have been demonstrated to be acutely toxic to various cell types. At submicromolar concentrations, Abeta peptides have been suggested to inhibit cellular metabolic activity, due to their inhibition of the ability of cells to metabolize the oxidoreductase substrate 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT). Here we show, first, that MTT reduction surprisingly leads to a breakdown in PC12 cell membrane integrity and cell death, presumably through the formation of a crystalline formazan product, and, second, that pretreatment of PC12 cells with nanomolar concentrations of Abeta peptide, rather than inhibiting their metabolic activity, increases the susceptibility of these cells to the secondary toxic effect of formazan crystal formation. These results suggest that low nanomolar concentrations of Abeta render membranes more susceptible to damage by a secondary insult, in this case, MTT reduction. It is plausible that such an effect, when combined with additional risk factors, could contribute to the neurodegeneration that occurs in Alzheimer's disease.
Amyloid beta-Peptides↗
The neuroprotective effect of the glycine site antagonist 3R-(+)-cis-4-methyl-HA966 (L-687,414) in a rat model of focal ischaemia.
3R-(+)-cis-4-Methyl-HA966 (L-687,414) is a novel and selective, low intrinsic activity, partial agonist at the glycine site of the N-methyl-D-aspartate (NMDA) receptor. Thus, while it acts primarily to block NMDA receptor function in the presence of glycine, it fails to produce a complete block of NMDA receptor activation. In this study, we have investigated its neuroprotective effects in a rat model of focal ischaemia, involving permanent occlusion of the left middle cerebral artery. L-687,414 was administered as a bolus dose of 17.6 mg/kg i.v. straight after the occlusion or as a bolus dose + infusion for 4 h. The doses of L-687,414 used for the infusion studies were 7 mg/kg i.v. + 7 mg/kg/h, 14 mg/kg + 14 mg/kg/h, or 30 mg/kg + 30 mg/kg/h. The 17.6-mg/kg dose gave an estimated peak plasma level of 24 micrograms/ml, which decayed with a t1/2 of 56 min. The three infusion dosing regimens gave mean plasma levels over the 4 h of 11, 25 and 61 micrograms/ml plasma, respectively. The 17.6-mg/kg dose of L-687,414 gave no significant protection against the volume of hemispheric, cortical, or caudate damage when compared with the control group of animals. The lowest infusion dosing regimen of L-687,414 which gave a plasma level of 11 micrograms/ml over the 4 h was also ineffective against the volume of infarction measured in the different brain regions.(ABSTRACT TRUNCATED AT 250 WORDS)
Animals↗
Identification of amino acids in the N-methyl-D-aspartate receptor NR1 subunit that contribute to the glycine binding site.
The mammalian N-methyl-D-aspartate (NMDA) receptor complex is though to consist of an NR1 subunit in combination with one or more of the four NR2 subunits (A, B, C, and D). When corresponding cDNAs are expressed in Xenopus oocytes, ion channels with the characteristic profile of NMDA receptors are formed. The receptor is unique in requiring two coagonists, glutamate and glycine, for activation of the channel. We have used site-directed mutagenesis to study amino acids in the human NR1 subunit that contribute to the glycine binding site of the NMDA receptor without affecting the agonist site for glutamate. Mutations to D481 and K483 produced receptors with up to 160-fold lower affinities for glycine, as well as other agonists and partial agonists, without affecting maximum current size or the degree of agonist efficacy. The D481A mutation also led to 40-50-fold lower affinities for two structurally diverse glycine site antagonists. From these data we propose that the carboxyl group of this aspartate interacts with the amino moiety of glycine and the equivalent group contained in other agonists and antagonists.
Amino Acid Sequence↗
Subtypes of NMDA receptor in neurones cultured from rat brain.
The non-competitive N-methyl-D-aspartate (NMDA) antagonist, ifenprodil, discriminates two receptor populations, each of which shows a reciprocal abundance in cultured cortical and cerebellar granule cells. Thus approximately 70% of NMDA-gated membrane current was antagonized with high affinity (IC50 = 1.4 +/- 0.9 microM) in cortical neurones whereas only approximately 20% was antagonized with high affinity (IC50 = 1.3 +/- 0.3 microM) in granule cells. Inhibition curves for CGS 19755 appeared relatively monophasic: this competitive NMDA antagonist had a significantly higher affinity for the granule cell receptor (Ki = 0.8 +/- 0.2 microM) compared with that on cortical neurones (Ki = 2 +/- 0.6 microM). The data suggest that these two antagonists may be of value in identifying the expression of subpopulations of native NMDA receptors in other brain regions.
Animals↗
A second generation of non-peptide cholecystokinin receptor antagonists and their possible therapeutic potential.
The profile of an acidic series of benzodiazepine CCK-B receptor antagonists is described. The tetrazolyl urea derivative L-368,935 had high affinity (CCK-B IC50 0.1 nM) and was one of the most selective (CCK-B/CCK-A 10,000) CCK-B antagonists known. L-368,935 was a CCK-B antagonist with high affinity on the rat ventromedial hypothalamic slice preparation (Kb 0.6 nM) and also blocked pentagastrin-induced calcium mobilization in GH3 cells. L-368,935 had potent in vivo activity and antagonized pentagastrin-induced gastric acid secretion in the anesthetized rat and CCK-8S-induced aspartate release using microdialysis in the striatum of conscious rats. Activity within the central nervous system was confirmed by a mouse ex vivo binding assay and by direct measurement of the compound within the central nervous system using an HPLC assay. A second generation of CCK-B receptor antagonists such as L-368,935 will be important in determining the therapeutic potential of this class of compound in man.
Animals↗
High-affinity and potent, water-soluble 5-amino-1,4-benzodiazepine CCKB/gastrin receptor antagonists containing a cationic solubilizing group.
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Animals↗
Second-generation benzodiazepine CCK-B antagonists. Development of subnanomolar analogs with selectivity and water solubility.
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Animals↗
A novel allosteric modulatory site on the GABAA receptor beta subunit.
Cloning of cDNAs that code for GABAA receptor subunits has revealed multiple receptor populations constructed from different subunit combinations. On native rat and cloned human GABAA receptors, the anticonvulsant compound loreclezole strongly potentiated GABA-mediated chloride currents. Using different combinations of human GABAA receptor subunits expressed in Xenopus oocytes and transfected 293 cells, loreclezole was highly selective for receptors containing the beta 2 or beta 3 subunit over those containing the beta 1 subunit. Loreclezole was demonstrated to act at a site distinct from the benzodiazepine, barbiturate, and steroid sites with a unique subunit dependence. These results describe a previously unidentified modulatory site on the GABAA receptor beta subunit that allows pharmacological discrimination of different GABAA receptor subpopulations in the brain and provides a new target for putative anticonvulsant/anxiolytic drugs.
Allosteric Site↗
Selective non-peptide ligands for an accommodating peptide receptor. Imidazobenzodiazepines as potent cholecystokinin type B receptor antagonists.
A series of imidazobenzodiazepines, non-peptide antagonists of the peptide hormone cholecystokinin (CCK), are described. Derived by chemical modification of the benzodiazepine ring system embedded within the CCK-B antagonist L-365,260, these compounds display CCK-B/CCK-A selectivity and some analogs have receptor binding affinities in the subnanomolar range. This group of novel imidazobenzodiazepines, among which N-[(2S,4R)-methyl-6-phenyl-2,4-dihydro-1H-imidazo[1,2- alpha][1,4]benzodiazepin-4-yl]-N'-[3-methylphenyl]-urea (12) is the principal compound, expands the structural diversity of the collection of non-peptide CCK-B antagonists and will be useful in further delineating the function of CCK in the central nervous system.
Animals↗
Cloning, functional coexpression, and pharmacological characterisation of human cDNAs encoding NMDA receptor NR1 and NR2A subunits.
Using expression cloning, and more recently using polymerase chain reaction cloning approaches, a family of rat N-methyl-D-aspartate (NMDA) receptor subunit cDNAs has been described (NR1, NR2A, NR2B, NR2C, and NR2D). Here we report cloning and sequencing of cDNAs encoding isoforms of the human NR1 subunit (NR1a, NR1d, and NR1e) that differ at their C-terminal end as a result of alternative splicing and also of a cDNA encoding the human NR2A subunit. The deduced amino acid sequences of the human NR1 subunit isoforms differed from the published rat NR1 subunit sequences at only eight positions, all of which were N-terminal to the alternatively spliced domains. The human NR2A subunit deduced amino acid sequence differed from the published rat NR2A subunit sequence at 81 of the 1,464 amino acids, with most of the substitutions being located in the C-terminal half of the subunit. The gene for NR2A has been localised to human chromosome 16. We also report the expression and pharmacological characterisation of recombinant human NR1a/NR2A heteromeric receptors in Xenopus oocytes. These receptors had EC50 values of 2.14 and 2.05 microM for glutamate and glycine, respectively, and an IC50 of 46.8 microM for Mg2+. Responses were antagonised by D-2-amino-5-phosphonovalerate, L-689,560, pH 6.3, zinc, and MK-801. No modulatory effect was observed on application of ifenprodil, confirming previous observations with rat NR1 + NR2A recombinant receptors.
Amino Acid Sequence↗
Effect of hypochlorhydria due to omeprazole treatment or atrophic gastritis on protein-bound vitamin B12 absorption.
OBJECTIVE: To investigate the effects of hypochlorhydria and acidic drink ingestion on protein-bound vitamin B12 absorption in elderly subjects. METHODS: Absorption of protein-bound vitamin B12 was examined in elderly normal subjects (n = 8), and in hypochlorhydric subjects due to omeprazole treatment (n = 8) or with atrophic gastritis (n = 3). Subjects underwent absorption tests of protein-bound vitamin B12 ingested with water, cranberry juice and 0.1 N hydrochloric acid. RESULTS: Protein-bound vitamin B12 absorption was lower in the omeprazole-treated group (0.50%) compared to the normal group (1.21%; p < 0.001). With cranberry juice ingestion, the omeprazole-treated group showed an increase in absorbed protein-bound vitamin B12 (p = 0.025). With dilute hydrochloric acid ingestion, there was a further increase in vitamin B12 absorption (p < 0.001). CONCLUSION: Omeprazole causes protein-bound vitamin B12 malabsorption, and ingestion of an acidic drink improves protein-bound vitamin B12 absorption.
Absorption↗
Modulation of cyclic AMP formation by putative metabotropic receptor agonists.
1. The effects of various metabotropic glutamate receptor agonists on [3H]-cyclic AMP accumulation and phosphoinositide hydrolysis were investigated in guinea-pig cerebral cortical slices prelabelled with [3H]-adenine or [3H]-inositol. 2. 1-Aminocyclopentane-1S,3R-dicarboxylate (1S,3R-ACPD), L-2-amino-4-phosphonobutanoate (L-AP4) and (2S,3S,4S)-alpha-(carboxycyclopropyl)glycine (L-CCG-I), elicited concentration-dependent inhibitions of forskolin-stimulated [3H]-cyclic AMP accumulation, with IC50 values of 2.1 +/- 0.3, 71 +/- 17 and 0.2 +/- 0.1 microM respectively. 3. 1S,3R-ACPD and L-CCG-I increased the cyclic AMP responses to histamine H2 receptor stimulation with EC50 values of 7 +/- 2 microM and 19 +/- 2 microM respectively. 1S,3R-ACPD (EC50 values 17 +/- 2 microM) and L-CCG-I (EC50 value 15 +/- 3 microM) potentiated the cyclic AMP responses to the adenosine receptor agonist 5'-N-ethylcarboxamidoadenosine (NECA, 10 microM). This potentiating effect of L-CCG-I was reduced in the presence of a protein kinase C inhibitor, and also in the absence of extracellular calcium. In contrast, L-AP4 inhibited the NECA response in a concentration-dependent manner, with an IC50 value of 120 +/- 20 microM. 4. L-AP4 (at concentrations up to 1 mM) failed to stimulate phosphoinositide hydrolysis in guinea-pig cerebral cortical slices, but both 1S,3R-ACPD (EC50 value 35 +/- 6 microM) and L-CCG-I (approximately 160 microM) elicited concentration-dependent stimulations of phosphoinositide turnover. 5. These results confirm the existence of at least two distinct subtypes of metabotropic receptor in guinea-pig cortex. The data also substantiate previous studies indicating the importance of the agonist L-CCG-I as a pharmacological tool for distinguishing metabotropic receptor subtypes.
Adenosine↗
Functional expression of human D3 dopamine receptors in differentiated neuroblastoma x glioma NG108-15 cells.
This study describes the depression of calcium currents caused by activation of human D3 dopamine receptors which have been stably expressed in the neuroblastoma x glioma NG108-15 cell line. Transfected cells, which had been differentiated with prostaglandin E1 and isobutylmethylxanthine, exclusively expressed D3 receptor mRNA, which was demonstrated by reverse transcription polymerase chain reaction techniques. Transfected cells had high affinity binding sites for iodosulpiride, with a Kd of 0.8 nM and receptor density of 240 fmol mg-1 protein. Calcium currents were recorded using nystatin-perforated patch clamp techniques. In contrast to untransfected cells that had been differentiated, high-threshold calcium currents in differentiated hD3-NG108-15 cells were depressed by application of dopamine and quinpirole. These responses were abolished by the dopamine receptor antagonist S-(-)-sulpiride (1 microM), demonstrating that they were caused by the activation of the transfected dopamine receptors. Coupling of human D3 receptors to calcium currents was sensitive to the action of pertussis toxin, suggesting the involvement of G-proteins of the Gi and/or G(o) subtype. These results demonstrate that human D3 receptors represent a functional class of dopamine receptor.
Brain Neoplasms↗