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Biomedical subjects

J A Kemp

Publications and source records attributed to J A Kemp.

At least 19 recordsLinked to original sources
PubMed1995

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↗
PubMed1995

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↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1994

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↗
PubMed1994

Block of human voltage-sensitive Na+ currents in differentiated SH-SY5Y cells by lifarizine.

1. The ability of lifarizine (RS-87476) to block human voltage-sensitive Na+ channel currents was studied by use of whole cell patch clamp recording from differentiated neuroblastoma cells (SH-SY5Y). 2. The Na+ conductance in differentiated SH-SY5Y cells (24.0 +/- 2.4 nS, n = 11) was half-maximally activated by 10 ms depolarizations to -37 +/- 2 mV and was half-maximally inactivated by predepolarizing pulses of 200 ms duration to -86 +/- 3 mV (n = 11). 3. At low stimulus frequencies (0.1 to 0.33 Hz) voltage-dependent sodium currents were completely blocked, in a concentration-dependent manner, by extracellular application of either tetrodotoxin (EC50 = 4 +/- 1 nM, n = 12) or by lifarizine (EC50 = 783 +/- 67 nM, n = 9). The onset of block by lifarizine (tau = 91 +/- 14 s at 10 microM) was considerably slower than that of tetrodotoxin (tau = 16 +/- 3 s at 100 nM). 4. Lifarizine (1 microM) reduced the peak sodium conductance in each cell (from 26.4 +/- 2.0 nS to 15.1 +/- 2.7 nS, n = 4) without changing the macroscopic kinetics of sodium current activation or inactivation (V1/2 = -35 1 mV and -87 +/- 4 mV respectively, n = 4). Similarly, lifarizine (1 microM) did not affect the reversal potential of the macroscopic sodium current (+14 +/- 5 mV in control and +16 +/- 2 mV in 1 microM lifarizine; n = 4) or reactivation time-constant (tau = 14.0 +/- 4.4 ms). 5. Block of the sodium channel open state by tetrodotoxin (30 nM) did not prevent the inhibition caused by a subsequent application of lifarizine (3 micro M). In contrast the depression caused by lifarizinewas readily reversible after pretreatment of cells with the local anaesthetic, lignocaine (1O mM).6. These data demonstrate that lifarizine is a use- and voltage-dependent antagonist of human voltage sensitive sodium currents. The slow kinetics and pharmacology of the block by lifarizine indicate that access of this drug to the channel is more restricted than that of tetrodotoxin and may involve an allosteric site or state of the channel that is also regulated by local anaesthetics.

Cell Differentiation↗
PubMed1994

Kinetic study of the interactions between the glutamate and glycine recognition sites on the N-methyl-D-aspartic acid receptor complex.

The N-methyl-D-aspartate (NMDA) receptor is unique among the ligand-gated ion channels, in that the gating process requires the binding of two independent coagonists, glutamate and glycine. Receptor binding experiments have suggested that the coagonist recognition sites interact with one another in an allosteric manner, and previous work in this laboratory has provided additional functional support in favor of an allosteric coupling; the affinity of glutamate for its recognition site was reduced when a partial agonist, (+)-HA-966, occupied the glycine site, compared with the affinity when glycine itself was bound to the receptor. The present experiments have taken these observations a step further and compare the effects of several glycine site ligands with different affinities and intrinsic activities (determined from equilibrium concentration-response curves) on glutamate off-rate. Thus, the dissociation rate for the decay of glutamate-activated membrane currents in voltage-clamped rat cortical neurons was fastest (160 +/- 28 msec) in the presence of saturating concentrations of (+)-HA-966 and progressively slower in the presence of D-cycloserine (258 +/- 27 msec), aminocyclopropanecarboxylic acid (330 +/- 21 msec), L-alanine (375 +/- 28 msec), and glycine (502 +/- 42 msec). We have also measured the affinities and intrinsic activities of several NMDA receptor ligands and report that a reciprocal interaction exists, such that the off-rate of glycine is influenced by the properties of the agonist occupying the glutamate coagonist site. Thus, the time constant for current decay after a brief exposure to glycine was fastest in the presence of a saturating concentration of cis-2,3-piperidinedicarboxylic acid (449 +/- 26 msec) and progressively slower in the presence of quinolinate (689 +/- 73 msec), NMDA (721 +/- 36 msec), and L-glutamate (1260 +/- 36 msec). The data suggested that the extent of the modulation of one site by the other is related to the intrinsic activity of the agonist, rather than its affinity. Specifically, we suggest that a partial agonist occupying one of the agonist recognition sites produces a conformational change that results in an accelerated off-rate for coagonist dissociation from the receptor; the lower the intrinsic activity, the greater is the effect on coagonist off-rate.

Animals↗
PubMed1994

Biological properties of the benzodiazepine amidine derivative L-740,093, a cholecystokinin-B/gastrin receptor antagonist with high affinity in vitro and high potency in vivo.

A novel series of 5-amino-1,4-benzodiazepin-2-one derivatives (amidines), which contain a cationic solubilizing group and which are antagonists for the cholecystokinin (CCK)-B receptor, have been identified. Optimization of this series led to the identification of an azabicyclononane amidine, L-740,093 [N-[(3R)-5-(3-azabicyclo[3.2.2]nonan-3-yl)-2,3-dihydro-1-methyl-2- oxo- 1H-1,4-benzodiazepin-3-yl]-N'-(3-methylphenyl)urea], that bound with high affinity of CCK-B receptors from guinea pig cerebral cortex (IC50 of 0.1 nM) and had a CCK-B/CCK-A receptor selectivity of 16,000. In comparison, L-365,260 had 85-fold lower affinity (8.5 nM) and was only 87-fold selective for CCK-B over CCK-A receptors. L-740,093 bound with high affinity to guinea pig gastrin receptors in vitro (IC50 of 0.04 nM). Electrophysiological studies on slices of rat ventromedial hypothalamic nucleus showed that L-740,093 produced rightward shifts of the concentration-response curve for the CCK-B receptor agonist pentagastrin (Kb of 0.06 nM). L-740,093 blocked pentagastrin-induced gastric acid secretion in anesthetized rats with a 50% inhibitory dose of 0.01 mg/kg, intraperitoneally, showing 100-fold greater activity, compared with L-365,260 (50% inhibitory dose of 1 mg/kg, intraperitoneally). An ex vivo binding assay in mice was used to investigate the interaction of L-740,093 with central CCK binding sites. After intravenous administration, L-740,093 inhibited ex vivo binding dose dependently, with a 50% effective dose of 0.2 mg/kg. These studies demonstrate that L-740,093 is the most potent and selective CCK-B antagonist yet described and that it has excellent central nervous system penetration.

Animals↗
PubMed1994

Molecular pharmacology of gamma-aminobutyric acid type A receptor agonists and partial agonists in oocytes injected with different alpha, beta, and gamma receptor subunit combinations.

Using systematic combination of alpha 1, alpha 3, and alpha 5 with beta 1, beta 2, and beta 3, together with gamma 1, gamma 2, and gamma 3, we have investigated the contributions of the various alpha, beta, and gamma subunits to the pharmacology of gamma-aminobutyric acid (GABA)A agonists. We have characterized GABA, (RS)-dihydromuscimol, piperidine-4-sulfonic acid, and 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol with recombinant human GABAA receptors expressed in Xenopus oocytes. Our observations indicate that the alpha subunit is the major determinant of efficacy for partial GABAA agonists. When alpha 1 and alpha 3 or alpha 1 and alpha 5 are coexpressed, the alpha 1 subunit determines the maximum efficacy, whereas the affinity is determined by the entire combination of subunits. Thus, the results of the present study demonstrate that the pharmacology of GABAA agonists is dependent on the subunit composition of the GABAA receptor complex. Functional GABAA receptors containing two different alpha subunits show pharmacological profiles distinctly different from those of receptors containing a single alpha subtype, indicating that two different alpha subunits can be coexpressed in one functional GABAA receptor complex.

Animals↗
PubMed1993

Preferential co-assembly of recombinant NMDA receptors composed of three different subunits.

cDNAs ENCODING NMDA receptor subunits have recently been cloned and been shown to have different distributions in the CNS. However, no studies on the possible in vivo combinations or subunit stoichiometry have yet been carried out. By combining human NR1 with rat NR2A and NR2C we have studied the pharmacological properties of three possible NMDA receptor subtypes; NR1 + NR2A, NR1 + NR2C and NR1 + NR2A + NR2C. By performing glycine concentration-response curves and comparing EC50s, it was possible to show that the NR1 + NR2A + NR2C receptor preferentially co-assembled when all three subunit cDNAs were present. This receptor had an affinity for glycine intermediate between that of NR1 + NR2A and NR1 + NR2C, but a similar Hill coefficient. Thus, two different NR2 subunits can combine in the same receptor, conferring unique pharmacological properties, suggesting that it is likely that two or more NR2 subunits co-assemble together in the same NMDA receptor complex.

Animals↗
PubMed1993

Agonist response kinetics of N-methyl-D-aspartate receptors in neurons cultured from rat cerebral cortex and cerebellum: evidence for receptor heterogeneity.

Accumulating evidence from both radioligand binding and molecular cloning experiments has led to the suggestion that there are heterogeneous populations of N-methyl-D-aspartate (NMDA) receptors. In particular, the NMDA receptor associated with cerebellar neurons has been suggested to be different from that in higher brain regions. With these observations in mind, we considered the possibility that the NMDA receptors on cultured neurons from rat cortex and cerebellum may show differences in their affinity for the coagonists, glutamate and glycine, and the ion channel blocker ketamine. A kinetic approach was used to derive the association and dissociation constants for each of the ligands from their respective macroscopic time constants of binding and unbinding. The constants were assessed electrophysiologically by measuring the onset and decay of whole-cell currents in response to drug applications to voltage-clamped neurons. In addition, differences in coagonist affinity were examined using conventional equilibrium concentration-response curve analysis. These experiments revealed that current relaxations after fast applications of either glutamate or glycine decayed more rapidly in cerebellar neurons, compared with cortical neurons. Thus, assuming two independent binding sites per receptor, the microscopic decay time constants (tau off) for glutamate were 341 +/- 47 (n = 12) and 934 +/- 76 msec (n = 11, p < 0.0001) for granule cells and cortical neurons, respectively. The resulting apparent microscopic dissociation constant (mKd) for glutamate at cerebellar granule cells, calculated from the forward and reverse rate constants, was > 2-fold lower than that for cerebral cortex receptors (496 nM, compared with 251 nM). The difference between the two cell types in the tau off for glycine was more substantial, i.e., 558 +/- 53 (n = 15) and 2214 +/- 125 msec (n = 19, p < 0.0001) for cerebellum and cortex, respectively. Corresponding apparent mKd values for glycine differed by > 4-fold, i.e., 189 nM and 45 nM for cerebellar granule and cortical neurons, respectively. Analysis of data obtained from equilibrium concentration-response curves also revealed differences in coagonist affinity between the two cell populations. The mean mKd values for glutamate at cerebellum and cortical neurons were 1260 nM and 630 nM, respectively, and those for glycine were 316 nM and 63 nM, respectively. No obvious differences were found between the two cell types with respect to the ion channel-blocking kinetics of the dissociative anesthetic ketamine.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1992

Stable expression of mammalian type A gamma-aminobutyric acid receptors in mouse cells: demonstration of functional assembly of benzodiazepine-responsive sites.

The differential sensitivity of type A gamma-aminobutyric acid (GABAA) receptors to benzodiazepine ligands seen in the mammalian nervous system is thought to be generated by the existence of a number of different receptor subtypes, assembled from a range of closely related subunits (alpha 1-6, beta 1-3, gamma 1-3, and delta) encoded by discrete genes. The characteristics of a given subtype can be determined by the coexpression of cloned cDNAs encoding the subunits of interest. Two transient expression systems have so far been employed in the study of the ligand-binding characteristics and chloride channel properties of such GABAA receptors--Xenopus oocytes and transfected mammalian cells. Here we report on the use of a steroid-inducible promoter expression system for the production of a permanently transfected clonal cell line expressing the alpha 1 beta 1 gamma 2L GABAA receptor subtype. Using both immunoprecipitation by subunit-specific antisera and gel-exclusion chromatography, we have shown that the alpha 1, beta 1, and gamma 2L subunits coassemble to form receptor macromolecules that are of the same size as native GABAA receptors. Additionally, the recombinant receptors have the same benzodiazepine pharmacology as native alpha 1-containing GABAA receptors and function as GABA-gated chloride channels. Such cell lines expressing individual GABAA receptor subtypes will prove important tools in the study of the structure, function, and pharmacology of GABAA receptors and in the development of subtype-specific drugs.

Allosteric Regulation↗
PubMed1992

2-Carboxytetrahydroquinolines. Conformational and stereochemical requirements for antagonism of the glycine site on the NMDA receptor.

2-Carboxy-1,2,3,4-tetrahydroquinoline derivatives, derived from kynurenic acid, have been synthesized and evaluated for in vitro antagonist activity at the glycine site on the NMDA receptor. 2,3-Dihydrokynurenic acids show reduced potency relative to the parent lead compounds (Table I) possibly as a result of conformational effects. Removal of the 4-oxo group results in further reduced potency, but introduction of a cis-carboxymethyl group to the 4-position restores antagonist activity (Tables III and IV). Replacement of the keto group of 5,7-dichloro-2,3-dihydrokynurenic acid with other alternative H-bonding groups, for example cis- and trans-benzyloxycarbonyl and cis- and trans-carboxamido (Table V), gives comparable activity, but there is negligible stereoselectivity. A significant increase in potency and stereoselectivity is seen within the 4-acetate series (Table VI). The trans-4-acetic acid is significantly more potent than the corresponding lead kynurenic acid and has 100-fold greater affinity than the cis isomer. The results are consistent with a requirement in binding for a pseudoequatorially placed 2-carboxylate and clearly demonstrate the importance for binding of a correctly positioned hydrogen-bond-accepting group at the 4-position. The high-affinity binding of an anionic group in the 4-substituent binding pocket suggests that the glycine site and the neurotransmitter recognition (NMDA) site may have some features in common.

Animals↗
PubMed1992

Inhibition of forskolin-stimulated cyclic AMP formation by 1-aminocyclopentane-trans-1,3-dicarboxylate in guinea-pig cerebral cortical slices.

The effects of the selective metabotropic glutamate receptor agonist 1-aminocyclopentane-trans-1,3-dicarboxylate (t-ACPD) on forskolin-stimulated cyclic AMP formation in guinea-pig cerebral cortex slices were determined. t-ACPD inhibited the accumulation of [3H]cyclic AMP by approximately 80%, with an IC50 value of 35 +/- 4 microM. The effect was reversible and stereoselective, with the 1S,3R isomer being approximately 400-fold more potent than the 1R,3S isomer. L-Glutamate (over a restricted concentration range) also partially inhibited the response to forskolin, but quisqualate, alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA), and N-methyl-D-aspartate (NMDA) were ineffective. The effect of t-ACPD was not blocked by antagonists of the phospholipase C-linked metabotropic glutamate receptor, the AMPA ionotropic glutamate receptor, or the NMDA receptor. In summary, our results indicate the presence of a glutamate receptor in guinea-pig brain that is activated selectively by t-ACPD and that is negatively linked to adenylyl cyclase.

Amino Acids↗
PubMed1992

The pharmacology of recombinant GABAA receptors containing bovine alpha 1, beta 1, gamma 2L sub-units stably transfected into mouse fibroblast L-cells.

1. Responses to gamma-aminobutyric acid (GABA) were evoked in mouse fibroblast L-cells stably transfected with bovine, alpha 1, beta 1, gamma 2L sub-units of the GABAA receptor. Expression was stimulated via a steroid-inducible promoter system. 2. In near symmetrical intracellular and extracellular chloride concentrations, GABA evoked inward currents at negative holding potentials that reversed at +5 mV and displayed slight outward rectification. Concentration-response curves were fitted well by the logistic equation. GABA had a pEC50 = 5.1 +/- 0.1 and the curves had a slope of 1.9 +/- 0.1. 3. Responses to GABA were antagonized by bicuculline, picrotoxin and penicillin. The action of bicuculline was competitive (pA2 = 6.4) whilst the block by picrotoxin was uncompetitive and strongly agonist-dependent. 4. Benzodiazepine receptor agonists potentiated responses to 3 microM GABA. The rank order of potency was FG 8205 > flunitrazepam > zolpidem > C1218872. FG 8205 and C1218872 produced markedly lower maximal potentiations with efficacies 0.4 and 0.6 x that of flunitrazepam, respectively. The potencies of zolpidem and C1218872 observed are in agreement with the BZ1 type pharmacology of this sub-unit combination. The potentiation of GABA by flunitrazepam was antagonized by flumazenil with a Ki of 3.8 nM. 5. GABA responses were potentiated in the presence of pentobarbitone and alphaxalone. The response was also noticeably broadened by these compounds due to a decrease in the response decay rate. Concentrations of pentobarbitone of 100 microM and above evoked an inward current in the absence of GABA. Alphaxalone up to 10 microM did not evoke a direct response. 6. This expression system produced functional receptors that behaved in a fashion analogous to those found endogenously in other preparations. Thus, this system appears to provide a useful and versatile preparation for the analysis of sub-unit regulation of GABAA receptor pharmacology.

Animals↗