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P J Whiting

Publications and source records attributed to P J Whiting.

At least 19 recordsLinked to original sources
PubMed1999

Special relationship of gamma-aminobutyric acid to the ventromedial nucleus of the hypothalamus during embryonic development.

The ventromedial nucleus of the hypothalamus (VMH) is a key nucleus for regulating homeostatic, neuroendocrine, and behavioral functions. We conducted immunocytochemical analyses by using antisera directed against gamma-aminobutyric acid (GABA), its synthetic enzyme glutamic acid decarboxylase (GAD67), GABA-A receptor subunits (alpha2, beta3, epsilon), estrogen receptor-alpha, and Neuropeptide Y (NPY) in the region of the VMH in embryonic mice to identify potential patterning elements for VMH formation. Cells and fibers containing GABA and GAD67 encircled the primordial VMH as early as embryonic day 13 (E13) when the cytoarchitecture of the VMH was not recognizable by Nissl stain. At E16-17 the cytoarchitecture of the VMH became recognizable by Nissl stain as GABAergic fibers invaded the nucleus, continued postnatally, and by adulthood the density of GABAergic fibers was greater inside than outside the VMH. GABA-A receptor subunit expression (beta3 by E13 and alpha2 by E15) within the primordial VMH suggested potential sensitivity to the surrounding GABA signal. Brain slices were used to test whether fibers from distal or proximal sites influenced VMH development. Coronal Vibratome slices were prepared and maintained in vitro for 0-3 days. Nissl stain analyses showed a uniform distribution of cells in the region of the VMH on the day of plating (E15). After 3 days in vitro, cellular aggregation suggesting VMH formation was seen. Nuclear formation in vitro suggests that key factors resided locally within the coronal plane of the slices. It is suggested that either GABA intrinsic to the region nearby the VMH directly influences the development and organization of the VMH, or along with other markers provides an early indicator of pattern determination that precedes the cellular organization of the VMH.

Animals↗
PubMed1999

Delineation of the structural determinants of the N-methyl-D-aspartate receptor glycine binding site.

In this study, we have further delineated the domains of the N-methyl-D-aspartate receptor NR1 subunit that contribute to the glycine co-agonist binding site. Taking an iterative approach, we have constructed truncation mutants of the NR1 subunit, transiently expressed them in HEK-293 cells, and determined the binding of the glycine site antagonist [3H]L-689,560. Amino acids 380-811 were sufficient to form a glycine binding site with affinities for [3H]L-689,560 and glycine that were not significantly different from wild-type NR1. More extensive deletions, from either the amino- or the carboxy-terminal end, resulted in loss of ligand binding. Additional constructs were made starting from amino acids 380-843 of NR1, replacing the transmembrane (TMI-TMIII) domain with intervening linker sequences while retaining the TMIV domain so as to anchor the polypeptide to the membrane. Although robust amounts of polypeptides were synthesised by transfected cells, only low levels of [3H]L-689,560 binding sites could be detected. This suggests that only a small proportion of the synthesised polypeptide folds in the appropriate manner so as to form a ligand binding site. These data indicate that although it is possible to reduce the glycine binding site to minimal so-called S1 and S2 domains, efficient folding of the polypeptide so as to form a ligand binding site may require sequences within the TMI-TMIII domain.

Amino Acid Sequence↗
PubMed1998

Functional characteristics of recombinant human GABA(A) receptors containing the epsilon-subunit.

(1) This paper further examines the functional characteristics of recombinant human GABA(A) receptors containing the epsilon-subunit expressed in Xenopus oocytes. (2) Alpha1beta1epsilon receptors are not modulated by benzodiazepine ligands or by a number of hypothalamic hormones. (3) The intravenous anaesthetic agents pentobarbital, propofol and etomidate all potentiate sub-maximal GABA currents (EC20) to a similar degree in alpha1beta1epsilon and alpha1beta1gamma2s receptors. (4) Direct activation by pentobarbital produced a similar maximum response on alpha1beta1epsilon and alpha1beta1gamma2s, however, both the EC50 and slope were lower on alpha1beta1epsilon compared to alpha1beta1gamma2s. (5) These results describe a novel pharmacology for recombinant alpha1beta1epsilon receptors.

Anesthetics, Intravenous↗
PubMed1998

Expression of 10 GABA(A) receptor subunit messenger RNAs in the motor-related thalamic nuclei and basal ganglia of Macaca mulatta studied with in situ hybridization histochemistry.

In situ hybridization histochemistry technique with [35S]UTP-labelled riboprobes was used to study the expression pattern of 10 GABA(A) receptor subunit messenger RNAs in the basal ganglia and motor thalamic nuclei of rhesus monkey. Human transcripts were used for the synthesis of alpha2, alpha4, beta2, beta3, gamma1 and delta subunit messenger RNA probes. Rat complementary DNAs were used for generating alpha1, alpha3, beta1 and gamma2 subunit messenger RNA probes. Nigral, pallidal and cerebellar afferent territories in the ventral tier thalamic nuclei all expressed alpha1, alpha2, alpha3, alpha4, beta1, beta2, beta3, delta and gamma2 subunit messenger RNAs but at different levels. Each intralaminar nucleus displayed its own unique expression pattern. In the thalamus, gamma1 subunit messenger RNA was detected only in the parafascicular nucleus. Comparison of the expression patterns with the known organization of GABA(A) connections in thalamic nuclei suggests that (i) the composition of the receptor associated with reticulothalamic synapses, except for those in the intralaminar nuclei, may be alpha1alpha4beta2delta, (ii) receptors of various other subunit compositions may operate in the local GABAergic circuits, and (iii) the composition of receptors at nigro- and pallidothalamic synapses may differ, with those at nigrothalamic probably containing beta1 and gamma2 subunits. In the medial and lateral parts of the globus pallidus, the subthalamic nucleus and the substantia nigra pars reticularis, the alpha1, beta2 and gamma2 messenger RNAs were co-expressed at a high level suggesting that this subunit composition was associated with all GABAergic synapses in the direct and indirect striatal output pathways. Various other subunit messenger RNAs were also expressed but at a lower level. In the substantia nigra pars compacta the most highly expressed messenger RNAs were alpha3, alpha4 and beta3; all other subunit messenger RNAs studied, except for gamma1, alpha1 and alpha2, were expressed at a moderate to high level. In the striatum, the following subunit messenger RNAs were expressed (listed in order of decreasing signal intensity): alpha4, beta3, alpha2, alpha3, beta2, delta, gamma2, alpha1. The expression patterns found in the monkey were similar to those described in comparable nuclei in the rat by Wisden et al. [J. Neurosci. (1992), 12, p. 1040]; however, the monkey nuclei displayed a much greater variety of GABA(A) receptor subunit messenger RNAs.

Animals↗
PubMed1998

Adaptation of gamma-aminobutyric acid type A receptors to alcohol exposure: studies with stably transfected cells.

We studied the adaptation of gamma-aminobutyric acid type A (GABAA) receptor function to chronic ethanol exposure in cells stably transfected with the following GABAA receptor subunits: alpha-1 beta-2 gamma-2L, alpha-1 beta-2 gamma-2S, alpha-1 beta-3 gamma-2S, alpha-1 beta-1, alpha-5 beta-3 gamma-3 and alpha-6 beta-3 gamma-2S. Chronic exposure to ethanol resulted in a decrease in muscimol-stimulated 36Cl- flux and a decrease in modulation of that flux by ethanol, flunitrazepam, methyl-6,7-4-dimethoxy-4-ethyl-beta-carboline-3-carboxylate and pregnanolone without any change in the modulation by pentobarbital or zinc. Direct activation of the GABAA receptor by pentobarbital was enhanced by chronic ethanol treatment. Reduction of the action of muscimol, ethanol and flunitrazepam differed in the duration and amount of ethanol required to see an effect. Reduction of the action of ethanol of alpha-1 beta-2 gamma-2L cells occurred within 15 min and was near-maximal for 25 mM ethanol, whereas reduction of the actions of muscimol and flunitrazepam actions required hours of exposure and higher concentrations of ethanol. Chronic ethanol exposure produced a reduction in the Emax value for the action of muscimol for all six subunit combinations, but quantification of surface receptors by immunolabeling showed no change in GABAA receptor density. The differences in alcohol sensitivity and time courses for different effects of ethanol indicate multiple mechanisms of adaptation of GABAA receptors. Use of stably transfected cells rules out "subunit substitution" as a mechanism for these changes and points to post-translational changes (e.g., phosphorylation, receptor assembly) as the most likely mechanisms. These in vitro findings are compared with results from in vivo studies.

Animals↗
PubMed1997

The interaction of the general anesthetic etomidate with the gamma-aminobutyric acid type A receptor is influenced by a single amino acid.

The gamma-aminobutyric acid type A (GABAA) receptor is a transmitter-gated ion channel mediating the majority of fast inhibitory synaptic transmission within the brain. The receptor is a pentameric assembly of subunits drawn from multiple classes (alpha1-6, beta1-3, gamma1-3, delta1, and epsilon1). Positive allosteric modulation of GABAA receptor activity by general anesthetics represents one logical mechanism for central nervous system depression. The ability of the intravenous general anesthetic etomidate to modulate and activate GABAA receptors is uniquely dependent upon the beta subunit subtype present within the receptor. Receptors containing beta2- or beta3-, but not beta1 subunits, are highly sensitive to the agent. Here, chimeric beta1/beta2 subunits coexpressed in Xenopus laevis oocytes with human alpha6 and gamma2 subunits identified a region distal to the extracellular N-terminal domain as a determinant of the selectivity of etomidate. The mutation of an amino acid (Asn-289) present within the channel domain of the beta3 subunit to Ser (the homologous residue in beta1), strongly suppressed the GABA-modulatory and GABA-mimetic effects of etomidate. The replacement of the beta1 subunit Ser-290 by Asn produced the converse effect. When applied intracellularly to mouse L(tk-) cells stably expressing the alpha6beta3gamma2 subunit combination, etomidate was inert. Hence, the effects of a clinically utilized general anesthetic upon a physiologically relevant target protein are dramatically influenced by a single amino acid. Together with the lack of effect of intracellular etomidate, the data argue against a unitary, lipid-based theory of anesthesia.

Amino Acids↗
PubMed1997

Neuronally restricted RNA splicing regulates the expression of a novel GABAA receptor subunit conferring atypical functional properties [corrected; erratum to be published].

We report the isolation and characterization of a cDNA encoding a novel member of the GABA receptor gene family, epsilon. This polypeptide is 506 amino acids in length and exhibits its greatest amino acid sequence identity with the GABAA receptor gamma3 subunit (47%), although this degree of homology is not sufficient for it to be classified as a fourth gamma subunit. The epsilon subunit coassembles with GABAA receptor alpha and beta subunits in Xenopus laevis oocytes and transfected mammalian cells to form functional GABA-gated channels. alpha1beta1epsilon GABAA receptors, like alpha1beta1gamma2s receptors, are modulated by pentobarbital and the steroid 5alpha-pregnan-3alpha-ol-20-one but, unlike alpha1beta1gamma2s receptors, are insensitive to flunitrazepam. Additionally, alpha1beta1epsilon receptors exhibit rapid desensitization kinetics, as compared with alpha1beta1 or alpha1beta1gamma2s. Northern analysis demonstrates widespread expression of a large epsilon subunit transcript in a variety of non-neuronal tissues and expression of a smaller transcript in brain and spinal cord. Sequence analysis demonstrated that the large transcript contained an unspliced intron, whereas the small transcript represents the mature mRNA, suggesting regulation of expression of the epsilon subunit via neuronally restricted RNA splicing. In situ hybridization and immunocytochemistry reveal a pattern of expression in the brain restricted primarily to the hypothalamus, suggesting a role in neuroendocrine regulation, and also to subfields of the hippocampus, suggesting a role in the modulation of long term potentiation and memory.

Amino Acid Sequence↗
PubMed1997

Alpha subunit isoform influences GABA(A) receptor modulation by propofol.

We have investigated the role of the alpha subunit in the modulation of gamma-aminobutyric acid type A (GABA(A)) receptors by the general anesthetic propofol, using whole-cell patch clamp recordings made from distinct stable fibroblast cell lines which expressed only alpha1beta3gamma2 or alpha6beta3gamma2 GABA(A) receptors. At clinically relevant anesthetic concentrations, propofol potentiated submaximal GABA currents in alpha1beta3gamma2 receptors to a far greater degree than those in alpha6beta3gamma2 receptors. The alpha subunit influenced the efficacy of propofol for modulation, but not its potency. In contrast, direct gating of the ion channel by propofol, in the absence of GABA, was significantly larger in the alpha6 than the alpha1 containing receptors. The potentiation of submaximal GABA by trichloroethanol, and the potentiation and direct gating by methohexital was also studied, and showed the same relative trends as propofol.

Anesthetics, Intravenous↗
PubMed1997

Ethanol, flunitrazepam, and pentobarbital modulation of GABAA receptors expressed in mammalian cells and Xenopus oocytes.

GABAA receptors composed of human alpha 1 beta 2 gamma 2L, alpha 1 beta 2 gamma 2S, alpha 1 beta 3 gamma 2S, alpha 6 beta 3 gamma 2S, and alpha 5 beta 3 gamma 3 subunits as well as bovine alpha 1 beta 1 gamma 2L and alpha 1 beta 1 subunits were stably expressed in mammalian L(tk-) cells and transiently expressed in Xenopus oocytes. Effects of muscimol, ethanol, flunitrazepam, and pentobarbital on receptor function were compared for the two expression systems using a 36Cl- flux assay for cells and an electrophysiological assay for oocytes. Muscimol activated all receptors in both expression systems but was more potent for L(tk-) cells than oocytes; this difference ranged from 2.6-to 26-fold, depending upon subunit composition. The most pronounced differences between receptors and expression systems were found for ethanol. In L(tk-) cells, low (5-50 mM) concentrations of ethanol potentiated muscimol responses only with receptors containing the gamma 2L subunit. In oocytes, concentrations of 30-100 mM produced small enhancements for most subunit combinations. Flunitrazepam enhanced muscimol responses for all receptors except alpha 6 beta 3 gamma 2S and alpha 1 beta 1, and this enhancement was similar for both expression systems. Pentobarbital also enhanced muscimol responses for all receptors, and this enhancement was similar for L(tk-) cells and oocytes, except for alpha 6 beta 3 gamma 2S where the pentobarbital enhancement was much greater in oocytes than cells. The alpha 6 beta 3 gamma 2S receptors were also distinct in that pentobarbital produced direct activation of chloride channels in both expression systems. Thus, the type of expression/assay system markedly affects the actions of ethanol on GABAA receptors and also influences the actions of muscimol and pentobarbital on this receptor. Differences between these expression systems may reflect posttranslational modifications of receptor subunits.

Animals↗
PubMed1997

Key amino acids in the gamma subunit of the gamma-aminobutyric acidA receptor that determine ligand binding and modulation at the benzodiazepine site.

Pharmacological analyses of gamma-aminobutyric acidA (GABAA) receptor subtypes have suggested that both the alpha and gamma subunits, but not the beta subunit, contribute to the benzodiazepine binding site. We took advantage of the different pharmacological properties conferred by the inclusion of different gamma subunits in the receptor macromolecule to identify amino acids gamma2Phe77 and gamma2Met130 as key determinants of the benzodiazepine binding site. gamma2Phe77 was required for high affinity binding of the benzodiazepine site ligands flumazenil, CL218,872, and methyl-beta-carboline-3-carboxylate but not flunitrazepam. This amino acid was, however, required for allosteric modulation by flunitrazepam, as well as other benzodiazepine site ligands. In contrast, gamma2Met130 was required for high affinity binding of flunitrazepam, clonazepam, and triazolam but not flumazenil, CL218, 872, or methyl-beta-carboline-3-carboxylate and did not affect benzodiazepine efficacy. Introduction of the phenylalanine and methionine into the appropriate positions of gamma1 was not sufficient to confer high affinity for the benzodiazepine site ligand zolpidem. These data show that gamma2Phe77 and gamma2Met130 are necessary for high affinity binding of a number of benzodiazepine site ligands. Although most previous studies have focused on the contribution of the alpha subunit, we demonstrated a critical role for the gamma subunit at the benzodiazepine binding site, indicating that this modulatory site is located at the interface of these two subunits. Furthermore, gamma2Phe77 is homologous to alpha1Phe64, which has been previously shown to be a key determinant of the GABA binding site, suggesting a conservation of motifs between different ligand binding sites on the GABAA receptor.

Amino Acid Sequence↗
PubMed1996

Platelet-derived growth factor induces a long-term inhibition of N-methyl-D-aspartate receptor function.

Platelet-derived growth factor (PDGF) is a multifunctional protein that plays important roles in many tissues, including the mammalian central nervous system. PDGF and PDGF receptors (PDGFRs) are expressed in virtually every region of the central nervous system where they are involved in the development, survival, growth, and differentiation of both neuronal and glial cells. We now report that a brief activation of PDGFRs produced a long-lasting inhibition of N-methyl-D-aspartate (NMDA)-dependent excitatory postsynaptic currents in CA1 pyramidal neurons in rat hippocampal slices. PDGF also inhibited NMDA receptors (NMDA-Rs) in cultured hippocampal neurons by a mechanism that involves a decrease in single channel open probability. Non-NMDA receptor function was not affected by PDGF in hippocampal neurons. Experiments with mutant PDGFRs and chelation of intracellular Ca2+ in Xenopus oocytes indicate that this inhibition depends on a phospholipase C-gamma-induced elevation of intracellular Ca2+ levels. The PDGF-induced inhibition of NMDA-Rs is produced by a mechanism different than the well characterized phenomenon of Ca2+-dependent NMDA-R run down because the effect of PDGF was blocked by the phosphatase inhibitor, calyculin A, and was not affected by the microtubule polymerizing agent, phalloidin. Because elevations of PDGF levels are associated with neurological trauma or disease, we propose that PDGF can exert neuroprotective effects by inhibiting NMDA-R-dependent excitotoxicity.

Analysis of Variance↗
PubMed1996

Cell surface expression of the human N-methyl-D-aspartate receptor subunit 1a requires the co-expression of the NR2A subunit in transfected cells.

Cell surface expression of the NR1a subunit has been examined in mouse L cell lines permanently transfected with the complementary DNA for human NR1a or with the complementary DNAs for NR1a and NR2A. The expression of the subunits was under the control of the murine mammary tumour virus promoter and following induction of expression by dexamethazone both cell lines expressed high levels of the NR1a subunit as determined by immunofluorescence using permeabilized cells and immunoblotting of cell membranes with subunit specific antibodies. However, cell surface expression of the NR1a subunit was found only in the cells expressing both the NR1a and NR2A subunits. This was confirmed by cell surface biotinylation of the two cell lines and affinity isolation of the receptor subunits. To determine if this result was solely due to the use of a particular cell line and or the choice of expression vector, Cos-7 cells were transiently transfected with either NR1a or NR1a plus NR2A. Here too cell surface expression was only found following co-transfection of both subunits.

Animals↗
PubMed1996

The messenger RNAs for the N-methyl-D-aspartate receptor subunits show region-specific expression of different subunit composition in the human brain.

The expression of the messenger RNAs encoding N-methyl-D-aspartate receptor subunits in neurologically normal post-mortem human brain was studied by in situ hybridization. In the caudate, putamen and nucleus accumbens strong hybridization signals were observed for N-methyl-D-aspartate R1-1 messenger RNA but much weaker signals for N-methyl-D-aspartate R1-3 and N-methyl-D-aspartate R1-4, N-Methyl-D-aspartate R1-2 was not detectable. N-methyl-D-aspartate R2B was the only N-methyl-D-aspartate R2 subunit detected in these nuclei. In the hippocampus the messenger RNAs for both N-methyl-D-aspartate R1-1 and N-methyl-D-aspartate R1-4 were strongly expressed in the dentate gyrus, CA3-CA1 pyramidal cells, subiculum, entorhinal cortex and perirhinal cortex. Much lower expression was seen for N-methyl-D-aspartate R1-2 and N-methyl-D-aspartate R1-3. The messenger RNAs for both N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2B, but not N-methyl-D-aspartate R2C, subunits were expressed in the hippocampus. In the temporal cortex all N-methyl-D-aspartate RI isoforms were expressed (N-methyl-D-aspartate R1-1 and N-methyl-D-aspartate R1-4 being the most abundant) and N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2B but not N-methyl-D-aspartate R2C were also moderately expressed. In the brain stem N-methyl-D-aspartate R1-4 was strongly expressed in various nuclei including the locus coeruleus, nucleus centralis superior and deep pontine nuclei. Only weak expression was seen for N-methyl-D-aspartate RI-1 and N-methyl-D-aspartate R1-3 but not N-methyl-D-aspartate RI-2; of the N-methyl-D-aspartate R2 subunits only N-methyl-D-aspartate R2C was found to be expressed in these nuclei. In the cerebellum all the N-methyl-D-aspartate I isoforms were expressed (mostly N-methyl-D-aspartate R1-4) in the Purkinje layer which also expressed N-methyl-D-aspartate R2A and N-methyl-D-aspartate R2C. In the molecular layer cells were found expressing N-methyl-D-aspartate R1-4 and N-methyl-D-aspartate R2B and cells in the granule layer were found to express N-methyl-D-aspartate R1-1, N-methyl-D-aspartate R1-3 and N-methyl-D-aspartate R1-4 and N-methyl-D-aspartate R2C only. Preliminary studies indicated that the messenger RNA for the N-methyl-D-aspartate R2D subunit was not expressed in the above areas of brain. These results give the first demonstration of the distribution of N-methyl-D-aspartate receptor subunit messenger RNAs in the human brain. The region-specific expression of subunit combinations suggests a heterogeneity of N-methyl-D-aspartate receptors with diverse physiological/pathophysiological roles and provides a rationale for the development of discriminatory N-methyl-D-aspartate receptor antagonists to target selective neuronal populations.

Aged↗
PubMed1996

Identification of domains in human recombinant GABAA receptors that are photoaffinity labelled by [3H]flunitrazepam and [3H]Ro15-4513.

We have used [3H]flunitrazepam and [3H]Ro15-4513 as photoaffinity labelling agents in combination with a chemical cleavage technique to localize the benzodiazepine recognition sites of specific human recombinant alpha 1 beta 1 gamma 2, alpha 1 beta 3 gamma 2 and alpha 6 beta 3 gamma 2 GABAA receptor subtypes. The chemical agent utilized was hydroxylamine, whose substrate is a relatively rare asparagine-glycine amide bond that occurs only in the alpha subunits of the receptors examined in this study. Cleavage products were resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of these experiments show that, in the alpha 1 subunit-containing receptors, incorporation of [3H]flunitrazepam occurs within residues 1-103 of the alpha 1 subunit, while incorporation of [3H]Ro15-4513 occurs within the region of the alpha 1 subunit that lies between residue 104 and the C-terminus. Photolabelling of membranes prepared from the alpha 6 beta 3 gamma 2-expressing cell line with [3H]Ro15-4513 resulted in the incorporation of radiolabel into two major protein species of M(r) 56,000 and M(r) 48,000, indicating incorporation into the alpha 6 subunit and possibly also the gamma 2 subunit. Hydroxylamine cleavage of alpha 6-containing receptors labelled with [3H]Ro15-4513 produced a gel profile consistent with the incorporation of the label occurring between residue 125 and the C-terminal. Thus, we have shown that the recognition sites for the agonist [3H]flunitrazepam and the inverse agonist [3H]Ro15-4513 occur within distinct domains of the human GABAA receptor.

Affinity Labels↗
PubMed1996

Which GABAA-receptor subtypes really occur in the brain?

GABAA receptors are a heterogeneous family of ligand-gated ion channels responsible for mediating inhibitory neurotransmission in the CNS. Since the identification of mammalian cDNAs encoding 13 GABAA-receptor subunits, the composition of native receptor molecules and their localization in the brain has been an area of intense study. We conclude that the number of major subtypes is probably less than ten but their physiological roles have yet to be clearly defined and this represents the next step in GABAA-receptor research.

Brain Chemistry↗
PubMed1996

Microtubule depolymerization inhibits ethanol-induced enhancement of GABAA responses in stably transfected cells.

We studied whether microtubule organization is important for actions of ethanol on GABAA ergic responses by testing the effects of microtubule depolymerization on ethanol enhancement of GABA action in mouse L(tk-) cells stably transfected with GABAA receptor alpha 1 beta 1 gamma 2L subunits. The microtubule-disrupting agents colchicine, taxol, and vinblastine completely blocked ethanol-induced enhancement of muscimol-stimulated chloride uptake. beta-Lumicolchicine, a colchicine analogue that does not disrupt microtubules, had no effect on ethanol action. Colchicin did not alter the potentiating actions of flunitrazeparn or pentobarbital on muscimol-stimulated chloride uptakes. Thus, colchicine specifically inhibited the potentiating action of ethanol. From these findings, we conclude that intact microtubules are required for ethanol-induced enhancement of GABAA responses and suggest that a mechanism involving microtubules produces posttranslational modifications that are necessary for ethanol sensitivity in this cell system.

Animals↗
PubMed1996

Generation and characterisation of stable cell lines expressing recombinant human N-methyl-D-aspartate receptor subtypes.

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.

2-Amino-5-phosphonovalerate↗