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R W Olsen

Publications and source records attributed to R W Olsen.

At least 109 records · Page 6Linked to original sources
PubMed1988

A novel GABAA antagonist [3H]SR 95531: microscopic analysis of binding in the rat brain and allosteric modulation by several benzodiazepine and barbiturate receptor ligands.

Recent reports have demonstrated that the synthetic gamma-aminobutyric acid (GABA)-derivative, SR 95531 [2-(3'-carbethoxy-2'-propyl)-3-amino-6-paramethoxy-phenyl-pyrid azinium bromide], possesses selective GABAA antagonistic properties. Because of its potency for recognition of GABAA sites, this agent has been used to identify GABAA receptors. In the present investigation, we studied the binding of [3H]SR 95531 to tissue sections of rat brain using microscopic analysis of receptor localization. The appropriate binding conditions for defining GABAA receptors with this radioligand were obtained by determining the dissociation and association kinetics, and performing saturation and displacement studies. Using membrane preparations from whole rat brain (or brain regions representing cortex, striatum, hippocampus, midbrain-thalamus, medulla-pons and cerebellum), saturation and displacement studies were analyzed, and allosteric modulation of [3H]SR 95531 binding was examined by including several benzodiazepine and barbiturate receptor ligands in the incubation media. To assess the stereoselective properties of [3H]SR 95531 binding in rat membranes, numerous barbiturates were added during the incubation. The binding of [3H]SR 95531 was demonstrated to be saturable, specific and to bind with relatively high affinity to low-affinity GABAA sites. Scatchard analysis performed on saturation data of binding to tissue sections showed a dissociation constant (KD) of 42.4 nM and a maximum number of binding sites (Bmax) of 105.8 fmol/mg tissue. Microscopic analysis showed that intermediate to high densities of [3H]SR 95531 binding occurred in brain regions containing intermediate to high densities of low-affinity GABAA receptor sites. The binding of [3H]SR 95531 to membranes also appeared to occur at low-affinity GABAA sites. Results from competition studies demonstrated that [3H]SR 95531 is displaceable by GABAA agents and displaced preferentially by GABAA antagonists. Scatchard analysis of saturation experiments from membrane preparations indicated that the KD and Bmax from the centrifugation assay was 53.0 nM and 4.26 pmol/mg protein, respectively. Using the filtration assay, binding to membranes yielded a KD value of 45.6 nM and a Bmax of 0.77 pmol/mg protein. The allosteric modulation data demonstrated that numerous benzodiazepine and barbiturate agents inhibited [3H]SR 95531 binding and this varied according to brain region. Several barbiturates included in the incubation media exhibited a stereoselective inhibition of [3H]SR 95531 binding to whole rat brain membranes.(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗
PubMed1988

Barbiturates.

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Animals↗
PubMed1988

Pyrethroid insecticides: stereospecific allosteric interaction with the batrachotoxinin-A benzoate binding site of mammalian voltage-sensitive sodium channels.

Pyrethroid insecticides are synthetic neurotoxins patterned after the naturally occurring pyrethrins. Their mechanism of action is thought to involve effects primarily at the voltage-sensitive sodium channel of both insect and mammalian neurons, although recent studies have raised the possibility that these compounds may also act at the gamma-aminobutyric acid receptor-chloride ionophore complex. Here we show that active pyrethroids of the alpha-cyano-3-phenoxybenzyl class allosterically enhance the binding of [3H]batrachotoxinin-A 20-alpha-benzoate to voltage-sensitive sodium channels of rat brain in a dose-dependent and stereospecific manner. Comparison of the rank order of potency for enhancement of [3H]batrachotoxinin-A 20-alpha-benzoate binding and insecticidal activity in a series of toxic stereoisomers of cypermethrin, representative of the class, reveals a correlation between the two measures. These results support a sodium channel site model for pyrethroid action and suggest a useful and practical method to help evaluate the relationship between effects at the sodium channel and insecticidal potency for members of this class of compounds.

Allosteric Regulation↗
PubMed1988

Immunohistochemical localization of benzodiazepine/GABAA receptors in the human hippocampal formation.

Monoclonal antibodies to a purified benzodiazepine/GABAA receptor complex from bovine cerebral cortex were used to determine the localization of this receptor in immunohistochemical preparations of the human hippocampal formation. The regional localization of the receptor was compared with the autoradiographic distribution of binding sites for a benzodiazepine antagonist, Ro 15-1788, and very similar patterns were observed. The highest levels of labeling were present in the molecular layer of the dentate gyrus, where much of the immunoreaction product was localized on the dendrites of granule cells. Lower levels of staining were observed in the granule cell layer, where reaction product was distributed around the perimeter of granule cell somata. The staining patterns varied among the different fields of the hippocampus. Labeling was lowest in the CA3 field, increased somewhat in the CA2 field, and was highest in the CA1 field. Within CA1, a laminar pattern was observed. Moderate to high levels of staining were present in strata oriens, pyramidale, radiatum, and moleculare, whereas less staining was observed in stratum lacunosum. The cellular localization of the receptor also differed among the hippocampal fields. Very little reaction product was observed on pyramidal neurons in the CA3 field, but labeling of pyramidal neurons appeared to increase progressively throughout CA2, CA1, and the subiculum. Nonpyramidal neurons were prominently labeled in all hippocampal fields. These results indicate that the benzodiazepine/GABAA receptor is heterogeneously distributed among the different fields and on different neuronal cell types of the human hippocampus.

Antibodies, Monoclonal↗
PubMed1988

Osmotic shock: a method to eliminate endogenous gamma-aminobutyric acid and account for the influence on benzodiazepine binding affinity in autoradiographic studies.

Inasmuch as the presence of endogenous gamma-aminobutyric acid (GABA) may affect benzodiazepine binding to tissue sections in autoradiographic studies, a protocol designed to check for this influence has been investigated. [3H]Flunitrazepam (1 nM) was used to label benzodiazepine receptors for autoradiographic localization. Bicuculline was added to the incubation medium of an additional set of tissue sections to antagonize any potential effect of endogenous GABA. Binding in these sections was compared to that occurring in another set in which excess GABA was added to "create" further GABA enhancement. Binding also was compared to adjacent sections which were treated similarly but also preincubated in distilled-deionized water to burst the cells by osmotic shock and eliminate endogenous GABA, thereby preventing any effect on benzodiazepine binding. The results indicated that endogenous GABA is indeed present in the slide-mounted tissue sections and is affecting benzodiazepine receptor binding differentially in various regions of the brain depending on the density of GABAergic innervation. Scatchard analysis of saturation data demonstrated that the alteration in benzodiazepine binding due to GABA was a result of a change in the affinity rather than number of receptors present. These experiments have been compared to the binding of the imidazodiazepine, [3H] Ro15-1788. We also show that the treatments affect endogenous GABA and not the receptors themselves. This suggests strongly that, when using a single nonsaturating concentration of radiolabeled benzodiazepine antagonist, autoradiographic studies to date may have been subject to erroneous interpretation due to the differential effects of endogenous GABA on benzodiazepine binding (increased affinity).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1987

Convulsant/barbiturate activity on the soluble gamma-aminobutyric acid-benzodiazepine receptor complex.

Cage convulsant t-butyl bicyclophosphoro[35S]thionate binding activity in rat brain membrane homogenates was solubilized with the zwitterionic detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (Chaps) and shown to co-purify with the benzodiazepine--gamma-aminobutyric acid (GABA) receptor complex on gel filtration and affinity chromatography. Whereas convulsant binding activity, but not GABA and benzodiazepine receptor binding, was eliminated by solubilization in other detergents like sodium deoxycholate or Triton X-100, or by addition of Triton X-100 to the extracts solubilized in the zwitterionic detergent, convulsant activity was not irreversibly lost or selectively unstable, but could be restored by exchanging the protein back into the detergent Chaps. The GABA-benzodiazepine receptor activity solubilized in Chaps alone, containing convulsant activity, and a sample in Chaps supplemented with Triton X-100 and lacking convulsant activity, did not differ in size as measured by gel filtration column chromatography or by radiation inactivation target size analysis. This suggests that convulsant binding activity does not require any additional protein subunits or other macromolecules nor any unique aggregation state relative to GABA and benzodiazepine receptor binding, and that all three activities reside on the same protein complex. As in intact brain, the target size for convulsant binding activity was 3-5 times that of benzodiazepine binding activity, suggesting that an oligomeric protein structure of the receptor complex with intact strong subunit interactions present in the native membrane environment is needed for convulsant activity, and that this and other properties are more preserved in Chaps than in other detergents.

Animals↗
PubMed1987

The gamma-aminobutyric-acid/benzodiazepine-receptor protein from rat brain. Large-scale purification and preparation of antibodies.

The gamma-aminobutyric-acid-receptor protein complex from rat brain was solubilized in high yield, purified in milligram amounts by benzodiazepine affinity chromatography and used to generate a high-titer rabbit antiserum. High concentrations of Triton X-100 detergent plus KCl solubilized about 90% of the membrane-bound gamma-aminobutyric acid receptor (assayed by [3H]muscimol binding) and benzodiazepine receptor (assayed by [3H]flunitrazepam binding) activities. Both activities were retained on an affinity column using an immobilized benzodiazepine ligand, and most of the column-absorbed receptor could be eluted by a solution of free benzodiazepine plus 4 M urea. The purified protein bound [3H]muscimol and [3H]flunitrazepam with receptor-like pharmacological specificity and specific activities of about 1700 pmol and 700 pmol bound/mg protein, respectively, for the two ligands. This corresponds to a purification of over 600-fold and a near theoretical purity, with a yield of milligram quantities from 100 g brain. Four peptide bands were observed on gel electrophoresis in sodium dodecyl sulfate, with molecular mass values of 31, 47, 52 and 57 kDa. The latter two were most significantly stained, and identified as receptor subunits by photolabeling with [3H]flunitrazepam (52 kDa) and [3H]muscimol (57 kDa), and by reaction on Western blots with monoclonal antibodies to this protein produced by Schoch et al. [(1985) Nature (Lond.) 314, 168-171]. Rabbit antiserum was raised to the purified protein and could, at high dilutions, both coprecipitate soluble gamma-aminobutyric-acid/benzodiazepine-receptor-binding activities and stain the receptor subunits (principally 52-kDa band) on Western blots.

Animals↗
PubMed1987

[3H]AMPA binding to glutamate receptor subpopulations in rat brain.

The glutamate analog (RS)-alpha-amino-3-hydroxy-5-methyl-isoxazole-4-propionic acid (AMPA), displaced 11% of the binding of L-[3H]glutamate to rat brain membranes, amounting to 22% of the specific binding displaceable by excess non-radioactive glutamate. AMPA-sensitive L-[3H]glutamate binding was additive with that displaced by kainic acid (1 microM) plus N-methyl-D-aspartate (10 microM) when low concentrations of non-radioactive AMPA (1 microM) were employed to determine non-specific background, but partially overlapped when higher concentration of AMPA (100 microM) were used. [3H]AMPA binding was 21% specific (displaceable by non-radioactive 0.1 mM AMPA) in sodium-, calcium- and chloride-free buffer, but increased to over 30% in the presence of 0.1 M chloride. AMPA-sensitive glutamate binding and AMPA binding were both stimulated dramatically by thiocyanate and by several other anions. [3H]AMPA binding activity was resistant to freezing and thawing, optimal at 0-4 degrees C, and detectable at slightly reduced levels by filtration assays and in tissue section autoradiography. AMPA showed a heterogeneous affinity in displacement of L-[3H]glutamate, and [3H]AMPA binding showed heterogeneity with respect to AMPA, quisqualate, and glutamic acid diethyl ester. Scatchard plots gave a best fit for two sites with Kd values of 28 and 500 nM and Bmax values of 200 and 1800 fmol/mg protein, respectively. [3H]AMPA was inhibited by quisqualate (IC50 = 60 nM), L-glutamate (2 microM), (RS)-3-hydroxy-4,5,6,7-tetrahydroisoxazolo-[5,4-c]-pyridine-7-carboxylic acid (7-HPCA, 5 microM), kainic acid (20 microM) and glutamic acid diethyl ester (21 microM) but insensitive to L-aspartate, ibotenic acid, N-methyl-D-aspartate, (RS)-2-amino-phosphonobutyric acid and (RS)-2-amino-phosphonovaleric acid. This is consistent with labeling of a quisqualate-specific subpopulation of glutamate receptors. The high affinity (28 nM) and intermediate affinity (0.5 microM) AMPA sites had similar pharmacological specificity and brain regional distribution as determined by autoradiography. The latter revealed high densities of [3H]AMPA binding in the superficial layers of the cerebral cortex; stratum pyramidale, stratum radiatum, and stratum oriens of the hippocampus; and stratum moleculare of the dentate gyrus. Within the cerebellum, higher densities of binding were observed in the molecular layer than in the granule cell layer. In many regions, [3H]AMPA binding had a similar distribution to that of L-[3H]glutamate binding displaced by AMPA (1 microM).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1987

Triethyllead inhibits gamma-aminobutyric acid binding to uptake sites in synaptosomal membranes.

Triethyllead (TEL), the active metabolite of tetraethyllead, was shown previously to inhibit selectively high-affinity Na+-dependent uptake of gamma-aminobutyric acid (GABA) into cerebrocortical synaptosomes. Such inhibition was not related to the Na+ gradient, Na+,K+-ATPase activity, [Cl-], or energy charge. We report here that TEL inhibits GABA binding to the presynaptic transporter involved in Na+-dependent uptake. Scatchard plot analysis of Na+-dependent [3H]GABA binding to a highly purified synaptic plasma membrane preparation revealed that 25 microM TEL reduced the Bmax by 44%, leaving the KD unchanged. This binding was reversible and predominantly involved membrane uptake sites, as characterized by pharmacological specificity to GABA ligands. Approximately 85% of specific GABA binding was considered membrane uptake site binding, as indicated by sensitivity to nipecotic acid and diaminobutyric acid, with relative insensitivity to muscimol, bicuculline methiodide, baclofen, and beta-alanine. With respect to previous data, these finding suggest that TEL inhibits Na+-sensitive high-affinity GABA uptake by interfering with GABA binding to its presynaptic transporter.

Animals↗
PubMed1987

gamma-Aminobutyric acid receptor-regulated 36Cl- flux in mouse cortical slices.

An improved neurochemical assay for gamma-aminobutyric acid (GABA) function has been developed using tracer-radioactive chloride efflux in mouse cortical slices. Careful maintenance of the brain slice viability resulted in a 3-fold stimulation of 36Cl- efflux rate by the GABA agonist muscimol (EC50 = 3 microM), comparable to electrophysiologic and other chloride flux preparations. The shape of the muscimol dose-response curve was shallow, suggestive of negative cooperativity or heterogeneous receptors, but tissue uptake of agonist, possible diffusion barriers and apparent functional desensitization complicated these results. The response to muscimol was inhibited by GABAA receptor antagonists such as RU5135 and was enhanced by barbiturates and benzodiazepines. As observed previously, barbiturates stimulated 36Cl- efflux rate on their own and potentiated the response (potency and maximal effect) to muscimol in a stereospecific and picrotoxin-sensitive manner. Benzodiazepine receptor ligands alone did not alter 36Cl- flux, but agonists such as flunitrazepam enhanced the response to muscimol, an effect sensitive to the antagonist Ro15-1788. The inverse agonist methyl 6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate did not inhibit muscimol-activated 36Cl- flux. The anthelminthic-insecticide avermectin B1a stimulated 36Cl- flux by itself, and this response was apparently additive with that of muscimol. This brain slice chloride flux assay is therefore suitable for the assessment of activity including dose-response curves for GABAA agonists, antagonists and modulators including benzodiazepines.

Androstanes↗
PubMed1987

gamma-Aminobutyric acid receptor binding in fresh mouse brain membranes at 22 degrees C: ligand-induced changes in affinity.

Binding of [3H]muscimol to mouse brain gamma-aminobutyric acid receptors has been assayed under more physiological conditions (never-frozen membranes, 22 degrees) than in previous studies (0-4 degrees on frozen-thawed membranes). Binding affinities were lower and agreed more closely with physiological dose response curves. In addition, heterogeneity in affinity was still present, apparently due to a mixture of non-interconvertible subpopulations and ligand-induced changes in kinetics. Super-high affinity sites (Kd less than 10 nM) observed in frozen membranes were not observed in fresh membranes under equilibrium binding conditions, and high affinity sites (Kd congruent to 25 nM) seen at 0 degree had lower affinity (Kd congruent to 250 nM) at 22 degrees. Despite an apparent best fit single-component Scatchard plot for the latter data, cold ligand displacement and association and dissociation rates demonstrated heterogeneity of affinities. Pentobarbital greatly increased the amount of high affinity sites at the expense of low affinity sites in equilibrium binding at 0 degree and slightly increased affinity at 22 degrees. The kinetics of [3H]muscimol association at 22 degrees (kapp = 1-2/min) were virtually independent of the ligand concentration, suggesting either negative cooperativity or an agonist binding-dependent receptor isomerization. Dissociation triggered by addition of excess cold ligand to membranes equilibrated with [3H]muscimol at different concentrations revealed two off-rates with t1/2 values of under 10 sec and 1-3 min; unexpectedly, the ratio of these two populations did not vary with ligand concentration but was constant at 50:50. Dissociation triggered by infinite dilution gave two off-rates, but the slowest component had a t1/2 of about 20 min; including cold muscimol in the dilution buffer increased the off-rate toward that observed in the excess cold ligand method (t1/2 = 1-2 min). The very slow off-rate was only observed following removal of agonist from a previously occupied receptor and suggests receptor isomerization into a high affinity state; this state is similar to that observed at 0 degree. Pentobarbital also favored the production of the receptor state showing very slow [3H]muscimol dissociation upon infinite dilution, opposing the action of high receptor occupancy with cold muscimol. Thus, [3H]muscimol-binding sites observed at 22 degrees with fresh membranes do not show artificially high affinity and have a Kd of 0.2-0.3 microM, closer to the EC50 for chloride channel activation (approximately 2-3 microM and 0.4 microM with pentobarbital).(ABSTRACT TRUNCATED AT 400 WORDS)

Allosteric Regulation↗
PubMed1986

Barbiturate and benzodiazepine modulation of GABA receptor binding and function.

The inhibitory neurotransmitter gamma-aminobutyric acid (GABA) acts primarily on receptors that increase chloride permeability in postsynaptic neurons. These receptors are defined by sensitivity to the agonist muscimol and the antagonist bicuculline, and are also subject to indirect allosteric inhibition by picrotoxin-like convulsants and enhancement by the clinically important drugs, the benzodiazepines and the barbiturates. All of these drugs modulate GABA-receptor regulated chloride channels at the cellular level assayed by electrophysiological or radioactive ion tracer techniques. Specific receptor sites for GABA, benzodiazepines, picrotoxin/convulsants, and barbiturates can be assayed in vitro by radioactive ligand binding. Mutual chloride-dependent allosteric interactions between the four receptor sites indicate that they are all coupled in the same membrane macromolecular complex. Indirect effects of barbiturates on the other three binding sites define a pharmacologically specific, stereospecific receptor. All of the activities can be solubilized in the mild detergent 3-[(3-cholamidopropyl)-dimethylammonio]propane sulfonate (CHAPS) and co-purify as a single protein complex.

Allosteric Regulation↗
PubMed1986

The actions of avermectin on crayfish nerve and muscle.

Avermectin is a potent anthelmintic, insecticide and acaricide, which is thought to act by opening GABA (gamma-amino butyric acid)-mediated chloride channels. The effects of avermectin on a crayfish nerve cell (stretch receptor neuron) were compared with those on a muscle (dactyl abductor). Concentrations above 10(-7) M caused a conductance increase and a slight reduction in GABA sensitivity in both nerve and muscle. Lower concentrations potentiated the effect of GABA in muscle, but not in the nerve cell.

Animals↗
PubMed1986

[3H]muscimol photolabels the gamma-aminobutyric acid receptor binding site on a peptide subunit distinct from that labeled with benzodiazepines.

Affinity column-purified GABA-benzodiazepine receptor protein from bovine brain was photoaffinity labeled with both [3H]flunitrazepam and [3H]muscimol. Gel electrophoresis in sodium dodecyl sulfate revealed that the benzodiazepine binding site labeled with [3H]flunitrazepam was primarily associated with a major peptide subunit revealed by protein staining with Mr = 52 kiloDaltons, with minor labeling of a second peptide of Mr = 57 kiloDaltons, corresponding to a second major stained band. Covalent incorporation of [3H]muscimol was limited to the 57 kiloDalton band, with no labeling of the 52 kiloDalton peptide, showing that the GABA binding site is carried by a subunit distinct from that carrying the benzodiazepine binding site.

Affinity Labels↗
PubMed1986

Opioid receptor alterations in a genetic model of generalized epilepsy.

Autoradiography was used to examine opioid receptor binding in the Mongolian gerbil, a genetic model of the epilepsies. Coronal brain sections of seizure-resistant (SR) and seizure-sensitive (SS) (both pre- and post-seizure conditions) gerbils were labeled with [3H]dihydromorphine. SS (pre-seizure) gerbils demonstrated overall greater brain opioid binding when compared to SR animals. The periaqueductal gray, substantia nigra and medial geniculate body were specific areas in SS (pre-seizure) gerbils which demonstrated highly significant increases in opioid binding compared to SR animals (% increase vs SR were 98%, 91.3% and 42.9%, respectively). Scatchard analysis demonstrated that the increase in opioid binding was due to an increase in the total number of receptors without a significant change in receptor affinity (i.e. periaqueductal gray area: total number of binding sites was 12.7 (SR) and 18.0 fmol/mg tissue (SS pre-seizure), while Kd values were 4.0 (SR) and 4.0 mM (SS pre-seizure). Opioid binding was also increased in the SS (post-seizure) animals when compared to SR animals, especially in the substantia nigra. However, when compared to SS (pre-seizure) gerbils, there was a general but not significant, decrease in opioid binding in SS post-seizure gerbils. The increased opioid binding in the SS (pre-seizure) gerbil compared to SR gerbils could reflect an up-regulation due to a deficit in endogenous ligand (e.g. a deficit in synthesis or decreased release) which could underlie the seizure diathesis in the gerbil.

Animals↗