Search PubMed⌕ Search

Biomedical subjects

R W Olsen

Publications and source records attributed to R W Olsen.

At least 91 records · Page 5Linked to original sources
PubMed1991

The GABAA receptor family in the mammalian brain.

The GABAA-benzodiazepine receptor protein from bovine brain was purified by affinity chromatography and the subunit composition examined by gel electrophoresis in sodium dodecyl sulfate. Protein staining revealed a doublet at 51-53 kDa, a band at 55 kDa, and a broad band at 57-59 kDa. The 51 and 53 kDa bands co-migrated with the alpha 1 and alpha 2 gene products identified by Western blotting with subtype-specific antibodies. These two bands were also photoaffinity labeled by [3H]flunitrazepam, as was a breakdown product at 44 kDa. Partial sequencing of proteolytic fragments of these polypeptides yielded sequences found in all alpha clones, and identified the benzodiazepine binding site within residues 8-297 and probably between 106-297 of alpha 1; the 44 kDa and 31 kDa bands yielded fragments containing alpha 3 sequence. The native alpha 3 polypeptide was identified with subtype-specific antibody at 57 kDa overlapping with the two major bands photolabeled with [3H]muscimol at 55 and 58 kDa. Antisera to a beta-selective peptide recognized four bands at 60, 58, 57 and 55 kDa. Thus, one can identify 6-8 distinct polypeptides with the possibility of another 4-6 in purified GABAA receptor proteins, depending on brain region, consistent with the family of gene products suggested by molecular cloning.

Affinity Labels↗
PubMed1991

Pharmacological and biochemical properties of the gamma-aminobutyric acid-benzodiazepine receptor protein from codfish brain.

The gamma-aminobutyric acidA (GABAA) receptor of codfish brain has been purified to homogeneity and contains a single polypeptide band of 56 kDa molecular mass. Polyacrylamide gel electrophoresis in sodium dodecyl sulfate (SDS-PAGE) of codfish GABA receptor photoaffinity-labeled by both [3H]flunitrazepam ([3H]Flu) and [3H]muscimol showed a single radioactive peak with molecular mass of 56 kDa, in contrast to the multiple subunits found in other vertebrate species. The codfish receptor, purified using benzodiazepine (BZ, Ro 7-1986/1) affinity chromatography, contains an apparent single band both by isoelectric focussing and on a silver-stained SDS gel. The receptor density and affinity constants for [3H]muscimol and [3H]Flu binding are comparable to those in mammalian brain, and the specific activity (greater than 1,000 pmol/mg of protein) is comparable to that of preparations purified from those sources. The pharmacological specificity of the codfish GABA-BZ receptor is generally similar to that of mammalian brain, including GABA-BZ coupling. The BZ binding exhibits homogeneous kinetic properties resembling those of the mammalian BZ2 receptor type, and shows strong GABA enhancement of [3H]Flu binding and weaker pentobarbital potentiation. This is consistent with other observations of an earlier phylogenetic, as well as ontogenetic, emergence in mammals of the BZ2 receptor subtype than the BZ1. Codfish GABA receptor is postulated to be a homo-oligomer in which the conformation of GABA and BZ recognition sites is very similar to that in the mammalian hetero-oligomeric GABAA receptor. The codfish receptor appears to be encoded by an ancestral gene and indicates an early development of BZ-GABA coupling.

Animals↗
PubMed1991

Cyclic AMP-dependent protein kinase decreases gamma-aminobutyric acidA receptor-mediated 36Cl- uptake by brain microsacs.

The effect of cyclic AMP (cAMP)-dependent protein phosphorylation on gamma-aminobutyric acidA (GABAA) receptor function was examined using isolated brain membrane vesicles (microsacs). Muscimol-stimulated 36Cl- uptake was studied in mouse brain microsacs permeabilized to introduce the catalytic subunit of cAMP-dependent protein kinase (PKA). At both submaximal and maximally effective concentrations of muscimol, PKA inhibited muscimol-stimulated 36Cl- uptake by approximately 25%. In parallel experiments, PKA and [gamma-32P]ATP were introduced into the microsacs, and we attempted to immunoprecipitate the entire GABAA receptor complex, under nondenaturing conditions, using an anti-alpha 1-subunit antibody. Data from such experiments show that PKA increases the phosphorylation of several microsac proteins, including a 66-kDa polypeptide specifically immunoprecipitated with the GABAA receptor anti-alpha 1 subunit antibody. Phosphopeptide mapping of the 66-kDa polypeptide demonstrated a 14-kDa fragment similar to that obtained with the purified, PKA-phosphorylated GABAA receptor. These results provide evidence that the catalytic subunit of PKA inhibits the function of brain GABAA receptors and demonstrate that this functional change is concomitant with an increase in protein phosphorylation.

Animals↗
PubMed1991

Ethanol has no effect on cAMP-dependent protein kinase-, protein kinase C-, or Ca(2+)-calmodulin-dependent protein kinase II-stimulated phosphorylation of highly purified substrates in vitro.

The actions of ethanol on kinase stimulated phosphorylation were examined using highly purified protein kinases and a variety of purified substrates. Ethanol (25-200 mM) failed to alter the phosphorylation of histone IIa and histone IIIs by cAMP-dependent protein kinase (PKA) and protein kinase C (PKC), respectively. Moreover, ethanol (25-200 mM) did not affect the phosphorylation of synapsin I by Ca(2+)-calmodulin-dependent protein kinase II (CAM kinase II). Finally, neither PKA nor PKC stimulated phosphorylation of the GABAA receptor (GABAA-R) was modulated by ethanol at any concentration of ethanol tested. These results suggest that ethanol, in pharmacological concentrations, has no direct actions on the ability of these kinases to catalyze the phosphorylation of specific substrate proteins. In particular, ethanol does not appear to directly influence GABAA-R phosphorylation by either PKA or PKC.

Animals↗
PubMed1991

The benzodiazepine/alcohol antagonist Ro 15-4513: binding to a GABAA receptor subtype that is insensitive to diazepam.

The imidazobenzodiazepinone Ro 15-4513 has been shown previously to bind to central benzodiazepine receptors as well as to a second, uncharacterized class of sites that do not bind diazepam, differentiating them from the normal benzodiazepine-binding site on the gamma-aminobutyric acid (GABA)-A (GABAA) receptor. This study describes the characterization of these unique diazepam-insensitive (DZ-IS) sites. Ro 15-4513 binding to DZ-IS sites was abundant in cerebellum from cow, rat and human and detectable in cortex, hippocampus and striatum by autoradiography on rat brain sections. These sites represented approximately 20% of the total binding in bovine cerebellar membranes, but only 2 to 3% of the total in cortex. Ro 15-4513 binds with the same affinity (Kd approximately 4.5 nM) to both diazepam-sensitive and DZ-IS sites in the cerebellum. A number of compounds which bind to the classical benzodiazepine receptors also bind to the DZ-IS sites. These compounds include: the pyrazoloquinoline CGS 8216, the beta-carbolines methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate, ZK 95962, ZK 94326 and ZK 93126, as well as the classical benzodiazepine receptor antagonist, Ro 15-1788. Besides binding diazepam poorly, the DZ-IS sites demonstrate a very low affinity for other benzodiazepines. Ligands which bind to the various drug receptor sites on the GABA receptor complex do not directly modulate the binding of Ro 15-4513 to DZ-IS sites nor does ethanol. However, antagonism of Ro 15-4513 binding to the DZ-IS sites by methyl-6,7-dimethoxy-4-ethyl-beta-carboline-3-carboxylate and by CGS 8216 is modulated by the presence of GABA.(ABSTRACT TRUNCATED AT 250 WORDS)

Allosteric Regulation↗
PubMed1990

Protein kinase C and cAMP-dependent protein kinase phosphorylate the beta subunit of the purified gamma-aminobutyric acid A receptor.

A number of recent studies have suggested that phosphorylation of the gamma-aminobutyric acid A (GABAA) receptor could modulate receptor function. Activators of protein kinase C and cAMP-dependent protein kinase have been shown to influence GABAA receptor function. In addition, Sweetnam et al. [Sweetnam, P. M., Lloyd, J., Gallombardo, P., Malison, R. T., Gallager, D. W., Tallman, J. F. & Nestler, E. J. (1988) J. Neurochem. 51, 1274-1284] have reported that a kinase associated with a partially purified preparation of the receptor could phosphorylate the alpha subunit of the receptor. Moreover, Kirkness et al. [Kirkness, E. F., Bovenkerk, C. F., Ueda, T. & Turner, A. J. (1989) Biochem. J. 259, 613-616] have recently shown that cAMP-dependent protein kinase could phosphorylate a muscimol binding polypeptide of the GABAA receptor. To explore the issue further, we have examined the ability of specific kinases to catalyze significant phosphorylation of the GABAA receptor that has been purified to near homogeneity. The GABAA receptor was purified as previously described using benzodiazepine affinity chromatography. The purified receptor possessed no detectable kinase activity. Protein kinase C and cAMP-dependent protein kinase catalyzed the phosphorylation of the beta and alpha subunits of the receptor. However, most of the phosphate incorporation was associated with the beta subunit. Two muscimol binding polypeptides designated beta 58 (Mr 58,000) and beta 56 (Mr 56,000) were present in the preparation. The higher molecular weight polypeptide, beta 58, was phosphorylated specifically by cAMP-dependent protein kinase. beta 56 was phosphorylated specifically by protein kinase C. beta 58 and beta 56 gave distinct patterns in a one-dimensional phosphopeptide analysis. The stoichiometry of phosphorylation (mol of phosphate/mol of muscimol binding) catalyzed by cAMP-dependent protein kinase was 0.52 and that catalyzed by protein kinase C was 0.38. Taken together these data confirm that there are two forms of the beta subunit of the GABAA receptor and suggest that these two forms of the beta subunit are phosphorylated by distinct kinases.

Animals↗
PubMed1990

Molecular biology of GABAA receptors.

The major type of receptor for the inhibitory neurotransmitter gamma-aminobutyric acid (GABA), called the GABAA receptor, is a member of a gene superfamily of ligand-gated ion channels. This receptor is a hetero-oligomeric protein composed of several distinct polypeptide types (alpha, beta, gamma, and delta). Molecular cloning of these polypeptides reveals that they show 20-40% identity with each other, and 10-20% identity with polypeptides of the nicotinic acetylcholine receptors and strychnine-sensitive glycine receptor. Each polypeptide type is also represented by a family of genes whose members have 60-80% amino acid sequence identity. Regions of conserved and variable amino acid sequence suggest structural and functional domains within each polypeptide. All of the polypeptides when expressed in heterologous cells produce GABA-activated chloride channels, and the different subtypes express different pharmacological properties. The distributions of mRNAs for the different GABAA receptor polypeptides and their subtypes show significant brain regional variation consistent with pharmacological and biochemical evidence for receptor heterogeneity. Subpopulations of GABAA receptors with different cellular and regional locations show differential sensitivity to GABA, to modulators like steroids, to physiological regulation, to disease processes, and to pharmacological manipulation by drugs such as benzodiazepines. The properties of the different subpopulations of GABAA receptors are determined by which one or more of the different polypeptides and their subtypes are expressed in a given cell to produce a variety of different oligomeric protein structures. Molecular cloning techniques have produced rapid advances in understanding the GABAA receptor protein family.

Amino Acid Sequence↗
PubMed1990

Multiple distinct subunits of the gamma-aminobutyric acid-A receptor protein show different ligand-binding affinities.

The purified gamma-aminobutyric acid/benzodiazepine receptor protein from mammalian brain contains at least four discrete polypeptides (Mr 51,000, 53,000, 55,000 and 58,000) by a variety of visualization techniques and in three species (rat, cow, and human). These polypeptide bands vary in their affinity for gamma-aminobutyric acid analogs as shown by inhibition of [3H]muscimol binding, demonstrated by photoaffinity labeling and gel electrophoresis in sodium dodecyl sulfate. One-dimensional peptide maps of proteolytic digests revealed that distinct fragments were produced, indicating that the four polypeptides represent discrete sequences. The four bands were identified by Western blotting with subunit-specific monoclonal antibodies as two species each of previously identified alpha and beta subunits. [3H]Muscimol photolabeled all four bands (beta and alpha) to varying degrees not proportional to the extent of protein staining. The Mr 58,000 beta subunit subtype showed a higher affinity for 4,5,6,7-tetrahydro-isoxazolo-[5,4-c]pyridin-3-ol than the Mr 56,000 beta subtype, whereas the Mr 56,000 beta and Mr 51,000 alpha bands were more enhanced by pentobarbital than the Mr 58,000 band. Furthermore, the alpha subunit pattern revealed by photoaffinity labeling with [3H]flunitrazepam was significantly different for three regions of bovine brain, showing only one major band in cerebellum at Mr 51,000, two major bands in cortex at Mr 53,000 and 51,000, and three bands in hippocampus at Mr 55,000 as well as Mr 53,000 and 51,000. Because the ratio of the amounts of the various polypeptides varies with brain region and the pharmacological properties of the peptides vary, it is likely that a family of oligomeric gamma-aminobutyric acid/benzodiazepine receptors exists in the brain. This is consistent with the reported variable expression of different subunit subtype mRNAs and with brain region-dependent variation in pharmacology and binding behavior.

Affinity Labels↗
PubMed1989

MPTP is proconvulsant acutely but has no long-term effect in rodent models of seizure and epilepsy.

1-Methyl-4-phenyl-1,2,5,6-tetrahydropyridine (MPTP) was used to lesion the substantia nigra of rodents to look for changes in various animal models of epilepsy and seizures. MPTP, acutely administered to C57BL/6J mice, could cause seizures at high doses and enhanced maximal electroshock seizures at lower doses. Older mice were more sensitive to MPTP toxicity. MPTP given over 1 week to produce a 75% drop of striatal dopamine had no effect on seizure thresholds to pentylenetetrazol or picrotoxin and did not change the maximal electroshock seizure. Epileptic gerbils given maximally tolerated doses of MPTP had only a slight striatal dopamine reduction (32%) while seizures remained unaltered. The data are consistent with the hypothesis that chronic dysfunction of dopamine containing substantia nigra neurons have no significant influence on seizures in these animal models.

Animals↗
PubMed1989

Anticonvulsant effect of muscimol injected into the thalamus of spontaneously epileptic Mongolian gerbils.

Injections of muscimol (12.5 or 25 ng bilaterally), a GABAA agonist, into the posterior nuclei of the thalamus suppressed generalized convulsive seizures in the spontaneously epileptic Mongolian gerbil. This anticonvulsant effect was dose-dependent and was reversed by picrotoxin (10 ng bilaterally), a GABAA antagonist. Bilateral intrathalamic injections of 1-baclofen (50 ng), an agonist of the GABAB receptor, were ineffective in suppressing seizures in this model. These results suggest that GABAergic transmission within the thalamus is involved in the control or the genesis of some generalized convulsive seizures.

Animals↗
PubMed1989

A novel alpha subunit in rat brain GABAA receptors.

Two cDNAs (alpha 1 and alpha 4) from rat brain cDNA libraries encode isoforms of the alpha subunit of the GABA/benzodiazepine receptor, which differ at 30% of their amino acid residues. Northern blot analysis and in situ hybridization histochemistry show that alpha 1 and alpha 4 mRNAs have distinct sizes and distinct regional and cellular distributions in rat brain: both mRNAs are found in the cortex and hippocampus; however, only the alpha 1 mRNA is detected in the cerebellum. We injected RNA transcribed from alpha 1 and alpha 4 cDNAs into Xenopus oocytes, together with an RNA for a rat beta subunit. We obtained GABA-dependent inward currents that were reversibly blocked by picrotoxin. Picrotoxin alone, applied to oocytes producing the alpha and beta polypeptides, elicited an outward current. We suggest that these polypeptides together produce GABA-gated ion channels that can also open spontaneously.

Amino Acid Sequence↗
PubMed1989

t-[35S]butylbicyclophosphorothionate binding sites in invertebrate tissues.

Specific high affinity binding of the cage convulsant t-[35S]butylbicyclophosphorothionate (TBPS) was observed in membrane homogenates of housefly heads and crayfish abdominal muscles. [35S]TBPS binding in these two invertebrate tissues was inhibited by biologically active cage convulsants, picrotoxin analogs, and barbiturates. The housefly binding sites were inhibited most potently by several insecticides. Approximately 50% of total binding was displaceable by excess (0.1 mM) nonradioactive TBPS, picrotoxinin, ethyl bicyclophosphate, or dieldrin. Optimal binding assay conditions for housefly homogenates included pH 7.5, 22 degrees C temperature, 0.3 M chloride concentration, and incubation for 60 min; for crayfish homogenates, 4 degrees C temperature and 150-min incubations were optimal. Scatchard plots of equilibrium binding indicated one site in both tissues (KD = 50 nM, Bmax = 250 fmol/mg protein in housefly; KD = 25 nM, Bmax = 100 fmol/mg protein in crayfish). Association kinetics in housefly were consistent with one rate constant (k+1 = 8 X 10(6) M-1 min-1), but dissociation was described better by two rate constants (k-1 = 0.28 min-1 and 0.042 min-1; calculated KD values of 80 nM and 12 nM). Displacement by cage convulsants showed Hill numbers near 0.5, also consistent with two populations of affinity, while displacement by other drugs showed Hill numbers near 1.0. [35S]TBPS binding in insects was most potently inhibited by the insecticides dieldrin (IC50 = 50 nM), aldrin, and lindane (200 nM), in a stereospecific manner, consistent with this binding site being the receptor for biological toxicity. [35S]TBPS binding was also inhibited by relatively high concentrations of some pyrethroid insecticides, such as deltamethrin and cypermethrin (1-2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1989

Steroid anesthetics and naturally occurring analogs modulate the gamma-aminobutyric acid receptor complex at a site distinct from barbiturates.

The steroid anesthetic alphaxalone and a series of naturally occurring analogs were compared in potency and efficacy with each other and the hypnotic barbiturate pentobarbital for interaction with gamma-aminobutyric acid (GABA) receptors:binding sites in rat brain membranes and functional activity in 36Cl- flux measurements with rat hippocampal slices. The steroids enhanced [3H]muscimol binding to GABA receptor sites, enhanced [3H] flunitrazepam binding to benzodiazepine receptors and inhibited [35S]t-butyl bicyclophosphorothionate binding to picrotoxin/convulsant binding sites on the GABA receptor-chloride channel complex. The same steroids that were active in modulating the binding of ligands to the various receptor sites on the GABA receptor complex at micromolar concentrations enhanced muscimol-stimulated 36Cl- flux in rat hippocampal slices. The steroids, like the barbiturates, increased the maximal response to muscimol but produced little or no potentiation of basal 36Cl- flux in the absence of GABA agonist. Although the effects of steroids and barbiturates were similar, separate sites of action were demonstrated conclusively by the observation that the two classes of compounds, when included together, gave additive or synergistic effects on binding, as well as on 36Cl- flux in the absence of GABA agonist. Structure-activity studies showed that the synthetic steroid anesthetic alphaxalone was the most potent compound tested, followed by the naturally occurring steroids tetrahydro-deoxycorticosterone, allo-tetrahydrocorticosterone, cis-androsterone and 5 alpha-androstan-17 beta-ol-3-one. The ability of several naturally occurring steroids to enhance GABA-mediated inhibition in the brain suggests the possibility of an endogenous steroid modulator of neuronal function.

Anesthetics↗
PubMed1988

gamma-Aminobutyric acid/benzodiazepine receptor protein carries binding sites for both ligands on both two major peptide subunits.

Affinity column-purified GABA-benzodiazepine receptor proteins from human, cow, and rat brain were photoaffinity labeled with both [3H]flunitrazepam and [3H]muscimol and examined by gel electrophoresis in sodium dodecyl sulfate. Using high receptor protein concentrations (1 microM), the benzodiazepine ligand [3H]flunitrazepam was incorporated covalently primarily into the expected 52 kiloDalton major subunit but also significantly into a second 57 kiloDalton peptide. Likewise the GABA ligand [3H]muscimol photolabeled primarily the 57 kiloDalton peptide but also to some extent the 52 kiloDalton peptide. This cross-labeling suggests strongly that both major subunits carry binding sites for both GABA and benzodiazepine.

Affinity Labels↗