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Biomedical subjects

R W Olsen

Publications and source records attributed to R W Olsen.

At least 145 records · Page 8Linked to original sources
PubMed1982

Physicochemical characterization of detergent-solubilized gamma-aminobutyric acid and benzodiazepine receptor proteins from bovine brain.

[3H]Muscimol and [3H]flunitrazepam binding activities have been solubilized from bovine cortex using the ionic detergent sodium deoxycholate. The soluble receptor proteins were shown to bind [3H]muscimol with a dissociation constant, Kd, of 12 nM and a binding capacity (Bmax value) of 1.56 pmol/mg protein; gamma-amino[3H]-butyric acid with a Kd of 50 nM and Bmax of 1.55 pmol/mg protein; and [3H]flunitrazepam with a Kd of 8 nM and a Bmax of 0.8 pmol/mg protein. Gel filtration of the soluble receptor proteins showed that the gamma-amino[3H]butyric acid and [3H]flunitrazepam binding activities comigrated with a Stokes radius of 6.8 nm. The two binding activities were also found to comigrate after sedimentation in a sucrose density gradient. The hydrodynamic properties of the assumed protein-detergent complexes were determined by gel filtration and sedimentation through gradients of sucrose in H2O or 2H2O. Under the conditions employed, the parameters for both the putative gamma-aminobutyric acid and benzodiazepine receptors were: partial specific volume, 0.73 ml g-1; sedimentation coefficient, 12.5 S; molecular weight, 355000; and frictional ratio 1.46. These observations are consistent with the conclusion that the majority of both binding activities solubilized in deoxycholate reside in a single macromolecular complex. However, Triton X-100 selectively solubilized the benzodiazepine binding activity. This suggests that the two binding activities can be at least partially separated.

Animals↗
PubMed1982

Solubilization by CHAPS detergent of barbiturate-enhanced benzodiazepine-GABA receptor complex.

Barbiturates enhance the binding of [3H]flunitrazepam to benzodiazepine receptors solubilized with the detergent 3-[(3-cholamidopropyl)-dimethylammonio]propanesulfonate (CHAPS) from bovine cortex. The enhancement by the barbiturates is seen as a decrease in the dissociation constant, KD, for specific benzodiazepine binding, with no effect on the number of binding sites. The effect of the barbiturates is facilitated by chloride ions, is concentration-dependent, and has a specificity that correlates well with the anesthetic potency of barbiturates. [3H]Flunitrazepam binding activity is stable with storage at 4 degrees C, but barbiturate enhancement of soluble benzodiazepine binding activity decayed rapidly (t 1/2 = 48 h). [3H]Muscimol binding (GABA receptor) activity was also enhanced by barbiturates. Agarose gel filtration column chromatography of the CHAPS-solubilized receptor proteins showed the same elution profile as receptors solubilized with sodium deoxycholate, and enhancement by barbiturates was observed for both the benzodiazepine and GABA binding activities.

Animals↗
PubMed1982

gamma-Aminobutyric acid receptors in normal human brain and Huntington disease.

GABA receptor binding curves were measured in well-washed membrane homogenates from eleven regions of normal human brain and four regions from brains of Huntington disease patients. Computer analysis suggested two populations of receptor sites of different affinity (KD = 10 nM and 240 nM) present in all brain regions but in variable quantity (cerebellar cortex and cerebral cortex greater than basal ganglia greater than deeper brain structures). The number of GABA receptor sites in the caudate-putamen region of Huntington brain was less than normal, but the number of sites was increased in Huntington disease substantia nigra. No differences from normal were found in cerebellar cortex or frontal cortex from Huntington disease, and no significant changes in binding affinity were observed for any of the four regions tested under the conditions used.

Brain↗
PubMed1981

The GABA postsynaptic membrane receptor-ionophore complex. Site of action of convulsant and anticonvulsant drugs.

The function of the inhibitory neurotransmitter, gamma-aminobutyric acid (GABA), has been implicated in the mode of action of many drugs which excite or depress the central nervous system. Many convulsant agents appear to block GABA action whereas anticonvulsants enhance GABA action. Some of these drug effects involve altered GABA-mediated synaptic transmission at the level of GABA biosynthesis, release from nerve endings, uptake into cells, and metabolic degradation. A greater number of agents of diverse classes appear to affect GABA action at the postsynaptic membrane, as determined from both electrophysiological and biochemical studies. The recently developed in vitro radioactive receptor binding assays have led to a wealth of new information about GABA action and its alteration by drugs. GABA inhibitory transmission involves the regulation, by GABA binding to its receptor site, of chloride ion channels. In this GABA receptor-ionophore system, other drug receptor sites, one for benzodiazepines and one for barbiturates/picrotoxinin (and related agents) appear to form a multicomponent complex. In this complex, the drugs binding to any of the three receptor categories are visualized to have an effect on GABA-associated chloride channel regulation. Available evidence suggests that the complex mediates many of the actions of numerous excitatory and depressant drugs showing a variety of pharmacological effects.

Anticonvulsants↗
PubMed1981

Dihydropicrotoxinin binding sites in mammalian brain: interaction with convulsant and depressant benzodiazepines.

The specific binding of [3H] alpha-dihydropicrotoxinin to rat brain membranes was inhibited competitively and potently (IC50 congruent to 100 nM) by a convulsant benzodiazepine drug, RO5-3663. This compound did not inhibit high affinity flunitrazepam binding to the same tissue under similar conditions, and its reported pharmacological activity as an antagonist of GABAergic synaptic transmission, which resembles that of picrotoxinin, appears to involve the picrotoxinin binding sites. Other benzodiazepines such as diazepam, in micromolar concentrations, inhibited picrotoxinin binding in a stereospecific and chemically specific manner. However, the order of potency of a series of depressant benzodiazepines did not correlate well with pharmacological activities nor with reported activities for displacement of high affinity benzodiazepine 'receptor' binding sites (although heterogeneity of both picrotoxinin and benzodiazepine binding site populations may make difficult such comparisons). A comparison of benzodiazepine-displaceable benzodiazepine binding and benzodiazepine-displaceable picrotoxinin binding for different brain regions and subcellular fractions revealed a very similar though not identical distribution of these two classes of drug receptor, again suggesting that the two are not identical. Both classes of drug binding site also showed a very similar distribution to sodium-independent GABA receptor binding sites, which is consistent with other evidence that at least part of these 3 receptor types may be found at least sometimes coupled together in the postsynaptic membrane GABA receptor-ionophore complex.

Animals↗
PubMed1981

Interaction of anticonvulsants with the barbiturate-benzodiazepine-GABA receptor complex.

Unlike the anesthetic barbiturate pentobarbital and the anxiolytic pyrazolopyridine etazolate, which enhance [3H]diazepam binding to rat brain membranes, the anticonvulsant barbiturates phenobarbital and metharbital, and also chlormethiazole, at therapeutic concentrations (10-1000 muM), do not stimulate [3H]diazepam binding, but instead block the enhancement by both pentobarbital and etazolate. The same anticonvulsants at similar concentrations inhibit [3H]alpha-dihydropicrotoxinin (DHP) binding suggesting that these anticonvulsants compete for the same receptor sites as pentobarbital and etazolate, designated the barbiturate-picrotoxinin receptor component of the benzodiazepine-GABA receptor complex.

Animals↗
PubMed1981

Characterization of the binding of the GABA agonist [3H]piperidine-4-sulphonic acid to bovine brain synaptic membranes.

The binding of radioactive piperidine-4-sulphonic acid ([3H]P4S) to thoroughly washed, frozen, and thawed membranes isolated from cow and rat brains has been studied. Quantitative computer analysis of the binding curves for four regions of bovine brain revealed the general presence of two binding sites. In these brain regions less satisfactory computer fits were obtained for receptor models showing one or three binding sites or negative cooperativity. With the use of Tris-citrate buffer at 0 degree C the two affinity classes for P4S in bovine cortex membranes revealed the following binding parameters: KD = 17 +/- 7 nM (Bmax = 0.15 +/- 0.07 pmol/mg protein) and KD = 237 +/- 100 nM (Bmax = 0.80 +/- 0.20 pmol/mg protein). Heterogeneity was also observed for association and dissociation rates of [3H]P4S. The slow binding component (kon = 5.6 X 10(7) or 8.8 X 10(7) M-1 min-1, koff = 0.83 min-1, and KD = 14.7 or 9.4 nM, determined by two different methods in phosphate buffer containing postassium chloride) corresponds to the high-affinity component of the equilibrium binding curve (KD = 11 nM, Bmax = 0.12 pmol/mg protein in the same buffer system). The association and dissociation rates for the subpopulation of rapidly of dissociating sites, apparently corresponding to the low-affinity sites, were too rapid to be measured accurately. The binding of [3H]P4S appears to involve the same two populations of sites with Bmax values similar to those for [3H]GABA binding to the same tissue, although the kinetic parameters for the two ligands are somewhat different. Furthermore, comparative studies on the inhibition of [3H]P4S and [3H]GABA binding by various GABA analogues, strongly suggest that P4S binds to the GABA receptors. The different effects of P4S and GABA on benzodiazepine binding are discussed.

Animals↗
PubMed1980

Endogenous inhibitors of picrotoxinin-convulsant binding sites in rat brain.

The binding of [3H]-alpha-dihydropicrotoxinin (DHP) to rat brain membranes was inhibited by aqueous extracts of the same tissue, as well as by several naturally occurring substances such as pyrimidines, purines, and their metabolites. The 200 000 X g supernatant fraction of cortex homogenates inhibited DHP binding competitively, reversibly, without preincubation, at 0 degrees C, with 40 microgram protein inhibiting 50%. Compounds such as adenine, hypoxanthine, inosine, cytosine, allantoin and uric acid at about 0.1 mM also inhibited DHP binding competitively, suggesting a relationship between DHP binding sites, benzodiazepine binding sites, and GABA receptor binding sites.

Animals↗