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K W Gee

Publications and source records attributed to K W Gee.

At least 55 records · Page 3Linked to original sources
PubMed1990

Heterotropic cooperativity between putative recognition sites for progesterone metabolites and the atypical benzodiazepine Ro 5-4864.

The binding of the cage convulsant t-butylbicyclophosphorothionate (TBPS) and 36Cl- uptake by synaptoneurosomes were used to test the ability of progesterone metabolites to modulate allosterically the Ro 5-4864 (4'-chlorodiazepam) binding site that is functionally coupled to the gamma-aminobutyric acid (GABA)/benzodiazepine receptor complex (GBRC) in rat brain. Dose-dependent enhancement of [35S]TBPS binding by Ro 5-4864 occurs in rat cerebral cortex in the presence of the progesterone metabolites 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) and 5 alpha-pregnan-3 alpha, 20 alpha-diol (pregnanediol). The pregnanediol effect is completely GABA dependent, whereas that of 3 alpha-OH-DHP is not. Conversely, Ro 5-4864 opposed the action of 3 alpha-OH-DHP by increasing the IC50 for 3 alpha-OH-DHP inhibition of [35S]TBPS binding. In cortical synaptoneurosomes, Ro 5-4864 antagonized both 3 alpha-OH-DHP and pregnanediol enhancement of GABA-stimulated 36Cl- uptake. In both binding and functional studies, pregnanediol showed limited efficacy relative to 3 alpha-OH-DHP, as previously reported. These findings provide the initial evidence that the GBRC-linked Ro 5-4864 binding site is allosterically coupled to the putative progesterone metabolite recognition site and confirm the GABA-mimetic properties of 3 alpha-OH-DHP and pregnanediol.

Animals↗
PubMed1989

5 alpha-pregnan-3 alpha,20 alpha-diol behaves like a partial agonist in the modulation of GABA-stimulated chloride ion uptake by synaptoneurosomes.

In rat cortical synaptoneurosomes, the maximum potentiation of GABA-stimulated 36Cl uptake produced by 5 alpha-pregnan-3 alpha,20 alpha-diol (5 alpha-pregnanediol) is significantly less than that elicited by 5 alpha-pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP). This observation suggests that 5 alpha-pregnanediol may be a partial agonist whereas 3 alpha-OH-DHP acts as a full agonist at a common site on or near the GABAA/benzodiazepine receptor-chloride ionophore complex (GBRC). This hypothesis is supported by the finding that 5 alpha-pregnanediol will antagonize in a dose-dependent manner the enhancement of GABA-stimulated 36Cl uptake produced by 3 alpha-OH-DHP under certain conditions. Collectively, these findings support the hypothesis that GBRC-active progesterone metabolites with varying degrees of efficacy exist as reflected by their differential ability to potentiate 36Cl uptake in brain synaptoneurosomes.

Animals↗
PubMed1989

Anticonvulsant profile of the progesterone metabolite 5 alpha-pregnan-3 alpha-ol-20-one.

5 alpha-Pregnan-3 alpha-ol-20-one (3 alpha-OH-DHP) is a naturally occurring metabolite of progesterone that can modulate brain excitability through a specific steroid recognition site on the GABA/benzodiazepine receptor-chloride ionophore complex. The anticonvulsant properties of 3 alpha-OH-DHP were determined using standardized anticonvulsant screening tests in mice. This steroid was found to be effective against metrazol-, (+)-bicuculline- and picrotoxin-induced seizures. The steroid has maximum potency against (+)-bicuculline-induced convulsions and no activity against maximal electroshock and strychnine-induced seizures. These findings support the hypothesis that therapeutically useful anticonvulsant steroids active at the putative steroid recognition site associated with the GABA/benzodiazepine receptor-chloride ionophore complex can be identified.

Animals↗
PubMed1989

Complex interactions between pregnenolone sulfate and the t-butylbicyclophosphorothionate-labeled chloride ionophore in rat brain.

Detailed studies of the interactions between the 'neuroactive' steroid, pregnenolone sulfate (PS), and the gamma-aminobutyric acid (GABA)/benzodiazepine receptor-linked chloride ionophore (GBRC) labeled by [35S]t-butylbicyclophosphorothionate ([35S]TBPS) in the rat brain reveal a site of action allosteric to the TBPS-labeled site as demonstrated by the ability of PS to accelerate TBPS-initiated dissociation of [35S]TBPS. In contrast to previous findings, PS modulates [35S]TBPS binding in a GABA 'agonist'-like fashion with micromolar potencies. The role of PS in the regulation of neuronal excitability through the GBRC is questioned, based upon the observation that brain concentrations of PS are 2-3 orders of magnitude less than that necessary for the modulation of chloride conductance in vitro.

Animals↗
PubMed1989

Effects of chronic delta-9-tetrahydrocannabinol (THC) administration on neurotransmitter concentrations and receptor binding in the rat brain.

THC is the major psychoactive constituent of marijuana and is also known as an hallucinogenic compound. Numerous reports have shown that large doses of THC produce significant alterations in various neurotransmitter systems. The present study was designed to determine whether chronic exposure to THC produces significant alterations in selected neurotransmitter systems (dopamine, serotonin, acetylcholine, GABAergic, benzodiazepine, and opiate) in the rat brain. In Experiment 1, male Sprague-Dawley rats were gavaged with vehicle, 10 or 20 mg THC/kg body weight daily, 5 days/week for 90 days. Animals were killed either 24 hours or two months after the last dose. Brains were dissected into different regions for neurochemical analyses. Two months after the cessation of chronic administration, there was a significant decrease in GABA receptor binding in the hippocampus of animals in the high dose group. However, no other significant changes were found in neurotransmitter receptor binding characteristics in the hippocampus or in neurotransmitter concentrations in the caudate nucleus, hypothalamus or septum after chronic THC administration. In an attempt to replicate the GABA receptor binding changes and also to determine the [35S]TBPS binding in hippocampus, we designed Experiment 2. In this experiment, we dosed the animals by gavage with 0, 5, 10 or 20 mg THC/kg daily, 5 days/week or with 20 mg THC/kg Monday through Thursday and 60 mg/kg on Friday for 90 days. Results from this experiment failed to replicate the dose-dependent effect of THC on GABA receptor binding in hippocampus. Modulation of [35S]TBPS binding by GABA or 3 alpha-OH-DHP or inhibition by cold TBPS in frontal cortex did not show any significant dose-related effects. Results from these experiments suggest that chronic exposure to THC does not produce significant alterations in catecholamine or indoleamine neurotransmitter systems or in opiate or GABA receptor systems in the rat brain.

Animals↗
PubMed1988

Steroid modulation of the GABA/benzodiazepine receptor-linked chloride ionophore.

Recent findings suggest that steroids with sedative-hypnotic properties interact specifically with the gamma-aminobutyric acidA/benzodiazepine receptor-chloride ionophore complex (GBRC). They show positive heterotropic cooperativity by allosterically enhancing the binding of GABA agonists and the clinically useful benzodiazepines (BZs) to their respective recognition sites. These steroids have stringent structural requirements for activity at the GBRC, with the essential requirements for high potency being a 3 alpha-hydroxyl group and a 5 alpha-reduced A-ring. Some of these steroids are naturally occurring metabolites of progesterone and deoxycorticosterone and have nanomolar potencies as potentiators of chloride channel conductance. These 3 alpha-hydroxylated, 5 alpha-reduced steroids do not act through any known sites on the GBRC. Thus, the exact site and mechanism of action remain to be determined. Together with the observation that physiological levels of these metabolites are sufficient to influence the function of the GBRC, the evidence clearly suggests a role for these steroids in the normal regulation of brain excitability by potentiating the postsynaptic effects of gamma-aminobutyric acid (GABA). Pharmacological studies of the GBRC-active steroids show that they possess anxiolytic and anticonvulsant activities. The potential therapeutic application of these steroids in the treatment of mood disorders and catamenial exacerbation of seizures associated with the menstrual cycle is discussed. Collectively, the evidence from the studies of these steroids imply that another mechanism by which the endocrine system influences brain function has been identified. Its characterization will provide important insight into how steroids modulate brain excitability under normal and pathophysiological states.

Animals↗
PubMed1988

Regional distribution of a Ro5 4864 binding site that is functionally coupled to the gamma-aminobutyric acid/benzodiazepine receptor complex in rat brain.

The hypothesis that a novel drug binding site linked to a gamma-aminobutyric acid (GABA)-regulated chloride ionophore mediates the excitatory effects of the atypical benzodiazepine (BZ) Ro5 4864 is further evaluated in the present study. Dose-dependent inhibition of [3H]flunitrazepam to the central BZ receptor in rat cerebral cortex by the cage convulsant t-butylbicyclophosphorotionate (TBPS) is modulated by Ro5 4864 and the isoquinoline PK 11195 in a manner consistent with their reported pro/anticonvulsant effects. The ability of Ro5 4864 to enhance the binding of [35S]TBPS to a GABA-regulated chloride ionophore in rat cortex is unchanged after the irreversible labeling of the central BZ receptor by the photoaffinity label Ro15 4513. Together, these observations further suggest that 1) the effect of Ro5 4864 on [35S]TBPS is not mediated by the central BZ receptor and 2) the Ro5 4864 binding site is allosterically coupled to the GABA/BZ receptor-chloride ionophore complex in rat cerebral cortex. Anatomical localization of Ro5 4864-stimulated [35S]TBPS binding in rat brain by autoradiography reveals a distribution of chloride ionophore-coupled Ro5 4864 sites which is in many instances similar to that of the GABA/BZ receptor-chloride ionophore complex. These studies lend additional support to the postulate that this drug binding site represents an additional locus for the regulation of GABAergic neurotransmission in the central nervous system.

Affinity Labels↗
PubMed1988

Steroid modulation of the chloride ionophore in rat brain: structure-activity requirements, regional dependence and mechanism of action.

Further in vitro studies of steroids active at the gamma-aminobutyric acidA (GABAA) receptor regulated Cl- channel labeled by [35S]-t-butylbicyclophosphorothionate ([35S]TBPS) reveal additional structural requirements necessary for activity. Evaluation of selected steroids for activity against TBPS-induced convulsions show similar requirements for activity. Interestingly, steroids (e.g., 5 alpha-pregnan-3 alpha, 20 alpha-diol) were identified that have high potency but limited efficacy as modulators of [35S]TBPS binding. These characteristics are reminiscent of the clinically useful benzodiazepines (BZs) such as clonazepam. However, interactions between the prototypical anesthetic-barbiturate, sodium pentobarbital, and steroids active at the Cl- channel suggest that they do not share a common site of action as allosteric modulators of [35S]TBPS and BZ receptor binding. The most potent steroid evaluated, 5 alpha-pregnan-3 alpha-ol-20-one, modulates [35S]TBPS binding at low concentrations (IC50 approximately 17 nM) in a regionally dependent manner. All [35S]TBPS binding sites appear to be functionally coupled to a steroid "modulatory site." Because several of the active steroids are metabolites of progesterone, their ability to inhibit the binding of [3H]promegestrone to the cytosolic progestin receptor in rat uterus was evaluated. Those steroids showing potent activity at the GABAA receptor-Cl- ionophore were inactive at the intracellular progestin receptor. Such specificity coupled with their high potency provide additional support for the hypothesis that some of these steroids may be involved in the homeostatic regulation of brain excitability via the GABAA-BZ receptor complex.

Androstane-3,17-diol↗
PubMed1987

GABA-dependent modulation of the Cl- ionophore by steroids in rat brain.

Steroids inhibit the binding of [35S]t-butylbicyclophosphorothionate ([ 35S]TBPS) to the GABAA-benzodiazepine receptor (GBR) linked Cl- ionophore in a GABA dependent manner but not through the GABAA receptor. The most potent steroid evaluated is a naturally occurring metabolite of progesterone, 3 alpha-hydroxy,5 alpha-dihydroprogesterone with an IC50 of approximately 17 nM. Structural requirements necessary for inhibitory activity coincide with those reported for anticonvulsant and anesthetic actions. Coupled with earlier evidence that these steroids do not act directly at the benzodiazepine receptor nor the [35S]TBPS labeled site to modulate the Cl- ionophore, the possibility is proposed that a distinct membrane-bound 'steroid site' coupled to the GBR-Cl- ionophore complex exists.

Animals↗
PubMed1987

Ro15-4513 differentially affects ethanol-induced hypnosis and hypothermia.

Recent reports of in vivo antagonism of the acute anti-conflict, ataxic, and lethal effects of ethanol with the partial inverse agonist imidazobenzodiazepine Ro15-4513 implicate the GABAa/benzodiazepine receptor in mediating several acute effects of ethanol. These data raise the question as to whether all of the acute effects of ethanol can be antagonized by Ro15-4513. The present study addressed this issue by investigating the effect of Ro15-4513 on ethanol-induced loss of righting reflex and hypothermia in mice. We found that Ro15-4513 antagonized the hypnotic, but not the hypothermic, effects of ethanol when tested under identical conditions. These results suggest that the GABAa/benzodiazepine receptor may not be involved in mediating all pharmacological effects of ethanol.

Animals↗
PubMed1987

Phenylquinolines PK 8165 and PK 9084 allosterically modulate [35S]t-butylbicyclophosphorothionate binding to a chloride ionophore in rat brain via a novel Ro5 4864 binding site.

The binding of the cage convulsant, [35S]t-butylbicyclophosphorothionate ([35S]TBPS), to a picrotoxin-sensitive site in rat cerebral cortical homogenates was used to identify and characterize the site of action of the phenylquinolines PK 8165 and PK 9084, the isoquinoline PK 11195 and the atypical benzodiazepine (BZ) Ro5 4864. These agents were found to allosterically modulate the binding of 2 nM [35S]TBPS in a pharmacologically relevant fashion. Evidence is presented to suggest that these compounds share a common site of action as modulators of [35S]TBPS binding. The relative potencies of these compounds in vitro are in the submicromolar to micromolar concentration range and correlate well with the concentrations reported to elicit specific responses in behavioral and electrophysiologic studies. Modulation of [35S]TBPS binding in vitro is affected by micromolar quantities of gamma-aminobutyric acid in a (+)-bicuculline-sensitive fashion and is unaffected by the central BZ receptor "antagonist" Ro15 1788. Collectively, the evidence presented suggests the existence of a novel drug binding site that is functionally coupled to a gamma-aminobutyric acid-A receptor and a [35S]TBPS-labeled chloride ionophore. Moreover, this site is distinct from the central BZ receptor recognized by clonazepam and the high-affinity peripheral BZ binding site labeled by [3H] Ro5 4864. The hypothesis is proposed that the novel "Ro5 4864 site" identified in the present study is a functionally relevant binding site that mediates some of the pharmacologic effects of Ro5 4864, PK 8165, PK 9084 and PK 11195 in the mammalian central nervous system.

Animals↗
PubMed1986

Modulation of the chloride ionophore by benzodiazepine receptor ligands: influence of gamma-aminobutyric acid and ligand efficacy.

t-Butylbicyclophosphorothionate (TBPS) produces dose-dependent enhancement of [3H]propyl beta-carboline-3-carboxylate ([3H]PCC, 40 pM) binding to the benzodiazepine1 (BZ1) receptor subtype in hippocampus. Furthermore, TBPS enhancement of [3H]PCC binding was antagonized by micromolar concentrations of gamma-aminobutyric acid (GABA) in a way reversible by bicuculline. BZ receptor ligands that are "GABA positive" (i.e., enhance GABA neurotransmission) allosterically inhibited [35S]TBPS binding, whereas "GABA-negative" ligands (i.e., inhibit GABA neurotransmission) produced the opposite effect. The efficacy of the ligands as modulators of [35S]TBPS binding was consistent with their reported in vivo pharmacology. The effects of positive and negative ligands on [35S]TBPS binding were modulated by micromolar concentrations of GABA. Examination of the kinetics of [35S]TBPS binding suggested the presence of slowly and rapidly dissociating components. The GABA-positive clonazepam stabilized the rapidly dissociating component of [35S]TBPS binding, whereas methyl beta-carboline-3-carboxylate had a similar effect on the slowly dissociating component. It is speculated that the slowly dissociating component of [35S]TBPS binding is associated with a closed chloride channel, whereas the opposite is proposed for the rapidly dissociating component. The differential effects of GABA-positive versus GABA-negative ligands on [35S]TBPS binding and the modulatory effect of GABA provide further evidence to suggest that [35S]TBPS labels a site near the chloride ionophore linked to the GABA-BZ receptor complex.

Animals↗
PubMed1985

Characterization of [3H][2-D-penicillamine, 5-D-penicillamine]-enkephalin binding to delta opiate receptors in the rat brain and neuroblastoma--glioma hybrid cell line (NG 108-15).

Specific binding properties of the tritium-labeled delta opiate receptor agonist [3H][2-D-penicillamine, 5-D-penicillamine]enkephalin [( 3H][D-Pen2, D-Pen5]enkephalin) were characterized in the rat brain and in a mouse neuroblastoma-rat glioma hybrid cell line (NG 108-15). Saturation isotherms of [3H][D-Pen2, D-Pen5]enkephalin binding to rat brain and NG 108-15 membranes gave apparent Kd values of 1-6 nM. These values are in good agreement with the Kd value obtained from the kinetic studies. The Bmax value in NG 108-15 membranes was 235.3 fmol/mg of protein. An apparent regional distribution of [3H][D-Pen2, D-Pen5]enkephalin binding was observed in the rat brain. A number of enkephalin analogues inhibited [3H][D-Pen2, D-Pen5]enkephalin binding with high affinity (IC50 values of 0.5-5.0 nM) in both NG 108-15 and rat brain membranes. However, putative mu receptor-selective ligands such as morphine, [D-Ala2, MePhe4, Gly5-ol]enkephalin, [MePhe3, D-Pro4]morphiceptin, and naloxone were less effective inhibitors of [3H][D-Pen2, D-Pen5]enkephalin binding in both systems tested. These data suggest that [3H][D-Pen2, D-Pen5]enkephalin is a potent and selective ligand for the delta opioid receptor.

Animals↗
PubMed1985

t-[3H]butylbicycloorthobenzoate: new radioligand probe for the gamma-aminobutyric acid-regulated chloride ionophore.

t-[3H]Butylbicycloorthobenzoate [( 3H]TBOB; 22 Ci/mmol) was prepared by reductive dechlorination of its 4-chlorophenyl analog with tritium gas. This new radioligand binds reversibly to fresh washed rat brain P2 membranes in 500 mM NaCl plus 50 mM sodium-potassium phosphate buffer (pH 7.4) at 25 degrees C, with 80-90% specific relative to total binding, a KD of 61 +/- 15 nM, and a Bmax of 1.6 +/- 0.5 pmol/mg of protein. [3H]TBOB association with its binding site(s) is monophasic, but its dissociation is biphasic. The binding characteristics of [3H]TBOB are essentially identical to those of t-[35S]butylbicyclophosphorothionate [( 35S]TBPS) with respect to pH dependence, stimulation by anions, regional distribution in the brain, and pharmacological profile. Saturation analyses and dissociation studies further indicate that TBOB and TBPS have a common binding site. However, binding of the two radioligands differs in respect to temperature effects. In contrast to [35S]TBPS, which exhibits negligible binding at 0 degrees C, [3H]TBOB binds to rat brain membranes at 0, 25, and 37 degrees C with similar KD values. [3H]TBOB with its long radioactive half-life and temperature-independent KD is a valuable supplement to [35S]TBPS in further biochemical and pharmacological characterization of the gamma-aminobutyric acid receptor-ionophore complex.

Androstanes↗
PubMed1984

Benzodiazepine anticonvulsant action: gamma-aminobutyric acid-dependent modulation of the chloride ionophore.

Brain-specific benzodiazepine receptors are allosterically coupled to chloride ionophore-associated binding sites for sulfur-35-labeled t-butylbicyclophosphorothionate. The specific binding of t-butylbicyclophosphorothionate to fresh unwashed rat cortical membranes is inhibited by nanomolar concentrations of five benzodiazepine agonists but not by the antagonist Ro 15-1788. Their inhibitory potencies in this assay are closely related to their antimetrazol activities. Studies with solubilized receptor-complex preparations establish an absolute requirement for gamma-aminobutyric acid (3 to 10 microM), strongly suggesting that the antagonism of metrazol-induced seizures by the benzodiazepines involves an action on the chloride ionophore mediated through the low affinity gamma-aminobutyric acid receptor.

Animals↗