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Biomedical subjects

A M Allan

Publications and source records attributed to A M Allan.

At least 37 records · Page 2Linked to original sources
PubMed1991

gamma-Aminobutyric acid-activated chloride channels in rats selectively bred for differential acute sensitivity to alcohol.

Effects of various sedative hypnotic agents on GABA-mediated chloride flux were evaluated in whole brain membrane vesicles (microsacs) prepared from rats selectively bred for high (HAS) and low sensitivity (LAS) to an acute hypnotic dose of alcohol. The HAS rats were more sensitive to the effects of pentobarbital, phenobarbital, flunitrazepam, and ethanol on GABA-mediated chloride flux compared with the LAS rats. No differences between the lines in GABA-stimulated chloride flux were observed. Modulation of 1-[3H]-phenyl-4-butyl-2,6,7-trioxabicyclo(2.2.2)octane ([3H]-TBOB) and [3H]-diazepam binding also was measured. The lines did not differ in inhibition of [3H]-TBOB binding by pentobarbital, phenobarbital, muscimol or picrotoxin. Although the lines displayed almost identical KD and Bmax for [3H]-diazepam binding, the GABA agonist, muscimol, was a more potent stimulator of [3H]-diazepam binding in membranes prepared from HAS rats than from LAS rats. These findings are discussed in light of previous work using other selected lines.

Alcohol Drinking↗
PubMed1991

Ethanol-induced changes in chloride flux are mediated by both GABA(A) and GABA(B) receptors.

Low concentrations of ethanol (10-30 mM) in the presence of a GABAB receptor agonist, baclofen, promoted 36Cl- uptake into membrane vesicles (microsacs) prepared from mouse cortex. Neither ethanol nor baclofen alone altered chloride influx. The GABAB antagonists, phaclofen and 2-hydroxy-saclofen, completely blocked the increase in chloride flux produced by ethanol in the presence of either baclofen or GABA. Ethanol increased the chloride conductance produced by the GABAA agonists muscimol, isoguvacine, imidazolacetic acid and amino-propane sulfonic acid and this action of ethanol was blocked by phaclofen. The specific GABAA antagonist, bicuculline, blocked ethanol-induced increase in chloride flux in the presence of either baclofen or GABA. GABA-activated chloride channels were also studied in Xenopus oocytes expressing mouse brain mRNA. In this preparation, GABA action was enhanced by ethanol, pentobarbital, and diazepam, and 2-hydroxy-saclofen partially antagonized the action of ethanol without altering the effects of pentobarbital or diazepam. These results suggest that ethanol enhancement of GABAA receptor-chloride channel function also requires activation of GABAB receptors.

Alcohol Drinking↗
PubMed1989

Genetic differences in coupling of benzodiazepine receptors to chloride channels.

The affinity and density of [3H]flunitrazepam binding sites and the ability of flunitrazepam to augment GABA-activated chloride flux were determined using membrane vesicles prepared from cortex of long-sleep (LS, alcohol-sensitive), short-sleep (SS, alcohol-resistant) and individual heterogeneous stock (HS) mice. LS membranes were more sensitive than SS membranes to the effects of flunitrazepam (and ethanol) on chloride flux but did not differ in the binding of [3H]flunitrazepam. Likewise, individual HS mice displayed marked differences in enhancement of chloride flux by flunitrazepam, but little difference in binding of flunitrazepam. These results suggest that there is little genetic variation in the binding of benzodiazepines to receptors, but there is a marked diversity in the coupling of these receptors to chloride channels. This implies the existence of 'spare receptors' in LS and most HS mice, but not in SS mice. These findings also suggest that genetic differences in alcohol sensitivity in vivo may be related to coupling of benzodiazepine binding to chloride channel function.

Animals↗
PubMed1989

Fluidization of brain membranes by A2C does not produce anesthesia and does not augment muscimol-stimulated 36Cl- influx.

Intravenous administration of 2-[2-methoxyethoxy]-ethyl 8-[cis-2-n-octylcyclopropyl]-octanoate (A2C) was found to disorder brain membranes but did not produce intoxication or anesthesia in mice. The abilities of A2C and an anesthetic (benzyl alcohol) to inhibit [35S]t-butylbicyclophosphorothionate (TBPS) binding, and modify gamma-aminobutyric acid (GABA) receptor-mediated 36Cl- influx into brain vesicles were then compared. Both of the perturbants inhibited [35S]TBPS binding at the same concentrations at which they reduced membrane order; however, the anesthetic was nearly 4 times more effective in reducing [35S]TBPS binding than was A2C. Muscimol-stimulated 36Cl- uptake was enhanced by benzyl alcohol at a concentration which produced little or no change in membrane order. Concentrations of both A2C and benzyl alcohol which reduced membrane order inhibited muscimol-stimulated 36Cl- influx. Similarly, membrane order and muscimol-activated 36Cl- uptake were reduced in brain vesicles prepared from mice which had received A2C in vivo. The effects of anesthetics on the GABAA receptor-chloride channel complex were analyzed by a two site model of action in which a 'perturbant' site is responsible for decreased 36Cl- uptake; but a distinct 'anesthetic' site is responsible for augmentation of chloride flux and anesthesia.

Anesthesia↗
PubMed1989

A new alcohol antagonist: phaclofen.

The ability of the GABA(B) receptor antagonist, phaclofen to alter behavioral effects of ethanol was evaluated by loss of righting reflex (sleep time), motor incoordination (bar holding), spontaneous locomotion (open field activity) and hypothermia. Pretreatment with phaclofen significantly decreased the effects of ethanol on motor incoordination, locomotor activity and hypothermia. However, phaclofen had no effect on either pentobarbitalor diazepam-induced motor incoordination. Phaclofen slightly increased the ED50 for loss of the righting reflex but did not alter either the duration of reflex loss produced by ethanol or blood ethanol levels at awakening. Our results suggest phaclofen is rapidly inactivated resulting in difficulty in observing antagonism of long duration ethanol effects. These findings suggest that the GABA(B) system may play a role in mediating several important actions of ethanol.

Animals↗
PubMed1989

Alcohol intoxication: ion channels and genetics.

Acute in vitro exposure to ethanol and other intoxicant-anesthetics activates gamma-aminobutyric acid (GABA)-stimulated chloride channels and inhibits voltage-dependent calcium and sodium channels of isolated brain membranes. The question of whether these neurochemical actions are responsible for intoxication in vivo has been addressed using animal populations displaying genetic differences in sensitivity to alcohol and benzodiazepine intoxication. These genetic approaches include inbred strains, selected lines, recombinant inbred strains, and heterogeneous stocks. Genetic differences in ion channel function provide strong evidence for a role of the GABA-stimulated chloride channel in ethanol and benzodiazepine intoxication; the role of calcium and sodium channels is less clear.

Alcoholic Intoxication↗
PubMed1989

Sensitivity to ethanol hypnosis and modulation of chloride channels does not cosegregate with pentobarbital sensitivity in HS mice.

Several findings suggest that barbiturates and alcohol produce their sedative effects through a common neural and possibly a common genetic mechanism. We tested this hypothesis by examining the correlation between ethanol and pentobarbital sedative effects in individual animals from a genetically heterogeneous population. The duration of pentobarbital-induced hypnosis (sleep-time) was unrelated to the sleep-time produced by ethanol in heterogeneous stock (HS) mice. Therefore, the present study also examined the effect of ethanol, pentobarbital, and flunitrazepam on muscimol-stimulated chloride flux into brain membranes prepared from HS mice selected for differences in pentobarbital- and ethanol-induced sleep-time. Brain membranes from mice selected for differences in ethanol sleep-time were differentially responsive to ethanol- and flunitrazepam-, but not to pentobarbital-induced augmentation of muscimol-stimulated chloride flux. No differences in augmentation of chloride flux by ethanol, pentobarbital, or flunitrazepam were found in membranes prepared from mice differentially sensitive to pentobarbital hypnosis. The ability of muscimol to stimulate chloride uptake was not related to ethanol or pentobarbital sensitivity. These findings suggest that sensitivity to ethanol is not likely to be genetically linked to pentobarbital sensitivity.

Animals↗
PubMed1988

Genetic selection for benzodiazepine ataxia produces functional changes in the gamma-aminobutyric acid receptor chloride channel complex.

The gamma-aminobutyric acid (GABA) receptor-operated chloride channel complex was evaluated in mice selected for differential sensitivity to the ataxic effects of diazepam (diazepam-sensitive (DS) and diazepam-resistant (DR) lines). The ataxic effects of several drugs purported to produce some of their actions through the benzodiazepine-GABA receptor complex were examined using the rotarod test. The duration of impairment produced by diazepam, ethanol, 4,5,6,7-tetrahydroisoxazol[5,4-C]pyridine-3-ol (THIP) and phenobarbital was greater in the diazepam-sensitive than in the diazepam-resistant mice. In contrast, pentobarbital produced an equivalent duration of ataxia in the two lines. Muscimol-stimulated 36Cl- influx and the binding of [35S]t-butylbicyclophosphorothionate (TBPS) and [3H]flunitrazepam were measured using isolated brain membrane vesicles (microsacs). Depolarization-dependent 45Ca2+ uptake was measured in whole brain synaptosomes. Muscimol was a more potent stimulator of 36Cl- flux in the DS compared to the DR mice, although no difference between the lines was found in muscimol-stimulation of [3H]flunitrazepam binding. Flunitrazepam augmented the muscimol-stimulated 36Cl- uptake in the DS but not in the DR mice. However, no differences between the lines of mice were found in either density or affinity of [3H]flunitrazepam binding sites. Similarly, no differences in either the density or affinity of [35S]TBPS binding sites was found. Ethanol (10-45 mM) potentiated the muscimol-stimulation of 36Cl- in DS, with no effect in DR mice. However, ethanol inhibition of [35S]TBPS binding was equivalent in the two lines of mice. Pentobarbital produced an equal potentiation of the muscimol-stimulated 36Cl- flux in the two lines, but phenobarbital potentiated the muscimol-induced 36Cl- influx slightly more in DS mice.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1988

gamma-Aminobutyric acid-activated chloride channels: relationship to genetic differences in ethanol sensitivity.

We demonstrated recently that low concentrations of ethanol enhanced the muscimol-stimulated chloride influx in cerebellar membranes from long sleep (LS-ethanol sensitive) mice, but had no effect on membranes from short sleep (SS-ethanol resistant) mice. The LS and SS were selected from a heterogeneous stock (HS) of mice for differential sensitivity to the hypnotic effects of ethanol as measured by the duration of the loss of the righting reflex (sleep time). In the present study, we tested 100 HS for ethanol sleep time. The mice with the shortest sleep time (HS-SS) and the mice with the longest sleep time (HS-LS) were selected and tested for the effect of ethanol and muscimol on chloride flux in cerebellum. The effects of ethanol and muscimol on both cerebellar and cortical chloride flux were also examined in rats from the 7th generation selected for differential sensitivity to the hypnotic effects of ethanol (high acute ethanol sensitive rats-HAS and low acute ethanol sensitive rats-LAS). Low concentrations of ethanol (10-30 mM) potentiated muscimol stimulation of 36Cl- uptake in both cortical and cerebellar membranes prepared from ethanol-sensitive animals (HS-LS and HAS). None of the ethanol concentrations tested altered stimulated chloride uptake in ethanol-resistant animals (HS-SS and LAS). No differences in muscimol stimulation of chloride uptake were observed between the pairs of selected lines. These findings strongly suggest that genetic differences in ethanol hypnosis are related to differences in the sensitivity of gamma-aminobutyric acid-operated chloride channels to ethanol.

Animals↗
PubMed1988

Antagonism of ethanol and pentobarbital actions by benzodiazepine inverse agonists: neurochemical studies.

The benzodiazepine inverse agonist Ro 15-4513 has been shown to antagonize several behavioral effects of ethanol and to block the effects of ethanol on chloride flux across brain membranes. We used isolated mouse brain membranes to test whether Ro 15-4513 would reduce the effects of ethanol on membrane fluidity, voltage-dependent calcium channels, microsomal calcium release or binding of t-[35S]butylbicyclophosphorothionate. None of these actions of ethanol were altered by Ro 15-4513. The enhancement of gamma-aminobutyric acid (GABA)-activated chloride flux produced by ethanol or pentobarbital was antagonized partially by Ro 15-4513. Another inverse agonist, FG 7142, was more effective than Ro 15-4513 as an antagonist of ethanol actions on chloride flux. These results demonstrate that the ethanol antagonist action of Ro 15-4513 is specific for GABA-activated chloride flux and does not extend to other neurochemical actions of ethanol. The inverse agonist action (i.e., inhibition of GABA-activated chloride flux tested in the absence of ethanol) of Ro 15-4513 and FG 7142 was revealed by pretreatment of mice in vivo with ethanol. This raises the possibility that ethanol exposure increases the inverse agonist actions of Ro 15-4513 and related drugs and that these inverse agonist actions contribute to the ethanol antagonism observed in vivo and in vitro.

Animals↗
PubMed1987

Involvement of neuronal chloride channels in ethanol intoxication, tolerance, and dependence.

Studies of the role of neuronal chloride channels in ethanol action have focused on chloride channels coupled to gamma-aminobutyric acid (GABA) receptors. Ethanol intoxication is hypothesized to result from enhancement of GABA action, leading to increased chloride conductance and decreased neuronal excitability. Chronic ethanol treatment is suggested to produce a subsensitivity to GABA, leading to decreased action of ethanol and hyperexcitability on withdrawal of ethanol. Behavioral, electrophysiological, and biochemical studies of acute and chronic ethanol treatments on the GABA-regulated chloride channels of brain are reviewed.

Alcoholic Intoxication↗
PubMed1987

Acute and chronic ethanol treatments alter GABA receptor-operated chloride channels.

The effect of ethanol exposure in vitro on the GABA receptor-operated chloride channel was evaluated by monitoring 36Cl- influx in a membrane vesicle suspension (microsacs) prepared from mouse cerebellum. These experiments directly demonstrate ethanol augmentation of muscimol-stimulated chloride flux. DBA/2J mice were made tolerant to and dependent on ethanol by administration of an ethanol containing liquid diet for 7 days. Exposure to physiologically relevant concentrations of ethanol (10-45 mM) in vitro potentiated muscimol stimulation of 36Cl- uptake in control (pair-fed) membranes, but had no effect on cerebellar microsacs from tolerant/dependent mice. Muscimol stimulation of 36Cl- uptake was not different for pair-fed and ethanol-treated mice. Augmentation of muscimol-induced 36Cl- flux by in vitro ethanol was abolished by a single 4 g/kg injection of ethanol. This "acute tolerance" occurred within 5 min and disappeared within 24 hr after ethanol treatment. The reduced sensitivity of ethanol treated (chronic and acute) mice to ethanol potentiation of muscimol stimulated 36Cl- uptake offers a biochemical correlate to the phenomenon of ethanol tolerance. Moreover, the findings suggest that this biochemical tolerance develops rapidly following a single hypnotic dose of ethanol.

Animals↗
PubMed1987

Neurochemical actions of anesthetic drugs on the gamma-aminobutyric acid receptor-chloride channel complex.

Interaction of intoxicant-anesthetic drugs with the gamma-aminobutyric acid (GABA) receptor-chloride channel complex of mouse brain was studied using the binding of [35S]t-butylbicyclophosphorothionate (TBPS) to isolated membranes and the GABA-stimulated uptake of 36Cl- by membrane vesicles. Anesthetic drugs, including barbiturates, chloroform, diethylether and ethanol, inhibited the binding of TBPS and enhanced the GABA-dependent influx of chloride. In the presence of bicuculline, barbiturates increased the binding of TBPS, but this action was not shared by other anesthetic agents. Inhibition of TBPS binding was found with drug concentrations that produce anesthesia in vivo, whereas augmentation of GABA action occurred at subanesthetic concentrations. Effects of a series of n-alcohols (methanol to decanol) were studied on TBPS binding and membrane fluidity (using 1,6-diphenyl-1,3,5-hexatriene as a fluorescent probe). The potencies of the alcohols for inhibiting TBPS binding and fluidizing synaptic membranes were similar to their anesthetic potencies, but there were differences in the relative potencies of the drugs for inhibition of TBPS binding and membrane fluidization. These results, together with effects of assay temperature, suggest that effects of anesthetics on the GABA receptor-chloride channel complex were not due to changes in bulk membrane fluidity. Correlation of anesthetic potencies of chemically diverse agents with both inhibition of TBPS binding and augmentation of GABA-dependent chloride flux suggests a role for the GABA complex in anesthesia.

Alcohols↗
PubMed1987

Alcohol and the GABA receptor-chloride channel complex of brain.

Several lines of research suggest that the depressant effects of intoxicant-anesthetic compounds are mediated through the GABA receptor-chloride ionophore. We have recently demonstrated a procedure that allows direct measurement of GABA-mediated chloride influx in neural tissue. Using this approach in conjunction with receptor-binding techniques, we examined the role of the GABAergic system using mice selected for genetic differences in ethanol hypnosis. A low concentration of ethanol (15 mM) enhanced the muscimol-stimulated chloride influx in cerebellar membranes from Long-sleep (LS-ethanol sensitive) mice, but had no effect on membranes from Short-sleep (SS-ethanol resistant) mice. In addition, muscimol was more potent in stimulating chloride flux and in inhibiting 35S-TBPS binding in LS membranes than in SS membranes. Mice from a heterogeneous stock were selected for differences in ethanol-induced loss of righting reflex. Cerebellar membranes from these mice also showed differences in ethanol augmentation of muscimol-stimulated chloride flux in vitro that were related to their differential sensitivity to ethanol in vivo. This combined genetic-neurochemical approach provides strong evidence for the importance of GABA-operated chloride channels in ethanol intoxication.

Animals↗
PubMed1986

Gamma-aminobutyric acid and alcohol actions: neurochemical studies of long sleep and short sleep mice.

Effects of ethanol and pentobarbital on the GABA receptorchloride channel complex were evaluated in mice selected for differential sensitivity to the hypnotic effects of ethanol (long sleep and short sleep lines). 36Cl- influx, [35S]tbutylbicyclophosphorothionate (TBPS) and [3H]muscimol binding were measured in a membrane vesicle suspension (microsacs) from cerebellum or forebrain. Muscimol was found to be a more potent stimulator of 36Cl- flux in the LS cerebellum, as compared to the SS cerebellum, but a similar maximal level of uptake was achieved in the two lines. Muscimol displaced [35S]TBPS (a ligand for the convulsant site) from cerebellar microsacs, and LS mice were also more sensitive than SS mice to this action of muscimol. However, the number or affinity of high affinity [3H]muscimol binding sites did not differ between the lines. Physiologically relevant concentrations of ethanol (15-50 mM) potentiated muscimol stimulation of 36Cl- uptake in LS cerebellum but had no effect in SS cerebellum. Ethanol failed to alter stimulated chloride flux hippocampal microsacs from either line. Both the LS and SS lines responded similarly to pentobarbital potentiation of muscimol stimulated chloride uptake regardless of brain region. The demonstrated difference between the LS and SS mice in muscimol stimulated chloride uptake as well as in muscimol displacement of [35S]TBPS binding offers a biochemical explanation for the line differences in behavioral responses to GABAergic agents. Moreover, the findings suggest that genetic differences in ethanol hypnosis are related to differences in the sensitivity of GABA-operated chloride channels to ethanol.

Animals↗
PubMed1986

Anesthetic and convulsant barbiturates alter gamma-aminobutyric acid-stimulated chloride flux across brain membranes.

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, increases membrane chloride conductance. Previously, the authors reported that GABA increases 36Cl- uptake by membrane vesicles (microsacs) prepared from mouse brain. In the present study, we examined the actions of barbiturates on basal and GABA-stimulated chloride influx by brain vesicles. The anesthetic barbiturates pentobarbital, phenobarbital, mephobarbital, amobarbital, hexobarbital and R-(-)-1-methyl-5-phenyl-5-propyl barbiturate enhanced GABA-dependent chloride flux. Barbiturate enhancement of GABA action was seen at concentrations that are subanesthetic in vivo (e.g., 10 microM pentobarbital was effective). Pentobarbital was about 10 times more potent than pentobarbital, suggesting that chloride flux is related to the sedative rather than anticonvulsant actions of barbiturates. Pentobarbital (1 mM) prevented the antagonism of GABA-stimulated 36Cl- produced by picrotoxinin. The barbiturates generally produced no change in GABA-independent flux, although large concentrations of pentobarbital or hexobarbital produced a slight enhancement of chloride flux in the absence of GABA. The convulsant barbiturate S-(+)-1-methyl-5-phenyl-5-propyl barbiturate inhibited GABA-stimulated chloride flux, an action opposite to that of its anesthetic enantiomer. These experiments provide evidence for a functional coupling among GABA and barbiturate receptors and the chloride ionophore and suggest that the GABA-activated chloride channel is a site of action for intoxicant-anesthetic and convulsant barbiturates.

Animals↗
PubMed1986

gamma-Aminobutyric acid agonists and antagonists alter chloride flux across brain membranes.

gamma-Aminobutyric acid (GABA), the major inhibitory neurotransmitter in the mammalian brain, increases membrane chloride conductance. Previously, we reported that GABA increases 36Cl- uptake by membrane vesicles (microsacs) prepared from mouse brain. Employing this technique, we found that the GABAA agonists, muscimol, isoguvacine, 4,5,6,7-tetrahydroisoxazolo(5,4-C)pyridine-3-ol, and 3-amino-1-propane sulfonate, all produced a concentration-dependent increase in 36Cl- influx, but baclofen, a GABAB agonist, failed to alter 36Cl- flux. Inhibition of GABA-dependent 36Cl- influx was produced by the convulsant drugs, bicuculline, picrotoxin, and pentylenetetrazole. Ion specificity was demonstrated by a failure of GABA agonists to stimulate influx of 45Ca2+, 86Rb+, 22Na+, or 35SO4(2). GABA-stimulated uptake of 36Cl- was largest in cortex and cerebellum and smaller in hippocampus and striatum. There was little difference in sensitivity to GABA among the areas. Analysis of subcellular fractions prepared from mouse brain demonstrated that the GABA-dependent 36Cl- influx was enriched in the synaptosomal fraction. The nonspecific (GABA-independent) uptake of 36Cl- was enriched in the myelin fraction. These experiments provide evidence for a functional coupling among GABA receptors and the chloride ionophore and suggest that the GABA-activated chloride channel is a site of action for several convulsant compounds.

4-Acetamido-4'-isothiocyanatostilbene-2,2'-disulfo↗