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Biomedical subjects

P R Dodd

Publications and source records attributed to P R Dodd.

At least 37 records · Page 2Linked to original sources
PubMed1997

Zolpidem binding sites on the GABA(A) receptor in brain from human cirrhotic and non-cirrhotic alcoholics.

The displacement of [3H]flunitrazepam by unlabelled flunitrazepam or zolpidem was used to assess the affinity and density of sub-types of GABA(A) receptors in the superior frontal and primary motor cortices of ten alcoholic, seven alcoholic-cirrhotic and ten matched control cases. The binding was best fitted by a model with a single site for flunitrazepam, but two sites for zolpidem. Neither the patients' age nor the post-mortem interval were significantly correlated with the affinity or density of any of the binding sites. The affinity of all ligands did not differ either between cortical regions or across case groups. Hence, the density of each binding site was analyzed at constant affinity. The densities of flunitrazepam and high-affinity zolpidem binding sites were invariant across cortical regions and case groups. Low-affinity zolpidem binding sites were significantly more dense in the frontal than in the motor cortex of alcoholic cases irrespective of cirrhosis, whereas this regional difference was not significant in control cases.

Adult↗
PubMed1997

Expression of the alpha 1, alpha 2 and alpha 3 isoforms of the GABAA receptor in human alcoholic brain.

The expression of the alpha 1, alpha 2 and alpha 3 isoforms of the GABAA receptor was studied in the superior frontal and motor cortices of 10 control, 10 uncomplicated alcoholic and 7 cirrhotic alcoholic cases matched for age and post-mortem delay. The assay was based on competitive RT/PCR using a single set of primers specific to the alpha class of isoform mRNA species, and was normalized against a synthetic cRNA internal standard. The assay was shown to be quantitative for all three isoform mRNA species. Neither the patient's age nor the post-mortem interval significantly affected the expression of any isoform in either cortical area. The profile of expression was shown to be significantly different between the case groups, particularly because alpha 1 expression was raised in both groups of alcoholics of controls. The two groups of alcoholics could be differentiated on the basis of regional variations in alpha 1 expression. In frontal cortex, alpha 1 mRNA expression was significantly increased when uncomplicated alcoholics were compared with control cases whereas alcoholic-cirrhotic cases were not significantly different from either controls or uncomplicated alcoholic cases. In the motor cortex, alpha 1 expression was elevated only when alcoholic-cirrhotic cases were compared with control cases. There was no significant difference between case groups or areas for any other isoform.

Adult↗
PubMed1997

A method for the quantitation of the alpha 1, alpha 2, and alpha 3 isoforms of the GABAA receptor in human brain using competitive PCR.

The GABAA receptor is the site of action of the inhibitory neurotransmitter GABA, as well as a number of pharmacologically important drugs such as benzodiazepines, barbiturates, and ethanol. The GABAA receptor is a pentameric complex composed of distinct polypeptides, which have been divided into five subunit classes on the basis of sequence homology. To date, 17 isoforms of the receptor have been identified and cloned in mammalian brain, and designated alpha 1-6, beta 1-4, gamma 1-4, delta and rho 1-2. In addition, several isoforms exist in alternatively spliced forms (for review see ref.). Studies on recombinant receptors have revealed that receptors constituted from different isoforms exhibit distinct pharmacological properties. For example, the alpha subunit class appears to be responsible for GABA enhancement of benzodiazepine binding. GABAA receptor function is modulated by benzodiazepine agonists such as flunitrazepam and diazepam, barbiturates, anaesthetics, neurosteroids, and ethanol. Chronic treatment of animals with many of these compounds can bring about profound changes in receptor expression and pharmacology. The RT/PCR assay described here was developed to quantify the alpha 1, alpha 2 and alpha 3 isoforms in the same assay. The amount of each isoform was quantified on the basis of a standard curve generated under identical PCR conditions to the target sequences. In this way it is possible to quantify multiple samples in each RT/PCR assay, thereby reducing inter-assay variability. The assay can be applied to quantify the expression of these isoforms in response to acute and chronic drug administration, or in particular disease states. Altered expression may reflect a corresponding change in protein synthesis, or an alteration of the subtype composition of GABAA receptor.

Brain↗
PubMed1996

Receptor binding sites and uptake activities mediating GABA neurotransmission in chronic alcoholics with Wernicke encephalopathy.

Superior frontal cortex (SFC) and primary motor cortex tissue was obtained at autopsy from thirteen severe chronic alcoholics with neuropathologically confirmed Wernicke Encephalopathy (WE) and 22 controls. Cases with both WE and cirrhosis showed markedly fewer neurones in SFC than did WE cases without cirrhosis. The extent of the apparent neuronal loss corresponded to an increase in post-synaptic GABAA receptor sites, as assessed by the binding of [3H]muscimol to synaptic membranes. Increased [3H]muscimol binding was not accompanied by an increase in 'central-type' benzodiazepine binding sites: as assessed by [3H]flunitrazepam binding, these sites were apparently unaltered, while as assessed by [3H]diazepam binding, they were decreased. The affinities of the two benzodiazepine ligands varied differently with disease. These discrepancies between [3H]flunitrazepam and [3H]diazepam binding could not be accounted for, either by the presence of a second, diazepam-preferring, 'central-type' benzodiazepine binding site, or by loss of 'peripheral-type' sites. The changes in the post-synaptic GABAA-benzodiazepine receptor sites did not reflect any regional, disease-related deficit of afferent GABAergic terminals, as assessed by synaptosomal high-affinity [3H]GABA uptake. On a number of indices, it appears most likely that the data reflect both a loss of receptor sites, and a change in the population of receptor sub-types.

Adult↗
PubMed1996

The neurochemical pathology of thiamine deficiency: GABAA and glutamateNMDA receptor binding sites in a goat model.

Synaptic plasma membranes were prepared from four cerebrocortical areas from six male Angora goats made chronically thiamine deficient (TD) by the administration of AmproliumTM (600-900 mg/kg daily for 38-44 d). Four male controls were matched for age (27-30 mo). Four different radioligands were used to characterise GABAA and Glu-RNMDA receptor binding sites. There were marked, localised and contrasting changes in motor cortex, with an increase in GABAA and a decrease in Glu-RNMDA binding site densities. Less clearcut changes of a similar nature were seen in visual cortex. There was no variation in the parameters of GABA-activated [3H]diazepam binding between cortical areas in control goats, but there was a reduction in the maximal response to GABA in all areas in TD goats. There were regional variations in glutamate-activated [3H]MK-801 binding in control goat brain, and a non-selectively reduced maximal response in TD. Alterations in these indices of GABA- and glutamate-mediated neurotransmission may underlie the neurological signs of acute thiamine deficiency in these animals.

Alcoholism↗
PubMed1996

Alcohol, alcoholic brain damage, and GABAA receptor isoform gene expression.

Selective variations in cerebral GABAA receptor pharmacology and function are observed in experimental animals subjected to a number of alcohol-treatment and -withdrawal paradigms, and where human alcoholics with and without a range of concomitant diseases are compared with non-alcoholic cases. Recombination studies have shown that variations in GABAA receptor pharmacology and function can result from altering its subunit isoform composition. This commentary examines the rôle of subunit isoform expression in the response to long-term alcohol administration in animals, and in the pathogenesis of alcoholism-related brain damage in human cases.

Alcoholism↗
PubMed1995

Developmental rearrangements of cortical glutamate-NMDA receptor binding sites in late human gestation.

NMDA-preferring glutamate receptor biding sites were characterized using the site-selective ligand [3H]MK801, in synaptic membranes prepared from cerebral cortex tissue obtained postmortem from human infants who had died with minimal neurological and neuropathological impairment between 22 and 42 weeks' gestation. It proved necessary to modify the assay protocol used with adult tissue before reliable data could be obtained. In the four cortical region studied (prefrontal, motor, occipital, temporal), [3H]MK801 bound to a single class of sites which showed significant variations in affinity only in motor cortex. The density of [3H]MK801 binding sites (calculated at constant affinity) showed marked increases in all cortical regions over this period. The extent to which glutamate could enhance [3H]MK801 binding became significantly lower in prefrontal and motor cortex as gestation progressed, so that at term, little activation was apparent. In occipital and temporal cortex, this parameter was low throughout late gestation. The evidence suggests that Glutamate-NMDA binding sites may undergo structural rearrangements which alter their ability to interact with ligands during the later stages of human gestation, and that such changes are regionally variable.

Binding Sites↗
PubMed1995

Benzodiazepine binding sites in alcoholic cirrhotics: evidence for gender differences.

Synaptic plasma membranes were prepared from superior frontal gyrus and motor cortex obtained at autopsy from 17 chronic alcoholics not differentiated on thiamine status, of whom 8 had pathologically confirmed cirrhosis of the liver, and 10 controls. Three of the cirrhotic alcoholic cases were female, as was one control. Cases were closely matched for age at death and post-mortem delay. The affinity of "central-type" benzodiazepine sites for [3H]diazepam tended to be lower in both brain regions of both groups of alcoholics of cf controls, but the reverse was true for [3H]flunitrazepam, especially in cirrhotic cases. [3H]Diazepam affinity was invariant across all males and the female control, but lower in the female cirrhotic alcoholics. Affinity for [3H]flunitrazepam tended to be the reverse of that for [3H]diazepam. [3H]Diazepam Bmax was markedly lower in female cirrhotic alcoholics, especially in superior frontal gyrus, whereas this region showed a much higher Bmax in the female control case. A small regional difference in [3H]flunitrazepam Bmax was the reverse of that for [3H]diazepam Bmax and was seen in all groups. GABA-mediated neurotransmission may be selectively altered in a pathologically abnormal region of cerebral cortex in cirrhotic alcoholics, and the sexes may show differing susceptibilities to change.

Adult↗
PubMed1995

[3H]MK-801 binding asymmetry in the IMHV region of dark-reared chicks is reversed by imprinting.

Glutamate NMDA-type receptor binding in the intermediate medial hyperstriatum ventrale (IMHV) of dark-hatched chicks is lateralized. This lateralization was found to be markedly influenced by imprinting. In dark-reared chicks the binding of the selective ligand [3H]MK-801 was two-fold higher in the right IMHV than in the left IMHV. In contrast, imprinted chicks have significantly higher levels of [3H]MK-801 binding in the left IMHV region than in the right IMHV. Imprinting results in 2a learning-related increase in NMDA-type receptor binding levels in the left IMHV, whereas [3H]MK-801 binding levels in the right IMHV remain unchanged by imprinting. Thus, the plasticity present in the NMDA-type receptor system and associated with imprinting appears to occur in the left hemisphere only.

Animals↗
PubMed1995

Variant forms of neuronal glutamate transporter sites in Alzheimer's disease cerebral cortex.

The displacement of Na(+)-dependent D-[3H]-aspartate binding by unlabeled D-aspartate or the inhibitors DL-threo-beta-hydroxyaspartate, L-cysteate, L-glutamate, dihydrokainate, DL-alpha-aminoadipate, alpha-methyl-DL-glutamate, and 1-aminocyclobutane-cis-1,3-dicarboxylate was used to characterize the high-affinity glutamate/aspartate uptake site in human cerebral cortex. Synaptosomal membranes were prepared from tissue obtained at autopsy from nondemented control, Alzheimer's disease (AD), and diffuse Lewy body disease (DLBD) cases. Areas that are damaged in AD (midtemporal, frontal, caudal cingulate, and hippocampal cortices) were compared with those that are spared (occipital and motor cortices). Profiles of the affinities (Ka values) of the ligands showed that areas spared from damage in AD cases differed significantly from equivalent areas in control (p < 0.001) and DLBD (p < 0.001) cases and also from areas susceptible to damage in the same AD cases (p < 0.001). Areas susceptible to damage in AD showed comparable profiles across the three case groups (p = 0.980). The glutamate/aspartate uptake site may be regionally variant in AD cases, and this may underlie local excitotoxicity. D-[3H]Aspartate binding site density was significantly lower in both dementia groups (control vs. AD, p < 0.001; control vs. DLBD, p = 0.009; but AD vs. DLBD, p = 0.528); within-group differences were not significant (control, p = 0.874; AD, p = 0.285; DLBD, p = 0.741).

ATP-Binding Cassette Transporters↗
PubMed1994

Characterization of non-conventional opioid binding sites in rat and human lung.

Indirect evidence suggests that nebulized morphine relieves dyspnoea and bronchoconstriction via opioid receptors within the lung. This study used equilibrium binding studies to characterize opioid binding sites in lung membrane preparations. [3H]Morphine and [3H]naloxone were incubated separately with homogenates of Wistar rat brain and lung, and human lung. Binding affinities for both morphine and naloxone in rat and human lung were two orders of magnitude lower than those in brain. However, opioid binding site densities in lung were up to 100 times greater than that in brain. The addition of Na+ or GTP to lung homogenate preparations caused atypical effects on opioid binding. Na+ (50 mM) decreased the specific binding of [3H]naloxone 50% viz-à-vis a 20% increase in binding in the brain. GTP (100 microM) caused a 200% increase in the apparent capacity of morphine binding in the lung compared with a marked decrease in binding in the brain.

Animals↗
PubMed1994

Excitotoxic mechanisms in the pathogenesis of dementia.

Alzheimer disease and related dementias, in common with most major neurological diseases, are characterized by localized brain damage. An abundance of senile plaques and neurofibrillary tangles in certain brain areas is pathognomic of the disease: of the two, the density of tangles may correlate more closely with disease severity ante mortem. Clinical manifestation of the disease also results from a locally severe loss of neurones. This might be caused by over-stimulation by excitant amino acid transmitters such as glutamate, which would promote cell death. Mechanisms which might give rise to the localization of Alzheimer pathogenesis include hypersensitivity to damage because a cell carries a particular sub-set of post-synaptic receptors; local variations in the efficiency of excitatory amino acid transport; and, possibly, local exacerbation of toxicity by substances such as beta-amyloid. Elucidation of such mechanisms could lead to new pharmacotherapies of dementia.

Alzheimer Disease↗
PubMed1994

Pharmacology of morphine and morphine-3-glucuronide at opioid, excitatory amino acid, GABA and glycine binding sites.

Morphine in high doses and its major metabolite, morphine-3-glucuronide, cause CNS excitation following intrathecal and intracerebroventricular administration by an unknown mechanism. This study investigated whether morphine and morphine-3-glucuronide interact at major excitatory (glutamate), major inhibitory (GABA or glycine), or opioid binding sites. Homogenate binding assays were performed using specific radioligands. At opioid receptors, morphine-3-glucuronide and morphine caused an equipotent sodium shift, consistent with morphine-3-glucuronide behaving as an agonist. This suggests that morphine-3-glucuronide-mediated excitation is not caused by an interaction at opioid receptors. Morphine-3-glucuronide and morphine caused a weak inhibition of the binding of 3H-MK801 (non-competitive antagonist) and 125I-ifenprodil (polyamine site antagonist), but at unphysiologically high concentrations. This suggests that CNS excitation would not result from an interaction of morphine-3-glucuronide and high-dose morphine with these sites on the NMDA receptor. Morphine-3-glucuronide and morphine inhibited the binding of 3H-muscimol (GABA receptor agonist). 3H-diazepam and 3H-flunitrazepam (benzodiazepine agonists) binding very weakly, suggesting the excitatory effects of morphine-3-glucuronide and high-dose morphine are not elicited through GABAA receptors. Morphine-3-glucuronide and high-dose morphine did not prevent re-uptake of glutamate into presynaptic nerve terminals. In addition, morphine-3-glucuronide and morphine did not inhibit the binding of 3H-strychnine (glycine receptor antagonist) to synaptic membranes prepared from bovine spinal cord. It is concluded that excitation caused by high-dose morphine and morphine-3-glucuronide is not mediated by an interaction with postsynaptic amino acid receptors.

Animals↗
PubMed1994

GABAA receptors in damaged cerebral cortex areas in human chronic alcoholics.

Chronic alcoholics without complicating disease showed greater densities of GABA agonist sites (labelled with the selective ligand [3H]muscimol) on the GABAA receptor in the superior frontal gyrus, in comparison with both precentral gyrus in the same cases, and with superior frontal gyrus in matched controls. Whereas cases with concomitant Wernicke encephalopathy may also have had greater numbers of superior frontal [3H]muscimol binding sites than controls, alcoholics with cirrhosis of the liver showed more muted differences. Since the GABAA receptor is a multimeric complex which also possesses binding sites for "central-type" benzodiazepine ligands, it would be expected that data obtained with these compounds should mimic that obtained with [3H]muscimol. This was not so. [3H]Flunitrazepam binding sites showed little variation between case groups, although they showed clear regional differences. [3H]Diazepam sites followed those for [3H]muscimol in uncomplicated alcoholics and alcoholics with cirrhosis of the liver, but were at lower density in superior frontal gyrus in Wernicke cases. Differential expression of GABAA receptor subunit isoform genes may give rise to these disparities.

Adult↗
PubMed1993

The regeneration of d,l-fenfluramine-destroyed serotonergic nerve terminals.

The regeneration of serotonergic nerve terminals subsequent to their destruction by high-dose fenfluramine administration was examined. Treating rats with fenfluramine (80 mg/kg over 2 days) destroyed 80% of serotonergic nerve terminals, indicated by reduced maximal [3H]paroxetine binding to 5-hydroxytryptamine (5-HT) uptake sites on synaptic membranes (Bmax) and maximal [14C]5-HT uptake rate into synaptosomes (Vmax). 25 weeks later, these indices of serotonergic nerve terminals had returned to 72% of control. Maximal synaptosomal loading (alpha) with [14C]5-HT also recovered (to 79% of control), reflecting an increased number of serotonergic synaptosomes. This suggests that the rebound in 5-HT uptake site density found after fenfluramine illustrates the regeneration of 5-HT-containing nerve endings.

Animals↗
PubMed1993

Brain extracts containing a Huntington disease antigen inhibit [3H]kainate binding and block synaptosomal amino acid transport.

Fractions isolated from mammalian brain which had previously been shown to inhibit the rate of migration of peripheral blood leukocytes taken from Huntington disease cases, and also to inhibit [3H]kainic acid binding, were characterized further. By use of repeated ultrafiltration onto a 1000D MW cutoff filter, and by the isolation and extensive washing of an enriched ammonium sulfate fraction, their activity was shown not to be due to the presence of endogenous glutamate, and to be relatively selective for brain glutamate receptor binding sites. Inhibitory activity at [3H]GABA, 5-[3H]hydroxytryptamine 5HT1 and dopamine D1 or D2 binding sites was much weaker or absent. Factor extracts were also shown to act as non-competitive inhibitors of synaptosomal amino acid transport: increasing concentrations of the factor had no significant effect on the KM for the uptake of either [3H]glutamate or [3H]GABA, but at a final concentration of 66 micrograms protein x ml-1 had reduced the VMAX for [3H]glutamate uptake to approximately 20% of control, and the VMAX for [3H]GABA uptake to approximately 40% of control. This may enhance the factor's potential excitotoxicity.

Amino Acids↗