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Biomedical subjects

P R Dodd

Publications and source records attributed to P R Dodd.

At least 19 recordsLinked to original sources

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

[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

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

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

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

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

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

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

Amino acid neurotransmitter receptor changes in cerebral cortex in alcoholism: effect of cirrhosis of the liver.

Gamma-aminobutyric acidA/benzodiazepine receptor binding sites and the N-methyl-D-aspartate subclass of glutamate receptor sites were assessed in synaptic plasma membrane homogenates of cerebral cortex tissue obtained at autopsy from cirrhotic and noncirrhotic alcoholic patients and matched control subjects. The alcoholic patients consumed an average of greater than 80 g of ethanol/day, the control subjects less than 20 g/day. Postmortem delays up to approximately 100 h caused no significant loss of any of the binding sites; the patient and subject groups were closely matched for age. The affinities (KD) of the receptor sites did not differ between the patient and subject groups, nor between cortical regions. Using three different radioligands ([3H]muscimol, [3H]flunitrazepam, and [3H]diazepam), the gamma-aminobutyric acidA/benzodiazepine receptor complex was found to have greater density (Bmax) in superior frontal gyrus in alcoholic patients (which selectively shows morphological change in alcoholic patients), but was unchanged in motor cortex. Alcoholic patients with cirrhosis had much less pronounced changes. The density of the N-methyl-D-aspartate subclass of glutamate receptors, assessed with [3H]MK-801, did not vary across patient and subject groups.

Adult

Glutamate and gamma-aminobutyric acid neurotransmitter systems in the acute phase of maple syrup urine disease and citrullinemia encephalopathies in newborn calves.

Cerebral cortex tissue was obtained at autopsy from neonatal Poll Hereford calves with clinically confirmed maple syrup urine disease (MSUD), neonatal Holstein-Friesian calves with clinically confirmed citrullinemia, and matched controls. From this, synaptosomes were prepared for studies of neurotransmitter amino acid uptake and stimulus-induced release, and synaptic plasma membranes were obtained for studies of associated postsynaptic receptor binding sites. As well as having abnormal brain tissue concentrations of the pathognomic plasma amino acids (markedly increased levels of the branched-chain compounds valine, isoleucine, and leucine in MSUD; marked elevation of citrulline levels in citrullinemia), both groups of diseased animals showed reduced brain tissue concentrations of each of the transmitter amino acids glutamate, aspartate, and gamma-aminobutyric acid (GABA). Nontransmitter amino acids were generally unaffected in either disease. Citrullinemic calves showed a marked increase in brain glutamine concentration; in calves with MSUD, the glutamine concentration was raised, but to a much lesser extent. The Na(+)-dependent synaptosomal uptake of both glutamate and GABA was markedly reduced (to less than 50% of control values in both cases) in citrullinemic calves but was unaltered in calves with MSUD. Whereas synaptosomes from normal calves showed the expected stimulus-coupled release of transmitter amino acids, especially glutamate and aspartate, and no response to stimulus of nontransmitter amino acids, there was no increased release of transmitter amino acids in response to depolarization in synaptosomes from citrullinemic calves.(ABSTRACT TRUNCATED AT 250 WORDS)

Acute-Phase Reaction

[3H]MK-801 binding in immature and mature chicken forebrain.

Previous studies have shown that the rate of calcium uptake stimulated by N-methyl-D-aspartate (NMDA) increased during the maturation phase of synapse development in chicken forebrain. To investigate whether this change in function is due to a change in the properties of NMDA receptor associated ion channels, we measured the binding of [3H]MK-801 (a ligand which binds to the NMDA receptor associated ion channel) to membranes from immature and mature chicken brain. The binding properties of MK-801 in chicken brain were similar to those in mammalian brain. There was no significant difference in any of the binding parameters measured at the two ages, i.e. KD, Bmax and optimal glutamate concentration for and maximal enhancement by glutamate of MK-801 binding. These results suggest that there is no change in the NMDA operated ion-channels during maturation. Thus the maturational change in NMDA receptor function could be due to: a change in the agonist portion of the NMDA receptor, a change in the regulation of the receptor/ionophore complex, perhaps by the postsynaptic density whose structure and composition changes during the same period, or a change in the number of voltage-sensitive calcium channels recruited as a result of NMDA receptor activation.

Adenosine Triphosphate

The interaction of a Huntington disease factor with receptors for the neurotoxin kainic acid.

A factor from mammalian and human brain, which inhibits the rate of migration of leukocytes obtained from sufferers from Huntington disease (Walls and Ruwoldt, 1984), inhibited the specific binding of the neurotoxin [3H]kainic acid to rat brain synaptic membranes. The factor was present in sucrose-particulate but not in soluble fractions from rat sub-cortical tissue, and was destroyed by tryptic digestion. Whereas an ammonium sulfate fraction of direct saline extracts of brain (Walls and Ruwoldt, 1984) gave poor chromatography on HPLC, prior separation of a sucrose-particulate fraction resulted in much improved chromatography. There was a good concordance between leukocyte migration inhibitory activity and [3H]kainic acid binding inhibitory activity. The factor may be an endogenous modulator of the kainic acid subset of receptors for the excitatory neurotransmitter glutamic acid.

Animals

Mechanisms of synaptic plasticity. Changes in postsynaptic densities and glutamate receptors in chicken forebrain during maturation.

We have shown that the synapse maturation phase of synaptogenesis is a model for synaptic plasticity that can be particularly well-studied in chicken forebrain because for most forebrain synapses, the maturation changes occur slowly and are temporally well-separated from the synapse formation phase. We have used the synapse maturation phase of neuronal development in chicken forebrain to investigate the possible link between changes in the morphology and biochemical composition of the postsynaptic density (PSD) and the functional properties of glutamate receptors overlying the PSD. Morphometric studies of PSDs in forebrains and superior cervical ganglia of chickens and rats have shown that the morphological features of synapse maturation are characteristic of a synaptic type, but that the rate at which these changes occur can vary between types of synapses within one animal and between synapses of the same type in different species. We have investigated, during maturation in the chicken forebrain, the properties of the N-methyl-D-aspartate (NMDA) subtype of the glutamate receptors, which are concentrated in the junctional membranes overlying thick PSDs in the adult. There was no change in the number of NMDA receptors during maturation, but there was an increase in the rate of NMDA-stimulated uptake of 45Ca2+ into brain prisms. This functional change was not seen with the other ionotropic subtypes of the glutamate receptor and was NMDA receptor-mediated. The functional change also correlated with the increase in thickness of the PSD during maturation that has previously been shown to be due to an increase in the amount of PSD associated Ca(2+)-calmodulin stimulated protein kinase II (CaM-PK II). Our results provide strong circumstantial evidence for the regulation of NMDA receptors by the PSD and implicate changing local concentrations of CaM-PK II in this process. The results also indicate some of the ways in which properties of existing synapses can be modified by changes at the molecular level.

Animals

Alterations in cortical [3H]kainate and alpha-[3H]amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid binding in a spontaneous canine model of chronic hepatic encephalopathy.

Excitatory amino acid receptor binding parameters were investigated in a spontaneous dog model of chronic hepatic encephalopathy. L-[3H]Glutamate, (+)-[3H]-5-methyl-10,11-dihydro-5H-dibenzo[a,d]cyclohepten-5,10-im ine maleate ([3H]MK-801), [3H]kainate, and alpha-[3H]-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA) binding experiments were performed using crude cerebrocortical synaptosomal membrane preparations from dogs with congenital portosystemic encephalopathy (PSE) and control dogs. There was no change in the affinity or density of L-[3H]-glutamate or [3H]MK-801 binding sites in dogs with congenital PSE compared with control dogs. However, in the PSE dogs there was a significant reduction in the density of [3H]kainate binding sites compared with control dogs and abolition of the low-affinity [3H]AMPA binding site. The relative binding capacity of PSE synaptosomal membranes for [3H]kainate and [3H]AMPA was expressed as the ratio Bmax/KD. There was a significant inverse correlation between the Bmax/KD ratio for [3H]AMPA binding and the worst grade of encephalopathy experienced by each dog. These results suggest that there is a significant perturbation of cerebrocortical non-N-methyl-D-aspartate receptor binding in dogs with congenital PSE which may have relevance to the pathogenesis of hepatic encephalopathy.

Animals

Plasma GABA-like activity in rats with hepatic encephalopathy is due to GABA and taurine.

Significant discrepancy exists between radioreceptor and high-performance liquid chromatographic estimates of plasma GABA concentrations in animal models of hepatic encephalopathy. A possible explanation for this discrepancy is the presence in plasma of a substance that can inhibit [3H]-GABA binding but is not GABA itself. The aim of this study was to determine whether any of the amino acids that are increased in the plasma of animal models of acute and chronic hepatic encephalopathy (glutamine, glutamate, phenylalanine, tyrosine, citrulline and taurine) can significantly inhibit [3H]-GABA binding and contribute to the GABA-like activity of plasma aliquots. At final assay concentrations equivalent to plasma concentrations found in hepatic encephalopathy, only taurine was found to significantly inhibit [3H]-GABA binding to whole rat brain synaptosomal membranes (36% inhibition at 30 mumol/L final assay concentration). The concentration of taurine that resulted in 50% inhibition (final assay concentration) was 158 mumol/L, corresponding to a plasma concentration of 1.58 mmol/L. Taurine inhibition of [3H]-GABA binding was competitive because the receptor density was unaltered, but the receptor affinity decreased with increasing concentration of taurine. Plasma GABA-like activity (determined by radioreceptor assay) and plasma GABA and taurine concentrations (determined by high-performance liquid chromatography) were measured in 18 rats with acute or chronic hepatic encephalopathy and 9 control rats. Plasma GABA-like activity and plasma GABA and taurine concentrations were significantly increased in rats with hepatic encephalopathy compared with control rats.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Increased gamma-aminobutyric acid receptor function in the cerebral cortex of myoclonic calves with an hereditary deficit in glycine/strychnine receptors.

Inherited congenital myoclonus (ICM) of Poll Hereford cattle is a neurological disease in which there are severe alterations in spinal cord glycine-mediated neurotransmission. There is a specific and marked decrease, or defect, in glycine receptors and a significant increase in neuronal (synaptosomal) glycine uptake. Here we have examined the characteristics of the cerebral gamma-aminobutyric acid (GABA) receptor complex, and demonstrate that the malfunction of the spinal cord inhibitory system is accompanied by a change in the major inhibitory system in the cerebral cortex. In synaptic membrane preparations from ICM calves, both high-and low-affinity binding sites for the GABA agonist [3H]muscimol were found (KD = 9.3 +/- 1.5 and 227 +/- 41 nM, respectively), whereas only the high-affinity site was detectable in controls (KD = 14.0 +/- 3.1 nM). The density and affinity of benzodiazepine agonist binding sites labelled by [3H]diazepam were unchanged, but there was an increase in GABA-stimulated benzodiazepine binding. The affinity for t-[3H]butylbicyclo-o-benzoate, a ligand that binds to the GABA-activated chloride channel, was significantly increased in ICM brain membranes (KD = 148 +/- 14 nM) compared with controls (KD = 245 +/- 33 nM). Muscimol-stimulated 36Cl- uptake was 12% greater in microsacs prepared from ICM calf cerebral cortex, and the uptake was more sensitive to block by the GABA antagonist picrotoxin. The results show that the characteristics of the GABA receptor complex in ICM calf cortex differ from those in cortex from unaffected calves, a difference that is particularly apparent for the low-affinity, physiologically relevant GABA receptors.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals

Uptake of gamma-aminobutyric acid and L-glutamic acid by synaptosomes from postmortem human cerebral cortex: multiple sites, sodium dependence and effect of tissue preparation.

The uptake of gamma-aminobutyric acid (GABA) and L-glutamic acid by synaptosomes prepared from frozen postmortem human brain was shown to be effected via distinct high and low affinity sites. At approximately 17 h postmortem delay, the kinetic parameters for GABA uptake were: high affinity site, Km 7.1 +/- 2.5 microM, Vmax 18.7 +/- 4.8 nmol.min-1 per 100 mg protein; low affinity site, Km 2 +/- 1 mM, Vmax 425 +/- 250 nmol.min-1 per 100 mg protein (means +/- S.E.M., n = 13). Kinetic parameters for L-glutamate uptake were: high affinity site, Km 7.5 +/- 1.0 microM, Vmax 85 +/- 8 nmol.min-1 per 100 mg protein; low affinity site, Km 1.8 +/- 1.2 mM. Vmax 780 +/- 175 nmol.min-1 per 100 mg protein (n = 11). A detailed kinetic analysis of high affinity GABA uptake was performed over a range of sodium ion concentrations. The results were consistent with a coupling ratio of one Na+ ion to one GABA molecule; a similar result was found with rat brain synaptosomes. However, rat and human synaptosomes differed in the degree to which the substrate affinity of the high affinity GABA uptake site varied with decreasing Na+ ion concentration. High affinity GABA uptake was markedly affected by the method used to freeze and divide the tissue, but did not vary greatly in different cortical regions. There was some decline of high affinity GABA uptake activity with postmortem delay, apparently due to a loss of sites rather than a change in site affinity.

Adult

Cortical dihydropyridine binding sites are unaltered in human alcoholic brain.

The density and affinity of sites labeled by the 1,4-dihydropyridines, [3H]nitrendipine and (+) [3H]PN 200-110, were not significantly different in superior frontal and parietal cortical membranes from alcoholic patients and nonalcoholic control patients. This is in contrast to the increased number of dihydropyridine receptors and increased functional activity of calcium channels reported in brain from rats treated chronically with ethanol and in neural cell lines grown in the presence of ethanol. These results indicate that 1,4-dihydropyridine-sensitive calcium channels (L type) in the brain, despite possible acute changes, are unaltered following long-term ethanol exposure in humans.

Alcoholism