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.

Synaptosomal and brain slice cerebrocortical [3H]L-glutamate uptake in a rat model of chronic hepatic encephalopathy.

Cerebrocortical [3H]L-glutamate uptake was examined using brain slices and synaptosomes obtained from rats with portal vein and bile duct ligation. In addition, the effect of in vitro addition of 5 mM ammonia on glutamate uptake parameters was determined. There was no significant difference in brain slice or synaptosomal glutamate uptake in rats with portal vein and bile duct ligation compared to control rats. In vitro addition of ammonia had no effect on uptake kinetics in either brain slices or synaptosomes. These results suggest that glutamate uptake kinetics are not perturbed in this animal model of chronic hepatic encephalopathy.

L-glutamate and gamma-aminobutyric acid uptake in synaptosomes from the cerebral cortex of dogs with congenital chronic hepatic encephalopathy.

High affinity [3H]gamma-aminobutyric acid (GABA) and [3H]L-glutamate uptake were determined in synaptosomes prepared from the cerebral cortex of dogs with congenital hepatic encephalopathy and control dogs. The Km value for GABA uptake was increased by 35% but there was a concomitant 34% increase in Vmax suggesting that GABA uptake capacity was not changed in HE dogs. In contrast, mean Vmax for glutamate uptake in HE dogs was 85% greater than mean Vmax in control dogs; mean Km was increased by 25% in HE dogs. Therefore, overall synaptosomal high affinity glutamate uptake capacity was increased in HE dogs compared to controls.

Glutamatergic neurotransmission in hepatic encephalopathy.

There is increasing evidence that glutamatergic neurotransmission is perturbed in hepatic encephalopathy (HE). Studies of the glutamate receptor system suggest that glutamate receptor density is reduced in different animal models of HE. Glutamate release and/or uptake is also believed to be altered in the disorder resulting in increased glutamate concentrations in the synaptic cleft. The relevance of these findings to the pathogenesis of HE remains to be clarified.

CNQX binding to non-NMDA glutamate receptors in canine cerebro-cortical crude synaptosomal membranes: pharmacological characterization and comparison of binding parameters in dogs with congenital portosystemic encephalopathy and control dogs.

The pharmacological profile and binding characteristics of the non-NMDA antagonist of glutamate receptors [3H]6-cyano-7-nitro-quinoxaline-2,3-dione (CNQX), were investigated in triton-washed crude synaptosomal membranes prepared from canine cerebral cortex. [3H]CNQX binding was inhibited by various glutamate agonists and antagonists, the rank order of potency being CNQX greater than alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) = quisqualate = kainate greater than glutamate. Two binding sites for [3H]CNQX were apparent when non-specific binding (NSB) was defined with unlabelled CNQX. In contrast, when NSB was defined with saturating concentrations of unlabelled AMPA and kainate, only one binding site was identified which corresponded to the high affinity site identified when CNQX was used to define NSB. No physiologically relevant differences were found in binding parameters for [3H]CNQX membranes from dogs with congenital portosystemic encephalopathy (PSE) when compared with control dogs. The affinity constant (Ki) of AMPA displacement of [3H]CNQX binding was not significantly different in PSE dogs compared with control dogs. These results suggest that the antagonist site on cortical non-NMDA receptors is not perturbed in dogs with congenital PSE.

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.

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.

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.

Neurochemical studies on quinolone antibiotics: effects on glutamate, GABA and adenosine systems in mammalian CNS.

Quinolone antibiotics, which can be proconvulsant in susceptible patients, were found to inhibit the specific binding of the adenosine receptor ligands L-3H-N6-phenylisopropyladenosine (L-3H-PIA) and 3H-N-ethylcarboxamidoadenosine (3H-NECA) to rat brain synaptic membranes. The inhibitions were concentration dependent, and for both ligands the order of potency was rosoxacin greater than nalidixic acid greater than oxolinic acid greater than or equal to ciprofloxacin greater than norfloxacin greater than enoxacin: IC20 values (concentrations causing a 20% inhibition of specific binding) ranged from 30-35 microM to 1-3 mM. Hill coefficients were approximately 0.5, suggesting that the compounds are probably antagonists at these sites. Most of the compounds did not alter 3H-diazepam binding directly, although rosoxacin showed relatively strong, and enoxacin weak, concentration-dependent inhibition. At 50 microM the compounds enhanced the maximal gamma-aminobutyric acid (GABA) activation of 3H-diazepam binding to varying degrees, without altering the EC50 of activation, whereas at 200 microM they tended to reduce GABA activation. Most noteworthy was the large increase in GABA-stimulated 3H-diazepam binding caused by 50 microM nalidixic acid. The compounds did not alter the Ca2+/Cl- -dependent binding of 3H-glutamate, nor of the binding of the glutamate site-selective ligands 3H-kainate and alpha-3H-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (3H-AMPA); the uptake of the non-metabolized glutamate analogue D-3H-aspartate by cortical homogenates was also unaffected. The CNS side effects of these antibiotics may result, in part, from interaction with sites which mediate the inhibitory neurotransmission of adenosine and, possibly, GABA.

Deficit of spinal cord glycine/strychnine receptors in inherited myoclonus of Poll Hereford calves.

Inherited myoclonus of Poll Hereford calves is characterized by hyperesthesia and myoclonic jerks of the skeletal musculature, which occur spontaneously and in response to sensory stimuli. The disease shows autosomal recessive inheritance, and significant proportions of the Poll Hereford herds in many countries are thought to be carriers of the mutant gene. Studies revealed a specific and marked (90 to 95 percent) deficit in [3H]strychnine binding sites in spinal cord membranes from myoclonic animals compared to controls, reflecting a loss of, or defect in, glycine/strychnine receptors. Spinal cord synaptosomes prepared from affected animals showed a significantly increased ability to accumulate [3H]glycine, indicating an increased capacity of the high-affinity neuronal uptake system for glycine. In contrast, stimulus-induced glycine release and spinal cord glycine concentrations were unaltered.

Necropsy study of GABA/benzodiazepine receptor binding sites in brain tissue from chronic alcoholic patients.

Neurological and cognitive deficits occur in many chronic alcoholic patients. Quantitative studies reveal a significant reduction in the number of neurons in the superior frontal gyrus of chronic alcoholics, with no change in the motor cortex. Our studies are aimed at delineating which neurotransmitter systems are altered in alcoholics and to correlate these alterations with morphometry and, where possible, clinical signs. Much evidence suggests both short- and long-term effects of ethanol on GABA-mediated neurotransmission. GABA and benzodiazepines bind to distinct, but allosterically linked, sites on the GABA/benzodiazepine receptor complex. In the absence of any detected difference in the number of benzodiazepine receptors in both the frontal and motor cortex from alcoholic and control patients, a significant increase in the amount of GABA enhancement of [3H]diazepam binding was measured in frontal cortex membranes from patients with Wernicke's encephalopathy relative to non-alcoholic controls. This finding suggests an increased coupling of the GABA and benzodiazepine recognition sites in this area, which may be due to ethanol-induced conformational changes in the receptor complex.

Educating physicians and treating patients in the ambulatory setting. Where are we going and how will we know when we arrive?

We evaluated 15 group practices in general internal medicine in university hospitals with regard to access to and quality of care, patients' satisfaction with that care, and quality of residency education provided. We used these data to speculate about potential changes in ambulatory care programs in university teaching hospitals. All 15 practices participated for 4 years. One third of their patient population had no medical insurance. Practice patients had twice as many chronic illnesses as did the general population, and two fifths of patients stayed at least 2 years in the practice. Few faculty members spent more than 14 hours weekly in the practices, and housestaff worked an average of 4 hours per week. Patient waiting times did not meet ideal standards, but patient satisfaction was higher than in a general population. Compliance with quality of care criteria was not exceptional; for example, 10% of eligible patients received an annual influenza vaccination. Housestaff assigned a relatively low ranking to their educational experience in the practices. We recommend the institution of additional experimental programs in ambulatory care and housestaff education to improve the quality of care in the ambulatory setting.

Adenosine receptors in post-mortem human cerebral cortex and the effect of carbamazepine.

Representative A1 site- and A2 site-selective ligands for adenosine receptors were found to bind to a single class of non-co-operative sites in synaptic membranes prepared from pieces of normal human temporal lobe cerebral cortex obtained at autopsies performed within 24 h post-mortem. The known adenosine antagonists theophylline and 1-isobutyl-3-methylxanthine also bound to these sites and could completely displace either ligand. Computer analysis combining data from 21 separate experiments which used membranes prepared from seven different autopsy cases gave the following kinetic parameters for the site: equilibrium dissociation constants (KD) for L-PIA, 4.1 +/- 0.9 nmol/l; KD for NECA, 26 +/- 4 nmol/l; KD for theophylline, 28 +/- 5 mumol/l; receptor number (Bmax), 510 +/- 77 fmol/mg protein. Carbamazepine could also displace either radioligand from the sites, although solubility limits for this drug were reached at 50% receptor occupancy. The KD for carbamazepine was 138 +/- 18 mumol/l. Other anticonvulsants tested were generally ineffective at their therapeutic concentrations, although phenytoin showed a small amount of ligand binding inhibition. The results are in line with earlier studies in rodent tissue preparations, and suggest a possible purinergic component in the anticonvulsant activity of carbamazepine in a man.

Actions of angiotensin on area postrema of the rat.

Previous studies have reported that rats drink more saline after area postrema has been removed. The results presented here indicate that prolonged administration of angiotensin II into area postrema of unrestrained rats at 4 pmol/h also caused them to drink more saline. They drank more when angiotensin was released in the anterolateral part of the organ than when it was released anteromedially. Diurnal variation of drinking was not disordered. Dose-response curves showed that rats lacking area postrema drank more saline in response to systemic angiotensin than sham operated animals. The very large 'spontaneous' consumption of saline by rats lacking area postrema was not diminished by saralasin, an angiotensin antagonist. It is concluded that area postrema is a site where systemic angiotensin can act to promote sodium consumption: and that although removing area postrema increases the sensitivity of the drinking response to systemic angiotensin, this enhanced sensitivity is not the cause of the increased sodium consumption.

Optimization of freezing, storage, and thawing conditions for the preparation of metabolically active synaptosomes from frozen rat and human brain.

Samples of rat and human cerebral cortex were frozen, stored, and thawed under a variety of conditions to define further the optimal procedure for storing human brain samples for subsequent metabolic and functional studies that use incubated synaptosomes. Tissue samples were best preserved by immersing them in isotonic sucrose prior to slow freezing, but there was no advantage in first chopping up the material. High concentrations of sucrose, rather than exerting a cryoprotective effect, were detrimental to subsequent synaptosomal performance (oxygen uptake, K+ accumulation, stimulus-induced release of amino acid neurotransmitters). However, good activity was observed in preparations from rat brain frozen in the absence of fluid. This result was confirmed by studies on the uptake of gamma-aminobutyrate (GABA) into an osmotically sensitive compartment in synaptosomes prepared from frozen human autopsy material transshipped from the brain tissue collection ("brain bank") at Harvard Medical School, MA, USA, to Sydney, Australia. Although activity was slowly lost over a 3-mo period in rat tissue samples stored at -20 degrees C, there was little or no such loss at -70 degrees C.

The effect of removing area postrema on the sodium and potassium balances and consumptions in the rat.

Previous studies have indicated that rats lose weight and develop a taste preference for solutions of sodium chloride after area postrema has been removed; results in other species have suggested that area postrema may be concerned with regulating the excretion of sodium and potassium. The results reported here demonstrate that in rats the reduction of weight can be explained adequately by anorexia, that diminished excretion of sodium and potassium results from anorexia, and that the consequent slower rate of gain of weight causes diminution of sodium and potassium balances. The results also show that the rats do not drink more of a solution of sodium chloride because they eat less, and conversely that when they can drink a saline solution they do not eat more: and that they do not drink more saline because of salt loss. Furthermore, individual rats show no significant correlation between the severity of anorexia, and the increased consumption of saline. Taken together the results suggest that primary disorder of regulation of intake occurs in rats after area postrema has been removed, and that the reduced intake of food and the increased intake of sodium chloride occur independently.

Electron spin resonance studies of an animal model of human congenital myotonia: increased erythrocyte membrane fluidity in rats with 20,25-diazacholesterol-induced myotonia.

Electron spin resonance experiments have been performed on erythrocyte membranes from rats with myotonia induced by treatment with 20,25-diazacholesterol. The results suggest that erythrocyte membranes in this animal model of human congenital myotonia possess a highly significantly increased surface membrane fluidity compared to that of controls. Alterations in the physical state of membrane proteins were not apparent. These findings, also present in human congenital myotonia [Butterfield, Chesnut, Roses & Appel, 1976, Nature (London) 263:159; Butterfield, 1977 (Submitted for publication)], strengthen the concepts that increased membrane fluidity is associated with the presence of myotonia and that congenital myotonia may be a diffuse membrane disease.

Hemisphere-hand interactions for the matching of letters.

Young, middle aged, and older subjects were given a Posner-type physical-name matching task, in which stimuli were presented to one or both hemispheres, and a simple discrimination reaction time (DRT) task. Reaction time differed between young and middle aged groups for both tasks with shorter RT for the DRT task. Reaction time for the older group did not differ significantly from that of the middle aged group for either task, although the reaction time for the older group was longer. Reaction time was faster for all age groups when Stimulus 1 and Stimulus 2 were presented to different hemispheres for the matching task than when both stimuli were presented to the same hemisphere. Ipsilateral finger responses (hand relative to hemisphere) were slower than contralateral responses but this was true only with regard to the visual field of presentation for stimulus 2. These data indicate that sharing of information between hemispheres under certain circumstances increases the efficiency of information processing relative to that observed with unilateral visual half-field (hemisphere) presentations and that hand or finger response relative to visual field of presentation is an important factor which must be controlled and examined relative to visual field of presentation.