Wendy E. J. Watson

Alterations in Cortical [3H]Kainate and α-[3H]Amino-3-Hydroxy-5-Methyl-4-Isoxazolepropionic Acid Binding in a Spontaneous Canine Model of Chronic Hepatic Encephalopathy

Abstract: 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‐imine maleate ([3H]MK‐801), [3H]kainate, and α‐[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.

ADENOSINE RECEPTORS IN POST-MORTEM HUMAN CEREBRAL CORTEX AND THE EFFECT OF CARBAMAZEPINE

1. 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.

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 γ-aminobutyric acid uptake in synaptosomes from the cerebral cortex of dogs with congenital chronic hepatic encephalopathy

High affinity [3H]γ-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.

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>α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA)=quisqualate=kainate>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.

Cerebellar [3H]kainate and [3H]ampa binding in dogs with congenital portosystemic encephalopathy

Abstract Previous studies have indicated that kainate and AMPA receptors are altered in cerebral cortex of dogs with chronic hepatic encephalopathy (HE). To ascertain whether receptors in dog cerebellum are similarly altered in HE [3H]kainate and [3H]α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) binding assays were performed on crude synaptosomal membranes prepared from cerebellar tissue from dogs with congenital portosystemic encephalopathy (PSE) and control dogs. There was no pathophysiologically relevant difference in the affinity or density of kainate or AMPA binding sites in PSE cerebellar tissue compared with control dogs. The failure to demonstrate alterations in these binding parameters in cerebellar tissue was expected as clinical signs of HE reflect cortical rather than cerebellar dysfunction.

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.