Differential effects of ammonia on the benzodiazepine modulatory site on the GABA-A receptor complex of human brain

Abstract

Ammonia is a key factor in the pathogenesis of encephalopathies associated with liver failure. A direct effect of ammonia on GABAergic neurotransmission was proposed as a mechanism that may explain its neurotoxic effect on the basis of electrophysiological and biochemical studies performed in animal models of liver failure. In the present study, we investigated using a radiometric assay the effect of ammonia on the binding of GABA-A receptor ligands to membranes from normal human brains. Ammonium tartrate significantly decreased the maximal binding of [ 3H]flunitrazepam to well-washed frontal cortical membranes (366 ± 63 fmol/mg protein in absence of ammonia versus 294.1 ± 51 fmol/mg protein in presence of 2 mM ammonia; p < 0.05). The efficacy of the effects of ammonia was within the millimolar range (IC 50 = 4.8 mM). This effect was not seen in cerebellum or hippocampus. Ammonia exposure decreased the maximal binding of [ 3H]flumazenil (284.9 ± 24.2 fmol/mg protein in absence of ammonia versus 146.4 ± 15.6 fmol/mg protein in presence of 2 mM ammonia; p < 0.01). This effect was seen with a greater potency ( I max = 32.4%) and a lower IC 50 (0.1 mM). Inhibition of [ 3H]flumazenil binding was significant in all brain regions. The apparent ammonia-induced decrease of [ 3H]flunitrazepam and [ 3H]flumazenil binding was due to a decrease in the binding affinities of these ligands for the benzodiazepine site. In contrast, ammonium tartrate exposure did not cause significant changes to the binding of [ 3H]muscimol in any brain region. These findings demonstrate that ammonia interacts negatively with components of the benzodiazepine-associated site at the GABA-A receptor complex in human brain in contrast to previous reports in the rat, and thus, does not support the notion that ammonia directly activates the GABA-A receptor complex resulting in increased GABAergic neurotransmission in human hepatic encephalopathy. These findings also suggest that positron emission tomography studies in cirrhotic patients using [ 11C]flumazenil may be underestimating GABA-A receptor sites depending upon the degree of hyperammonemia of the patient.