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

doi:10.1016/j.neuint.2005.04.007

Neurochemistry International

Volume 47, Issues 1–2, July 2005, Pages 58-63

https://doi.org/10.1016/j.neuint.2005.04.007 Get rights and content

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 (IC₅₀ = 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 IC₅₀ (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.

Introduction

Ammonia is well known for its neurotoxicity and is considered to be a key factor in the pathogenesis of encephalopathies associated with either liver failure or congenital hyperammonemias (Raabe, 1988). An effect of ammonia on the GABA-A receptor complex was suggested as one of the pathophysiologic mechanisms that explain the neuroinhibition characteristic of end-stage liver failure in patients with hepatic encephalopathy (HE) (Basile and Jones, 1997). GABAergic neurotransmission is mediated by GABA that activates the post-synaptic GABA-A receptor complex, a specific ligand-gated ion channel selective for chloride, which following activation allows chloride to enter and inhibit the post-synaptic neuron. GABA-A receptors form a pentameric (hetero-oligomeric) transmembrane assembly of several subunits to which drugs such as barbiturates, benzodiazepines as well as neurosteroids bind and elicit their effects (Mehta and Ticku, 1999). Ammonia, in concentrations that commonly occur in patients with acute and chronic liver failure (less than 1 mM), facilitates GABA-A receptor-gated Cl⁻ currents in rat cultured cortical neurons (Takahashi et al., 1993) and enhances synergistically the binding of ligands to the GABA recognition ([³H]muscimol) and the benzodiazepines-associated ([³H]flunitrazepam) sites on the GABA-A receptor complex in rat brain membranes (Ha and Basile, 1996). Based on these studies performed in material from experimental animals, it was postulated that ammonia could enhance the binding of accumulated endogenous benzodiazepine ligands in HE patients (Mullen et al., 1990, Olasmaa et al., 1990) and increase GABAergic neurotransmission (Basile and Jones, 1997).

In the present study, we investigated the effect of ammonia on the binding of GABA-A receptor ligands to membranes from human brain preparations using a radiometric assay. The effect of various ammonium salts was evaluated in relation to GABA-A receptor sites (GABA recognition and benzodiazepine sites) in three brain regions (frontal cortex, cerebellum, and hippocampus).

Section snippets

Chemicals

[³H]muscimol (specific activity 28.5 Ci/mmol), [³H]flunitrazepam (specific activity 84.5 Ci/mmol), and [³H]flumazenil (specific activity 78.6 Ci/mmol) were purchased from Perkin-Elmer (Life Sciences Inc., Boston, MA) and dissolved in 50 mM Tris–HCl buffer, pH 7.4. Ammonium tartrate, ammonium chloride, and ammonium acetate were purchased from Sigma–Aldrich (Oakville, Ont., Canada).

Materials

Post-mortem brain tissue was obtained from four subjects who were free from systemic, neurologic, or psychiatric

Results

Ammonium tartrate significantly decreased the binding of [³H]flunitrazepam to well-washed frontal cortical membranes from normal human subjects in a dose-dependent manner. A significant effect was seen with a concentration as low as 1 mM (p < 0.01, ANOVA). The maximal inhibition (I_max) was 18.5% (Table 1; Fig. 1). Binding site densities in the absence of ammonia were 366.0 ± 63 fmol/mg protein compared to 294.1 ± 51 fmol/mg protein in presence of 2 mM ammonia (p < 0.05, t-test). Exposure to ammonium salts

Discussion

Results of the present study show that ammonia has no significant effects on the binding of [³H]muscimol, but inhibits with millimolar affinities the binding of [³H]flunitrazepam, or with submillimolar affinities the binding of [³H]flumazenil to brain membrane preparations from normal human subjects. These findings contrast with previous studies in rodent brain. Ha and Basile (1996), with a comparable radiometric procedure to that used in the present study, showed a biphasic effect of ammonia

Acknowledgements

The authors thank the Canadian Institute of Health Research (CIHR) for funding and the University of Maryland Human Brain Bank for some of the samples used in this study.

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

Abstract

Introduction

Section snippets

Chemicals

Materials

Results

Discussion

Acknowledgements

Neurochem. Int.

Neurochem. Int.

Eur. J. Pharmacol.

Brain Res.

Brain Res. Rev.

Lancet

J. Steroid Biochem. Mol. Biol.

Neurosci. Lett.

Hepatology

Unequivocal evidence for a role of neurosteroids with positive allosteric modulatory properties at the GABA-A receptor in the pathogenesis of hepatic encephalopathy

Hepatology

Increased brain concentrations of endogenous (non-benzodiazepine) GABA-A receptor ligands in human hepatic encephalopathy

Metab. Brain Dis.

Ammonia and GABA-ergic neurotransmission: interrelated factors in the pathogenesis of hepatic encephalopathy

Hepatology

Affinities and densities of high-affinity [3H]muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy

Hepatology

Affinities and densities of high-affinity [³H]muscimol (GABA-A) binding sites and of central benzodiazepine receptors are unchanged in autopsied brain tissue from cirrhotic patients with hepatic encephalopathy