Normal coupling of brain benzodiazepine and neurosteroid modulatory sites on the GABA-A receptor complex in human hepatic encephalopathy
Introduction
GABA-A receptors are ligand-gated ion channels made up of transmembrane hetero-oligomeric proteins. They form a pentameric assembly of various subunits, to which several drugs including barbiturates and benzodiazepines as well as neurosteroids bind and elicit their effects (Mehta and Ticku, 1999). Increase of GABA-A receptors and their associated benzodiazepine modulatory sites have been described in brains of animals with galactosamine-induced acute liver failure (Schafer et al., 1983, Baraldi et al., 1984). However, these findings were not confirmed in other experimental animal models of liver failure (Roy et al., 1988, Mans et al., 1992) or in human hepatic encephalopathy (HE; Butterworth et al., 1988). On the other hand, substances with allosteric agonist properties at both the benzodiazepine (Olasmaa et al., 1990, Basile et al., 1994) and the neurosteroid (Itzhak et al., 1995, Ahboucha et al., 2002) sites have been described in brain in HE, and the benzodiazepine receptor antagonist flumazenil has consistently been found to be effective in reversing HE albeit in a subset of patients (Grimm et al., 1988, Pomier-Layrargues et al., 1994). The mechanism whereby flumazenil exerts this beneficial effect is unclear but could result from an inhibitory effect on the action of endogenous benzodiazepines or on altered coupling between the benzodiazepine/GABA-A/neurosteroid sites on the receptor complex.
In order to address this latter issue, the present study was undertaken to assess the functional coupling between the benzodiazepine and neurosteroid modulatory sites to the GABA recognition site on the GABA-A receptor complex in autopsied brain tissue from cirrhotic patients who died in hepatic coma compared to material from appropriately-matched controls. The effect of the benzodiazepine agonist flunitrazepam on the binding of muscimol was investigated together with the effect of the neurosteroid agonist (5α-pregnan-3α-ol-20-one; allopregnanolone) on the binding of both muscimol and flunitrazepam.
Section snippets
Chemicals
flunitrazepam (specific activity 84.5 Ci/mmol), flumazenil (specific activity 78.6 Ci/mmol) and muscimol (specific activity 28.5 Ci/mmol) were purchased from Perkin-Elmer (Life Sciences Inc., Boston, MA) and dissolved in 50 mM Tris–HCl buffer, pH 7.4. Allopregnanolone (5α-pregnan-3α-ol-20-one) was purchased from Sigma–Aldrich (Oakville, Ontario, Canada).
Materials
Postmortem brain tissue was obtained from six cirrhotic patients who died in hepatic coma and six control subjects who were free from
Results
Patient characteristics (cirrhotics and controls) are summarized in Table 1. Groups were rigorously matched for age, gender, and time between death and sample freezing. The differences between these parameters in the two groups were not statistically significant by Student’s t-test.
The binding of muscimol to the GABA recognition site in the frontal cortex and flunitrazepam and flumazenil to the benzodiazepine site revealed comparable values for both Kd and Bmax parameters between
Discussion
Results of the present study reveal unaltered densities and affinities of binding sites for the GABA recognition site agonist muscimol and the benzodiazepine modulatory site agonist flunitrazepam and antagonist flumazenil in frontal cortical samples obtained at autopsy from cirrhotic patients who died in hepatic coma compared to similar samples from appropriately-matched controls. Furthermore, the present study demonstrates that the functional coupling between the benzodiazepine
Acknowledgements
Studies described were funded by a grant from CIHR (Canada).
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