Resistance to 3-mercaptopropionic acid-induced seizures in hepatic encephalopathy.

BACKGROUND/AIMS: To determine if a model of hepatic encephalopathy (HE) exhibits decreased sensitivity to the neuronal effects of a drug that induces seizures as a consequence of decreasing GABA-mediated inhibitory neurotransmission. MATERIALS AND METHODS: 3-Mercaptopropionic Acid (MPA) is an inhibitor of L-glutamate decarboxylase which catalyzes the synthesis of GABA from glutamate. MPA was administered, either by intraperitoneal or intracerebroventricular injection, into rats with stage III HE due to thioacetamide-induced fulminant hepatic failure and into normal control rats. RESULTS: When MPA was administered by intraperitoneal injection, seizure-inducing doses were similar for rats with HE and control rats. However, when a constant dose of MPA (330 micrograms) was administered by intracerebroventricular injection, rats with HE took significantly longer to develop seizures than control rats (16.2 vs. 7.3 minutes; p < 0.0005). CONCLUSIONS: In a model of HE: (i) There is increased resistance to the convulsive effects of MPA; and (ii) This phenomenon is apparent when MPA is given centrally, but not when it is given peripherally. Increased resistance to the development of a complication of reduced GABA-mediated neurotransmission induced by MPA in the model provides support for the hypothesis that HE is associated with increased GABA-mediated inhibitory neurotransmission.

Brain concentrations of benzodiazepines are elevated in an animal model of hepatic encephalopathy.

Brain extracts from rats with hepatic encephalopathy due to thioacetamide-induced fulminant hepatic failure contained 4- to 6-fold higher concentrations of substances that inhibit radioligand binding to benzodiazepine receptors than corresponding control rat extracts. Both isocratic and gradient-elution HPLC indicated that this inhibitory activity was localized in 3-8 peaks with retention times corresponding to deschlorodiazepam, deschlorolorazepam, lorazepam, oxazepam, diazepam, and N-desmethyldiazepam. The presence of diazepam and N-desmethyldiazepam was confirmed by mass spectroscopy. Both mass spectroscopic and radiometric techniques indicated that the concentrations of N-desmethyldiazepam and diazepam in brain extracts from encephalopathic rats were 2-9 and 5-7 times higher, respectively, than in control brain extracts. While benzodiazepines have been identified previously in mammalian and plant tissues, this report demonstrates that concentrations of these substances are increased in a pathophysiological condition. These findings provide a rational basis for the use of benzodiazepine receptor antagonists in the management of hepatic encephalopathy in humans.

Reversal of the behavioral and electrophysiological abnormalities of an animal model of hepatic encephalopathy by benzodiazepine receptor ligands.

Behavioral and electrophysiological evidence implicating the GABA-benzodiazepine receptor complex in the pathogenesis of hepatic encephalopathy was obtained using an improved rat model of hepatic encephalopathy caused by thioacetamide-induced fulminant hepatic failure. After the administration of thioacetamide together with supportive therapy, acute hepatocellular failure developed in rats as a result of massive hepatocellular necrosis without evidence of renal failure or hypoglycemia. The evolution of hepatic encephalopathy in this model was sufficiently slow to readily permit the staging of the syndrome. Prominent features of the encephalopathy include a marked reduction in open field activity and an abnormal visual evoked response. Both the deficits in spontaneous motor function and visual evoked response abnormalities of rats in stages III to IV hepatic encephalopathy were significantly improved after the administration of the benzodiazepine receptor ligands flumazenil or Ro 15-4513. Doses of flumazenil or Ro 15-4513 that produced these effects in rats with hepatic encephalopathy had no detectable action on either the behavior or the visual evoked responses of normal rats. The ability of benzodiazepine receptor ligands to ameliorate both the behavioral depression and the visual evoked response abnormalities associated with hepatic encephalopathy in the thioacetamide-induced rat model suggest an involvement of the GABA/benzodiazepine receptor complex in the pathogenesis of hepatic encephalopathy. In addition, the similarity of these observations to those in rabbits with hepatic encephalopathy caused by galactosamine-induced fulminant hepatic failure is compatible with the hypothesis that the mechanisms of hepatic encephalopathy in these two distinct models share a common final pathway, the allosteric enhancement of GABAergic tone through the benzodiazepine receptor.(ABSTRACT TRUNCATED AT 250 WORDS)

The GABAA receptor complex in hepatic encephalopathy. Autoradiographic evidence for the presence of elevated levels of a benzodiazepine receptor ligand.

Autoradiographic analysis was used to examine radioligand binding to benzodiazepine (BZ) and GABAA receptors in the brains of rabbits with hepatic encephalopathy (HE). Thin sections of whole brain from normal rabbits and rabbits with HE were mounted on slides and subdivided into two groups. One group was washed before incubation with radioligand, while the second group was not prewashed. [3H]Flunitrazepam binding to BZ receptors was decreased by 22% to 42% (p less than 0.05) in the cerebral cortex, superior and inferior colliculi, and cerebellum of unwashed sections from rabbits with HE compared to all other groups. The binding of [3H]Ro 15-1788 to unwashed sections from rabbits with HE was reduced by a similar degree (18% to 37%, p less than 0.05) in the cerebral cortex, hippocampus, superior colliculus, and cerebellar cortex. Incubation of sections with the GABA-mimetic muscimol and NaCl produced an additional decrease in [3H]flunitrazepam binding to the cortex and hippocampus (25% to 31%, p less than 0.05) in unwashed HE rabbit brain, but increased radioligand binding (27% to 71%, p less than 0.05) to several regions in control rabbits. No changes in radioligand binding to either GABAA or peripheral benzodiazepine receptors was observed between HE and control rabbit sections. These findings are consistent with previous electrophysiologic and neurochemical observations indicating no significant changes in either the function or density of GABAA or BZ receptors in this model of HE. Further, they indicate that a reversible BZ receptor ligand with agonist properties is present in the brain in HE. This substance may contribute to the enhancement of GABAergic tone observed in this syndrome.

The involvement of the benzodiazepine receptor in hepatic encephalopathy: evidence for the presence of a benzodiazepine receptor ligand.

The involvement of GABAergic systems in the pathogenesis of HE was supported by electrophysiologic studies of single Purkinje neurons from rabbits with HE which demonstrated the hypersensitivity of these neurons to depression by GABA and BZ receptor agonists. In contrast, these neurons were excited by BZ receptor antagonists. At concentrations which had no effect on neuronal activity, BZ receptor antagonists also reversed the hypersensitivity of HE neurons to depression by muscimol. This combination of neuronal responses is consistent with an increase in the concentration or availability of a ligand for the BZ receptor with agonist properties in the brains of rabbits with HE. Subsequent neurochemical studies support these electrophysiologic observations. Autoradiographic techniques indicated the presence of a reversible inhibitor of [3H]Ro 15-1788 and [3H]flunitrazepam binding to the cerebral and cerebellar cortices of rabbits with HE. The ability of this substance to inhibit [3H]flunitrazepam binding to HE rabbit brain sections was further enhanced in the presence of NaCl and GABA. The autoradiographic studies suggested that the density and affinity of the components of the GABA-BZ receptor complex are unaltered in this animal model of HE. This inference is fully supported by the subsequent studies of radioligand binding to well-washed membrane preparations. Finally, extracts of HE rabbit brains yielded a family of substances with the properties of BZ receptor agonists. These substances may include, but are not limited to, diazepam, oxazepam and desmethyldiazepam, but do not include substances commonly elevated in the plasma and CSF of patients with HE4. The positive identification of these substances awaits confirmation by mass-spectroscopic analysis. However, the precedent for the presence of a family of benzodiazepines in animals that were not administered these drugs has been set. The origin of these substances is a matter of ongoing research. Several studies have shown the presence of benzodiazepines in plant and animal materials. It is possible that these "endogenous" benzodiazepines are the result of contamination of the food chain. A normally functioning liver would capture and metabolize these compounds after their absorption from the gut. This function of the liver would be impaired in liver failure, thus allowing sufficient levels of BZ receptor agonists to accumulate in the CNS, contributing to the pathogenesis of HE. However, studies by DeBlas and coworkers have reported that 1,4 benzodiazepines are present in human brains preserved prior to the commercial use of these compounds. Further, they have found benzodiazepines in cell lines cultured without potential exogenous sources of benzodiazepines.(ABSTRACT TRUNCATED AT 400 WORDS)

GABAA receptor complex in an experimental model of hepatic encephalopathy: evidence for elevated levels of an endogenous benzodiazepine receptor ligand.

The involvement of the gamma-aminobutyric acidA (GABAA) receptor complex in the pathogenesis of hepatic encephalopathy was examined in thioacetamide-treated rats with fulminant hepatic failure. Partially purified extracts from encephalopathic rat brain were approximately three times more potent in inhibiting [3H]Ro 15-1788 binding to benzodiazepine receptors than identically prepared extracts from control rats. High levels of inhibitory activity were also found in extracts of plasma, heart, and liver from thioacetamide-treated rats. The inhibition of [3H]Ro 15-1788 binding by brain extracts appeared to be competitive and reversible and was unaffected by treatment with either proteolytic enzymes or boiling. Further, GABA significantly enhanced the potency of these extracts in inhibiting [3H]flunitrazepam binding. In contrast, no differences were found in radioligand binding to the constituent recognition sites of the GABAA receptor complex in well-washed brain membranes prepared from control and encephalopathic animals. These findings suggest that the recognition-site qualities of the constituent proteins of the GABAA receptor complex are unchanged in an experimental model of hepatic encephalopathy. However, significant elevations in the level of a substance or substances with neurochemical properties characteristic of a benzodiazepine receptor agonist may contribute to the electrophysiological and behavioral manifestations of hepatic encephalopathy.

Hepatic encephalopathy.

Hepatic encephalopathy (HE) is a complex reversible syndrome that can progress to coma. Recently, behavioral and electrophysiologic ameliorations of HE have been reported to occur in animal models of fulminant hepatic failure (FHF) and, in uncontrolled studies, in a majority of patients with FHF or cirrhosis following the intravenous administration of the benzodiazepine (BZ) receptor antagonist, flumazenil. These observations, while not excluding a role for other mechanisms in the mediation of HE, raise the possibility that an endogenous BZ receptor ligand with agonist properties may contribute to the manifestations of HE by allosterically potentiating GABA-mediated neurotransmission.

NIH conference. The gamma-aminobutyric acid A (GABAA) receptor complex and hepatic encephalopathy. Some recent advances.

Increased neural inhibition appears to be an important component of the syndrome of hepatic encephalopathy. The pathways subserved by the gamma-aminobutyric acid (GABA)-benzodiazepine receptor complex are the principal inhibitory systems in the mammalian brain. Hyperpolarization of neural membranes is accomplished by an increase in transmembrane chloride flux through a GABA-gated chloride channel in the complex. The opening of the chloride channel is induced by the binding of GABA to its receptors, and it is potentiated by barbiturates or benzodiazepines that act at distinct recognition sites on the complex. Involvement of the GABA neurotransmitter system in hepatic encephalopathy is suggested by several findings in animal models of fulminant hepatic failure. For example, hepatic encephalopathy resembles encephalopathies induced by drugs (including benzodiazepines) that potentiate GABAergic neurotransmission. In addition, neurons from animals with hepatic encephalopathy show increased sensitivity to benzodiazepine and GABA receptor agonists. Moreover, these neurons are excited by benzodiazepine receptor antagonists at concentrations that do not affect control neurons. Also, elevated levels of a substance that inhibits radioligand binding to benzodiazepine receptors have been found in cerebrospinal fluid from animals with hepatic encephalopathy. Furthermore, manifestations of hepatic encephalopathy can be ameliorated by benzodiazepine receptor antagonists. The relevance of these findings to hepatic encephalopathy in human beings is supported by clinical observations showing that a benzodiazepine receptor antagonist can lessen the degree of hepatic encephalopathy. These findings suggest that an endogenous substance with benzodiazepine-like properties contributes to the neuropsychiatric manifestations of hepatic encephalopathy by augmenting GABAergic neurotransmission.

Differential responsiveness of cerebellar Purkinje neurons to GABA and benzodiazepine receptor ligands in an animal model of hepatic encephalopathy.

The role of the GABA-benzodiazepine receptor complex in the pathogenesis of hepatic encephalopathy was investigated by recording the electrophysiological responses of single cerebellar Purkinje neurons from rabbits with hepatic encephalopathy due to galactosamine-induced fulminant hepatic failure. Both the GABAmimetic muscimol and the benzodiazepine receptor agonist flunitrazepam were 3-4 times more potent in depressing the spontaneous activity of Purkinje neurons from rabbits with hepatic encephalopathy than from control animals. Furthermore, qualitatively different responses of Purkinje neurons to benzodiazepine receptor antagonists (Ro 15-1788 and Ro 14-7437) were found in controls and rabbits with hepatic encephalopathy. These compounds markedly excited Purkinje neurons from rabbits with hepatic encephalopathy, but had either no effect (Ro 14-7437) or partially suppressed (Ro 15-1788) the spontaneous activity of neurons from control animals. In addition, incubation of Purkinje neurons from rabbits with hepatic encephalopathy with subthreshold concentrations of Ro 14-7437 reduced their sensitivity to muscimol, whereas treatment of control neurons with Ro 14-7437 had no effect on their sensitivity to muscimol. Finally, Purkinje neurons from hepatic encephalopathy and control rabbits displayed no difference in sensitivity to the depressant actions of the alpha-adrenoceptor agonist phenylephrine. These findings demonstrate a differential responsiveness of Purkinje neurons from an animal model of hepatic encephalopathy to ligands that interact with the GABA-benzodiazepine receptor complex. Furthermore, the observations made in this experimental model are consistent with the involvement of the GABA-benzodiazepine receptor complex in mediating hepatic encephalopathy, and provide a potential explanation for the reported efficacy of benzodiazepine receptor antagonists in ameliorating this syndrome.