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Abstract
GABA is the major inhibitory neurotransmitter in the mammalian CNS. Dysfunction of the GABA system has been implicated in a wide variety of diseases. The GABA$\sb{\rm A}$ receptor chloride channel complex contains binding sites for GABA, benzodiazepines, barbiturates, neurosteroids, and picrotoxin.
Previous binding studies have reported binding affinities for GABA and the agonist muscimol to be in the low nanomolar range (K$\sb{\rm d}$ $<$ 200 nM). This is inconsistent with the approximately micromolar concentrations typically required to elicit a functional response. Using a novel tissue preparation, synaptoneurosomes, a new ligand binding protocol was developed to address this inconsistency. Synaptoneurosomes are vesicular structures containing both pre- and post-synaptic elements that have the ability to maintain a membrane potential. This protocol represents a more physiologically relevant system as compared to standard methodologies using simple brain membrane fragments. A significant advantage of this new protocol is the improved correlation observed between binding affinities for GABA agonists and antagonists and concentrations which elicit functional activity.
The synaptoneurosomal binding protocol was used in conjunction with recently developed methods for measuring GABA-mediated $\sp{36}$Cl$\sp-$ flux in the same tissue preparation (Harris and Allan, 1985). Combination of these methodologies provides a convenient and accessible means to quantify drug/receptor interactions within a framework of physiological relevance. This approach is applied here in the re-evaluation of the pharmacology of the GABA$\sb{\rm A}$ receptor with respect to anxiety and anesthesia, the mechanisms of which are believed to involve the GABA$\sb{\rm A}$ receptor complex. Using the synaptoneurosomal binding protocol, it was possible to correlate benzodiazepine binding with anxiolytic activity relative to the effect temperature has on the binding constants for the benzodiazepines diazepam, hydazepam, and BD-798.
Anesthetic agents were evaluated by binding and $\sp{36}$Cl$\sp-$ uptake in synaptoneurosomes. Combination of diazepam and pentobarbital demonstrated synergism in both induction of anesthesia and enhancement of ($\sp3$H) muscimol binding. However, benzodiazepine-barbiturate combinations failed to produce supra-additive enhancement of $\sp{36}$Cl$\sp-$ flux. Since the interaction between benzodiazepines and barbiturates does not display synergistic enhancement of Cl$\sp-$ conductance, the mechanistic basis for anaesthetic synergism may involve other non-GABAergic components.
