Gamma-hydroxybutyrate (GHB) has gradually gained a foothold as a substance of abuse in the U.S. and has a reputation as being used as a """"""""date-rape"""""""" drug, particularly in the presence of alcohol. Increasingly, incidents of untoward reactions and even death have been reported with GHB misuse. Administration of this drug results in a marked central nervous system (CNS) depressant action manifested by sedation and coma at higher doses. These effects can be accompanied by seizures, particularly in experimental animals. Owing to its pronounced CNS actions and to the fact that it is an endogenous brain substance, GHB is considered to be a neurotransmitter or a substance with a significant role in regulating neuronal function. Part of GHB action appears intimately tied to the GABAergic system. For this cross-disciplinary pilot project we propose to study the process of GHB signal transduction in both cultured neurons and animal nervous tissue.
In Aim 1, we plan to use neuroblastoma hybrid NCB-20 cultures to investigate the effects of GHB on forskolin-induced cAMP production and the ability of GHB to activate pertussis toxin sensitive G-proteins. These studies are expected to lead to the identification of a subfamily of G-proteins associated with the GHB receptor.
In Aim 2, this GHB receptor-expressing cell line will be used to study the involvement of plasma membrane Ca2+ regulatory mechanisms (calcium channels, sodium-calcium exchanger, Ca2+ pump, passive Ca2+ permeability) in GHB signal transduction.
For Aim 3, we will map the regional distribution of GHB transport sites in the brain and ascertain the kinetic specificity of the GHB transporter for its substrate. Finally in Aim 4, based on the hypothesis that GHB activity at the GABAA receptors is dependent on their subunit composition, we will study the effects of GHB on GABA-stimulated chloride influx in different brain areas and in cultured cells expressing GABAA receptors composed of different subunits. These studies may reveal the GABAA receptor subtype specificity for the effects of GHB and possibly new targets for drugs to treat GHB dependence and toxicity. The exploratory, four-pronged approach of this project promises to expand our current, limited understanding of how GHB functions as a neuronal regulatory chemical and how its pharmacological effects are linked to cellular events. In turn, this will help to develop therapeutic and preventive strategies resulting in a lessened abuse potential of GHB.
