Beta-adrenergic receptor activation increases GABA uptake in adolescent mice frontal cortex: Modulation by cannabinoid receptor agonist WIN55,212-2.

GABA transporters regulate synaptic GABA levels and dysfunctions in this system might result in psychiatric disorders. The endocannabinoid system (ECS) is the main circuit breaker in the nervous system and may alter noradrenaline (NA) communication, which in turn modulates the release of GABA. However, a close relationship between these systems has not been recognized. We asked whether NA and ECS might control extracellular GABA levels in slices of frontal cortex (FC) of adolescent Swiss mice with 40 days after birth (PN40). Here we show that NA and isoproterenol (ISO), a beta-adrenergic agonist, increased [3H]-GABA uptake in mice FC, while alpha1-adrenergic agonist phenylephrine had no effect. As GAT-1 is expressed and fully functional at the FC, addition of NO-711, a GAT-1 inhibitor, dose dependently blocked [3H]-GABA uptake. The increase of [3H]-GABA uptake induced by ISO was also blocked by NO-711. [3H]-GABA release induced by 80 mM KCl was reduced by NO-711, but not by removal of Ca2+. ISO also increased cyclic AMP (cAMP) levels and addition of WIN 55,212-2, a mixed CB1/CB2 receptor agonist, inhibited the effect of ISO in GABA uptake increase, GAT-1 expression and cAMP levels compared to control. Our data show that GABA transport increased by NA and ISO is negatively regulated by cannabinoid receptor agonist WIN55,212-2.

Evaluation of anxiolytic-like, anticonvulsant, antidepressant-like and antinociceptive properties of new 2-substituted 4-hydroxybutanamides with affinity for GABA transporters in mice.

PURPOSE: The inhibition of plasma membrane GABA transporters (GATs) is responsible for anxiolytic-like, anticonvulsant, antinociceptive and antidepressant-like effects in mice. It also influences animals' motor coordination and their sensitivity to ethanol. The aim of this study was to assess the pharmacological activity of two novel 2-substituted 4-hydroxybutanamides (BM 130 and BM 131) in some screening models. An attempt has been made to establish the relationship between the inhibition of GAT subtype and the observed in vivo activity. METHODS: The affinity for GAT subtypes was evaluated by means of [(3)H]GABA uptake assay. It indicated that BM 130 inhibited GAT1 and GAT2, whereas BM 131 inhibited GAT1 and GAT3. In mice anxiolytic-like, antidepressant-like, anticonvulsant and antinociceptive properties of the test compounds were assessed. Their influence on motor coordination, locomotor activity and the ability to potentiate effects of subnarcotic doses of ethanol was also tested. RESULTS: Both compounds administered intraperitoneally exerted a significant anxiolytic-like effect in the four plate test with ED50 values 3.4 and 7.9 mg/kg, respectively. At 30 mg/kg they reduced duration of immobility in the forced swim test for 33% and 19%, respectively. They had no effect on electroconvulsive threshold or pain reactivity in the hot plate assay but they were antinociceptive in the acetic acid-induced writhing test (ED50 values were 12.7 and 18.6 mg/kg, respectively) and in both phases of the formalin test (ED50 values in the first phase were 10.2 and 2.1 mg/kg for BM 130 and BM 131, respectively). No motor adverse effects were observed in mice pretreated with the test compounds in the rotarod or chimney tests but BM 131 caused a transient but statistically significant decrease of animals' locomotor activity. CONCLUSIONS: In mice BM 130 and BM 131 have anxiolytic-like, antidepressant-like and antinociceptive properties which can be attributed to their affinity for not only mGAT1 but also mGAT2-4.

The four human gamma-aminobutyric acid (GABA) transporters: pharmacological characterization and validation of a highly efficient screening assay.

The neurotransmission mediated by gamma-aminobutyric acid (GABA) in the mammalian brain is terminated by a family of four GABA transporters (GATs). Inhibition of GATs is currently used in the treatment of epilepsy and these proteins are generally considered as important drug targets. In this study, we perform the first elaborate pharmacological characterization of all four human GAT subtypes. We conduct the experiments in parallel in a [3H]GABA uptake assay using 14 standard GAT substrates and inhibitors. This setup enables direct comparison of the absolute values of inhibitory activities of the compounds between the different GAT subtypes. The results are overall in agreement with data reported by other groups for the orthologous murine GATs. However, there do seem to be some minor variations among species. In contrast to the several subtype selective ligands identified for the GAT-1 subtype, no subtype selective ligands have been reported for the three remaining GATs. Given the potential therapeutic relevance of the individual GAT subtypes, a search for novel structures displaying selectivities for specific GAT subtypes is important. In this study, we validate our [3H]GABA uptake assay for use in high throughput screening. We find that the assay is categorized by high Z'-factors (Z' > 0.5) for all four GAT subtypes, demonstrating that the assay is excellent for a high throughput screen. This [3H]GABA uptake assay therefore enables future high throughput screening of compound libraries at the four human GATs.