The specific mechanisms underlying general anesthesia are primarily unknown. The intravenous general anesthetic etomidate acts by potentiating GABA(A) receptors, with selectivity for beta2 and beta3 subunit-containing receptors determined by a single asparagine residue. We generated a genetically modified mouse containing an etomidate-insensitive beta2 subunit (beta2 N265S) to determine the role of beta2 and beta3 subunits in etomidate-induced anesthesia. Loss of pedal withdrawal reflex and burst suppression in the electroencephalogram were still observed in the mutant mouse, indicating that loss of consciousness can be mediated purely through beta3-containing receptors. The sedation produced by subanesthetic doses of etomidate and during recovery from anesthesia was present only in wild-type mice, indicating that the beta2 subunit mediates the sedative properties of anesthetics. These findings show that anesthesia and sedation are mediated by distinct GABA(A) receptor subtypes.

Aims: The NADPH oxidase (NOX) family of enzymes catalyzes the formation of reactive oxygen species (ROS). NOX enzymes not only have a key role in a variety of physiological processes but also contribute to oxidative stress in certain disease states. To date, while numerous small molecule inhibitors have been reported (in particular for NOX2), none have demonstrated inhibitory activity in vivo. As such, there is a need for the identification of improved NOX inhibitors to enable further evaluation of the biological functions of NOX enzymes in vivo as well as the therapeutic potential of NOX inhibition. In this study, both the in vitro and in vivo pharmacological profiles of GSK2795039, a novel NOX2 inhibitor, were characterized in comparison with other published NOX inhibitors. Results: GSK2795039 inhibited both the formation of ROS and the utilization of the enzyme substrates, NADPH and oxygen, in a variety of semirecombinant cell-free and cellbased NOX2 assays. It inhibited NOX2 in an NADPH competitive manner and was selective over other NOX isoforms, xanthine oxidase, and endothelial nitric oxide synthase enzymes. Following systemic administration in mice, GSK2795039 abolished the production of ROS by activated NOX2 enzyme in a paw inflammation model. Furthermore, GSK2795039 showed activity in a murine model of acute pancreatitis, reducing the levels of serum amylase triggered by systemic injection of cerulein. Innovation and Conclusions: GSK2795039 is a novel NOX2 inhibitor that is the first small molecule to demonstrate inhibition of the NOX2 enzyme in vivo. Antioxid. Redox Signal. 23, 358-374.

In the mammalian central nervous system, transporter-mediated reuptake may be critical for terminating the neurotransmitter action of D-serine at the strychnine insensitive glycine site of the NMDA receptor. The Na(+) independent amino acid transporter alanine-serine-cysteine transporter 1 (Asc-1) has been proposed to account for synaptosomal d-serine uptake by virtue of its high affinity for D-serine and widespread neuronal expression throughout the brain. Here, we sought to validate the contribution of Asc-1 to D-serine uptake in mouse brain synaptosomes using Asc-1 gene knockout (KO) mice. Total [(3)H]D-serine uptake in forebrain and cerebellar synaptosomes from Asc-1 knockout mice was reduced to 34 +/- 5% and 22 +/- 3% of that observed in wildtype (WT) mice, respectively. When the Na(+) dependent transport components were removed by omission of Na(+) ions in the assay buffer, D-serine uptake in knockout mice was reduced to 8 +/- 1% and 3 +/- 1% of that measured in wildtype mice in forebrain and cerebellum, respectively, suggesting Asc-1 plays a major role in the Na(+) independent transport of D-serine. Potency determination of D-serine uptake showed that Asc-1 mediated rapid high affinity Na(+) independent uptake with an IC(50) of 19 +/- 1 microm. The remaining uptake was mediated predominantly via a low affinity Na(+) dependent transporter with an IC(50) of 670 +/- 300 microm that we propose is the glial alanine-serine-cysteine transporter 2 (ASCT2) transporter. The results presented reveal that Asc-1 is the only high affinity D-serine transporter in the mouse CNS and is the predominant mechanism for D-serine reuptake.