γ-Hydroxybutyrate Reduces Mitogen-activated Protein Kinase Phosphorylation via GABAB Receptor Activation in Mouse Frontal Cortex and Hippocampus

Xiuhai Ren,Istvan Mody

doi:10.1074/jbc.m304238200

Abstract

gamma-Hydroxybutyrate (GHB) naturally occurs in the brain, but its exogenous administration induces profound effects on the central nervous system in animals and humans. The intracellular signaling mechanisms underlying its actions remain unclear. In the present study, the effects of GHB on the activation (phosphorylation) of mitogen-activated protein kinases (MAP kinases), extracellular signal-regulated kinase 1 and 2 (ERK1/2), were investigated. Acute administration of GHB (500 mg/kg, intraperitoneal) induced a fast and long lasting inhibition of MAP kinase phosphorylation in both frontal cortex and hippocampus. The reduced MAP kinase phosphorylation was observed in the CA1 and CA3 areas but not in the dentate gyrus. Pretreatment with the specific gamma-aminobutyric acid, type B (GABAB), receptor antagonist CGP56999A (20 mg/kg, intraperitoneal) prevented the action of GHB, and the effect of GHB was mimicked by baclofen, a selective GABAB receptor agonist, whereas the high affinity GHB receptor antagonist NCS-382 (200 mg/kg, intraperitoneal) had no effect on GHB-inhibited MAP kinase phosphorylation. Moreover, the GHB dehydrogenase inhibitor valproate (500 mg/kg, intraperitoneal), which inhibits the conversion of GHB into GABA, failed to block the effect of GHB on MAP kinase phosphorylation. Altogether, these data suggest that GHB, administered in vivo, reduces MAP kinase phosphorylation via a direct activation of GABAB receptors by GHB. In contrast, GHB (10 mm for 15 min) was found ineffective on MAP kinase phosphorylation in brain slices, indicating important differences in the conditions required for the second messenger activating action of GHB.

Highlights

Readily cross the blood-brain barrier and produces significant behavioral, electrophysiological, and biochemical effects
We have demonstrated that GHB reduces neuronal excitability and synaptic activity in neocortical and hippocampal neurons via GABAB receptor activation [31]
The Effect of GHB on MAP Kinase Phosphorylation Is Not Mediated through the High Affinity GHB Receptor—Because certain effects of GHB have been proposed to occur via high affinity GHB receptors [9, 37, 38], we addressed this point by using the specific GHB receptor antagonist NCS-382 in a separate set of experiments

Summary

EXPERIMENTAL PROCEDURES

In Vivo Treatment and Slice Preparation—C57Black mice, purchased from Harlan Sprague-Dawley (Indianapolis, IN), were maintained in the UCLA Division of Laboratory Animal Medicine (DLAM) vivarium facilities on a 12-h light/12-h dark cycle. The dose of GHB (purchased from Sigma), 500 mg/kg (intraperitoneally), was chosen based on previous reports [11, 17, 18] and data from GHB abuse in humans. For phosphorylation analysis of MAP kinases, 5–10 ␮g of protein was boiled in the 2ϫ sample buffer (125 mM Tris-HCl, pH 6.8, 4% SDS, 20% glycerol, 12% ␤-mercaptoethanol, 0.004% bromphenol blue), applied onto a 10% polyacrylamide gel, subsequently transferred to a nitrocellulose membrane (Osmonics Inc.), and blocked with 5% milk in TBS-T (10 mM Tris-HCl, pH 7.6, 150 mM NaCl, and 0.1% Tween 20). For the visualization of total MAP kinases, the membrane was stripped with a stripping buffer (0.2 M glycine, pH 2.2, 0.1% SDS, 0.1% Tween 20) at 37 °C for 30 min, re-labeled with the primary antibody against total ERK1/2 (1:1000 dilution; Cell Signaling Technology, New England Biolabs), and detected as described above. Significance level was set at p Ͻ 0.05 (two-tailed test)

RESULTS

GHB Reduces MAP Kinase Phosphorylation via GABAB Receptors

DISCUSSION