Neurochemical and electrophysiological evidence for the existence of a functional γ-hydroxybutyrate system in NCB-20 neurons

Abstract

Clonal neurohybridoma NCB-20 cells express a valproate-insensitive succinic semialdehyde reductase activity that transforms succinic semialdehyde into γ-hydroxybutyrate. This activity (1.14±0.16 nmol/min/mg protein) was similar to the lowest activity existing in adult rat brain. [ 3H] γ-Hydroxybutyrate labels a homogeneous population of sites on NCB-20 cell membranes ( K d =250±44.4 nM, B max=180±16.2 fmol/mg protein) that apparently represents specific γ-hydroxybutyrate binding sites characterized previously on brain cell membranes. Finally, an Na +-dependent uptake of [ 3H] γ-hydroxybutyrate was expressed in NCB-20 cells with a K m of 35+21.1 μM and a V max of 80±14.2 pmol/min/mg protein. A three-day treatment with 1 mM dibutyryl-cyclic-AMP induced a three-fold increase in the cellular succinic semialdehyde reductase activity. In parallel, a K +-evoked release of [ 3H] γ-hydroxybutyrate occurred. This release was Ca 2+ dependent and was not present in undifferentiated cells. Cyclic-AMP treatment induced a decrease of [ 3H] γ-hydroxybutyrate binding sites, which could be due to spontaneous γ-hydroxybutyrate release. Patch-clamp experiments carried out on differentiated NCB-20 cells revealed the presence of Ca 2+ conductances which were partially inhibited by 50 μM γ-hydroxybutyrate. This γ-hydroxybutyrate-induced effect was blocked by the γ-hydroxybutyrate receptor antagonist NCS-382, but not by the GABA B antagonist CGP-55 845. These results demonstrate the presence of an active γ-hydroxybutyratergic system in NCB-20 cells which possesses the ability to release γ-hydroxybutyrate. These cells express specific γ-hydroxybutyrate receptors which modulate Ca 2+ currents independently of GABA B receptors.