Neurochemical and electrophysiological evidence for the existence of a functional gamma-hydroxybutyrate system in NCB-20 neurons.
Clonal neurohybridoma NCB-20 cells express a valproate-insensitive succinic semialdehyde reductase activity that transforms succinic semialdehyde into gamma-hydroxybutyrate. This activity (1.14+/-0.16 nmol/min/mg protein) was similar to the lowest activity existing in adult rat brain. [3H]gamma-Hydroxybutyrate labels a homogeneous population of sites on NCB-20 cell membranes (Kd=250+/-44.4nM, Bmax=180+/-16.2fmol/mg protein) that apparently represents specific gamma-hydroxybutyrate binding sites characterized previously on brain cell membranes. Finally, an Na+-dependent uptake of [3H]gamma-hydroxybutyrate was expressed in NCB-20 cells with a Km of 35+21.1 microM and a Vmax 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]gamma-hydroxybutyrate occurred. This release was Ca2+ dependent and was not present in undifferentiated cells. Cyclic-AMP treatment induced a decrease of [3H]gamma-hydroxybutyrate binding sites, which could be due to spontaneous gamma-hydroxybutyrate release. Patch-clamp experiments carried out on differentiated NCB-20 cells revealed the presence of Ca2+ conductances which were partially inhibited by 50 microM gamma-hydroxybutyrate. This gamma-hydroxybutyrate-induced effect was blocked by the gamma-hydroxybutyrate receptor antagonist NCS-382, but not by the GABA(B) antagonist CGP-55845. These results demonstrate the presence of an active gamma-hydroxybutyratergic system in NCB-20 cells which possesses the ability to release gamma-hydroxybutyrate. These cells express specific gamma-hydroxybutyrate receptors which modulate Ca2+ currents independently of GABA(B) receptors.
Laboratoire de Neurobiologie Moléculaire des Interactions Cellulaires, UPR 416 CNRS, Centre de Neurochimie, Strasbourg, France.
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V
Kemmel
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Taleb
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A
Perard
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Andriamampandry
J C
JC
Siffert
J
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Mark
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Maitre
D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D001921:Brain / Q000201:enzymology; D002118:Calcium / Q000378:metabolism; D015220:Calcium Channels / Q000502:physiology*; D002460:Cell Line; D006224:Cricetinae; D003412:Cricetulus; D006825:Hybridomas; D006884:Hydroxybutyrate Dehydrogenase / Q000378:metabolism*; D007150:Immunohistochemistry; D007700:Kinetics; D008564:Membrane Potentials; D051379:Mice; D009447:Neuroblastoma; D009474:Neurons / Q000502:physiology*; D018408:Patch-Clamp Techniques; D051381:Rats; D012978:Sodium Oxybate / Q000378:metabolism*; D014635:Valproic Acid / Q000494:pharmacology
D015220:Calcium Channels; D014635:Valproic Acid; D012978:Sodium Oxybate; D006884:Hydroxybutyrate Dehydrogenase; C019416:4-hydroxybutyrate dehydrogenase; D002118:Calcium