Synthesis and catabolism of gamma-hydroxybutyrate in SH-SY5Y human neuroblastoma cells

R.C. Lyon, S.M. Johnston, D.G. Watson, G. McGarvie, E.M. Ellis

Research output: Contribution to journalArticle

34 Citations (Scopus)

Abstract

Gamma-Hydroxybutyrate (GHB) is an endogenous metabolite synthesized in the brain. There is strong evidence to suggest that GHB has an important role as a neurotransmitter or neuromodulator. The human aldo-keto reductase AKR7A2 has been proposed previously to catalyze the NADPH-dependent reduction of succinic semialdehyde (SSA) to GHB in human brain. In this study we have used RNA interference to evaluate the role of AKR7A2 in GHB biosynthesis in human neuroblastoma SH-SY5Y cells. Quantitative reverse transcription-PCR analysis and immunoblotting revealed that short interfering RNA molecules directed against AKR7A2 led to a significant reduction in both AKR7A2 transcript and protein levels 72 h post-transfection. We have shown that reduced expression of AKR7A2 results in a 90% decrease in SSA reductase activity of cell extracts. Furthermore, we have shown using gas chromatography-mass spectrometry that a decrease in the level of AKR7A2 was paralleled with a significant reduction in intracellular GHB concentration. This provides conclusive evidence that AKR7A2 is the major SSA reductase in these cells. In contrast, short interfering RNA-dependent reduction in AKR7A2 levels had no effect on the GHB dehydrogenase activity of the extracts, and inhibitor studies suggest that another enzyme characteristic of an NAD-dependent alcohol dehydrogenase may be responsible for catalyzing this reverse reaction. Together these findings delineate pathways for GHB metabolism in the brain and will enable a better understanding of the relationship between GHB biosynthesis and catabolism in disease states and in drug overdose.
Original languageEnglish
Pages (from-to)25986-25992
Number of pages6
JournalJournal of Biological Chemistry
Volume282
Issue number36
DOIs
Publication statusPublished - 7 Sep 2007

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Sodium Oxybate
Neuroblastoma
Brain
Biosynthesis
Small Interfering RNA
Neurotransmitter Agents
Drug Overdose
Alcohol Dehydrogenase
Transcription
Metabolites
RNA Interference
Cell Extracts
NADP
Immunoblotting
Metabolism
Gas chromatography
NAD
Gas Chromatography-Mass Spectrometry
Reverse Transcription
Transfection

Keywords

  • hydroxyacid-oxoacid transhydrogenase
  • succinic semialdehyde reductase
  • B-1 aldehyde reductase
  • alcohol-dehydrogenase
  • semi-aldehyde
  • human brain
  • rat brain
  • gas chromatography
  • energy metabolism
  • acid

Cite this

Lyon, R.C. ; Johnston, S.M. ; Watson, D.G. ; McGarvie, G. ; Ellis, E.M. / Synthesis and catabolism of gamma-hydroxybutyrate in SH-SY5Y human neuroblastoma cells. In: Journal of Biological Chemistry. 2007 ; Vol. 282, No. 36. pp. 25986-25992.
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Synthesis and catabolism of gamma-hydroxybutyrate in SH-SY5Y human neuroblastoma cells. / Lyon, R.C.; Johnston, S.M.; Watson, D.G.; McGarvie, G.; Ellis, E.M.

In: Journal of Biological Chemistry, Vol. 282, No. 36, 07.09.2007, p. 25986-25992.

Research output: Contribution to journalArticle

TY - JOUR

T1 - Synthesis and catabolism of gamma-hydroxybutyrate in SH-SY5Y human neuroblastoma cells

AU - Lyon, R.C.

AU - Johnston, S.M.

AU - Watson, D.G.

AU - McGarvie, G.

AU - Ellis, E.M.

PY - 2007/9/7

Y1 - 2007/9/7

N2 - Gamma-Hydroxybutyrate (GHB) is an endogenous metabolite synthesized in the brain. There is strong evidence to suggest that GHB has an important role as a neurotransmitter or neuromodulator. The human aldo-keto reductase AKR7A2 has been proposed previously to catalyze the NADPH-dependent reduction of succinic semialdehyde (SSA) to GHB in human brain. In this study we have used RNA interference to evaluate the role of AKR7A2 in GHB biosynthesis in human neuroblastoma SH-SY5Y cells. Quantitative reverse transcription-PCR analysis and immunoblotting revealed that short interfering RNA molecules directed against AKR7A2 led to a significant reduction in both AKR7A2 transcript and protein levels 72 h post-transfection. We have shown that reduced expression of AKR7A2 results in a 90% decrease in SSA reductase activity of cell extracts. Furthermore, we have shown using gas chromatography-mass spectrometry that a decrease in the level of AKR7A2 was paralleled with a significant reduction in intracellular GHB concentration. This provides conclusive evidence that AKR7A2 is the major SSA reductase in these cells. In contrast, short interfering RNA-dependent reduction in AKR7A2 levels had no effect on the GHB dehydrogenase activity of the extracts, and inhibitor studies suggest that another enzyme characteristic of an NAD-dependent alcohol dehydrogenase may be responsible for catalyzing this reverse reaction. Together these findings delineate pathways for GHB metabolism in the brain and will enable a better understanding of the relationship between GHB biosynthesis and catabolism in disease states and in drug overdose.

AB - Gamma-Hydroxybutyrate (GHB) is an endogenous metabolite synthesized in the brain. There is strong evidence to suggest that GHB has an important role as a neurotransmitter or neuromodulator. The human aldo-keto reductase AKR7A2 has been proposed previously to catalyze the NADPH-dependent reduction of succinic semialdehyde (SSA) to GHB in human brain. In this study we have used RNA interference to evaluate the role of AKR7A2 in GHB biosynthesis in human neuroblastoma SH-SY5Y cells. Quantitative reverse transcription-PCR analysis and immunoblotting revealed that short interfering RNA molecules directed against AKR7A2 led to a significant reduction in both AKR7A2 transcript and protein levels 72 h post-transfection. We have shown that reduced expression of AKR7A2 results in a 90% decrease in SSA reductase activity of cell extracts. Furthermore, we have shown using gas chromatography-mass spectrometry that a decrease in the level of AKR7A2 was paralleled with a significant reduction in intracellular GHB concentration. This provides conclusive evidence that AKR7A2 is the major SSA reductase in these cells. In contrast, short interfering RNA-dependent reduction in AKR7A2 levels had no effect on the GHB dehydrogenase activity of the extracts, and inhibitor studies suggest that another enzyme characteristic of an NAD-dependent alcohol dehydrogenase may be responsible for catalyzing this reverse reaction. Together these findings delineate pathways for GHB metabolism in the brain and will enable a better understanding of the relationship between GHB biosynthesis and catabolism in disease states and in drug overdose.

KW - hydroxyacid-oxoacid transhydrogenase

KW - succinic semialdehyde reductase

KW - B-1 aldehyde reductase

KW - alcohol-dehydrogenase

KW - semi-aldehyde

KW - human brain

KW - rat brain

KW - gas chromatography

KW - energy metabolism

KW - acid

UR - http://dx.doi.org/10.1074/jbc.M702465200

U2 - 10.1074/jbc.M702465200

DO - 10.1074/jbc.M702465200

M3 - Article

VL - 282

SP - 25986

EP - 25992

JO - Journal of Biological Chemistry

JF - Journal of Biological Chemistry

SN - 0021-9258

IS - 36

ER -