Functional intracellular glutaminase activity in intact astrocytes

Area of Science:

• Biochemistry
• Cellular Metabolism
• Neuroscience

Background:

• Phosphate-activated glutaminase activity is influenced by various cellular metabolites.
• Measuring enzyme activity in cell extracts may not accurately represent in vivo metabolic flux.
• Astrocytes play crucial roles in brain metabolism, including glutamine processing.

Purpose of the Study:

• To measure the functional intracellular phosphate-activated glutaminase activity in intact astrocytes.
• To compare intracellular activity with enzyme levels in cell extracts.
• To investigate the effects of dibutyryl c-AMP (DiBcAMP) and other metabolites on astrocyte glutaminase activity.

Main Methods:

• Intact astrocyte cultures were used to measure 3H2O formation from L-[2-3H]glutamine.
• Enzyme activity in cell extracts was determined under optimal conditions.
• 14CO2 release from L-[1-14C]glutamine was measured to assess glutaminase flux.

Main Results:

• Intracellular glutaminase activity (88-153 nmol/mg protein/h) was substantially lower than in cell extracts (~1,750 nmol/mg protein/h).
• DiBcAMP treatment increased intracellular glutaminase activity.
• Intracellular activity was sensitive to glutamine concentration, glutamate inhibition, glucose availability, and serum presence.

Conclusions:

• Intact cell measurements provide a more physiologically relevant assessment of glutaminase activity than cell extracts.
• Astrocyte glutaminase activity is tightly regulated by intracellular metabolic conditions.
• DiBcAMP influences astrocyte glutamine metabolism, impacting neurotransmitter homeostasis.