Colorimetric Detection of Glutamine Synthetase-Catalyzed Transferase Activity in Glucocorticoid-Treated Skeletal Muscle Cells

• Published in: Analytical biochemistry 2001-02, Vol.289 (1), p.18-25
• Main Authors: Santoro, Joseph C., Harris, Georgianna, Sitlani, Ayesha
• Format: Article
• Language: English
• Subjects: Animals; Catalysis; Cell Line; colorimetric assay; Colorimetry; corticosteroid; Dexamethasone - metabolism; Dexamethasone - pharmacology; gamma-Glutamyltransferase - metabolism; Glutamate-Ammonia Ligase - metabolism; glutamine synthetase; Muscle, Skeletal - drug effects; Muscle, Skeletal - enzymology; Muscle, Skeletal - metabolism; Rats; Receptors, Glucocorticoid - metabolism; Reproducibility of Results; skeletal muscle cells; γ-glutamyltransferase
• ISSN: 0003-2697; 1096-0309
• DOI: 10.1006/abio.2000.4911

Induction of the enzyme glutamine synthetase (GS) by corticosteroids correlates with muscle wasting and gluconeogenesis, characteristic side effects of chronic glucocorticoid treatment. This highlights the importance of developing robust high-throughput assays to measure drug-induced GS in whole cells. We have optimized a colorimetric method to measure GS-catalyzed γ-glutamyltransferase (GT) activity in rat L6 skeletal muscle cells (96-well-plate format) and human skeletal muscle cells (24-well-plate format). We observe a fourfold increase in GT activity in dexamethasone treated L6 cells, as compared to untreated cells, with good reproducibility in the measurements (errors of less than 5%). This assay can distinguish between partial agonists such as halopredone acetate and complete agonists such as prednisolone and measure the potency of known glucocorticoid receptor (GR) antagonists like mifepristone. Importantly, the ability of corticosteroids to induce GS-catalyzed GT activity correlates well with their whole cell GR binding potency, indicating a GR-specific effect. Interestingly, in general, induction of GT activity by commonly administered anti-inflammatory corticosteroid drugs is comparable in rat and human skeletal muscle cells, which emphasizes the potential of a rat model system to study GS induction and muscle wasting by these drugs in humans.