Abstract
Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in the regulation of Ca2+ release from inositol 1,4,5-triphosphate-sensitive stores. U73122 (1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) has been extensively used as a pharmacological inhibitor of PLC to elucidate the importance of this enzyme family in signal transduction pathways. U73122 has an electrophilic maleimide group, which readily reacts with nucleophiles such as thiols and amines. In the current study the conjugation of U73122 to common components of cell culture medium, namely l-glutamine, glutathione, and bovine serum albumin (BSA), was demonstrated. The half-life of U73122 on incubation with phosphate-buffered saline (PBS), Hanks' buffered saline solution (with 2 mM glutamine), optimized basal nutrient medium (MCDB131, without BSA), complete medium, Dulbecco's modified Eagle's medium (with 2 mM l-glutamine) was ∼150, 60, 32, 30, and 18 min, respectively. However, U73122 was not recoverable from medium supplemented with 0.5% BSA. U73122 underwent hydrolysis of the maleimide group when incubated with PBS. Glutamine conjugates of U73122 were identified in cell culture medium. Furthermore, the inhibition of epidermal growth factor-stimulated Ca2+ release in a human epidermoid carcinoma cell line (A431) by U73122 was substantially reduced by the presence of BSA in a time-dependent manner. In complex cellular assays, the availability of U73122 to inhibit PLC may be limited by its chemical reactivity and lead to the misinterpretation of results in pharmacological assays.
Footnotes
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This work was supported by Cancer Research UK [CUK] Programme Grant C309/A8274.
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Article, publication date, and citation information can be found at http://dmd.aspetjournals.org.
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doi:10.1124/dmd.106.014498.
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ABBREVIATIONS: PLC, phosphoinositide-specific phospholipase C; PIP2, phosphatidylinositol 4,5-bisphosphate; U73122, 1-(6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione; U73343, 1-[6-((17β-3-methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl]-2,5-pyrrolidinedione; BSA, bovine serum albumin; DMSO, dimethyl sulfoxide; LC/MS, liquid chromatography-mass spectrometry; EGF, epidermal growth factor; DTT, dithiothreitol; NEM, N-ethylmaleimide; HBSS, Hanks' buffered saline solution; PBS, phosphate-buffered saline; DMEM, Dulbecco's modified Eagle's medium without phenol red and l-glutamine; A431, human epithelial carcinoma cell line; MCDB131, optimized basal nutrient medium; GFR, growth factor receptor.
- Received December 21, 2006.
- Accepted March 28, 2007.
- The American Society for Pharmacology and Experimental Therapeutics
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