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SHORT COMMUNICATION

The Phosphoinositide-Specific Phospholipase C Inhibitor U73122 (1-(6-((17ß-3-Methoxyestra-1,3,5(10)-trien-17-yl)amino)hexyl)-1H-pyrrole-2,5-dione) Spontaneously Forms Conjugates with Common Components of Cell Culture Medium

Cancer Research UK Centre for Cancer Therapeutics, The Institute of Cancer Research, Belmont, Surrey, United Kingdom (N.E.W., W.J.C., R.R., Y.M.N., W.A., P.W.S., S.A.E., F.I.R.); and Cancer Research UK Centre for Cell and Molecular Biology, Chester Beatty Laboratories, The Institute of Cancer Research, London, United Kingdom (N.P.J., M.K.)

Phosphoinositide-specific phospholipase C (PLC) is a key enzyme in the regulation of Ca²⁺ release from inositol 1,4,5-triphosphate-sensitive stores. U73122 [GenBank] (1-(6-((17ß-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 [GenBank] has an electrophilic maleimide group, which readily reacts with nucleophiles such as thiols and amines. In the current study the conjugation of U73122 [GenBank] to common components of cell culture medium, namely L-glutamine, glutathione, and bovine serum albumin (BSA), was demonstrated. The half-life of U73122 [GenBank] 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 [GenBank] was not recoverable from medium supplemented with 0.5% BSA. U73122 [GenBank] underwent hydrolysis of the maleimide group when incubated with PBS. Glutamine conjugates of U73122 [GenBank] 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 [GenBank] was substantially reduced by the presence of BSA in a time-dependent manner. In complex cellular assays, the availability of U73122 [GenBank] to inhibit PLC may be limited by its chemical reactivity and lead to the misinterpretation of results in pharmacological assays.