RT Journal Article
SR Electronic
T1 The Phosphoinositide-Specific Phospholipase C Inhibitor U73122 (1-(6-((17β-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
JF Drug Metabolism and Disposition
JO Drug Metab Dispos
FD American Society for Pharmacology and Experimental Therapeutics
SP 1017
OP 1022
DO 10.1124/dmd.106.014498
VO 35
IS 7
A1 Nicola E. Wilsher
A1 Will J. Court
A1 Ruth Ruddle
A1 Yvette M. Newbatt
A1 Wynne Aherne
A1 Peter W. Sheldrake
A1 Neil P. Jones
A1 Matilda Katan
A1 Suzanne A. Eccles
A1 Florence I. Raynaud
YR 2007
UL http://dmd.aspetjournals.org/content/35/7/1017.abstract
AB 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. The American Society for Pharmacology and Experimental Therapeutics