Abstract

The ability of rat brain (minus cerebellum) homogenates to deamidate arachidonyl ethanolamide (anandamide) was determined with a custom-synthesized substrate, arachidonyl ethanolamide-[1-³H] ([³H]anandamide). Conditions whereby initial velocities were measured were established. The homogenates deamidated anandamide with a K_m value of 0.8 μM and a V_max value of 1.73 nmol · (mg protein)⁻¹ · min⁻¹. The deamidation of 2 μM [³H]anandamide was inhibited by phenylmethylsulfonyl fluoride and arachidonyl trifluoromethyl ketone with IC₅₀ values of 3.7 and 0.23 μM, respectively. Ibuprofen inhibited anandamide deamidation in a mixed fashion, withK_i and K′_i values of 82 and 1420 μM. At an anandamide concentration of 2 μM, the IC₅₀ values (in μM) of a series of compounds related in structure to ibuprofen were as follows: suprofen, 170; ibuprofen, 270; fenoprofen, 480; naproxen, 550; ketoprofen, 650; diclofenac, ∼1000. Sulindac produced 27% inhibition at a concentration of 1000 μM, whereas isobutyric acid, hydrocinnamic acid, acetylsalicylic acid and acetaminophen were essentially inactive at concentrations ≤ 1 mM. We conclude that ibuprofen inhibits anandamide deamidation at pharmacologically relevant concentrations and that there is some specificity to the inhibition produced by ibuprofen and suprofen.