Effects of pH on the inhibition of fatty acid amidohydrolase by ibuprofen

1 The pharmacological properties of fatty acid amidohydrolase (FAAH) at dif ferent assay pH values were investigated using [H-3]-anandamide ([H-3]-AEA) as substrate in rat brain homogenates and in COS-7 cells transfected with wild type and mutant FAAH. 2 Rat brain hydrolysis of [H-3]-AEA showed pH dependency with an optimum ar ound pH 8-9. Between pH 6.3 and 8.2, the difference in activity was due to differences in the V-max, rather than the K-M values. 3 For inhibition of rat brain [H-3]-AEA metabolism by a series of known FAA H inhibitors, the potencies of the enantiomers of ibuprofen and phenylmethy lsulphonyl fluoride (PMSF) were higher at pH 5.28 than at pH 8.37, whereas the reverse was true for oleyl trifluoromethylketone (OTMK) and arachidonoy lserotonin. At both pH values, (-)ibuprofen was a mixed-type inhibitor of F AAH. The K-i(slope) and K-i(intercept) values for(-)ibuprofen at pH 5.28 we re 11 and 143 muM, respectively. At pH 8.37, the corresponding values were 185 and 3950 muM, respectively. 4 The pH dependency for the: inhibition by OTMK and (-)ibuprofen was also s een in COS-7 cells transiently transfected with either wild type, S152A or C249A FAAH. No differences in potencies between the wild type and mutant en zymes were seen. 5 It is concluded that the pharmacological properties of FAAH are highly pH -dependent. The higher potency of ibuprofen at lower pH values raises the p ossibility that in certain types of inflamed tissue, the concentration of t his compound following oral administration may be sufficient to inhibit FAA H.