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

• The pharmacological properties of fatty acid amidohydrolase (FAAH) at different assay pH values were investigated using [(3)H]-anandamide ([(3)H]-AEA) as substrate in rat brain homogenates and in COS-1 [corrected] cells transfected with wild type and mutant FAAH. Rat brain hydrolysis of [(3)H]-AEA showed pH dependency with an optimum around 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. For inhibition of rat brain [(3)H]-AEA metabolism by a series of known FAAH inhibitors, the potencies of the enantiomers of ibuprofen and phenylmethylsulphonyl fluoride (PMSF) were higher at pH 5.28 than at pH 8.37, whereas the reverse was true for oleyl trifluoromethylketone (OTMK) and arachidonoylserotonin. At both pH values, (-)ibuprofen was a mixed-type inhibitor of FAAH. The K(i)((slope)) and K(i)((intercept)) values for (-)ibuprofen at pH 5.28 were 11 and 143 microM, respectively. At pH 8.37, the corresponding values were 185 and 3950 microM, respectively. The pH dependency for the inhibition by OTMK and (-)ibuprofen was also seen in COS-1 [corrected] cells transiently transfected with either wild type, S152A or C249A FAAH. No differences in potencies between the wild type and mutant enzymes were seen. It is concluded that the pharmacological properties of FAAH are highly pH-dependent. The higher potency of ibuprofen at lower pH values raises the possibility that in certain types of inflamed tissue, the concentration of this compound following oral administration may be sufficient to inhibit FAAH.

Nyckelord

Anandamide

fatty acid amidohydrolase

ibuprofen

pH dependency

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