Anandamide metabolism by fatty acid amide hydrolase in intact C6 glioma cells. Increased sensitivity to inhibition by ibuprofen and flurbiprofen upon reduction of extra- but not intracellular pH

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Abstract
The metabolism of anandamide by fatty acid amidohydrolase (FAAH) at different intra- and extracellular pH values has been investigated in intact C6 rat glioma cells. The cellular uptake of anandamide at 37°C was found to decrease by 28% when the extracellular pH (pHe) was reduced from pH 7.4 to pH 6.2. In contrast, a selective decrease in intracellular pH (pHi), accomplished by acidifying the cells followed by incubation in sodium-free buffer at pH 7.4, did not affect the uptake. Anandamide uptake was inhibited by (R)-ibuprofen, with pI50 values of 3.05±0.57, 3.66±0.23 and 3.94±0.88 at pHe values of 7.4, 6.8 and 6.2, respectively. In the presence of phenylmethylsulfonyl fluoride, however, (R)-ibuprofen failed to inhibit the uptake of anandamide. A reduction in pHe from 7.4 to 6.2 produced a 17% reduction in the FAAH-catalyzed metabolism of anandamide in the intact C6 cells. However, an increased sensitivity of FAAH activity to inhibition by (R)-ibuprofen as well as (R,S)-flurbiprofen and (S)-flurbiprofen was seen at a lower pHe. For (R)-ibuprofen, pI50 values of 3.57±0.08, 4.04±0.05 and 4.59±0.04 were found at pHe values of 7.4, 6.8 and 6.2, respectively. For (R,S)- and (S)-flurbiprofen, the pI50 values at pHe 7.4 were 4.02±0.05 and 4.13±0.18, respectively at a pHe of 7.4, and 4.81±0.11 and 4.84±0.10, respectively, at a pHe of 6.2. In contrast, intracellular acidification did not affect either the rate of anandamide metabolism or its inhibition by (R)-ibuprofen or (S)-flurbiprofen. It is concluded that a reduction of extracellular pH produces an enhanced accumulation of the acidic NSAIDs ibuprofen and flurbiprofen into C6 glioma cells and thereby an inhibition of anandamide metabolism.

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