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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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Fig. 4A, B.

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Acknowledgements

The authors are grateful to Dr. William Wechter for his gift of (S)-flurbiprofen. This study was supported by grants from the Swedish Research Council (Grant no. 12158, medicine), Konung Gustav V's and Drottning Victorias Foundation, the Swedish Asthma and Allergy Association's Research Foundation, and the Research Funds of the Medical Odontological Faculty, Umeå University.

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Correspondence to Sandra Holt.

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All experiments in this study comply with Swedish laws

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Holt, S., Fowler, C.J. 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. Naunyn-Schmiedeberg's Arch Pharmacol 367, 237–244 (2003). https://doi.org/10.1007/s00210-002-0686-z

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