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.
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 degrees C was found to decrease by 28% when the extracellular pH (pH(e)) was reduced from pH 7.4 to pH 6.2. In contrast, a selective decrease in intracellular pH (pH(i)), 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 pI(50) values of 3.05+/-0.57, 3.66+/-0.23 and 3.94+/-0.88 at pH(e) 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 pH(e) 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 pH(e). For (R)-ibuprofen, pI(50) values of 3.57+/-0.08, 4.04+/-0.05 and 4.59+/-0.04 were found at pH(e) values of 7.4, 6.8 and 6.2, respectively. For (R,S)- and (S)-flurbiprofen, the pI(50) values at pH(e) 7.4 were 4.02+/-0.05 and 4.13+/-0.18, respectively at a pH(e) of 7.4, and 4.81+/-0.11 and 4.84+/-0.10, respectively, at a pH(e) 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.
Holt
Sandra
S
Department of Pharmacology and Clinical Neuroscience, Umeå University, 901 87 Umeå, Sweden. Sandra.Holt@pharm.umu.se
Fowler
Christopher J
CJ
D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology*; D001095:Arachidonic Acids / Q000037:antagonists & inhibitors / Q000378:metabolism*; D063388:Endocannabinoids; D005110:Extracellular Space / Q000378:metabolism; D005480:Flurbiprofen / Q000494:pharmacology*; D005910:Glioma; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007052:Ibuprofen / Q000494:pharmacology*; D042541:Intracellular Space / Q000378:metabolism; D053284:Polyunsaturated Alkamides; D051381:Rats; D013237:Stereoisomerism; D013997:Time Factors; D014407:Tumor Cells, Cultured; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D000894:Anti-Inflammatory Agents, Non-Steroidal; D001095:Arachidonic Acids; D063388:Endocannabinoids; D005480:Flurbiprofen; D007052:Ibuprofen; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide