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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References
Andersson SE, Lexmüller K, Johansson A, Ekström GM (1999) Tissue and intracellular pH in normal periarticular soft tissue and during different phases of antigen induced arthritis in the rat. J Rheumatol 26:2018–2024
Avdeef A, Box KJ, Comer JEA, Gilges M, Hadley M, Hibbert C, Patterson W, Tam KY (1999) PH-metric log P 11. pKa determination of water-insoluble drugs in organic solvent–water mixtures. J Pharm Biomed Anal 20:631–641
Beltramo M, Stella N, Calignano A, Lin SY, Makriyannis A, Piomelli D (1997) Functional role of high-affinity anandamide transport, as revealed by selective inhibition. Science 277:1094–1097
Bisogno T, Maurelli S, Melck D, De Petrocellis L, Di Marzo V (1997) Biosynthesis, uptake, and degradation of anandamide and palmitoylethanolamide in leukocytes. J Biol Chem 272:3315–3323
Cravatt BF, Demarest K, Patricelli MP, Bracey MH, Giang DK, Martin BR, Lichtman AH (2001) Supersensitivity to anandamide and enhanced endogenous cannabinoid signaling in mice lacking fatty acid amide hydrolase. Proc Natl Acad Sci USA 98:9371–9376
Day TA, Rakhshan F, Deutsch DG, Barker EL (2001) Role of fatty acid amide hydrolase in the transport of the endogenous cannabinoid anandamide. Mol Pharmacol 59:1369–1375
De Petrocellis L, Bisogno T, Davis JB, Pertwee RG, Di Marzo V (2000) Overlap between the ligand recognition properties of the anandamide transporter and the VR1 vanilloid receptor: inhibitors of anandamide uptake with negligible capsaicin-like activity. FEBS Letts 483:52–56
Deutsch DG, Chin SA (1993) Enzymatic synthesis and degradation of anandamide, a cannabinoid receptor agonist. Biochem Pharmacol 46:791–796
Deutsch DG, Glaser ST, Howell JM, Kunz JS, Puffenbarger RA, Hillard CJ, Abumrad N (2001) The cellular uptake of anandamide is coupled to its breakdown by fatty acid amide hydrolase (FAAH). J Biol Chem 276:6967–6973
Di Marzo V, Fontana A, Cadas H, Schinelli S, Cimino G, Schwartz JC, Piomelli D (1994) Formation and inactivation of endogenous cannabinoid anandamide in central neurons. Nature 372:686–691
Fowler CJ, Tiger G, Stenström A (1997) Ibuprofen inhibits rat brain deamidation of anandamide at pharmacologically relevant concentrations. Mode of inhibition and structure-activity relationship. J Pharmacol Exp Ther 283:729–734
Fowler CJ, Jansson U, Johnsson RM, Wahlström G, Stenström A, Norström Å, Tiger G (1999) Inhibition of anandamide hydrolysis by the enantiomers of ibuprofen, ketorolac and flurbiprofen. Arch Biochem Biophys 362:191–196
Fowler CJ, Holt S, Tiger G Acidic nonsteroidal anti-inflammatory drugs inhibit rat brain fatty acid amide hydrolase in a pH-dependent manner. J Enzyme Inhib Med Chem 18:55–58
Glaser ST, Studholme KM, Fatade F, Yazulla S, Abumrad N, Deutsch DG (2002) Is there an anandamide transporter? 2002 Symposium on the Cannabinoids, Burlington Vermont, International Cannabinoid Research Society, p 9
Graf P, Glatt M, Brune K (1975) Acidic nonsteroid anti-inflammatory drugs accumulating in inflamed tissue. Experientia 31:951–953
Häbler C (1929) Über den K- und Ca-Gehalt von Eiter und Exsudaten und seine Beziehungen zum Entzündungsschmerz. Klin Wochenschr 8:1569–1572
Hillard CJ, Wilkison DM, Edgemond WS, Campbell WB (1995) Characterization of the kinetics and distribution of N-arachidonylethanolamine (anandamide) hydrolysis by rat brain. Biochim Biophys Acta 1257:249–256
Hillard CJ, Edgemond WS, Jarrahian A, Campbell WB (1997) Accumulation of N-arachidonoylehanolamine (anandamide) into cerebellar granule cells occurs via facilitated diffusion. J Neurochem 69:631–638
Holt S, Nilsson J, Omeir R, Tiger G, Fowler CJ (2001) Effects of pH on the inhibition of fatty acid amidohydrolase by ibuprofen. Br J Pharmacol 133:513–520
Jacobsson SOP, Fowler CJ (2001) Characterization of palmitoylethanolamide transport in mouse Neuro-2a neuroblastoma and rat RBL-2H3 basophilic leukaemia cells: comparison with anandamide. Br J Pharmacol 132:1743–1754
Jonsson K-O, Vandevoorde S, Lambert DM, Tiger G, Fowler CJ (2001) Effects of homologues and analogues of palmitoylethanolamide upon the inactivation of the endocannabinoid anandamide. Br J Pharmacol 133:1263–1275
Karttunen P, Saano V, Paronen P, Peura P, Vidgren M (1990) Pharmacokinetics of ibuprofen in man: a single-dose comparison of two over-the-counter, 200 mg preparations. Int J Clin Pharmacol Ther Toxicol 28:251–255
Kitai R, Kabuto M, Kubota T, Kobayashi H, Matsumoto H, Hayashi S, Shioura H, Ohtsubo T, Katayama K, Kano E (1998) Sensitization to hyperthermia by intracellular acidification of C6 glioma cells. J Neurooncol 39:197–203
Lambert DM, Vandevoorde S, Jonsson K-O, Fowler CJ (2002) The palmitoylethanolamide family: a new class of anti-inflammatory agents? Curr Med Chem 9:663–674
Maurelli S, Bisogno T, De Petrocellis L, Luccia AD, Marino G, Di Marzo V (1995) Two novel classes of neuroactive fatty acid amides are substrates for mouse neuroblastoma 'anandamide amidohydrolase'. FEBS Letts 377:82–86
Natarajan V, Schmid PC, Reddy PV, Schmid HHO (1984) Catabolism of N-acylethanolamine phospholipids by dog brain preparations. J Neurochem 42:1613–1619
Pargal A, Kelkar MG, Nayak PJ (1996) The effect of food on the bioavailability of ibuprofen and flurbiprofen from sustained release formulations. Biopharm Drug Dispos 17:511–519
Paria BC, Deutsch DD, Dey SK (1996) The uterus is a potential site for anandamide synthesis and hydrolysis: differential profiles of anandamide synthase and hydrolase activities in the mouse uterus during the periimplantation period. Mol Reprod Dev 45:183–192
Patricelli MP, Cravatt BF (2001) Proteins regulating the biosynthesis and inactivation of neuromodulatory fatty acid amides. Vitam Horm 62:95–131
Patricelli MP, Lashuel HA, Giang DK, Kelly JW, Cravatt BF (1998) Comparative characterization of a wild type and transmembrane domain-deleted fatty acid amide hydrolase: identification of the transmembrane domain as a site for oligomerization. Biochemistry 37:15177–15187
Rakshan F, Day TA, Blakely RD, Barker EL (2000) Carrier-mediated uptake of the endogenous cannabinoid anandamide in RBL-2H3 cells. J Pharmacol Exp Ther 292:960–967
Rice ASC (2001) Cannabinoids and pain. Curr Opin Invest Drugs 2:399–414
Roberts LJ, Morrow JD (2001) Analgesic-antipyretic and antiinflammatory agents and drugs employed in the treatment of gout. In: Hardman JG, Limbird LE, Goodman Gilman A (eds) Goodman and Gilman's the pharmacological basis of therapeutics, 10th edn. McGraw-Hill, New York, pp 687–731
Schmid PC, Zuzarte-Augustin ML, Schmid HHO (1985) Properties of rat liver N-acylethanolamine amidohydrolase. J Biol Chem 260:14145–14149
Shrode LD, Putnam RW (1994) Intracellular pH regulation in primary rat astrocytes and C6 glioma cells. Glia 12:196–210
Steen AE, Reeh PW, Geisslinger G, Steen KH (2000) Plasma levels after peroral and topical ibuprofen and effects upon low pH-induced cutaneous and muscle pain. Eur J Pain 4:195–209
Steen KH, Reeh PW, Anton F, Handwerker HO (1992) Protons selectively induce lasting excitation to mechanical stimulation of nociceptors in rat skin, in vitro. J Neurosci 12:86–95
Thomas JA, Buchsbaum RN, Zmniak A, Racker E (1979) Intracellular pH measurements in Ehrlich ascites tumor cells utilizing spectroscopic probes generated in situ. Biochemistry 18:2210–2218
Tiger G, Stenström A, Fowler CJ (2000) Pharmacological properties of rat brain fatty acid amidohydrolase in different subcellular fractions using palmitoylethanolamide as substrate. Biochem Pharmacol 59:647–653
Tsou K, Nogueron MI, Muthian S, Sañudo-Peña MC, Hillard CJ, Deutsch DG, Walker JM (1998) Fatty acid amide hydrolase is located preferentially in large neurons in the rat central nervous system as revealed by immunochemistry. Neurosci Letts 254:137–140
Ueda N, Kurahashi Y, Yamamoto S, Tokunaga T (1995) Partial purification and characterization of the porcine brain enzyme hydrolyzing and synthesizing anandamide. J Biol Chem 270:23823–23827
Voilley N, De Weille J, Mamet J, Lazdunski M (2001) Nonsteroid anti-inflammatory drugs inhibit both the activity and the inflammation-induced expression of acid-sensing ion channels in nociceptors. J Neurosci 21:8026–8033
Watanabe K, Ogi H, Nakamura S, Kayano Y, Matsunaga T, Yoshimura H, Yamamoto L (1998) Distribution and characterization of anandamide amidohydrolase in mouse brain and liver. Life Sci 62:1223–1229
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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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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DOI: https://doi.org/10.1007/s00210-002-0686-z