Human Platelets and Polymorphonuclear Leukocytes Synthesize Oxygenated Derivatives of Arachidonylethanolamide (Anandamide): Their Affinities for Cannabinoid Receptors and Pathways of Inactivation
- William S. Edgemond1,
- Cecilia J. Hillard1,
- J. R. Falck2,
- Christopher S. Kearn1 and
- William B. Campbell1
- 1Department of Pharmacology and Toxicology, Medical College of Wisconsin, Milwaukee, Wisconsin 53226 (W.S.E., C.J.H., C.S.K., W.B.C.), and Departments of 2Pharmacology and Biochemistry, The University of Texas Southwestern Medical Center, Dallas, Texas 75235 (J.R.F.)
Abstract
Arachidonylethanolamide (AEA), the putative endogenous ligand of the cannabinoid receptor, has been shown to be a substrate for lipoxygenase enzymes in vitro. One goal of this study was to determine whether lipoxygenase-rich cells metabolize AEA. [14C]AEA was converted by human polymorphonuclear leukocytes (PMNs) to two major metabolites that comigrated with synthetic 12(S)- and 15(S)-hydroxy-arachidonylethanolamide (HAEA). Human platelets convert [14C]AEA to 12(S)-HAEA. 12(S)-HAEA binds to both CB1 and CB2 receptors with approximately the same affinity as AEA. 12(R)-HAEA, which is not produced by PMNs, has 2-fold lower affinity for the CB1 receptor and 10-fold lower affinity for the CB2 receptor than 12(S)-HAEA. 15-HAEA has a lower affinity than AEA for both receptors, with Ki values of 738 and >1000 nm for CB1 and CB2 receptors, respectively. The addition of a hydroxyl group at C20 of AEA resulted in a ligand with the same affinity for the CB1 receptor but a 4-fold lower affinity for the CB2 receptor than AEA. 12(S)-HAEA and 15-HAEA are poor substrates for AEA amidohydrolase and do not bind to the AEA uptake carrier. In conclusion, the addition of a hydroxyl group at C12 of the arachidonate backbone of AEA does not affect binding to CB receptors but is likely to increase its half-life. The addition of hydroxyl groups at other positions affects ligand affinity for CB receptors; both the position of the hydroxyl group and the configuration of the remaining double bonds are determinants of affinity.
Footnotes
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Send reprint requests to: Cecilia J. Hillard, Ph.D., Department of Pharmacology, Medical College of Wisconsin, 8701 Watertown Plank Road, Milwaukee, WI 53226. E-mail:chillard{at}mcw.edu
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This work was supported by United States Public Health Service Grants DA09155, HL51055 (W.B.C.), and GM31278 (J.R.F.).
- Abbreviations:
- AEA
- N-arachidonylethanolamine
- AA
- arachidonic acid
- BSA
- bovine serum albumin
- CB1
- cannabinoid receptor subtype 1
- CB2
- cannabinoid receptor subtype 2
- CHO
- Chinese hamster ovary
- GC
- gas chromatography
- MS
- mass spectroscopy
- HAEA
- hydroxyarachidonylethanolamide
- HEPES
- 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid
- HETE
- hydroxyeicosatetraenoic acid
- PMN
- polymorphonuclear leukocyte
- RP
- reverse phase
- HPLC
- high pressure liquid chromatography
- TMS
- trimethylsilyl
- TME
- Tris/MgCl2/EDTA
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- Received September 10, 1997.
- Accepted March 30, 1998.
- The American Society for Pharmacology and Experimental Therapeutics
