title: Confocal microscopy and biochemical analysis reveal spatial and functional separation between anandamide uptake and hydrolysis in human keratinocytes.
abstract: The signaling activity of anandamide (AEA) is terminated by its uptake across the cellular membrane and subsequent intracellular hydrolysis by the fatty acid amide hydrolase (FAAH). To date, the existence of an AEA membrane transporter (AMT) independent of FAAH activity remains questionable, although it has been recently corroborated by pharmacological and genetic data. We performed confocal microscopy and biochemical analysis in human HaCaT keratinocytes, in order to study the cellular distribution of AMT and FAAH. We found that FAAH is intracellularly localized as a punctate staining partially overlapping with the endoplasmic reticulum. Consistently, subcellular fractionation and reconstitution of vesicles from membranes of different compartments demonstrated that FAAH activity was localized mainly in microsomal fractions, whereas AMT activity was almost exclusively in plasma membranes. These results provide the first morphological and biochemical evidence to support the view that transport and hydrolysis are two spatially and functionally distinct processes in AEA degradation.
authors.affiliation: Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy.
authors.forename: S
authors.initials: S
authors.lastname: Oddi
authors.forename: M
authors.initials: M
authors.lastname: Bari
authors.forename: N
authors.initials: N
authors.lastname: Battista
authors.forename: D
authors.initials: D
authors.lastname: Barsacchi
authors.forename: I
authors.initials: I
authors.lastname: Cozzani
authors.forename: M
authors.initials: M
authors.lastname: Maccarrone
mesh_terms: D000581:Amidohydrolases / Q000502:physiology; D001095:Arachidonic Acids / Q000032:analysis* / Q000493:pharmacokinetics / Q000502:physiology*; D001692:Biological Transport / Q000187:drug effects / Q000502:physiology; D002460:Cell Line; D002462:Cell Membrane / Q000502:physiology; D063388:Endocannabinoids; D004721:Endoplasmic Reticulum / Q000502:physiology; D006801:Humans; D006868:Hydrolysis; D015603:Keratinocytes / Q000166:cytology* / Q000187:drug effects / Q000502:physiology*; D018613:Microscopy, Confocal / Q000379:methods; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases
chemical_list: D000581:Amidohydrolases; D001095:Arachidonic Acids; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide
references.citation: J Am Coll Cardiol. 2008 Sep 30;52(14):1134-40
references.pmid: 18804739
references.citation: Cardiovasc Interv Ther. 2017 Jan;32(1):24-35
references.pmid: 26979599
references.citation: J Am Coll Cardiol. 2008 Mar 11;51(10):986-90
references.pmid: 18325436
references.citation: J Interv Cardiol. 2012 Apr;25(2):118-25
references.pmid: 22324820
references.citation: J Am Coll Cardiol. 2013 May 7;61(18):1853-9
references.pmid: 23500262
references.citation: Am J Cardiol. 2014 Jan 15;113(2):229-35
references.pmid: 24188893
references.citation: JACC Cardiovasc Interv. 2011 Jan;4(1):30-8
references.pmid: 21251626
references.citation: Lancet. 2011 Jun 25;377(9784):2193-204
references.pmid: 21665265
references.citation: EuroIntervention. 2012 Jan;7(9):1021-9
references.pmid: 22207227
references.citation: J Am Coll Cardiol. 2013 Jan 29;61(4):404-10
references.pmid: 23265346
references.citation: Circ J. 2014;78(1):101-9
references.pmid: 24189504
references.citation: JACC Cardiovasc Interv. 2011 Jan;4(1):14-23
references.pmid: 21251624
references.citation: Circulation. 2009 Feb 10;119(5):687-98
references.pmid: 19171852
references.citation: Int J Cardiol. 2013 Apr 30;165(1):134-41
references.pmid: 21864917
references.citation: Eur Heart J. 2012 Oct;33(20):2569-619
references.pmid: 22922416
references.citation: J Invasive Cardiol. 2011 Aug;23(8):336-41
references.pmid: 21828397
references.citation: Circ J. 2010 Jun;74(6):1111-20
references.pmid: 20453391
references.citation: Am Heart J. 1999 Feb;137(2):313-21
references.pmid: 9924166
references.citation: Eur J Heart Fail. 2011 Feb;13(2):148-53
references.pmid: 21037250
references.citation: Am J Cardiol. 2008 Jun 15;101(12):1716-22
references.pmid: 18549846
references.citation: JACC Cardiovasc Interv. 2013 Jun;6(6):548-53
references.pmid: 23683734
references.citation: J Am Coll Cardiol. 2008 Jun 24;51(25):2396-402
references.pmid: 18565395
references.citation: JAMA. 2007 May 2;297(17):1892-900
references.pmid: 17473299
references.citation: Int J Cardiol. 2011 Oct 6;152(1):70-7
references.pmid: 20684999
references.citation: JACC Cardiovasc Interv. 2011 Jan;4(1):24-9
references.pmid: 21251625
references.citation: EuroIntervention. 2013 Feb 22;8(10):1199-206
references.pmid: 23425544
references.citation: Eur Heart J. 2012 Oct;33(20):2551-67
references.pmid: 22922414
references.citation: Int J Cardiol. 2008 May 7;126(1):37-44
references.pmid: 17544528
references.citation: J Am Coll Cardiol. 2014 Nov 18-25;64(20):2101-8
references.pmid: 25457398
references.citation: Circulation. 2007 May 1;115(17):2344-51
references.pmid: 17470709
references.citation: Circulation. 2010 Jul 6;122(1):52-61
references.pmid: 20566955
references.citation: Heart. 2007 Feb;93(2):210-5
references.pmid: 16940389
references.citation: Circ J. 2013;77(6):1453-60
references.pmid: 23535195