Confocal microscopy and biochemical analysis reveal spatial and functional separation between anandamide uptake and hydrolysis in human keratinocytes.
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.
Oddi
S
S
Department of Biomedical Sciences, University of Teramo, Piazza A. Moro 45, 64100 Teramo, Italy.
Bari
M
M
Battista
N
N
Barsacchi
D
D
Cozzani
I
I
Maccarrone
M
M
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
D000581:Amidohydrolases; D001095:Arachidonic Acids; D063388:Endocannabinoids; D053284:Polyunsaturated Alkamides; D000099252:Fatty Acid Amide Hydrolases; C078814:anandamide
J Am Coll Cardiol. 2008 Sep 30;52(14):1134-40
18804739
Cardiovasc Interv Ther. 2017 Jan;32(1):24-35
26979599
J Am Coll Cardiol. 2008 Mar 11;51(10):986-90
18325436
J Interv Cardiol. 2012 Apr;25(2):118-25
22324820
J Am Coll Cardiol. 2013 May 7;61(18):1853-9
23500262
Am J Cardiol. 2014 Jan 15;113(2):229-35
24188893
JACC Cardiovasc Interv. 2011 Jan;4(1):30-8
21251626
Lancet. 2011 Jun 25;377(9784):2193-204
21665265
EuroIntervention. 2012 Jan;7(9):1021-9
22207227
J Am Coll Cardiol. 2013 Jan 29;61(4):404-10
23265346
Circ J. 2014;78(1):101-9
24189504
JACC Cardiovasc Interv. 2011 Jan;4(1):14-23
21251624
Circulation. 2009 Feb 10;119(5):687-98
19171852
Int J Cardiol. 2013 Apr 30;165(1):134-41
21864917
Eur Heart J. 2012 Oct;33(20):2569-619
22922416
J Invasive Cardiol. 2011 Aug;23(8):336-41
21828397
Circ J. 2010 Jun;74(6):1111-20
20453391
Am Heart J. 1999 Feb;137(2):313-21
9924166
Eur J Heart Fail. 2011 Feb;13(2):148-53
21037250
Am J Cardiol. 2008 Jun 15;101(12):1716-22
18549846
JACC Cardiovasc Interv. 2013 Jun;6(6):548-53
23683734
J Am Coll Cardiol. 2008 Jun 24;51(25):2396-402
18565395
JAMA. 2007 May 2;297(17):1892-900
17473299
Int J Cardiol. 2011 Oct 6;152(1):70-7
20684999
JACC Cardiovasc Interv. 2011 Jan;4(1):24-9
21251625
EuroIntervention. 2013 Feb 22;8(10):1199-206
23425544
Eur Heart J. 2012 Oct;33(20):2551-67
22922414
Int J Cardiol. 2008 May 7;126(1):37-44
17544528
J Am Coll Cardiol. 2014 Nov 18-25;64(20):2101-8
25457398
Circulation. 2007 May 1;115(17):2344-51
17470709
Circulation. 2010 Jul 6;122(1):52-61
20566955
Heart. 2007 Feb;93(2):210-5
16940389
Circ J. 2013;77(6):1453-60
23535195