Measurement of saturable and non-saturable components of anandamide uptake into P19 embryonic carcinoma cells in the presence of fatty acid-free bovine serum albumin.
There is considerable controversy at present concerning the mechanisms responsible for the cellular uptake of anandamide. One particular issue concerns whether fatty acid-free bovine serum albumin should be used in the assays, it having been argued that such a presence effectively prevents the specific uptake of anandamide. In the present study, it has been demonstrated that in the presence of a low (0.1%, w/v) concentration of fatty acid-free bovine serum albumin, a temperature-dependent and saturable (K(m) approximately 1 microM) uptake of anandamide into P19 embryonic carcinoma cells can be demonstrated using an incubation time of 4 min. Under these conditions, the uptake of anandamide at 4 degrees C is low at a substrate concentration of 100 nM. The uptake at 37 degrees C was not significantly reduced following treatment of the cells with either methyl-beta-cyclodextrin (50 microM) or mevinolin (1 microM), but was reduced by the FAAH inhibitor URB597 (1 microM) and inhibited by the transport inhibitor cum FAAH substrate AM404 with an IC(50) value of 12 microM. When a 45 s incubation time was used, the uptake of anandamide was not saturable at 37 degrees C over the concentration range tested (0.1-1 microM). Analysis of the data at 37 degrees C obtained with 45 s, 4 min and 15 min incubation times revealed a very rapid (i.e. complete by 45 s) non-saturable component followed by a slower saturable (K(m) approximately 1 microM) component of the uptake. It is concluded that the presence of a low concentration of fatty acid-free bovine serum albumin at a suitable concentration reduces non-specific binding (and release) of anandamide to cell culture wells, greatly reduces the cellular accumulation seen at 4 degrees C, and allows the visualisation of both non-saturable and saturable components of the uptake to be seen at 37 degrees C.
Sandberg
Anna
A
Department of Pharmacology and Clinical Neuroscience, Umeå University, SE-901 87 Umeå, Sweden.
Fowler
Christopher J
CJ
D000581:Amidohydrolases / Q000378:metabolism; D000818:Animals; D000924:Anticholesteremic Agents / Q000494:pharmacology; D001095:Arachidonic Acids / Q000378:metabolism*; D002277:Carcinoma; D045744:Cell Line, Tumor; D002784:Cholesterol / Q000378:metabolism; D004622:Embryo, Mammalian; D063388:Endocannabinoids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors / Q000494:pharmacology; D008148:Lovastatin / Q000494:pharmacology; D051379:Mice; D053284:Polyunsaturated Alkamides; D012710:Serum Albumin, Bovine / Q000378:metabolism; D047392:beta-Cyclodextrins / Q000494:pharmacology; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D000924:Anticholesteremic Agents; D001095:Arachidonic Acids; D002784:Cholesterol; D063388:Endocannabinoids; D019161:Hydroxymethylglutaryl-CoA Reductase Inhibitors; D008148:Lovastatin; D053284:Polyunsaturated Alkamides; D012710:Serum Albumin, Bovine; D047392:beta-Cyclodextrins; D000099252:Fatty Acid Amide Hydrolases; C108732:methyl-beta-cyclodextrin; C078814:anandamide