Inhibition of the cellular uptake of anandamide by genistein and its analogue daidzein in cells with different levels of fatty acid amide hydrolase-driven uptake.
OBJECTIVE
Genistein, a tyrosine kinase inhibitor used to block caveolae dependent endocytosis, reduces the cellular uptake of anandamide in RBL2H3 basophilic leukaemia cells. However, genistein is also a competitive inhibitor of fatty acid amide hydrolase, the enzyme responsible for anandamide hydrolysis. Here we have investigated whether inhibition of fatty acid amide hydrolase rather than inhibition of endocytosis is the primary determinant of genistein actions upon anandamide uptake.


METHODS
Cellular uptake of anandamide, labelled in the arachidonoyl part of the molecule was assessed in four different cell lines using a standard method. Fatty acid amide hydrolase activity in homogenates and intact cells was measured using anandamide labelled in the ethanolamine part of the molecule.


RESULTS
The fatty acid amide hydrolase inhibitor URB597 inhibited anandamide uptake into RBL2H3 cells and R3327 AT-1 prostate cancer cells, but not into 3T3-L1 preadipocytes or PC-3 prostate cancer cells. An identical pattern was seen with genistein. The related compound daidzein inhibited anandamide hydrolysis in homogenates and intact cells, and reduced its uptake into RBL2H3 and R3327 AT-1, but not PC-3 cells. Anandamide hydrolysis by cell homogenates was in the order RBL2H3 > R3327 AT-1 > PC-3 approximately 3T3-L1.


CONCLUSIONS
The ability of genistein to inhibit anandamide uptake is mimicked by daidzein (which does not affect tyrosine kinase), and is only seen in cells that show sensitivity to URB597. This indicates that blockade of fatty acid amide hydrolase is the primary determinant of the effects of genistein on cellular anandamide uptake.
Thors
L
L
Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden.
Eriksson
J
J
Fowler
C J
CJ
D041721:3T3-L1 Cells; D000581:Amidohydrolases / Q000378:metabolism*; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism* / Q000493:pharmacokinetics; D001549:Benzamides / Q000494:pharmacology; D001692:Biological Transport / Q000187:drug effects; D002219:Carbamates / Q000494:pharmacology; D045744:Cell Line, Tumor; D004305:Dose-Response Relationship, Drug; D063388:Endocannabinoids; D019833:Genistein / Q000737:chemistry / Q000494:pharmacology*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis / Q000187:drug effects; D007529:Isoflavones / Q000737:chemistry / Q000494:pharmacology*; D008297:Male; D051379:Mice; D015394:Molecular Structure; D053284:Polyunsaturated Alkamides / Q000378:metabolism* / Q000493:pharmacokinetics; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D051381:Rats; D014316:Tritium; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D001549:Benzamides; D002219:Carbamates; D063388:Endocannabinoids; D019833:Genistein; D007529:Isoflavones; D053284:Polyunsaturated Alkamides; D047428:Protein Kinase Inhibitors; D014316:Tritium; D000099252:Fatty Acid Amide Hydrolases; C500528:cyclohexyl carbamic acid 3'-carbamoylbiphenyl-3-yl ester; C004742:daidzein; C078814:anandamide