The 'specific' tyrosine kinase inhibitor genistein inhibits the enzymic hydrolysis of anandamide: implications for anandamide uptake.
OBJECTIVE
The cellular uptake of anandamide is reduced by inhibitors of fatty acid amide hydrolase (FAAH) and by agents disrupting endocytotic mechanisms. However, it is not clear if these events occur over the same time frame and if they occur to the same extent in different cells. We have therefore investigated the effects of such compounds in three cell lines of different origins using different assay incubation times and temperatures.


METHODS
FAAH activity and cellular uptake of anandamide was measured using anandamide, radio-labelled either in the ethanolamine or arachidonoyl part of the molecule.


RESULTS
The FAAH inhibitor URB597 inhibited the uptake of anandamide into C6 glioma, RBL2H3 basophilic leukaemia cells and P19 embryonic carcinoma cells at incubation time 4 min. However, a time-dependent and temperature-sensitive residual uptake remained after URB597 treatment. The combination of progesterone and nystatin reduced the uptake, but also decreased the amount of anandamide retained by the wells. Genistein inhibited anandamide uptake in a manner that was not additive to that of URB597. However, genistein was a potent competitive inhibitor of FAAH (K(i) value 8 microM).


CONCLUSIONS
The reduction of anandamide uptake by genistein can be explained by its ability to inhibit FAAH with a potency which overlaps that for inhibition of tyrosine kinase. The FAAH- resistant but time-dependent uptake of anandamide is seen in all three cell lines studied and is thus presumably a generally occurring process.
Thors
L
L
Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden. lina.thors@pharm.umu.se
Alajakku
K
K
Fowler
C J
CJ
D000581:Amidohydrolases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D000818:Animals; D001095:Arachidonic Acids / Q000378:metabolism*; D001549:Benzamides / Q000494:pharmacology; D001921:Brain / Q000201:enzymology; D002219:Carbamates / Q000494:pharmacology; D045744:Cell Line, Tumor; D063388:Endocannabinoids; D019833:Genistein / Q000494:pharmacology*; D006868:Hydrolysis; D051379:Mice; D009761:Nystatin / Q000494:pharmacology; D053284:Polyunsaturated Alkamides / Q000378:metabolism*; D011374:Progesterone / Q000494:pharmacology; D047428:Protein Kinase Inhibitors / Q000494:pharmacology; D011505:Protein-Tyrosine Kinases / Q000037:antagonists & inhibitors; D051381:Rats; D000099252:Fatty Acid Amide Hydrolases
D000581:Amidohydrolases; D001095:Arachidonic Acids; D001549:Benzamides; D002219:Carbamates; D063388:Endocannabinoids; D019833:Genistein; D009761:Nystatin; D053284:Polyunsaturated Alkamides; D011374:Progesterone; D047428:Protein Kinase Inhibitors; D011505:Protein-Tyrosine Kinases; D000099252:Fatty Acid Amide Hydrolases; C500528:cyclohexyl carbamic acid 3'-carbamoylbiphenyl-3-yl ester; C078814:anandamide