Thors L, Alajakku K, Fowler C J
Department of Pharmacology and Clinical Neuroscience, Umeå University, Umeå, Sweden.
Br J Pharmacol. 2007 Apr;150(7):951-60. doi: 10.1038/sj.bjp.0707172. Epub 2007 Feb 26.
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.
FAAH activity and cellular uptake of anandamide was measured using anandamide, radio-labelled either in the ethanolamine or arachidonoyl part of the molecule.
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).
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.
脂肪酸酰胺水解酶（FAAH）抑制剂以及破坏内吞机制的试剂均可降低花生四烯酸乙醇胺的细胞摄取。然而，尚不清楚这些事件是否在同一时间范围内发生，以及它们在不同细胞中的发生程度是否相同。因此，我们使用不同的测定孵育时间和温度，研究了此类化合物对三种不同来源细胞系的影响。
使用在分子的乙醇胺或花生四烯酰部分进行放射性标记的花生四烯酸乙醇胺来测量FAAH活性和花生四烯酸乙醇胺的细胞摄取。
FAAH抑制剂URB597在孵育4分钟时抑制了花生四烯酸乙醇胺进入C6胶质瘤细胞、RBL2H3嗜碱性白血病细胞和P19胚胎癌细胞。然而，URB597处理后仍存在时间依赖性和温度敏感性的残留摄取。孕酮和制霉菌素的组合降低了摄取，但也减少了孔中保留的花生四烯酸乙醇胺量。染料木黄酮抑制花生四烯酸乙醇胺摄取的方式与URB597的抑制方式无相加作用。然而，染料木黄酮是FAAH的有效竞争性抑制剂（抑制常数K(i)值为8 microM）。
染料木黄酮对花生四烯酸乙醇胺摄取的降低可归因于其抑制FAAH的能力，其效力与抑制酪氨酸激酶的效力重叠。在所有研究的三种细胞系中均观察到花生四烯酸乙醇胺对FAAH耐药但具有时间依赖性的摄取，因此推测这是一个普遍发生的过程。
