Figure 2. The recycling of AEA to form new AEA and 2-AG molecules requires intact lipid rafts and is blocked with AM404.

RBL-2H3 cells were incubated with 1 nM [³H] AEA for 24 hours. Cells were washed and then treated with nystatin (25 μg/mL)/progesterone (10 μg/mL) (N/P) or KRH buffer without nystatin and progesterone for 30 min at 37 °C. One of the indicated treatment conditions included a 100 μM AM404 treatment (10 min at 37 °C). Cells were then treated either with buffer or 1 μM ionomycin for 10 min at 37 °C in order to stimulate AEA (A) and 2AG (B) synthesis and release, or ARA (C) release. The extracellular reaction buffer was collected, and the lipids were separated and analyzed as described in Materials and Methods. The results obtained with TLC analysis were confirmed using an HPLC method. The identity of the peak corresponding to AEA on the UV spectrum corresponded to the [³H] AEA that eluted from the HPLC (inset). One-way ANOVA with Newman-Keul’s post-hoc test was performed for statistical analysis. * p<0.05, ionomycin vs. AM404+ionomycin or ionomycin vs. N/P+ionomycin. Data represent mean ± SEM from three separate experiments performed in duplicate. CPM values (means ± SEM) for [³H] AEA synthesis and release were: buffer = 60 ± 18; ionomycin = 689 ± 157; AM404+ionomycin = 253 ± 38; N/P+buffer = 37 ± 5; and N/P+ionomycin = 152 ± 35. For [³H] 2-AG synthesis and release: buffer = 31 ± 5; ionomycin = 395 ± 170; AM404+ionomycin = 195 ± 65; N/P+buffer = 31 ± 7; and N/P+ionomycin = 58 ± 13. For [³H] ARA release: buffer = 56 ± 15; ionomycin = 308 ± 186; AM404+ionomycin = 345 ± 178; N/P+buffer = 86 ± 46; and N/P+ionomycin = 174 ± 147.