Fig. 2.
Exosomes are carriers of GTPγS-activatable phospholipases A2. A: HPLC profiles of exosome phospholipase A2 activity in the presence of GTPγS. Intact RBLwt exosomes were preincubated with MAFP for 10 min at room temperature. The reaction was started by adding BODIPY-PC in the presence of GTPγS at the indicated concentrations. Incubations were for 1 h at 37°C in Ca2+/Mg2+ free-PBS. B: Concentration-dependent effect of GTPγS on exosome MAFP-insensitive PLA2. Activity is expressed in pmol/h/mg of protein from HPLC profiles obtained in Fig. 2A. Inset: Double reciprocal plot: 1/(activity) versus 1/[GTPγS]. C: Effect of class-specific PLA2 inhibitors on total exosome PLA2 activity. Sonicated exosomes from RBLwt cells were preincubated for 10 min at room temperature with 50 µM of various class inhibitors—MAFP (cPLA2 > iPLA2 inhibitor), pyrrolidine (cPLA2 inhibitor), BEL (iPLA2 inhibitor), and Me-indoxam (sPLA2 inhibitor)—in the presence of 100 µM GTPγS. The reaction was started by addition of BODIPY-PC. The respective inhibitions are expressed as percentages of the total activity measured without inhibitor. The sum of inhibitions triggered by pyrolidine, BEL, and indoxam is indicated by the right bar (Total b-d). Results are means of four independent experiments +/−SEM for pyrolidine, BEL and indoxam treatments, and six independent experiments +/−SEM for MAFP treatment. E: Immunodetection of PLA2 class members in exosomes. The calcium-dependent cPLA2−IVA, calcium-independent iPLA2-VIA, and secreted sPLA2-IIA and V were detected in RBLwt-derived exosomes by Western blotting. Exos = exosomes; sPLA2 IIA = recombinant sPLA2-IIA; sPLA2 V = recombinant sPLA2-V.