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.