A2a receptors block mTORC2 and mitochondrial activation. (A and B) Freshly isolated PMNs were loaded with Rhod2 as described in Fig. 2 and stimulated simultaneously with 10 nM fMLP and 1 µM of the A2a receptor agonist CGS21680 at t = 0 s, and mitochondrial Ca²⁺ uptake was assessed by flow cytometry. (B) Mitochondrial Ca²⁺ uptake in cells treated with the indicated concentrations of CGS21680. (C) PMNs loaded with Rhod2 were incubated with or without 10 µM H89 for 30 min and stimulated with 10 nM fMLP ± 1 µM CGS21680, and mitochondrial Ca²⁺ uptake was assessed with flow cytometry. (D) PMNs loaded with Rhod2 were incubated for 30 min with or without 1 µM cAMP-AM, and mitochondrial Ca²⁺ uptake in response to fMLP stimulation (1 nM) was monitored with fluorescence microscopy (see also Video 9). (E) Differentiated HL-60 cells were pretreated with 1 µM cAMP-AM and stimulated with 100 nM fMLP, or exposed to the indicated concentrations of CGS21680 and simultaneously stimulated with 100 nM fMLP. After 5 min, mTOR and MAPK p38 activation was determined with immunoblotting as described in Fig. 5. Results are expressed as means ± SD (error bars) of at least three independent experiments; *, P < 0.05; Student’s t test.