Figure 7

8-Br-cAMP stimulated capsaicin responses are not inhibited by morphine. (A) FLAG-MOP/TRPV1 double stable HEK293 cells (Colony 21) were loaded with the fluorescent Ca²⁺ dye Fluo-3 and Ca²⁺ responses to injection of 300 nM capsaicin were monitored using a fluorescent Microplate reader. Cells were pre-incubated for 15 min with IBMX (100 μM) as well as varying concentrations of the direct PKA activator 8-Br-cAMP (1 mM, 300 μM, 100 μM, 30 μM, 10 μM, 3 μM, 1 μM or PSS). Capsaicin (300 nM) was injected and Ca²+ responses plotted as the maximum increase in ΔF/F after capsaicin injection. Pre-incubation with 8-Br-cAMP and IBMX potentiated Ca²⁺ responses to 300 nM capsaicin in an 8-Br-cAMP-concentration-dependent manner. (B) Morphine does not inhibit capsaicin responses potentiated by the direct PKA activator 8-Br-cAMP. Fluo-3 loaded FLAG-MOP/TRPV1 cells were incubated with PSS for untreated cell (◆) or morphine (1 μM) for treated cells (○) for 15 min, followed by potentiated with IBMX (100 μM) and 8-Br-cAMP (300 μM) for 15 min. Untreated control responses are denoted by (*). Pre-incubation with morphine did not inhibit 8-Br-cAMP potentiated capsaicin responses (○). Data are presented as mean ± SEM for n = 4–8. (C) Morphine does not inhibit capsaicin responses potentiated by the PKC activator PMA. Fluo-3 loaded FLAG-MOP/TRPV1 cells were incubated with PSS for untreated cell (*). PMA-potentiation was achieved by incubation of cells for 10 minutes with PMA (100 nM), preceded by 15 min incubation with PSS for control responses (○) or morphine (1 μM) for treated cells (▼). Pre-incubation with morphine did not inhibit 8-Br-cAMP potentiated capsaicin responses.