Figure 5

Morphine inhibits FSK-potentiated capsaicin responses in FLAG-MOP/TRPV1 double stable HEK293 cells but not capsaicin responses in absence of FSK. Double stable FLAG-MOP/TRPV1 colonies were loaded with Fluo-3 and pre-incubated with PSS for untreated or morphine (1 μM) for treated cells for 15 min followed by further incubation with buffer containing either no added drugs for unstimulated cells or FSK (50 μM) and IBMX (100 μM) stimulated cells. Ca²⁺ responses after injection of capsaicin were monitored for 45 s using a fluorescent microplate reader. (A and D) Capsaicin dose-response curves for colony 21 (A) and colony 13 (D) without pre-treatment (capsaicin, ▼), after morphine incubation (capsaicin+morphine, x), after pre-incubation with FSK+IBMX (capsaicin+FSK+IBMX, ●) and after treatment with FSK, IBMX and morphine (capsaicin+FSK+IBMX+morphine, ◊). A 4-parameter Hill equation was fitted using GraphPad Prism. Data was plotted as maximum increase in fluorescence (ΔF/F) after injection of capsaicin. FSK-potentiated capsaicin responses were inhibited by morphine (1 μM), while capsaicin responses in the absence of FSK were not affected by morphine. B and C, E and F: Representative Ca²⁺ responses evoked by capsaicin (300 nM) in FLAG-MOP/TRPV1 expressant 21 (B and C) and 13 (E and F). FLAG-MOP/TRPV1 colony 21 and 13 responded to injection with 300 nM capsaicin with a characteristic increase in Ca²⁺, represented as an increase in ΔF/F (B and E). Pre-incubation with morphine (1 μM) did not affect the magnitude of Ca²⁺ responses (B and E). Pre-incubation with FSK (50 μM) and IBMX (100 μM) increased capsaicin-evoked Ca²⁺ responses (C and F) and treatment with morphine (1 μM) inhibited FSK-potentiated capsaicin responses (C and F). Data are presented as mean ± SEM with n = 8 for each colony and capsaicin concentration and statistical significance were determined at peak Ca²⁺ responses after addition of capsaicin. * p < 0.05 compared to FSK alone.