FIGURE 2.

NK1R activation by SP leads to reduced NE transport and reduced cell surface expression of NET in parallel with reduced surface NK1R in transfected HTR cells. HTR cells coexpressing hNET and hNK1R were preincubated with vehicle or 10 μm EMEND (Em) at 37 °C for 10 min. Incubations were continued in the presence or absence of 0.25 μm SP for 15 min. A, NE uptake: drug-treated cells were used for NE uptake assays as described under “Experimental Procedures.” Data were derived from three separate experiments, each in triplicate are given as mean ± S.E. * indicates significant change (p < 0.01) in NE transport (one-way analysis of variance; Dunnett's test: F_(4,8) = 53.12; p < 0.01). B and C, surface biotinylation: cells treated with drugs as above were biotinylated and biotinylated NET or NK1R were isolated and analyzed as described under “Experimental Procedures.” Total (T), non-biotinylated (UB), and biotinylated (B) hNET or hNK1R were analyzed using hNET monoclonal antibody or hNK1R antibody. Representative blots show hNET-specific bands at ∼85 and ∼48 kDa (upper panel) and the hNK1R band at ∼55 kDa. Densities of biotinylated NET (∼85 kDa) or NK1R (∼55 kDa) from three separate experiments are given as mean ± S.E. (bar graphs). * indicates significant change (p < 0.05) in surface hNET or hNK1R immunoreactivity following SP treatment compared with vehicle-control (one-way analysis of variance; Dunnett's test: F_(4,8) = 19.28, p = 0.01 for hNET and F_(4,8) = 50.95, p < 0.01 for hNK1R). Calnexin immunoblots corresponding to the total are shown for equal protein loading. Densities of avidin unbound fractions of NET (∼85 kDa) or NK1R (∼55 kDa) from three separate experiments are given as mean ± S.E. (bar graphs). * indicates a significant change (p < 0.05) in intracellular hNET or hNK1R immunoreactivity following SP treatment compared with vehicle-control (one-way analysis of variance; Dunnett's test: F_(4,8) = 7.37, p = 0.01 for hNET and F_(4,8) = 7.86, p < 0.01 for hNK1R).