FIGURE 1.

NK1R activation by GR73632 leads to reduced NE transport and reduced surface expression of NET along with reduced NK1R surface expression in rat VST synaptosomes. Rat VST synaptosomes were preincubated with vehicle or 10 μm EMEND (Em) at 37 °C for 10 min. Incubations were continued in the presence or absence of 10 μm GR73632 for 10 min. A, NE uptake: drug-treated synaptosomes 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) = 10.99; p < 0.01). B and C, surface biotinylation: synaptosomes 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) rNET or rNK1R were analyzed using mNET monoclonal antibody or rNK1R polyclonal antibody. Representative blots show a rNET-specific band at ∼70 kDa (upper panel) and rNK1R at ∼50 kDa. Densities of biotinylated rNET (∼70 kDa) or rNK1R (∼50 kDa) from three separate experiments are given as mean ± S.E. (bar graphs). * indicates significant change (p < 0.05) in surface rNET or rNK1R immunoreactivity following SP treatment compared with vehicle-control (one-way analysis of variance; Dunnett's test: F_(4,8) = 11.90, p = 0.01 for NET and F_(4,8) = 17.71, p = 0.01 for NK1R). Calnexin immunoblots corresponding to the total are shown for equal protein loading. Densities of avidin unbound (intracellular) fractions of rNET (∼70 kDa) or rNK1R (∼50 kDa) from three separate experiments are given as mean ± S.E. (bar graphs). * indicates significant change (p < 0.05) in intracellular rNET or rNK1R immunoreactivity following GR73632 treatment compared with vehicle-control (one-way analysis of variance; Dunnett's test: F_(4,8) = 7.121, p = 0.01 for NET and F_(4,8) = 9.308, p = 0.01 for NK1R).