FIGURE 1.

Cocaine-induced NET up-regulation and phosphorylation in the mouse PFC are p38 MAPK-dependent. Mouse PFC synaptosomes were preincubated with vehicle (Veh) or 20 μm PD169316 (PD) at 37 °C for 15 min. Incubations were continued in the presence or absence of 50 μm cocaine (Coc) for 60 min. A, NE uptake. Data derived from three separate experiments, each in triplicate, are given as mean ± S.E. (error bars). * indicates a significant change in NE transport (one-way analysis of variance; Dunnett's test: F_(4,44) = 5.31, p < 0.05). B, NET surface biotinylation. Synaptosomes treated with drugs as above were biotinylated, and biotinylated NETs were isolated by avidin binding. Equal aliquots from total (T) and avidin-unbound fractions (UB) and entire eluates from avidin beads representing bound fractions (B) were loaded onto gels, and the blots were probed with commercially available mouse NET monoclonal antibody as described under “Experimental Procedures.” Representative blots show a NET-specific band at ∼85 kDa. Increased avidin-bound NET in cocaine-treated synaptosomes compared with vehicle-, PD169316-, or PD169316 + cocaine-treated synaptosomes is shown. Quantified surface NET densities (normalized to TH levels in the total blot) from three separate experiments are given in the bar graph as mean ± S.E. (error bars). * indicates a significant change from vehicle control (one-way analysis of variance; Dunnett's test: F_(4,8) = 4.16, p < 0.05). A TH-reprobed immunoblot shows relative equivalent loading of total protein. C, NET phosphorylation. Following metabolic labeling with ³²P, synaptosomes were treated with drugs as described above and solubilized. The synaptosomal extract was subjected to immunoprecipitation with NET-82 polyclonal antibody and autoradiography as described under “Experimental Procedures.” A representative autoradiogram shows enhanced density of phospho-NET following cocaine treatment compared with vehicle, PD169316, or PD169316 + cocaine treatments. Parallel immunoprecipitations (IP) using unlabeled synaptosomes show pulldown of an equal amount of NET following drug treatments as well as no NET protein in the unbound fractions (shown in the immunoblots (IB) below the autoradiogram). Mean values of quantified phospho-NET (p-NET) density (normalized to NET pulldowns from unlabeled synaptosomes) from two experiments are given below in the bar graph. D, NET-p38 MAPK interaction. Synaptosomes treated as indicated were solubilized, and equal aliquots of synaptosome extracts were subjected to pulldown experiment using phospho-p38 MAPK (p-p38MAPK) antibody-immobilized beads or immunoprecipitation with NET antibody followed by SDS-PAGE and immunoblotted with NET, DAT, SERT, or phospho- or total p38 MAPK antibodies. Representative NET-, DAT-, SERT-, and total or phospho-p38 MAPK-immunoreactive bands from two independent experiments are shown. Total extracts subjected to SDS-PAGE and immunoblotting with TH antibody show input levels, and bar graphs below the immunoblots show mean values of quantified phospho-p38 MAPK-associated NET, DAT, and SERT densities (normalized to TH input) or NET-associated phospho-p38 MAPK densities (normalized to TH input) from two experiments. Amph, amphetamine.