Figure 3.
KOR activation increases KOR-P staining in astrocyte culture. A–I, Astrocyte cultures were pretreated for 1 h at 37°C with vehicle (A, D, G), 1 μm U50,488 (B, E, H), or 10 μm norBNI with 1 μm U50,488 (C, F, I). Cells were fixed, double labeled with affinity-purified KOR-P antibody (10 μg/ml) and anti-GFAP antibody (1:1000), and then visualized with a confocal microscope. The KOR-P antibody did not label untreated cells (A), whereas incubation with U50,488 (1 μm) for 1 h produced an increase in KOR-P antibody labeling that colocalized with the GFAP labeling (B, H). Coincubation with the KOR-selective antagonist norBNI (10 μm) blocked the U50,488-induced increase of KOR-P labeling (C, I). GFAP staining did not change significantly after 1 h U50,588 treatment (D–F). J, Mean ± SEM pixel intensity of KOR-P staining in astrocyte cultures. Image analysis of the astrocytes expressing KOR-P treated with U50,488 demonstrated a 2.3-fold higher signal intensity (62.48 ± 4.52 AU; n = 4) than that of untreated cells (27.56 ± 1.2 AU; n = 4) (*p < 0.001). Image analysis of the astrocytes expressing KOR-P coincubated with both U50,488 and norBNI showed no significant difference in signal intensity (23.8 ± 2.04; n = 4) compared with untreated cells. K, Dual labeling of anti-A2B5 (green) and anti-GFAP (red) showed that these astrocytes were type II astrocytes. Scale bars: A–I, 100 μm; K, 50 μm.