Figure 3
Agonist-induced Ca2+mobilization. (A,B) HEK-293 cells and NG108-15 neuronal cells were transfected with expression plasmids for EGFP-tagged hOXTR-376R (common type) and visualized by fluorescence microscopy. Scale bar, 10 μm. (C,D) Images of fluorescence changes in HEK-293 cells (C) and NG108-15 cells (D) expressing hOXTR-376R-EGFP (top), hOXTR-376G-EGFP (middle), and hOXTR-376C (bottom) are shown before and after treatment with 100 nM OXT. The ratio of fluorescence intensities detected at 340 and 380 nm was used to determine [Ca2+]i. Emission ratio values ((F/F0) were pseudo-colored to represent calcium concentrations according to the scale bar on the right. Black arrows indicate application with 100 nM OXT; and white arrows show large [Ca2+]i changes in the cells after stimulation. Insets show larger images of the cells indicated by arrows. Scale bar, 100 μm. (E,F) Average time courses of changes in [Ca2+]i elicited with 100 nM OXT were measured in HEK-293 cells (E) and NG108-15 neuronal cells (F) expressing hOXTR-376R-EGFP, hOXTR-376G-EGFP, or hOXTR-376C-EGFP and in mock-transfected cells. Values are mean ± standard error of the mean (n = 24 to 30 cells from three or four independent cultures; six to ten cells were analyzed in each culture). Statistical significance was evaluated by the Student’s t test (* P <0.05, **P <0.01; compared with hOXTR-376R-EGFP).