KISS1R trafficking is essential for the KP10-induced sustained second-phase calcium response. A–C, CHO-KISS1R (A), GT1–7-KISS1R (B), and CHO-TRHR (C) cells were treated with 10−7 M KP10 or 10−7 M TRH, respectively, in the presence or absence of 80 μM dynasore, a dynamin inhibitor, or 5 μM PAO, an endocytosis inhibitor, to block KISS1R internalization. The values shown are depicted as RFU. D and E, CHO-KISS1R cells were treated with 10−7 M KP10 in the presence or absence of 5 μg/mL BFA, a receptor recycling inhibitor. The [Ca2+]i responses were recorded every 10 to 30 seconds for 5 to 10 minutes. The values shown are depicted as percent maximum response. All experiments were repeated 3 times, each in triplicate. Means ± SEM are shown. Significant differences were present among/between the curves in A, B, and D, as determined by two-way ANOVA (P < .001).