Figure 1.
KP10 stimulates a biphasic intracellular calcium response that requires the continued presence of extracellular KP10. A, CHO-KISS1R cells were treated with increasing concentrations (10−9–10−6 M) of KP10. [Ca2+]i was measured as RFU every 30 seconds for 30 to 60 minutes. A single representative experiment is shown, which was repeated 3 times with similar results. B, CHO-KISS1R and CHO-TRHR cells were treated with 10−7 M KP10 and 10−7 M TRH, respectively. [Ca2+]i was recorded every 30 seconds for 10 minutes and is depicted as RFU. A representative experiment is shown, which was repeated 3 times with similar results. C, CHO-KISS1R cells were treated with 10−8 M KP10. After 5 minutes, some cells (marked KP10 removal and KP10 removal + KP10) were washed 5 times with the same calcium assay medium to remove ligand. After the final wash, calcium assay medium without KP10 (for the KP10 removal group) or calcium assay medium containing 10−8 M KP10 (for the KP10 removal + KP10 group) was added. The total time for the washes was less than 20 seconds. After 30 minutes, the medium in the KP10 removal and KP10 removal + KP10 was again removed and replaced with medium containing 10−8 M KP10. D, GT1–7-myc-KISS1R cells were treated with 10−8 M KP10. After 5 minutes, some cells (marked KP10 removal and KP10 removal + KP10) were washed 5 times with the same calcium assay medium in the absence or presence of KP10 and further treated as described for C, except that the interval between treatments was 10 minutes. A representative plot of 3 independent experiments with similar results is shown.