Figure 9.

Influence of IN3 and cetrorelix (Cet.) on GnRH-stimulated NFAT-EFP translocation in gonadotroph-lineage cells. LβT2 cells were transduced with the NFAT-EFP reporter (10 pfu/nl) and were stimulated 45 min with the indicated concentrations of GnRH as above (Fig. 7) except that they were not transduced with Ad GnRHR. Panel A shows representative images for control and 10⁻⁷ m GnRH-stimulated wells, and the lower images illustrate image segmentation and the application of a filter to define the proportion of cells in which the N:C NFAT-EFP ratio is more than 1.25. Effects of IN3 and cetrorelix were also determined by coincubation of the indicated doses of agonists and antagonists (panels B and C). GnRH concentration-response curves were also generated in cells preincubated (18 h) with the indicated concentrations of IN3 or cetrorelix and then coincubated (45 min) with the indicated concentration of GnRH and the same antagonist concentration as used in the pretreatment (panels D and E). The effect of the preincubation is illustrated for single antagonist concentrations by comparison of curves with antagonist coincubation alone and those with co- and preincubation with 10⁻⁸ m IN3 (panel F; data from panels B and D) or 10⁻⁹ m cetrorelix (panel G; data from panels C and E). The figures show the proportion of cells in which the N:C NFAT-EFP ratio was more than 1.25, normalized (as a percent of the internal control maximal response). Values shown are means ± sems (n = 3–4) from four separate experiments, each with triplicate wells.