Fig. 2.
Gpr116 activation by exogenous synthetic agonist peptide mobilizes calcium in vitro and in split-open cortical collecting ducts. (A–E) Stimulation of heterologous murine and human Gpr116 in HEK293 cells with p16 synthetic agonist peptide (200 μM), but not with the control peptide (F3A), causes an increase in [Ca2+]i as measured by ΔF340/F380. Nontransfected control is shown in E. In A and C, traces are the mean ± SEM of 20 individual p16-responsive cells; in B, D, and E traces are the mean ± SEM of 16 regions of an entire field of view. (F) Representative images of Fura-2 loaded murine split-open collecting ducts demonstrating identification of V-ATPase expressing ICs. As these are split-open tubules viewed from above, V-ATPase stain can be used to identify cell types (but not for subcellular localization). Magnification: 40×. (G) Calcium mobilization in principal cells following stimulation with p16 (200 μM) and then ATP (50 μM) in split-open collecting ducts. Trace is mean ± SEM of 24 cells from three collecting ducts. (H) Calcium mobilization in V-ATPase expressing ICs following stimulation with p16 and then ATP. Trace is mean ± SEM of 21 cells from 3 collecting ducts.