Fig. 3.

Sustained stimulation with PTH(1-34) reduces carbachol-evoked increases in cytosolic IP₃ concentration. (A) Cytosolic IP₃ was measured in single HEK-PR1 cells using a FRET sensor during stimulation (3 min) with 1 mM carbachol (CCh) (S1) and then, after washing, with 30 µM carbachol added 30 min later (S2). The trace shows typical results from a cell with no intervening PTH treatment. FRET is denoted as CFP:YFP fluorescence ratio, so that an increased signal (decreased FRET) corresponds to an increase in IP₃ concentration (see Materials and Methods). The inset shows the IP₃ sensor with excitation and emission (italics) wavelengths in nm. IBC, IP₃-binding core. (B) Summary results (mean±s.e.m. for 62 cells from six coverslips) showing ΔFRET (stimulated/basal signal) for cells stimulated with the indicated carbachol concentrations presented as either the first (S1) or second stimulus (S2, i.e. after 1 mM carbachol). (C,D) Typical results from single cells subject to similar treatments to those in A, but with PTH(1-34) (100 nM) added 1 min (C) or 30 min (D) before, and then during, the second addition of carbachol. (E,F) Summary results show ΔFRET for the first and second carbachol stimulation (S1 and S2) as mean±s.e.m. for 36 and 34 cells from five (E) and seven (F) coverslips. (G) For each cell, ΔFRET measurements for the first (S1, 1 mM carbachol) and second stimulus (S2, 30 µM carbachol) were used to calculate S2/S1 for the indicated treatments. Results are mean±s.e.m. for 28–36 cells.