198 G.E. Morris et al. Figure 3 Time-course of UTP-induced P2Y2-receptor desensitization. MSMCs were transfected with eGFP-PH (0.5 mg), or loaded with Fluo4 and subjected to the following desensitization protocol: cells were challenged with UTP (R1, 10 mM, 30 s) 5 min before a desensitizing UTP concentration (Rmax ¼ 100 mM for 30 s), and again after a variable washout period (2, 3, 5, 10, or 15 min) (R2, 10 mM, 30 s). Representative traces and images from single cells either expressing eGFP-PH (A and B) or loaded with Fluo4 (C and D) with either a 3 (A and C ), 10 (B), or 15 min (D) wash period are shown. P2Y2 receptor desensitization was determined as the relative (%) change in R2 response compared with R1 for both eGFP-PH and Fluo4 signals. (E) Cumulative data are presented as means + SEM for ﬁve to nine cells (eGFP-PH), or 21 – 45 cells (Fluo4) at each time-point. MSMCs used were generated from preparations from three or more different animals.