Fig 1: Sig-1R and PLD Contribute to Ca2+ Signals Evoked by Agonists of GPCRs(A) Typical pseudocolor images show peak Ca2+ signals (F340/F380) evoked by ATP (50 μM) in Fura-2-loaded NG108-15 cells transfected with control shRNA or shRNA to PLD1 and PLD2, or Sig-1R, each tagged with RFP. Calibration code (F340/F380) and scale bar (20 μm) apply to all panels.(B) Time course of response to ATP (bar; n = 6).(C) Summary (mean ± SD; n = 6) shows Δ[Ca2+]i evoked by ATP. ∗p < 0.05, ANOVA with Bonferroni test, relative to untransfected cells.(D) Δ[Ca2+]i evoked by bradykinin in populations of NG108-15 cells. Histogram (which shares the y axis) compares responses to bradykinin (10 μM) after treatment with scrambled or Sig-1R shRNA. Results are means ± SEM; n = 3 with duplicate determinations. ∗p < 0.05, Student’s t test.(E) WB shows effects of indicated shRNA, each tagged with RFP, on expression of PLD1 and PLD2 in NG108-15 cells. Mr markers (kDa) are shown. Results, typical of 3 WBs, underestimate knockdowns in the cells used for Ca2+ measurements, which used only cells shown to be transfected by expression of RFP (see A).(F and G) Intracellular concentrations of choline (F) and IP3 (G) during stimulation of NG108-15 cells with ATP (50 μM, bar) show the effects of shRNA for PLD1 and PLD2. Results show means ± SD; n = 6.(H) GPCRs that activate PLC and phospholipase D (PLD) initiate two parallel signaling pathways that converge at IP3Rs. IP3 from PLC directly activates IP3R. Choline from PLD activates Sig-1R, which potentiates IP3-evoked Ca2+ release.