A second stimulus produces a Ca²⁺ drop followed by a new Ca²⁺ rise. (A) Ca²⁺ signals were evoked by cGMP. Single 25% flash (blue) and two identical 25% flashes (red; Δt = 1 s) are shown. (inset) The second Ca²⁺ signal was shifted by −Δt to the left and superposed on the first Ca²⁺ signal. (B) V_m signals evoked by paired cGMP stimuli (Δt = 1 s). (inset) Kinetics of the Ca²⁺ drop (blue) and the hyperpolarization (red) evoked by the second stimulus. Downward peaks of the Ca²⁺ drop and the hyperpolarization were normalized. (C and D) Ca²⁺ (C) and V_m (D) signals. Paired stimuli (caged resact of 100 nM) were delivered. Intensity of the first flash was kept constant at 50%, and intensity of the second flash (Δt = 1 s) was varied (in percentage): 10 (green), 25 (magenta), 50 (blue), and 100 (red). For comparison, a 50% flash signal without second flash is shown (black). (E) Relationship between ΔV_m after the second flash and slope of the Ca²⁺ drop. The plot was constructed from experiments as in C and D (V_m signals, n = 4; Ca²⁺ signals, n = 3). Absolute values in millivolts for ΔV_m were obtained from ΔR signals as previously described in Strünker et al. (2006). A linear regression was fitted through data points. Numbers near data points indicate the intensity of the second flash. Error bars show SDs. (F) Scheme of the delivery of a second stimulus at different Δt along a Ca²⁺ signal. (G) Paired cGMP flashes (10% each) were delivered with Δt (in seconds) of 2 (black), 4 (red), 6 (blue), 14 (green), and 28 (magenta). (H) Comparison of Ca²⁺ drops evoked by the second flash at various Δt as in G. The second flashes were aligned to t = 0 by shifting the x axis to the left by the respective −Δt. The fluorescence before t = 0 shows the [Ca²⁺]_i at the time of the second flash. The Ca²⁺ signal evoked by the first flash is shown in orange. (I) Ca²⁺ drop amplitude (ΔF_drop) versus Ca²⁺ level (F_norm) at the time of the second flash (blue symbols, n = 21). To compare across experiments, Ca²⁺ signals were normalized to 0 (baseline Ca²⁺ signal) and 1 (Ca²⁺ signal amplitude after the first flash). A linear regression was fitted (R² = 0.98). Arrows indicate time of flashes.