FIGURE 5.

IP₃R up-regulation sensitizes IP₃-dependent Ca²⁺ release and VSM contraction. A, PE concentration-response curve for Ca²⁺ release in control (Con) A7r5 cells or similar cells depolarized by K20 for 24 h to up-regulate IP₃R. Cells were loaded with Fura2-AM and bathed in Ca²⁺-containing HBS, and images were acquired using an epifluorescence microscope. At low PE concentrations, the two groups of cells had similar fluorescence amplitudes. At PE concentrations above 5 μm, K20-pretreated cells showed higher fluorescence amplitude than control. Values are the mean of fluorescence intensity of 15–20 individual cells for each group given as the relative change in Fura2 fluorescence ratio (ΔF₃₄₀/F₃₈₀) (n = 3–5, mean ± S.E.; *, p < 0.05; **, p < 0.01). B–E, kinetics of IP₃-dependent Ca²⁺ release evoked by UV photorelease of ciIP₃ in A7r5 cells bathed in Ca²⁺-containing HBS and loaded with ciIP₃ and Ca-Green-1. ciIP₃ was uncaged by repeated exposure of cells to UV illumination (100 ms every 1.5 s), and the fluorescence of Ca-Green was recorded in real time. B, in a subset of cells, ciIP₃ uncaging caused an initial global sustained Ca²⁺ transient followed by Ca²⁺ oscillations in both control and 20K-pretreated cells. Cells in both groups also responded to ciIP₃ uncaging by a monotonic Ca²⁺ rise without Ca²⁺ oscillations. C, the maximal IP₃-dependent Ca²⁺ release was increased in K20-pretreated cells exhibiting enriched IP₃R (n = 8; *, p < 0.05). D, the percentage of oscillating cells (n = 8–10; ***, p < 0.0001), oscillation frequency/min (n = 5–8; ***, p < 0.0001), and duration of oscillations (n = 5–7; *, p = 0.05) were enhanced in K20-pretreated cells. E, the time to threshold Ca²⁺ signal was shortened in K20-pretreated cells compared with control (n = 8; **, p < 0.001). F, contractile responses of A7r5 cells to PE (20 μm) in Ca²⁺-containing HBS were evaluated by imaging of contractile fiber formation. PE results in a transient contractile response in control cells, whereas the contractile response in 20K-pretreated cells is more pronounced and sustained. G, statistical analysis of the maximal contractile response to PE of control and K20-pretreated A7r5 cells (n = 4; **, p < 0.05). Error bars, S.E.