FIGURE 4.

A–D, Ca²⁺ imaging experiments, showing involvement of endogenous H₂O₂ in AVP-stimulated Ca²⁺ entry. A, representative traces, showing the AVP-evoked Ca²⁺ response in HEK293T cells expressing TRPC6 and an empty vector (T6+vector) or in cells co-transfected with TRPC6 and AVP V1R in the presence (T6+V1R+Catalase) or absence (T6+V1R) of PEG-catalase (250 units/ml for 30 min). Ca²⁺ concentration in the bathing solution is indicated by the numbers inside the top open bar. Application of 100 nm AVP is indicated by the top solid bar. [Ca²⁺]_B indicates the Ca²⁺ concentration in the bathing solution. B, summary data, showing Ca²⁺ entry responses in different groups. *, p < 0.05, compared with T6 + vector; †, p < 0.05, compared with T6 + vector and T6 + V1R. C, effect of catalase on AVP-induced Ca²⁺ release in cells co-transfected with TRPC6 and AVP V1R. D, effect of catalase on normalized Ca²⁺ entry (ratio of Ca²⁺ entry to Ca²⁺ release) in response to AVP stimulation. *, p < 0.05, compared with T6 + V1R. B–D, n indicates the number of cells analyzed from 4 to 5 sets of transfection. E–G, in A7r5 cells. E, whole-cell patch clamp, showing inward currents in response to 100 μm H₂O₂ in A7r5 with (siRNA-T6) and without (Scramble) knockdown of TRPC6 at a holding potential of −60 mV. The dashed lines indicate zero currents. Application and removal of H₂O₂ were indicated by the vertical lines. F, Western blot, showing TRPC6 expression levels in A7r5 cells transfected with TRPC6 siRNA sequence (siRNA-T6) and scrambled sequence (Scramble). Actin served as a loading control. UT, untransfected. G, summary data, showing the H₂O₂ effect on whole-cell currents in A7r5 cells with (SiRNA-T6) and without (Scramble) knockdown of TRPC6. The responses were measured by the difference between the membrane current before application of H₂O₂ and the peak current after application of H₂O₂. The whole-cell currents were expressed as current density (pA/pF), normalized to the cell membrane capacitance. n indicates the number of cells analyzed from three sets of transfection. Asterisk indicates statistically significant difference compared with Scramble (Student's t test).