Figure 4

BK-induced Ca²⁺ release from internal TG-sensitive Ca²⁺ store plays a role in BK-induced MMP-9 expression. (A) Cells were pretreated without or with BAPTA/AM (30 μM) or TG (1 μM) for 1 h before exposure to 10 nM BK for the indicated time intervals. The conditioned media were collected for zymographic analysis of MMP-9 expression. (B) Cells were pretreated with or without BAPTA/AM (30 μM) or TG (1 μM) for 1 h before exposure to 10 nM BK for 16 h. The total RNA was collected and analyzed by RT-PCR. (C) For Ca²⁺ mobilization, confluent cultures of RBA-1 cells on glass coverslips were loaded with Fura-2/AM and fluorescent measurement of [Ca²⁺]_i was carried out in a dual excitation wavelength spectrofluorometer, with excitation at 340 nm and 380 nm. Cells were incubated in Ca²⁺-containing normal buffer (solid line) or Ca²⁺-free buffer (dot line) and then exposed to BK at 50 sec. In a Ca²⁺-free buffer, cells were pretreated with or without TG (1 μM), Apo (10 μM), or DPI (1 μM) for 30 min, exposed to BK at 50 sec, and then 2 mM Ca²⁺ was added to the cells. (D) RBA-1 cells were pretreated without or with BAPTA/AM (30 μM) or TG (1 μM) for 1 h and then incubated with 10 nM BK for 5 min. The Nox activity and ROS generation were analyzed. Data are expressed as the mean ± SEM (n = 3). ^*P < 0.05; ^#P < 0.01, as compared with the respective values of cells stimulated with BK only. The figure represents one of three similar experiments.