Figure 2

Effects of calcimimetics on Cd induced PLC activity and intracellular Ca²⁺ level elevation in mRTEC cells. (a) Effects of Cd and R-467 on PC-PLC activity in mRTEC cells. After post-exposed to Cd treatment (5 μM) for 1, 2, 5 min or co-treated with R-467 (1 μM) for 2 min, the PC-PLC activity of mRTEC cells were detected. Cd induced the activity of PC-PLC in mRTEC cells in a time-dependent manner. R-467 increased PC-PLC activity in presence of Cd. Result is presented as mean ± SD (n = 4). (b) Calcium imaging shows Cd induced elevation of intracellular Ca²⁺ level in mRTEC cells was eliminated by U73122, 2-APB, BAPTA and R-467. The mRTEC cells incubated with 1 μM Fluo-3 AM dye for 30 min, then pre-treated with U73122 (1 μM), 2-APB (50 μM), and BAPTA (10 μM) for another 30 min, then treated with Cd (5 μM) or Cd (5 μM) + R-467 (1 μM) for 1 hour and observed by Olympus IX73 microscopy. Scale bar: 20 μM. (c) Western blotting shows effects of U73122, 2-APB and BAPTA on Cd-induced apoptosis. Cells pretreated with U73122 (1 μM), 2-APB (50 μM), and BAPTA (10 μM) for 30 min, followed Cd treatment (5 μM) for 24 h, total proteins were extracted for western blotting analysis of expressions of cleaved caspase-3. (d) Effects of U73122, 2-APB and BAPTA on Cd-reduced cytotoxicity in mRTEC. Cells pretreated with U73122 (1 μM), 2-APB (50 μM), and BAPTA (10 μM) for 30 min, followed Cd treatment (5 μM) for 24 h, cell viability was evaluated by MTT assay. *Statistical significance between control and treatments, or Cd treatment and in presence of inhibitors, *P < 0.05, using Student’s t-test and one-way ANOVA followed by Duncan’s multiple range tests.