Figure 10.

Cav-1 regulates intracellular Ca²⁺ concentration through NR2B. A, RT-PCR assay showed that Cav-1 mRNA expression was significantly increased in the cultured ACC neurons treated with 50 mm KCl for 12 h. ***p < 0.001, compared with the KCl group. Total RNA was extracted from the cultured ACC neurons at 12 h after KCl treatment. The mRNA expression levels of Cav-1 were then measured in the total RNA by performing real-time RT-PCR, using β-actin as an internal control. These levels were expressed as a percentage of those in naive controls. RT-PCR assay was performed at three independent experiments.***p < 0.001, compared with the KCl group. B, The representative Fluo-3 fluorescence (left) and the quantitative data (right) showed that knockdown of Cav-1 with Cav-1 siRNA, but not control scramble siRNA, significantly inhibited KCl-induced increase of intracellular Ca²⁺ concentration in cultured ACC neurons. The cultured ACC neurons were treated with 0.8 μg/ml Cav-1 siRNA or its control scramble siRNA for 72 h before treatment with KCl. Then neurons in culture were loaded with Fluo-3 AM (5 μm for 30 min at 37°C) to measure changes in free intracellular Ca²⁺. The fluorescence changes determined by Fluo-3 represent the cytoplasmic calcium [Ca²⁺]i changes (n = 25–30 cells in 3 independent experiments). **p < 0.01, compared with control-scramble siRNA group or KCl-Cav-1 siRNA group. C, The representative Fluo-3 fluorescence (left) and the quantitative data (right) showed that overexpression of Cav-1 with Lenti-Cav-1, but not control Lenti-empty, significantly increased the intracellular Ca²⁺ concentration in cultured ACC neurons. The cultured ACC neurons were treated with 6 × 10⁷ TU/ml Lenti-Cav-1 or its control Lenti-empty for 72 h before loading with Fluo-3 AM (n = 25–30 cells in 3 independent experiments). **p < 0.01. D, The representative Fluo-3 fluorescence (left) and the quantitative data (right) showed that NR2B inhibitor ifenprodil (6 μm) inhibited the increase of intracellular Ca²⁺ concentration induced by overexpression of Cav-1. The cultured ACC neurons were treated with 6 × 10⁷ TU/ml Lenti-Cav-1 and ifenprodil (6 μm) or its vehicle for 72 h before loading with Fluo-3 AM (n = 25–30 cells in 3 independent experiments). **p < 0.01. E, The representative Fluo-3 fluorescence (left) and the quantitative data (right) showed that NR2B inhibitor ifenprodil (6 μm) inhibited the increase of intracellular Ca²⁺ concentration induced by KCl. The cultured ACC neurons at 7 d were pretreated in 6 μm ifenprodil 10 min before treatment by 50 mm KCl, then testing with Fluo-3 AM (n = 25–30 cells in 3 independent experiments), **p < 0.01. Data are shown as means ± SEM. Scale bar, 50 μm. Ifen, ifenprodil.