蛋白激酶C活性下调对钙库操纵的钙通道活性和气道平滑肌细胞增殖的作用

目的:研究蛋白激酶C(PKC)活性下调对大鼠气道平滑肌细胞(ASMCs)的Ca2+库操纵的Ca2+通道(SOC)和ASMCs增殖的影响。方法:分离培养大鼠支气管平滑肌细胞;利用激光共聚焦显微镜测定ASMCs的fluo-3/AM的荧光信号;利用长时间暴露于PKC活化剂诱导PKC活性下调,观察PKC活性下调对ASMCs的SOC通道和增殖的影响;Alamar blue还原率测定法测定ASMCs的增殖。结果:PKC激活剂PMA(10μmol/L)和PDBu(1μmol/L)作用24h下调PKC活性后可以抑制ASMCs的增殖,PKC抑制剂chelerythrine同样抑制ASMCs的增殖;PKC活性下调和chelerythrine均可以抑制ASMCs的SOC通道活性;小剂量PKC激活剂PMA(100nmol/L)可以促进ASMCs的增殖,这种作用被SOC通道抑制剂SKF-96365抑制。结论:PKC活性下调或抑制导致SOC通道的活性下降,表明PKC可能参与了SOC通道功能调控;SOC通道开放可能参与了PKC促进ASMCs增殖的作用。

Effects of down-regulation of protein kinase C on activation of storeoperated Ca~(2+) channels and the proliferation of airway smooth muscle cells

AIM: To investigate the effects of down-regulation of protein kinase C (PKC) on the activity of store-operated Ca2+ channels (SOC) and the proliferation of airway smooth muscle cells (ASMCs). METHODS: Rat bronchial smooth muscle cells were isolated and cultured. Fluo-3/AM fluorescence was measured by laser confocal microscope to assessing intracellular Ca2+. Down-regulation of PKC activity was achieved by incubation of ASMCs with PKC activator phorbol-12-myristate-13-acetate (PMA, 10 μmol/L) or phorbol 12, 13-dibutyrate (PDBu, 1 μmol/L) for 24 h. The proliferation of ASMCs was assayed by calculating the reduction rates of Alamar blue. RESULTS: Down-regulation of PKC activity by long-term exposure of PMA or PDBu inhibited the proliferation of ASMCs, the similar results were obtained by using PKC inhibitor chelerythrine. Both down-regulation of PKC activity and inhibition of PKC activity by chelerythrine reduced Ca2+ entry through SOC channels. Low concentration of PMA (0.1 μmol/L) promoted the proliferation of ASMCs, and this effect was inhibited by SOC blocker SKF-96365. CONCLUSION: Inhibition or down-regulation of PKC activity results in the inhibition of SOC channels, suggesting that PKC is involved in the activation of these channels. Ca2+ entry through SOC channels might contribute to PKC-promoted proliferation of ASMCs.