Analysis of calcium channels roles in cancer cells proliferation

TRPV2通道在多种组织中表达，包括神经元、神经内分泌细胞、肾脏和肝脏的上皮细胞以及巨噬细胞等血细胞。在本研究中，我们研究了巨噬细胞中TRPV2通道的调节。巨噬细胞在无血清条件下培养时，TRPV2免疫反应主要分布在胞浆中。加入血清可诱导部分TRPV2转位至质膜。趋化性多肽fMLP进一步诱导TRPV2转位到质膜。根据这一观察结果，fMLP增加巨噬细胞Cs电流，这一电流可被Ru红和显性负性突变体TRPV2的转染所抑制。PI3-激酶抑制剂和百日咳毒素预处理可阻断fMLP诱导的TRPV2转位。用Fura-2监测细胞内钙离子浓度([Ca~(2+)]_c)时，fMLP可引起细胞内[Ca~(2+)]_c快速持续升高，这种升高可被细胞外钙离子清除所抑制。加入Ru红或转染显性负性突变体TRPV2不影响初始[Ca~(2+)]_c的升高，但可减弱fMLP诱导的[Ca~(2+)]c升高的持续期。最后，Ru红或转染显性负性突变体TRPV2可抑制fMLP诱导的巨噬细胞趋化作用。这些结果表明，fMLP通过PI3-激酶依赖的机制诱导TRPV2的易位，这种易位对巨噬细胞内[Ca~(2+)]c的持续升高是重要的。

The TRPV2 channel is expressed in various tissues including neurons, neuroendocrine cells, epithelial cells in the kidney and liver and blood cells such as macrophages. In the present study, we investigated the regulation of the TRPV2 channel in macrophages. When macrophages were cultured in a serum-free condition, immunoreactivity of TRPV2 was detected largely in cytoplasm. Addition of serum induced translocation of some of the TRPV2 to the plasma membrane. A chemotactic peptide fMLP further induced translocation of the TRPV2 to the plasma membrane. In accordance with this observation, fMLP increased the Cs current in macrophage, which was inhibited by ruthenium red and the transfection of the dominant-negative mutant of TRPV2. fMLP-induced translocation of the TRPV2 was blocked by PI 3-kinase inhibitors and pretreatment with pertussis toxin. When cytoplasmic calcium concentration ([Ca2+]c)was monitored by using fura-2, fMLP induced a rapid and sustained elevation of [Ca2+]c, the latter of which was abolished by removal of extracellular calcium. Addition of ruthenium red or transfection of the dominant-negative mutant of TRPV2 did not affect the initial rise in [Ca2+]c but attenuated the sustained phase of fMLP-induced elevation of [Ca2+]c. Finally, addition of ruthenium red or transfection of dominant-negative mutant of TRPV2 inhibited chemotaxis of macrophage induced by fMLP. These results indicate that fMLP induces translocation of TRPV2 by a PI 3-kinase-dependent mechanism and this translocation is important for sustained elevation of [Ca2+]c in macrophage.