雄激素可与骨髓巨噬细胞膜结合引起细胞外钙离子的内流

Testosterone Could Induce a Rapid Rise in Intracellular Free Ca^(2+) Concentration Through Binding to the Membrane Surface of Bone Marrow-derived Macrophages

目的:探讨雄激素作用于骨髓巨噬细胞(BMMs)的途径,研究雄激素与BMMs结合后对其产生的功能性影响。方法:体外诱导培养BMMs,提取细胞总RNA和蛋白,用RT-PCR和Western印迹方法观察BMMs中雄激素经典受体的表达情况。激光共聚焦显微镜观察T-BSA-FITC结合BMMs的情况。雄激素作用下,用Fura-2方法观察BMMs内钙离子浓度水平及特异性钙离子通道拮抗剂NiCl2对这种作用的影响,并用膜片钳方法观察BMMs膜表面离子电流的变化。结果:RT-PCR和Western印迹法都未检测到巨噬细胞中的雄激素受体,而阳性对照组(睾丸组织)的条带显著。用激光共聚焦显微镜观察到BSA-FITC能结合于巨噬细胞的细胞膜上。雄激素作用于BMMs后能迅速引起胞内游离钙离子浓度增加,这种作用可被NiCl2阻断。用膜片钳方法观察到雄激素作用于BMMs后引起胞外的阳离子内流,与Fura-2方法得出的结果一致。结论:雄激素可能作用于BMMs膜表面的非经典受体,诱发细胞外钙离子快速内流,使细胞内钙离子浓度升高,从而对巨噬细胞产生功能性影响。

Objective： To investigate the ways testosterone influences the murine bone marrow-derived maerophages （BMMs） and how testosterone affects the function of BMMs after bound to their membrane surface. Methods： BMMs were cultured in vitro, their total RNA and proteins isolated, and the expression of intraeellular androgen receptor （AR） detected through RT-PCR and Western blotting. The binding site of testosterone （T） to the membrane surface of BMMs was observed by confocal laser scanning microscopy after T-BSA-FITC incubation. Moreover, the intracellular Ca^2＋ was tested by Fura-2 method, and the influence of ionic currents on BMMs plasma membrane induced by testosterone was examined by the whole cell patch-clamp. Results ： RT-PCR and Western blotting failed to detect intracellular ARs in BMMs, but confocal laser scanning microscopy showed testosterone to be bound to the membrane surface of BMMs by impermeable T-BSA-FITC, inducing a rapid rise in the intracellular free Ca^2＋ concentration （ [ Ca^2＋ ] i） of Fura-2 loaded BMMs, predominantly due to the influx of extracellular Ca^2＋ through Ni^2＋ -blockable Ca^2＋ cannels in the plasma membrane. Similarly, the patch-clamp technique revealed T-induced calcium influx in BMMs. Conclusion： It is reasonable to assume that the testosterone receptor exists on the plasma membranes, and testosterone act through unconventional plasma membrane receptors, induce Ca^2＋ influx and a rapid rise in the intracellular Ca^2＋ concentration, and influence the function of BMMs.