Sun F, Berry D J, Leong D A, Veldhuis J D
Department of Internal Medicine, University of Virginia Health Sciences Center, Charlottesville 22908, USA.
Endocrine. 1997 Oct;7(2):219-26. doi: 10.1007/BF02778144.
We have investigated at the single-cell level how the human LH receptor mediates a dose-responsive increase in intracellular free calcium-ion concentrations ([Ca2+]i). In human embryonic kidney cells (293 cells) stably transfected with the full-length human LH receptor cDNA. Intact dimeric LH, but not LH beta- or alpha-subunits, evoked specific [Ca2+]i signals. High-resolution fluorescence (fura-2) video-microscopy demonstrated cell-to-cell variability in [Ca2+]i signaling responses in individual cells, viz., an all-or-none spike (9%), spike-and-plateau (25%), or plateau (52%) types of temporal signal. Oscillatory [Ca2+]i responses were observed in 12-14% of LH-stimulated cells unrelated to LH concentration. The LH dose-response originated by higher concentrations of LH recruiting more individually responding cells (rather than altering [Ca2+]i signal amplitude), and eliciting a [Ca2+]i rise more rapidly, i.e., at reduced latency. Cobalt did not abolish the LH-stimulated [Ca2+]i spike-and-plateau response, but decreased the percentage of cells with a plateau pattern. Quench experiments demonstrated influx of Mn2+ following the [Ca2+]i spike, thus directly documenting divalent cation inflow during the plateau phase. Adenylyl-cyclase activation with forskolin or treatment with a cAMP analog failed to elicit the biphasic [Ca2+]i response, and pertussis toxin (PTX) did not alter LH-stimulated [Ca2+]i signaling. However, overnight preincubation with LH reduced the percentage of [Ca2+]i-responding cells following re-exposure to LH to 5.7% (vs 72% in control), suggesting LH-induced desensitization of the LH-receptor directed [Ca2+]i signal. In summary, the present studies of human LH receptor signal transduction at the single-cell level show that increasing concentrations of LH achieve a dose-dependent intracellular Ca2+ signaling response by recruiting an increasing number of [Ca2+]i-responding cells, while concomitantly decreasing the temporal latency of the biphasic [Ca2+]i signal without altering the amplitude of its spike phase. Prolonged exposure to LH appears to desensitize the LH receptor-driven [Ca2+]i signal.
我们已在单细胞水平研究了人促黄体生成素（LH）受体如何介导细胞内游离钙离子浓度（[Ca2+]i）的剂量依赖性增加。在稳定转染了全长人LH受体cDNA的人胚肾细胞（293细胞）中进行研究。完整的二聚体LH可诱发特异性的[Ca2+]i信号，而LH的β或α亚基则不能。高分辨率荧光（fura-2）视频显微镜显示，单个细胞中[Ca2+]i信号反应存在细胞间差异，即全或无尖峰型（9%）、尖峰-平台型（25%）或平台型（52%）的时间信号类型。在12%-14%受LH刺激的细胞中观察到振荡性[Ca2+]i反应，且与LH浓度无关。LH的剂量反应源于较高浓度的LH招募了更多单独产生反应的细胞（而非改变[Ca2+]i信号幅度），并更快速地引发[Ca2+]i升高，即潜伏期缩短。钴并未消除LH刺激的[Ca2+]i尖峰-平台反应，但降低了出现平台模式的细胞百分比。淬灭实验表明，在[Ca2+]i尖峰后有Mn2+内流，从而直接证明了平台期有二价阳离子流入。用福斯可林激活腺苷酸环化酶或用cAMP类似物处理均未能引发双相性[Ca2+]i反应，百日咳毒素（PTX）也未改变LH刺激的[Ca2+]i信号传导。然而，用LH预孵育过夜后，再次暴露于LH时，产生[Ca2+]i反应的细胞百分比降至5.7%（对照组为72%），提示LH诱导了LH受体介导的[Ca2+]i信号脱敏。总之，目前在单细胞水平对人LH受体信号转导的研究表明，LH浓度增加通过招募越来越多产生[Ca2+]i反应的细胞来实现剂量依赖性的细胞内Ca2+信号反应，同时在不改变其尖峰相幅度的情况下，双相性[Ca2+]i信号的时间潜伏期缩短。长时间暴露于LH似乎会使LH受体驱动的[Ca2+]i信号脱敏。
