Civitelli R, Bacskai B J, Mahaut-Smith M P, Adams S R, Avioli L V, Tsien R Y
Division of Bone and Mineral Diseases, Washington University School of Medicine, Jewish Hospital of St. Louis, Missouri.
J Bone Miner Res. 1994 Sep;9(9):1407-17. doi: 10.1002/jbmr.5650090912.
We previously demonstrated that the [Ca2+]i response to PTH is heterogeneous in single UMR-106-01 osteogenic sarcoma cells. To verify whether response heterogeneity is a universal feature of PTH signal transduction, cAMP production was monitored in monolayer cultures of UMR-106-01 cells and human trabecular bone osteoblasts (HOB) using the cAMP-sensitive fluorescent indicator FlCRhR. FlCRhR was microinjected into single cells, and the 500-530/> 560 nm fluorescence ratio was monitored by confocal laserscanning video imaging as a measure of cAMP concentration ([cAMP]). Virtually all UMR-106-01 cells exposed to bovine PTH(1-34) (10(-7) M) exhibited an increase in intracellular [cAMP], with an average fluorescence ratio change of 145 +/- 17% of baseline (n = 15), corresponding to nearly maximal dissociation of protein kinase A. In the continued presence of the hormone (10(-7) M), [cAMP] remained elevated for at least 30 minutes. This effect was accompanied by a slow translocation of the fluorescein-labeled catalytic subunit of protein kinase A from the cytoplasm to the nucleus. In contrast, PTH(1-34) caused no detectable increase in [cAMP] in HOB cells, although PGE2 (3 x 10(-6) M) stimulation was able to increase the FlCRhR ratio (154 +/- 27%, n = 10). The truncated fragment PTH(2-34) was only 67% as potent at PTH(1-34), but deletion of the first two amino acids at the N terminus abolished the hormone's ability to stimulate cAMP production in UMR-106-01 cells. Brief exposure to 10(-7) M of either PTH(3-34) or PTH(7-34) did not affect the amplitude of the fluorescence ratio change induced by equimolar doses of PTH(1-34). Thus, in osteoblast-like cells stimulated with PTH, the [cAMP] response is much more homogeneous from cell to cell than the [Ca2+]i response.
我们之前证明，在单个UMR-106-01骨肉瘤细胞中，细胞内钙离子浓度（[Ca2+]i）对甲状旁腺激素（PTH）的反应具有异质性。为了验证反应异质性是否是PTH信号转导的普遍特征，我们使用对环磷酸腺苷（cAMP）敏感的荧光指示剂FlCRhR，监测了UMR-106-01细胞和人小梁骨成骨细胞（HOB）单层培养物中的cAMP生成。将FlCRhR显微注射到单个细胞中，并通过共聚焦激光扫描视频成像监测500-530/＞560nm荧光比率，以此作为cAMP浓度（[cAMP]）的指标。实际上，所有暴露于牛PTH(1-34)（10^(-7)M）的UMR-106-01细胞，其细胞内[cAMP]均升高，平均荧光比率变化为基线的145±17%（n = 15），这对应于蛋白激酶A几乎最大程度的解离。在激素（10^(-7)M）持续存在的情况下，[cAMP]至少30分钟保持升高。这种效应伴随着蛋白激酶A的荧光素标记催化亚基从细胞质缓慢转位至细胞核。相比之下，PTH(1-34)未导致HOB细胞中[cAMP]出现可检测到的增加，尽管前列腺素E2（PGE2，3×10^(-6)M）刺激能够增加FlCRhR比率（154±27%，n = 10）。截短片段PTH(2-34)的效力仅为PTH(1-34)的67%，但N端前两个氨基酸的缺失消除了该激素刺激UMR-106-01细胞中cAMP生成的能力。短暂暴露于10^(-7)M的PTH(3-34)或PTH(7-34)，均不影响等摩尔剂量PTH(1-34)诱导的荧光比率变化幅度。因此，在用PTH刺激的成骨样细胞中，[cAMP]反应在细胞间比[Ca2+]i反应更为均匀。
