Department of Pharmacology, School of Dentistry, Aichi-Gakuin University, 1-100 Kusumoto-cho, Chikusa-ku, Nagoya 464-8650, Japan.
Biochem Biophys Res Commun. 2013 Jul 26;437(2):239-44. doi: 10.1016/j.bbrc.2013.06.047. Epub 2013 Jun 24.
Recent studies have revealed that the sympathetic nervous system is involved in bone metabolism. We previously reported that noradrenaline (NA) suppressed K(+) currents via Gi/o protein-coupled alpha1B-adrenergic receptor (α1B-AR) in human osteoblast SaM-1 cells. Additionally, it has been demonstrated that the intracellular Ca(2+) level ([Ca(2+)]i) was increased by NA via α1B-AR. In this study, we investigated the signal pathway of NA-induced [Ca(2+)]i elevation by using Ca(2+) fluorescence imaging in SaM-1 cells. NA-induced [Ca(2+)]i elevation was suppressed by pretreatment with a PLC inhibitor, U73122. This suggested that the [Ca(2+)]i elevation was mediated by Gq protein-coupled α1B-AR. On the other hand, NA-induced [Ca(2+)]i elevation was completely abolished in Ca(2+)-free solution, which suggested that Ca(2+) influx is the predominant pathway of NA-induced [Ca(2+)]i elevation. Although the inhibition of K(+) channel by NA caused membrane depolarization, the [Ca(2+)]i elevation was not affected by voltage-dependent Ca(2+) channel blockers, nifedipine and mibefradil. Meanwhile, NA-induced [Ca(2+)]i elevation was abolished following activation of store-operated Ca(2+) channel by thapsigargin. Additionally, the [Ca(2+)]i elevation was suppressed by store-operated channel inhibitors, 2-APB, flufenamate, GdCl3 and LaCl3. These results suggest that Ca(2+) influx through store-operated Ca(2+) channels plays a critical role in the signal transduction pathway of Gq protein-coupled α1B-AR in human osteoblasts.
近期研究揭示，交感神经系统参与骨代谢。我们先前报道，去甲肾上腺素（NA）通过 Gi/o 蛋白偶联的α1B-肾上腺素能受体（α1B-AR）抑制人成骨细胞 SaM-1 细胞中的 K+电流。此外，已证实 NA 通过 α1B-AR 增加细胞内 Ca2+水平([Ca2+]i)。在这项研究中，我们通过 SaM-1 细胞中的 Ca2+荧光成像来研究 NA 诱导的[Ca2+]i 升高的信号通路。用 PLC 抑制剂 U73122 预处理可抑制 NA 诱导的[Ca2+]i 升高。这表明[Ca2+]i 升高是由 Gq 蛋白偶联的 α1B-AR 介导的。另一方面，在无 Ca2+溶液中，NA 诱导的[Ca2+]i 升高完全被消除，这表明 Ca2+内流是 NA 诱导的[Ca2+]i 升高的主要途径。尽管 NA 抑制 K+通道导致膜去极化，但电压依赖性 Ca2+通道阻滞剂硝苯地平和米贝地尔对[Ca2+]i 升高没有影响。同时，在用 thapsigargin 激活储存操纵性 Ca2+通道后，NA 诱导的[Ca2+]i 升高被消除。此外，储存操纵性通道抑制剂 2-APB、flufenamate、GdCl3 和 LaCl3 抑制[Ca2+]i 升高。这些结果表明，Ca2+通过储存操纵性 Ca2+通道的内流在人成骨细胞中 Gq 蛋白偶联的α1B-AR 的信号转导通路中起关键作用。
