Twort C H, van Breemen C
Division of Medicine, United Medical School, St Thomas' Hospital, London, UK.
Pulm Pharmacol. 1989;2(1):45-53. doi: 10.1016/s0952-0600(89)80009-2.
The release of intracellularly stored calcium (Ca2+) contributes to the rise in cytoplasmic Ca2+ concentration during agonist-induced activation of airway smooth muscle. We describe a novel preparation--human airway smooth muscle cells cultured in monolayers--which has enabled the investigation of the identity and the function of this intracellular Ca2+ store. Cells, enzymatically dispersed from surgically resected human bronchi, were cultured in monolayers and confirmed as smooth muscle by positive immunocytochemical staining for actin and myosin. The release of intracellularly stored Ca2+ in response to bronchoconstrictor agonists, histamine and carbachol, was demonstrated by stimulation of Ca2+ efflux from 45Ca-labelled cells. The technique of permeabilisation of the plasmalemmal membrane by saponin allowed the measurement of the Ca2+ content of the intracellular store in 45Ca-labelled cells. Uptake of Ca2+ by the store was energy-dependent and was enhanced by cyclic AMP. The effects of inhibitors of sarcoplasmic reticulum and mitochondrial function on Ca2+ uptake identified the store as sarcoplasmic reticulum. Inositol 1,4,5-trisphosphate released stored Ca2+ in a time and dose-dependent manner, supporting its putative role as an intracellular messenger for Ca2+ release in human airway smooth muscle.
细胞内储存钙（Ca2+）的释放导致激动剂诱导气道平滑肌激活过程中细胞质Ca2+浓度升高。我们描述了一种新的制备方法——单层培养的人气道平滑肌细胞，这使得对这种细胞内Ca2+储存库的特性和功能的研究成为可能。从手术切除的人支气管中酶解分散得到的细胞进行单层培养，并通过肌动蛋白和肌球蛋白的免疫细胞化学阳性染色确认为平滑肌细胞。通过刺激45Ca标记细胞的Ca2+外流，证实了对支气管收缩激动剂组胺和卡巴胆碱的反应中细胞内储存Ca2+的释放。皂角苷使质膜通透的技术可用于测量45Ca标记细胞内储存库的Ca2+含量。储存库对Ca2+的摄取依赖能量，并被环磷酸腺苷增强。肌浆网和线粒体功能抑制剂对Ca2+摄取的影响表明该储存库为肌浆网。肌醇1,4,5-三磷酸以时间和剂量依赖的方式释放储存的Ca2+，支持其作为人气道平滑肌中Ca2+释放的细胞内信使的假定作用。
