Nishimura J, Kolber M, van Breemen C
Department of Pharmacology, University of Miami School of Medicine, Florida 33101.
Biochem Biophys Res Commun. 1988 Dec 15;157(2):677-83. doi: 10.1016/s0006-291x(88)80303-6.
A new method for preparing permeabilized smooth muscle fibers from rabbit mesenteric artery has been developed using alpha-toxin, a transmembrane pore-making exo-protein produced by Staphylococcus aureus. After alpha-toxin treatment the fibers developed tension as a function of Ca2+ concentration (EC50 = 890 nM). But they could not contract without added ATP, indicating ATP is permeable. When the sarcoplasmic reticulum was loaded with 5 X 10(-7) M Ca2+ solution, NE induced a transient contraction in 2 mM EGTA 0 M Ca2+ solution and a transient and maintained contraction in 5 X 10(-7) M Ca2+ solution. GTP-gamma-S, a non-hydrolyzable analogue of GTP, substituted for NE in producing these contractile effects. The analysis of the relationship between Ca2+ and maintained tension revealed that NE and GTP-gamma-S cause increases in Ca2+ sensitivity of myofilament shifting the EC50 to 280 nM and 160 nM, respectively. We conclude that NE or GTP-gamma-S causes an increase in myofilament Ca2+ sensitivity and that G protein may be involved in receptor signal transduction system. alpha-Toxin is a useful tool to permeabilize the smooth muscle tissue to ions and small molecules without any damage of receptor and signal transduction system.
一种利用α毒素从兔肠系膜动脉制备通透化平滑肌纤维的新方法已被开发出来，α毒素是一种由金黄色葡萄球菌产生的跨膜造孔外毒素。经α毒素处理后，纤维产生的张力是Ca2+浓度的函数（半数有效浓度[EC50]=890纳摩尔）。但在不添加ATP的情况下它们无法收缩，这表明ATP是可通透的。当肌浆网用5×10−7摩尔/升Ca2+溶液加载时，去甲肾上腺素（NE）在2毫摩尔乙二醇双四乙酸（EGTA）、0摩尔/升Ca2+溶液中诱导出瞬时收缩，在5×10−7摩尔/升Ca2+溶液中诱导出瞬时且持续的收缩。鸟苷-5′-三磷酸γ-硫酯（GTP-γ-S），一种不可水解的GTP类似物，在产生这些收缩效应时可替代NE。对Ca2+与持续张力之间关系的分析表明，NE和GTP-γ-S会导致肌丝Ca2+敏感性增加，使EC50分别移至280纳摩尔和160纳摩尔。我们得出结论，NE或GTP-γ-S会导致肌丝Ca2+敏感性增加，并且G蛋白可能参与受体信号转导系统。α毒素是使平滑肌组织对离子和小分子通透而不损伤受体和信号转导系统的有用工具。
