Tanimura A, Turner R J
Clinical Investigations and Patient Care Branch, NIDR, National Institutes of Health, Bethesda Maryland 20892, USA.
J Biol Chem. 1996 Nov 29;271(48):30904-8. doi: 10.1074/jbc.271.48.30904.
Oscillations in intracellular Ca2+ concentration ([Ca2+]i) are thought to play an important role in phosphoinositide-linked Ca2+ signaling events. We demonstrate corresponding inositol 1,4, 5-trisphosphate (IP3)-dependent oscillations of Ca2+ concentration within the lumen of the IP3-sensitive stores ([Ca2+]L) of saponin-permeabilized HSY cells by monitoring [Ca2+]L with the fluorescent Ca2+ indicator Mag-fura-2. The associated openings and closings of the IP3-sensitive Ca2+ release channel were detected via quenching of Mag-fura-2 fluorescence due to the entry of Mn2+, a Ca2+ surrogate, into the stores. Evidence for complimentary Ca2+ oscillations at the external surface of the stores was provided by the membrane-bound Ca2+ probe Calcium Green C18. The permeabilization of saponin-treated HSY cells to macromolecules was confirmed by demonstrating that permeabilized cells readily took up and lost (t1/2 approximately 46 s) a fluorescently tagged 70-kDa dextran. Our results impose a number of constraints on possible mechanisms for [Ca2+]i oscillations. In addition, they support recent proposals that [Ca2+]i oscillations arise directly from the (biphasic) effects of Ca2+ itself on IP3-sensitive Ca2+ channel activity.
细胞内钙离子浓度（[Ca2+]i）的振荡被认为在磷酸肌醇连接的钙离子信号事件中起重要作用。我们通过用荧光钙离子指示剂Mag-fura-2监测[Ca2+]L，证明了在皂素通透的HSY细胞的IP3敏感储存库内腔（[Ca2+]L）中存在相应的依赖于肌醇1,4,5-三磷酸（IP3）的钙离子浓度振荡。由于钙离子替代物Mn2+进入储存库导致Mag-fura-2荧光淬灭，从而检测到IP3敏感的钙离子释放通道的相关开闭。膜结合的钙离子探针Calcium Green C18提供了储存库外表面存在互补钙离子振荡的证据。通过证明通透细胞能够轻易摄取并丢失（半衰期约为46秒）荧光标记的70 kDa葡聚糖，证实了皂素处理的HSY细胞对大分子的通透性。我们的结果对[Ca2+]i振荡的可能机制施加了一些限制。此外，它们支持了最近的观点，即[Ca2+]i振荡直接源于钙离子本身对IP3敏感钙离子通道活性的（双相）影响。
