Wiemann M, Büsselberg D, Schirrmacher K, Bingmann D
Institut für Physiologie, Universität-GH Essen, Germany.
Calcif Tissue Int. 1998 Aug;63(2):154-9. doi: 10.1007/s002239900507.
Osteoblast-like (OBL) cells in primary culture were tested for their ability to generate a calcium release activated calcium flux (CRAC). Influx of Ca2+ was optically detected by fura-2. Intracellular calcium stores (ICS) were emptied in the absence of extracellular calcium ([Ca2+]e) by 5 microM thapsigargin (TG) or 2 microM A23187. Readdition of 1.8 mM [Ca2+]e increased the free intracellular Ca2+ ([Ca2+]i) after a delay of 30-60 seconds at a rate of 2.3 nM/s due to CRAC. This rate depended on [Ca2+]e and was substantially lowered if readdition of 1.8 mM [Ca2+]e was preceded by, e.g., 0.72 mM [Ca2+]e. CRAC-induced [Ca2+]i peaks were correlated (r = 0.543) with [Ca2+]i peaks during the complete depletion of ICS with A23187. Ca2+ influx due to CRAC could be blocked by flufenamic acid (100 microM) but not verapamil (20 microM). Ni2+ (1 mM), although reversibly blocking CRAC, accelerated the initial [Ca2+]i influx rate. Induction of CRAC enhanced the influx of Mn2+ 4.3-fold, as measured by quenching of fura-2 fluorescence. In summary, OBL cells exhibit a CRAC which allows for the permeation of ions other than Ca2+. This Ca2+ flux may be activated by transmembraneous gradients of Ca2+ and Ni2+.
对原代培养的成骨样（OBL）细胞产生钙释放激活钙流（CRAC）的能力进行了测试。通过fura-2光学检测Ca2+的流入。在无细胞外钙（[Ca2+]e）的情况下，用5微摩尔毒胡萝卜素（TG）或2微摩尔A23187排空细胞内钙库（ICS）。重新添加1.8毫摩尔[Ca2+]e后，由于CRAC，在延迟30 - 60秒后，细胞内游离Ca2+（[Ca2+]i）以2.3纳摩尔/秒的速率增加。该速率取决于[Ca2+]e，如果在重新添加1.8毫摩尔[Ca2+]e之前先添加例如0.72毫摩尔[Ca2+]e，则该速率会大幅降低。CRAC诱导的[Ca2+]i峰值与用A23187完全耗尽ICS期间的[Ca2+]i峰值相关（r = 0.543）。CRAC引起的Ca2+流入可被氟芬那酸（100微摩尔）阻断，但不能被维拉帕米（20微摩尔）阻断。Ni2+（1毫摩尔）虽然可逆地阻断CRAC，但加速了初始[Ca2+]i流入速率。通过fura-2荧光淬灭测量，CRAC的诱导使Mn2+流入增加了4.3倍。总之，OBL细胞表现出一种CRAC，它允许除Ca2+之外的离子渗透。这种Ca2+流可能由Ca2+和Ni2+的跨膜梯度激活。
