Ross P E, Cahalan M D
Department of Physiology and Biophysics, University of California, Irvine 92717, USA.
J Gen Physiol. 1995 Sep;106(3):415-44. doi: 10.1085/jgp.106.3.415.
We used fura-2 video imaging to characterize two Ca2+ influx pathways in mouse thymocytes. Most thymocytes (77%) superfused with hypoosmotic media (60% of isoosmotic) exhibited a sharp, transient rise in the concentration of intracellular free Ca2+ ([Ca2+]i). After a delay of approximately 70 s, these swelling-activated [Ca2+]i (SWAC) transients reached approximately 650 nM from resting levels of approximately 100 nM and declined from a time constant of 20 s. Peak [Ca2+]i during transients correlated with maximum volume during swelling. Regulatory volume decrease (RVD) was enhanced in thymocytes exhibiting SWAC transients. Three lines of evidence indicate that Ca2+ influx, and not the release of Ca2+ from intracellular stores, underlies SWAC transients in thymocytes. First, thymocytes swollen in Ca2+-free media failed to respond. Second, Gd3+ and La3+ inhibited SWAC influx with Kd's of 3.8 and 2.4 microM, respectively. Finally, the depletion of Ca2+ stores with thapsigargin (TG) before swelling did not inhibit the generation, nor decrease the amplitude, of SWAC transients. Cell phenotyping demonstrated that SWAC transients are primarily associated with immature CD4-CD8- and CD4+CD8+ thymocytes. Mature peripheral lymphocytes (mouse or human) did not exhibit SWAC transients. SWAC influx could be distinguished from the calcium release-activated Ca2+ (CRAC) influx pathway stimulated by store depletion with TG. In TG-treated thymocytes, [Ca2+]i rose steadily for approximately 100 s, peaked at approximately 900 nM, and then declined slowly. Simultaneous activation of both pathways produced an additive [Ca2+]i profile. Gd3+ and La3+ blocked Ca2+ entry during CRAC activation more potently (Kd's of 28 and 58 nM, respectively) than Ca2+ influx during SWAC transients. SWAC transients could be elicited in the presence of 1 microM Gd3+, after the complete inhibition of CRAC influx. Finally, whereas SWAC transients were principally restricted to immature thymocytes. TG stimulated the CRAC influx pathway in all four thymic CD4/CD8 subsets and in mature T cells. We conclude that SWAC and CRAC represent separate pathways for Ca2+ entry in thymocytes.
我们使用fura-2视频成像技术来表征小鼠胸腺细胞中的两种钙离子内流途径。大多数胸腺细胞（77%）用低渗培养基（等渗的60%）进行灌流时，细胞内游离钙离子浓度（[Ca2+]i）会出现急剧、短暂的升高。延迟约70秒后，这些肿胀激活的[Ca2+]i（SWAC）瞬变从约100 nM的静息水平达到约650 nM，并以20秒的时间常数下降。瞬变过程中的[Ca2+]i峰值与肿胀过程中的最大体积相关。在表现出SWAC瞬变的胸腺细胞中，调节性容积减小（RVD）增强。三条证据表明，胸腺细胞中SWAC瞬变的基础是钙离子内流，而非细胞内钙库释放钙离子。第一，在无钙培养基中肿胀的胸腺细胞无反应。第二，钆离子（Gd3+）和镧离子（La3+）分别以3.8和2.4 microM的解离常数抑制SWAC内流。最后，在肿胀前用毒胡萝卜素（TG）耗尽钙库，并不抑制SWAC瞬变的产生，也不降低其幅度。细胞表型分析表明，SWAC瞬变主要与未成熟的CD4-CD8-和CD4+CD8+胸腺细胞相关。成熟的外周淋巴细胞（小鼠或人）不表现出SWAC瞬变。SWAC内流可与TG耗尽钙库刺激的钙释放激活钙离子（CRAC）内流途径区分开来。在TG处理的胸腺细胞中，[Ca2+]i稳定上升约100秒，在约900 nM处达到峰值，然后缓慢下降。同时激活这两种途径会产生相加的[Ca2+]i图谱。Gd3+和La3+在CRAC激活过程中比在SWAC瞬变过程中更有效地阻断钙离子进入（解离常数分别为28和58 nM）。在完全抑制CRAC内流后，在1 microM Gd3+存在的情况下可引发SWAC瞬变。最后，虽然SWAC瞬变主要局限于未成熟胸腺细胞，但TG在所有四个胸腺CD4/CD8亚群和成熟T细胞中刺激CRAC内流途径。我们得出结论，SWAC和CRAC代表胸腺细胞中钙离子进入的不同途径。
