Hirono M, Takamura K, Ito Y, Nakano Y, Chikaoka Y, Suzuki N, Yoshioka T
Department of Molecular Neurobiology, School of Human Sciences, Waseda University, Tokorozawa, Japan.
Cell Calcium. 1999 Feb;25(2):125-35. doi: 10.1054/ceca.1998.0015.
The relative contribution of voltage-sensitive Ca2+ channels, Ca(2+)-ATPases, and Ca2+ release from intracellular stores to spontaneous oscillations in cytosolic free Ca2+ concentration ([Ca2+]i) observed in secretory cells is not well characterized owing to a lack of specific inhibitors for a novel thapsigargin (Tg)-insensitive Ca(2+)-ATPase expressed in these cells. We show that spontaneous [Ca2+]i oscillations in GH3 cells were unaffected by Ca2+ depletion in inositol-1,4,5-trisphosphate (IP3)-sensitive Ca2+ stores by the treatment of Tg, but could be initiated by application of caffeine. Moreover, we demonstrate for the first time that these spontaneous [Ca2+]i oscillations were highly temperature dependent. Decreasing the temperature from 22 to 17 degrees C resulted in an increase in the frequency, a reduction in the amplitude, and large inhibition of [Ca2+]i oscillations. Furthermore, the rate of ATP-dependent 45Ca2+ uptake into GH3-derived microsomes was greatly reduced at 17 degrees C. The effect of decreased temperatures on extracellular Ca2+ influx was minor because the frequency and amplitude of spontaneous action potentials, which activate L-type Ca2+ channels, was relatively unchanged at 17 degrees C. These results suggest that in GH3 secretory cells, Ca2+ influx via L-type Ca2+ channels initiates spontaneous [Ca2+]i oscillations, which are then maintained by the combined activity of Ca(2+)-ATPase and Ca(2+)-induced Ca2+ release from Tg/IP3-insensitive intracellular stores.
由于缺乏针对分泌细胞中表达的一种新型毒胡萝卜素（Tg）不敏感的Ca(2+)-ATP酶的特异性抑制剂，电压敏感性Ca2+通道、Ca(2+)-ATP酶以及细胞内钙库释放Ca2+对分泌细胞中观察到的胞质游离Ca2+浓度（[Ca2+]i）自发振荡的相对贡献尚未得到很好的表征。我们发现，用Tg处理使肌醇-1,4,5-三磷酸（IP3）敏感的Ca2+库中的Ca2+耗竭，并不会影响GH3细胞中[Ca2+]i的自发振荡，但应用咖啡因可引发这种振荡。此外，我们首次证明这些[Ca2+]i自发振荡高度依赖温度。将温度从22℃降至17℃会导致频率增加、振幅减小以及[Ca2+]i振荡受到极大抑制。此外，在17℃时，GH3衍生微粒体中依赖ATP的45Ca2+摄取速率大幅降低。温度降低对细胞外Ca2+内流的影响较小，因为激活L型Ca2+通道的自发动作电位的频率和振幅在17℃时相对未变。这些结果表明，在GH3分泌细胞中，通过L型Ca2+通道的Ca2+内流引发了[Ca2+]i的自发振荡，然后由Ca(2+)-ATP酶的联合活性以及来自Tg/IP3不敏感的细胞内钙库的Ca(2+)-诱导Ca2+释放来维持。
