Goldman W F, Yarowsky P J, Juhaszova M, Krueger B K, Blaustein M P
Department of Physiology, University of Maryland School of Medicine, Baltimore 21201.
J Neurosci. 1994 Oct;14(10):5834-43. doi: 10.1523/JNEUROSCI.14-10-05834.1994.
Regulation of the cytosolic free Ca2+ concentration ([Ca2+]cyt) by an Na/Ca exchanger was studied in primary cultured rat cortical astrocytes. [Ca2+]cyt was measured by digital imaging in cells loaded with fura-2. The resting [Ca2+]cyt, approximately 150 nM, was only slightly increased by reducing the extracellular Na+ concentration ([Na+]o) to 6.2 mM, or by treating the cells with ouabain for 15 min (to raise cytosolic Na+). Following treatment with ouabain, however, lowering [Na+]o caused [Ca2+]cyt to rise rapidly to approximately 1300 nM. When Ca2+ sequestration in intracellular stores was blocked by thapsigargin, lowering [Na+]o increased [Ca2+]cyt to approximately 1500 nM in the absence of ouabain. The low-[Na+]o-stimulated rise in [Ca2+]cyt was abolished by removal of external Ca2+, but was not blocked by the Ca2+ channel blocker verapamil, or by caffeine or ryanodine, which deplete an intracellular Ca2+ store responsible for Ca(2+)-induced Ca2+ release. These data suggest that Na+ gradient reduction promotes net Ca2+ gain via Na/Ca exchange. Normally, however, a large rise in [Ca2+]cyt is prevented by sequestration of the entering Ca2+; this buffering of cytosolic Ca2+ can be circumvented by blocking sequestration with thapsigargin, or overwhelmed by enhancing net Ca2+ gain by pretreating the cells with ouabain. The presence of Na/Ca exchanger protein and mRNA in the astrocytes was confirmed by Western and Northern blot analyses, respectively. Immunohistochemistry revealed that exchanger molecules are distributed in a reticular pattern over the astrocyte surface. We suggest that the Na/Ca exchanger plays a role in regulating both [Ca2+]cyt and the intracellular stores of Ca2+ in astrocytes, and may thus contribute to the control of astrocyte responsiveness to neurotransmitters and neurotoxins.
在原代培养的大鼠皮层星形胶质细胞中研究了钠钙交换体对胞质游离钙离子浓度（[Ca2+]cyt）的调节作用。通过数字成像测量加载了fura-2的细胞中的[Ca2+]cyt。静息[Ca2+]cyt约为150 nM，通过将细胞外钠离子浓度（[Na+]o）降至6.2 mM，或用哇巴因处理细胞15分钟（以提高胞质钠离子浓度），其仅略有升高。然而，在用哇巴因处理后，降低[Na+]o会使[Ca2+]cyt迅速升至约1300 nM。当用毒胡萝卜素阻断细胞内钙库的钙螯合时，在无哇巴因的情况下降低[Na+]o会使[Ca2+]cyt升高至约1500 nM。去除细胞外钙离子可消除低[Na+]o刺激引起的[Ca2+]cyt升高，但不受钙离子通道阻滞剂维拉帕米、咖啡因或雷诺丁的阻断，后三者可耗尽负责钙诱导钙释放的细胞内钙库。这些数据表明，钠离子梯度降低通过钠钙交换促进净钙内流。然而，正常情况下，进入的钙离子被螯合可防止[Ca2+]cyt大幅升高；用毒胡萝卜素阻断螯合可规避这种对胞质钙的缓冲作用，或通过用哇巴因预处理细胞增强净钙内流使其作用失效。分别通过蛋白质印迹法和Northern印迹法证实了星形胶质细胞中存在钠钙交换体蛋白和mRNA。免疫组织化学显示，交换体分子以网状模式分布在星形胶质细胞表面。我们认为，钠钙交换体在调节星形胶质细胞的[Ca2+]cyt和细胞内钙库方面发挥作用，因此可能有助于控制星形胶质细胞对神经递质和神经毒素的反应性。
