Augmentation of L-type calcium current by hypoxia in rabbit carotid body glomus cells: evidence for a PKC-sensitive pathway.
Augmentation of L-type calcium current by hypoxia in rabbit carotid body glomus cells: evidence for a PKC-sensitive pathway.
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DOI：
10.1152/jn.2000.84.3.1636
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发表时间：
2000-09
影响因子：
2.5
通讯作者：
B. A. Summers;J. L. Overholt;Nanduri R. Prabhakar
中科院分区：
文献类型：
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作者：
B. A. Summers;J. L. Overholt;Nanduri R. Prabhakar
以往的研究表明，血管球细胞内电压门控性Ca（2+）内流在颈动脉体化学感受器的感觉转导中起着关键作用。本研究旨在观察缺氧对血管球细胞钙电流的影响，并探讨其机制。实验在新鲜分离的兔颈动脉体球细胞上进行。采用全细胞膜片钳技术，以Ba（2+）为电荷载体监测Ca（2+）电流。缺氧（pO（2）= 40 mmHg）以电压非依赖性方式使Ca（2+）电流增加24 +/- 3%（n = 42，0 mV）。这种效应在CO（2）/HCO（3）（-）-中观察到，但在pH 7.4的HEPES缓冲细胞外溶液中未观察到（n = 6）。当HEPES缓冲的细胞外溶液的pH从7.4降低到7时。0时，缺氧使钙电流增加20 ± 5%（n = 4，0 mV）。尼索地平，一种L-型Ca（2+）通道阻断剂（2 μ M，n = 6），阻止了缺氧引起的Ca（2+）电流的增加，而ω-芋螺毒素MVIIC（2 μ M，n = 6），一种N和P/Q型Ca（2+）通道抑制剂，不能阻止缺氧引起的Ca（2+）电流的增加，这意味着低氧影响血管球细胞中的L-型Ca（2+）通道。蛋白激酶C（PKC）抑制剂，星形孢菌素（100 nM，n = 6）和双吲哚马来酰亚胺（2 μ M，n = 8，0 mV），防止，而蛋白激酶A抑制剂（4 nM PKAi，n = 10）不能防止缺氧诱导的Ca（2+）电流的增加。佛波醇12-肉豆蔻酸酯13-乙酸酯（PMA，100 nM），一种PKC激活剂，使Ca（2+）电流增加20 +/- 3%（n = 8，0 mV）。在用PMA过夜（100 nM）处理的血管球细胞中，缺氧不增加Ca（2+）电流（-3 +4%，n = 5，0 mV）。免疫细胞化学分析显示PKCdelta样免疫反应在球细胞的胞质溶胶。在缺氧（6%O（2）5分钟）后，PKC δ样免疫反应性在87 +/- 3%的细胞中转移到质膜，表明PKC激活。这些结果表明，缺氧通过PKC敏感的机制增加L型Ca（2+）通道的Ca（2+）电流。
Previous studies have suggested that voltage-gated Ca(2+) influx in glomus cells plays a critical role in sensory transduction at the carotid body chemoreceptors. The purpose of the present study was to determine the effects of hypoxia on the Ca(2+) current in glomus cells and to elucidate the underlying mechanism(s). Experiments were performed on freshly dissociated glomus cells from rabbit carotid bodies. Ca(2+) current was monitored using the whole cell configuration of the patch-clamp technique, with Ba(2+) as the charge carrier. Hypoxia (pO(2) = 40 mmHg) augmented the Ca(2+) current by 24 +/- 3% (n = 42, at 0 mV) in a voltage-independent manner. This effect was seen in a CO(2)/HCO(3)(-)-, but not in a HEPES-buffered extracellular solution at pH 7.4 (n = 6). When the pH of a HEPES-buffered extracellular solution was lowered from 7.4 to 7. 0, hypoxia augmented the Ca(2+) current by 20 +/- 5% (n = 4, at 0 mV). Nisoldipine, an L-type Ca(2+) channel blocker (2 microM, n = 6), prevented, whereas, omega-conotoxin MVIIC (2 microM, n = 6), an inhibitor of N and P/Q type Ca(2+) channels, did not prevent augmentation of the Ca(2+) current by hypoxia, implying that low oxygen affects L-type Ca(2+) channels in glomus cells. Protein kinase C (PKC) inhibitors, staurosporine (100 nM, n = 6) and bisindolylmaleimide (2 microM, n = 8, at 0 mV), prevented, whereas, a protein kinase A inhibitor (4 nM PKAi, n = 10) did not prevent the hypoxia-induced increase of the Ca(2+) current. Phorbol 12-myristate 13-acetate (PMA, 100 nM), a PKC activator, augmented the Ca(2+) current by 20 +/- 3% (n = 8, at 0 mV). In glomus cells treated with PMA overnight (100 nM), hypoxia did not augment the Ca(2+) current (-3 + 4%, n = 5, at 0 mV). Immunocytochemical analysis revealed PKCdelta-like immunoreactivity in the cytosol of the glomus cells. Following hypoxia (6% O(2) for 5 min), PKCdelta-like immunoreactivity translocated to the plasma membrane in 87 +/- 3% of the cells, indicating PKC activation. These results demonstrate that hypoxia augments Ca(2+) current through L-type Ca(2+) channels via a PKC-sensitive mechanism.