REGULATION OF THE INTRACELLULAR FREE CALCIUM-CONCENTRATION IN SINGLE-RAT DORSAL-ROOT GANGLION NEURONS INVITRO
REGULATION OF THE INTRACELLULAR FREE CALCIUM-CONCENTRATION IN SINGLE-RAT DORSAL-ROOT GANGLION NEURONS INVITRO
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DOI：
10.1113/jphysiol.1990.sp018094
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发表时间：
1990-06-01
影响因子：
5.5
通讯作者：
MILLER, RJ
中科院分区：
文献类型：
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作者：
THAYER, SA;MILLER, RJ
1.用全细胞膜片钳和Fura-2荧光显微镜同时记录大鼠背根神经节原代培养神经元的钙电流（伊卡）和细胞内游离钙浓度（[Ca ~（2+）]i）。2.将细胞保持在-80 mV并去极化至0 mV引起伊卡，导致[Ca 2 +]i瞬变，其在电压阶跃期间未被显著缓冲，并在细胞复极化和电流停止后持续很长时间。细胞缓冲[Ca 2 +]i回到基础水平的过程可以用单指数方程来描述。3.膜电位与伊卡和[Ca ~（2+）]i的关系显示，在给定的测试电位下诱发的[Ca ~（2+）]i瞬变的峰值与伊卡的幅度接近，这表明电压依赖性或Ca ~（2+）诱导的胞内钙库Ca ~（2+）释放对[Ca ~（2+）]i瞬变没有显著贡献。4.当通过改变测试脉冲的持续时间或电位用不同幅度的Ca 2+负荷挑战细胞时，[Ca 2 +]i缓冲对于较大的Ca 2+负荷更有效。积分伊卡和[Ca 2 +]i瞬态峰值之间的关系在大的Ca 2+负荷下达到渐近线，表明Ca 2+依赖性过程变得更有效或低亲和力过程已被招募。5.用神经肽Y抑制Ca 2+内流表明，抑制大的伊卡在[Ca 2 +]i瞬变的峰值中产生微小的改变，而抑制较小的电流在[Ca 2 +]i瞬变中产生相应的降低。因此，伊卡的抑制仅在向细胞施加次最大Ca 2+负荷时由峰[Ca 2 +]i的变化反映，这意味着[Ca 2 +]i的调节依赖于细胞的活化状态。6.在全细胞膜片钳实验中，用线粒体Ca 2+摄取阻断剂钌红进行细胞内透析，去除了缓冲组分，当将大的Ca 2+负荷施加于细胞时，观察到缓冲组分负责更有效地去除[Ca 2 +]i。7.当细胞灌流50 mM-K+时，从细胞索马记录的[Ca 2 +]i瞬变非常缓慢地恢复到对照水平。药理学研究表明，在[Ca 2 +]i持续升高期间，线粒体正在循环Ca 2+。相反，从细胞过程记录的[Ca 2 +]i瞬变相对迅速地返回到基础水平。8.细胞外Na+依赖性Ca ~（2+）流出对背根神经节神经元胞体缓冲[Ca ~（2+）]i瞬变无显著影响。此外，钙调素拮抗剂和原钒酸钠通过贴片移液器应用对Ca 2+缓冲没有影响。因此，Na+-Ca 2+交换系统不参与背根神经节神经元胞体中的Ca 2+缓冲，并且可能参与从胞质溶胶中去除Ca 2+的ATP依赖性过程似乎不以与先前从红细胞表征的Ca 2 +-Mg 2 +-ATP酶相同的方式进行调节。
1. Simultaneous whole-cell patch-clamp and Fura-2 microfluorimetric recordings of calcium currents (ICa) and the intracellular free Ca2+ concentration ([Ca2+]i) were made from neurones grown in primary culture from the dorsal root ganglion of the rat. 2. Cells held at -80 mV and depolarized to 0 mV elicited a ICa that resulted in an [Ca2+]i transient which was not significantly buffered during the voltage step and lasted long after the cell had repolarized and the current ceased. The process by which the cell buffered [Ca2+]i back to basal levels could best be described with a single-exponential equation. 3. The membrane potential versus ICa and [Ca2+]i relationship revealed that the peak of the [Ca2+]i transient evoked at a given test potential closely paralleled the magnitude of the ICa suggesting that neither voltage-dependent nor Ca2+-induced Ca2+ release from intracellular stores made a significant contribution to the [Ca2+]i transient. 4. When the cell was challenged with Ca2+ loads of different magnitude by varying the duration or potential of the test pulse, [Ca2+]i buffering was more effective for larger Ca2+ loads. The relationship between the integrated Ica and the peak of the [Ca2+]i transient reached an asymptote at large Ca2+ loads indicating that Ca2+-dependent processes became more efficient or that low-affinity processes had been recruited. 5. Inhibition of Ca2+ influx with neuropeptide Y demonstrated that inhibition of a large ICa produced minor alterations in the peak of the [Ca2+]i transient, while inhibition of smaller currents produced corresponding decreases in the [Ca2+]i transient. Thus, inhibition of the ICa was reflected by a change in the peak [Ca2+]i only when submaximal Ca2+ loads were applied to the cell, implying that modulation of [Ca2+]i is dependent on the activation state of the cells. 6. Intracellular dialysis with the mitochondrial Ca2+ uptake blocker Ruthenium Red in whole-cell patch-clamp experiments removed the buffering component which was responsible for the more efficient removal of [Ca2+]i observed when large Ca2+ loads were applied to the cell. 7. When cells were superfused with 50 mM-K+, [Ca2+]i transients recorded from the cell soma returned to control levels very slowly. Pharmacological studies indicated that mitochondria were cycling Ca2+ during this sustained elevation in [Ca2+]i. In contrast, [Ca2+]i transients recorded from cell processes returned to basal levels relatively rapidly. 8. Extracellular Na+-dependent Ca2+ efflx did not significantly contribute to buffering [Ca2+]i transients in dorsal root ganglion neurone cell bodies. Furthermore, calmodulin antagonists and sodium orthovanadate applied via the patch pipette were without effect on Ca2+ buffering. Thus, the Na+-Ca2+ exchange system does not participate in Ca2+ buffering in the cell bodies of dorsal root ganglion neurones and the ATP-dependent processes with presumably participate in removing Ca2+ from the cytosol do not seem to be regulated in the same manner as the Ca2+-Mg2+-ATPase previously characterized from erythrocytes.