MICRO-INJECTION OF CATALYTIC SUBUNIT OF CYCLIC AMP-DEPENDENT PROTEIN-KINASE ENHANCES CALCIUM ACTION-POTENTIALS OF BAG CELL NEURONS IN CELL-CULTURE
MICRO-INJECTION OF CATALYTIC SUBUNIT OF CYCLIC AMP-DEPENDENT PROTEIN-KINASE ENHANCES CALCIUM ACTION-POTENTIALS OF BAG CELL NEURONS IN CELL-CULTURE
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DOI：
10.1073/pnas.77.12.7487
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发表时间：
1980-01-01
期刊：
影响因子：
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通讯作者：
GREENGARD, P
中科院分区：
文献类型：
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作者：
KACZMAREK, LK;JENNINGS, KR;GREENGARD, P
本文报道了腹神经节袋细胞神经元的Ca动作电位[A. cAMP-dependent protein kinase的催化亚基的细胞内显微注射可以增强cAMP-dependent protein kinase的催化亚基的细胞内显微注射。催化亚基从牛心脏中纯化，并在10-50 nM的浓度下有效刺激匀浆中袋细胞蛋白的磷酸化。细胞内注射到保持在原代培养物中的分离的袋细胞神经元中是通过向在尖端填充有催化亚基（5-22 μ M）的微电极施加压力来进行的。在16个注射细胞中的11个中，由恒定去极化电流诱发的动作电位的上升相的斜率和高度相对于注射前对照显著增强（平均增加，分别为73%和35%）。这种效应可能在静息电位或去极化脉冲开始时的动作电位潜伏期无变化的情况下发生。用溶解在3种不同盐溶液（磷酸钠、磷酸钾或KCl）中的酶观察效果。在2个实验中，加入到细胞外培养基中的河豚毒素（50 μ M）对增强的动作电位没有影响。随后加入钙拮抗剂Co ~（2+）减弱或消除了这种尖峰。在超过一半的实验中，催化亚基的注入伴随着通过施加小的超极化电流脉冲测量的细胞的输入电阻的增加。在3个实验中，膜电位的阈下振荡由注射引起。对照注射（24个细胞），进行单独的载体培养基或与热灭活的酶制剂，没有产生尖峰增强，增加输入电阻或振荡。cAMP依赖性蛋白激酶的催化亚基刺激细胞内蛋白磷酸化可能通过改变Ca和K通道或电流而增强囊细胞神经元的兴奋性。
The Ca action potentials of bag cell neurons from the abdominal ganglion of Aplysia [A. californica] may be enhanced by intracellular microinjection of the catalytic subunit of cAMP-dependent protein kinase. The catalytic subunit was purified from bovine heart and was effective in stimulating the phosphorylation of bag cell proteins in homogenates at concentrations of 10-50 nM. Intracellular injection into isolated bag cell neurons maintained in primary culture was through pressure applied to microelectrodes filled at the tip with catalytic subunit (5-22 .mu.M). In 11 of 16 injected cells, the slope of the rising phase and the height of the action potentials evoked by a constant depolarizing current were markedly enhanced relative to the pre-injection control (mean increases, 73 and 35%, respectively). This effect could occur with no change in resting potential or in the latency of the action potential from the onset of the depolarizing pulse. The effect was observed with enzyme dissolved in 3 different salt solutions (Na phosphate, K phosphate or KCl). In 2 experiments, tetrodotoxin (50 .mu.M) added to the extracellular medium had no effect on the enhanced action potentials. Subsequent addition of the Ca antagonist Co2+ diminished or abolished the spikes. In more than half of the experiments the injection of catalytic subunit was accompanied by an increase in the input resistance of the cells as measured by applying small hyperpolarizing current pulses. In 3 experiments, subthreshold oscillations in membrane potential resulted from the injections. Control injections (24 cells), carried out either with carrier medium alone or with heat-inactivated enzyme preparations, did not produce spike enhancement, increased input resistance or oscillations. The stimulation of intracellular protein phosphorylation by the catalytic subunit of cAMP-dependent protein kinase probably enhances the excitability of bag cell neurons by modifying Ca and K channels or currents.