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ACh对大鼠皮层体感区神经元延迟整流钾电流的抑制作用

崔岚巍, 李玉荣, 杨雷, 贾淑伟, 曲丽辉, 姚坤, 金宏波

哈尔滨医科大学生理学教研室. 哈尔滨 150086

摘要

利用全细胞膜片钳技术研究乙酰胆碱(acetylcholine, ACh)对大鼠皮层体感区神经元延迟整流钾电流(I_(K))的调制作用。结果表明：ACh (0.1、1、10、100 #mu#mol/L)对大鼠皮层体感区神经元I_(K)有抑制作用，并具有剂量依赖性关系({sl P}<0.01)。ACh可使I_(K)激活曲线的斜率变大，并使激活曲线向超极化方向移动。I_(K)激活曲线的半数激活电压(V_(1/2))和斜率因子(k)分别由给药前的(--41.8±9.7) mV和(30.7±7.2) mV变为给药后的(--122.4±38.6) mV和(42.4±7.0) mV。 100 #mu#mol/L的N受体拮抗剂筒箭毒碱(tubocurarine)可减弱ACh对I_(K)的抑制作用，在指令电压+60 mV时tubocurarine+ACh组的I_(K)幅度下降了(16.9±13.8)% ({sl n}=8)，与10 #mu#mol/L ACh组引起的(36.5±7.8)%的I_(K)下降幅度相比，有极显著差异({sl P}<0.01)。10 #mu#mol/L的M_(1)受体拮抗剂哌仑西平(pirenzepin)拮抗ACh对I_(K)的抑制作用不明显 ({sl n}=7, {sl P} >0.05); 而10 #mu#mol/L的M_(3)受体拮抗剂 4--DAMP可部分拮抗ACh对I_(K)的抑制作用，并且4--DAMP+ACh组使I_(K)的电流值下降了(26.8±4.7)% ({sl n}=6)，与ACh组引起的I_(K)电流下降相比，有显著差异({sl P}<0.05)。蛋白激酶C (Protein kinase C, PKC)阻断剂chelerythrine拮抗ACh对I_(K)的抑制作用，PKC激动剂PDBu可增强ACh对I_(K)的抑制作用({sl P}<0.05)。综上所述，ACh对大鼠皮层体感区神经元I_(K)的抑制作用主要是通过烟碱受体(nAChRs)和M_(3)受体介导，并经过PKC信号途径。

关键词： 乙酰胆碱受体; 皮层; 延迟整流钾电流; 全细胞膜片钳; 蛋白激酶C

Inhibition of ACh on the delayed rectifier--like potassium current in acutely isolated cerebral cortical neurons of rats

Cui Lanwei, Li Yurong, Yang Lei, Jia Shuwei, Qu Lihui, Yao Kun, Jin Hongbo

Department of Physiology, Harbin Medical University. Harbin 150086, China

Abstract

The modulation of ACh on delayed rectifier-like potassium currents (I_(K)) was studied in freshly dissociated cerebral cortical neurons using the whole-cell patch-clamp technique. Wistar rats between 10- and 14-day old of both sexes were used. After rats were decapitated, their brains were quickly removed, iced, and then manually cut into 400 µm slices. Slices were then incubated for 0.5 h at 32℃ in a buffered artificial cerebrospinal fluid (ACSF) bubbled with 95% O_(2), 5% CO_(2). Slices were then removed into buffered ACSF containing protease (0.5 mg/ml) at 32℃. After 30 min of enzyme digestion, tissue was rinsed three times in the buffered saline. Then the enzyme-treated slices were mechanically dissociated with a graded series of fire-polished Pasteur pipettes. The cell suspension was then plated into a 35 mm dish and placed on the stage of a Olympus inverted microscope. For whole-cell recordings of currents, standard voltage-clamp techniques were used. Neurons were held at -80 mV, and the I_(K) was evoked by 2 000 ms depolarizing voltage commands to potential between -40 mV and +60 mV in 10 mV steps applied at a frequency of 0.5 Hz. It was found that the inhibitory effect of ACh (0.1, 1, 10, 100 µmol/L) on I_(K) was dose-dependent. It was also found that ACh affected the activation process of I_(K) significantly, i.e., the activation curve of I_(K) was characterized by half-activation potential of (-41.8±9.7) mV and a slope factor of (30.7±7.2) mV in the cortical neurons and they were changed to (-122.4±38.6) mV and (42.4±7.0) mV, respectively, after giving ACh (10µmol/L). Tubocurarine (100 µmol/L) antagonized the inhibitory effect of ACh on I_(K), and the drop of currents varied from the control value of (36.5±7.8)% to (16.9±13.8)% (n=8, P<0.01). 4-DAMP (10 µmol/L) blocked the inhibitory effect of ACh on I_(K), and the currents reduced from the control value of (36.5±7.8)% to (26.8±4.7)% (n=6, P<0.05). Pirenzepin did not antagonize the inhibition of ACh on I_(K) (n=7, P>0.05). Chelerythrine (20 #mu#mol/L) blocked the inhibitory effect of ACh on I_(K) and the currents reduced from the control value of (36.5±7.8)% to (11.7±17.3)% (n=6, P<0.05). On the contrary, PDBu (10 #mu#mol/L) strengthened the inhibition of ACh on I_(K) and the drop of currents changed from the control value of (36.5±7.8)% to (59.2±14.0)% (n=5, P<0.05). PDBu abolished the antagonism of chelerythrine on ACh in cortical neurons. It is suggested that the ACh-induced depolarization of neurons in the cortex is attributed to the inhibition of I_(K) that is most likely evoked by the activation of nicotinic ACh receptors and muscarinic M_(3) receptor via protein kinase C (PKC) signal transduction pathway.

Key words: Acetylcholine receptors;Cortexin;Delayed rectifier-like potassium currents;Whole-cell recording;Protein kinase C

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引用本文：

崔岚巍, 李玉荣, 杨雷, 贾淑伟, 曲丽辉, 姚坤, 金宏波 . ACh对大鼠皮层体感区神经元延迟整流钾电流的抑制作用 [J]. 生理学报 2006; 58 (1): .

Cui Lanwei, Li Yurong, Yang Lei, Jia Shuwei, Qu Lihui, Yao Kun, Jin Hongbo. Inhibition of ACh on the delayed rectifier--like potassium current in acutely isolated cerebral cortical neurons of rats . Acta Physiol Sin 2006; 58 (1): (in Chinese with English abstract).