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钠钾氯共转运体在γ-氨基丁酸引起的肠神经元兴奋中的作用

刘素梅^1,2,*, 郑丽飞³, NEITZEL Kayla¹, 纪托³, REN Wei², 曲梅花²

¹美国威斯康星大学拉克罗斯分校科学与健康学院生物学系，威斯康星州拉克罗斯市 54601；²美国俄亥俄州立大学医学院生理学与细胞生物学系，俄亥俄州哥伦布市 43210；³首都医科大学基础医学院生理学与病理生理学系，北京 100069

摘要

γ-氨基丁酸(γ-aminobutyric acid, GABA)是成年个体中枢神经系统(central nervous system, CNS)的一种抑制性神经递质，但对未成熟CNS神经元有兴奋作用。出生后GABA对CNS神经元的作用由去极化逐渐转为超极化，这种转变主要是由于大脑Na+-K+-2Cl−共运体1 (Na+-K+-2Cl− symporter 1, NKCC1)表达逐渐减少和K+-Cl−共运体2 (K+-Cl− cotransporter 2, KCC2)表达增加。与CNS神经元不同，肠神经系统(enteric nervous system, ENS)中未成熟和成体神经元都可被GABA去极化。GABA激发ENS神经元兴奋的分子机制尚不清楚，但与ENS神经元内的高Cl−浓度有关。本研究目的是检验一个假设，即ENS神经元的细胞内高Cl−浓度是由NKCCs活动维持的。结果显示，NKCC2免疫反应性(immunoreactivity, IR)在出生后第1天(P1)大鼠结肠ENS中出现，随后持续升高，在P14达到稳定的高水平，并在成年后保持在这一水平。NKCC1的IR出现在P14大鼠的ENS中，并维持到成年。在任何发育阶段的大鼠ENS中都检测不到KCC2的IR。在ENS神经元中，NKCC1和NKCC2均与GABAA受体共同表达。外源性GABA (1 mmol/L)可引起ENS神经元膜去极化。GABA诱导的去极化逆转电位约为−16 mV。NKCC抑制剂布美他尼(50 μmol/L)或速尿(300 μmol/L)可显著抑制GABA诱导的去极化。布美他尼(50 μmol/L)使GABA诱导的去极化逆转电位向超极化方向移动。无论是KCC抑制剂DIOA (20 μmol/L)还是Cl−/HCO3−交换抑制剂DIDS (200 μmol/L)对GABA诱发的去极化都没有明显影响。以上结果提示，ENS表达NKCC1和NKCC2，但是不表达KCC2。NKCC1和NKCC2在GABA引起的肠神经元兴奋中起重要作用。

关键词： 钾氯共转运体; 钾氯共转运体; γ-氨基丁酸; 肠神经系统; 胃肠道

分类号：Q48

Na+-K+-2Cl− symporter contributes to γ-aminobutyric acid-evoked excitation in rat enteric neurons

LIU Sumei^1,2,*, ZHENG Lifei³, NEITZEL Kayla¹, JI Tuo³, REN Wei², QU Mei-Hua²

¹Department of Biology, College of Science and Health, University of Wisconsin-La Crosse, La Crosse, WI, USA；²Department of Physiology and Cell Biology, College of Medicine, The Ohio State University, Columbus, OH, USA；³Department of Physiology and Pathophysiology, School of Basic Medical Science, Capital Medical University, Beijing 100069, China

Abstract

Gamma-aminobutyric acid (GABA) is an inhibitory neurotransmitter in the adult central nervous system (CNS), however, it causes excitation in the immature CNS neurons. The shift from GABA-induced depolarization to hyperpolarization in postnatal brain is primarily due to progressive decrease in the expression of the Na+-K+-2Cl− symporter 1 (NKCC1) and increased expression of the K+-Cl− cotransporter 2 (KCC2). Unlike CNS neurons, both immature and mature neurons in the enteric nervous system (ENS) are depolarized by GABA. Molecular mechanisms by which GABA excites ENS neurons are unclear. It is understood, however, that the excitatory action depends on elevated intraneuronal Cl−. We aimed to test a hypothesis that high intracellular Cl− in ENS neurons is maintained by activity of the NKCCs. We found that NKCC2 immunoreactivity (IR) was expressed in the ENS of the rat colon on postnatal day 1 (P1). The expression level of NKCC2 continuously increased and reached a steady high level on P14 and maintained at that level in adulthood. NKCC1 IR appeared in ENS on P14 and maintained through adulthood. KCC2 IR was not detectable in the ENS in any of the developmental stages. Both NKCC1 IR and NKCC2 IR were co-expressed with GABAA receptors in ENS neurons. Exogenous GABA (1 mmol/L) caused membrane depolarization in the ENS neurons. The reversal potential of GABA-induced depolarization was about −16 mV. Blockade of NKCC by bumetanide (50 μmol/L) or furosemide (300 μmol/L) suppressed the depolarizing responses to GABA. Bumetanide (50 μmol/L) shifted the reversal potential of GABA-induced depolarization in the hyperpolarizing direction. Neither the KCC blocker DIOA (20 μmol/L) nor the Cl−/HCO3− exchanger inhibitor DIDS (200 μmol/L) suppressed GABA-evoked depolarization. The results suggest that ENS neurons continuously express NKCC2 since P1 and NKCC1 since P14, which contribute to the accumulation of Cl− in ENS neurons and GABA-evoked depolarization in neonate and adult ENS neurons. These results provide the first direct evidence for the contribution of both NKCC2 and NKCC1 to the GABAA-mediated depolarization.

Key words: Na+-K+-2Cl− symporter; K+-Cl− cotransporter; γ-aminobutyric acid; enteric nervous system; intestine

收稿日期：2019-10-21　　录用日期：2020-01-29

通讯作者：刘素梅　　E-mail: sliu@uwlax.edu

DOI: 10.13294/j.aps.2020.0035

引用本文：

刘素梅, 郑丽飞, NEITZEL Kayla, 纪托, REN Wei, 曲梅花. 钠钾氯共转运体在γ-氨基丁酸引起的肠神经元兴奋中的作用[J]. 生理学报 2020; 72 (3): 263-273.

LIU Sumei, ZHENG Lifei, NEITZEL Kayla, JI Tuo, REN Wei, QU Mei-Hua. Na+-K+-2Cl− symporter contributes to γ-aminobutyric acid-evoked excitation in rat enteric neurons. Acta Physiol Sin 2020; 72 (3): 263-273