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瞬时感受器电位A1 通道参与冷刺激引起的离体大鼠结肠平滑肌收缩

董杨, 石海莲, 施建蓉, 吴大正

上海中医药大学 中药研究所，上海市复方中药重点实验室；教学实验中心，上海 201203

摘要

瞬时感受器电位A1 (transient receptor potential A1, TRPA1)通道是一种可以被伤害性冷刺激激活的TRP 家族离子通道。本研究的目的是探讨TRPA1 通道是否参与冷刺激引起的大鼠结肠平滑肌收缩及其可能的机制。分离雄性Wistar 大鼠升结肠和降结肠的平滑肌，制备成肌条，灌流不同温度Krebs 液给予冷刺激，然后17 °C 冷刺激条件下用不同药物灌流，用张力换能器记录张力变化。逆转录PCR (RT-PCR)检测上述平滑肌中TRPA1、TRPM8 和TRPV1 的mRNA表达。结果显示，在大鼠升结肠和降结肠的平滑肌层有TRPA1 的表达，但无TRPM8 和TRPV1 的表达。肌张力研究显示，不同温度(32、25、17、12 °C)的冷刺激可以引起大鼠升结肠和降结肠纵行平滑肌的收缩，并且收缩张力随着温度降低而增强，17 °C 冷刺激可引起最大收缩(P<0.01)。TRPA1 的阻断剂钌红(30 μmol/L)可以抑制冷刺激引起的升、降结肠平滑肌的收缩(P<0.05)。TRPA1 激动剂异硫氰酸烯丙酯(AITC, 300 μmol/L)和桂皮醛(CA, 1 mmol/L)预处理肌条后，可降低或几乎取消冷刺激引起的升结肠(P<0.01,P<0.05)和降结肠(均P<0.001)平滑肌的收缩。去除细胞外Ca2+ (EGTA, 1 mmol/L)、PLC 阻断剂 U73122 (10 μmol/L)、IP3 受体阻断剂2-APB (30 μmol/L)均可抑制17 °C 冷刺激引起的升、降结肠平滑肌的收缩(P<0.001, P<0.05, P<0.001)。阿托品(1 μmol/L)则对17 ºC 冷刺激引起的升、降结肠平滑肌的收缩均无影响。L- 型钙通道阻断剂硝苯地平(1 μmol/L)和神经毒素河豚毒素(2 μmol/L)可抑制降结肠平滑肌的收缩(P<0.01, P<0.05)，而对升结肠没有作用。综上所述，TRPA1 通道参与冷刺激引起的大鼠升结肠和降结肠平滑肌的收缩，其胞内信号转导机制可能涉及PLC/IP3/Ca2+ 途径的激活。另外， L- 型钙通道和非M 受体介导的神经机制部分参与冷刺激引起的降结肠平滑肌收缩，这可能是降结肠收缩张力大于升结肠的原因。

关键词： 结肠; 平滑肌; 收缩; TRPA1

分类号：R333

Transient receptor potential A1 is involved in cold-induced contraction in the isolated rat colon smooth muscle

DONG Yang, SHIHai-Lian, SHI Jian-Rong, WU Da-Zheng

Institute of Chinese Materia Medica, Shanghai Key Laboratory of Complex Prescription； Experimental Teaching Centre, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China

Abstract

Transient receptor potential (TRP) A1, a member of TRP channel family, is activated by noxious cold. The aims of this studywere to determine if TRPA1 contributed to cold-induced contractions in the isolated rat colon preparations and explore the potentialmechanisms. The colon smooth muscle layers were surgically isolated from the male Wistar rats and changes in isotonic tension oflongitudinal muscle under various treatments were recorded as colonic motilities. Cold stimuli were obtained by the reperfusion withKrebs-Henseleit solution at given temperature using Constant Flow Pump. The mRNA expressions of TRPA1, TRPV1 and TRPM8in rat colon smooth muscle layer were examined by using reverse transcription-polymerase chain reaction (RT-PCR) techniques. Theresults showed that the contractions induced by cold stimuli (from 37 °C to 12 °C stepwise) were inversely proportional to thetemperature with a maximum contraction at 17 °C in both proximal and distal colons (P<0.01). RT-PCR analysis revealed theexpression of TRPA1, but not TRPM8 and TRPV1, in the rat proximal and distal colon smooth muscle layers. Cold-induced coloniccontractions were specially inhibited by TRPA1 blocker, ruthenium red (30 μmol/L), in the proximal and distal colon (P<0.05). Thecold-induced contractions of proximal (P<0.01, P<0.05) and distal colons (both P<0.001) were almost abolished or inhibited by thepretreatments of TRPA1 agonists, Allyl isothiocyanate (AITC, 300 μmol/L) and cinnamaldehyde (CA, 1 mmol/L). Extracellularcalcium removal (EGTA, 1 mmol/L), PLC blocker (U73122, 10 μmol/L) and IP3 receptor blocker (2-aminoethoxydiphenyl borate,2-APB, 30 μmol/L) all decreased the contractions evoked by the cooling at 17 °C in the proximal and distal colon (P<0.001, P<0.05,P<0.001). Atropine (1 μmol/L) had no effects on these contractions. L-type Ca2+ channels blocker nifedipine (1 μmol/L) and neurotoxintetrodotoxin (TTX, 2 μmol/L) decreased the contractile response in the distal colon (P<0.01, P<0.05), but not in the proximal colon.In conclusion, TRPA1 contributes to cold-induced contractions of the rat colon smooth muscle, and the mechanism of TRPA1activation involves PLC/IP3/Ca2+ pathway. L-type Ca2+ channel and neurogenic mechanism other than muscarinic receptor might bepartially involved in cold-induced contraction of the distal colon, which probably resulted in higher contraction of distal coloncompared with that of proximal colon.

Key words: Colon; smooth muscle; contraction; TRPA1

收稿日期：2010-05-11　　录用日期：2010-06-24

通讯作者：吴大正　　E-mail: wdazheng@hotmail.com

引用本文：

董杨, 石海莲, 施建蓉, 吴大正. 瞬时感受器电位A1 通道参与冷刺激引起的离体大鼠结肠平滑肌收缩[J]. 生理学报 2010; 62 (4): 349-356.

DONG Yang, SHIHai-Lian, SHI Jian-Rong, WU Da-Zheng. Transient receptor potential A1 is involved in cold-induced contraction in the isolated rat colon smooth muscle. Acta Physiol Sin 2010; 62 (4): 349-356 (in Chinese with English abstract).