Dong Yang, Shi Hai-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, China.
Sheng Li Xue Bao. 2010 Aug 25;62(4):349-56.
Transient receptor potential (TRP) A1, a member of TRP channel family, is activated by noxious cold. The aims of this study were to determine if TRPA1 contributed to cold-induced contractions in the isolated rat colon preparations and explore the potential mechanisms. The colon smooth muscle layers were surgically isolated from the male Wistar rats and changes in isotonic tension of longitudinal muscle under various treatments were recorded as colonic motilities. Cold stimuli were obtained by the reperfusion with Krebs-Henseleit solution at given temperature using Constant Flow Pump. The mRNA expressions of TRPA1, TRPV1 and TRPM8 in rat colon smooth muscle layer were examined by using reverse transcription-polymerase chain reaction (RT-PCR) techniques. The results showed that the contractions induced by cold stimuli (from 37 degrees C to 12 degrees C stepwise) were inversely proportional to the temperature with a maximum contraction at 17 degrees C in both proximal and distal colons (P<0.01). RT-PCR analysis revealed the expression of TRPA1, but not TRPM8 and TRPV1, in the rat proximal and distal colon smooth muscle layers. Cold-induced colonic contractions were specially inhibited by TRPA1 blocker, ruthenium red (30 μmol/L), in the proximal and distal colon (P<0.05). The cold-induced contractions of proximal (P<0.01, P<0.05) and distal colons (both P<0.001) were almost abolished or inhibited by the pretreatments of TRPA1 agonists, Allyl isothiocyanate (AITC, 300 μmol/L) and cinnamaldehyde (CA, 1 mmol/L). Extracellular calcium removal (EGTA, 1 mmol/L), PLC blocker (U73122, 10 μmol/L) and IP(3) receptor blocker (2-aminoethoxydiphenyl borate, 2-APB, 30 μmol/L) all decreased the contractions evoked by the cooling at 17 degrees 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 Ca(2+) channels blocker nifedipine (1 μmol/L) and neurotoxin tetrodotoxin (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 TRPA1 activation involves PLC/IP(3)/Ca(2+) pathway. L-type Ca(2+) channel and neurogenic mechanism other than muscarinic receptor might be partially involved in cold-induced contraction of the distal colon, which probably resulted in higher contraction of distal colon compared with that of proximal colon.
瞬时受体电位（TRP）A1是TRP通道家族的成员之一，可被有害冷刺激激活。本研究旨在确定TRPA1是否参与离体大鼠结肠标本中冷诱导的收缩，并探讨其潜在机制。从雄性Wistar大鼠手术分离结肠平滑肌层，记录不同处理下纵行肌等张张力的变化作为结肠动力。使用恒流泵在给定温度下用Krebs-Henseleit溶液再灌注以获得冷刺激。采用逆转录-聚合酶链反应（RT-PCR）技术检测大鼠结肠平滑肌层中TRPA1、TRPV1和TRPM8的mRNA表达。结果显示，冷刺激（从37℃逐步降至12℃）诱导的收缩与温度呈反比，在近端和远端结肠中，17℃时收缩最大（P<0.01）。RT-PCR分析显示，在大鼠近端和远端结肠平滑肌层中存在TRPA1的表达，但未检测到TRPM8和TRPV1的表达。TRPA1阻滞剂钌红（30μmol/L）可特异性抑制近端和远端结肠中冷诱导的结肠收缩（P<0.05）。TRPA1激动剂异硫氰酸烯丙酯（AITC，300μmol/L）和肉桂醛（CA，1mmol/L）预处理后，近端结肠（P<0.01，P<0.05）和远端结肠（均P<0.001）的冷诱导收缩几乎被消除或抑制。细胞外钙去除（EGTA，1mmol/L）、PLC阻滞剂（U73122，10μmol/L）和IP(3)受体阻滞剂（2-氨基乙氧基二苯硼酸，2-APB，30μmol/L）均降低了17℃冷却诱导的近端和远端结肠收缩（P<0.001，P<0.05，P<0.001）。阿托品（1μmol/L）对这些收缩无影响。L型钙通道阻滞剂硝苯地平（1μmol/L）和神经毒素河豚毒素（TTX，2μmol/L）可降低远端结肠的收缩反应（P<0.01，P<0.05），但对近端结肠无影响。总之，TRPA1参与大鼠结肠平滑肌的冷诱导收缩，TRPA1激活机制涉及PLC/IP(3)/Ca(2+)途径。L型钙通道和除毒蕈碱受体外的神经源性机制可能部分参与远端结肠的冷诱导收缩，这可能导致远端结肠收缩高于近端结肠。
