M1 is a major subtype of muscarinic acetylcholine receptors on mouse colonic epithelial cells
M1 is a major subtype of muscarinic acetylcholine receptors on mouse colonic epithelial cells
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DOI：
10.1007/s00535-012-0718-5
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发表时间：
2013-08
影响因子：
6.3
通讯作者：
Md. Rafiqul Islam Khan;A. Anisuzzaman;S. Semba;Yanju Ma;Junsuke Uwada;H. Hayashi;Yuichi Suzuki;T. Takano;H. Ikeuchi;M. Uchino;A. Maemoto;F. Ushikubi;I. Muramatsu;T. Taniguchi
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作者：
Md. Rafiqul Islam Khan;A. Anisuzzaman;S. Semba;Yanju Ma;Junsuke Uwada;H. Hayashi;Yuichi Suzuki;T. Takano;H. Ikeuchi;M. Uchino;A. Maemoto;F. Ushikubi;I. Muramatsu;T. Taniguchi
背景毒蕈碱乙酰胆碱受体（mAChR）是肠道上皮功能的主要调节因子。然而，精确的亚型组成尚未明确。方法我们采用[3H]-N-甲基氯化东莨菪碱作为放射性配体和几种亚型选择性化学物质，表征了小鼠结肠隐窝中mAChRs的药理学特征，并通过测量Ussing室中的短路电流（Isc）和评估小鼠结肠粘膜片中的MAP激酶磷酸化来表征其功能。结果在隐窝上检测到了mAChRs （Kd= 163.2 ± 32.3 pM，Bmax= 47.3 ± 2.6 fmol/mg 总细胞蛋白）。毒蕈碱毒素 7（MT-7，M1 亚型选择性）给出了具有高亲和力的位移曲线，但有一部分对 MT-7 不敏感（总特异性结合的 18.8 ± 0.4%）。 MT-7 不敏感成分被达非那新（M3 选择性）以高亲和力完全取代。 ACh 诱导 Isc 增加，MT-7 显着增强该增加，但达非那新或阿托品完全抑制该增加。结肠炎诱导导致 mAChR 密度显着降低，这主要发生在 MT-7 敏感成分（M1 亚型）中。免疫学实验显示结肠炎诱导后 M1 信号减少，但 M3 信号没有减少。在小鼠结肠上皮中，毒蕈碱刺激诱导 MAP 激酶磷酸化增加，这种磷酸化被 MT-7 完全抑制，并因炎症而减弱。结论这些结果表明，小鼠结肠上皮细胞中的 mAChR 由两种亚型组成：M1 (80%) 和 M3 (20%)。主要的 M1 亚型可能对上皮氯分泌产生负面影响，并且容易受到炎症的影响，可能与炎症性肠道功能障碍有关。
BackgroundMuscarinic acetylcholine receptors (mAChRs) are major regulators of gut epithelial functions. However, the precise subtype composition has not been clarified.MethodsWe characterized the pharmacological profile of mAChRs on mouse colonic crypts, employing [3H]-N-methyl scopolamine chloride as a radioligand and several subtype-selective chemicals, and the functional aspect by measuring short-circuit current (Isc) in Ussing chambers and by evaluating MAP kinase phosphorylation in mouse colonic mucosal sheets.ResultsThe mAChRs were detected on the crypts (Kd= 163.2 ± 32.3 pM,Bmax= 47.3 ± 2.6 fmol/mg of total cell protein). Muscarinic toxin 7 (MT-7, M1 subtype selective) gave a displacement curve with high affinity, but there was a part insensitive to MT-7 (18.8 ± 0.4 % of the total specific binding). The MT-7-insensitive component was displaced completely by darifenacin (M3 selective) with high affinity. ACh induced an increase inIsc, which was significantly enhanced by MT-7 but was completely inhibited by darifenacin or atropine. Colitis induction resulted in a significant decrease in the density of mAChRs, which occurred mainly in the MT-7-sensitive component (M1 subtype). Immunological experiments exhibited a reduction of M1 but not of M3 signal after colitis induction. Muscarinic stimulation induced an increase in MAP kinase phosphorylation, which was completely suppressed by MT-7 and was attenuated by inflammation, in mouse colonic epithelium.ConclusionsThese results suggest that mAChRs in mouse colonic epithelial cells consist of two subtypes, M1 (80 %) and M3 (20 %). The major M1 subtype was likely to regulate epithelial chloride secretion negatively and was susceptible to inflammation and may be relevant to inflammatory gut dysfunction.