In vitro measurements of tracheal constriction using mice.
In vitro measurements of tracheal constriction using mice.
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DOI：
10.3791/3703
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发表时间：
2012-06
期刊：
影响因子：
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通讯作者：
I. Semenov;J. Herlihy;R. Brenner
中科院分区：
文献类型：
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作者：
I. Semenov;J. Herlihy;R. Brenner
转基因和基因敲除小鼠已成为研究气道生理和病理生理的有力工具（1,2）。离体气管制剂的体外张力测定已被证明是一种有效的转基因小鼠气道平滑肌（ASM）收缩反应测定方法。这些体外气管制剂相对简单，提供强大的反应，并保留功能胆碱能神经末梢和肌肉反应，即使经过长时间的孵育。气管张力法还提供了一种功能分析来研究各种影响平滑肌收缩的第二信使信号通路。气管收缩主要由副交感神经和胆碱能神经介导，这些神经将乙酰胆碱释放到ASM（图1）。ASM乙酰胆碱受体主要为毒蕈碱M2和M3，分别为G（i/o;）和Gq偶联受体（3,4,5）。M3受体通过偶联Gq激活磷脂酶C，增加IP3的产生和IP3介导的肌浆网钙释放，从而引起收缩（3,6,7）。M2/G（i/o;）信号被认为通过抑制腺苷酸环化酶导致cAMP水平降低来增强收缩（5,8,9,10）。这些通路构成了所谓的气道平滑肌的“药物-收缩耦合”（11）。此外，通过M2受体传递的胆碱能信号（由M3信号调节）涉及ASM去极化的通路，进而激活l型电压依赖性钙通道（图1）和钙内流（所谓的“兴奋-收缩耦合”）（4,7）。可以找到控制气道收缩的信号通路的更详细的综述（4,12）。上述途径在小鼠和其他物种之间似乎是保守的。然而，小鼠的气管在某些信号通路上与其他物种不同。最突出的是它们对组胺和腺苷缺乏收缩反应（13,14），两者都是人类和其他物种中众所周知的ASM调节剂（5,15）。在这里，我们提出了分离小鼠气管环和在体外测量其收缩输出的方案。包括设备配置，气管环隔离和收缩测量的说明。本文给出了通过高钾刺激神经间接引起收缩和通过ASM肌去极化直接激活电压依赖性钙内流的例子(1)。高K(+)，图1)。此外，还提出了使用电场刺激单独刺激神经的方法(2)。EFS，图1)，或者使用外源性神经递质直接刺激ASM肌(3)。外源性乙酰胆碱，图1)。这种灵活性和制备的便捷性使得分离气管环模型对气道平滑肌收缩中涉及的一些信号级联反应具有强大的功能分析。
Transgenic and knockout mice have been powerful tools for the investigation of the physiology and pathophysiology of airways(1,2). In vitro tensometry of isolated tracheal preparations has proven to be a useful assay of airway smooth muscle (ASM) contractile response in genetically modified mice. These in vitro tracheal preparations are relatively simple, provide a robust response, and retain both functional cholinergic nerve endings and muscle responses, even after long incubations. Tracheal tensometry also provides a functional assay to study a variety of second messenger signaling pathways that affect contraction of smooth muscle. Contraction in trachea is primarily mediated by parasympathetic, cholinergic nerves that release acetylcholine onto ASM (Figure 1). The major ASM acetylcholine receptors are muscarinic M2 and M3 which are G(i/o ;)and Gq coupled receptors, respectively(3,4,5). M3 receptors evoke contraction by coupling to Gq to activate phospholipase C, increase IP3 production and IP3-mediated calcium release from the sarcoplasmic reticulum(3,6,7). M2/G(i/o ;)signaling is believed to enhance contractions by inhibition of adenylate cyclase leading to a decrease in cAMP levels(5,8,9,10). These pathways constitute the so called "pharmaco-contraction coupling" of airway smooth muscle(11). In addition, cholinergic signaling through M2 receptors (and modulated by M3 signaling) involves pathways that depolarize the ASM which in turn activate L-type, voltage-dependent calcium channels (Figure 1) and calcium influx (so called "excitation-contraction coupling")(4,7). More detailed reviews on signaling pathways controlling airway constriction can be found(4,12). The above pathways appear to be conserved between mice and other species. However, mouse tracheas differ from other species in some signaling pathways. Most prominent is their lack of contractile response to histamine and adenosine(13,14), both well-known ASM modulators in humans and other species(5,15). Here we present protocols for the isolation of murine tracheal rings and the in vitro measurement of their contractile output. Included are descriptions of the equipment configuration, trachea ring isolation and contractile measurements. Examples are given for evoking contractions indirectly using high potassium stimulation of nerves and directly by depolarization of ASM muscle to activate voltage-dependent calcium influx (1. high K(+), Figure 1). In addition, methods are presented for stimulations of nerves alone using electric field stimulation (2. EFS, Figure 1), or for direct stimulation of ASM muscle using exogenous neurotransmitter applied to the bath (3. exogenous ACH, Figure 1). This flexibility and ease of preparation renders the isolated trachea ring model a robust and functional assay for a number of signaling cascades involved in airway smooth muscle contraction.