Semenov Iurii, Herlihy Jeremiah T, Brenner Robert
Department of Physiology, UT Health Science Center, San Antonio, TX, USA.
J Vis Exp. 2012 Jun 25(64):3703. doi: 10.3791/3703.
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.
转基因小鼠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)。离体气管制备物的体外张力测定已被证明是一种用于检测转基因小鼠气道平滑肌（ASM）收缩反应的有用方法。这些体外气管制备相对简单，能产生强烈反应，即使在长时间孵育后仍保留功能性胆碱能神经末梢和肌肉反应。气管张力测定还提供了一种功能检测方法，用于研究影响平滑肌收缩的各种第二信使信号通路。气管收缩主要由副交感胆碱能神经介导，这些神经将乙酰胆碱释放到ASM上（图1）。ASM的主要乙酰胆碱受体是毒蕈碱M2和M3受体，它们分别是与G(i/o ;)和Gq偶联的受体(3,4,5)。M3受体通过与Gq偶联激活磷脂酶C，增加IP3生成并介导IP3介导的肌浆网钙释放来引起收缩(3,6,7)。M2/G(i/o ;)信号传导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)。这些通路构成了气道平滑肌所谓的“药物收缩偶联”(11)。此外，通过M2受体的胆碱能信号传导（并受M3信号传导调节）涉及使ASM去极化的通路，这反过来又激活L型电压依赖性钙通道（图1）和钙内流（所谓的“兴奋收缩偶联”）(4,7)。有关控制气道收缩的信号通路的更详细综述可见(4,12)。上述通路在小鼠和其他物种之间似乎是保守的。然而，小鼠气管在某些信号通路方面与其他物种不同。最显著的是它们对组胺和腺苷缺乏收缩反应(13,14)，而组胺和腺苷在人类和其他物种中都是众所周知的ASM调节剂(5,15)。在此，我们介绍分离小鼠气管环及其收缩输出体外测量的方法。包括设备配置、气管环分离和收缩测量的描述。给出了通过高钾刺激神经间接诱发收缩以及通过ASM肌肉去极化直接激活电压依赖性钙内流来诱发收缩的示例（1. 高钾，图1）。此外，还介绍了单独使用电场刺激刺激神经的方法（2. 电场刺激，图1），或使用施加到浴液中的外源性神经递质直接刺激ASM肌肉的方法（3. 外源性乙酰胆碱，图1）。这种灵活性和易于制备使得离体气管环模型成为一种用于研究气道平滑肌收缩中涉及的许多信号级联反应的强大且实用的检测方法。
