Cobine C A, Hannah E E, Zhu M H, Lyle H E, Rock J R, Sanders K M, Ward S M, Keef K D
Department of Physiology and Cell Biology, University of Nevada, Reno School of Medicine, Reno, NV, 89557, USA.
Department of Anatomy, UCSF School of Medicine, San Francisco, CA, 94143, USA.
J Physiol. 2017 Mar 15;595(6):2021-2041. doi: 10.1113/JP273618. Epub 2017 Feb 14.
The internal anal sphincter develops tone important for maintaining high anal pressure and continence. Controversy exists regarding the mechanisms underlying tone development. We examined the hypothesis that tone depends upon electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca -activated Cl channels (ANO1, encoded by Ano1) and voltage-dependent L-type Ca channels (Cav , encoded by Cacna1c). Measurement of membrane potential and contraction indicated that ANO1 and Cav have a central role in SW generation, phasic contractions and tone, independent of stretch. ANO1 expression was examined in wildtype and Ano1 mice with immunohistochemical techniques. Ano1 and Cacna1c expression levels were examined by quantitative PCR in fluorescence-activated cell sorting. ICC-IM were the predominant cell type expressing ANO1 and the most likely candidate for SW generation. SWs in ICC-IM are proposed to conduct to smooth muscle where Ca entry via Cav results in phasic activity that sums to produce tone.
The mechanism underlying tone generation in the internal anal sphincter (IAS) is controversial. We examined the hypothesis that tone depends upon generation of electrical slow waves (SWs) initiated in intramuscular interstitial cells of Cajal (ICC-IM) by activation of Ca -activated Cl channels (encoded by Ano1) and voltage-dependent L-type Ca channels (encoded by Cacna1c). Phasic contractions and tone in the IAS were nearly abolished by ANO1 and Cav antagonists. ANO1 antagonists also abolished SWs as well as transient depolarizations that persisted after addition of Cav antagonists. Tone development in the IAS did not require stretch of muscles, and the sensitivity of contraction to ANO1 antagonists was the same in stretched versus un-stretched muscles. ANO1 expression was examined in wildtype and Ano1 mice with immunohistochemical techniques. Dual labelling revealed that ANO1 expression could be resolved in ICC but not smooth muscle cells (SMCs) in the IAS and rectum. Ano1, Cacna1c and Kit gene expression were the same in extracts of IAS and rectum muscles. In IAS cells isolated with fluorescence-activated cell sorting, Ano1 expression was 26.5-fold greater in ICC than in SMCs while Cacna1c expression was only 2-fold greater in SMCs than in ICC. These data support a central role for ANO1 and Cav in the generation of SWs and tone in the IAS. ICC-IM are the probable cellular candidate for ANO1 currents and SW generation. We propose that ANO1 and Cav collaborate to generate SWs in ICC-IM followed by conduction to adjacent SMCs where phasic calcium entry through Cav sums to produce tone.
肛门内括约肌产生的张力对于维持较高的肛管压力和控便功能很重要。关于张力产生的机制存在争议。我们检验了这样一种假说，即张力取决于由钙激活氯离子通道（由Ano1编码的ANO1）和电压依赖性L型钙通道（由Cacna1c编码的Cav ）激活，在肌间 Cajal 间质细胞（ICC-IM）中引发的电慢波（SWs）。膜电位和收缩的测量表明，ANO1和Cav在SWs产生、相位性收缩和张力中起核心作用，与牵张无关。采用免疫组织化学技术检测了野生型和Ano1基因敲除小鼠中ANO1的表达。通过荧光激活细胞分选技术的定量PCR检测了Ano1和Cacna1c的表达水平。ICC-IM是表达ANO1的主要细胞类型，也是产生SWs最可能的候选细胞。推测ICC-IM中的SWs传导至平滑肌，通过Cav进入的钙离子导致相位性活动，这些活动叠加产生张力。
肛门内括约肌（IAS）张力产生的机制存在争议。我们检验了这样一种假说，即张力取决于由钙激活氯离子通道（由Ano1编码）和电压依赖性L型钙通道（由Cacna1c编码）激活，在肌间 Cajal 间质细胞（ICC-IM）中引发的电慢波（SWs）。ANO1和Cav拮抗剂几乎消除了IAS中的相位性收缩和张力。ANO1拮抗剂也消除了SWs以及在加入Cav拮抗剂后持续存在的瞬时去极化。IAS中张力的产生不需要肌肉牵张，并且在牵张和未牵张的肌肉中，收缩对ANO1拮抗剂的敏感性相同。采用免疫组织化学技术检测了野生型和Ano1基因敲除小鼠中ANO1的表达。双重标记显示，在IAS和直肠中，ANO1表达可在ICC中分辨出来，但在平滑肌细胞（SMCs）中则分辨不出。IAS和直肠肌肉提取物中Ano1、Cacna1c和Kit基因表达相同。在通过荧光激活细胞分选分离的IAS细胞中，ICC中Ano1的表达比SMCs高26.5倍，而SMCs中Cacna1c的表达仅比ICC高2倍。这些数据支持ANO1和Cav在IAS中SWs产生和张力形成中起核心作用。ICC-IM可能是ANO1电流和SWs产生的细胞候选者。我们推测ANO1和Cav协同作用在ICC-IM中产生SWs，随后传导至相邻的SMCs，在那里通过Cav进入的相位性钙离子叠加产生张力。
