ANO1 in intramuscular interstitial cells of Cajal plays a key role in the generation of slow waves and tone in the internal anal sphincter

Abstract

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.

The cells and conductance mediating cholinergic neurotransmission in the murine proximal stomach

“…; Cobine et al . ). We reasoned that, if post‐junctional electrical responses (EJPs) were compromised in animals made null for Ano1 , this would be strong support for the innervation of ICC‐IM by cholinergic motor neurons and, if post‐junctional electrical and mechanical responses were reduced or blocked in fundus muscles of Ano1 −/− mice, this would suggest that ICC‐IM are the dominant post‐junctional cell receiving and transducing inputs from cholinergic enteric motor neurons in wild‐type muscles.…”

Rhythmic calcium transients in smooth muscle cells of the mouse internal anal sphincter

“…Contractile data were collected, stored, and analyzed using AcqKnowledge 3.9.1 software (Biopac Systems Inc). As previously described, 13 the average peak and trough values for phasic contractions were determined for 60 seconds of spontaneous activity.…”

“…We have hypothesized that phasic Ca 2+ entry events give rise to tone. 13,14 One mechanism that may be involved relates to SW frequency. The frequency of SWs in the IAS is significantly greater than that of other gastrointestinal (GI) muscles that do not generate tone (eg, 30 cpm vs 8 cpm for the dog IAS vs rectum 9 ).…”

“…Contraction and SWs in the IAS are dependent upon Ca 2+ entry through Cav L because both are abolished by Cav L blockers or by hyperpolarizing membrane potential below the Cav L activation threshold with the K ATP channel activator pinacidil 9,[12][13][14]. AdditionalF I G U R E 3 Examples of the origin and spread of Ca 2+ transients.…”

Changes in murine anorectum signaling across the life course

“…As in other parts of the gastrointestinal tract (GI tract), contraction and relaxation of the anorectum is regulated by intrinsic enteric and extrinsic autonomic neurons and by the actions of other cell types, including Interstitial cells of Cajal and PDGFRα + cells. [4][5][6][7][8] Here, we focus on changes in the function of excitatory and inhibitory motor neurons in the mouse anorectum during aging.…”