AB285. SPR-12 Evidence supporting a pivotal role for intramuscular interstitial cells of Cajal in the generation of pacemaker activity, phasic contractions and tone in the internal anal sphincter

Objective: A fundamental property of the internal anal sphincter (IAS) is its ability to generate tone. However, the mechanism underlying tone generation is still controversial. In other GI regions, interstitial cells of Cajal (ICC) have been demonstrated to be the pacemaker cells that generate slow waves (SWs). We have shown that SWs are also present in the IAS along with a population of intramuscular ICC (ICC-IM). However, the identity of the cell that generates SWs in the IAS is still uncertain. We hypothesize that SWs are generated by ICC-IM via a rise in intracellular calcium, activation of calcium-activated chloride channels (ANO1) and voltage-dependent L-type calcium channels (Cav_L). We further propose that SWs play a critical role in tone development in the IAS through the generation and summation of phasic contractile activity.

Methods: Cell specific protein expression was examined with immunohistochemistry while gene expression was determined with qPCR on whole muscles and cells isolated with FACS. An inducible Cre/loxP technique was used to express a genetically-encoded Ca²⁺ biosensor (GCaMP3) in a cell-specific manner. Calcium transients were imaged from the IAS of mice expressing GCaMP3 in ICC (Kit-GCaMP3) and smooth muscle cells (SMC; smMHC-GCaMP3) with an Olympus DSU.

Results: Dual labeling immunohistochemistry revealed ANO1 expression in ICC but not SMC while gene expression of ANO1 (Ano1) was 26× greater in FACS-sorted ICC than in sorted SMC. In contrast, gene expression of Cav_L (Cacna1c) was only 2× greater in SMC than in ICC. Calcium transients were visualized in Kit-GCaMP3⁺ cells in the circular muscle layer of the IAS. Kit-GCaMP3⁺ cells exhibited rhythmic whole cell Ca²⁺ transients that occurred at the same frequency as the SWs we have previously recorded from this muscle. The activity of adjacent Kit-GCaMP3⁺ cells (<200 µm) was synchronized suggesting coupling between ICC-IM. Rhythmic calcium transients in Kit-GCaMP3⁺ cells were abolished by removal of extracellular calcium, by the Cav_L blocker nicardipine (1 µM) and the ANO1 blocker CaCCinh-A01 (3–10 µM). SWs and phasic contractions were also abolished by these blockers. Like SWs, Ca²⁺ transients in both smMHC-GCaMP3⁺ and Kit-GCaMP3⁺ cells were greatest in frequency at the distal extremity of the IAS and declined in the proximal direction. However, when the distal edge of the IAS was removed, both SWs and Ca²⁺ transients persisted in the remaining muscle although there was a reduction in frequency.

Conclusions: This study provides the first direct evidence that ICC-IM are the pacemaker cells of the IAS. The properties of Ca²⁺ transients in Kit-GCaMP3⁺ and smMHC-GCaMP3⁺ cells are commensurate with the electrical and contractile properties of this muscle. The ability of nicardipine and CaCCinh-A01 to abolish tone, SWs and rhythmic calcium transients in Kit-GCaMP3⁺ cells suggests a causal relationship between these events. The importance of the distal edge of the IAS in regulating phasic contractions in this muscle is underscored by the decline in both SW and Ca²⁺ transient frequency and amplitude in the proximal direction indicating that like the heart there is a dominant site of pacemaker activity in the IAS. The observation that the greatest frequency of these events is at the distal edge suggests an important function in ensuring fecal continence.

Funding Source(s): NIH DK078736

Keywords: Interstitial cells; internal anal sphincter (IAS); gene expression; immunostaining; Ano1