Introduction

Interstitial cells of Cajal (ICC) are Kit positive cells found throughout the gastrointestinal tract. ICC generate rhythmic intracellular Ca2+ signals that drive phasic contractions of the gut and maintain key digestive processes, like peristalsis and maintenance of tone (Koh et al., 2022). Studies in rodents identified the Ca2+-activated Cl- channel, Anoctamin (Ano1), as a key driver for the rhythmic ICC signalling responsible for gut pacemaking (Hwang et al., 2009).  However, functional significance of Ano1 in human gastrointestinal tract is yet to be revealed. Gut motility disorders affect millions worldwide, therefore understanding the molecular mechanisms controlling gut motility in humans, in comparison to other mammals, may drastically improve clinical outcomes for sufferers of these conditions (Wetherill & Sutcliffe, 2014).

Aims

This research aims to investigate the role of ICC and Ano1 in human gut through comparisons of expression and functional activity of Ano1 in human left colon and rat proximal colon.

Methods

Samples of smooth muscle from left colon of 3 paediatric patients were obtained from stoma closure surgeries. Proximal colon samples from 9 Wistar rats of both genders were also taken. Circular sections with dimensions of 2x5mm were cut and placed into a 50ml organ bath at 37ºC for isometric contraction measurements and the effects of voltage-gated Na2+ channel blocker, tetrodotoxin (TTX; 1µM) and a selective Ano1 inhibitor, Ani9 (≤10µM) were recorded. Sections of smooth muscle from the same areas in both human and rat were fixed onto slides, and immunostained using anti-Kit and anti-Ano1 antibodies. Ano1 transcript levels in human and rat proximal colon were analysed using reverse-transcription polymerase chain reaction (RT-PCR). Human tissue experiments are covered under REC reference 14/NS/0018 and IRAS number 92423.

Results

In rats, application of TTX removed enteric modulation and significantly increased (p<0.05) contractile intensity. The addition of Ani9 in increasing concentrations (0.1-3µM) led to a concentration-dependent decrease in the intensity and frequency of contractions, until contractions stopped completely. In human tissue, adding Ani9 also caused a significant (p<0.02) decrease in the frequency of contractions, however the effects did not manifest until higher concentrations of Ani9 (3µM) and there was no significant change in the intensity of contractions. The blocking effect by Ani9 in human tissue was incomplete even at 10µM, unlike in rodents. The contraction frequency was reduced from 4.3±1.0mHz (control) to 1.8±0.1mHz (10µM Ani9; n=3; p<0.02).

Imaging studies revealed similar patterns of Kit positive ICC in human and rodent tissue with a strong colocalization between Kit and Ano1 immunoreactivity. Presence of Ano1 in the colon of either species was confirmed by RT-PCR.

Conclusion

This study reports functional importance of Ano1 for rhythmic contractility of human colon. Immunofluorescence imaging revealed strong similarities between localisation and expression patterns of ICC and Ano1 in human and rodent tissue. The contractility measurements uncovered strong contributions of Ano1 to human colon rhythmicity, albeit it not as strong as in rat. This study paves way towards better understanding of molecular mechanisms of human gut contractility.